Large Animal Review - Year 26, Number 2, April 2020

0A_Copert LAR 2_2020_ok 27/04/20 16:47 Pagina 2

Poste Italiane spa - Spedizione in A.P. - D.L. 353/2003 (conv. in L. 27/02/2004 N. 46) art. 1, comma 1, DCB Piacenza - Concessionaria esclusiva per la pubblicità: E.V. Soc. Cons. a r.l. - Cremona

02/20

Bimonthly, Year 26, Number 2, April 2020

LAR Large Animal Review

ISSN: 1124-4593

LARGE ANIMAL REVIEW is ranked in Citation Index (SciSearch®) Journal Citation Reports/Science Edition and CAB ABSTRACTS

ORIGINAL ARTICLES BOVINE • Metabolic adaptation in first week after calving and early prediction of ketosis type I and II in dairy cows • Effects of different mineral supplementation programs on beef cattle serum Se, Zn, Cu, Mn concentration, health, growth performance and meat quality OVINE • The effect of final weight on slaughtering and carcass quality characteristics of lambs in concentrate-based or pasturebased production systems • Enzootic pneumonia in sheep: ewe and lamb immune response after Mannheimia haemolytica vaccine administration under field condition in Italy • Infezione da Ovine herpesvirus tipo 2 in allevamenti ovini in Umbria EQUINE • Cuboni Reaction: non-invasive late pregnancy test in Martina Franca jennies REVIEWS BOVINE • Genomics of subacute ruminal acidosis CASE REPORTS CAMELID • A case of epiploic foramen entrapment of jejunal intestinal tract in an alpaca (Vicugna pacos) cria

SOCIETÀ ITALIANA VETERINARI PER ANIMALI DA REDDITO ASSOCIAZIONE FEDERATA ANMVI

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INDEX

ORIGINAL ARTICLES

Anno 26, numero 2, Aprile 2020 Rivista indicizzata su: CAB ABSTRACTS e GLOBAL HEALTH IF (2017/2018): 0.26

N

BILJANA DELIĆ, BRANISLAVA BELIĆ, MARKO CINCOVIĆ, RADOJICA DJOKOVĆ, IVANA LAKIĆ

Editor in chief: Massimo Morgante

Technical Editor: Enrico Fiore

CARLO ANGELO SGOIFO ROSSI, SILVIA GROSSI, RICCARDO COMPIANI, GIANLUCA BALDI, MARIA AGOVINO, LUCIANA ROSSI 57

OVINE

l

LARGE ANIMAL REVIEW è una rivista bimestrale pubblicata per favorire l’aggiornamento dei veterinari che si dedicano alla prevenzione e alla cura delle malattie degli animali da reddito e alla qualità e salubrità dei prodotti derivati.

Direttore Responsabile Antonio Manfredi

The effect of final weight on slaughtering and carcass quality characteristics of lambs in concentrate-based or pasture-based production systems BULENT EKIZ, ALPER YILMAZ, HULYA YALCINTAN, OMUR KOCAK, MUSTAFA OZCAN

67

Enzootic pneumonia in sheep: ewe and lamb immune response after Mannheimia haemolytica vaccine administration under field condition in Italy

Consiglio direttivo SIVAR 2020-2023 Mario Facchi (Presidente) Daniele Gallo (Presidente Senior) Alberto Ferrero (Vice-Presidente) Michela Conterbia (Segretario) Vito Loconte (Tesoriere) Alessandro Federici (Consigliere) Osvaldo Parolin (Consigliere) Chiara Musella (Consigliere) Mattia Bottacini (Consigliere) Giuseppe Argiolas (Consigliere) Edizioni SCIVAC Palazzo Trecchi - 26100 Cremona Tel. 0372/460440 Iscrizione registro stampa del Tribunale di Cremona n. 299 del 25/9/1995

51

Effects of different mineral supplementation programs on beef cattle serum Se, Zn, Cu, Mn concentration, health, growth performance and meat quality

Editorial Board 2019-2021: Anna Rita Attili - Roberto Bardini Francesca Bonelli - Marta Brscic Marco Colombo - Vincenzo Cuteri Antonella Dalle Zotte - Enrico Fiore Giovanni Franzo - Matteo Gianesella Elisabetta Giudice - Paolo Moroni Davide Ranucci - Antonia Ricci Giuseppe Stradaioli - Erminio Trevisi Managing Editor: Matteo Gianesella

BOVINE Metabolic adaptation in first week after calving and early prediction of ketosis type I and II in dairy cows

CRISTINA PESCA, KATIA FORTI, ANDREA FELICI, ELEONORA SCOCCIA, CLAUDIO FORTE, PIETRO ANTENUCCI, SABINA MUNTONI, LUCIA ANZALONE, ANTONELLA DI PAOLO, SILVIA CROTTI 73

Infezione da Ovine herpesvirus tipo 2 in allevamenti ovini in Umbria ORIANA RAFFAELE, EMILIA DEL ROSSI, LORENZO CASTELLI, STEFANO PIGNANI, STEFANO PETRINI, MARIA TERESA MANDARA, MARIA LUISA MARENZONI 79

z

EQUINE Cuboni Reaction: non-invasive late pregnancy test in Martina Franca jennies

Stampa Press Point - Via Cagnola, 35 20081 Abbiategrasso (MI) - Tel. 02/9462323

ROBERTA BUCCI, BRUNELLA ANNA GIANGASPERO, MICHELA D’ANGELO, DOMENICO ROBBE, PATRIZIA PONZIO, ANNA CHIARA MANETTA, LORELLA DI GIUSEPPE, IPPOLITO DE AMICIS 87

Spedizione Poste Italiane SPA - Spedizione in A.P. D.L. 353/2003 (Conv. in L. 27/02/2004 N. 46) Art. 1, Comma 1, DCB Piacenza

REVIEWS

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Palazzo Trecchi - 26100 Cremona Ufficio Pubblicità: Paola Orioli Tel. 0372/403539 - E-mail: info@sivarnet.it Prezzo di copertina: € 10,00.

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BOVINE Genomics of subacute ruminal acidosis MUHAMMAD IRFAN MALIK, MUHAMMAD AFZAL RASHID

93

CASE REPORTS CAMELID

A case of epiploic foramen entrapment of jejunal intestinal tract in an alpaca (Vicugna pacos) cria LAKAMY SYLLA, MARTINA CROCIATI, DOMENICO CAIVANO, VASILICA FLORY PETRESCU, LORENZO PISELLO, CALOGERO STELLETTA 99

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IL PORTALE DEL VETERINARIO

DI FIDUCIA

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Tutti i dati vengono trattati ai sensi della normativa sulla privacy. Il portale contiene: – notizie – materiali didattici – DES (Database Epidemiologico Sanitario) – DDD (Database per il Monitoraggio degli Antibiotici) – forum di discussione

DES

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DEFINED DAlLY DOSE Software sperimentale che consente di calcolare le quantità di antibiotici somministrati sui propri allevamenti

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B. Delić et al. Large Animal Review 2020; 26: 51-55

Metabolic adaptation in first week after calving and early prediction of ketosis type I and II in dairy cows

51

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BILJANA DELIĆ1, BRANISLAVA BELIĆ1, MARKO CINCOVIĆ1, RADOJICA DJOKOVĆ2, IVANA LAKIĆ1 1

Department of Veterinary Medicine, Laboratory of pathophysiology, Faculty of Agriculture, University of Novi Sad, Trg Dositeja Obradovića 8, Novi Sad, Serbia 2 Department of Animal Science, Faculty of Agronomy, Čačak, University of Kragujevac, Cara Dušana 34, Čačak, Serbia

SUMMARY The aim of this study is to determine differences in metabolic adaptation in first week after calving between healthy and cows that develop ketosis type I (diagnosed 3-6 weeks after calving) or II (diagnosed 2-3 weeks after calving) after calving. Experiment included 50 healthy cows, 50 ketosis I type cows and 50 ketosis II cows. Animals were selected retrospectively form 435 Holstein cows that were constantly monitored. Monitoring is regular procedures during all season. Blood samples were taken in first week after calving. Concentration of ketone body was measured every two day by test stripes from the end of 1st until the end of 6th week. Any color change in test strip (5 mg/dL, trace, or higher) was indicator of ketosis in cows. Cows were clinically evaluated to determine any clinical symptoms (reduced appetite, rumen atony, behavioral changes). In cows with ketosis type I were noted higher concentrations of nonesterified fatty acid (NEFA) and lower concentrations of glucose and insulin compared to control group. Higher concentrations of beta hydroxybutyrate (BHB), tumor necorsis factor alpha (TNF-α) and total bilirubin were noted in cows with ketosis type II compared to control group. Value of revised quantitative insulin sensitivity check index (RQUICKI) was lower, and aspartate aminotransferase (AST) was higher in blood of ketosis II cows compared to ketosis I cows. Prediction of ketosis type I is significant by logistic regression model which include insulin, NEFA and glucose as independent predictor (area under ROC curve, AUC=0.78, p<0.05). Possibility of ketosis I development increases with NEFA increase and decrease of glucose and insulin concentrations. Prediction of ketosis type II development is significant by logistic regression model which include BHB, TNF-α and total bilirubin where increase of these parameters indicates higher possibility of ketosis II development (AUC=0.87, p<0.01). Differentiation of ketosis type I and II is significant by logistic regression model which include value of RQUICKI and AST (AUC=0.74, p<0.05), so with increasing of AST and decreasing of RQUICKI in first week of lactation increase risk for type II ketosis. Percentage of variation in the metabolic parameters that is predictable from the BHB in first week after calving was significantly higher in ketotic (10.9-18.5%) then in healthy cows (2.5-9.1%). Cows in ketosis type I and II show different metabolic adaptations in first week after calving. These differences allow prediction of development a exact type of ketosis. Metabolic adaptation in function of ketogenesis was developed in first week after calving, early before manifestation of ketosis.

KEY WORDS Cows, ketosis, inflammation, insulin resistance, liver function.

INTRODUCTION Ketosis is significant metabolic disorder of cows that is developed as a consequence of negative energetic balance and it is presented with higher concentrations of ketone bodies in blood, milk and urine. Subclinical ketosis may be diagnosed when blood serum BHB concentrations are above 1.2 mmol/L, and clinical ketosis with blood BHB level above 2.6 mmol/L as gold standard test1.

Basal metabolic adaptations in cows that develop ketosis are: higher concentrations of BHB, higher concentrations of NEFA, lower concentrations of glucose, higher concentrations of liver enzymes and bilirubin, disturbances in macro and micro elements, greater values of inflammatory markers, oxidative stress and insulin resistance. It can be followed

Corresponding Author: Marko R. Cincović (mcincovic@gmail.com).

with abomasal dislocation, mastitis, metritis, laminitis and behavioral changes2-4.

On pathohysiologic aspect, ketosis can be divided into ketosis type I and II5-7. Type I is developed 3-6 weeks after calving while type II develops 1-3 weeks after calving and it is tightly related to fatty liver. Ketosis I develops as a consequence of underfeeding and increase in milk production, while ketosis II is developed as a consequence of homeorhetic processes in early lactation. Use of some biochemical markers from blood and milk can be predicted development of ketosis in cows. Milk parameters may be also used for objective ketosis diagnosis8-10. Chuang et al.11 have been determined differences in values of some laboratory parameters in blood between ketosis I and II measured from moment of calving until fourth week of lactation.

Early prediction of ketosis in the first week after calving may be possible. Classification of cows according to lipolysis, ketogenesis or activity of anabolic hormones in first week after calving shows metabolic differences between the classes for seve-

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52

Metabolic adaptation in first week after calving and early prediction of ketosis type I and II in dairy cows

ral consecutive weeks after calving. Kessel et al.12 noted that retrospectively classified cows with high BHB concentration (over 1 mmol/L) have higher concentrations of BHB, NEFA, AST, GGT and lower concentrations of IGF-I in first 14 weeks after calving. Cincović et al.13 founded that intensive lipolysis and ketogenesis in first week before and after calving cause lower concentrations of glucose, total proteins, cholesterol and urea and higher concentrations of bilirubin and AST compared to cows in control group. Cows loaded with metabolic stress (high catabolic and low anabolic blood indicators) showed significant difference in metabolic adaptation in relation to control group: higher values of STH, BHB, higher values of bilirubin, AST, ALT, GGT, AP and MDA and lower levels of glucose, total proteins, albumin and body condition14.

The aim of this study is to determine differences in metabolic adaptations in first week after calving in ketotic cows type I and II and to determine possibility of prediction of ketosis development and type of ketosis with metabolic parameters in first week after calving. The aim is, also, to determine if there is any correlation of metabolic parameters and BHB and to determine differences in function of type of ketosis in cows in first week after calving.

termined values were read on reader RT-2100 C (Rayto, PRC).

Insulin resistance indices were calculated by standard methodology16: RQUICKI = 1/[log (conc.t0 glucose mg/dL) + log (conc.t0 insulin mmol/L)+log (conc.t0 NEFA mmol/L)].

Statistics: Differences in concentrations of metabolic parameters and indexes of insulin resistance between groups were determined by ANOVA analysis and post hock LSD-test. Possibility of ketosis diagnosis was determined by multiple logistic regressions in which model was included metabolic parameters that were showed significant differences in LSD-test.

Odds ratio (OR) and area under ROC curve (AUC) were determined for every model. Model that questions possibility of differential diagnosis of ketosis type I and II was made with metabolic parameters that are different in ketosis I and II by multiple logistic regressions. Early changes in metabolism in function of ketogenesis were determined by coefficient of correlation and determination (percentage of variation) in linear model between BHB and other metabolic parameters concentrations in first week after calving. Using the Fisher rto-z transformation, will be calculate a value of z that can be applied to assess the significance of the difference between two correlation coefficients (healthy vs. ketosis type I; healthy vs. ketosis type II and ketosis type I vs. ketosis type II).

MATERIALS AND METHODS RESULTS Animals and model: 150 animals were included in experiment: 50 healthy, 50 cows in type I ketosis and 50 cows in type II ketosis. Animals were selected retrospectively form 435 Holstein cows that were constantly monitored. Monitoring is regular procedures during all season. Cows were from second to fourth parity. During the early lactation the daily ration consisted of 4 kg alfalfa hay, 15 kg maize silage (30%DM), 8 kg alfalfa haylage, 4 kg ear maize silage (68%DM), 2 kg dry sugar beet pulp, 2 kg extruded soybean meal, and 4.5 kg concentrate (18%CP), NEL 153 MJ. Energy balance was evaluated according to body weight, offered meal and average milk production using NRC [2001] standards15. Blood samples were taken in first week after calving.

From the end of the 1st to the end of the 6th week concentration of ketone body in urine was measured every two day by test stripes (Ketostix, Bayer, GER). Any color change in test strip (5 mg/dL, trace, or higher) was indicator of ketosis in cows. Cows were clinically evaluated to determine any clinical symptoms (reduced appetite, rumen atony, behavioral changes). Healthy cows did not show clinical symptoms of ketosis and urinary ketone bodies were not diagnosed in those cows. In cows with clinical symptoms and moderate or large color change on Ketostix was determined blood BHB concentration. Concentration of BHB >1.2 mmol/L in blood was cut-off value for final diagnosis of ketosis1. Based on the time when ketosis occurred, cows were classified into ketosis type I (diagnosed 3-6 weeks after calving), ketosis type II cows (diagnosed 2-3 weeks after calving) and healthy cows.

Blood sample analyses: In first week after calving concentrations of BHB, NEFA, glucose, total proteins, albumins, bilirubin, AST, GGT, cholesterol, triglycerides were determined by biochemical colorimetric kits (Biosystem, SP ans Randox, UK). Concentrations of observed parameters were determined by the Chemray analyzer (Rayto, PRC). Insulin concentration was determined by ELISA method (Cusabio, PRC) just like TNF-α concentration (Cloud-Clone Corp., USA). De-

In first week after calving differences in metabolic adaptations in cows with different type of ketosis were found (Table 1). Higher concentrations of BHB, TNF-α and total bilirubin were founded in cows that developed ketosis type II in comparison to control group. Higher concentrations of NEFA and lower concentrations of glucose and insulin were noted in cows that developed ketosis type I compared to control group during first week after calving. Determination of ketosis type I or II by metabolic parameters can be done by lower RQUICKI index and higher AST levels in blood of cows that develop ketosis type II. Prediction of ketosis type I is significant by logistic regression model which include insulin, NEFA and glucose as independent predictor (area under ROC curve, AUC=0.78, p<0.05).

Possibility of ketosis I development increases with NEFA increase and decrease of glucose and insulin concentrations.

Prediction of ketosis type II development is significant by logistic regression model which include BHB, TNF-α and total bilirubin where increase of these parameters indicates higher possibility of ketosis II development (AUC=0.87, p<0.01).

Differentiation of ketosis type I and II is significant by logistic regression model which include value of RQUICKI and AST (AUC=0.74, p<0.05), so with increasing of AST and decreasing of RQUICKI in first week of lactation increase risk for type II ketosis. Results are presented in Table 2, Figure 1 and 2.

Linear correlation analysis between BHB and metabolic parameters was analysed in first week after calving. Percentage of variation in the metabolic parameters that is predictable from the BHB in first week after calving was significantly higher in ketotic (10.9-18.5%) then in healthy cows (2.59.1%). We found statistically significant difference in coefficient correlation between healthy and ketotic cows. Those results indicated that metabolic pathways are changed in function of ketogenesis in first week after calving and before ketosis manifestation.

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B. Delić et al. Large Animal Review 2020; 26: 51-55

DISCUSSION Ketosis development in cows and its early prediction is researched with different models. All models showed that great significance in ketosis prediction have values of NEFA, AST, glucose and other parameters which is in compliance with

our results. Asl et al.8 founded that cut-off point for NEFA concentrations of 0.26 mmol/L can be used during early lactation for diagnosis of subclinical ketosis in first 8 weeks after calving. Sun et al.9 founded that thresholds were more than 0.76 mmol/L for NEFA, more than 104 U/L for AST, less than 140 U/L for cholinesterase and more than 3.3 µmol/L

Table 1 - Blood parameters in healthy cows and cows with ketosis type I and II. Parameter

Healthy control

Ketosis I

Ketosis II

Significance

BHB (mmol/L)

0.52±0.19a

0.71±0.16b

0.89±0.14b

* ** ** * **

a

b

NEFA (mmol/L)

0.49±0.18

0.71±0.2

0.54±0.19ab

Glucose (mmol/L)

2.51±0.35a

2.13±0.36b

2.4±0.32a

a

a

T. bilirubin (µmol/L)

5.6±2.1

AST (IU/L)

94.5±20.5ab

8.9±2.4b

6.2±2.9

89±19.1a

a

a

119.3±21.2b

GGT (IU/L)

33.5±2.5

32.4±1.7

34.3±3.2a

NS

Albumin (g/L)

33.6±2.1a

32.4±1.7a

31.2±2.6a

NS

a

a

a

T. Protein (g/L)

62±4.4

59±3.9

58±3.8

Cholesterol (mmol/L)

3.01±0.29a

2.92±0.18a

2.86±0.24a

a

a

NS NS

a

Triglycerides (mmol/L)

0.14±0.014

0.14±0.017

0.13±0.016

NS

TNF-α (ng/mL)

0.78±0.18a

0.83±0.19ab

1.01±0.17b

Insulin (µIU/L)

4.5±1.4a

3.1±1.5b

4.3±1.6ab

RQUICKI

0.51±0.015ab

0.52±0.016a

0.50±0.014b

** * **

NS: p > 0.05; *: p < 0.05; **: p < 0.01. Means followed by different letters differ significantly at p < 0.05.

Table 2 - Combination of biomarkers in evaluation of ketosis I and II development.

Estimated

OR

% of explained variance

Model significance

Ketosis type I from Healthy control Insulin

-0.2

1.1-1.6

NEFA

0.14

1.5-2.8

Glucose

-0.78

0.9-1.8

76%

**

82%

***

71%

*

Ketosis type II from Healthy control BHB

0.21

1.8-3.1

TNF-α

0.52

1.9-2.8

T. bilirubin

1.3

1.5-2.6 Ketosis type I from Ketosis type II

RQUICKI AST

-0.092

1.05-1.7

8.9

1.2-1.8

53

*: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 1 - Prediction of ketosis I and II development and prediction of ketosis.

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54

Metabolic adaptation in first week after calving and early prediction of ketosis type I and II in dairy cows

Figure 2 - RQUICKI and AST in cows in ketosis I and II.

for total bilirubin. Cao et al.17 confirmed significance of glucose, AST and NEFA levels in evaluation of healthy and ketotic cows (GLU >3.04 mmol/L, AST <100 U/L, and NEFA <0.82 mmol/L). In listed experiments cut-off values are calculated on the moment of ketosis diagnosis no matter the type of ketosis. Unlike these studies, our experiment examined possibility of ketosis prediction based on values of blood parameters in first week after calving. Comparison of AUC value in our and mentioned studies showed that our model (that considers values of parameters in first week after calving) with similar efficiency determines ketotic cows just like model that includes single blood parameters (measured in the moment of ketosis diagnosis). Parity effect on metabolic parameters was not significant in our research. The lack of influence of parity is in compliance with the results found by Fiore et al.18.

Cows in ketosis type I have higher concentrations of BHB and NEFA and lower concentration of insulin and glucose compared to control group. Ketosis type I is developed 3-6 weeks after calving as a consequence of reduced food intake.

Experiment of Bjerre-Harpøth et al.19 showed significant increase of NEFA and reduction of insulin and glucose in cows that had food restriction in early lactation. Similar metabolic changes were found in our trial. More recent results show that decreased dry matter intake in the prepartum period is a significant predictor of ketosis in cows, with the additional finding that cows in ketosis had lower dry matter intake du-

ring postpartal period20. Our results are consistent with characteristics of ketosis I that are described in classification of the authors cited6-8.

Cows in ketosis type II have higher concentration of BHB, total bilirubin and TNF-α compared to healthy cows and/or ketosis type I type group. Oetzel7 showed that fatty liver is the main characteristic of ketosis II type. Mostafavi et al.21 noted that concentrations of BHB, NEFA, AST and NEFA/cholesterol relation are very specific in evaluation of fat liver development in cows. The increase in AST and NEFA concentrations during the postpartum period is considered a suitable indicator of ketosis associated to hepatic steatosis or liver damage22,23.

Recent studies showed that cows in ketosis have higher concentrations of TNF-α24. Aplication of ketoprofen as non-steroid anti-inflammatory drug in cows during early lactation decrease sign of liver inflammation and AST activity in blood25. Concentration of TNF-α was higher in monocyte cell culture, during cultivation with BHB26. High expression of TNF-α in the liver is upregulated by interleukin-8 (IL-8) and IL-1β secreted from placenta, which is liver-related metabolic disorder mechanism27. Excessive inflammation in periparturient period is in basis of ketosis in dairy cows28.

Comparison of ketosis type showed that cows that developed ketosis type II had greater activity of AST and lover value of RQUICKI index compared to ketosis type I group of cows.

Higher degree of insulin resistance (reflected in lower RQUICKI index) with higher AST level indicated development of ketosis II type. Chuang et al.11 founded higher concentrations of BHB, NEFA, glucose, bilirubin, AST and ALT in ketosis II type compared to ketosis type I. Xu et al.29 noted lower RQUICKI index in ketotic cows compared to control group. Djoković et al.3 showed that insulin resistance indexes that are measured in basal conditions significantly affect dynamical changes in concentrations of insulin, glucose and NEFA in ketotic cows.

Possibility of early prediction of ketosis development and type of ketosis in cows by metabolic markers in first week after calving was proved in this research. In model of logistic regression given markers showed higher risk for ketosis development and question of correlation of these markers with

Table 3 - Coefficient of determination of significant ketosis predictors with BHB in first week after calving.

Type 2

Healthy control

Ketosis type I

Ketosis type II

TNF-α

2.5%

15.2%

11.3%

T. bilirubin

6.4%

14.5%

12.6%

Insulin

8.4%

14.6%

16.4%

NEFA

9.1%

15.4%

18.5%

Glucose

2.4%

13.1%

15.7%

RQUICKI

6.3%

11.3%

13.8%

AST

5.2%

10.9%

11.7%

NS: p > 0.05; *: p < 0.05; **: p < 0.01.

Ketosis type I: Healthy control

Ketosis type II: Healthy control

Ketosis type I: Ketosis type II

** ** ** * ** ** **

* ** ** ** ** ** **

NS NS NS NS NS NS NS

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B. Delić et al. Large Animal Review 2020; 26: 51-55

BHB had appeared. Healthy cows have lower degree of commonly explained variation between BHB and parameters in healthy then in ketotic cows. That was founded in this research and indicates early adaptation of metabolism in ketosis function in cows. Results of Djoković et al.30 showed that in early lactation correlation of BHB with insulin and other indicators of functional status of liver is not in function of energetic balance in cows that indicates importance of BHB in metabolic adaptations of cows in early lactation.

10.

11.

12.

13.

CONCLUSIONS Cows in ketosis type I and II show different metabolic adaptations in first week after calving. These differences allow prediction of development a exact type of ketosis. Ketosis type I is determined by values of glucose, insulin and NEFA (lipolysis and insulin secretion). Ketosis type II is determined by values of BHB, TNF-α and bilirubin (ketogenesis, inflammation and functional status of liver). Differentiation of ketosis type I and II is possible by RQUICKI and AST (insulin resistance and hepatocytes damage). Proportion of the variation of metabolic parameters that is predictable from the BHB was higher in ketotic than in healthy cows. Metabolic adaptation in function of ketogenesis was developed in first week after calving, early before manifestation of ketosis.

14.

15.

16.

17.

18.

19.

20.

ACKNOWLEDGEMENT This research is supported by projects “Influence and clinical evaluation of blood serum tumor necrosis factor alpha (TNF-α) in inflammatory response of ruminants and dogs” - Provincial secretariat of high education and science, Vojvodina, Serbia.

21.

22.

23.

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3. Djoković R., Dosković V., Cincović M., Belić B., Fratrić N., Jašović B., Lalović M. (2017). Estimation of Insulin Resistance in Healthy and Ketotic Cows during an Intravenous Glucose Tolerance Test. Pak Vet J, 37: 387-392.

4. Rodriguez-Jimenez S., Haerr K.J., Trevisi E., Loor J.J., Cardoso F.C., Osorio J.S. (2018). Prepartal standing behavior as a parameter for early detection of postpartal subclinical ketosis associated with inflammation and liver function biomarkers in peripartal dairy cows. J Dairy Sci, 101: 8224-8235.

5. Holtenius P., Holtenius K. (1996). New aspects of ketone bodies in energy metabolism of dairy cows: a review. J Vet Med Series A, 43: 579-587.

6. Herdt TH. (2000). Ruminant adaptation to negative energy balance: Influences on the etiology of ketosis and fatty liver. Vet Clin North Am Food Anim Pract, 16: 215-230.

7. Oetzel G.R. (2007). Herd-level ketosis-diagnosis and risk factors. In Proceedings of the 40th annual conference of bovine practitioners, Vancouver, Canada. pp. 67-91.

8. Asl A.N., Nazifi S., Rowshan Ghasrodashti A., Olyaee A. (2011). Prevalence of subclinical ketosis in dairy cattle in the Southwestern Iran and detection of cutoff point for NEFA and glucose concentrations for diagnosis of subclinical ketosis. Prev.Vet. Med., 100: 38-43.

