Large Animal Review - Anno 25, Numero 3, Giugno 2019 by E.V. Soc. Cons. a r.l.

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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

03/19

Bimestrale, Anno 25, Numero 3, Giugno 2019

LAR Large Animal Review

ISSN: 1124-4593

LARGE ANIMAL REVIEW è indicizzata su Science Citation Index (SciSearch®) Journal Citation Reports/Science Edition e CAB ABSTRACTS

ORIGINAL ARTICLES BOVINE • Effects of Saccharomyces cerevisiae as dead yeast culture on feed supplement in fattening cattle CAPRINE • Small ruminant lentivirus and Mycobacterium avium subsp. paratuberculosis SWINE • Natural occurrence of ochratoxin A in confined reared and grazing pigs derived products REVIEWS FOOD HEALTH • Fermentation as a strategy to increase conjugated linoleic acid in dairy products CASE REPORTS BOVINE • Evaluation of marsupialization combined with long-term administration of antibacterial agents in calves with omphalophlebitis and secondary liver abscess SWINE • Severe outbreak of pasteurellosis in sows: a case description

SOCIETÀ ITALIANA VETERINARI PER ANIMALI DA REDDITO ASSOCIAZIONE FEDERATA ANMVI

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INDEX

ORIGINAL ARTICLES

Anno 25, numero 3, Giugno 2019 Rivista indicizzata su: CAB ABSTRACTS e GLOBAL HEALTH IF (2017/2018): 0.26

Editor in chief: Massimo Morgante Editorial Board 2019-2021: Anna Rita Attili - Roberto Bardini Marco Colombo - Vincenzo Cuteri Enrico Fiore - Matteo Gianesella Elisabetta Giudice - Paolo Moroni Davide Ranucci - Antonia Ricci Giuseppe Stradaioli - Erminio Trevisi

OMAR MAAMOURI, SAMIA MABROUK, LINDA MAJDOUB MATHLOUTHI

Managing Editor: Matteo Gianesella Technical Editor: Enrico Fiore

Edizioni SCIVAC Palazzo Trecchi - 26100 Cremona Tel. 0372/460440 Iscrizione registro stampa del Tribunale di Cremona n. 299 del 25/9/1995

CAPRINE Small ruminant lentivirus and Mycobacterium avium subsp. paratuberculosis: co-infection prevalence and preliminary investigation on genetic resistance to both infections in a Garfagnina goat flock FRANCESCA CECCHI, FILIPPO FRATINI, DOMENICO CERRI, PATRIZIA BANDECCHI, CARLO CANTILE, MAURIZIO MAZZEI

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.

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BOVINE Effects of Saccharomyces cerevisiae as dead yeast culture on feed supplement in fattening cattle on growth, intake parameters and nutrient digestibility

SWINE Natural occurrence of ochratoxin A in confined reared and grazing pigs derived products VALENTINA MEUCCI, ALESSANDRO PISTOIA, SIMONE BERTINI, ALESSANDRO MENOZZI, LUIGI INTORRE

REVIEWS

쓼

FOOD HEALTH Fermentation as a strategy to increase conjugated linoleic acid in dairy products LINA COSSIGNANI, FRANCESCA BLASI

101

Direttore Responsabile Antonio Manfredi

CASE REPORTS

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BOVINE Evaluation of marsupialization combined with long-term administration of antibacterial agents in calves with omphalophlebitis and secondary liver abscess SATO REIICHIRO, SHINOZUKA YASUNORI, ONDA KEN, OCHIAI HIDEHARU, YAMADA KAZUTAKA

107

SWINE Severe outbreak of pasteurellosis in sows: a case description GIUSEPPE MARRUCHELLA, CRISTINA ESMERALDA DI FRANCESCO, ABIGAIL ROSE TRACHTMAN, FRANCESCO MOSCA, ANDREA DI PROVVIDO, CARLA SEBASTIANI, PIETRO GIORGIO TISCAR, CHIARA MAGISTRALI

111

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La SIVAR ringrazia i colleghi per la numerosa affluenza, i relatori, gli sponsor e gli espositori che hanno contribuito alla migliore riuscita dell’evento 21° Congresso Internazionale SIVAR - www.sivarnet.it

ARRIVEDERCI AL 22° CONGRESSO: 13-14-15 MAGGIO 2020

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O. Maamouri et al. Large Animal Review 2019; 25: 83-87

Effects of Saccharomyces cerevisiae as dead yeast culture on feed supplement in fattening cattle on growth, intake parameters and nutrient digestibility

OMAR MAAMOURI1, SAMIA MABROUK2, LINDA MAJDOUB MATHLOUTHI2 1

National Institute of Agronomic Research Tunisia, Street Hedi Karray, 1004, El Menzah 1, University of Carthage, Tunisia 2 High Institute of Agriculture Sciences of Chott Mariem, University of Sousse Tunisia

SUMMARY This study was carried out to study the interest of the incorporation of a dead yeast culture Saccharomyces cerevisiae in the concentrate and to see its effect on the growth, the ingestion and the digestibility in vitro of the cattle of fattening. The trial involved a fattening farm containing 20 fattening cattle divided into two homogeneous groups based on initial body weight of 396.4 ± 69.7 kg and 404.6 ± 97.8 kg (Pr. > F) respectively for the control group (C) and the experimental group (Y). The ration used is wheat straw and concentrate. This same ration was distributed for the group Y plus a quantity of 10 g / head / day powder in the concentrate yeast culture. Amount of feed distributed was 3 kg DM wheat straw and 8 kg DM concentrate. This trial lasted 112 days (including adaptation period). The weights are calculated every two weeks with a cattle scale. The refused amounts of wheat straw are also weighed at each control. A significant (P <0.01) increase in the mean total daily gain (ADGT) during the trial was noted 450 g / head. And a significant (P <0.01) increase in the final weight gain (FWG) of 51.6 kg / head for the “yeast” group compared to the “control” group. Feed Intake does not differ with yeast intake. Voluntary feed intake increased for group (Y) at third control. For food conversion, it was similar for group Y and group C with 2.6 ± 0.003, P <0.05, respectively.

KEY WORDS Bulls, acidogenic diet, yeast, productivity, rumen.

INTRODUCTION In Tunisia, cattle’s breeding is an important component of agricultural production and the national economy. As a result of population growth, the state has always invested in improving the beef sector to meet the ongoing need for red meat. The increase in the number of cattle was at the expense of the available food. This intensification of livestock production has led to excessive use of concentrated feeds and cereals in animal feeds, specifically in the fattening of young bulls. Nevertheless, to succeed fattening, certain conditions must be respected and a minimum of knowledge in breeding is necessary. In order to value their products and improve their incomes, these feeders increase the proportions of concentrated feeds in animal feed without taking into account the risks of metabolic diseases such as acidosis led by this misuse, leading to decreased performance. To prevent this risk, several studies have shown that the use of food additives seems to be an effective solution to limit the risk of latent acidosis in ruminants. In particular, yeast Saccharomyces cerevisiae has been widely studied (Chaucheyras-Durand et al., 20081; Desnoyers et al., 20062; Chaucheyras-Durand & Durand., 20103). They make it possible to maintain good animal health following digestive

Corresponding Author: Omar Maamouri (omar_maamouri@yahoo.fr).

comfort and thus improve their zootechnic performance. The objective of this study is to explore the effect of the addition of Saccharomyces cerevisiae yeast culture in the feeding of cattle’s on in vitro digestibility and zootechnic performance in intensify system.

MATERIALS AND METHODS Experiment design and measurements The trial was conducted in north eastern Tunisia for 112 days out of 20 Holstein cattle that were split into two equal groups (10 cattle per group) according to age (15 months), body weight (400 ± 5.8 kg) fed the same ration (composed of wheat straw and concentrate). Each bull of the yeast group (Y) also received 10 g / head / day of yeast Saccharomyces cerevisiae powder on the concentrate. The ration consists of wheat straw (5 kg DM / head / day) and 8 kg DM concentrate for the control group (C). For group Y he also received 10 g / head / day of S. cerevisiae. The weights were measured every two weeks with a cattle scale. We also calculated the average daily gain (ADG), the total daily gain (ADGT), the final weight gain (FWG) and the feed conversion (FC). The refused quantities of wheat straw are also weighed each control with a balance. It should be noted that the entire amount of concentrate is ingested. Yeast Culture is Saccharomyces cerevisiae yeast grown on a media of sucrose and cane molasses.