9. Sun Y., Wang B., Shu S., Zhang H., Xu C., Wu L., Xia C. (2015). Critical thresholds of liver function parameters for ketosis prediction in dairy

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Cincović M.R., Belić B., Radojičić B., Hristov S., Ðoković R. (2012). Influence of lipolysis and ketogenesis to metabolic and hematological parameters in dairy cows during periparturient period. Acta veterinaria (Beograd). 62: 429-444.

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Mostafavi M., Seifi H.A., Mohri M., Jamshidi A. (2013). Optimal thresholds of metabolic indicators of hepatic lipidosis in dairy cows. Rev Méd Vét, 164: 564-571.

Fiore E., Barberio A., Morgante M., Rizzo M., Giudice E., Piccione G., Marcello L., Gianesella M. (2015). Glucose infusion response to some biochemical parameters in dairy cows during the transition period.

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Fiore E., Perillo L., Piccione G., Gianesella M., Bedin S., Armato L., Giudice E., Morgante M. (2016). Effect of combined acetylmethionine, cyanocobalamin and α-lipoic acid on hepatic metabolism in high-yielding dairy cow. J Dairy Res, 83: 438-441.

Zhang G., Hailemariam D., Dervishi E., Goldansaz S.A., Deng Q., Dunn S.M., Ametaj B.N. (2016). Dairy cows affected by ketosis show alterations in innate immunity and lipid and carbohydrate metabolism during the dry off period and postpartum. Res Vet Sci, 107: 246-256.

Kovačević Z., Belić B., Cincović M.R., Stojanac N., Stevančević O., Erdeljan M., Davidov I., Stojanovic, D. (2019). Effects of Ketoprofen Administration on Relation between Acute Phase Proteins and Metabolic Parameters in Cows during Early Lactation. Acta Sci Vet, 47: 1663. DOI: 10.22456/1679-9216.91667.

Jain S.K., Kannan K., Lim G., McVie R., Bocchini J.A. (2002). Hyperketonemia increases tumor necrosis factor-α secretion in cultured U937 monocytes and type 1 diabetic patients and is apparently mediated by oxidative stress and cAMP deficiency. Diabetes, 51: 2287-2293.

Loor J.J., Dann H.M., Everts R.E., Oliveira R., Green C.A., Guretzky N.A.J., Rodriguez-Zas S.L., Lewin H.A., Drackley J.K. (2005). Temporal gene expression profiling of liver from periparturient dairy cows reveals complex adaptive mechanisms in hepatic function. Physiol. Genomics., 23: 217-226.

Mezzetti M., Minuti A., Piccioli-Cappelli F., Amadori M., Bionaz M., Trevisi, E. (2019). The role of altered immune function during the dry period in promoting the development of subclinical ketosis in early lactation. J Dairy Sci, 102: 9241-9258.

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C.A. Sgoifo Rossi et al. Large Animal Review 2020; 26: 57-64

Effects of different mineral supplementation programs on beef cattle serum Se, Zn, Cu, Mn concentration, health, growth performance and meat quality

57

N

CARLO ANGELO SGOIFO ROSSI1, SILVIA GROSSI1, RICCARDO COMPIANI1, GIANLUCA BALDI1, MARIA AGOVINO2, LUCIANA ROSSI1 1

University of Milan, Faculty of Veterinary Medicine, Department of Veterinary Science for Health, Animal Production and Food Safety, Milan 20133, Italy 2 European ruminant manager, Alltech, Casalecchio di Reno 40033, Italy

SUMMARY The effects of different mineral sources (organic vs inorganic) on beef cattle serum Se, Zn, Cu, Mn concentration, health, growth performance and meat quality (hot carcass weight, dressing percentage, conformation score, fattening score and pH) were assessed in 156 intact Charolaise males. The aim of the study was to investigate a possible ameliorative action of the organic sources of those minerals compared to the inorganic ones, essential for animal welfare and productivity.

The animals were allotted to three treatment groups, receiving different sources (organic or inorganic) of minerals at different levels. H-In group received a mineral supplementation from an inorganic source according to the average mineral inclusion recommended by the European beef system. The TRT group received an organic source of minerals at the doses recommended by the producer (TRT, Total Replacement Technology, Alltech). The In group received minerals from an inorganic source, but following the same doses employed in the TRT group.

Growth performance and health status were monitored throughout the fattening period (186 days). Blood samples were evaluated in relation to mineral serum concentrations, antioxidant status and immunity reactions. After slaughtering, carcass characteristics and meat quality of Longissimus dorsi samples were evaluated.

Growth performance and health status were improved in the TRT group (P<0.05). The incidence of Bovine Respiratory Disease was also lower in this group. The animals in the TRT group had a better immune response (P<0.05) due to the higher circulating mineral concentrations which positively affected the immune function. In addition, carcass characteristics were positively affected in the TRT group because of the enhanced myogenesis (P<0.05). Meat quality was improved in the TRT group due to the higher level of antioxidants in the meat (P<0.05).

The results suggest that organic sources have a strong impact on animalsâ&#x20AC;&#x2122; metabolism and immune function, which led to an improvement in growth performance, health and antioxidant status together with carcass and meat quality.

KEY WORDS Cattle, minerals, performance, health, meat quality.

INTRODUCTION Intensive beef cattle production is mainly based on fattening weaned young cattle, which have been moved from the farms of origin. In Europe, these farms are often located in other countries, which are specialized in extensive suckler cow breeding. In Italy young cattle are mainly imported from France. This procedure causes several stressors other than weaning, such as long-distance transport, group mixing, feed and water restrictions. Young cattle also have to adapt to a new environment and feeding conditions in the fattening units1. All these factors promote immunosuppression which can lead to pathogen colonization and proliferation, and cause various diseases. Bovine respiratory disease (BRD) is currently the most common one. In stressful situations, se-

Corresponding Author: Silvia Grossi (silvia.grossi1994@libero.it).

veral nutritive substances such as minerals become essential and supplementation is recommended1.

Mineral supplementation can help cattle recover quickly from transport fever and reactivate ruminal activity, thereby limiting oxidative stress and promoting the reactivity of their immune system2. Supplementation of the main micro-minerals is also required in the fattening period in order to satisfy the animalsâ&#x20AC;&#x2122; requirements and optimize ruminal and metabolic functions; consequently, rumen efficiency and growth performance improve.

Several events can lead to pro-inflammatory conditions in fattening cattle, particularly when the diet is characterized by energy levels that enhance the risk of sub-clinical acidosis and connected pathologies3,4. Mineral supplementation can limit these problems and help the cattle recover quickly, reducing the negative effect of stress on animal performance and growth. Trace minerals such as zinc (Zn), copper (Cu), manganese (Mn) and cobalt (Co), as well as selenium (Se), play an important role in stress, health and growth responses in cattle5. Normally, they are required in very small

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Effects of different mineral supplementation programs on beef cattle serum Se, Zn, Cu, Mn concentration

amounts. However, under stressful situations and in farms with high production standards, they are needed in increasing amounts. Consequently, supplementing the diets with these minerals is necessary to avoid deficiencies.

The type of mineral sources used also has different biological effects. Minerals can in fact be provided as inorganic mineral salts or in organic forms such as chelates or enriched yeast. Due to the higher bioavailability of minerals, supplementing organic complexed minerals, such as Zn, Se, Cu, Mn, Co, may be of further benefit based on their enhanced absorption, retention and biological activity compared to inorganic sources6.

The hypothesis was that the use of organic mineral will lead to an improvement in growth performance and health status of highly stressed cattle in contrast to inorganic supplementation at different levels, due to the higher bioavailability of organic minerals and their immune and antioxidant abilities, since little is known about the effects of organic vs inorganic sources of several basic minerals such as selenium, zinc, copper and manganese. The aim of the trial was to evaluate the effects of different dietary mineral sources (organic or inorganic) and doses on absorption, health, growth performance and meat quality.

Table 1 - Mineral and vitamin supplementation.

Nutrient

H-In

TRT

In

Mn, mg/d

550

180

180

Cu, mg/d

250

140

140

Zn, mg/d

1000

280

280

Se, mg/d

4

3

3

TRT and IN Almost in line with NCR requirements and according to manufacturing recommendations.

H-In comes from an average of several mineral mixes used in Europe and with levels of trace minerals similar to that recommended by NRC for stressed animals.

neral inclusion recommended by the European beef system.

Animals in the TRT group received an organic source of minerals at the doses recommended by the producer (TRT, Total Replacement Technology, Alltech). Animals in the inorganic (In) group received minerals from an inorganic source at the same doses employed in the TRT group the same minerals as the inorganic source but following the same amount as the TRT group.

PARAMETERS MATERIAL AND METHODS Nutritional value of the diets Animals and Farm The study was carried out in an intensive beef fattening unit in northern Italy and involved 156 intact Charolaise males.

The animals were monitored from their arrival from France throughout the entire fattening period of 186 days. Upon arrival, animals were submitted to a typical prophylaxis protocol: vaccination against respiratory diseases, antibiotic treatment if necessary, and treatment against endo- and ecto-parasites.

Samples of the experimental diets (100 g/each) were collected every two weeks, pooled and analysed in relation to dry matter, crude protein, ether extract, starch and ash, according to the AOAC (1990) methods6. Neutral detergent fibre was determined according to Van Soest et al. (1991)8.

The net energy content of the diets, was calculated using the reference values for all feed ingredients reported by NRC (2016)2.

Minerals titration in TMR Animal assignment to treatment groups Three trucks of animals arrived simultaneously. The animals from each truck were randomly allotted to the three different treatment groups in order to avoid the bias due to the batch effect. Each truck was considered as a block, the random assignment to one of the three treatment groups was performed according to the order of that the animals went into the weighing chute.

Feed macro- and trace mineral concentrations (Zn, Se, Mn, Cu) were determined by standard wet chemistry techniques using the titration procedure.

The titration analysis consists in an inductively coupled plasma emission spectroscopy. The selenium concentration was determined using fluoro-metric methods, according to AOAC (1990)6.

Growth performance and health Housing and feeding The cattle were group-fed and housed in pens of 5-6 animals each on a slatted floor. The animals from the three experimental groups were submitted to the same nutritional management and diet. During the entire fattening period two diets were administered, based on the same feedstuffs: silomais, corn meal, brewers, wheat straw, wheat bran, sunflower meal, rice bran, coconut cake, DDGS (distillers dried grains solubles) and rape cake. The first diet was formulated for the adaptation phase (0,68 Mcal/kg; 13,20 CP %DM; 34,73 Starch %DM; 4,40 EE %DM) and the second for the fattening phase (0,89 Mcal/kg; 14,66CP %DM; 46,68 Starch %DM; 4,69 EE %DM). The diets differed only for the mineral premixes employed as reported in Table 1.

Animals in the H-In group received a mineral supplementation from an inorganic source according to the average mi-

On day 0 (arrival), and at days 21, 54 and 186, before the morning feeding, the cattle were individually weighed and the average daily gain (ADG) was calculated. Once a week the average daily feed intake (ADFI) was calculated: before further feed administration, the feed intake was recorded by weighing the TMR offered and the residue in the manger 24h later. The pen feed conversion ratio (FRC) was calculated.

Animals were inspected twice a day by the farm veterinary staff for sickness, including BRD symptoms. Animals were considered affected by BRD if the rectal temperature was â&#x2030;Ľ40.0°C and if both the depression and respiratory character scores differed from the normal health status - score 0 of Baggot et al., 20119. The animals diagnosed as affected by BRD were treated appropriately and registered. The mortality was also recorded.

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Blood parameters Blood samples were collected before the morning feeding from 14 animals on days 0, 21 and 105 to evaluate the immune status. Blood samples were collected by jugular venipuncture. Immunity reaction was evaluated by a BHV-1 (Bovine Herpesvirus-1) serum neutralization test, performed according to OIE (2012)10. The neutralizing antibody titre was measured against the homologous vaccine virus. Antibodies were titrated by the constant virus-varying serum dilution from ½ onward. The data of the neutralizing antibody titres were transformed as log(1/x), where x was the serum dilution. Serum bactericidal activity was performed according to Amadori et al. (1997)11.

Blood samples were also collected on days 0, 21, 105 and 186 to evaluate mineral (Se, Zn, Cu and Mn) serum concentrations and antioxidant status. Blood samples were collected by jugular venipuncture into 10 ml EDTA tubes and 10 ml glass tubes and immediately cooled in ice. Two tubes were collected for each animal, one was used for serum extraction and one for the evaluation of the antioxidant condition. Serum samples were obtained by centrifugation at 3000 rpm for 10 min at 4°C and stored at -20°C. Mineral concentrations were determined in serum samples using an inductively coupled plasma source. The antioxidant condition was evaluated using glutathione peroxidase (GPx activity) titration. GPx activity was measured indirectly by a coupled reaction with glutathione reductase (GR), using the glutathione peroxidase assay kit12.

59

concentration of minerals (Se, Zn, Cu, Mn) was then determined by flame atomic absorption spectrometry, using a spectrometer.

Drip loss analysis was carried out following Honikel (1998)14. After the meat samples were cut from the carcass, they were immediately weighed and placed in the netting and then suspended in an inflated watertight nylon bag. The meat samples were stored at chill temperatures (4°C). In the following seven days, the samples were taken out of the bags, dried by a paper towel and then weighed again. The drip loss was expressed as a percentage of the initial weight.

Every day the instrumental colour parameter, such as L (lightness), a (redness), b (yellowness) were measured for seven days, using a tristimulus colorimeter. Before each measurement, the colorimeter was standardized.

Meat cooking loss was estimated by the Shilling method15.

The samples were weighed and cooked, achieving a temperature of 75°C inside. The samples were then weighed again.

Cooking loss was expressed as a percentage of the initial weight. Thawing loss was also assessed, evaluating the difference in weight before and after thawing. The thawing loss was expressed as a percentage of the initial weight. The tenderness of the samples was also examined, using the WarnerBratzler Shear Force procedure, which measures the force required to cut a piece of meat, according to the AMSA guidelines (1995)16.

The shelf life of the different samples was also evaluated.

Statistical analysis Carcass characteristics and meat quality The main carcass characteristics (hot carcass weight, dressing percentage, conformation score, fattening score and pH) were also evaluated at the slaughterhouse.

Hot carcass weight (HCW) was obtained after the removal of the head, hide, intestinal tract and intestinal organs.

Dressing percentage is the percentage of the animals that ends up as a carcass, and was computed from the following formula: Dress % = (HCW ÷ live weight) × 100% The carcasses were classified according to the official carcass classification scheme, which includes carcass conformation and fattening score. The carcass conformation score was evaluated using the SEUROP classification. The fattening score was evaluated using a 1 to 5 scale (1 - very low, 5 - very high), according to the EU Beef Carcass Classification Scheme.

The pH was measured 24h post-mortem.

In addition, meat quality was evaluated in 10 longissimus dorsi samples per experimental group. 2,5 cm thick steaks were cut from the ribeye (between the 12th thoracic and 5th lumbar vertebra) to evaluate centesimal composition, meat mineral continent, drip loss, instrumental colour, thawing loss, cooking loss, tenderness and shelf life.

The samples were examined for the centesimal composition: moisture, ash, lipid and protein composition were determined, according to official methods6.

Before analysing the meat mineral content, the muscle samples were digested using an ETHOS 900 microwave digestion system. The sample digestion procedure was performed according to the NF EN 13805 standard “Foodstuffs Determination of trace elements -Pressure digestion”13. The

Data were analyzed with the GLM (General Linear Model) procedure of SAS. Student “t” and Tukey tests were used to compare the means of each group. The level of significance to indicate differences stated in the ANOVA model were P<0.05; levels of P≤0.1 were considered a tendency. The d0 values were used as a covariate in the statistical analysis. Weight at d0 was set as a covariate for growth performance and hot carcass weight; meat samples weight at d0 was set as a covariate for meat weight and drip loss.

RESULTS AND DISCUSSIONS Mineral titration in TMR Mineral titration in the different experimental TMR diets, in all the different experimental diets, are reported in Table 2. All the mineral concentrations were higher in the H-IN group, both in the adaptation and the fattening diets. In fact, the mineral quantities supplemented were higher in the H-In group.

Growth performance and health condition The cattle growth performances are reported in Table 3. Average live weight was higher for the TRT group compared to the inorganic (In) group starting from d21 until the end of the fattening cycle. TRT fed cattle showed a higher live weight compared to H-In at d105 and d186. At d186 a statistical difference was also found between H-In and In live weights.

During the critical moment of the adaptation phase, the administration of minerals from an organic source led to a better ADG compared to animals fed with the same amount of minerals but from an inorganic source (ADG d0-21 TRT vs

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Effects of different mineral supplementation programs on beef cattle serum Se, Zn, Cu, Mn concentration

Table 2 - Mineral titration in TMR. Mineral concentrations

H-In adaptation

H-In fattening

TRT adaptation

TRT fattening

IN adaptation

IN fattening

Se mg/kg DM

0.79

0.54

0.62

0.49

0.65

0.46

Zn mg/kg DM

184.24

124.52

82.39

60.12

80.94

62.45

Cu mg/kg DM

43.66

28.64

26.52

21.08

27.88

19.16

Mn mg/kg DM

111.39

68.17

57.39

34.12

58.3

36.28

Table 3 - Growth performance.

n°

Table 4 - Health status.

H-In

TRT

In

52

52

52

H-In

Weight, kg d0

430.48

430.48

430.48

d21

456.19

a

b

d105

b

d186

576.16

b

704.12

458.04

a

586.22

a

714.87

13.46 (7)

5.77 (3)

19.23a (10)

BRD relapses, % (n)

9.61a (5)

0.00b (0)

0.00b (0)

Lameness, % (n)

1.92 (2)

1.92 (2)

1.92 (1)

Mortality, % (n)

5.77 (3)

0.00 (0)

0.00 (0)

572.60

a, b, c

b

In

BRD first pull, % (n)

454.80

b

TRT

on the same raw: P<0.05.

c

693.54

ADG, kg/d 0-21

1.224

1.312a

1.158b

21-105

1.445b

1.544a

1.419b

105-186

1.530a,x

1.578a,y

1.476b

0-186

1.473b

1.531a

1.416c

0-21

y

6.86

7.36a,x

6.57b

AFI, kg/d

21-105

10.24

10.29

10.20

105-186

11.62

11.62

11.62

0-186

10.44

10.52

10.39

FCR, kg/d 0-21

5.60

5.61

5.67

21-105

7.09

6.66

7.19

105-186

7.59

7.36

7.87

0-186

7.09

6.87

7.34

a, b, c

on the same row: P<0.05. x, y, z on the same row: P<0.1.

such animals may return to normal health quite rapidly, for several days they may reduce their feed consumption and consequently their weight gain. In fact, there is a strong relation between health condition, feed intake and ADG. This delay in growth in the first part of the fattening cycle is rarely compensated in the following weeks a statistical difference in the overall ADG was in fact found between TRT and the inorganic mineral source at the two levels examined in this study. These results are in line with Richeson and Kegley who found that highly stressed, newly received cattle treated with supplemental trace minerals had a reduced influence of BRD and an improvement in ADG18,19.

As reported in Table 4, no difference was detected regarding the incidence of lameness which was generally low and in line with the average prevalence in this farming system in well-managed beef cattle farms4. On the other hand, a higher mortality was found in H-In group though even with no statistical difference. Two out of the three animals died because they failed to recover from BRD after several relapses and one because of enterotoxaemia.

Blood parameters IN; P<0.05). The same growth results were achieved only by increasing the level of inorganic supplementation. These findings could be related to the improvement in ruminal and immunity functions promoted by a higher bioavailability of some basic minerals administered such as selenium, zinc, copper and manganese17. In fact, the animals in the TRT group showed a better health status during adaptation. Data in Table 4 reports how animals supplemented with minerals from the inorganic source showed a higher incidence of respiratory disease (BRD first pull: In 19.23% vs TRT 5.77%; P<0.05), while those supplemented with a high level of inorganic minerals presented a higher incidence of BRD relapses (BRD relapses: H-In 9.61% vs TRT 0%; P<0.05). The higher incidence of BRD in the inorganic (In) group influenced the weight gain in the adaptation phase because of a reduction in feed intake as reported in Table 3.

During the adaptation phase, several animals incurred mild undetected forms of BRD but recovered spontaneously and efficiently through the natural immune defense. Although

The better health condition shown by TRT group animals was confirmed by a better immune reaction after vaccination. Micronutrients such as Se, Zn, Cu, Mn are involved in maintaining the correct functionality of the immune system.

Supplementation with these nutrients can improve the functionality of the immune system and also the production of antibodies. Arthington and Havenga found that the use of an injectable solution of trace minerals (Cu, Zn, Mn, and Se) at the same time as the vaccination, enhanced the antibody response to Bovine Herpes Virus 120. In their study, the animals were vaccinated on arrival against Bovine Herpes Virus 1 of Infectious Bovine Rhinotracheitis among other pathogens.

Since in France no animals are vaccinated and the virus has almost been eradicated, this parameter is a good index to evaluate the immune reaction. In our study, as expected, on arrival the animals presented no serum antibody (Figure 1).

At d21, the TRT animals showed a higher presence of antibodies compared to the In group, and a trend for a difference was also found at d105. The activity and functionality of the

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C.A. Sgoifo Rossi et al. Large Animal Review 2020; 26: 57-64

Figure 1 - Specific immunity serum indicator. a, b: P<0.05 - x, y: P<0.1.

immune system, under different mineral supplementations, was evaluated with the seroneutralization test, as reported in Figure 1. This test is used to quantify the titer of neutralizing antibodies for a virus, in this case for BHV-1. The graph shows an improvement in antibody production in the TRT group, which may reflect a higher lymphocyte B activity, due to a higher GSH-Px activity. This result could be due to the higher bioavailability of organic minerals, especially of Se, supplemented in this group.

These results agree with the findings of Nicholson et al. and Arthur et al.21,22. Indeed, selenium supplementation improves the antioxidant status and especially gluthatione peroxidase activity. Organic selenium sources have better effects on the antioxidant status. Calves supplemented with organic sources of selenium (Se-yeast) have been shown to have better in vitro macrophage phagocytosis compared with Na selenite supplementation23. As reported by Arthur et al.

(2003)20, Se improves the vitality and activity of the immune cells, including lymphocyte B. The better immune reaction after vaccination is likely due to the higher serum availability of minerals which are essential for the immune system. In fact, as reported in Table 5, starting from d21, the circulating mineral concentration of selenium, zinc, copper and manganese was higher in the TRT group compared to the In and H-In groups.

These findings are in line with other studies. Chirase found that calves supplemented with Zn-methionine tend to recover from diseases more rapidly than calves supplemented with ZnO, due to the higher effect of the organic source used24. Also, in steers challenged with infectious bovine rhinotracheitis virus, supplementation with Zn-methionins has been shown to increase the feed intake and lower the rectal temperature, compared with the control. These are signs of a better immune function25.

Copper levels are also involved in the immune response to bovine herpes virus 1. Arthington found that copper deficiency altered the acute-phase protein response in cattle in

Table 5 - Mineral bioavailability.

n°

H-In

TRT

In

14

14

14

Se, g/L d0

31.36

30.46

31.15

d21

b

26.48

a

30.65

27.46b

d105

31.83

33.15a

31.05b

d186

31.39

32.59a

30.14b

Zn, mol/L d0

11.58

11.77

11.79

d21

13.57b

15.46a

12.63b

d105

13.41y

14.83x

13.44y

d186

b

13.31

a

14.55

13.26b

d0

7.08

7.28

7.14

d21

8.54b

9.88a

d105

b

d186

Cu, mol/L

7.39c

9.05

a

11.06

8.88b

9.11b

11.54a

8.63b

Mn, g/L d0

2.57

2.50

2.52

d21

b

2.92

a

3.34

2.79b

d105

3.53b

3.93a

3.03c

d186

3.58b

4.08a

3.18c

GSH-Px, U/g Hg

a, b, c x, y, z

d0

176.57

176.43

174.57

d21

b

174.86

a

183.19

175.57b

d105

164.27b

177.57a,x

170.68y

d186

168.71b,z

177.93a,x

173.50y

on the same row: P<0.05. on the same row: P<0.1.

61

Sgoifo Rossi_imp_ok 24/04/20 13:40 Pagina 62

62

Effects of different mineral supplementation programs on beef cattle serum Se, Zn, Cu, Mn concentration

terms of infection20. Copper is a component of ceruloplasmin, one of the acute phase proteins. The average serum level was higher with the same trend also throughout the fattening cycle. Serum minerals levels of animals supplemented with high levels of inorganic minerals never reached those of the TRT group but were higher compared to the In group for Cu at d21 and Mn at d105 and d186.

The body’s ability to react to oxidative stress was higher in the TRT group. As reported in Table 5, glutathione peroxidase activity (GSH-Px) was higher in the TRT group starting from d21, with a similar trend to the observed serum selenium concentration. This effect could be attributed to the supplementation with an organic form of selenium, which is more available and has a greater effect on the oxidant status. In fact, selenium is a component of glutathione peroxidase, an important water-soluble antioxidant. This data agrees with Juniper et al., who found higher blood glutathione peroxidase and higher glutathione peroxidase activities in beef cattle fed with organic Se, compared to those fed sodium selenites.

Selenium deficiency affects the neutrophil bactericidal activity26. A deficient Se status is associated with a decline in the immune response against infectious diseases, because of this decreased neutrophil bactericidal activity17.

Table 6 - Carcass characteristics.

n°

The results of slaughtering performance and meat quality seem to be related to animals’ live performance, health status and blood parameters. As reported in Table 6, animals’ hot carcass weight was statistically different with the same ADG trend but better results were found in the TRT group.

More carcasses from the inorganic (In) group were assigned to a lower conformation category compared to the H-In and TRT groups. These findings are closely related to animal health. In fact, several authors have reported a worsening in slaughtering performance for animals with BRD27. Regarding the fattening score, the results are in contrast with Gardner et al. 199928, who reported a lower cover fat in animals with BRD. In the present study, the higher bioavailability of minerals may have improved both the metabolic and ruminal activities, enhancing myogenesis rather than adipogenesis, in agreement with the observed improved ADG. In the present experimental conditions, supplementation with organic minerals seemed to lead to leaner carcasses than the other two groups.

Meat mineral contents were consistent with the different levels of bioavailability of minerals with higher levels of Se, Zn, Cu and Mn in the TRT group compared to the inorganic supplemented animals, as reported in Table 7. H-In samples did not obtain the mineral levels of the TRT group but were higher compared to the inorganic group. The absence of an effect of mineral sources on meat centesimal composition is consistent with other studies on fattening cattle29,30.

The potential different antioxidant action from the different supplemented minerals influenced the meat characteristics.

TRT supplementation statistically improved the meat drip loss, color and overall shelf life. These findings are a consequence of the improved stability of cellular membranes due to higher antioxidant properties.

This increase in muscular antioxidant ability is consistent with the findings of several authors30. During ruminal de-

TRT

In

49

52

52

Hot carcass weight, kg 419.46b

426.33a

411.39c

Dressing percentage, % 59.57

59.63

59.32

Conformation, % (n°) S

0.00 (0)

0.00 (0)

0.00 (0)

E

93.88a (46)

94.23a (49)

76.92b (40)

b

U

4.08 (2)

5.77 (3)

23.08a (12)

b

R

2.04 (1)

0.00 (0)

0.00 (0)

O

0.00 (0)

0.00 (0)

0.00 (0)

P

0.00 (0)

0.00 (0)

0.00 (0)

Fattening score, % (n°) 5

0.00 (0)

0.00 (0)

0.00 (0)

4

0.00 (0)

0.00 (0)

0.00 (0)

3

18.37a,x (9)

5.77b (3)

28.84a,y (15)

b,x

2

81.63

1

Carcass characteristics and meat quality

H-In

a

(40)

0.00 (0)

94.23 (49)

71.15b,y (37)

0.00 (0)

0.00 (0)

a, b, c

on the same row: P<0.05. x, y, z on the same row: P<0.1.

Table 7 - Meat chemical composition.

n°

H-In

TRT

In

10

10

10

5.71

5.74

pH 5.74

Centesimal composition Humidity, %

73.35

73.05

73.44

Fat, %

2.52

2.59

2.59

Protein, %

23.14

23.39

23.01

Ash, %

0.96

0.98

0.97

Meat mineral content Se, mg/100 g DM

0.092b

0.133a

0.069c

Zn, mg/100 g DM

16.41b

17.95a

11.90c

Cu, mg/100 g DM

0.29

0.32

0.19b

Mn, µ/100g DM

29.44b

31.95a

20.99c

a, b, c

a

a

on the same row: P<0.05.

gradation and intestinal digestion, organic minerals are submitted to different chemical processes from inorganic minerals, leading to higher absorption and storage at several tissue levels. The increase in muscular antioxidant activity might have reduced the extent of enzymatic and myofibrillar protein oxidation, thus promoting higher membrane stability. This results in better water holding with a lower drip loss in TRT meat samples, as reported in Table 8. This hypothesis is supported by Rowe et al.31, who reported that dietary antioxidant supplementation in beef (vitamin E) reduced protein oxidation, and positively promoted post mortem pro-

Sgoifo Rossi_imp_ok 24/04/20 13:40 Pagina 63

C.A. Sgoifo Rossi et al. Large Animal Review 2020; 26: 57-64 Table 8 - Drip loss.

Table 9 - Meat color.

H-In

TRT

In

H-In

TRT

In

n°

10

10

10

n°

10

10

10

d0

135.32

135.32

135.32

d1

45.68b

49.42a

42.00c

d1

132.78b

d3

b

133.49a

132.64b

d2

45.01b

48.86a

42.56c

131.02

a

131.96

b

130.91

d3

b

43.05

a

47.98

40.53c

d5

128.50b

129.81a

128.68

d4

38.69b

46.97a

35.62c

d7

125.05b

126.27a

126.04

d5

38.80b

45.75a

35.69c

d6

34.32b

40.54a

32.93b

d7

b

34.60

a

32.62b

Meat weight, g

Lightness, L*

Drip loss, %

a, b, c

63

d1

a

1.93

b

1.43

2.15

d1-3

1.32

1.17

1.36

d3-5

2.01

1.72

1.90

d1

23.88b,y

25.71a

25.20x

d5-7

2.72a

2.77a

2.10b

d2

23.69

23.10b

24.79a

d1-7

a

b

d3

a

20.85

a

21.72

17.53b

d4

19.80

21.11a

17.57b

d5

17.32b

20.35a

15.40c

d6

12.69b

16.71a

11.21b

d7

b

13.52

a

11.70c

d1

13.71b

14.78a,x

13.97y

d2

13.50b

14.46a,x

13.80y

d3

b

7.99

7.10

a

Redness, a*

7.51

on the same row: P<0.05.

teolysis. In our study, higher oxidant stability led to a better meat color from the first day after cutting and in the following seven days, as reported in Table 9, with a better lightness, redness and yellowness index.

An improvement in lightness due to the supplementation of organic selenium was also reported by Cozzi et al.30, who found an increased L* value after 6 and 11 days of vacuum packaged ageing. Taylor et al. (2008)29 also found that meat from cattle fed a selenium-enriched diet had a higher Se content and tended to have a higher average a* and b* during 12 days on the shelf, compared with animals fed an unenriched diet.

In addition, in our study TRT meat samples showed a better overall shelf life of more than one day compared to In and H-In groups (Table 10). Several statistical differences were also found between the H-In and In groups. H-In meat samples were characterized by better lightness from d1 to d5, redness at d1, d3, d5 and d7, yellowness at d5 and d7 and shelf life.

These findings demonstrate that the mineral supplementation level also improves meat quality. Some improvements with a high inorganic mineral supplementation are possible compared to a low supplementation but not as high as the level achieved with the organic supplementation.

No statistical differences were observed regarding thawing loss, cooking loss and tenderness as reported in Table 10.

a, b, c

12.37

a

13.72

11.73b

d4

11.77b

13.65a

11.88b

d5

9.11b

13.23a

6.82c

d6

7.74b

10.92a

6.48b

d7

b

a

10.67

5.97c

H-In

TRT

In

10

10

10

6.10a

4.54b,y

7.42

on the same row: P<0.05.

Table 10 - Meat quality.

n°

Shelf life, d b,x

5.00

Thawing loss, % 1.72

1.62

1.72

Cooking loss, % 34.29

33.79

33.89

2

WBSH, kg/cm

a, b, c x, y, z

Under our study conditions, the supplementation of minerals from an organic source to beef cattle, led to better animal health conditions and growth performance that probably relates to the high bioavailability of essential cofactors of basic metabolic and immune processes. These conditions also led to an improvement in meat quality. The administration of minerals from inorganic sources can lead to good but not excellent farming results only if administered at high concentrations.

16.87

Yellowness, b*

3.36

CONCLUSIONS

39.70

3.32

3.31

on the same row: P<0.05. on the same row: P<0.1.

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Effects of different mineral supplementation programs on beef cattle serum Se, Zn, Cu, Mn concentration

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Meat Sci. Assoc., Chicago, IL. 17. Enjalbert F., Lebreton P., Salat O., 2006. Effects of copper, zinc and selenium status on performance and health in commercial and dairy beef herds: retrospective study. J. Anim. Physiol. Anim. Nutr. 90, 459-466, https://doi.org/10.1111/j.1439-0396.2006.00627.x.

18. Richeson J.T., Kegley E.B., 2011. Effect of supplemental trace minerals

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Arthington J.D., Hevenga L.J., 2012. Effect of injectable trace minerals on the humoral immune response to multivalent vaccine administration in beef cattle. J. Anim. Sci. 90, 1966-1971, https://doi.org/10.2527/ jas.2011-4024.

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Arthur J.R., McKenzie R.C., Beckett G.J., 2003. Selenium in the immune system. J. Nutr. 113, 1457S-1459S, https://doi.org/10.1093/jn/ 133.5.1457S. Beck P.A., Wistuba T.J., Davis M.E., Gunter S.A., 2005. Effects of feeding supplemental organic or inorganic selenium to cow-calf pairs on selenium status and immune responses of weaned beef calves. Prof. Anim.

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Health of finishing steers: effects on performance, carcass traits, and meat tenderness. J. Anim. Sci. 77, 3168-3175, https://doi.org/10.2527/ 1999.77123168x.

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Rowe L.J., Maddock K.R., Lonergan S.M., Huff-Lonergan E., 2004. Influence of early post-mortem protein oxidation on beef quality. J. Anim. Sci. 82, 785-793, https://doi.org/10.2527/2004.823785.

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B. Ekiz et al. Large Animal Review 2020; 26: 67-72

The effect of final weight on slaughtering and carcass quality characteristics of lambs in concentrate-based or pasture-based production systems

67

l

BULENT EKIZ, ALPER YILMAZ, HULYA YALCINTAN, OMUR KOCAK, MUSTAFA OZCAN Department of Animal Breeding and Husbandry, Istanbul University-Cerrahpaşa, Faculty of Veterinary Medicine, 34320 Avcilar, Istanbul, Turkey

SUMMARY In lamb finishing for meat production, determination of optimal slaughter weight is an important issue for the provision of a profitable and high-quality lamb production. Aim of the study was to investigate the effect of final weight (LOW: 25-26 kg, MEDIUM: 30-31 kg and HIGH: 35-36 kg) on the slaughtering and carcass quality characteristics of Kivircik lambs, which were finished with concentrate-based diet (CON) or on pasture (PAS). CON lambs (n = 27) were weaned at 76-d and then finished on concentrate feed and alfalfa hay until slaughter in sheepfold. PAS lambs (n = 25) were on native pasture in the day-time with their mothers and allowed to suck their mothers until the slaughter. In the CON system, commercial and real dressing percentages were lower in the LOW group compared to MEDIUM and HIGH groups. In MEDIUM and HIGH weight groups, CON system yielded higher commercial dressing than PAS system. CON lambs had higher liver percentage than PAS lambs in all final weight groups. LOW lambs had lower values for carcass measures and indices, except chest roundness index than HIGH lambs. CON lambs a had higher buttock width and circumference, carcass width, carcass compactness and chest roundness index in all final weight groups. Lambs from LOW group in PAS system had a higher yellowness than that in CON system. In conclusion, CON system can be preferred to produce a higher amount of lamb meat in a shorter time. Final weight of 35-36 kg might be preferred in both production systems to increase lamb production, without any adverse effect on carcass quality.

KEY WORDS Carcass quality, fat colour, lamb, production system.

INTRODUCTION Most of the sheep farms in Turkey are small-scale family farms as in other the Middle East and some of the Mediterranean countries. Sheep farming is usually conducted in rural areas by low-income villagers. Lamb sales constitute an important part of the income generated in these enterprises1.

Moreover, lamb and sheep meat are consumed with pleasure in Middle Eastern countries and it also makes an important contribution to meeting the animal-based food needs of low-income people2.

Feed cost forms an important part of a livestock enterpriseâ&#x20AC;&#x2122;s expenditures. If the pasture areas are sufficient and pasture conditions are favourable, grazing on pasture is usually the cheapest way for lamb feeding. Therefore, in the traditional production systems, lambs are kept in the sheepfold until 7590 days of age and then finished at natural pastures in the daytime by supplementing with roughage at nights1. However, the amount of milk sold is reduced as the lamb sucks their mothers in this production system. On the other hand, pasture areas have gradually decreased, and uncontrolled and excessive grazing has caused the quality of existing pastures to decline in recent years. As a result, natural pastures are no longer sufficient to meet the nutritional requirements

Corresponding Author: Bulent Ekiz (bekiz@istanbul.edu.tr).

of lambs for finishing nowadays. These adversities led sheep farmers to search for alternative systems for lamb finishing.

Nowadays, many sheep farmers prefer the concentratesbased lamb finishing system, in which lambs are weaned at 75-90 days of age and fed with concentrates until slaughter1.

Slaughtering characteristics and carcass quality of lambs may vary due to production systems3,4,5,6. Final or slaughter weight may also influence these characteristics7,8,9,10. Therefore, the deciding the appropriate production system and the final weight of lambs in commercial lamb production is of great importance in obtaining high-quality lamb carcasses.

Many sheep breeds are bred in Turkey, and the majority of these breeds are fat-tailed. However, the most preferred sheep breed by consumers is Kivircik, which is a thin-tailed breed.

Kivircik and its crossbreds are also bred in Greece, Bulgaria and some Balkan countries11. The aim of the current study was to determine the growth rate, slaughtering and carcass quality traits of Kivircik lambs at three final weights (LOW: 25-26 kg, MEDIUM: 30-31 kg and HIGH: 35-36 kg) in pasture-based and concentrate-based production systems.

MATERIALS AND METHODS Handling, feeding and housing procedures applied in the current study were approved by the local Ethics Committee of Istanbul University Faculty of Veterinary Medicine (No: 05-109).

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The effect of final weight on slaughtering and carcass quality characteristics of lambs in concentrate-based

Animal material and production systems The study was conducted at sheep farm of the Veterinary Faculty. The first week after birth the lambs were fed only with dam’s milk. Thereafter, the lambs were given alfalfa hay in addition to their mother’s milk until 76-day of age. Fiftyfour male lambs born as a single in early February were selected for the animal material of the study when the average lamb age attained about 76 days (76.5 ± 0.6 days). These lambs were randomly divided into two production systems with 27 lambs in each group. Afterwards, lambs of each production system were randomly allocated into LOW (25-26 kg), MEDIUM (30-31 kg) and HIGH (35-36 kg) final weight groups. Handling, housing and feeding procedures in production systems were: Concentrate-based (CON) lamb production

The lambs, which selected for the CON system, were weaned at 76-day immediately after grouping. Afterwards, three separate pens (5.0 × 3.2 m each) were built in the sheepfold for LOW, MEDIUM and HIGH final weight groups, and lambs were placed in these pens. During the whole finishing period, lambs had free access to the concentrate feed, alfalfa hay and fresh clean water. Concentrate feed consumptions of each weight group was determined at weekly basis. Mean daily concentrate consumption was 0.73 kg, 0.92 kg and 1.01 kg in LOW, MEDIUM and HIGH final weight groups, respectively.

Pasture-based (PAS) lamb production

PAS lambs were not weaned until slaughter. These lambs were grazed with their dams in the natural pasture during the day and fed free access with alfalfa hay in the sheepfold during the night. Composition of pasture (on dry matter basis) was: 52% Gramineae (Lolium spp. and Festuca spp.), 22% Leguminosae (Vicia spp., Medicago spp. and Trifolium spp.) and 26% other families (mainly Conium spp., Viola spp., Geranium spp., Plantago spp. and Rumex spp.)1. The composition of the alfalfa hay, concentrate feed and pasture are given in Table 1.

Table 1 - Chemical composition of concentrate feed, alfalfa hay and pasture used in the study.

Concentrate feed3

Alfalfa hay

Pasture

Dry matter (%)

90.30

92.14

37.95

1

Crude protein (%)

16.90

15.85

11.5

Ether extract1 (%)

3.14

2.10

5.46

Crude cellulose1 (%)

14.65

26.50

24.4

Ash (%)

6.92

8.49

10.43

Neutral detergent fibre1 (%)

29.41

46.16

42.67

Acid detergent fibre1 (%)

10.30

33.92

35.97

Metabolizable energy2, (MJ ME/kg DM)

11.81

8.67

9.21

Chemical composition

1

1

As a percentage of dry matter.

Calculated values.

3 Concentrate feed contained: wheat bran 40%, barley 15.5%, corn 15.3%, soybean meal 8%, sunflower meal 6%, DDGS (dried distillers grains with solubles) 5%, molasses 4%, Safflower meal 3%, CaCO3 2.5%, NaCl 0.5%, vitamin/mineral premix 0.2%.

2

Two lambs in HIGH group of PAS system were excluded from the study since they had laminitis and therefore could not graze on the pasture. Hence, 25 lambs in PAS system and 27 lambs in CON system were slaughtered in the study.

All lambs were weighed every Wednesday morning before feeding. Lambs reaching the target weight in weekly weighings were transferred to Istanbul University experimental slaughterhouse for slaughtering.

Slaughter procedures and determination of carcass characteristics Lambs were kept overnight at lairage unit of the slaughterhouse with free access to water but not feed. After recording the pre-slaughter live weight, the lambs were electrically stunned and then slaughtered. Afterwards, head, skin, feet, lungs and trachea, liver, heart, spleen, testicles and gastro-intestinal tract were removed and weights of these tissues were recorded. After the removal of these tissues, hot carcass weight was recorded. Afterwards, the carcasses were kept in cold storage (4oC) for 24 h.

At 24 h post-mortem, colour variables of subcutaneous fat were measured by using a colorimeter (Minolta CR-400) from the tail root. Afterwards, following carcass measurements were also determined on the intact carcass as reported by Ekiz et al.12: buttock width, buttock circumference, leg length, carcass length, carcass width, chest depth and chest circumference. Next, carcasses were split along the vertebral column into right and left parts. Left parts of the carcasses were used to measure hind limb length and internal carcass length12. Once the carcass measurements have been determined, hind limb compactness, carcass compactness and chest roundness index were calculated as reported by Ekiz et al.12. After all carcass measurements were recorded, the right sides of carcasses were separated into ribs, thoracic and pelvic limbs, neck and flank joints at 24 h post-mortem13.

Statistical analyses The effects of production system, final weight group and their interaction on slaughtering and carcass characteristics were analysed using the General Linear Model (GLM) procedures in SPSS 13.0 programme. Moreover, one-way ANOVA and Duncan’s multiple range test were applied to compare the final weight groups for each production system. The comparison of PAS and CON systems for each final weight group was performed by independent sample t-test.

RESULTS As planned in the experimental design, initial ages and weights of lambs in six sub-groups (2 production system × 3 final weight groups) were similar (Table 2). Finish duration was lower in lambs of CON system in both LOW and HIGH weight groups than lambs of the PAS system. In the CON system, commercial and real dressing percentages were lower in the LOW group compared to MEDIUM and HIGH groups. However, differences among final weight groups in terms of commercial and real dressing percentages were not significant in the PAS system. In MEDIUM and HIGH weight groups, CON system yielded higher commercial dressing than PAS system, but such a difference was

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Table 2 - Performance traits and slaughtering characteristics of lambs in different final weights1 due to production systems.

Characteristics

Concentrate-based system

Pasture-based system

P-Value

SEM

LOW

MEDIUM

HIGH

LOW

MEDIUM

HIGH

WG

PS

WG×PS

Initial age, days Initial weight, kg Finishing duration, days

76.67 17.37 43.78 c,x

76.11 17.93 75.67 b

76.78 17.91 94.67 a,x

77.89 17.49 60.33 f,y

75.56 17.80 85.00 e

77.43 17.91 107.57 d,y

1.539 0.230 2.555

0.914 0.573 <0.001

0.887 0.878 0.015

0.970 0.912 0.840

Pre-slaughter weight, kg Hot carcass weight, kg Commercial dressing2, % Real dressing3, %

24.09 c 11.56 c 48.03 b 54.69 b

28.46 b 14.26 b,x 50.10 a,x 56.11 a

33.26 a 16.82 a,x 50.57 a,x 56.37 a,x

24.11 f 11.24 f 46.68 53.87

27.98 e 13.19 e,y 47.05 y 54.24

32.47 d 15.07 d,y 46.46 y 54.20 y

0.161 0.131 0.346 0.272

<0.001 <0.001 0.267 0.261

0.071 <0.001 <0.001 0.005

0.173 0.035 0.272 0.571

Non-carcass parts, % Head Feet Skin Lungs and trachea Liver Heart Spleen Empty stomachs Empty intestines Gastro-intestinal content

6.62 y 3.04 a,y 10.23 1.84 a 2.39 x 0.52 y 0.23 3.57 6.83 a 13.96

6.52 y 2.81 b,y 10.66 1.56 b,y 2.29 x 0.50 0.21 3.45 6.04 b 12.02 y

6.80 2.68 c,y 10.77 1.49 b,y 2.18 x 0.50 y 0.20 3.34 5.42 c 11.51 y

7.08 x 3.22 d,x 10.92 1.80 2.06 y 0.56 x 0.22 3.55 6.38 d 15.59

7.10 x 3.02 e,x 11.09 1.73 x 2.02 y 0.53 0.24 3.55 5.83 e 15.46 x

6.91 2.86 f,x 11.47 1.73 x 2.05 y 0.54 x 0.22 3.53 5.41 f 16.83 x

0.048 0.019 0.141 0.022 0.028 0.005 0.005 0.049 0.086 0.455

0.905 <0.001 0.297 <0.001 0.269 0.110 0.209 0.584 <0.001 0.635

0.001 <0.001 0.037 0.007 <0.001 <0.001 0.165 0.392 0.206 <0.001

0.140 0.962 0.903 0.210 0.340 0.850 0.245 0.687 0.572 0.271

1

Final weight groups: LOW = 25-26 kg, MEDIUM = 30-31 kg, HIGH = 35-36 kg.

WG: Final weight group; PS: Production system.

Mean values with different superscripts in the same line for concentrate-based system are significantly different (P < 0.05). d, e, f Mean values with different superscripts in the same line for pasture-based system are significantly different (P < 0.05). x, y Mean values for CON and PAS systems with different superscripts within the same final weight group are significantly different (P < 0.05).

2 (Hot dressing percentage / pre-slaughter weight) × 100.

3 (Hot dressing percentage / empty body weight) × 100. a, b, c

not observed in LOW weight group. A decrease in percentages of feet and empty intestines were determined with the increase in final weight in both CON and PAS systems.

Lambs of CON system had higher liver percentage than PAS lambs in all final weight groups.

In both CON and PAS systems, LOW lambs had lower mean values in terms of carcass measures and indices, except chest

roundness index than HIGH lambs (Table 3). MEDIUM group had also higher values than LOW group for the majority of carcass measures. Lambs in CON system a had higher buttock width and circumference, carcass width, carcass compactness and chest roundness index in all final weight groups compared with PAS lambs.

Lightness and redness values of subcutaneous fat were not

Table 3 - Carcass measures and indices of lambs in different final weights1 due to production systems.

Characteristics

Concentrate-based system LOW

Carcass length, cm

MEDIUM

65.32 55.06

c

Leg length, cm

19.44

b

Hind limb length, cm

26.94 b

Internal carcass length, cm

66.11 57.46

b

20.39

a

27.81 ab

28.58 a 20.94

a,x

60.51

a,x

20.98

a,x

56.10

f

19.84

e

27.02 e 18.10

e,y

53.56

f,y

17.73

e,y

68.93

d

58.42

e

20.78

d

28.13 de

72.13

0.557

0.002

0.091

0.251

60.09

d

0.212

<0.001

0.110

0.594

21.01

d

0.095

<0.001

0.120

0.794

28.46 d

0.182

0.005

0.800

0.886

19.80

d,y

0.107

<0.001

<0.001

0.972

58.79

d,y

0.204

<0.001

<0.001

0.520

19.64

d,y

19.02

0.146

<0.001

<0.001

0.380

Chest depth, cm

23.60 c

24.64 b

26.40 a

23.87 e

25.24 d

26.07 d

0.099

<0.001

0.371

0.175

Chest circumference, cm

64.38 c

69.08 b

72.67 a

65.47 f

68.38 e

70.97 d

0.218

<0.001

0.322

0.039

217.30 e,y 236.85 d,y

1.969

<0.001

<0.001

0.045

69.46 c

Chest roundness index

0.74 x

82.06

b,x

0.78 x

18.51

de,y

WG×PS

Carcass width, cm

Hind limb compactness, g/cm

55.80

e,y

PS

d

b,x

203.34 c,x 241.33 b,x 272.65 a,x 192.55 f,y

18.96

de,y

WG

54.99

1

20.68

a,x

20.90

a

64.64

e

P-Value

SEM

HIGH

c,x

Carcass compactness, g/cm

58.32

b,x

60.02

a

MEDIUM

19.20

Buttock circumference, cm

20.18

68.50

Pasture-based system LOW

c,x

Buttock width, cm

b,x

HIGH

90.81 a,x

68.40 e

73.47 e,y

83.49 d,y

0.922

<0.001

0.004

0.203

0.75 x

0.69 y

0.68 y

0.71 y

0.006

0.482

<0.001

0.116

Final weight groups: LOW = 25-26 kg, MEDIUM = 30-31 kg, HIGH = 35-36 kg.

WG: Final weight group; PS: Production system. a, b, c Mean values with different superscripts in the same line for concentrate-based system are significantly different (P < 0.05). d, e, f Mean values with different superscripts in the same line for pasture-based system are significantly different (P < 0.05). x, y Mean values for CON and PAS systems with different superscripts within the same final weight group are significantly different (P < 0.05).

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The effect of final weight on slaughtering and carcass quality characteristics of lambs in concentrate-based

Table 4 - Fat colour characteristics and proportions of carcass joints of lambs in different final weights1 due to production systems.

Characteristics

Concentrate-based system

Pasture-based system

LOW

MEDIUM

HIGH

LOW

MEDIUM

HIGH

Fat colour Lightness Redness Yellowness Chroma Hue

69.96 4.64 6.81 y 8.26 55.55 b

69.58 3.89 7.31 8.29 61.96 a

69.09 4.09 7.48 8.54 61.46 a

70.04 4.40 7.75 x 8.96 60.82

69.99 4.20 7.81 8.88 61.56

69.64 4.01 7.96 8.92 63.11

Proportions of carcass joints, % Pelvic limb Ribs Thoracic limb Neck Flank Tail

34.24 a 25.38 19.23 y 7.47 b,y 10.32 1.17

33.23 ab 26.41 18.69 y 8.12 ab 10.01 1.26

32.20 b,y 26.95 x 18.78 y 8.58 a 9.91 1.33 x

34.03 d 24.79 e 20.30 x 8.33 x 10.05 1.01

32.64 e 26.44 d 20.14 x 8.81 9.40 1.04

34.18 d,x 24.73 e,y 20.28 x 8.65 9.73 0.96 y

P-Value

SEM WG

PS

WG×PS

0.148 0.100 0.119 0.129 0.572

0.230 0.089 0.315 0.890 0.010

0.246 0.980 0.010 0.036 0.063

0.801 0.486 0.667 0.888 0.123

0.146 0.198 0.086 0.116 0.144 0.037

0.003 0.024 0.228 0.043 0.360 0.748

0.186 0.023 <0.001 0.033 0.230 0.001

0.002 0.073 0.531 0.385 0.821 0.494

1

Final weight groups: LOW = 25-26 kg, MEDIUM = 30-31 kg, HIGH = 35-36 kg.

WG: Final weight group; PS: Production system. a, b Mean values with different superscripts in the same line for concentrate-based system are significantly different (P < 0.05). d, e Mean values with different superscripts in the same line for pasture-based system are significantly different (P < 0.05). x, y Mean values for CON and PAS systems with different superscripts within the same final weight group are significantly different (P < 0.05).

influenced from neither by the weight group nor by the production system (Table 4). Lambs from LOW group in PAS system had a higher yellowness than that in CON system. In CON system, hue value was lower in LOW group compared to MEDIUM and HIGH groups. In CON system, HIGH lambs had lower pelvic limb proportion and higher neck proportion than LOW lambs. Proportion of thoracic limb was higher in PAS lambs than CON lambs in all final weight groups.

DISCUSSION In lamb finishing for meat production, determination of optimal slaughter weight is an important issue for the provision of a profitable and high-quality lamb production. In Turkey, pricing of lamb carcasses is usually based on carcass weight, so marketing of overweight lambs may be considered by sheep farmers. In LOW and HIGH weight groups, lambs of CON system reached the final weight earlier than the PAS lambs. This may be due to the discrepancy between the two production systems in terms of food resources. CON lambs were fed ad-libitum with concentrate and alfalfa hay, while pasture grass, alfalfa and mothers’ milk were main feed resources of PAS lambs. Supporting the current results, Ekiz et al.2 for Kivircik lambs and Borton et al.14 for Targhee × Hampshire lambs found lower growth rate in pasture lambs than lambs fattened with concentrates. On the other hand, if pasture conditions are favourable, studies found similar growth rate for grazing and stall lambs were also available6,15.

In MEDIUM and HIGH weight groups, CON lambs had higher hot carcass weight than lambs reared under PAS system. One possible explanation for higher hot carcass weights of CON lambs might be their higher dressing percentages. The results that higher carcass weights in lambs finished with concentrates than pasture lambs were also observed for Targhee × Hampshire lambs14 and Kivircik lambs2, previously. In MEDIUM and HIGH weight groups, a higher commercial and real dressing percentages observed

in CON lambs might be due to the higher percentages of head, feet, lungs and trachea, and gastro-intestinal content in PAS lambs.