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Effects of Saccharomyces cerevisiae as dead yeast culture on feed supplement in fattening cattle on growth

Sampling and chemical analysis Chemical composition of various feed resources was determined in the animal nutrition laboratory at National Institute of Agronomic Research Tunisia (Table 1). Nutritive values of experimental aliments were determined following the method described by Sauvant (1981)4. Samples of diets were dried in a forced-air oven at 105 °C for 24 h to determine DM. Dried samples were then ground through a 1-mm screen. Ground samples were used to determine ash content (450 °C for 8 h), crude fiber (CF) by the method of Weende (AOAC, 1984)5. Fat matter was determined by Randhall (AOAC, 1984)5. Crude protein was determined by Kjeldahl method (AOAC, 1984)5.

Table 1 - Chemical composition and nutritive value of concentrate and Wheat straw. Diets

Concentrate

Wheat straw

DM (%)

89.61

89.51

TN (%DM)

1.90

0.64

CP (%DM)

11.9

CF (%DM)

6.3

29.3

Asch (%DM)

OM (%DM)

88.3

FM (%DM)

4.3

–

NEA (%DM)

68.5

–

In vitro fermentation parameters

PDIE (g/kg DM)

Determination of the total gas was performed on the contents of the rumen filtered from cattle just after slaughter. In syringes, were put 0.3 g of substrate (concentrate ground to 1 mm), 10 ml of rumen juice and 20 ml of artificial saliva. The syringes are then placed vertically in a water bath at 39 °C; the reading is done each two hours after mixing syringes until a bearing (Orskov and Mc. Donald., 1979)6.

PDIN (g/kg DM)

1.06

0.4

Statistical analysis The results of the effects of diets on the measured parameters (weights, adg, feed intake, fc) were subjected to analysis of variance with the GLM procedure of the statistical package SAS (2000)7 and compared by t-test diff. The statistical model was: Yij = µ + Ri + eij With: Yij: measured parameter. µ: overall mean. Ri: fixed effect of diet (i = 1, 2). eij: residual error term. Significance was declared at P <0.05 unless otherwise declared.

RESULTS AND DISCUSSION Chemical composition of food The chemical composition of foods is shown in Table 1. For wheat straw, it has a low crude protein (CP) content (4%) and fodder unit (UF) (0.4 UF / kg DM). The CP content could be considered deficient (Norton, 1994)8. For feed concentrate, CP and UF contents are 11.9% and 1.06 UF/kg DM respectively.

Growth (weight) and average daily gain (ADG) The results showed that supplementation of 10 g yeast Saccharomyces cerevisiae per head per day only increase significantly average daily gain: ADG1 P <0.01, ADG4 P <0.04 and ADG6 P <0.06 by 1180 g/d, 570 g/d and 980 g/d respectively for ADG1, ADG4 and ADG6. There was a significant (P <0.01) increase of ADGT during all trial by 450 g/head. And a significant (P <0.01) increase of final weight gain (FWG) by 51.6 kg/head for “yeast” group in comparison with “control” one (Table 2). Prebiotics can increase the weight gain of ruminants. Prebiotics isolated from healthy goat, when fed to goats for eight weeks, commencing at 75 days of age, resulted in improve-

UF (g/kg DM)

DM: dry matter; OM: organic matter; CP: crude protein; FM: fat matter; CF: crude fiber; UF: meat fodder unit: NEA: non extractif azote; PDIE: digestible protein in the liver of energy origin; PDIN: digestible proteins in the liver of microbial when the ration is deficient in degradable nitrogen.

ment in average body weight by 9% (Apas et al., 2010)9. Similar improved growth rate was obtained with a yeast-based commercial probiotic containing S. cerevisiae given to growing dairy heifers (Ghazanfar et al., 2015)10 when fed to pregnant white Dorper ewes on a palm kernel-based diet, increased DM intake and live weight gain during pregnancy, followed by better performance of the lambs during early lactation (Le et al., 2014)11. Likewise, a novel bacterial strain isolated in Australia, P. jensenii 702, significantly enhanced weight gain in Holstein calves by (25%) during the pre-weaning period and by (50%) during the weaning period (Adams et al., 2008)12. Frizzo et al. (2011)13, based on meta-analysis of 21 publications between 1985 and 2010, concluded that lactic acid prebiotics bacteria in comparisons with and without L. acidophilus, L. plantarum, L. salivarius, E. faecium, L. caseilparacasei or Bifidobacterium spp., increased body weight gain (standardized mean difference = 0.22822, 95% confidence interval = 0.1006 to 0.4638) and improved feed use efficiency (standardized mean difference = -8.141, 95% confidence interval = -1.2222 to -0.4059) in young calves compared with control groups when probiotics were added to milk replacer, but were ineffective when added to whole milk. In contrast, some studies have reported no effect on calf growth when the diet was supplemented with L. acidophilus (Abu-Tarbouch, Al-Saiady & El-Din, 1996)14; Cruywagen, Jordaan &Venter, 1996)15, a mixture of L. acidophilus and L. plantarum (Abu-Tarbouch, Al-Saiady & El-Din, 199614, B. subtilis (Galina et al., 2009)16, or a mixture of L. acidophilus, L. lactis and B. subtilis (Galina et al., 2009)16. The results found are in agreement with those of Cano Lopez et al. (2010)17 who found no significant differences between the two treatments at the level of the QGMs (p >0.05), even if numerically it is higher in the animals that received the yeasts. On the other hand, the tests carried out by El’Hassan et al. (1993)18 and Hancock et al. (1994)19 on young bulls reported a significant increase in GMQ when animals were fed an acidogenic diet and this could be the cause of the yeast effect which probably helps to limit fermentative disturbances in the rumen generally caused concentrated diets (Desnoyers, 2008)20.

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O. Maamouri et al. Large Animal Review 2019; 25: 83-87

Table 2 - Effect of yeast culture on growth [Weight (W)] and ADG. Group

MSE

Pr. > F

404.6±97.8

84.9

0.8

416.9±68.7

441.5±98.4

84.8

0.5

W2 (kg)

469±68.3

491.2±90.2

0.5

W3 (kg)

493.2±69

523.8±93.7

82.3

0.4

W4 (kg)

501.9±69.5

540.6±92.3

81.6

0.3

W5 (kg)

511.2±72.3

542.6±85

78.5

0.3

W6 (kg)

524.9±79.9

570±86.9

83.2

0.2

W7 (kg)

538.5±84.9

587.7±87.1

85.9

0.2

ADG1 (kg/d)

1.46 ±1.1

2.64 ±0.8

0.9

0.01

ADG 2 (kg/d)

3.72±2.6

3.56±1.4

2.07

0.8

Control

Yeast culture

W0 (kg)

396.4±69.7

W1 (kg)

ADG 3 (kg/d)

1.74±0.7

2.33±0.9

0.79

0.1

ADG 4 (kg/d)

0.62b±0.3

1.19a±0.8

0.6

0.04

ADG 5 (kg/d)

0.67±0.5

0.97±0.6

0.58

0.2

ADG 6 (kg/d)

1.07

0.06

0.99 ±0.7

1.97 ±1.3

ADG 7 (kg/d)

0.97±0.7

1.25±0.7

0.7

0.3

ADGT (total) (kg/d)

1.28b±0.4

1.73a±0.3

0.36

0.01

142.1b±46.4

193.7a±30.7

39.7

0.01

FWG (kg)

a, b: Mean values with different letters in the same row are significantly different; MSE: mean standard error; (±): standard deviation; ADGT: adg during all trial; FWG: final weight gain.

Feed intake and feed conversion (fc) Voluntary intake increased for the Y group from the third control, but this increase wasn’t mentioned a significant difference (P >0.05). For the feed conversion (FC), it was simi-

lar for the Y group and the C group which around 2.6±0.003; (P <0.05) (Table 3). Our results are consistent with those of Desnoyers et al. (2006)2, who found that the amount ingested does not differ

Table 3 - Effect of yeast culture on feed intake and feed conversion (FC). Group

MSE

Pr. > F

444.8±37.2

85.1

0.4

469±26.1

492.2 ±37.4

79.9

0.5

Inake3 (g DM/d)

493.2 ±26

526.4±34.3

81.7

0.3

Inake4 (g DM/d)

501.9±26.2

543.2±35.6

81.3

0.2

Inake5 (g DM/d)

511.2±26.4

548.6±35.1

79.1

0.3

Inake6 (g DM/d)

524.9±27.5

573.4±32.3

83.9

0.2

Inake7 (g DM/d)

538.5±30.4

585.2±33

85.6

0.2

Inake8 (g DM/d)

152.2±32.3

155.4±33.1

32.2

0.8

FC1

2.602 ±0.005

2.602±0.007

32.2

0.5

FC2

2.604±0.004

2.605±0.006

30.3

0.5

FC3

2.607±0.004

2.608±0.004

31.2

0.4

FC4

2.608±0.003

2.609±0.004

31.01

0.3

FC5

2.609±0.003

2.610±0.003

29.8

0.3

FC6

2.609±0.003

2.610±0.003

31.6

0.2

FC7

2.610±0.003

2.611±0.003

32.6

0.2

FC8

2.610±0.003

2.612±0.003

0.005

0.9

Control

Yeast culture

Inake1 (g DM/d)

416.9±26.5

Inake2 (g DM/d)

MSE: mean standard error. (±): standard deviation.

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Effects of Saccharomyces cerevisiae as dead yeast culture on feed supplement in fattening cattle on growth

Table 4 - The parameters a, b, c and a+b of non linear model of gas production and estimated parameters from gas produced at 24 hours: comparaison of the two trials diets (C) and (Y). Group

Control

Yeast

MSE

Pr < F

a (ml)

-0.6b ±1.6

1.8a ±1.02

0.4.10-4

<0.0001

b (ml)

140.4a ± 22.4

118.1b ±6.2

0.36.10-5

<0.0001

c (h-1) a + b (ml)

0.02 ±0.006 a

0.032 ±0.003

<0.0001

139.8 ±24

116.3 ±7.2

–

<0.0001

Prod gas 24 h (ml)

64b ±1.4

68.5a±0.7

1.11

0.05

DMO (%)

77.7b±1.2

81.7a±0.6

0.99

0.05

0.17

0.05

EM (MJ) VFA (mmol/syringue) EM (Kcal)

11.38 ±0.22

12.08 ±0.1

1.47±0.02

8.95±10.4

7.36

0.4

2719.4b±53

2888a±26.5

41.9

0.05

a, b: Mean values with different letters in the same row are significantly different; MSE: mean standard error. a: amount of gas product (ml) immediately from the substrate; b: potential of gas production; c: speed of gas production.

with the addition of yeast in the diet. This lack of difference can also be explained by the fact that the straw was not really distributed at libitum. The addition of yeast increases the voluntary intake of forage (Majdoub-Mathlouthi et al., 2011)21. Mutsvangwa et al. (1992)22 reported that the addition of yeast to an acidogenic diet contributes to the increase in dry matter intake in beef cattle. On the other hand, other work conducted by Moncoulon & Auclair (2001)23 even showed a significant decrease of 2.6% in the quantity of dry matter ingested. This trend can be explained by the fact that the yeast effect on ingestion is negligible with a diet rich in concentrated food (high energy intake) because of the metabolic satiety already established following the large production of VFA from carbohydrates quickly fermentable. Thus, the ingestion can probably increase in the case of a ration rich in fiber following the direct action of the yeast on the communities which degrade the fiber within the rumen by its action on the level of oxygen consumption (Marden et al., 2008)24 and promote fibrolytic activity by accelerating digestive transit and subsequently increasing the amount of dry matter ingested (Chaucheyras-Durand & Durand, 2010)3. As the ingested did not differ significantly between animals in two groups throughout the trial, the statistical analysis also showed that there was not a significant difference (P >0.05) in the feed conversion between the two groups respectively (Table 3).

1)] followed by concentrated feed) [0.02 (h-1)]. In vitro fermentation of two substrates is dependent on a latency phase, indicated by the negative value of the soluble fraction (a) (-0.6 ml / 0.3 g DM and 1.8 ml / 0.3 g DM respectively for “C” and “Y” group respectively), which partly explains its low degradation. This latency phase seems to be due to the time required for microorganisms to adhere and colonize dietary fiber. Regarding the other parameters, the values predicted mention that the digestibility of the organic matter (OMD) of the concentrated feed alone is 77.7% and 81.7% for the mixture (concentrate + Saccharomyces cerevisiae) respectively with a significant effect on this parameter (P <0.05). It’s the same for ME released by the different substrates (P <0.05) (11.4 MJ vs. 12.1 MJ respectively for “C” and “Y” group respectively). As well as VFA recorded, the respective values were 1.5 mmol / syringe for the concentrated feed alone vs. 8.9 mmol / syringe for concentrated feed + Saccharomyces cerevisiae with a significant difference (P <0.05). In general, the positive effect of yeast supplementation on rumen pH increased with the percentage of concentrate in the diet and with the Dry Matter Intake (DMI) level. Similarly, yeast increased the concentration of VFA with increased CP concentration and DM.

Parameters of rumen fermentative

This study allowed us to specify the interest of yeast culture as a food additive to modulate microbial fermentations of rumen and improve performance cattle in production. The results show that supplementation can improve moderately the performance animal (growth, feed conversion). And it appears crucial to explore the mechanisms of action of the Saccharomyces cerevisiae metabolic activities and intra-ruminal lipid and nitrogen metabolism of ruminants.

This study showed (Table 4) that supplementation with yeast Saccharomyces cerevisiae didn’t affect the facies’ parameters fermentation (OMD, VFA’s concentration and ME) and also the ammoniacal nitrogen (P >0.05). In vitro gas production in 100 glass syringes’ ml undergoes a rapid evolution after incubation. After 24 hours of incubation the (C) diet registers the lower significant (P >0.05) amount of gas (64 ml / 0.3 g DM) and is followed by the diet complemented by yeast which gives the largest amount (68.5 ml / 0.3 g). The kinetic parameters of the in vitro fermentation of different substrates, deduced from the exponential model of Orskov & Mc Donald (1979)6 are mentioned in the table 4. The mixture (concentrated feed + Yeast) is the most rapidly (P <0.0001) fermented by the microbiota ruminal [0.03 (h-

CONCLUSIONS

ACKNOWLEDGEMENTS The authors gratefully acknowledge the technical assistance of Mr Ben Othmane from OEP, Mr Amrawi from High School of Agriculture of Mateur and Mr Zied Maalaoui

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O. Maamouri et al. Large Animal Review 2019; 25: 83-87

Sales Manager in Northwest Africa and Middle East of Arm and Hammer Animal Nutrition Church and Dwight Co., Inc; to the conduct of this study, and for their assistance and logistic help.

12.

CONFLICT OF INTEREST STATMENT

13.

No potential conflict of interest was reported by the authors. 14.

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23. 24.

ghan M. (2014) Probiotic Bacillus amyloliquefaciens Strain H57 Improves the Performance of Pregnant and Lactating Ewes Fed a Diet Based on Palm Kernel Meal. In: ISNH/ISRP. International Conference, Canberra, Australia. https://espace.library.uq.edu.au/view/UQ:354091. Adams M., Luo J., Rayward D., King S., Gibson R., Moghaddam G. (2008) Selection of a novel direct-fed microbial to enhance weight gain in intensively reared calves. Anim Feed Sci Technol. 145, (1), 41-52. https://doi.org/10.1016/ j.ani feed sci.2007.05.035. Frizzo L., Zbrun M., Soto L., Signorini M. (2011) Effects of probiotics on growth performance in young calves: A meta-analysis of randomized controlled trials. Anim Feed Sci Techno. 169, (3), 147-156. https://doi.org/10.1016/j.anifeedsci.2011.06.009. Abu-Tarboush H.M., Al-Saiady M.Y., El-Din A.H.K. (1996) Evaluation of diet containing lactobacilli on performance, fecal coliform, and lactobacilli of young dairy calves. Anim Feed Sci Technol. 57, (1), 39-49. https://doi.org/10.1016/0377-8401(95)00850-0. Cruywagen C., Jordaan I., Venter L. (1996) Effect of Lactobacillus acidophilus supplementation of milk replacer on preweaning performance of calves. J. Dairy Sci. 79, (3), 483-486. https://doi.org/10.3168/jds. S0022-0302 (96)76389-0. Galina M., Ortiz-Rubio M., Delgado-Pertiñez M., Pineda L. (2009) Goat kid’s growth improvement with a lactic probiotic fed on a standard base diet. Options Méditerranéennes. Série A, Séminaires Méditerranéens. (85), 315-322. http://om.ciheam.org/article.php?IDPDF=801024. Cano Lopez G., Villalba Mata D., Philippe F. (2010) Comparaison de l’effet de l’apport en levures Saccharomyces cerevisiae vivantes ou inactivées sur les performances de croissance de bovins en engraissement. Renc. Rech. Ruminants, 2010, 17. El Hassan S.M., Newbold C.J., Wallace R.J. (1993) The effect of yeast in the rumen and the requirement for viable yeast cells. Anim. Prod. 54: 504 (Abstract). Hancock D.L., Brake A.C., Montgomery A.L., Dominey J.R., Mattingly C.A, Cecava M.J. (1994) Influence of yeast addition and Compudose implantation on feedlot performance and carcass characteristics of growing and finishing steers. J. Anim. Sci. 72 (Suppl 1):300. Desnoyers M. (2008) Intérêt de l’apport de levures sur la susceptibilité à l’acidose et lecomportement alimentaire du ruminant. Thèse doctorat. Institut des sciences et Industries duvivant et de l’environnement (AgroParisTech). 387 pages. Majdoub-Mathlouthi L., Chammaoui A., Kraiem K. (2011) Effet de la levure Saccharomyces cerevisiae sur les performances des taurillons à l’engraissemnt alimentés à base de fourrages pauvres. Lives Res for Rural Development. 23(11). Mutsvangwa T., Edwards I.E., Topps J.H., PATERSON G.F.M. (1992) The effect of dietary inclusion of yeast culture (Yea-Sacc) on patterns of rumen fermentation, food intake and growth of intensively fed bulls. Anim. Prod. 55:35-40. Moncoulon R., Auclair E. (2001) Utilisation du BIOSAF® Sc 47 pour la production de viande de taurillon. Rapport de Recherche. pp 17. Marden J P., Julien C., Monteils V., Auclair E., Moncoulon R., Bayourthe C. (2008) How does live yeast differ from sodium bicarbonate to stabilize ruminal pH in high-yielding dairy cows? J. Dairy Sci. 91:3528-3535.