In lambs of PAS system, commercial and real dressing percentages were not affected by final weight group. Similar results were reported for suckling lambs by Santos et al.16. On the other hand, in CON lambs, commercial and real dressing percentages were lower in lambs of the LOW group than that of MEDIUM and HIGH weight groups. MajdoubMathlouthi et al.17 also found an increase in dressing percentage with increasing slaughter weight in lambs fattened with concentrates. The authors explained this result by reduced gut content percentage with increasing slaughter weight. Oliveira et al.18 explained a higher dressing percentage in Sante Ines lambs, which slaughtered at heavier weights by higher deposition of muscle and fat in carcasses of these lambs. Borton et al.14 also found higher dressing percentage for concentrate-fed lambs than pasture lambs and attributed this result to a decrease in the size of gastrointestinal tract. Indeed, in the current study, there was a tendency in CON lambs to decrease in the percentage of gastrointestinal content with the increase in the final weight for lambs (P = 0.06).

Feet percentage dropped as final weight increased in both CON and PAS systems. These results might be explained by the fact that bone is the most precocious tissue followed by lean, while fat tissue develops later6. In the current study, lambs from different final weight groups were slaughtered at 4-6 months of age, therefore weight gains of lambs at these ages might have been based on mainly an increase in muscle and fat tissues rather than bone tissues. The higher head, feet, lungs and trachea, and heart percentage in PAS lambs were expected because CON lambs reached to the target final weight about 13 days earlier than PAS lambs. In the previous study, where Kivircik lambs were slaughtered at a constant weight of 30 kg, the percentages of feet and lungs and trachea of the older ones were found to be higher2.

CON lambs had higher liver percentages than PAS lambs in all final weight groups. McClure et al.19 also found higher

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B. Ekiz et al. Large Animal Review 2020; 26: 67-72

liver weight for lambs fed concentrate-based diet than lambs of grazing-based system. Similarly, Ekiz et al.2 reported a higher liver percentage for weaned lambs finished with concentrates compared with lambs of grazing-based systems.

Kochewad et al.20 also found higher liver percentage for Deccani lambs finished with concentrates than lambs finished at pasture.

When carcass measurements are evaluated, a significant increase in the majority of carcass measures and indices were observed with increased final weight group in both CON and PAS systems. Similar trend regarding carcass measurements were reported previously for several breeds7,10,16. Conformation and body/carcass measurements of healthy and well-fed animals are expected to rise with increasing age and weight until reaching the mature age7. Therefore, the current results regarding effects of final weight group on carcass measurements and indices might be evaluated as expected results.

Moreover, carcass measures and indices determined for LOW and MEDIUM groups were comparable with the previous results by Ekiz et al.21 for Kivircik lambs slaughtered at 26.6 and 30.3 kg, respectively.

Production system did not affect length measures (lengths of carcass, internal carcass, leg and hind limb), and depth and circumference of chest in all final weight groups. But, CON lambs had higher mean values regarding width measures (buttock width and carcass width) and buttock circumference in all final weight groups. Hence, a higher width and circumference measurements of CON lamb carcasses caused the objective carcass indices (carcass compactness, hind limb compactness and chest roundness index) in these lambs to be higher than in PAS lambs. Supporting the current result, Carrasco et al.5 reported lower transversal measures, and decreased hindquarters compactness, chest rounded index, pelvic limb compactness and carcass compactness for Churra Tensina light lambs reared under grazing compared with drylot lambs with dams fed in confinement. Gallo et al.22 found higher carcass compactness index in feedlot lambs that fed a diet composed of 20% of roughage and 80% of concentrate than lambs raised on pasture. In that study, internal carcass length and leg length were also similar in pasture and feedlot lambs.

Final weight group had no significant influence on subcutaneous fat colour parameters in both CON and PAS systems, except hue value in CON system. Ripoll et al.23 also reported no significant influence of carcass weight on subcutaneous fat lightness and chroma values. Fat of lamb carcasses in LOW group had lower hue value compared to that of MEDIUM and HIGH groups. Moreover, significant production system effect was only observed in LOW weight group for the yellowness of carcass fat. PAS lambs had more yellow fat colour with higher b* value compared with CON lambs. This may be attributed to the greater intake of herbage (and therefore pigments) in PAS lambs. Similarly, Carrasco et al.24 observed higher b* values in lambs from grazing groups than drylot lambs and explained this by more carotenoids deposition in grazed lambs due to pasture intake. In the other study, Dıaz et al.6 also found higher b* value for subcutaneous fat in lambs finished at pasture than that of sheepfold ones.

The proportion of thoracic limb was higher in PAS lambs.

With regard the pelvic limb proportion, PAS lambs in HIGH group had also higher mean values than that of

71

CON lambs. Ekiz et al.2 also observed lower proportions of thoracic and pelvic limbs in unweaned lambs finished with concentrates compared with lambs from grazing-based systems. Borton et al.14 noted that proportions of leg and foreshank joints, which are related to motor function, might be increased consistently in forage-finishing systems.

Higher proportions of ribs and tail in HIGH weight group of CON lambs may be related to the higher fatness level of these lambs and/or lower proportions of the thoracic limb in these lambs compared to PAS lambs. Proportions of carcass joints found in the current study were comparable with the results of Aksoy et al.25 for Kivircik lambs finished with concentrate based system.

CONCLUSIONS In the conditions of the current study, CON lambs were significantly younger at slaughter. Moreover, CON lambs had higher dressing percentage, better carcass conformation with greater width measurements. Therefore, CON system can be preferred to produce a higher amount of lamb meat in a shorter time. Final weight did not influence on dressing percentage and subcutaneous fat colour. Therefore, among the groups investigated in the current study, final weight of 3536 kg might be preferred to increase lamb production, without any adverse effect on carcass quality for Kivircik lambs.

ACKNOWLEDGEMENTS The study was supported by Scientific Research Projects Coordination Unit of Istanbul University-Cerrahpasa (BEK2017-24916).

References 1. Ekiz B., Yilmaz A., Ozcan M., Kocak O. (2012). Effect of production systems on carcass measurements and meat quality of Kivircik lambs.

Meat Sci, 90: 465-471.

2. Ekiz B., Yilmaz A., Demirel G., Yalcintan H., Kocak O., Ozcan M., Altinel A. (2013). Slaughter characteristics, carcass quality and fatty acid composition of lambs under four different production systems. Small Rum Res, 114: 26-34.

3. Boughalmi A., Araba A. (2016). Effect of feeding management from grass to concentrate feed on growth, carcass characteristics, meat quality and fatty acid profile of Timahdite lamb breed. Small Rum Res, 144, 158-163.

4. Cañeque V., Velasco S., Díaz M.T., de Huidobro F.R., Perez C., Lauzurica S. (2003). Use of whole barley with a protein supplement to fatten lambs under different management systems and its effect on meat and carcass quality. Anim Res, 52(3): 271-285.

5. Carrasco S., Ripoll G., Sanz A., Álvarez-Rodríguez J., Panea B., Revilla R., Joy M. (2009). Effect of feeding system on growth and carcass characteristics of Churra Tensina light lambs. Livest Sci, 121: 56-63.

6. Díaz M.T., Velasco S., Caneque V., Lauzurica S., Huidobro F., Perez C., Gonzales J., Manzanares C. (2002). Use of concentrate or pasture for fattening lambs and its effect on carcass and meat quality. Small Rum Res, 43: 257-268.

7. Abdullah A.Y., Qudsieh R.I. (2008). Carcass characteristics of Awassi ram lambs slaughtered at different weights. Livest Sci, 117: 165-175.

8. Díaz M.T., Velasco S., Pérez C., Lauzurica S., de Huidobro R.F., Cañeque V. (2003). Physico-chemical characteristics of carcass and meat Manchego-breed suckling lambs slaughtered at different weights. Meat Sci, 65(3): 1085-1093.

9. Lloyd W.R., Slyter A.L., Costello W.J. (1980). Effect of breed, sex and final weight on feedlot performance, carcass characteristics and meat palatability of lambs. J Anim Sci, 51: 316-320.

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10. Peña F., Cano T., Doménech V., Alcalde Ma J., Matos J., Garcia-Martinez A., Herrera M., Rodero E. (2005). Influence of sex, slaughter weight and carcass weight on “non-carcass” and carcass quality in Segureña lambs. Small Rum Res, 60: 247-254.

11. Anonymous (2009). Republic of Turkey, Ministry of Food, Agriculture and Livestock, General Directorate of Agricultural Research and Policies. Turkish Farm Animal Genetic Resources Catalogue. https://www.tarim.gov.tr/TAGEM/Belgeler/yayin/Katalog%20T%C3 %BCrk%C3%A7e.pdf 12. Ekiz B., Ozcan M., Yilmaz A., Tölü C., Sava T. (2010). Carcass measurements and meat quality characteristics of dairy suckling kids compared to an indigenous genotype. Meat Sci, 85: 245-249.

13. Colomer-Rocher F., Morand-Fehr P., Kirton A.H. (1987). Standard methods and procedures for goat carcass evaluation, jointing and tissue separation. Livest Prod Sci, 17: 149-159.

14. Borton R.J., Loerch S.C., McClure K.E., Wulf D.M. (2005). Comparison of characteristics of lambs fed concentrate or grazed on ryegrass to traditional or heavy slaughter weights. I. Production, carcass, and organoleptic characteristics. J Anim Sci, 83; 679-685.

15. Atti N., Mahouachi M. (2009). Effects of feeding system and nitrogen source on lamb growth, meat characteristics and fatty acid composition. Meat Sci., 81: 344-348.

16. Santos V.A.C., Silva S.R., Mena E.G., Azevedo J.M.T. (2007). Live weight and sex effects on carcass and meat quality of “Borrego terrinchoPDO” suckling lambs. Meat Sci, 77(4): 654-661.

17. Majdoub-Mathlouthi L., Saïd L., Say A., Kraiem K. (2013). Effect of concentrate level and slaughter body weight on growth performances, carcass traits and meat quality of Barbarine lambs fed oat hay based diet. Meat Sci, 93: 557-563.

18. Oliveira F.G., Sousa W.H., Cartoxa F.Q., Cunha MG.G., Ramos J.P.F., Cezar M.F., Menezes L.M., Oliveira A.B. (2018). Carcass characteristics of Santa Ines sheep with different biotypes and slaughtering weights.

Rev Bras Saúde Prod Anim Salvador, 19: 347-359.

19. McClure K.E., Solomon M.B., Loerch S.C. (2000). Body weight and tissue gain in lambs fed an all-concentrate diet and implanted with trenbolone acetate or grazed on alfalfa. J Anim Sci, 78: 1117-1124.

20. Kochewad S.A., Raghunandan T., Sarjan Rao K., Kondal Reddy K., Nalini Kumari N., Ramana D.B.V., Anil Kumar D., Kankarne Y, Kumar S., Meena L.R., Singh M. (2018). Productive performance, body condition score and carcass characteristics of Deccani lambs reared under different farming systems. Indian J Anim Res, 52: 444-448.

21. Ekiz B., Koçak O., Yalcintan H., Yilmaz A. (2016). Effects of suckling duration on growth, slaughtering and carcass quality characteristics of Kivircik lambs. Trop Anim Health Prod, 48: 395-401.

22. Gallo S.B., Arrigoni M.B., da Silva C. Lemos A.L., Haguiwara M.M.H., Bezerra H.V.A. (2019). Influence of lamb finishing system on animal performance and meat quality. Acta Scientiarum Anim Sci, v41i1.44742 (Doi: 10.4025/actascianimsci.v41i1.44742).

23. Ripoll G., Blanco M., Panea B., Joy M. (2019) The effect of carcass weight on fatness and muscle and fat colour of male Ojinegra de Teruel light lambs. Anim Prod Sci, 59: 1168-1175.

24. Carrasco S., Panea B., Ripoll G., Sanz A., Joy M. (2009). Influence of feeding systems on cortisol levels, fat colour and instrumental meat quality in light lambs. Meat Sci, 83: 50-56.

25. Aksoy Y., Uğurlu M., Önenç A., Şirin E., Şen U., Çiçek Ü., Ulutaş Z., Kuran M. (2018). Meat production characteristics of Turkish native breeds: I. Fattening, slaughter and carcass traits of lambs. S. Afr. J Anim Sci, 48: 665-672.

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C. Pesca et al. Large Animal Review 2020; 26: 73-76

Enzootic pneumonia in sheep: ewe and lamb immune response after Mannheimia haemolytica vaccine administration under field condition in Italy

73

l

CRISTINA PESCA1, KATIA FORTI1, ANDREA FELICI1, ELEONORA SCOCCIA1, CLAUDIO FORTE1, PIETRO ANTENUCCI2, SABINA MUNTONI2, LUCIA ANZALONE1, ANTONELLA DI PAOLO1, SILVIA CROTTI1 1

Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati”, Via G. Salvemini 1, 06126 Perugia, Italy 2 MSD Animal Health, Segrate, Milano, Italy

SUMMARY Mannheimia haemolytica related pneumonia is characterized by high morbidity and mortality in lambs and thus is a common cause of important economic losses in sheep industry due to reduction in lamb growing and decreased carcass value. Due to the frequency of the disease, vaccination is a very common practice in Italy but the extent and duration of colostral protection in Sarda lambs born from vaccinated dams is not known. In this study, the extent and length of colostral antibody protection in Sarda lambs born from M. haemolytica-vaccinated ewes was evaluated in field condition. The in-field trial took place in two different farms in Sardinia region (Italy). A total of forty-five adult healthy pregnant Sarda sheep were enrolled for the study (24 from the first flock and 21 from the second flock). Each flock in each farm was divided in two groups: unvaccinated control group (10 sheep from the first flock and 8 from the second flock) and vaccinated sheep (14 from the first flock and 13 from the second flock). A total of forty-five lambs born from both vaccinated (n=27) and unvaccinated (n=18) dams were included in the study. Colostrum was collected from all the dams within 6-12 hours after lambing. Blood samples were taken from all the lambs included in the study at 48 hours, 15, 30, 45, 60 days after parturition. Antibody responses following vaccinations were detected by ELISA test. In the trial, antibody titres both on serum of ewes and lambs and colostrum were comparatively examined and important differences were observed in vaccinated pregnant ewes where serum titres developed after vaccination resulted two times higher than in the unvaccinated group (p-value=0.0029); within the lamb groups, an increase of serum ELISA titres was found in lambs born from vaccinated ewes until 60 days after birth. Colostral titres didn’t show any significant difference. In conclusion, when the control group and the trial group were compared, vaccinating the lambs belonged to vaccinated dams showed significant differences concerning antibody responses against M. haemolytica.

KEY WORDS Vaccination; immunity, enzyme-linked immunosorbent assay; Sarda sheep.

INTRODUCTION Sheep farming is a significant industry in Italy. According to 2018 data reported by the National Data Bank (BDN), more than a half of the total amount of sheep reared belongs to Sarda breed. Pneumonia is responsible for important economic losses in sheep farming worldwide1. Mannheimia haemolytica is a Gram-negative, facultative anaerobic, non-motile, opportunistic pathogen bacteria and is one of the most important respiratory pathogens in the family Pasteurellaceae able to cause serious outbreaks of acute pneumonia in sheep of all ages including in neonatal, weaned and growing lambs.

M. haemolytica and the related pneumonia is common and is distributed worldwide although the prevalence of serotypes may vary by region and flock; it is a commensal of the upper respiratory tract of healthy sheep that can cause pneumonia

Corresponding Author: Cristina Pesca (c.pesca@izsum.it).

either alone or in conjunction with other organisms. The interaction of the host with respiratory pathogens such as respiratory syncytial virus, adenovirus, parainfluenza virus type 3, and Mycoplasma spp. in particular, under the influence of environmental factors and stressors are thought to break down the mucosal barrier integrity of the lower respiratory tract, allowing to M. haemolytica to colonize, proliferate and induce significant tissue damage2,3,4,5,6. The disease is also called “shipping fever” because it appears to occur most often in animals that have undergone recent stress such as weaning, transportation, changes in diet or cohabitation with new animals from different farms. Clinical signs include an acute onset of hyperthermia (>40,5°C), dyspnea, anorexia, depression, inappetance, lethargy. In lambs pneumonia causes mortality, decreased growth at considerable cost to the industry, along with animal welfare implications. Death losses are high in severely affected animals within 12 hours of the onset of any clinical signs7. Gross lesions typically show a fibrinous, necrotic pneumonia. The introduction of an effective antibiotic therapy is necessary when the disease outbreaks into a flock. Treatment shall begin at the rise of the pathology be-

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Enzootic pneumonia in sheep: ewe and lamb immune response after Mannheimia haemolytica vaccine administration

cause of the rapid progression of lung damage and endotoxin release. Administering prophylactic antibiotics to at-risk lambs may be beneficial even if this would bring towards the development of antimicrobial resistance. Management measures to reduce known stressors such as overcrowding, poor ventilation, high and cold temperatures should also be considered. In severely affected flocks response to therapy is often disappointing because of the widely use of antimicrobials and the related resistance. For these reasons, for the economic costs of treatment and reduction of lamb weight in survivors, application of prophylactic measures would be desirable. Due to the frequency of the disease, vaccination is a very common practice in Italy but the extent and duration of colostral protection in Sarda lambs born from vaccinated dams is not known. Due to the lack of studies on immunities of M. haemolytica in Sarda sheep lambs, a key question is how long lambs born from M. haemolytica-vaccinated ewes under field condition are protected. The aim of this study was to measure the extent and length of colostral antibody protection in Sarda lambs born from M. haemolytica-vaccinated ewes. The immune response of both ewes and lambs with until 60 days of life was evaluated by measuring specific serum and colostral antibody titres produced against the bacterium by ELISA test.

were taken from all the lambs included in the study at 48 hours, 15, 30, 45, 60 days after parturition. A total of 5 lambs came to death during the study period (Table 1).

Colostrum samples and serum samples from both dams and lambs were tested for M. haemolytica antibodies by ELISA home made test.

Vaccination The sheep were vaccinated subcutaneously with 2 ml per injection on the site around the neck. The vaccine was obtained from formalin killed cells of the epidemiologically most important serotypes of M. haemolytica (AI, A2, A6, A7 and A9), grown under iron restricted conditions: 5x108 cells per strain in buffered physiological saline adsorbed onto aluminum hydroxide. Thiomersal was included as a preservative.

ELISA assay Blood samples were taken lambs from the jugular vein using vacutainer tubes six times and sera were stored at −20°C until serological tests were performed.

The serum of a newborn lamb born from an unvaccinated ewe was used as negative control of the test. ELISA test was performed for the determination of antibody levels in lambs born from vaccinated and unvaccinated dams as follows: the vaccine was diluted in carbonate/bicarbonate buffer (0.05 M, pH 9.6) to a final concentration of 2.5x106 cells/well and used to coat 96-well plate with 100 µl/well (MaxiSorp). To normalize the effect of the adjuvant two lane were coated with adjuvant diluted in carbonate/bicarbonate buffer to 1.25%. After incubation over night at +4°C, the plate was washed three times in PBS containing 0.05% Tween 20 (PBST) and blocked by adding 200 µl/well of buffer containing 1% of casein in PBST (saturating buffer) and incubated at room temperature under stirring (300 rpm) for 1 h. After decanting the saturating buffer, 2fold serial diluted (1:100-1:12.800) in saturating buffer, of vaccinated and unvaccinated sera were added in duplicate to the plate at 100 µl/well. In each plate a negative control (serum taken from a lamb immediately after birth) was added. After 1 h of incubation at room temperature under stirring (300 rpm), the plate was washed with PBST and an antibody rabbit anti-sheep peroxidase-conjugated (Santa Cruz Biotechnology, Inc) was added at the dilution of 1: 5000 in saturating buffer for 1 h. The plate was washed with PBST and incubated in the dark, with a peroxidase substrate solution (1-step Ultra TMB-Elisa-Thermo Scientific) for 20 minutes at room temperature, and finally the reaction was blocked with a 0.5% sulfuric acid solution and the optical density was measured at 450 nm by spectrophotometer microplate reader (Sunrise™ Basic Absorbance Reader -Tecan Trading AG). The averages of the duplicated OD values subtracted from the adjuvant value were recorded.

MATERIALS AND METHODS Study design, sample size and animals The in-field trial took place in two different farms in Sardinia region (Italy). A total of forty-five adult healthy pregnant Sarda sheep were enrolled for the study (24 from the first flock and 21 from the second flock). Sheep selected were similar for body condition score, age (between 2 and 3 years) and at the same month of pregnancy. Each flock in each farm was divided in two groups: unvaccinated control group (10 sheep from the first flock and 8 from the second flock) and vaccinated sheep (14 from the first flock and 13 from the second flock). A total of eighteen unvaccinated sheep from the two flocks and the relative lambs were used as unvaccinated controls in the study. In total eighteen lambs were born from these ewes. Twenty-seven ewes from the two flocks were vaccinated for the first time six weeks prior to lambing. Vaccinated ewes were reinoculated at 2 week intervals. Serum was collected 2 weeks after the second vaccination (two weeks before lambing). In total twenty-seven lambs were born from these ewes. A total of forty-five lambs born from both vaccinated (n=27) and unvaccinated (n=18) dams were included in the study. The farmer checked carefully that all the lambs received colostrum. Colostrum was thus collected from all the dams within 6-12 hours after lambing. Serum samples

Table 1 - Number of lambs sampled from 48 hours until 60 days after birth in both farms.

Farm

Group

48 hours

15 days

30 days

45 days

60 days

1

Unvaccinated dams

10

10

10

10

10

1

Vaccinated dams

14

14

14

14

14

2

Unvaccinated dams

8

8

8

8

8

2

Vaccinated dams

13

12

11

11

10

45

44

43

43

42

Total

Statistical analysis The Pearson χ2 test was used to evaluate the difference between vaccinated and unvaccinated sheep and the presence of antibodies in dams, colostrum and lambs.

The non parametric Mann-Whitney test was used to assess at each time the quantitative difference between groups (vacci-

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nated vs. unvaccinated). A p-value ≤ 0.05 was considered statistically significant. Calculations were carried out in Stata 11.2 and Miscrosoft Excel 2013.

RESULTS Serum and colostrum antibodies titres on vaccinated and unvaccinated dams To determine ELISA titres we considered that if the vaccine was effective in inducing specific antibody, the vaccinated animals would have higher specific antibody titres than control animals. In order of that, in this ELISA test, the cut off values were calculated similar to the methods described by Mehmet et al.8 and on the unvaccinated control groups (sheep serum, colostrum, lambs at 48 hours, lambs at 15, 30, 60 days). The cut off values were calculated to the average values of the OD 450 nm obtained at the sample serum dilution 1:1600 adding 3 standard deviations. OD results were evaluated at the median serum diluition of 1:800 and each sample was considered “negative” when the value was below the cut-off and “positive” when was above.

Of the twenty-seven vaccinated ewes, 77.8% developed antibody titres >1: 800 detectable on serum by ELISA while of the eighteen unvaccinated ewes, 66.7% developed antibody titres ≤1: 800 (Table 2); a significant difference was found between these groups (p-value=0.0029).

Of the twenty-seven colostrum samples belonging to the vaccinated dams, only 18.5% developed antibody titres > 1:800 detectable by ELISA (Table 3). Colostrum samples did not showed any difference between the two groups (p-value=0.506).

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Table 2 - Number of serum samples below and above the cut off value evaluated at the median serum diluition of 1:800 in vaccinated and no vaccinated ewes against M. haemolytica (% calculated on the total column).

Groups

Serum samples ≤ 1: 800

> 1: 800

Total

Vaccinated ewes

6 (33.3%)

21 (77.8%)

27 (60%)

Unvaccinated ewes

12 (66.7%)

6 (22.2%)

18 (40%)

Total

18 (100%)

27 (100%)

45 (100%)

Table 3 - Number of colostrum samples below and above the cut off value evaluated at the median serum diluition of 1:800 in vaccinated and no vaccinated dams against M. haemolytica (% calculated on the total column).

Groups

Colostrum ≤ 1: 800

> 1: 800

Total

Vaccinated dams

22 (57.9%)

5 (71.4%)

27 (60%)

Unvaccinated dams

16 (42.1%)

2 (28.6%)

18 (40%)

Total

38 (100%)

7 (100%)

45 (100%)

from the unvaccinated dams (Figure 1). In particular, significant differences were demonstrated at 15 days (p-value=0.04) after birth, whereas no significant differences were found at 2 days (p-value=0.501), 30 days (p-value=0.43), 45 days (p-value=0.74) and 60 days (p-value=0.246).

Serum antibodies titres on lambs born from vaccinated and unvaccinated dams

DISCUSSION

The results on the total of the serum samples (n=118) of the lambs born from vaccinated dams showed higher anti-M. haemolytica titres time by time until sixty days after birth in comparison to the samples from the lambs (n=90) born

Infectious respiratory diseases of sheep and goats contribute to 5.6 percent of the total diseases of small ruminants9. M. haemolytica constitutes one of the most significant agents of Figure 1 Mean antibody titres in serum against M. haemolytica in lambs from vaccinated and no vaccinated ewes at each sampling time.

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respiratory infection, which is a major problem for sheep farmers. In a large Italian survey, 43.5% of the lambs between 25 and 35 days of life tested positive for M. haemolytica by PCR3. This result was in agreement with other reports in literature, which assess that lambs are most susceptible to the infection by this pathogen during the first months of life7,10.

Until now, there are no published data on Sarda sheep supporting the knowledge about the extent and length of colostral antibody protection against M. haemolytica in lambs born from vaccinated ewes under field condition. This could lead to ensure that farmers can vaccinate their ewes at the optimal time prior to lambing to achieve maximum levels of colostral antibody transfer, maximizing the duration of colostral immunity, while also indicating when lambs should then be newly immunized without interfering with colostral immunity. Prevention of M. haemolytica infection by using a good vaccination program both on ewes and lambs would be expected to decrease pneumonia in lambs by developing good maternal and passive colostral immunity.

In this study, the length and efficacy of immunization with a vaccine against M. haemolytica infection in Sarda sheep were investigated. Serological determination of antibody titres in serum and colostrum in a group of vaccinated dams and relative lambs and that one of unvaccinated dams and relative lambs was tested. Antibodies were detected among the pregnant ewes and their lambs bled at 48 hours, 15, 30, 45, 60 days after birth by ELISA test. To the authors knowledge, the serum ELISA titres obtained in this study could not be compared because no similar studies were performed in Italy on Sarda sheep; on the contrary, for the determination of antibody titres obtained after M. haemolytica vaccinations, many foreign studies have been performed and different antigens and techniques have been used11,12. Amongst these techniques, ELISA is widely used13. Mosier et al.14, showed a positive correlation between the ELISA titres and resistance to experimental pasteurellosis in cattle and reported that ELISA was a more feasible and faster technique considering the number of sera that could be tested readily. In this study, significant increase of serum ELISA titres following vaccination in pregnant ewes and its efficacy on immunity on lambs is shown. In vaccinated pregnant ewes, serum titres developed after vaccination resulted two times higher than in the unvaccinated group (Table 2), this difference was statistically significant (p-value=0.0029). On the contrary, colostral titres didn’t show any significant difference between the two groups examined (Table 3). This result suggested that in many cases colostrum couldn’t be sampled within the 6-12 hours after birth and it must be considered that this kind of matrix is highly degradable15. Within the lamb groups, an increase of serum ELISA titres was found in lambs born from vaccinated ewes until 60 days after birth (Figure 1). After this period, lamb passive immunity seems dangerously to decrease and this can lead to the development of pneumonia. The result is in agreement with the reports which assess that the major incidence of respiratory diseases (over 30%) is registered in sheep between 3 and 6 months of life7,10. The major differences regarding the concentration of antibody titres against M. haemolytica were found at 15 days (p-value=0.04). According to the results obtained in this investigation, M. haemolytica vaccination on pregnant ewes between six and four weeks prior to lambing seems to provide a good passive immunity to lambs until 60 days after birth.