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F. Cecchi et al. Large Animal Review 2019; 25: 89-92

Small ruminant lentivirus and Mycobacterium avium subsp. paratuberculosis: co-infection prevalence and preliminary investigation on genetic resistance to both infections in a Garfagnina goat flock

FRANCESCA CECCHI, FILIPPO FRATINI, DOMENICO CERRI, PATRIZIA BANDECCHI, CARLO CANTILE, MAURIZIO MAZZEI Department of Veterinary Science, University of Pisa, Viale delle Piagge 2, 56124 Pisa, Italy

SUMMARY Small ruminant lentiviruses (SRLVs) are a heterogeneous group of viruses of sheep, goats and wild ruminants, causing persistent infection and responsible of chronic degenerative disease of joints, lungs, udder and central nervous system in small ruminants. Mycobacterium avium subsp. paratuberculosis (MAP) is also a major production-limiting disease, which causes a chronic infection of ruminants. The disease causes persistent diarrhea, progressive weight loss, debilitation, anemia, and eventually death. Both SRLVs and MAP are widespread in small ruminants in many countries. The aim of this study was to estimate the prevalence of SRLVs and MAP co-infection in a Garfagnina goat flock consisting of 269 females and 20 males and located in Garfagnana district (Tuscany, Italy). All adult females were tested for MAP and SRLVs infection. Thirty-six out of 269 animals in total resulted positive for one or both infections. A total of 27 goats (10%) were positive to MAP and 21 goats (7.8%) were positive to SRLV. The apparent prevalence of co-infection was 4.5%, counting 12 goats positive for both infections. No significant association was found between subjects seropositive to SRLV and MAP. To investigate possible genetic influences on susceptibility or resistance of goats for both disease, all co-infected animals were compared with no infected animals (control group, 12 goats). Blood samples were collected and 12 STR markers (MAF65, SRCRSP5, INRA023, MCM527, CSRD247, SRCRSP23 OarFCB20, TGLA53, INRA005, INRA063, ETH10, ILSTS87) were investigated. For each marker, allele and genotypes frequencies between the two groups of animals were compared using the chi-square test and Fisher’s exact tests. No statistical differences in STR alleles or genotypes frequency were observed between healthy animals and co-infected animals. Future works may include replication of this study with a larger number of animals to try to identify candidates genes for the genetic resistance to both infections.

KEY WORDS Garfagnina goats, Paratuberculosis, small ruminant lentivirus, co-infection, STR, genetic resistance.

INTRODUCTION Maedi/Visna Virus (MVV) and Caprine Arthritis Encephalitis Virus (CAEV) are members of the Retrovidae family, genus Lentivirus. Both viruses are unequivocal closely related lentiviruses and thus designated as Small Ruminant Lentiviruses (SRLV)1. The majority of SRLV isolates can be classified in 3 main genotypes: A and B including isolates originally isolate from sheep and goats, respectively; and genotype E firstly identified in certain Italian caprine isolates2. SRLVs in small ruminants are responsible of persistent infection causing for a slow and progressive clinical disease involving lungs, mammary glands, joints and in young kids’ central nervous system. Mycobacterium avium subsp. paratuberculosis (MAP) is the bacterial etiologic agent which causes a chronic progressive granulomatous enteritis known as Paratuberculosis or Johne’s disease and it primarily affects wild and domestic ruminants3. Nowadays, Paratuberculosis represents not only an important animal

Corresponding Author: Francesca Cecchi (francesca.cecchi@unipi.it).

disease but also a questioned human health problem, since MAP has been isolated from intestinal tissues of Crohn’s disease patients, giving rise to a hypothesis of there being a possible link between these two diseases4. In addition, several reports of MAP isolation from sheep and goat cheeses both in Europe5,6 and also in our country7 have been published. Both SRLV and MAP are widespread in small ruminants in many countries8,9,10,11,12. Presently, for both disease, there are no effective vaccines neither treatments so an alternative strategy to reduce their incidence could be the selection of the most genetic resistant animals. Several attempts to locate loci associated with resistance to MAP and to SRLV have been made during the last 15 years13,14,15,16. In particular, on Garfagnina goats, association studies between STR markers and susceptibility/resistance to both SRLV and MAP infections have been made17,18. Moreover, attempt to identify genomic regions associated to the infection using a genome-wide scan19,20 have been carried out. The aims of this study were to estimate the prevalence of SRLV and MAP co-infection in a Garfagnina goat flock located in Garfagnana district (Tuscany, Italy), and to investigate the association between both infection-resistant or both infection-susceptible goats and STR markers.

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Small ruminant lentivirus and Mycobacterium avium subsp. paratuberculosis: co-infection prevalence and preliminary investigation

MATERIALS AND METHODS The study was performed in a Garfagnina goat breed flock. Garfagnina is an Italian native goat population registered on the Tuscan regional repertory of genetic resources at risk of extinction, with about 745 animals belonging to 17 flocks. The origin of this population is still uncertain, even if it seems to derive from crossings between native goats from Alpine Arc and from the Tuscan-Emilian Apennines; local breeders refer that the population was reared for generations for its milk and meat production. The flock under study consisted of 269 females and 20 males. Age ranged from 2 to 9 years. All animals were recorded in the herdbook, but genealogical information was not available. The flock was located in the Garfagnana district (Media Valle del Serchio, Lucca, Italy) and a semi-extensive farming was practiced. The goats grazed during the morning (feed supplements are given mainly over the winter), and were housed overnight, when they received an integration of forage and feed. Flock management was of a family farm type. Milking was practiced twice a day using a trolley milking and the milk was conveyed in refrigerated tanks. From 269 female goats, 5 mL of blood were collected into tubes (Vacutainer, BD Biosciences) without anticoagulant; the samples were transported, under refrigeration conditions, to the laboratories (Department of Veterinary Sciences, University of Pisa) and sera were separated from the clot by centrifugation at 2000 g for 10 min and frozen at −20 °C until performing serological analyses. Serum samples were analyzed by ELISA ID screen® Paratuberculosis Indirect screening test (ID.VET, Montpellier, France) and the positive samples were subsequently tested with ELISA ID screen® Paratuberculosis Indirect confirmation test (ID.VET, Montpellier, France). To evaluate the SRLV seroprevalence and characterize the SRLV antibody response sera samples were analyzed by a genotype specific ELISA test (Eradikit™ SRLV Genotyping kit, In3diagnostic, Torino, Italy). The genotyping ELISA plates were coated with a mix of gag and env peptides belonging to the three most divergent SRLV viral genotypes: genotype A, B, and E. The serum samples were examined according to the manufacturer’s instructions. Specific genotype was assigned when the OD value of a single well was >40% when compared to the OD value of the same sera in other wells coated with different antigens. Sera were considered as SRLV positive when positive on at least one genotype. Blood were collected from 48 goats (including all MAP and SRLV positive goats and twelve healthy goats as control groups). Blood samples were collected in Vacutainer tubes with K-EDTA as anticoagulant and stored at −20 °C until genomic DNA was extracted using Qiagen QIAamp DNA blood mini/midi kit (Qiagen, San Diego, CA, USA). Twelve microsatellites were considered: MAF65, SRCRSP5, INRA023, MCM527, CSRD247, SRCRSP23 OarFCB20, TGLA53, INRA005, INRA063, ETH10, ILSTS87; some of these markers belonged to a panel validated by the International Society of Animal Genetics (ISAG) and others routinely were used by the facilities of the Laboratorio di Genetica e Servizi (Associazione Italiana Allevatori, Migliaro, Italy). The markers were located in 12 chromosomes and amplified in one multiplex PCR reactions21. The association for each single animal between MAP and

SRLV serological results were verified by Chi-square test22. For each marker, the following parameters were computed using the Molkin v2.0 program23: number of alleles, effective allele size, observed heterozygosity and polymorphism information content (PIC). Allelic and genotype frequencies were estimated by direct counting. To investigate possible genetic influences on susceptibility or resistance of goats to both infections, for each marker, alleles and genotypes frequencies between co-infected animals and negative animals were compared using the Chi-square test and Fisher’s exact tests.

RESULTS Serological analysis revealed that 36 out of 269 animals were positive for one or both infections. Twenty-seven goats (10%) were positive to MAP and twenty-one goats (7.8%) were positive to SRLV. Genotype analysis for SRLV could classify 15 out of 21 seropositive sera into the 3 genotypes, in details four sera reacted specifically to genotype A antigens, five to genotype B and six to genotype E. Genotype characterization was not determined in six sera, although reactive to SRLV (Table 1). Table 2 reports the number of positive animals for MAP and SRLV or both infections according to the animals’ age. The greatest number of infected animals were aged between 5 and 7 years. The highest number of co-infected animals were aged between 5 and 6 years (50%). Analysis of the goat MAP/SRLV data revealed no significant associations between the two infections (Table 3). Detailed information of the used markers and the results of Table 1 - Serological results. Goat

OD vs A

OD vs B

OD vs E

Genotype

POS1

0.835

0.387

0.463

POS2

0.414

0.182

0.237

POS3

1.078

0.352

0.615

POS4

0.554

0.277

0.184

POS5

0.19

0.422

0.187

POS6

1.043

2.656

0.63

POS7

0.446

2.134

0.204

POS8

0.378

1.855

0.26

POS9

0.337

1.254

0.279

POS10

0.2

0.162

1.923

POS11

0.142

0.169

1.774

POS12

0.44

0.456

1.457

POS13

0.523

0.352

0.956

POS14

0.144

0.321

0.456

POS15

0.144

0.18

1.532

POS16

0.663

0.595

0.238

Ind

POS17

0.333

0.613

0.485

Ind

POS18

0.126

0.476

0.411

Ind

POS19

0.375

0.371

0.434

Ind

POS20

0.497

0.349

0.504

Ind

POS21

0.456

0.177

0.574

Ind

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F. Cecchi et al. Large Animal Review 2019; 25: 89-92

the microsatellite analysis in term of number of alleles observed, alleles size, PIC and observed heterozygosity of the total analyzed Garfagnina goat population were reported in a previous paper21. No statistical differences in STR alleles or genotypes frequency were observed between healthy animals and co-infected animals. Since in previous papers SRCRSP0518 and ETH10 markers17,18 showed significant association with the infections in this paper we report the percentage of each of the most frequent alleles (>15%) for both SRCRSP05 and ETH10 markers in co-infected group (SRLV and MAP seropositive) and control group (SRLV and MAP Seronegative) (Table 4). The co-infected group showed a higher frequency of allele 205bp of ETH10 marker (0.583 vs 0.458 in control group) and of allele 203 bp (0.250 vs. 0.125 in control group) and a less frequency of allele 207bp (0.167 vs 0.0.417 in control group). Consequently, the frequencies of the six genotypes of this marker were different in the two groups. Co-infected group presented a higher frequency of homozygous genotype 205205 of ETH10 marker (41.7% vs 16.7%) and a low frequency of heterozygous 205207 and heterozygous 207207 genotypes (8.30% vs 33.30% for the first genotype and 8.30% vs 25% for the second one).

Table 2 - Positive records according to the animalsâ&#x20AC;&#x2122; age. Age (years)

PosMAP

PosLentivirus

PosBoth

Tot

8-9

Tot

Table 3 - Representation of SRLV and MAP infection in goats. SRLV positive

SRLV negative

Total

MAP negative

233

242

MAP positive

Total

248

269

Regarding SRCRSP05 marker six different alleles were observed but only three alleles had a frequency higher than 15%. Co-infected animals had a lower frequency of 163bp (0.25 vs. 0.375) allele and 173bp (0.25 vs. 0.375) allele and a higher frequency of allele 179 (0.25 vs. 0.167). For this reasons differences were observed for 163173 genotype with a frequency of 41.7% in control group vs 16.7% in coinfected group.

DISCUSSION To our knowledge only a single research carried out by Stones et al.24 discussed SRLV and MAP co-infection considering 28 Ontario goats flock (about 20 goats per flock) recording a prevalence of 14.3%. Our co-infection prevalence of 4.5% was lower than that reported by these Authors. Moreover, the lack of association between the two infections highlighted in this work seems to be in contrast with the results obtained by Stones et al.24. Nevertheless, these Authors observed a significant association only when considering the total data deriving from 558 goats irrespectively to flock origin, while the associations carried out in each single flock was not significant, given the small number of subjects. Finally, despite the possible implication of the ETH10 marker in the resistance to the two infections, previously highlighted by the studies of association analysis between microsatellites and each infections17,18, it is possible to suppose that there is no correlation between genetic resistance to the two infections, in consideration of the evidence that the candidate genes for genetic resistance to MAP19 and to SRLV20 carried out with SNPs, were different and located on different chromosomes.

CONCLUSIONS The observed results were obtained from the analysis of animals from a single farm, indicating that few animals are coinfected and that there is not association between these two infections. For this reason, our results must be supposed completely preliminary. Future works may include replication of this study involving different flocks to increase the number of observations to better understand the role of the ETH10 marker and to try to reveal and identify candidates genes implicated in genetic resistance to both infections.

Table 4 - Percentage of each alleles for SRCRSP05 and ETH10 markers in Sero-Positive (SP) and Sero-Negative (SN) group. Group

ACKNOWLEDGEMENTS

NÂ°

Marker

SRCRSP05 163

173

179

0.375

0.167

0.250

203

205

207

Marker

ETH10

0.125

0.458

0.417

0.250

0.583

0.167

This work was supported by grants of the University of Pisa (PRA 2016 and Fondi Ateneo).

References 1. Zanoni R.G. (1998). Phylogenetic analysis of small ruminant lentiviruses. J Gen Virol, 79: 1951-1961. 2. Grego E., Bertolotti L., Quasso A., Profiti M., Lacerenza D., Muz D., Rosati S. (2007). Genetic characterization of small ruminant lentivirus in Italian mixed flocks: evidence for a novel genotype circulating in a local goat population. J Gen Virol, 88: 3423-3427.