CONCLUSIONS Nowadays, the Italian sheep industry is facing many difficulties because of the minimal public funds given to the farmers and the lowest profits in the agricultural industry.

Given the significant economic losses due to M. haemolytica infection and the increasing antibiotic resistance, it is necessary to adopt useful preventive tools for the benefits of the whole sheep industry. The results obtained from this investigation showed that immunization of ewes and lambs with a vaccine containing M. haemolytica AI, A2, A6, A7 and A9 bacterins, under field condition and between the sixth and the fourth week prior to lambing significantly increases serum antibody titres and provides good protection to lambs.

References 1. Brodgen K.A., Lehmkuhl H.D., Cutlip R.C. (1998) Pasteurella haemolytica complicated respiratory infections in sheep and goats. Vet Res, 29: 233-254.

2. Ackermann M.R. (2014) Lab model of respiratory syncytial virus-associated lung disease: insights to pathogenesis and novel treatments.

ILAR J, 55: 4-15. doi: 10.1093/ilar/ilu003.

3. Bottinelli M., Schnee C., Lepri E., Stefanetti V., Filippini G., Gobbi M., Sebastianelli M., Antenucci P., Rampacci E., Coletti M., Passamonti F. (2017) Investigation on mycoplasma populations in pneumonic dairy lamb lungs using a DNA microarray assay. Small Ruminant Res, 147: 96-100. doi: 10.1016/j.smallrumres.2016.12.038.

4. Lin Y.C., Miles R.J., Nicholas R.A.J., Kelly D.P., Wood A.P. (2008) Isolation and Immunological detection of and Immunological detection of Mycoplasma ovipneumoniae in sheep with atypical pneumonia, and lack of a role for Mycoplasma arginini. Res Vet Sci, 84: 367-373. doi: 10.1016/j.rvsc.2007.06.004.

5. Nicholas R., Ayling R., McAuliffe L. (2008) Respiratory Disease of Small Ruminants. In: Mycoplasma Diseases of Ruminants, Ed. Nicholas R., Ayling R., McAuliffe L., 1st ed., 169-198, CAB International, Oxforshire, UK. 6. Rodger J.L. (1989) Parainfluenza 3 vaccination of sheep Vet. Rec., 125: 453-456.

7. Alley M.R. (1991) Pneumonia in sheep. Veterinary Annual, 31: 51-58.

8. Mehmet A., Tanar O., Baris S., Rifki H., Osman Y., Zafer C. (2006).

Vaccination studies of lambs against experimental Mannheimia (Pasteurella) haemolytica infection. Small Rumin Res 65, 44-50. doi: 10.1016/j.smallrumres.2005.05.020 9. Amit K., Suresh K. Tikoo, Praveen Malik and Aruna T. Kumar (2014) Respiratory Diseases of Small Ruminants. Vet Med Int, 2014: 2. doi: 10.1155/2014/373642.

10. Radostits O.M., Clive C.G., Hinchcliff K.W., Constable P.D. (2007) In: Veterinary Medicine: A textbook of the diseases of cattle, horses, sheep, pigs and goats, Ed. 10th, 946-949, Elsevier, Saunders, Edinburgh.

11. Srinand S., Hsuan S.L., Yoo H.S., Maheswaran S.K., Ames T.R., Werdin R.E. (1996) Comparative evaluation of antibodies induced by commercial Pasteurella haemolytica vaccines using solid phase immunoassays.

Vet Microbiol, 49: 181-195. doi.org/10.1016/0378-1135(95)00187-5.

12. Tao S. (2009) Evaluation of a vaccine against Mannheimia haemolytica and Pasteurella multocida in sheep. Honors Thesis Presented to the College of Agriculture and Life Sciences, Department of Animal Science of Cornell University in Partial Fulfillment of the Requirements for the Research Honors Program.

13. Akan M., Öncel T., Sareyyüpoğlu B., Hazırŏglu R., Yaşar Tel O., Cantekin Z. (2006) Vaccination studies of lambs against experimental Mannheimia (Pasteurella) haemolytica infection. Small Ruminant Res, 65: 44-50. doi: 10.1016/j.smallrumres.2005.05.020.

14. Mosier D.A., Confer A.W., Hall S.M., Gentry M.J., Panciera R.J. (1986) Enzyme-linked immunosorbent assay for detection of serum antibodies to Pasteurella haemolytica cytotoxin (leukotoxin) in cattle. J Clin Microbiol, 24: 218-222.

15. Gautier J. M., Corbière F. (2017) Better knowledge for sheep colostrum quality and passive immune transfer, Ninth International Sheep Veterinary Congress, Harrogate, UK, 22-26.

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Infezione da Ovine herpesvirus tipo 2 in allevamenti ovini in Umbria

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ORIANA RAFFAELE1, EMILIA DEL ROSSI1, LORENZO CASTELLI2, STEFANO PIGNANI2, STEFANO PETRINI3, MARIA TERESA MANDARA1, MARIA LUISA MARENZONI1 1

Dipartimento di Medicina Veterinaria, Università degli Studi di Perugia Associazione Regionale Allevatori Umbria 3 Istituto Zooprofilattico Sperimentale Umbria e Marche “Togo Rosati” 2

RIASSUNTO Ovine gammaherpesvirus 2 (OvHV-2) è un gammaherpesvirus che causa un’infezione asintomatica nella pecora e una malattia sistemica molto grave in altre specie di ruminanti e nel maiale, denominata febbre catarrale maligna (FCM). Il controllo dell’infezione nella pecora risulta essere un fattore chiave nel contenimento della FCM. Inoltre non può essere escluso un ruolo di questo virus nella patologia ovina. Scopo del presente studio è stato quello di indagare la presenza di OvHV-2 negli allevamenti ovini in Umbria e di comprendere la relazione di alcuni fattori di rischio dell’infezione individuati negli allevamenti controllati. Sono stati analizzati, tramite nested PCR, 50 tamponi nasali prelevati rispettivamente da 39 pecore e da 11 capre, provenienti da 9 diverse aziende, per ricercare la presenza del DNA di OvHV-2. La specie, l’età, l’attitudine produttiva, la promiscuità con specie diverse (bovini e/o capre) e l’effetto del singolo allevamento sono stati valutati come possibili fattori di rischio dell’infezione mediante analisi statistica univariabile e multivariabile. Ventinove su 39 (74,4%) pecore e 3 su 11 (27,3%) capre sono risultate positive. I fattori di rischio presi in considerazione si sono dimostrati significativi nell’analisi univariabile, ma solo l’effetto dell’allevamento è rimasto statisticamente significativo nell’analisi multivariabile. Probabilmente il singolo allevamento contiene al suo interno altre variabili in grado di spiegare una modificazione nella prevalenza dell’infezione da OvHV-2. In futuro dovranno essere esaminati altri fattori tipici di ciascun allevamento al fine di comprendere meglio l’epidemiologia dell’infezione.

PAROLE CHIAVE Ovine gammaherpesvirus 2, Regione Umbria, pecora, PCR, allevamento ovino.

INTRODUZIONE Ovine gammaherpesvirus 2 (OvHV-2) è un gammaherpesvirus che causa un’infezione pressoché asintomatica nelle pecore, mentre è associato a forme cliniche, anche molto gravi, in specie affini ma non completamente permissive. In quest’ultimo caso la malattia è conosciuta come febbre catarrale maligna (FCM). Nonostante siano stati fatti passi avanti nelle conoscenze della patogenesi, ancora si è lontani dal capire il reale meccanismo che è alla base sia dell’infezione nella pecora sia della FCM nelle specie affini suscettibili. Si pensa che la trasmissione di OvHV-2 avvenga prevalentemente tramite la via inalatoria1,2, ma anche la via venerea è stata ritenuta possibile, in particolare da parte dell’ariete, condizione che potrebbe determinare una stagionalità dell’infezione3. Il sito di replicazione primaria del virus negli ovini è rappresentato dal polmone4, mentre la successiva fase di latenza è localizzata nei linfociti1,5. Per quanto riguarda la malattia nelle specie secondarie, sembra che l’evento chiave nel determinismo della FCM sia una disregolazione delle cellule T, determinata dal virus6,7.

Corresponding Author: Maria Luisa Marenzoni (marialuisa.marenzoni@unipg.it).

La FCM ha una manifestazione clinica abbastanza variabile.

Esiste una forma classica, acuta, costituita da una sindrome caratterizzata da linfoproliferazione sistemica, vasculite e lesioni infiammatorie distribuite in molti tessuti, e che presenta un tasso di letalità del 50-70%8. Esistono inoltre forme croniche, che possono esitare in guarigione parziale o completa, e anche infezioni asintomatiche, che probabilmente non vengono diagnosticate9,10. Questa stessa malattia, in realtà, può essere causata da diversi gammaherpesvirus del genere Macavirus: oltre a OvHV-2, anche alcelaphine herpesvirus-1 (AlHV-1), caprine herpesvirus-2 (CpHV-2), ibex malignant catarrhal fever virus, alcelaphine herpes virus-2like e un virus di origine ancora non definita, che ha causato la malattia nel cervo dalla coda bianca, sono stati associati a forme cliniche di FCM in diverse specie animali. Tutti questi virus, oltre a infettare in maniera asintomatica il rispettivo ospite primario (pecore, gnu, capre, ibex, alce, etc.), sono in grado di infettare specie affini, ma secondarie, dove causano appunto la FCM. Tra le specie più colpite da FCM vi sono i bovini, i bufali, il bisonte americano, varie specie di cervidi, nonché altri ruminanti selvatici, ma anche i suini11,12. Alcune specie di animali sono considerate altamente suscettibili11,12.

La FCM si manifesta con depressione, anoressia, febbre alta, linfoadenopatia, congiuntivite, opacità corneale, infiammazione, ulcerazione ed essudazione del tratto respiratorio e digestivo superiore, diarrea e deficit neurologici, fino ad arri-

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vare alla morte dell’animale colpito11,12. Le possibilità terapeutiche sono pressoché nulle, per cui l’infezione grave è quasi invariabilmente letale. Il virus è difficilmente isolabile13 e questo ha contribuito all’incapacità di sviluppare un vaccino, ragion per cui non è possibile effettuare neanche una profilassi indiretta. Al contempo le diagnosi differenziali che occorre considerare in questi casi includono malattie quali l’afta epizootica, la diarrea virale bovina, la rinotracheite infettiva bovina, la cheratocongiuntivite infettiva da herpesvirus, la cheratocongiuntivite infettiva da Moraxella bovis, la stomatite papulosa e la blue tongue, che prevedono anche l’adozione di misure sanitarie di prevenzione restrittive, quali sequestro dell’allevamento infetto, blocco delle movimentazioni, abbattimenti, etc.

In Italia, a parte i casi registrati nei giardini zoologici14,15, dove la possibilità che la specie di virus coinvolto sia più ampia, i virus che maggiormente vengono associati alla FCM sono OvHV-2 e CpHV-2. Gli episodi di FCM segnalati nel nostro territorio sono relativamente rari, occasionalmente con andamento epidemico, ma in generale probabilmente sottodiagnosticati. Generalmente questi casi sono riconducibili a situazioni di allevamenti promiscui di bovini/ovini o bovini/ovini/caprini16,17,18 o bufali/bovini/ovini19. In uno studio effettuato in Italia si è arrivati alla conclusione che la FCM, nella sua forma associata alle pecore, ossia da OvHV-2, è da considerare una malattia endemica20. Tuttavia, non esistono dati che indichino quale sia la reale prevalenza di infezione da OvHV-2 in Italia e anche a livello internazionale sono poche le indicazioni a tal proposito. La Tabella 1 riassume le prevalenze riportate per OvHV-2 nel mondo.

Tra i fattori di rischio indicati per l’infezione da OvHV-2 compaiono l’età delle pecore (gli agnelli di età compresa tra i 6 e i 9 mesi diffondono il virus molto più frequentemente e più intensamente degli adulti), gli allevamenti promiscui (bovini/ovini, bovini/ovini/caprini e bufali/bovini/ovini), la densità degli animali in allevamento (maggiore è la densità, mag-

giore è il rischio di contagio per gli animali suscettibili), la pratica della transumanza (maggior probabilità di contatto diretto tra animali infetti e suscettibili), i fattori climatici, la presenza di vettori e il livello di stress dovuto alle variazioni climatiche o alle pratiche di management10,12,16,17,18,19,21,22,23,24.

Non rientrerebbero invece tra i possibili fattori di rischio la razza e il sesso, né l’attitudine dell’allevamento (latte, carne o misto)22,25.

Per tale motivo è importante conoscere se e quanto questi virus circolano negli allevamenti ovini e caprini del territorio e i fattori che possono influenzare la loro diffusione, che costituiscono il punto chiave per contenere l’infezione.

Scopo del presente studio è stato quello di valutare la presenza di OvHV-2, potenziale trigger di FCM, in allevamenti ovini del territorio umbro e verificare se alcuni fattori di rischio sono presenti nei nostri allevamenti al fine di aumentare le conoscenze sull’epidemiologia di questa infezione nel territorio.

MATERIALI E METODI Nel periodo ottobre 2016 - febbraio 2017 sono stati prelevati, previo consenso informato degli allevatori, dei tamponi nasali (TN) da pecore in quanto considerati un campione indicativo della possibilità di trasmissione del virus, che avviene principalmente per via inalatoria, a seguito di contatto ravvicinato.

Gli allevamenti testati sono stati selezionati in quanto iscritti all’Associazione Regionale Allevatori (ARA) dell’Umbria e considerati rappresentativi della realtà produttiva ovina e caprina locale. Gli elementi che sono stati registrati al momento del prelievo erano il codice identificativo del soggetto campionato; la razza; l’età; il sesso; l’attitudine produttiva; la consistenza numerica dell’allevamento; la tipologia di allevamento (intensivo, semi-intensivo, estensivo); il numero ap-

Tabella 1 - Prevalenza dell’infezione da OvHV-2 riportata in precedenti studi nel mondo.

Area geografica

Test applicato

Prevalenza OvHV-2

Bibliografia

INDIA (Regione del Kashmir)

PCR da sangue

pecore: 84,4% (28/33) agnelli: 95,2% (20/21) capre: 61,5% (16/26)

Wani et al., 20068

INDIA (Regione del Karnataka)

PCR da sangue

pecore: 24,44% (87/356)

Premkrishnan et al., 201521

KENYA

PCR da sangue

pecore: 90,4% (161/178)

Mirangi et al., 199730

PORTOGALLO

PCR da sangue

pecore (allevamento monospecifico): 81,3% (65/80) allevamento solo bovini (allevamento monospecifico): 0% (0/18) pecore (allevamento misto bovini-pecore): 70,5% (31/44) bovini (allevamento misto bovini-pecore): 7,4% (2/27)

Cortez et al., 200810

USA

PCR da sangue CI-ELISA§

pecore: 99% (143/144) pecore (non associate a casi di FCM nel bovino): 53% (282/531) pecore (associate a casi clinici di FCM nel bovino): 59% (88/149) capre: 61% (177/291)

Li et al., 199625

SUD AFRICA

PCR da sangue

pecore: 75,3% (130/170)

Bremer et al., 201022

TURCHIA

PCR da sangue

pecore: 62,7% (37/59)

Kalayci et al., 201531

GERMANIA

PCR da sangue CI-ELISA§

pecore: 100% (20/20) pecore: 72% (36/50)

Frolich et al., 199840

GIAPPONE

SN*

pecore: 37,6% (56/154) capre: 40% (2/5)

Giangaspero et al., 201341

§

CI-ELISA: competitive inibition ELISA; * SN: sieroneutralizzazione.

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prossimativo di movimentazioni in entrata o in uscita dall’allevamento; l’ubicazione dell’allevamento; la densità degli animali nell’allevamento; l’effettuazione della pratica della transumanza; la gestione delle rimonte e l’età di separazione degli agnelli dalle madri; infine, se l’allevamento era promiscuo con caprini, bovini o altri ruminanti.

Da ogni allevamento sono stati campionati almeno 5 animali fino a un massimo di 10.

I TN sono stati effettuati utilizzando tamponi di cotone che sono stati strofinati all’interno delle narici delle pecore e successivamente posti in 500 µl di tampone fosfato salino (PBS).

Estrazione del DNA Il DNA è stato estratto partendo da 200 µl del liquido di sospensione del tampone nasale, utilizzando un kit commerciale, secondo le indicazioni del produttore (QIAamp DNA Mini kit, Qiagen).

Protocolli di PCR Al fine di identificare OvHV-2 sono stati utilizzati inizialmente tre protocolli di PCR. Il primo era un protocollo di PCR in grado identificare tutti i membri della famiglia Herpesviridae, avendo come gene target un frammento della DNA polimerasi comune a tutti gli herpesvirus26. In parallelo, nelle prime fasi, sono stati utilizzati due ulteriori protocolli di PCR OvHV-2-specifici, secondo quanto indicato dalla World Organisation for Animal Health (OIE)27,28,29. I protocolli di PCR sono stati sottoposti a un settaggio iniziale, tramite PCR a gradiente, che ha valutato temperature comprese tra 53 e 66°C per verificare se il profilo termico indicato nei protocolli pubblicati fosse quello ottimale o se la temperatura di annealing dei primer andasse modificata per ottenere un protocollo più sensibile. Le temperature di annealing sono state verificate tramite diluizioni seriali del controllo positivo (DNA di OvHV-2 sequenziato). I tre diversi protocolli di PCR sono stati poi comparati tramite diluizioni seriali del controllo positivo per valutare la diversa sensibilità. Ogni set di reazione allestito aveva un controllo positivo (DNA di OvHV-2 precedentemente sequenziato) e un controllo negativo (senza DNA di OvHV-2).

Analisi statistica La stima della prevalenza dell’infezione da OvHV-2 è stata definita come proporzione di animali positivi alla PCR da TN sul totale degli animali testati.

Le variabili indipendenti registrate al momento del prelievo e considerate potenziali fattori di rischio per l’infezione (specie, età, allevamento, attitudine produttiva, promiscuità tra specie) sono state analizzate separatamente per accertare la possibile relazione tra potenziale fattore di rischio e infezione da OvHV-2. L’età è stata analizzata categorizzandola in due gruppi: soggetti di età > di 1 anno oppure > di 2 anni. Il test F di Fisher o il test χ2 sono stati utilizzati secondo il metodo più appropriato ai dati in studio. Variabili che sono risultate con una probabilità (P) ≤0,20 nel modello univariabile sono state incluse nel modello multivariabile, inserendo manualmente le variabili ed eliminandole con la modalità backward.

La stima dell’Odds Ratio (OR) e dei corrispondenti intervalli di confidenza al 95% (95% CI) è stata ottenuta tramite regressione logistica. I dati sono stati analizzati tramite il software R, version 2.8.1 (R, Development Core Team 2007).

Un valore di P<0.05 è stato considerato significativo.

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RISULTATI Scelta del protocollo di PCR Dai risultati ottenuti con la PCR a gradiente, le temperature indicate nei protocolli pubblicati da Baxter et al. (1993)28 e Flach et al. (2002)29 non rappresentavano le migliori e quindi sono state utilizzate nuove temperature di annealing, come indicato nella Tabella 2.

Il protocollo più sensibile è risultato quello descritto da Flach et al., 200229, che è stato utilizzato per eseguire le analisi su tutti i soggetti.

Prevalenza e analisi dei fattori di rischio In totale sono stati campionati 50 animali, di cui 39 pecore e 11 capre. Sette animali erano sotto l’anno di età, 23 di età compresa tra 1 e 2 anni e 20 di età superiore ai 2 anni. Nel periodo dell’anno in cui è stato effettuato il campionamento non erano presenti soggetti di 6-9 mesi di età, che rappresentano la fascia di età più suscettibile all’infezione.

Gli allevamenti avevano caratteristiche uniformi per quanto riguarda la densità degli animali (conforme alle indicazioni del Decreto Legislativo 26 marzo 2001, n. 146, recante l’Attuazione della direttiva 98/58/CE sulla protezione degli animali negli allevamenti, GU n. 95 del 24-04-2001), la transumanza (che non era praticata da nessun allevamento), la separazione degli agnelli dalle madri (che avveniva a circa 30 giorni di vita) e l’introduzione delle rimonte nel gruppo degli adulti (che avveniva dopo il primo parto, a circa 15 mesi di età).

Gli allevamenti erano tutti di tipo semiestensivo, ad eccezione di uno intensivo e uno estensivo. L’attitudine produttiva era da latte per sei aziende e da carne per tre. La consistenza numerica degli allevamenti variava da 70 a 1200 capi, con un valore medio di 471 animali e una mediana di 460. Solo un’azienda aveva una consistenza inferiore ai 100 capi.

Il numero di movimentazioni era basso per tutte le aziende, da considerarsi occasionale, sebbene in alcuni casi una singola movimentazione poteva consistere anche nell’acquisizione di circa 100 nuovi soggetti. Tutti gli allevamenti erano ubicati vicino ad altri allevamenti, riserve naturali o zone di ripopolamento, in cui erano comunque presenti altri ruminanti selvatici o domestici.

Quattro aziende allevavano esclusivamente ovini, due avevano sia pecore che capre, due anche capre e bovini e una era promiscua con bovini. In genere erano associate pecore da carne e bovini da ingrasso. Un allevamento prima promiscuo con pecore e capre ha inserito i bovini da ingrasso nel periodo successivo allo studio.

La prevalenza totale dell’infezione riscontrata da TN era del 64% (32/50). La Tabella 3 riporta le prevalenze ottenute, considerando le singole variabili esaminate nell’analisi statistica.

All’analisi univariabile sono risultati significativi tutti i fattori indagati, ossia la specie (pecora vs capra), l’età superiore a 1 anno o superiore a 2 anni, l’allevamento, l’attitudine (latte vs carne), la promiscuità tra specie. L’unico fattore che invece è risultato significativo anche nell’analisi multivariabile è l’allevamento, che ha ottenuto un OR=0.55, CI 95%: 0.38-0.79, P=0.02. Pertanto, l’allevamento risulta un fattore più determinante rispetto agli altri, perché probabilmente assorbe in un unico parametro tutte le altre variazioni considerate.

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Infezione da Ovine herpesvirus tipo 2 in allevamenti ovini in Umbria

Tabella 2 - Primer utilizzati nello studio.

Nome primer

Gene target

DFA (F)

DNA polimerasi degli herpesvirus

ILK (F)

DNA polimerasi degli herpesvirus

KG1 (R)

DNA polimerasi degli herpesvirus

TGV (FN)

DNA polimerasi degli herpesvirus

IYG (RN)

DNA polimerasi degli herpesvirus

556 (F; FN)

DNA polimerasi dell’OvHV-2 e AIHV-1

755 (R)

DNA polimerasi dell’OvHV-2 e AIHV-1

555 (RN)

DNA polimerasi dell’OvHV-2 e AIHV-1

POL1 (F)

DNA polimerasi dell’OvHV-2 e AIHV-1

POL2 (R; RN)

DNA polimerasi dell’OvHV-2 e AIHV-1

OHVPol (FN)

DNA polimerasi dell’OvHV-2 e AIHV-1

Tipo di PCR

Bibliografia

Temperatura di annealing indicata sul manuale OIE27 (°C)

Temperatura di annealing utilizzata nello studio (°C)

VanDevanter et al., 199626

46

46

225

VanDevanter et al., 199626

46

46

422

Baxter et al., 199328

60

66

238

Baxter et al., 199328

60

66

386

Flach et al., 200229

60

54

172

Flach et al., 200229

60

54

Lunghezza bp

Sequenza primer

725 (DFA+KG1)

5’-GAYTTYGCNAGYYTNTAYCC-3’ convenzionale 5’-TCCTGGACAAGCAGCARNYSGCNMTNAA-3’ 5’-GTCTTGCTCACCAGNTCNACNCCYTT-3’

oppure 470 (ILK+KG1)

5’-TGTAACTCGGTGTAYGGNTTYACNGGNGT-3’ nested 5’-CACAGAGTCCGTRTCNCCRTADAT-3’ 5’AGTCTGGGTATATGAATCCAGATGGCTCTC-3’ convenzionale 5’-AAGATAAGCACCAGTTATGCATCTGATAAA-3’ nested

5’-TTCTGGGGTAGTGGCGAGCGAAGGCTTC-3’ 5’-GGC(CT)CA(CT)AA(CT)CTATGCTACTCCAC-3’

convenzionale 5’-ATT(AG)TCCACAAACTGTTTTGT-3’

seminested

5’-AAAAACTCAGGGCCATTCTG-3’

F: forward; R: reverse; FN: forward protocollo nested; RN: reverse protocollo nested.

DISCUSSIONE Tutti i 9 allevamenti testati sono risultati positivi all’infezione da OvHV-2 con almeno un soggetto positivo, confermando l’ampia diffusione del virus negli allevamenti ovini e ovini/caprini del territorio, condizione che era stata ipotizzata

sulla base dei dati internazionali. Sebbene esistano molte differenze tra gli studi di prevalenza per quanto riguarda test applicato, matrice biologica analizzata e area geografica, si può affermare che la prevalenza ottenuta nello studio è sovrapponibile a quelle riscontrate precedentemente a livello internazionale8,10,21,22,25,30,31.

Tabella 3 - Risultati dell’analisi univarabile e multivariabile dei fattori associati ad una variazione nella prevalenza dell’infezione da OvHV-2.

Analisi univariabile

Analisi multivariabile

Fattore

Categoria

Prevalenza

P

OR

95% IC

P

SPECIE

pecore capre

74.4% (29/39) 27.3% (3/11)

0.01

0.0

0.0

0.9

≤ 1 anno > 1 anno

100% (7/7) 58.1% (25/43)

0.04

0.0

0.0

0.9

≤ 2anni > 2 anni

83.3% (25/30) 35% (7/20)

0.01

0.0

0.0

0.9

da latte da carne

83.3% (25/30) 35% (7/20)

< 0.0001

0.06

0.001-5.67

0.23

solo pecore capre bovini capre-bovini

90% (18/20) 70% (7/10) 40% (2/5) 33.3% (5/15)

0.004

2.39

0.3-20.7

0.42

80% (4/5) 100% (5/5) 100% (5/5) 100% (5/5) 80% (4/5) 40% (2/5) 40% (4/10) 40% (2/5) 20% (1/5)

0.02

0.55

0.38-0.79

0.001

ETÀ

ATTITUDINE PRODUTTIVA

PROMISCUITÀ

ALLEVAMENTO

azienda azienda azienda azienda azienda azienda azienda azienda azienda

1 2 3 4 5 6 7 8 9

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Inoltre, seppur non statisticamente significativa nel modello multivariabile, la tendenza ad avere un’alta prevalenza nei soggetti più giovani, indicata dalla bibliografia, è confermata25.

Sono state fatte invece osservazioni differenti per quanto riguarda le altre variabili analizzate. La maggior prevalenza ottenuta nelle pecore rispetto alle capre appare giustificata dal fatto che gli ovini sono gli ospiti primari del virus12,15. Anche l’attitudine da latte, così come l’allevamento monospecifico delle pecore, sembrano favorire una maggior prevalenza di infezione da OvHV-2; quando invece aumenta la promiscuità con capre, bovini e capre associate a bovini, la prevalenza dell’infezione nella pecora diminuisce. Tutti questi fattori però perdono di significatività quando vengono valutati nel loro insieme nell’analisi multivaribile e di fatto vengono tutti assorbiti dalla variabile allevamento. Evidentemente ci sono altri elementi intrinseci all’allevamento, che andranno ulteriormente indagati, che influiscono sull’infezione. Ad esempio è possibile che l’organizzazione di un allevamento monospecifico di pecore, magari ad attitudine latte, sia caratterizzato da situazioni in cui vi è un maggior contatto tra animali, che favorisce la trasmissione dell’infezione. Quando entrano in analisi anche le capre e soprattutto i bovini, che probabilmente comportano anche una diversa disposizione e gestione dell’allevamento, allora la prevalenza di infezione nella pecora diminuisce. Il fatto che un diverso management dell’azienda possa influenzare l’andamento dell’infezione è stato già ipotizzato in precedenza12.

La promiscuità di specie, che comunque è il prerequisito nello sviluppo della FCM, è in ogni caso una realtà frequente negli allevamenti ovini e caprini in centro Italia: 5 dei 9 allevamenti considerati avevano promiscuità di specie allevate al loro interno; uno è diventato promiscuo con bovini successivamente allo studio e comunque tutti gli allevamenti si trovavano in prossimità di altri allevamenti o zone in cui erano presenti ruminanti selvatici o domestici. Questo è un elemento di rischio da considerare nelle realtà dell’Appennino Centrale per quanto riguarda la possibilità di occorrenza della FCM nelle diverse specie suscettibili. Tuttavia, la sola presenza di un ospite serbatoio non è considerata sufficiente per realizzare la trasmissione dell’infezione, soprattutto a specie secondarie, e probabilmente serve un contatto estremamente stretto tra di esse12. Powers et al. 20059 indicano una vicinanza di 70 metri come rischiosa per la trasmissione di OvHV-2 da ovini a bovini. Allo stesso modo, distanze di 100 metri sono state indicate efficaci per la trasmissione di AlHV-1 tra specie diverse27. La separazione a lunghe distanze è ritenuta comunque un rischio quando gli ospiti sono altamente suscettibili e la concentrazione virale è alta, come nei bisonti e negli agnelli in allevamento11,32. Per CpHV-2 è stata segnalata la possibilità di trasmissione fino a 5 km4.

Discorso a parte va fatto per la capra, che costituisce un ulteriore serbatoio dell’OvHV-2. Nel presente lavoro sono risultate positive 3 su 11 capre per la presenza di OvHV-2. Nella capra è stata riprodotta sperimentalmente l’infezione, che è risultata asintomatica33. Tuttavia la capra ospita anche CpHV-2, che è correlato antigenicamente all’OvHV-2. Sono state segnalate anche coinfezioni con i due virus12. CpHV-2 sembra meno patogeno rispetto a OvHV-2 ai fini della FCM12,34. Questi sono alcuni dei motivi per cui è necessario avere mezzi diagnostici in grado di distinguere i due virus, anche al fine di identificare le diverse fonti di virus in caso di FCM12. Il protocollo utilizzato nel presente studio è stato in

83

grado di distinguere i due virus in quanto capace di rilevare specificatamente OvHV-229. Un monitoraggio periodico del genoma di questi virus, o loro parti, con la caratterizzazione delle sequenze di virus provenienti da specie a diversa suscettibilità, differenti aree geografiche e anni, potrebbe essere utile per implementare banche dati tematiche, fondamentali in caso di epidemie per risalire all’origine dei focolai23,35.

Altro elemento da considerare in futuro per i prelievi è la stagione. Infatti nel presente studio il campionamento è stato condotto in un periodo limitato (ottobre-febbraio). L’allevamento ovino è già di per sé molto stagionalizzato e nel periodo preso in esame non erano presenti i soggetti di 6-9 mesi, che sono ritenuti i maggiori diffusori del virus. Inoltre, potrebbe essere ipotizzato un effetto stagionale del clima sull’infezione, potendo influire su stress, contatti, presenza o meno di pascoli, etc.

Sebbene la numerosità campionaria per allevamento del presente studio possa essere ritenuta sufficiente per la ricerca di OvHV-212, un’ulteriore implementazione dello studio andrà fatta sul numero di soggetti da sottoporre a prelievo all’interno dell’allevamento per aumentare la consistenza dei dati.

Al momento non è stato possibile neanche effettuare un campionamento randomizzato, che invece è un assunto importante per la generalizzabilità dei risultati.

Comunque, una volta identificati i fattori di rischio per l’infezione presenti nel territorio, risulta possibile mettere in atto alcune strategie per limitare la diffusione dell’infezione, come ad esempio separare le specie suscettibili da pecore e capre infette. È stato dimostrato che è addirittura possibile costituire greggi di pecore o capre non infette da OvHV-2 o CpHV-2, separando precocemente gli agnelli o i capretti (ad esempio a una settimana, più precoce, per i capretti e 2 mesi per gli agnelli) dalle madri e dagli adulti in generale. Infatti questi virus non vengono trasmessi per via verticale (diversamente da AlHV-1) e gli agnelli risultano anche protetti nel periodo neonatale, probabilmente grazie alla presenza dell’immunità passiva materna36. La separazione tra soggetti negativi e soggetti positivi va mantenuta nel tempo, creando due greggi distinti, e questo comporta che gli animali vadano periodicamente sottoposti a test per verificare il mantenimento della negatività37. I metodi più utilizzati per poter diagnosticare al momento l’infezione da OvHV-2 sono il test ELISA o la sieroneutralizzazione per la sierologia e la PCR per la diagnosi diretta. Mentre la sierologia è il metodo più rapido per evidenzare l’esposizione al virus in un allevamento, la PCR è il metodo più usato per ottenere una diagnosi di certezza, identificare soggetti diffusori del virus e per poter tracciare l’origine di un focolaio tramite metodi biomolecolari27.

Sempre per ridurre la possibilità di passaggio dell’infezione a specie affini, i bovini non dovrebbero pascolare negli stessi spazi dove pascolano pecore o capre o ruminanti selvatici sucettibili. Stessa condizione dovrebbe essere mantenuta negli zoo, assicurando la separazione delle specie recettive tra loro.

Ridurre il più possibile i fattori stressanti per gli animali può aiutare a prevenire lo sviluppo di forme cliniche. In caso di focolaio, gli animali suscettibili dovrebbero essere subito separati dai malati e dai sospetti infetti. Va considerato però che, potendo essere molto lungo il periodo di incubazione, possono presentarsi per mesi nuovi casi di infezione. Poiché i bovini e altri ospiti accidentali sono ospiti a fondo cieco, non è necessario l’abbattimento11.

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Infezione da Ovine herpesvirus tipo 2 in allevamenti ovini in Umbria

CONCLUSIONI

KEY WORDS Ovine gammaherpesvirus 2, Umbria Region, sheep, PCR.

L’infezione da OvHV-2 viene spesso studiata in funzione della FCM, mentre poco si sa del reale impatto che ha nell’allevamento ovino. Considerando che, seppur sporadicamente, in altre specie i gammaherpesvirus possono causare malattia anche nell’ospite primario6, ulteriori studi dovrebbero essere effettuati nella specie ovina. Questo virus andrebbe indagato in tutte le condizioni patologiche, soprattutto se a eziologia non ancora definita. Recenti studi hanno permesso di poter conoscere alcuni aspetti del meccanismo patogenetico dell’OvHV-2, ma si è lontani dalla comprensione completa dei meccanismi di infezione nella pecora e dell’innesco della FCM in altre specie7,38. Poiché non si conosce ancora del tutto l’azione che il virus svolge, pur essendo addirittura possibile eliminare l’infezione nell’ospite primario, non è detto che eliminare un’infezione comporti necessariamente dei benefici. Infatti, è stato riconosciuto come alcune infezioni, anche da herpesvirus, siano addirittura utili a stimolare l’immunità cellulare nell’ospite39.

Per una infezione poco conosciuta come questa, causa anche di malattia grave e per la quale non esistono terapie specifiche, né vaccini, occorre certamente migliorarne le conoscenze e identificarne quanto meno i fattori di rischio per poter controllare l’infezione ed evitare l’iperendemia, che è il maggior pericolo nello sviluppo della FCM.

RINGRAZIAMENTI Si ringraziano gli allevatori che hanno dato la disponibilità a partecipare allo studio.

❚ Ovine herpesvirus 2 infection in sheep flock of the Umbria Region SUMMARY Ovine gammaherpesvirus 2 (OvHV-2) is a gammaherpesvirus that causes an asymptomatic infection in sheep and a severe systemic disease in other ruminants and pigs, called malignant catarrhal fever (FCM). The control of the infection in sheep is the key to avoid the FCM. Moreover, a role of this virus in the pathology of the sheep can not be excluded. Aim of the present study was to investigate the presence of OvHV-2 in sheep flocks of the Umbria Region and understand the role of some characteristics of the farms for the infection. Nasal swabs of 39 sheep and 11 goats coming from 9 different farms were investigated for the presence of the OvHV-2 DNA by nested PCR. Twentynine out of 39 (74.4%) sheep and 3 out of 11 (27.3%) goats were positive. The role of the species, the age of the animals, the productive attitude, the breeding mixed with cattle and/or goats, and the effect of the single farm were evaluated as risk factors for the infection by univariable and multivariable statistical analysis. All these factors resulted significant by univariable analysis, whereas only the effect of the single farm remained significant in the multivariable analysis. Probably the farm contains other variables that are able to explain variation in the prevalence of OvHV-2. Further factors specific of each farm will have to be investigated to understand the epidemiology of the infection.

Bibliografia 1. Li H., Hua G., Snowder., Crawford T. B. (2001). Levels of ovine herpesvirus 2 DNA in nasal secretions and blood of sheep: implication for transmission. Vet Microbiol. 79: 301-310.

2. Taus N.S., Traul D.L., Oaks J.L., Crawford T.B., Lewis G.S., Li H. (2005).

Experimental infection of sheep with ovine herpesvirus 2 via aerosolization of nasal secretions. J Gen Virol, 86: 575-579.

3. Hussy D., Janett F., Albini S., Stauber N., Thun R., Ackermann M. (2002). Analysis of the pathogenetic basis for shedding and transmission of ovine gamma herpesvirus 2. J Clin Microbiol, 40: 4700-4704.

4. Li H., Cunha C.W., Davies C.J., Gailbreath K.L., Knowles D.P, Oaks J. L., Taus N. S. (2008). Ovine herpesvirus 2 replicates initially in the lung of experimentally infected sheep. J Gen Virol, 89: 1699-1708.

5. Baxter S.I.F., Wiyono A., Pow I., Reid H.W. (1997). Identification of ovine herpesvirus 2 infection in sheep. Arch Virol, 142: 823-831.

6. Ackermann M. (2006). Pathogenesis of Gammaherpesvirus infections.

Vet Microbiol, 113: 211-22.

7. Thonur L., Russell G., Stewart J.P., Haig D.M. (2005). Differential transcription of ovine herpesvirus 2 genes in lymphocytes from reservoir and susceptible species. Virus Genes, 32: 27-35.

8. Wani S.A., Samanta I., Pandit F., Buchoo B.A., Peer F., Bhat M.A. (2006). Molecular epidemiology of ovine herpesvirus type 2 infection in Kashmir, India. Vet Rec, 159: 587-590.

9. Powers J.G., VanMetre D.C., Collins J.K., Dinsmore R.P., Carman J., Patterson G., Brahmbhatt D., Callan R.J. (2005). Evaluation of ovine heresvirus type infections as detected by competitive inhibition ELISA and polymerase chain reaction assay, in dairy cattle without clinical signs of malignant catarrhal fever. J Am Vet Med Assoc. 227: 606-611.

10. Cortez P.P., Carvalheira J., Pauperio S., Thompson G. (2008). Prevalence of ovine herpesvirus type 2 in north-west Portugal. Vet Rec, 162: 282-284.

11. OIE technical disease cards, http://www.oie.int/fileadmin/Home/eng/ Animal_Health_in_the_World/docs/pdf/Disease_cards/MALIGNANT _CATHARRAL_FEVER.pdf 12. Stahel A.B.J., Baggenstos R., Engels M., Friess M., Ackermann M. (2001). Two different Macavirus, ovine herpesvirus-2 and caprine herpesvirus-2, behave differently in water buffaloes than in cattle or in their respective reservoir species. PLos ONE, 8, e83695.

13. Mushi, E. Z., Karstad, L., Jessett, D. M. (1980). Isolation of bovine malignant catarrhal fever virus from ocular and nasal secretions of wildebeest calves. Res Vet Sci, 29:168-171.

14. Campolo M., Lucente M.S., Mari V., Elia G., Tinelli A., Laricchiuta P., Caramelli M., Nava D., Buonavoglia C., Decaro N. (2008). Malignant catarrhal fever in a captive American bison (Bison bison) in Italy. J Vet Diagn Invest, 20: 843-6.

15. Modesto P., Grattarola C., Biolatti C., Varello K., Casalone C., Mandola M.L., Caruso C., Dondo A., Goria M., Rocca F., Decaro N., Leonardi C., Iulini B., Acutis P.L. (2015). First report of malignant catarrhal fever in a captive pudu (Pudu puda). Res Vet Sci, 99: 212-214.

16. Decaro N., Bozzo G., Tinelli A., Aliberti A., Buonavoglia D., Magrì V.M. (2003). Febbre catarrale maligna in due bovine in Sicilia. Large Animal Review, 1: 29-35.

17. Casalinuovo F., Cacia A., Scarpino P., Gualtieri G., Gagliardi G., Caparello G. (1996). Febbre catarrale maligna dei bovini: segnalazione di 2 casi clinici negli allevamenti calabresi. Praxis Vet, 17: 20-22.

18. Guarino A., Agrimi U., Fenizia D., Tollis M. (1993). Focolaio di Febbre catarrale maligna bovina in Italia. Atti del XLVII congresso S.I.S.Vet., 29 Settembre - 2 Ottobre 1993, Riccione (RN) pp. 1949-1950.

19. Martucciello A., Marianelli C., Capuano M., Astarita S., Alfano D., Galiero, G. (2006). Indagine su un focolaio di febbre catarrale maligna nella bufala mediterranea (bubalus bubalis). Large Animal Review, 5: 21-24.

20. Sconza S., Brunetti B., Gentile A., Benazzi C. (2003). La febbre catarrale maligna del bovino: una malattia trascurata? Atti del XXXV congresso della Società Italiana di Buiatria, pp. 219-231.

21. Premkrishnan G.N., Sood R., Hemadri D., Chanu Kh.V., Khandia R., Bhat S., Dimri U., Bhatia S. (2015). Cross-sectional study indicates nearly a quarter of sheep population in Karnataka state of India is infected with ovine herpesvirus 2. Vir Dis, 26: 180-188.

22. Bremer C.W. (2010). The prevalence of ovine herpesvirus-2 in 4 sheep breeds from different regions in South Africa. Jl S Afr Vet Ass, 81: 93-96.

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O. Raffaele et al. Large Animal Review 2020; 26: 79-85 23. Grattarola C., Decaro N., Amorisco F., Dondo A., Giorgi I., Varello K., Casalone C., Masoero L., Crescio M.I., Trisorio S., Peletto S., Acutis P.L. (2012). Analisi filogenetica di OvHV-2 isolati in Piemonte. XIV Congresso Nazionale SIDILV- Sorrento (NA), 24-26 Ottobre 2012.

24. Grattarola N.C., Iulini B., Zoppi S., Varello K., Dondo A., Rumello G., Baglivo T., Rondoletti M., Savini G., Amorisco F., Casalone C. (2011).

Indagine su tre focolai di febbre catarrale maligna nel bovino in Piemonte. Large Animal Review, 17: 49-55.

25. Li H., Shen D.T., Knowles D.P., Gorham J.R., Thorne T., O’Toole D., Crawford T.B. (1996). Prevalence of antibody to malignant catarrhal fever virus in wild and domestic ruminants by competitive-inhibition ELISA. J Wildl Dis, 32: 437-443.

26. VanDevanter D.R., Warrener P., Bennett L., Schultz E.R., Coulter S., Garber R.L., Rose T.M. (1996). Detection and analysis of diverse herpesviral species by consensus primer PCR.J Clin Microbiol, 34: 1666-71.

27. OIE, Terrestrial Manual 2018. Malignant catarrhal fever. Chapter 3.4.13. Pp. 1173-1184. http://www.oie.int/fileadmin/Home/eng/Health _standards/tahm/3.04.13_MCF.pdf 28. Baxter S.I., Pow I., Bridgen A., Reid H.W. (1993). PCR detection oft he sheep - associated agent of malignant catarrhal fever. Arch Virol, 132: 145-159.

29. Flach E.J., Reid H., Pow I., Klemt A. (2002). Gamma-herpesvirus carrier status of captive artiodactyls. Res.Vet Sci, 73: 93-99.

30. Mirangi K., Kang’ee F.M. (1997). Detection of ovine herpesvirus in Kenyan sheep by polymerase chain reaction. Vet Rec, 41: 76-177.

31. Kalayci G., Ekmez K., Kalan M., Ozkan B., Kucukali Y. (2015). Investigation of ovine herpesvirus- in sheep. ESVV05 - EPIZONE, 2-3 September 2015, Montpellier-France.

32. Li H., Snowder G., O’Toole D.T., Crawford T.B. (1998). Transmission of ovine herpesvirus 2 in lambs. J Clin Microbiol, 36: 223-226.

33. Campolo M., Desario C., Lorusso E., Elia G., Nava D., Decaro N., Buonavoglia C. (2005). Infezione sperimentale di capre con herpesvirus ovi-

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no tipo 2. URL: https://www.researchgate.net/profile/Donatella_Nava/ publication/242513351_infezione_sperimentale_di_capre_con_herpesvirus_ovino_tipo_2_experimental_infection_in_goats_with_ovine_ herpesvirus_type_2/links/0a85e53b2961e095a9000000/infezione-sperimentale-di-capre-con-herpesvirus-ovino-tipo-2-experimental-infection-in-goats-with-ovine-herpesvirus-type-2.pdf.

Zhu H., Huang Q., Hu X., Chu W., Zhang J., Jiang L., Yu X., Zhang X., Cheng S.(2018). Caprine herpesvirus 2-associated malignant catarrhal fever of captive sika deer (Cervus nippon) in an intensive management system. BMC Vet Res, 14: 38.

Taus N.S., Herndon D., Traul D.L., Stewart J.P., Ackermann M., Li H., Knowles D.P., Lewis G.S., Brayton K.A. (2007). Comparison of ovine herpesvirus 2 genomes isolated from domestic sheep (Ovis aries) and a clinically affected cow (Bos bovis). J Gen Virol, 88: 40-45.

Li H., Snowder G., O’Toole D., Crawford T.B. (1997). Transmission of Ovine Herpesvirus 2 in lambs. J Clin Microbiol, 36: 223-226.

Li H., Snowder G., Crawford T.B. (1999). Production of malignant catarrhal fever virus-free sheep. Vet Microbiol. 65: 167-172.

Riaz A., Dry I., Levy C.S., Hopkins J., Grey F., Shaw D.J., Dalziel R.G. (2014). Ovine herpesvirus-2- encoded microRNAs target virus genes involeved in virus latency. J Gen Virol, 95: 472-480.

Barton E.S., White D.W., Cathelyn J.S., Brett-McClellan K.A., Engle M., Diamond M.S., Miller V.L., Virgin H.W. (2007). Herpesvirus latency confers symbiotic protection from bacterial infection. Nature, 447: 326-329.

Frolich K., Li H., Muller-Doblies U. (1998). Serosurvey for antibodies to Malignant Catarrhal fever associated viruses in free-living and captive cervids in Germany. Journal of Wildlife Diseases, 34: 777-782.

Giangaspero M., Savini G., Osawa T., Harasawa R. (2013). Serological survey to determine the occurrence of malignant catarrhal fever infection in the Japanese small ruminant population from northern districts. J Vet Med Sci 75: 815-818.

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Cuboni Reaction: non-invasive late pregnancy test in Martina Franca jennies

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ROBERTA BUCCI1, BRUNELLA ANNA GIANGASPERO1, MICHELA D’ANGELO1, DOMENICO ROBBE1, PATRIZIA PONZIO2, ANNA CHIARA MANETTA1, LORELLA DI GIUSEPPE1, IPPOLITO DE AMICIS1 1

University of Teramo, Faculty of Veterinary Medicine, Veterinary University Teaching Hospital, 64100, Piano d’Accio (TE), Italy 2 University of Torino; Department of Veterinary Science; 10095, Grugliasco (TO), Italy

SUMMARY Martina Franca donkey is an Italian native breed originally from the Puglia region in South Italy. These donkeys are mostly raised in groups that live outdoors throughout the year.

The “Cuboni Reaction” is a test based on a chemical reaction that detects free estrogen in mares’ urine using hydrochloric acid, benzene and sulfuric acid; it allows for a late pregnancy diagnosis. To the authors’ knowledge, there is only one paper in literature reproducing this technique on asinine species. The purpose of this study is to apply the Cuboni Reaction test to the Martina Franca donkey to confirm the effectiveness of this method in asinine species as well.

18 Martina Franca jennies, whose pregnancy has been confirmed through transrectal palpations and ultrasound examinations, were subjected to urine collection to confirm the pregnancy diagnosis by the Cuboni Reaction.

Data collection were taken at G 120, G 135 and G 150, to identify the moment when the reaction becomes positive. Other samples were taken from G 270, every 15 days until a negative result was obtained. Furthermore, the test was carried out on 2 nonpregnant jennies in order to assess reaction specificity.

Two collection techniques were used: collection from spontaneous urination was performed in jennies not accustomed to the medical procedures; and collection from bladder catheterization in animals accustomed to the medical procedures.

This work shows that Cuboni Reaction is over 50% sensitive from 120th day of pregnancy and 100% sensitive from 150th to 300th day. However, the sensitivity of Cuboni Reaction is less than 40% over 315th day of pregnancy; it also loses specificity detecting false negatives from day 315.

Cuboni Reaction, despite having been overtaken by new technologies for pregnancy diagnosis, still proves useful, particularly in Martina Franca donkey farms, where animals are raised in fields and are not used to handling.

This method can still be used as a late pregnancy test in jennies for which the ovulation day is not known but in which there is a supposed date of mating; it is safe for the involved animals, easy to carry out by the breeder and provides results in a short time.

KEY WORDS Urine collection; animal welfare; pregnancy diagnosis; jennies; chemical test.

INTRODUCTION Martina Franca (MF) donkey is an Italian native breed, whose breeding originated in the Itria valley (Puglia, South Italy). Traditionally, these donkeys were used for fieldwork, but new technologies have diminished their usefulness1. Nowadays they are considered an endangered species

by FAO2;3.

The current resurgence in interest in donkey breeding originates from their usefulness in pet therapy4 and milk production1;3. In Puglia, for the protection of the native species, MF donkeys are mostly raised in groups, composed of 20-25 jennies and 2 jackasses1, that live outdoors in fields and wooded areas, separated by drywalls, throughout the year. The recovery center of the Puglia region for the MF donkey (Crispiano, Taranto, 40°37’55.3”N 17°16’31.2”E) corresponds to the characteristics listed.

Pregnancy diagnosis is important to ensure a better management of jennies, predict a precise date of delivery and guarantee animal welfare throughout the gestation period. It is generally known that, unlike mares, pregnancy in jennies lasts on average of 371 days5.

Many diagnostic techniques have been developed for pregnancy detection, from clinical ones like transrectal exploration6 and ultrasonography (US)5, to laboratory tests, less invasive and requiring minimum containment for the animals.

Ultrasonography has been used for over 40 years for pregnancy diagnosis in horses and, in the last 20 years, has been also used in donkeys7.

This method is elective for pregnancy detection in equids, nevertheless it is not always applicable in field conditions.

Furthermore, animals raised in fields may not be used to handling so, both for the animal’s welfare and operator’s safety, non-invasive techniques should be preferred.

Corresponding Author: Roberta Bucci (rbuccivet@gmail.com).

All the authors contributed equally to the study design, data recording, results, discussion, presentation and manuscript writing.

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Cuboni Reaction: non-invasive late pregnancy test in Martina Franca jennies

In particular, MF jennies are raised in groups in which the male is introduced from April to June and are not used in medical procedures.

Among non-invasive techniques, there is the Cuboni Reaction, a late pregnancy test based on a chemical reaction between free estrogen in pregnant mare’s urine and sulfuric acid8. The Cuboni Reaction was developed to be performed on the equine species, but in literature there are records of experimental trials also on other species, in particular camelids9. Recently, Kubatova et al.10 published the preliminary results of the tests on donkeys. To the authors’ knowledge, there are no other publications about application of Cuboni Reaction in donkeys.

Some advantages of the Cuboni test are: the minimal invasiveness of this procedure compared to others, stress reduction for the pregnant animal and consequently lower risk of miscarriages; in fact, urine collection can be performed without capturing the animal10;11.

The method respects animal welfare, as it reduces stress due to the restraint, thus is easily applicable without any risk for safety of both the breeder and the veterinary technician.

Moreover, breeders can easily perform urine collection and the test is performed quickly10.

The reliability of a test is determined by sensitivity (the likelihood of a positive test results in patients known to have the disease) and by specificity (the likelihood of a negative test results in a patient known to be free of diseases)12.

In jennies, the Cuboni test acquires greater reliability from the second 3rd of gestation (approximately 120th day onwards).

Before this date incidence of false negatives is too high and data is therefore not reliable10. This is due to low levels of estrogen present in small quantities in urine, making it difficult to detect. It is also known that Cuboni Reaction in mares is not effective over the 300th day of gestation due to the decrease in the concentration of estrogen in blood and urine6.

The choice of using the Cuboni test for pregnancy diagnosis in MF jennies depends on the sensitivity of the subjects to the handling and the breeding method.

The aim of this project is to assess the reliability of the Cuboni Reaction in jennies in the ranges 120-150 days and 300-345 days of pregnancy. During this time frame, it should be possible to observe a change in the test response in most of the animals in order to determine the range of reliability for the Cuboni Reaction in the asinine species.

A

MATERIAL AND METHODS The animals involved in the study were housed at the teaching farm of the University of Teramo, located in Chiareto di Bellante (Te) (42°43’36.3”N, 13°46’23.4”E), during the breeding seasons of 2016-2017. Laboratory tests were carried out at the digestibility laboratory of the zootechnics department of the same University.

18 Martina Franca jennies, in good health and free from reproductive diseases, were subjected to estrous cycle monitoring and to artificial insemination (AI). The time of ovulation was detected by US examination. The first pregnancy diagnosis and subsequent controls were carried out by transrectal palpation and US examination7.

Urine samples were collected to confirm the pregnancy diagnosis through the Cuboni Reaction.

Ovulation day was regarded as G 0 of pregnancy in order to determine the days for collecting samples. Samples were collected at G 120, G 135 and G 150, to identify the moment when the reaction becomes positive. Other samples were taken from G 270, every 15 days until a negative result was obtained.

Two nonpregnant jennies were subjected to collection to evaluate the specificity of this test.

The techniques used for the collection are the following: • jennies not accustomed to the medical procedures were moved to a clean box to stimulate urination; a telescopic stick, with a container fixed to the tip, was used for a noninvasive collection (Photo 1A); • in animals accustomed to medical procedures, urine samples were collected by bladder catheterization (Photo 1B): this method, although minimally invasive, is rapid and involves a minimum stress. A bladder catheter was inserted with the animal adequately contained in a gynecological reinforcement; the anogenital area was previously cleaned with water and betadine and the tail bandaged.

The freshly collected urines were filtered in a graduated cylinder and frozen until the time of analysis.

To check that freezing does not affect the test results, some randomly selected samples were collected in double rate.

One of the two samples was frozen and the other one chilled, then subjected to analysis.

The reactions were carried out exclusively under a chemical hood. Operators involved in the procedure were adequately equipped with Personal Protective Equipment (PPE).