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Small ruminant lentivirus and Mycobacterium avium subsp. paratuberculosis: co-infection prevalence and preliminary investigation

3. OIE (2014). Paratuberculosis (Johne’s disease). OIE Terrestrial Manual; Chapter 2.1.11. 4. Chiodini R.J., Chamberlin W.M., Sarosiek J., McCallum R.W. (2012) Crohn’s disease and the mycobacterioses: a quarter century later. Causation or simple association? Crit Rev Microbiol, 38: 52-93. 5. Ikonomopoulos J., Pavlik I., Bartos M., Svastova P., Ayele W.Y., Roubal P., Lukas J., Cook N., Gazouli M. (2005). Detection of Mycobacterium avium subsp. paratuberculosis in retail cheeses from Greece and the Czech Republic. Appl Environ Microbiol, 71: 8934-8936. 6. Williams A.G., Withers S.E. (2010). Microbiological characterisation of artisanal farmhouse cheeses manufactured in Scotland. Int J Dairy Technol, 63: 356-369. 7. Galiero A., Fratini F., Mataragka A., Turchi B., Nuvoloni R., Ikonomopoulos J., Cerri D. (2016) Detection of Mycobacterium avium subsp. paratuberculosis in cheeses from small ruminants in Tuscany. Int J Food Microbiol, 217: 195-199. 8. Windsor P.A. (2015). Paratuberculosis in sheep and goats. Vet Microbiol, http://dx.doi.org/10.1016/j.vetmic.2015.07.019. 9. Angelidou E., Kostoulas P., Leontides L. (2014). Flock-level factors associated with the risk of Mycobacterium avium subsp. paratuberculosis (MAP) infection in Greek dairy goat flocks. Prev Vet Med, 117: 233-241. 10. Liapi M., Botsaris G., Slana I., Moravkova M., Babak V., Avraam M., Di Provvido A., Georgiadou S., Pavlik I. (2015). Mycobacterium avium subsp. paratuberculosis Sheep Strains Isolated from Cyprus Sheep and Goats. Transbound Emerg Dis 62: 223-227. 11. Galiero A., Turchi B., Pedonese F., Nuvoloni R., Cantile C., Colombani G., Forzan M., Cerri D., Fratini F. (2017). Serological, culture and molecular survey of Mycobacterium avium paratuberculosis in a goat flock in Tuscany. Folia Microbiol, 62: 6, 471-477. 12. Minguijón E., Reina R., Pérez M., Polledo L., Villoria M., Ramírez H., Leginagoikoa I., Badiola J.J., García-Marín J.F., de Andrés D., Luján L., Amorena B., Juste RA. (2015). Small ruminant lentivirus infections and diseases. Vet Microbiol, 181:75-89. 13. Zare Y., Shook G.E., Collins M.T., Kirkpatrick B.W. (2014). Genomewide association analysis and genomic prediction of Mycobacterium avium subspecies paratuberculosis infection in US Jersey cattle. PLoS ONE, 9, e88380. 14. Moioli B., D’Andrea S., De Grossi L.C., Sezzi E., De Sanctis B., Catillo

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G., Steri R., Valentini A., Pilla F. (2016). Genomic scan for identifying candidate genes for paratuberculosis resistance in sheep. Animl Prod Sci, 56: 1046-1055. Clawson M.L., Redden R., Schuller G., Heaton M.P., Workman A., Chitko-McKown C.G., Smith T.P., Leymaster K.A. (2015). Genetic subgroup of small ruminant lentiviruses that infects sheep homozygous for TMEM154 frameshift deletion mutation A4 53. Vet Res, 46: 22. Wang W., Ye C., Di Zhang J.L., Kimata J.T., Zhou P. (2014). CCR5 Gene Disruption via Lentiviral Vectors Expressing Cas9 and Single Guided RNA Renders Cells Resistant to HIV-1 Infection. PLOS Published: December 26, https://doi.org/10.1371/journal.pone.0115987. Cecchi F., Russo C., Fratini F., Turchi B., Preziuso G., Cantile C.(2018). Preliminary association analysis of microsatellites and Mycobacterium avium subspecies paratuberculosis infection in the native Garfagnina goats. J Applied Anim Res, 46: 879-882. Cecchi F., Fratini F., Bandecchi P., Cantile C., Mazzei M. (2018). Investigation on goat lentiviral infections and preliminary association analysis with microsatellites in the native Garfagnina goats breed. Rend Lincei Sci Fis Nat, 29: 885-889. Cecchi F., Russo C., Iamartino D., Galiero A., Turchi B., Fratini F., Degl’Innocenti S., Mazza R., Biffani S., Preziuso G., Cantile C.(2017). Identification of candidate genes for paratuberculosis resistance in the native Italian Garfagnina goat breed. Trop anim health and prod, 49: 1135-1142. Cecchi F., Dadousis C., Bozzi R., Fratini F., Russo C., Bandecchi P., Cantile C., Mazzei M. (2019). Genome scan for the possibility of identifying candidate resistance genes for goat lentiviral infections in the Italian Garfagnina goat breed. Trop Anim Health and Prod, 51: 729-733. Cecchi F., Russo C., Cantile C., Preziuso G. (2017). Preliminary genetic variability analysis of the native Garfagnina goats based on microsatellite polymorphism. Large Animal Review 23: 191-193. SAS, JMP.User’s Guide, (2007). ver. 7.0 SAS Inst. Cary, NC, U.S.A. Gutièrrez J.P., Royo L.J., Alvarez I., Goyache F.(2005). MolKin v2.0:a computer program for genetic analysis of populations using molecular coancestry information. J Hered, 96: 718-721. Stonos N., Bauman C., Menzies P., Wooton S.K., Karrow N.A. (2017). Prevalence of small ruminant Lentivirus and Mycobacterium avium subsp. Paratuberculosis co-infection in Ontario dairy sheep and dairy goats. Can J Vet Res, 81: 155-159.

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V. Meucci et al. Large Animal Review 2019; 25: 95-99

Natural occurrence of ochratoxin A in confined reared and grazing pigs derived products

VALENTINA MEUCCIa, ALESSANDRO PISTOIAb, SIMONE BERTINIc, ALESSANDRO MENOZZIc, LUIGI INTORREa a

Department of Veterinary Science, University of Pisa, San Piero a Grado, Pisa, Italy Department of Agriculture, Food and Environment, University of Pisa, Pisa, Italy c Department of Veterinary Science University of Parma, Parma, Italy b

SUMMARY Ochratoxin A (OTA) is a well-known nephrotoxic and immunotoxic mycotoxin occurring in several foods and feeds. OTA residues have been detected as a carryover in pigs and poultry meats. In this study samples of pigs fresh tissues (muscle, backfat) and processed meat products (seasoned muscle-coppa, seasoned backfat-lard, mortadella and salami) were obtained from pigs raised following two breeding systems: conventional production systems (indoor) and alternative pig rearing systems (free outdoor access). Pigs tissues and meat products samples were analysed for OTA content by means of an HPLC-FLD method. OTA was found in all pigs tissues and meat products at concentration lower than the Italian recommended maximum level of 1 µg/kg, only a sample of mortadella of indoor group showed a concentration very close to this limit. This study suggests that the contribution from pig fresh tissues to the total intake of the toxin is small if compared to other sources. However, the OTA occurrence in some derived products, such as mortadella, seems to be of some concern.

KEY WORDS Ochratoxin A, pig, HPLC, meat, carry-over.

INTRODUCTION Ochratoxin A (OTA) is a mycotoxin produced as secondary metabolite by Aspergillus and Penicillium spp. fungi during storage. OTA is nephrotoxic, immunosuppressive and teratogenic and has been classified by the International Agency for Research on Cancer as possible carcinogen Group 2B to humans1,2. Several studies suggest the involvement of OTA in the etiology of human Balkan Endemic Nephropathy (BEN) and of human chronic interstitial nephropathy in North Africa3,4. BEN has similarities with mycotoxic porcine nephropathy, which has been associated with pigs ingestion of OTA in Denmark in the 1960s and 1970s5. Public health concerns regarding OTA human exposure are justified on the basis of its demonstrated toxic effects, coupled with its ability to be carried through the food chain6. OTA can be detected in cereal products, coffee, wine, beer, cheese, and in food of animal origin. The latter can be contaminated either directly with toxigenic moulds or following the carry-over from naturally contaminated animal feed7-10. OTA concentrations in the range of 0.1-103.69 µg/kg have been detected in foodstuffs of animal origin (pork meat, dry-cured ham, sausage, salami)10-12. OTA-producing fungi can be recovered from the environment, the air of ripening plant rooms13 and the surface of processed meat products14,15. The addition of flavouring materials, such as spices, may also be a supplementary source of mycotoxin contamination16. Non-rumi-

Corresponding Author: Valentina Meucci (valentina.meucci@unipi.it).

nant mammals such as swine tend to accumulate OTA because of a rather long serum half-life of 72-120 hours. Legislation regarding mycotoxins has been created to minimize the risk of food contamination by these compounds which are known to cause losses of quality in food products and adverse effects to human and animal health. Maximum levels of OTA in pigs meat or derived products have been set in some countries such as Denmark (pig kidney 10 µg/kg, pig blood 25 µg/ml), Estonia (pig liver 10 µg/kg), Romania (pig kidney, liver and meat 5 µg/kg) and Slovakia (meat 5 µg/kg, milk 5 µg/kg)10. Other countries such as Italy have developed guidelines for recommended maximum OTA levels (pig meat and derived products 1 µg/kg)17. On the contrary at the European level maximum limits of OTA in meat or other animal products has not been established. According to the EFSA18 more extensive occurrence data on OTA in animal tissues and derived products are required to assess the significance of residue levels. An increasing percentage of the population shows a preference for organically produced agricultural products due to the claimed absence of chemical contaminants within this mode of production. However, it is feared that products from organic agriculture could be more affected by mycotoxins than those from conventional agriculture, due to the lack of fungicide applications19. Very few data are available on the natural occurrence of OTA in products derived from pigs raised in organic conventional production systems (indoor) and alternative pig rearing systems (free outdoor access). Interest in outdoor pig production is growing in several countries because of better animal welfare and meat quality. The aim of the present study was to investigate the presence of OTA in tissues of pigs raised in an organic farm in

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Natural occurrence of ochratoxin A in confined reared and grazing pigs derived products

different conditions (indoor and outdoor) during the fattening period; then, typical Italian seasoned pork products were prepared using the tissues and seasoned in a manufacturing plant. At the end of seasoning, analysis for OTA content was carried out on the seasoned products.