B

Photo 1 - A) non-invasive urine collection; B) kit for bladder catheterization.

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R. Bucci et al. Large Animal Review 2020; 26: 87-91 Photo 2 Left, positive reaction (green); right, negative reaction (brown).

Distilled water was used as blank control to correct any errors related to the analysis procedure. The blank test was carried out in parallel with each set of analysis.

The protocol used for this analysis has been partially modified compared to the original Cuboni method8, as follows.

Briefly, 3 ml hydrochloric acid (HCl) were slowly added to 15 ml of filtered urine in a test tube. After 10 minutes in boiling water, the tube was cooled to room temperature. The mixture, poured through a separating funnel, was added to 18 ml benzene and shaken carefully for 60 seconds. After separation of the two phases, the lower layer was removed and the top one poured into a second separating funnel, shaken carefully with 10 ml of sulfuric acid (H2SO4) for 60 seconds and left to settle. The lower layer was transferred into a test tube and incubated in a water bath for 10 minutes at 90° C. After cooling, the test tube was exposed to sunlight: the sample resulted positive if the liquid assumed a greenish reflection, like lubricating oil (Photo 2, left tube). A homogeneous brownish colour instead indicated a negative reaction (Photo 2, right tube).

The results were defined as uncertain when the reflection of the solution was neither green nor brown.

Data analysis was performed using Manova on the entire dataset and post-hoc one-way ANOVAs were performed on first range and second, a was set as 0.05.

The study was approved by University of Bologna Ethical Committee for Animal Welfare (Prot. N. 62128, 23/04/2018).

RESULTS In this study, 7 out of 18 pregnant jennies were subjected to urine sampling collection by spontaneous urination, while the remaining 11, by bladder catheterization.

Subjects involved in this study gave birth to alive and viable

89

Table 1 - Results of the performed reactions.

Days of pregnancy

Positive results

Negative results

Uncertain results

G 120

55.56% (10/18)

0% (0/18)

44.43% (8/18)

G 135

77.78% (14/18)

0% (0/18)

22.21% (4/18)

G 150

100% (18/18)

0% (0/18)

0% (0/18)

G 300

100% (18/18)

0% (0/18)

0% (0/18)

G 315

38.89% (7/18)

11.10% (2/18)

50% (9/18)

G 330

0% (0/18)

61.11% (11/18)

38.39% (7/18)

G 345

0% (0/18)

100% (18/18)

0% (0/18)

foals; no negative effects have been reported following the urine collection.

Control tests performed on cooled and frozen samples showed that the freezing process does not affect the results of the Cuboni Reaction, in fact, the results were concordant in 100% of cases.

In non-pregnant jennies, test results were always negative.

Cuboni pregnancy test shows 100% positive results between the G 150 and G 300 of gestation. Before the 150th day, tests showed uncertain results: at G 120; 8 out of 18 samples resulted uncertain (44.33%), and only 4 out of 18 at G 135 (22.21%).

However, after 300th day, in addition to uncertain samples, the incidence of false negatives also increases (Table 1).

The data analysis performed on the entire data set using MANOVA show that the test on pregnancy is time locked, p < .00001 (F = 28.67), (Graph 1A). A series of post-hoc ANOVAs were performed on first and second range, which return in both case statistical significance, p < .00457 [F(2;51) = 6] and p < .00001 [F(2;119) = 6] respectively, (Graph 1B, C).

DISCUSSIONS AND CONCLUSIONS In equine pregnancy, estrogen, whose main source is represented by the foetal gonads, is detectable in the blood from 40th - 60th day of gestation, reaches its maximum values between 150th and 210th days and then decreases rapidly, allowing the preparation for birth13.

Cuboni Reaction is a chemical test that allows to carry out pregnancy diagnosis highlighting the estrogen in the urine of a mare supposedly pregnant8.

The bibliography shows that positive results can already be obtained from the 90th - 120th day of gestation6, but the test becomes reliable from approximately 150th to 300th day of pregnancy10. Data presented in this work agrees with other authors, in particular with Kubatova et al.10, who states that the test is 100% reliable in the third period of the gestation.

Furthermore, it is possible to state that the test is not completely reliable before 150th day. In fact, results are uncertain over 40% at G 120 but only over 20% at G 135. Results are clearly positive in 100% of the cases from G 150 to G 300 (Table 1).

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Cuboni Reaction: non-invasive late pregnancy test in Martina Franca jennies

A

B

C

Graph 1 - MANOVA analysis was performed on the entire data set (1 = G 120; 2 = G 135; 3 = G 150; 4 = G 300; 5 = G 315; 6 = G 330; 7 = G 345), the test on pregnancy result time locked, p < .00001 (F = 28.67), (Graph 1A). In order, to verify the hypothesis a post-hoc ANOVAs were performed on first and second range (Graph 1B and 1C), both are statistically significant.

The colorimetric reaction is dependent on the concentrations of estrogen and this can cause uncertain results, because, below a given concentration, the colour change to green is not clear enough. Therefore, it is possible to state that Cuboni Reaction is over 50% sensible from 120th day of pregnancy and 100% sensible from 150th to 300th day.

However, over the 300th gestation day, results show that the test loses reliability due to the occurrence of false negatives, never reported before in donkeys. This finding is justified by the decrease in estrogen secretion before birth14.

In fact, over 10% of false negatives and 50% of uncertain results are detected from G 315 up to 100% of false-negative at G 345 (Table 1).

Hence it can be stated that the sensitivity of the Cuboni Reaction is less than 40% over 315th day of pregnancy; it also loses specificity detecting false negatives after this date.

Test specificity is defined by the negative result in non-pregnant jennies. While Kubatova et al.10 reported over 86% specificity in their work, this work shows 100% specificity.

This discrepancy may be related to the smaller control group involved in this work. Moreover, this test proved to be easily applicable both in animals accustomed to human contact and in animals raised in the wild, causing minimal stress in jennies and having no negative effects on pregnancy. In fact, urine collection is less invasive than blood samples or transrectal US. Another advantage is represented by rapidity in the execution of the test that can also be done in studs provided there is adequate equipment (chemical hood, PPE) for trained operators, such as veterinary technicians.

Furthermore, as revealed by statistical analysis the field test for pregnancy is clearly time locked and return the best score on G 150 and G 300 to identify a pregnant subject, while fail

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R. Bucci et al. Large Animal Review 2020; 26: 87-91

in G 120, G 330 and G 345, could return unclear results at G 135 and G 315.

In conclusion, Cuboni Reaction, despite being overtaken by new technologies for pregnancy diagnosis, proved to be still useful particularly in Martina Franca donkey farms, where animals are usually raised in fields and are not used to handling.

This method can still be used as a late pregnancy test for jennies with an uncertain ovulation day but in which there is a supposed date of mating; it is safe for the animals involved, easily executable by the breeder and provides results in a short time.

References 1. Pinto F. (2009). Un eccezionale patrimonio genetico del territorio: rivalutazione dell’asino di Martina Franca attraverso l’innovativa produzione di latte. Riflessioni Umanesimo della pietra, 32, 169-178.

2. Food and Agriculture Organization Domestic Animal Diversity Information System 2014. Menu: Breeds, population structure and inbreeding (F) for a specific year; choose a breed: countries - Italy, species ass, breeds - Martina Franca/Italy; choose a year: 2014. Retrieved on 11 August 2016 from http://dad.fao.org/.

3. Carluccio A., Gloria A., Robbe D., Veronesi M.C., De Amicis I., Cairoli F., Contri A. (2017). Reproductive characteristics of foal heat in female donkeys. Animal, 11(3), 461-465.

4. Amendola S., Macchi E., Rasola M., Carluccio A., Marsilio F., Contri A., Ponzio P. (2012). Monitoraggio del comportamento e del benessere di asine in Attività e Terapie Assistite con gli Animali (TAA/AAA) simulate. Ippologia, 23(2), 9-16.

5. Carluccio A., Gloria A., Veronesi M.C., De Amicis I., Noto F., Contri, A. (2015). Factors affecting pregnancy length and phases of parturition in Martina Franca jennies. Theriogenology, 84(4), 650-655.

6. Richter J., Götze R., Rosenberger G., Tillmann H., Oliva O. (1994). Ostetricia veterinaria. Casa Editrice Ambrosiana.

7. Carluccio A., Villani M., Contri A., Tosi U., Veronesi, M.C. (2005).

Rilievi ecografici della gravidanza precoce nell’asina di Martina Franca.

Ippologia, 4(16), 31-35.

8. Cuboni E.. (1934) A rapid pregnancy diagnosis test for mares. Clinical Veterinarian (Milano) 57, 85-93.

9. Fedorova T., Brandlova K., Lukešová, D. (2015). Application of noninvasive pregnancy diagnosis in Bactrian camels (Camelus bactrianus) using Cuboni reaction and barium chloride test. Journal of Zoo and Wildlife Medicine, 46(2), 355-358.

10. Kubátová A., Fedorova T., Skálova I., Hyniová L. (2016). Non-invasive pregnancy diagnosis from urine by the Cuboni reaction and the barium chloride test in donkeys (Equus asinus) and alpacas (Vicugna pacos).

Polish journal of veterinary sciences, 19(3), 477-484.

11. Waits L.P., Paetkau D. (2005). Noninvasive genetic sampling tools for wildlife biologists: a review of applications and recommendations for accurate data collection. The Journal of Wildlife Management, 69(4), 1419-1433.

12. Smith, R.D. (2005). Veterinary clinical epidemiology. CRC press.

13. Conley AJ. (2011); “Review of the reproductive endocrinology of the pregnant mare” in McKinnon A.O., Squires E.L., Vaala W.E., Varner D.D. Equine reproduction, John Wiley and Sons.

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M.I. Malik et al. Large Animal Review 2020; 26: 93-98

Genomics of subacute ruminal acidosis

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MUHAMMAD IRFAN MALIK1, MUHAMMAD AFZAL RASHID1 1

Department of Animal Nutrition, University of Veterinary and Animal Sciences Lahore, 5400, Pakistan

SUMMARY The present review will present the most recent development in sub-acute ruminal acidosis (SARA) regarding high concentrate diet and its interaction on gene expression. The SARA is a common disorder in high producing ruminants. In addition SARA is a big challenge for ruminant nutritionists, therefore, the subject has been extensively studied in recent decades. In current decade nutrigenomic has been used to understand the cellular response influenced by high concentrate diet in ruminants. For sustainability of higher growth and production, animals are forced to feed on high concentrate and low fiber in diet. High concentrate diet leads to accumulation of higher concentration of VFA consequently ruminal pH decreases. Depression in pH for longer duration impairs ruminal epithelium tight junctions and hyperkeratosis. SARA is characterized by fewer tight junctions between cells, thinner cell layers, and an increased sloughing of the stratum corneum leading to a weakened permeability barrier. Ruminal epithelium serves as barrier for microbes, damages to ruminal epithelium leads to translocation of lipopolysaccharides (LPS) from compromised ruminal epithelium to blood circulation and different organs of the body. The LPS induce immunogenic response in ruminal papillae, epithelium, liver, and other organs. LPS induce a cascade mechanism of gene expression by binding with Tolllike receptor 4 (TLR4) a receptor for LPS, binding of LPS with TLR4 activate nuclear factor kappa B (NF-κB) consequently genes for interleukin 6 (IL), IL-8, tumor necrosis factor (TNF)-α and many more genes were up regulated or down regulated in rumen epithelium and other organs. In conclusion feeding high concentrate diets to ruminants’ triggered local and systemic inflammation response at molecular level. By exploring the mechanism of SARA and its effects on gene up regulation or down regulation can be a value able tools for understanding the SARA and its adverse effects on animal health and production performance.

KEY WORDS Gene expression, High concentrate, rumen epithelium, SARA, TLR-4.

INTRODUCTION

SUBACUTE RUMINAL ACIDOSIS

Nutrigenomics is an emerging science that is based on combination of two major sciences, nutrition and genomics. The study of the interaction of nutrients or other dietary bioactive substances at molecular level or gene expression1,2. Nutrition or nutrients have direct influence on organism at molecular level. The science of nutrigenomics tells us which type of nutrients we have consumed in past, by changing the gene expression level of certain genes. What we eat or feed to animals it is a message to an organism’s body. By learning the language or message that is given by nutrients we can control the genetic expression of desirable genes3. Nutrigenomics is an emerging field of research in animals and holds great potential to improve health and productivity4,5. In current decades animal nutritionists are trying to find alternative ways to explore the potential of diets and animals by evaluating the gene expression in response to various diets. To the best of author knowledge there is not a single review paper published in this area. The purpose of this review is to summarize the high concentrate diet and its effects on gene expression in rumen epithelium, liver, uterus and mammary gland.

In intensive ruminant production system for the sustainability of high production provision of highly fermentable carbohydrates in the form of cereal grains is common practice6.

Due to highly fermentable diets, production of SCFA increases, absorption and lower buffering capacity lead to accumulation of SCFA, consequently lower ruminal pH. Ruminal pH <5.6 for 180 m/days is considered as a threshold level for SARA7,8. The rumen is the first organ that is influenced by low ruminal pH. Depression in ruminal pH leads to induction of certain changes in the ruminal epithelium and compromises the epithelium integrity9. Additionally, depression in pH for long-term induces parakeratosis and hyperkeratosis10, consequently invasion of pathogenic microbes into portal vein drainage results into liver abscess and other diseases11. Lower ruminal pH or in case of SARA, lysis of gramnegative bacteria increases, consequently lipopolysaccharide (LPS) an endotoxin are free to translocate through damaged ruminal wall12,13. Lipopolysaccharide is a strong inflammatory inducer that can elicit inflammatory responses14. There is certain inflammatory response are induced during inflammation e.g Toll-like receptor 4 (TLR4) a receptor for LPS, binding of LPS with TLR4 activate nuclear factor kappa B (NF-kB) consequently genes for interleukin 6 (IL), IL-8, tumor necrosis factor (TNF)-α up regulated at inflammation site15. In current decade it is well established that SCFA has direct effects on gene expression in intestinal tissue16.

Corresponding Author: Muhammad Irfan Malik (muhammad.irfan@uvas.edu.pk).

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BUTYRIC ACID AS NUTRIGENOMIC In ruminants, butyrate is the preferred energy source for stomach wall and plays a major role in energy metabolism.

Ruminants utilize 70% of butyrate as energy sources for stomach wall, monogastric hindgut also utilizes butyrate as a major energy source17. In dairy cows, butyrate is one of the short-chain fatty acids which is extensively studied as nutrigenomics. Butyrate upregulates a large number of gene expression involved in rumen development, cell cycle arrest and immune response18. Butyrate is also considered as histone deacetylase inhibitor drug that can inhibit cell proliferation and cell apoptosis19 and play a role as antitumorigenic20. In a recent study, it is reported that relative expression of IL-1β, IL-8, IL-10 up-regulated in high concentrate (HC) diet in goats fed concentrate to forage ratio 60:40, whereas those supplemented with sodium butyrate IL-1β, IL-8, IL-10 were similar to those fed low concentrate (LC). Toll-like receptor-4 were similar in LC and those supplements with sodium butyrate with HC. Relative gene expression of matrix metalloproteinase-2 (MMP) and MMP-9 tight junction degrading enzymes were also higher in HC as compared to LC and those supplemented with sodium butyrate with HC. Down-regulation of MMP-2, MMP-9, IL-1β, IL-8, and IL-10 by supplementation of sodium butyrate can be helpful to ameliorate the adverse effects of the HC diet in ruminant21. In another experiment rumen gene expression was evaluated in dairy cow in which butyric acid was supplemented 2.5% of DM. In this experiment differential expression of 1191 genes were indicated by microarray, forty-nine of 1191 were differentially expressed (Table 1) by qRT-PCR. Out of 49, nine genes were up-regulated, 20 down regulated and 20 were unaffected by supplementation of butyrate22.

HIGH CONCENTRATE, SARA AND ITS EFFECTS ON RUMEN EPITHELIUM AND GENE EXPRESSION Rumen epithelium is stratified squamous epithelium, which is further divided into four distinct strata. From the luminal surface the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale. Each stratified layer performs a specific function and serves as syncytium23. After stratified corneum and stratified granulosum cells are connected with each other with the help of tight junctions. Tight junction proteins are multifunctional protein complex, there are more than 40 tight junction proteins, and the principal function of these proteins is to prevent leakage24. Claudin-1 and zonula occluden-1 are the two principle tight junction proteins23. In ruminal epithelium these two tight cell junction proteins form a barrier that prevents the translocation of larger molecules, pathogenic microbes25 and LPS26. Expression of tight cell junction proteins were evaluated in goats fed HC diet, Expression of mRNA for claudin-4, occludin, ZO-1 were down regulated in HC fed goats. However, the expression of claudin-1 gene was up regulated. Results revealed that the HC diet has profound effects on the ruminal epithelium. Expression of IL-1, IL-6, and IL-10 were similar in HC and hay fed goats, while TNF-α and IFN-γ were up regulated in HC fed goats. There is a positive correlation in mRNA expression

between claudin-1 and IFN-γ. While claudin-4 negatively correlated with TNF-γ27.

Highly fermentable carbohydrates have an influence on rumen papillae size, consequently more surface area for absorption of SCFA28. The molecular signaling mechanism is not completely understood. However, IGF-1 is thought to be the candidate gene responsible for rumen epithelium growth proliferation29. Cascade mechanisms of IGF-1 can be modulated by IGF-binding proteins30. Modulation of IGF-1 mechanism also observed in transition cows fed the HC diet. In this experiment expression of mRNA for IGFBP5 up-regulated, whereas, IGFBP3 and IGFBP6 were downregulated9.

Toll-like receptors are a group of receptors identified in monogastric gut tissue31. However, the presence of TLR-1-10 also confirmed in dairy calves, TLR recognizes the differential pathogenic-host immune interaction and trigger the immunogenic response32. A recent experiment in goats fed HC diet mRNA expression of TLR2, TLR3 and TLR5 were up regulated in rumen epithelial tissue. However, the relative expression of TLR4 was not affected by diet or low ruminal pH33.

On the contrary, several other studies reported the up regulation of TLR-4 mRNA expression21,34,37. Variation in the relative expression of TRL-4 may be associated with different location of sample collection or change in diet, because diet causes a shift in the bacterial population, shifting of the bacterial population may induce differential expression of TLR in different studies33.

SARA AND LIVER GENE EXPRESSION Translocation of LPS from ruminal fluid to the bloodstream during SARA38 activates the immune response which is stimulated by recognition of LPS from TLR-4 receptor39. TLR4 is a receptor for LPS, expression of TLR-4 was higher in those fed HC21,36. TLR4 is known to initiate a signal transduction mechanism consequently NF-kB activated15 and orchestrates of a pool of acute-phase proteins synthesis initiated40. Nuclear factor-kappa B is the most important transcription factor which is responsible for the production of pro-inflammatory cytokines and chemokines, such as TNFα, IL-1, and IL-6. These chemokines and cytokines travel to the liver through bloodstream induce pathological changes in the liver41 and up regulate the expression of acute-phase protein (AAP) mRNA36 consequently the production of APP increase41.

In a recent study expression of IL-10, IL-8, CCL5 and CCL20 in the liver were higher by 5, 11, 21 and 3 fold respectively in HC fed goats as compared to those fed LC. Expression of liver protein for primary pro inflammatory cytokines IL-1β, IL-6, TNFα was also higher in HC as compared to those fed LC diet. Expression of mRNA for APP, serum amyloid A3 (SAA), haptoglobin and lipopolysaccharide-binding protein was up regulated by 44, 29 and 8 fold respectively in HC fed goats as compared to those fed LC36.

The expression of immune genes in the liver in response to SARA is strengthened by a recent study. In this experiment mRNA expression of inflammatory mediators IL-1α, IL-1β, and TNF-α higher in HC fed dairy cow by 3 folds, IL-6 expression was higher by 47 fold. Acute-phase proteins such as LBP, SAA3, and HP expression were higher by 10.32, 8.64, and 3.97-fold respectively37. Serum amyloid A is a high-den-

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Table 1 - Expression of genes in various studies during subacute ruminal acidosis.

Observations

Rumen pH

Gene

Reference

Up regulation in those fed HC

IL-1β, IL-8, IL-10, TNF-α, TLR-4, MMP-2, MMP-9, MPO, CD68

Dai et al., 2017

Down regulation in those treated with sodium butyrate with HC

IL-1β, IL-8, IL-10, TNF-α, MMP-2, MMP-9, MPO

Dai et al., 2017

Up regulation HC diet (40:60 Concentrate: Forage)

pH <5.6 for >180 m/day

TRAF6, NF-κB, p38, MAPK, extracellular regulated protein kinases MAPK, IL-1 and SAA

Guo et al., 2017

Not influenced by experimental diet

pH <5.6 for >180 m/day

TLR-4

Guo et al., 2017

Up regulation in histamine treated

5.5

TNF-α, IL-6, and IL-1β

Sun et al., 2017

Up regulation in HC

<5.8 for >3 h

TLR4, IL6, 1β, MyD88, TRAF6 and NF-κB

Bilal et al., 2017

Down regulation in HC supplemented with sodium butyrate

<5.8 for >3 h

TLR4, MyD88, TRAF-6, NF-κB, IL-1β and IL-6

Bilal et al., 2017

IL-1β, IL-2, IL-22, CCL19, CCL8, CX3CR1, CXCL6, INHBE, LEPR, PRL, and TNFRSF9

Zhang et al., 2016

TLR4, MyD88, TRAF-6, NF-κB, TNF-α, IL-8, IL-1β and LBP

Bilal et al., 2016

TLR4, LBP, IL-1A, IL-1B, IL-6, TNF-α, IL-8, IL-10, CCL5, CCL20, SAA3

Chang et al., 2015

Up regulation in HC Up regulation in HC

<5.6 for 223 min/day

Up regulated in HC Up regulated in HC fed goats

5.33

Claudin-1, TNF-α, IFN-γ

Liu et., 2013

Down regulated in HC fed goats

5.33

Claudin-4, occludin, ZO-1

Liu et., 2013

Not influenced by HC or LC

5.33

IL-1, IL-6, and IL-10

Liu et., 2013

Up regulated in HC

<5.8 from 2 to 6 h

P45017α, 3β-HSD, HP and SAA

Jia et., al 2014

Up regulated in butyrate (2.5% DM) supplemented

5.67

LCN2, MMP1, MUC16, GPX2, CSTA, FUT1 SERPINE2, BCAM, RAC3

Dionissopoulos et al., 2013

Down regulated in butyrate (2.5% DM) supplemented

5.67

MTOR, AKIRIN2, NFKBIZ, ACVR2A, LAMB1, FRS2, PPARD NFKB2, LBP, NEDD4L, SGK1, DEDD2, MAP3K8, PARD6B, PLIN2, ADA, HPGD, FMO5, BMP6, TCHH

Dionissopoulos et al., 2013

Unaffected in butyrate (2.5% DM) supplemented and control treatment

5.67

CD14/TLR4/LY96, EGF, EGFR, ERK1/2, Fgf, Fgfr, FN1, IGFBP7, IL10, LY96, MIF, MLST8, NFKB1, PTGS1, PTGS2, SMAD1, STAT6, TGFB1I1, TLR4, TNFRSF6B

Dionissopoulos et al., 2013

Up regulation in HC fed goats

pH <5.6 for >180 m/day

TLR-4, IL-8, IL-10, CCL5, CCL20, HP, SAA3, LBP, IL-1β, IL-6, TNFα

Chang et al., 2015

Up regulation in HC fed dairy cow

pH <5.6 for >180 m/day

LAP, TNF-α, IL-8, IL-1β and IL-6

Jin et al., 2016

TLR-2,TLR-3, TLR-5

Liu et al., 2015

Up regulation in HC fed goats

sity lipoprotein which is responsible for migration of neutrophils and monocytes at the site of inflammation, HP binds with free hemoglobin released from damaged erythrocytes and serves as anti-inflammatory agent, while LBP form complex with LPS in blood or extracellular fluid facilitate the neutralization of LPS consequently initiate the release of inflammatory mediators, Such as TNF-α, IL-6 and IL1β42,43. Relative expression of TLR-4 was not influenced by HC diet (40:60 Concentrate: Forage), In this experiment expression of TLR-4 was slightly lower in HC fed dairy cows as

compared to those fed LC diet34. On the contrary several studies reported higher mRNA expression for TLR-421,35,36, lower expression of mRNA for TLR-4 may be associated with clearance of LPS from the liver into intestine along with bile secretion, subsequently, hypo responsiveness of liver cells to LPS makes it possible lower level of expression of TLR-444.

However, mRNA expression TNF receptor-associated factor 6 (TRAF6), NF-κB, p38, mitogen activated protein kinase (MAPK), extracellular regulated protein kinases (ERK) MAPK was higher in HC treatment. Similarly, the relative ge-

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ne expression of IL-1 and SARA was higher in HC fed cows.

By keeping in view the above-mentioned studies, it can be concluded that translocation of LPS during SARA induce pathological damages to liver cells, impairment of certain liver function, up regulation or down regulation of hepatic immune genes expression and activation of inflammatory signaling pathways34.

SARA AND ITS EFFECTS ON OTHER ORGANS Mammary gland Translocation of LPS induces a systematic inflammatory response and a cascade of gene expression activated in different organs of the body41. In a recent study, mammary gene expression was evaluated in dairy cow fed HC and LC with concentrate to forage ratio (60:40, 40:60) respectively. Expression of mRNA showed that lingual antimicrobial peptide (LAP), TNF-α, IL-8, IL-1β and IL-6 were up regulated in HC as compared to those fed LC. In this experiment, NF-κB was not affected by the HC diet. So, provision of HC for long-term not only has adverse effects on rumen epithelium, instead of translocation of rumen derived LPS from the rumen to bloodstream also initiate the synthesis of LAP synthesis via the NF-kB signaling pathway in mammary glands of lactating cows45.

Histone 3 acetylation is involved in DNA replication, transcription, repair, and various cellular functions, cell proliferation is also facilitated by histone acetylation46,47. A high concentrate diet induces epigenetic changes in mammary tissue.

Histone 3 acetylation was lower in dairy cows fed HC as compared to those fed LC. Lower concentration of histone 3 acetylation in mammary tissue may be associated with a higher concentration of LPS, However, the exact mechanism is not completely explored further research is warranted to identify the LPS mediated depression of histone acetylation48.

Adrenal gland It is a proven fact that there is a strong relationship between immune response and stress. Studies have shown that LPS activates the hypothalamic pituitary adrenal axis (HPA) in dairy cows53. Similarly, sheep injected with IL-6 stimulated secretion of cortisol and corticotropin hormone into peripheral circulating system. Secretion of cortisol and corticotropin indicating a cross-talk of the immune system and the HPA axis54. Relative gene expression of P45017α and 3βHSD in the adrenal cortex were up regulated in SARA goats fed HC (60:40, Concentrate: Forage) as compared to those fed LC43. For the synthesis of cortisol, cytochrome P450 catalyzes the conversion of cholesterol to form pregnenolone55.

Another key regulatory step for cortisol synthesis is the conversion of pregnenolone to form progesterone and catalyzing the conversion of 17-hydroxypregnenolone to 17-hydroxyprogesterone, which is catalyzed by 3β-HSD56.

HISTAMINE AS A SIGNALING MOLECULE Depression in ruminal pH induces a lytic process of bacteria which produce LPS and histamine, histamine is considered to be a potent inducer of rumenitis in SARA. A high concentration of histamine in ruminal fluid during SARA induce pathophysiological changes in the ruminal wall, therefore availability of energy becomes limited to animals57. Histamine is a potent inflammatory mediator, by doing so it works as a signaling molecule for up regulation of inflammatory cytokines58. The nuclear factor-κB (NF-κB) plays an important role in the regulation of inflammatory pro mediator gene expression59. Recently a study conducted to evaluate the relative gene expression in female Holstein cow for TNF-α, IL-6, and IL-1β, histamine treated group showed a higher level of mRNA expression for these inflammatory mediators as compare to control bovine ruminal epithelial cells60.

Uterus Similarly, in a recent study, it is evaluated that SARA had adverse effects on uterus gene expression. In this experiment TLR4, MyD88, TRAF-6, NF-κB, TNF-α, IL-8, IL-1β and LBP were up regulated in HC (40:60, Forage: Concentrate respectively). In conclusion, the authors stated that long-term provision of the HC diet causes a uterine inflammatory response in mid lactating dairy cows35. Similarly, expression of the uterine genes were also observed in goats fed HC. In this experiment TLR4, IL6, 1β, MyD88, TRAF-6 and NF-κB were up regulated in the HC group49. Adverse effects of SARA on rumen pH and uterine gene expression ameliorated by supplementation of sodium butyrate in lactating goat by similar authors in another work. Expression of mRNA of TLR4, MyD88, TRAF-6, NF-κB, IL-1β and IL-6 were lower in those supplemented sodium butyrate with HC as compared to those fed HC only50. Lower expression of mRNA for proinflammatory cytokines were associated with anti-inflammatory properties of sodium butyrate51. Another possible explanation is that sodium butyrate plays a role as a buffering agent in the rumen and increases ruminal pH52. Inflammatory response from uterine tissue may be associated with higher concentration LPS in the bloodstream of HC fed animals as compared to those fed LC45.

SARA AND ITS RELATION WITH LAMINITIS Laminitis, an aseptic inflammation of the dermal layers inside the hoof, one of the leading causes of lameness in dairy cows at commercial dairy farms62. Laminitis has been associated with many factors like traumatic, floor, bedding63, diet and feeding management specifically with acute and subacute ruminal acidosis64. However, the pathophysiological relationship between SARA and laminitis is still unclear65. Subacute ruminal acidosis could be one of the reasons for the onset of laminitis in high producing animals66,67. One of the proposed relationships is damaged epithelium may permit the absorption of various vasoactive substances such as histamine, biogenic amines and LPS. Translocation of vasoactive substances damages to the capillaries of the lamellae in the foot and causing hemorrhage, inflammation and lameness65,68. On the contrary, in dairy cattle LPS increased in rumen rather than peripheral blood circulation in grain induced SARA69, which disagrees with65,68. Another hypothesis has found that matrix metalloproteinase could break the junction of corium and horn in equine70. So far, this hypothesis has not been reproduced in dairy cows.

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CONCLUSIONS Keeping in view the above-mentioned studies it can be concluded that a high concentrate diet induces SARA. Depression in pH impairs the epithelial tight junctions consequently translocation of LPS occurs. Lipopolysaccharide activates the cascade mechanism and immunogenic expression of genes involved in the inflammatory process. The rumen is the first organ affected by low ruminal pH, however, liver, uterus, mammary gland and adrenal gland also affected by SARA. Relative gene expression in the rumen or other organs helps to understand the molecular mechanism responsible for SARA.

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DECLARATION OF INTEREST Author declare no conflict of interest.

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L. Sylla et al. Large Animal Review 2020; 26: 99-102

99

A case of epiploic foramen entrapment of jejunal intestinal tract in an alpaca (Vicugna pacos) cria LAKAMY SYLLA1, MARTINA CROCIATI1, DOMENICO CAIVANO1, VASILICA FLORY PETRESCU1, LORENZO PISELLO1, CALOGERO STELLETTA2 1 2

Department of Veterinary Medicine, University of Perugia, Via San Costanzo 4, 06126 Perugia - Italy Department of Animal Medicine, Production and Health, University of Padova, Viale dell’Università 16, 35020 Legnaro (Padova) - Italy

SUMMARY Signs of colic in camelids are generally vague and non-specific. Diagnostic techniques are mainly based on physical examination; however, a transabdominal ultrasonography could be helpful to evidence the most common surgical lesions. An exploratory laparotomy or laparoscopy should be considered as an extension of the physical examination.

In the present case report, a 6 month old, 20 Kg bodyweight Alpaca cria with colic symptoms secondary to small intestine entrapment was referred to the Teaching Veterinary Hospital of University (OVUD) at the Department of Veterinary Medicine University of Perugia - Italy, and subjected to an exploratory laparotomy. The cria was firstly treated medically for anorexia and depression; but, upon deterioration of the health status, a laparotomy was performed, leading to the definitive diagnosis of the epiploic herniation of the jejune tract.

On presentation at the OVUD, the patient appeared depressed, alternatively in sternal and lateral recumbency, with body temperature of 38.5°C, heart rate of 52 bpm; at auscultation of the abdomen the forestomach and intestinal motility were normal.

Abdominal ultrasonography revealed that small intestines were dilated, with intraluminal fluid accumulation but normal motility.

Late morning of the third day, the clinical condition worsened and the patient presented sinusal tachycardia (210 bpm), tachypnea (52) and dyspnoea. The Owner gave consent to proceed with an explorative laparotomy. At surgery there was no peritoneal fluid accumulation; all tracts of the gut were normal in term of colour and volume, except for a small area of jejunum which appeared dark reddish with fibrinous spots on its surface. The jejunum was hard in consistency at digital palpation and was entrapped in the epiploic foramen. Due to the necrotic lesions on the intestinal tract, the owner was informed and authorised the execution of the euthanasia.

At necropsy, topography of abdominal viscera was maintained, except for a 5 cm of small intestine that were entrapped within the epiploic foramen; it appeared with multifocal, brown-reddish, necrotic and haemorrhagic lesions.

Although herein the cria was positive to E. macusaniensis infection, there was any sign of diarrhoea in the previous weeks to hospitalization.

In conclusion, necropsy confirmed the diagnosis of small intestine herniation into the epiploic foramen with related severe acute necro-haemorrhagic enteritis.

KEY WORDS Alpaca cria; colic symptoms; intestinal entrapment; exploratory laparotomy.

CASE HISTORY Camelids are considered pseudo-ruminants as their gastrointestinal system is similar to but distinctly different from that of traditional four-chambered ruminants and have three anatomically and functionally distinct areas referred to as C1, C2, and C3; they rely on forestomach protozoal and bacterial fermentation to break down plant material to digest nutrients. Diseases of the gastrointestinal tract are considered to be the main causes of mortality in camelids; they include ulceration of C3, enteritis, intestinal wall perforation, intussusception, volvulus of the mesentery, meconium impaction, enteric impaction, ruptured gut, sep-

Corresponding Author: Lakamy Sylla (lakamy.sylla@unipg.it).

tic peritonitis, necrotizing enteritis and urinary bladder rupture1-7. Signs of colic in camelids are generally vague and non-specific, such as vocalizing, bruxism, getting up and lying down, refusing to stand, rolling, kicking or looking at the belly, peculiar stance, kyphosis, depression, pyrexia, anorexia, tachycardia, tachypnea, tenesmus, decreased fecal output, tense or painful abdomen, distended abdomen, pollakiuria, C1 atony, and regurgitation2-4,6,8. Therefore, camelid patients should be strictly and frequently monitored when abdominal condition is suspected, in order to ensure a detailed observation of the several clinical features5.

In case of small intestinal obstruction even more violent clinical signs, such as rolling and thrashing, could be seen while the severity of clinical signs associated with lesions affecting the large intestine tend to be more subtle2. Diagnostic techniques are mainly based on physical examination; a transabdominal ultrasonography could be helpful to evi-

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A case of epiploic foramen entrapment of jejunal intestinal tract in an alpaca (Vicugna pacos) cria

glycaemia decreased to 48 mg/dl, requiring a fast intravenous administration of a 33% dextrose solution diluted in 100 ml of saline solution over 15 min (Tables 1 and 2). General body condition was stationary and normal, except that the patient was still anorexic.

At day 1 post-recovery, the cria was subjected to an ophthalmological examination that evidenced a right corneal ulcer; a local therapy was instituted by application of Colbiocin® TID. Gastric antiulcer and anti-inflammatory treatments were started with Sucralfate® at the dose of 25 mg/kg per OS four times a day and Flunixin Meglumine 0.5 mg/kg IV once a day, respectively.

During the previous days, the cria continued to evacuate normal faeces, even if in small amount.

Figure 1 - Two crias of the same age, approximately six months, at presentation at the OVUD. The male sick cria (on the right) was under-grown compared to the healthy female of the same age. Painrelated behavior, such as frequent recumbence and stand-up together with depression and anorexia, were shown.

Table 1 - Blood cells count of the cria at day one of presentation at the OVUD. Value

Reference limits7,9

10.15

9.1 - 13.8

Hb (g/dL)

11.3

10.4 - 17.0

Htc (%)

20.1

24 - 36

MCV (fL)

19.8

21.8 - 28.9

MCH (pg)

11.1

10.6 - 12.7

MCHC (%)

56.2

39.3 - 46.8

WBC (x103/µL)

6.98

5.7 - 32.9

Neutrophils (%)

84.1

49 - 65

Lymphocytes (%)

14.8

21 - 25

RBC (x106/µL)

dence the most common surgical lesions2. An exploratory laparotomy or laparoscopy should be considered an extension of the physical examination. The outcome of gastrointestinal surgery is often reliant upon early diagnosis and timely surgical intervention.

This case report described the clinical findings and surgical approach of intestinal tract herniation in the epiploic foramen in an alpaca cria.

A 6-month-old, 20 Kg bodyweight, intact male alpaca was referred to the Teaching Veterinary Hospital of University (OVUD) at the Department of Veterinary Medicine University of Perugia - Italy, with a history of poor somatic growth compared to other crias of the same age, progressive weight loss, lethargy, dehydration and colic symptoms.

On presentation at the OVUD, the patient appeared depressed, alternatively in sternal and lateral recumbency, with body temperature of 38.5°C, heart rate of 52 bpm; at auscultation of the abdomen the forestomach and intestinal motility were normal, oral and conjunctival mucous membranes were pinkish, apparent lymph nodes were normal and a monolateral epiphora from the right eye was noted (Figure 1).

The patient was immediately subjected to medical treatment including intravenous fluid therapy (750 ml Ringer Lactate, 250 ml of 5% Dextrose solution, 2 ml Desadrexon and 5 ml Dobetin, intravenously, at a rate of 45 ml/Kg over one hour).

Abdominal ultrasonography was performed using an ultrasound machine equipped with a 3-8 MHz convex transducer (MyLab 30 Gold, Esaote, Genova, Italy) after clipping the fiber of interested areas. The cria was examined in lateral recumbence in order to check the gastrointestinal tract. Small intestine was dilated, with intraluminal fluid accumulation and normal motility. The urinary bladder was normal in appearance and fluid-filled; both kidneys presented normal echostructure. No peritoneal effusion was present, while other abdominal organs were unremarkable.

At presentation the glycaemia was 320 mg/dl; therefore, the cria was subjected to an IM injection of 40 UI of human insulin. One hour later the glycaemia decreased to 269 mg/dl.

The following morning, blood analysis was carried out; the

Monocytes (%)

1.1

0-5

Eosinophils (%)

0.0

6 - 22

Basophils (%)

0.0

0 - 0.5

PLT (x103/ L)

323

200 - 600

Table 2 - Biochemistry profile of the cria at day one of presentation at the OVUD. Value

Reference limits7,9

AST (IU/L)

230.0

137.0 - 391.0

ALP (IU/L)

466.0

32.0 - 167.0

GGT (IU/L)

12.0

13.0 - 50.0

Cholesterol (mg/dL)

31.0

15.5 - 88.9

Triglycerides (mg/dL)

5.0

10.62 - 45.14

BUN (mg/dL)

48.0

21.62 - 60.66

CPK (IU/L)

208.0

56.0 - 662.0

LDH (IU/L)

951.0

10.0 - 695.0

Creatinine (mg/dL)

1.56

1.0 - 2.4

Glucose (mg/dL)

48.0

90.0 - 149.0

Calcium (mg/dL)

9.1

4.2 - 9.0

Sodium (mEq/L)

152.0

144.0 - 155.0

2.1

4.0 - 5.7

117.0

97.0 - 111.0

Total proteins (g/dL)

5.3

5.7 - 7.2

Albumin (g/dL)

3.99

2.9 - 4.3

Potassium (mEq/L) Chloride (mEq/L)

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L. Sylla et al. Large Animal Review 2020; 26: 99-102

Late morning of the third day, the clinical condition worsened and the patient presented sinusal tachycardia (210 bpm), tachypnea (52) and dyspnoea (open mouth breathing). An additional abdominal ultrasound was performed, confirming the excessive fluid content in small intestine with normal peristalsis. Forestomachal compartments appeared dilated. A gastric tubing was performed in order to empty the C1 compartment; few hours later he started to evacuate a discrete amount of catarrhal faeces.

The farmer was informed about the deterioration of the clinical condition and was requested for the authorization to proceed with an explorative laparotomy, due to the suspicion of an obstructive ileus.

General anaesthesia was obtained with a mixture of Xylazine (0.46 mg/kg), Ketamine (4.6 mg/kg) and Butorphanol (0.046 mg/kg) administered intramuscularly. Then, anaesthesia was maintained with Isoflurane (MAC 1.1) and Oxygen through a tracheal intubation (size 8.5 length 13 cm). Surgical preparation and scrub of the ventro-caudal region was achieved and routine laparotomy procedure was performed between the navel scar and the prepubic tendon attachment. There was no peritoneal fluid accumulation; forestomachal compartments were all excessively dilated with fluid content.

Therefore, two different sites gastrotomy were performed on both C1 and C3 in order to evacuate abnormal liquid content mixed to ingesta. More in detail, gastrotomy site in C1 was identified in the dorsal aspect of the caudal sac, 2 cm above and in right-direction from the glandular saccules of the first compartment. The incision of C3 was performed on the ventral aspect of the terminal part, corresponding to the curvature before pyloric area. Almost all tracts of the gut were normal in term of colour and volume, except for a small area of jejunum which appeared dark reddish with fibrinous spots on its surface. The jejunum was hard in consistency at digital palpation and was entrapped in the epiploic foramen (Figure 2).

The intestinal tract was freed by small incision in the ring of the epiploon combined by gentle traction. Due to the necrotic lesions on the intestinal tract, the owner was informed and authorised the execution of the euthanasia. This intestinal tract has been later recognized as herniated and entrapped in epiploic foramen. Due to proximity to pylorus, presence of fibrin, and to involvement of pancreatic mesenteric area, the cria was euthanized.

At necropsy, body condition score was 2/5, mucous membranes appeared normal; a focal ulceration was present in the right conjunctiva. A moderate sero-haemorrhagic fluid peritoneal accumulation was observed in the abdominal cavity.

Topography of abdominal viscera was maintained, except for a 5 cm of small intestine that were entrapped within the epiploic foramen; it showed multifocal, brown-reddish, necrotic and haemorrhagic lesions (Figure 3).

Its content included fluid and semisolid material and brown-reddish in colour; the wall showed the typical aspect of necro-haemorrhagic enteritis. The C1 forestomach presented a fresh surgical suture line; moderate amount of ingesta was present in the digestive tract and the mucosa of C3 showed a segmental hyperaemia, corresponding to the second gastrotomy site. Mucosa of the ileus showed sub-mucosal haemorrhages which were compatible with haemomelasma ilei. Kidneys, spleen and liver gross anatomy appeared normal in position and morphology. Light sero-haemor-

101

Figure 2 - Intraoperative view of necrotic small intestinal loop found during explorative laparotomy of the male cria.

Figure 3 - Necropsy findings in the abdominal cavity of the sixmonths old cria. The topography of the gastrointestinal tract was conserved, except for a small intestinal loop, 5 cm long, which resulted entrapped within the epiploic foramen. The wall of the herniated organ was hemorrhagic and necrotic. In the picture, forestomaches were moved ventrally to allow visualization of the herniation area.

rhagic fluid accumulation was found in the thoracic cavity and post mortem congestion stasis was present in the right lung. Heart was normal in dimension and morphology.

Small intestinal tract histopathology showed severe necrosis in the sub-mucosa and lamina propria haemorrhages and occasionally in the muscular layers. Diffuse lymphocyte infiltration (Mucosa-Associated Lymphoid Tissue or MALT hyperplasia) was found associated to blood vessel congestion. In forestomach sections, slight MALT hyperplasia was evident; occasionally, necrosis and bacterial overgrowth were identified, probably due to post-mortem alteration. Lung sections revealed congestion and edema into the alveoli.

Spleen sections showed moderate but diffuse bloody congestion, while kidneys were normal in appearance.

In the present case report, a 6 month old Alpaca cria with colic symptoms secondary to jejune entrapment was subjected to an exploratory laparotomy. The cria was firstly treated medically for anorexia and depression; but, upon deteriora-

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tion of the health status, a laparotomy was performed, leading to the definitive diagnosis of the epiploic herniation of the jejune tract.

Secondary intestinal entrapments are more common in camelids that had previous abdominal surgery or infection; however, in the present case there was no history of previous abdominal surgery2,3,5,6. Intestinal entrapments generally involve the caudal flange of the jejunum, ileum, cecum, or ascending colon; these intestinal tracts may migrate through the epiploic foramen (as in the present study) or a defect in the body wall or diaphragm2,3,6. Risk factors for entrapments are very different from those for intraluminal obstructions such as congenital, acquired, or iatrogenic defects of the mesentery or walls of the peritoneal cavity.

Camelids with intestinal entrapments may continue to evacuate small quantities of normal or blood-tinged faeces whereas consistent passage of small amounts of diarrhoea in spite of on-going pain signs for up to 4 days is common with infection by Eimeria macusaniensis. Although herein the cria was positive to E. macusaniensis infection, there was no sign of diarrhoea before hospitalization.

Thorough examination and use of appropriate diagnostics including blood and biochemistry profiles, abdominocentesis and ultrasonography should help in reaching a diagnosis.

Exploratory surgery is warranted for crias showing severe abdominal pain and gross distension of intestinal loops, particularly if no faecal material is being passed or if the crias fail to improve on medical therapy.

In conclusion, necropsy confirmed the diagnosis of small intestine herniation into the epiploic foramen with related severe acute necro-haemorrhagic enteritis.

ACKNOWLEDGMENTS No third-party funding or support was received in connection with this study or the writing or publication of the manuscript.

CONFLICT OF INTEREST The authors declare that there were no conflicts of interest.

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2. Cebra CK, Cebra ML, Garry FB, Larsen RS, Baxter GM. Acute gastrointestinal disease in 27 New World camelids: clinical and surgical findings. Vet Surg 1998; 27:112-21.

3. St-Jean G, Anderson DE, Anderson NV, Hoskinson J. Abdominal pain associated with an umbilical abscess in a llama. Cornell Vet 1993; 83:77-81.

4. Costarella CE, Anderson DE. Ileocecocolic intussusception in a onemonth-old llama. J Am Vet Med Assoc 1999; 214:16C72-3, 1640.

5. Bickers RJ, Templer A, Cebra CK, Kaneps AJ. Diagnosis and treatment of torsion of the spiral colon in an alpaca. J Am Vet Med Assoc 2000; 216:380-2.

6. Black-Schultz LL, Hanson PD, Wilson DG, Markel MD. Diaphragmatic hernia in a llama. J Am Vet Med Assoc 1993; 1993:410-2.

7. Vencato J, Fiore E, Morgante M, Stelletta C. Gastric ulcers in south american camelids. Large Animal Review 2012; 18:123-127.

8. Smith JJ, Dallap BL. Splenic torsion in an alpaca. Vet Surg 2005; 34:1-4.

9. Foster A, Bidewell C, Barnett J, Sayers R. Haematology and biochemistry in alpacas and llamas. In practice 2009; 31:276-281.

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Il testo va redatto in Microsoft Word preferibilmente, oppure OpenOffice oppure Rich Text Format, con le linee e pagine numerate consecutivamente, con carattere Times New Roman 12 punti, margini laterali di 2 centimetri, interlinea singola e non deve superare il numero di caratteri (spazi inclusi) indicato nel paragrafo precedente per ciascun tipo di contributo.

Tabelle e Figure - Le tabelle, i grafici e le immagini devono essere annesse al testo del manoscritto e numerate con numeri arabi e corredate da titoli o didascalie concisi ma sufficientemente dettagliati, in modo che risultino comprensibili senza dovere fare riferimento al testo. Tabelle e figure devono essere posizionate alla fine del manoscritto.

Bibliografia - Le referenze bibliografiche ritenute essenziali (non oltre 30 ad eccezione delle review) devono essere richiamate nel testo con un numero progressivo fra parentesi ed elencate nello stesso ordine numerico nella bibliografia.

Per gli articoli tratti da riviste si dovranno indicare: cognome e iniziale del nome dell’Autore e dei Coautori, anno di pubblicazione, titolo dell’articolo, indicazione abbreviata della rivista (in accordo all’Index Medicus), numero del volume, numero della pagina iniziale e finale. Per citazioni bibliografiche di articoli o capitoli contenuti nei libri di testo, si dovranno indicare: cognome e iniziale del nome dell’Autore e dei Coautori, anno di pubblicazione, titolo del capitolo, titolo del libro, numero del volume (se più volumi), editori, edizione, pagina iniziale e finale del capitolo, casa editrice e sua sede. Si riportano due referenze a titolo di esempio: – Journals - Galey F.D., Terra R., Walker R., Adaska J., Etchebarne M.A., Puschner B., Fisher E., Whitlock R.H., Rocke T., Willoughby D., Tor E. (2000). Type C botulism in dairy cattle from feed contamined with a dead cat. J Vet Diagn Invest, 12: 204-209.

– Books - Gustafson D.P. (1986). Pseudorabies. In: Diseases of swine, Ed. Dunn H.W., 5th ed., 274-289, Iowa State University Press, Ames, IA. – Conferences - Barbano D. M. (1996). Mozzarella cheese yield: Factors to consider. Page 29 in Proc. Wisconsin Cheese Makers Mtg., Madison. Ctr. Dairy Res., Univ. Wisconsin, Madison.

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INVIO Il manoscritto deve essere sottomesso on-line al seguente link: www.largeanimalreview.com Per informazioni: Dr. Enrico Fiore - Technical Editor largeanimalreview@sivarnet.it

- S O C I E T À I TA L I A N A V E T E R I N A R I

PER

ANIMALI

DA

Centro Studi EV - Cremona - Tel. +39 0372 403539 - info@sivarnet.it - www.sivarnet.it

R E D D I TO

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LAR

Large Animal Review

GUIDELINES FOR AUTHORS Official scientific journal of SIVAR

Large Animal Review is a bimonthly magazine published by SIVAR (Italian Society of Farm Animals Veterinary Practitioners) for scientific updating of veterinarians who deal with animals in livestock production and the supply chain control in the production of food industry. The topics of main interest for the journal are those of internal medicine, surgery, obstetrics, animal nutrition, zootechnics, infectious and parasitic diseases, food safety and security, animal welfare, prevention and management.

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MANUSCRIPTS Large Animal Review publishes manuscripts in the form of reviews, original articles and case reports; manuscripts must comply with the guidelines below.

Review - This is a complete coverage of a specific topic accompanied by a detailed and updated bibliography. Authors interested in writing a review should contact the editor of Large Animal Review. The text should not have more than 48.000 characters (including spaces) and not be accompanied by more than 15 figures or tables.

Original Article - The papers published in Large Animal Review are short or full-length research articles related to the topic of the journal. The full text article should not exceed 32.000 characters (including spaces) and should not be accompanied by more than 10 figures or tables. Manuscripts in the form of short articles should not exceed 16.000 characters and no more than 4 figures or tables.

Case Report - Single clinical or herd case report should be presented in Large Animal Review. The manuscript must not exceed 10.000 characters (including spaces) and no more than 4 figures or tables.

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FORMAT All manuscripts (review, original articles and case reports) must have the following structure.

Language - English or Italian.

Title - The title of the manuscript has to be short and explicative and written on the front of the first page. Under the title, names of authors should be given indicating the surname and the name (e.g., Smith Tom). Institutional addresses are displayed below the author names; footnotes referring from author names to displayed addresses should be numbered. The full name, mailing address, phone number, and e-mail address of the corresponding author should appear directly below the affiliation lines on the title page. The corresponding author will be identified by a symbol footnote (e.g., Smith Tom*) and e-mail address below the affiliation lines on the first page of the published article (e.g., *Corresponding author: Smith Tom, University of …).

Abstract - Abstract has to be placed on the second page of the manuscript and written in English. Abstracts should be limited from 300 to 500 words. The abstract disseminates scientific information through abstracting journals and is a convenience for readers. Exclude references, figure or table.

Key Words - After the abstract, list 3 to 5 key words have to be placed. In case of manuscripts written in Italian, the key words have also be translated into English.

Body of the Paper - The manuscript of the original articles must show the following outline: introduction, materials and methods, results, discussion, conclusions, acknowledgments and bibliography. Regarding the reviews, the outline is not expected, but the topic must be clearly argued and divided in chapters.

The text should be typed in Microsoft Word preferably, or OpenOffice or Rich Text Format, with lines and pages numbered consecutively, using Times New Roman font at 12 points. All margins should be at least 2 centimeters, single spacing and cannot exceed the number of characters (including spaces) indicated in the previous section for each type of manuscript.

Tables and Figures - Tables, graphs and images must be included in the manuscript text and numbered (Arabic numerals).

The titles or captions should describe concisely the data shown, sufficiently detailed and comprehensible to the reader. Tables and figures should be placed in separate sections at the end of the manuscript.

References - The references must be selected by the authors (not more than 30, except in a review) and should be cited in the text with a serial number in round brackets and listed in the same numerical order in the bibliography.

For articles from journals you should indicate: surname and first initial of the author and co-author/s names, year of publication, article title, abbreviated indication of the magazine (Index Medicus), volume number, number of first and last pages.

For citations to articles or chapters contained in textbooks, you should indicate: surname and first initial of the Author and co-author/s names, year of publication, chapter’s title, book title, volume number (if more than one volume) editors, edition, first and last page of the chapter, publishing house and its location.

Examples: – Journals - Galey F.D., Terra R., Walker R., Adaska J., Etchebarne M.A., Puschner B., Fisher E., Whitlock R.H., Rocke T., Willoughby D., Tor E. (2000). Type C botulism in dairy cattle from feed contamined with a dead cat. J Vet Diagn Invest, 12: 204-209.

– Books - Gustafson D.P. (1986). Pseudorabies. In: Diseases of swine, Ed. Dunn H.W., 5th ed., 274-289, Iowa State University Press, Ames, IA. – Conferences - Barbano D. M. (1996). Mozzarella cheese yield: Factors to consider. Page 29 in Proc. Wisconsin Cheese Makers Mtg., Madison. Ctr. Dairy Res., Univ. Wisconsin, Madison.

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SUBMISSION OF MANUSCRIPTS The manuscripts have to be submitted exclusively on the following link: www.largeanimalreview.com Informations: Dr. Enrico Fiore - Technical Editor largeanimalreview@sivarnet.it

- I TA L I A N A S S O C I AT I O N

OF

FA R M A N I M A L P R AC T I T I O N E R S

Centro Studi EV - Cremona (I) - Tel. +39 0372 403539 - info@sivarnet.it - www.sivarnet.it

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