MATERIALS AND METHODS In the present study, the pig production chain was analyzed from the breeding to the slaughterhouse up to the production of seasoned meat.

Animals Ten Large White-Duroc crossbreed castrated male pigs (weight 30 ± 5 kg) were raised in an organic farm (Tuscany, Italy). During the fattening period 5 animals of the group were raised outdoors in a 5000 m2 extension land characterized by chestnut trees, holm-oaks, heather, bramble shrubs (OTD group); the others 5 were raised indoors in a pen (IND group). All the animals were fed with the same diet. Flaked protein pea, flaked corn, flaked wheat, flaked barley was the composition of the same diet of the experimental groups (Table 1) according to requirements of National Research Council20. Animals had free access to water and the feed was distributed twice a day; OTD had the possibility to integrate their ration with woodland feed resources when available (chestnut, grass, roots).

Feed collection A feed aliquot of flaked protein pea, flaked corn, flaked wheat and flaked barley for the analyses was collected at the beginning (T1) and end (T2) of the fattening period.

muscle cut according to local processed technique, all fats were removed. After dry salting samples were washed, dried, trimmed and bagged in plastic wrap be aged in ventilated room for five months. Lard was obtained using about 5 kg of fresh backfat divided in small pieces of about 0.3 kg and placed into marble boxes. A layer of salt, minced garlic and mixture of herbs were added. Boxes were closed with a marble cover to limit light and air exposure during 15 days. Mortadella were obtained from a quantitative pork meat (in natural bowel) triturated and mixed with little backfat cubes. Samples were produced in batches of 50 kg each by mincing deboned shoulders (40%), porcine stomachs (25%), lard cubes from jowl (25%), fat emulsion (10%); mixing with salt, saccarose, caseinate, polyphosphates, sodium nitrite, pepper, garlic and stuffing in artificial casings. Cooking took place immediately in dry ovens at 85°C room temperature up to 72°C core temperature and was followed by cooling under tap water sprays and refrigeration in cold rooms at 4°C. Salami were produced by mincing and mixing deboned shoulders (30%), ham trimmings (40%), backfat (15%) and belly (15%). During mixing the following additives were added: salt, dried skimmed milk, saccarose, potassium nitrate, sodium nitrite. The mixes were stuffed in reconstructed casings and seasoned at 11-12°C and 75-85% RH up to 3 months from manufacture. The samples were obtained from the core of each product; all samples were vacuum-packaged and stored at -20°C until the analyses.

High-performance liquid chromatography method

OTA (from Aspergillus ochraceus) (M 403.8) reference standard was purchased from Sigma (Milan, Italy). The OTA standard was dissolved in a toluene-acetic acid mixture (99:1%, v/v) to give a stock solution of 200 µg/ml which was Tissues collection stored at -20°C until use. Working solutions were prepared When the pigs reached 140 kg of live weight were transferred by diluting the stock solution with the mobile phase consistto local slaughterhouse. During the slaughter samples of ing of methanol-sodium phosphate buffer (pH 7.5) 50:50% fresh Longissimus lumborum muscle and fresh backfat were v/v. HPLC-grade water, methanol and acetonitrile were purcollected from individuals of each group (OTD and IND). chased from VWR (Milan, Italy). The immunoaffinity columns (IAC) OchraStar® were purchased from TecnaSrl Meat products collection (Trieste, Italy). The chromatographic system consisted of In a meat processing factory, meat derived products (seaJasco 880 pump and Jasco 821 fluorescence detector (Jasco, soned muscle (coppa), seasoned backfat (lard), mortadella Tokyo, Japan). JascoBorwin software was used for data proand salami), obtained from the same pigs sampled at the cessing. The excitation wavelength (λex) and emission waveslaughterhouse, were collected. Coppa was obtained from length (λem) were set at 380 and 420 nm. The reversedphase column was a Luna C18 ODS2, 3 µm (4.6 x 150 mm) (Phenomenex® Torrance, CA, USA). Table 1 - Ingredients and chemical composition of the diet. The column was kept at room temperature. The HPLC was operated with mobile phase system 30-50 kg 50-90 kg > 90 kg consisting of methanol-phosphate buffer soluFlaked protein pea g/kg 450 350 300 tion pH 7.5 (0.03 M Na2HPO4, 0.007M Flaked corn g/kg 200 300 350 NaH2PO4) 50/50% v/v at flow rate of 1 ml/min. Flaked barley g/kg 150 150 200 The HPLC method was validated according to international rules21: selectivity, linearity, limits of Flaked wheat g/kg 200 200 150 determination (LOD) and quantification (LOQ), Dry matter g/kg 867 868 869 repeatability and reproducibility were deterCrude protein g/kg DM 176 159 151 mined. Calibration curves were based on the Ether extract g/kg DM 20 21 22 analysis of triplicate standards solution at 7 concentration levels in matrix. Meat samples spiked NDF g/kg DM 166 165 165 with OTA at 0.02, 0.05, 0.1, 0.25, 0.5, 1 and 2 Ash g/kg DM 25 22 20 µg/kg were analysed using extraction and HPLC Digestible energy MJ/kg DM 16.47 16.54 16.58 method. Feed samples spiked with OTA at 0.2,

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V. Meucci et al. Large Animal Review 2019; 25: 95-99

0.5, 1.0, 2.5, 5, 10 and 20 µg/kg were analysed using extraction and HPLC method The experiment was repeated 5 times. Taking into account dilution and concentration steps, meat spiked samples corresponded to OTA standard concentrations of 0.2, 0.5, 1, 2.5, 5, 10 and 20 µg/l and feed spiked samples corresponded to OTA standard concentrations of 0.8, 2.0, 4.0, 10, 20, 40 and 80 µg/l. The repeatability was tested by analyzing samples of meat and feed spiked with OTA. Meat samples were spiked at the levels of 0.05 µg/kg (corresponding to 0.5 µg/l), 0.25 µg/kg (corresponding to 2.5 µg/l), and 2 µg/kg (corresponding to 20 µg/l). Feed samples were spiked at the levels of 0.5 µg/kg (corresponding to 2.0 µg/l), 2.5 µg/kg (corresponding to 10 µg/l), and 20 µg/kg (corresponding to 80 µg/l). All samples were measured in triplicates on the same day. For the within-laboratory reproducibility test, each of the contamination level was tested in triplicates in seven days. The results of these experiments were used also for the determination of the recovery. Selectivity studies have been expressed as the ability to asses unequivocally OTA in the presence of components which may be expected to be present: it has been evaluated by the comparison of free-OTA vs spiked samples.

Samples preparation Meat/Backfat/Meat products

acetic acid 98/2 v/v. The elute was evaporated to dryness under nitrogen stream. The residue was redissolved in 250 µl of HPLC mobile phase and injected into HPLC system.

Spiked samples Samples spiked before extraction were used to check the performance of the extraction and clean-up procedure and to obtain validation parameters. Spiking solutions of OTA were prepared daily by dilution with HPLC mobile phase. For samples of pigs meat, after thoroughly mixing for 30 min, the OTA fortified homogenate was left for at least 2 hours at room temperature to enable equilibration and used to assay the cleaning procedures prior to HPLC analysis. For samples of feed, spiked samples were prepared by adding appropriate volume of the spiking solution of OTA to the feed suspension (described above) and then processed as for meat samples.

Statistical analysis Statistical analysis was performed with GraphPad Prism (v. 6) software (La Jolla, CA, USA). All data were tested for normality by means of the Kolmogorov-Smirnov test. The data are reported as mean ± standard deviation (SD). The influence of the different rearing system and on OTA concentrations was detected by t-test. The difference in OTA concentrations between fresh and seasoned meat products was detected by ttest. A value of p < 0.05 was considered significant.

A 5 g sample aliquot was homogenized with 5 ml of phosphoric acid 1 M using an Ultra Turrax T25 homogenizer for a few minutes. A 2.5 g aliquot of the homogenate was transferred into a centrifuge tube, extracted with 5 ml of ethylacRESULTS AND DISCUSSIONS etate, vortexed for 1 min, shaked for 10 min on horizontal High-performance shaker and then centrifuged for 10 min at 3000 rpm. The orliquid chromatography method ganic phase was removed, the residue re-extracted, as above, and the organic phases combined. The volume of the orValidation parameters of HPLC method are reported in ganic phase was reduced to approximately 5 ml and backTable 2. The mean OTA recoveries in spiked samples ranged extracted with 5 ml of NaHCO3 pH 8.4, vortexed for 1 min from 79.9 to 89.4% for feeds and from 89.9 to 95.0% for and centrifuged for 10 min at 3000 rpm. The aqueous exmeat. The calculated LOD were 0.0125 and 0.1 µg/kg for tract was acidified to pH 2.5 with H3PO485% and briefly meat and feeds, respectively. A calibration curve peak area sonicated to strip the CO2 formed. OTA was finally back-exversus concentration was obtained using the linear squares tracted into 5 ml ethylacetate, vortexed for 1 min and cenregression procedure. The OTA linearity in the spiked samtrifuged for 10 min at 3000 rpm; the organic phase was evaporated to dryTable 2 - Validation parameters for HPLC methods of OTA in meat and feed samples. ness under nitrogen stream, reconstiParameters Meat Feed tuted in 250 µl of mobile phase and a 100 µl aliquot injected. LOD µg/kg 0.0125 0.1000 Feed

Ten grams of feed samples were mixed with 40 ml of extraction solution (water/methanol 50/50 v/v). Extraction was done by shaking for 10 min on horizontal shaker and centrifuging at 3000 rpm for 10 min. A 4 ml aliquot of the extract was diluted with 46 ml of phosphate-buffered saline (PBS, pH 7.4). The diluted sample was filtered through Whatman filter paper (Millipore Corporation, Maid Stone, UK). This solution (50 ml) was passed through IAC at flow-rate of 1-2 drops s-1. The column was washed with 20 ml of PBS (1-2 drops s-1). Elution was performed with 3 ml of methanol and

LOQ

µg/kg

0.0250

0.2500

0.9910

0.9920

CV % (intra-day)

0.05 µg/kg

n = 3 replicates

5.8

0.50 µg/kg

4.9

0.25 µg/kg

n = 3 replicates

8.9

2.50 µg/kg

9.5

2.00 µg/kg

n = 3 replicates

6.0

10.00 µg/kg

7.8

0.05 µg/kg

n = 21 replicates

9.7

0.50 µg/kg

8.9

0.25 µg/kg

n = 21 replicates

7.6

2.50 µg/kg

8.0

2.00 µg/kg

n = 21 replicates

5.8

10.00 µg/kg

7.0

0.05 µg/kg

n = 21 replicates

89.9 ± 1.8

0.50 µg/kg

79.9 ± 5.0

0.25 µg/kg

n = 21 replicates

91.2 ± 3.5

2.50 µg/kg

89.4 ± 0.8

2.00 µg/kg

n = 21 replicates

95.0 ± 1.2

10.00 µg/kg

85.9 ± 6.8

CV % (inter-day)

Recovery %

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Natural occurrence of ochratoxin A in confined reared and grazing pigs derived products

Table 3 - OTA concentrations (µg/kg) in analyzed feed samples.

Among these, OTA was reported as a natural contaminant in pepper and T1 T2 Significance nutmeg24. Spices, as black pepper, may Flaked corn µg/kg 2.03 ± 0.07 2.36 ± 0.42 NS be able to raise OTA levels in pork deFlaked protein pea µg/kg 2.50 ± 0.50 2.60 ± 0.26 NS rived products. The OTA concentration in mortadella samples could also Flaked wheat µg/kg 1.56 ± 0.42 2.12 ± 0.33 NS be the use of organs for its preparaFlaked barley µg/kg 7.17 ± 0.36 7.27 ± 0.68 NS tion. In fact, several studies in which NS: p > 0.05; T1 beginning the fattening period; T2 end of the fattening period. tissues and organs from pigs exposed to OTA contaminated feed were analysed, have shown that some tissues Table 4 - OTA concentrations (µg/kg) in analyzed pigs tissues and derived products. (such as lung and heart) had higher levels of OTA compared to other maIND OTD Significance trices such as muscle and fat, that are a a Muscle (fresh) µg/kg 0.055 ± 0.015 0.078 ± 0.011 * the main ingredients used in the Muscle (seasoned) µg/kg 0.088 ± 0.048b 0.178 ± 0.031b ** preparation of products as salami25-28. a a Backfat (fresh) µg/kg 0.079 ± 0.018 0.085 ± 0.025 NS This is the first study reporting OTA detection in mortadella samples, more Backfat (seasoned) µg/kg 0.170 ± 0.064b 0.204 ± 0.069b NS samples are needed to confirm this hySalami µg/kg 0.058 ± 0.015 0.064 ± 0.004 NS pothesis. Statistically significant differMortadella µg/kg 0.537 ± 0.042 0.558 ± 0.016 NS ences were observed for OTA content NS: p > 0.05; *: p < 0.05; **: p < 0.01. Means followed by different letters differ significantly at p < 0.05. in muscle samples of both OTD and IND groups between fresh and seasoned products (Table 4). Statistically ples at 5 determinations of 7 concentrations levels was excelsignificant differences were observed for OTA content in lent, as shown the correlation coefficient >0.99. The method backfat samples of OTD and IND groups between fresh and has shown to be suitable for accurate quantitative determiseasoned products (Table 4). These results could be related to nation of OTA in different tissues of pigs and feed samples. a concentration effect due to the loss of water during the seasoning of meat products.

Feed samples Concentrations of OTA in conventional diet are reported in Table 3. The OTA concentrations found at T1 were not statistically different from those found at T2. None of the feed samples analysed in the present study was contaminated with OTA at a level exceeding the European Commission recommended limit of 50 µg/kg in swine feed22.

Meat samples OTA concentrations in tissues and meat products samples are showed in Table 4. OTA was found in all pig’s tissues and meat products samples at concentrations lower than the Italian recommended maximum level of 1 µg/kg. Although, OTA content in the diet was much lower than 50 µg/kg, it was enough to determine after lengthy exposure, a final detectable amount in the muscle and backfat. This agrees with previous studies in which a diet naturally contaminated at levels of 0.3 µg/kg for 119 days resulted in final OTA concentrations in muscle of 0.88 µg/kg23. OTA concentrations in fresh and seasoned muscle (coppa) of OTD group were significantly higher than those of IND group. OTA concentrations in fresh and seasoned backfat (lard) of OTD group were not significantly different from those of IND group. OTA concentrations in salami and mortadella of OTD group were not significantly different from those of IND group. The highest OTA concentration was found in one mortadella sample of IND group showing a concentration of 0.889 µg/kg very close to the Italian recommended maximum level of 1 µg/kg. This can be related to the type of production of mortadella. Pepper was added only to mortadella samples and not in salami production. The most utilized spices in pork derived products are garlic, anise, cinnamon, cloves, coriander, fennel, nutmeg and pepper.

CONCLUSIONS The present study indicate that OTA exposure is a common phenomenon for which limitation is essential to increase controls on foodstuffs taking care of every stage of the production chain, given the growing concern for consumer health. The biggest problem in managing mycotoxin risk is represented by the difficulty of precisely identifying the phases in which contamination can occur, and the great variety of foods on which this can occur. Only a global vision of the problem involving all types of skills, such as agronomic, biological, pathophysiological, chemical and many others could lead to better assessment and management of mycotoxin risk and limit the presence of OTA in food.

ACKNOWLEDGEMENTS This paper was not supported by grants.

CONFLICT OF INTEREST The authors declare no conflict of interest.

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L. Cossignani et al. Large Animal Review 2019; 25: 101-104

Fermentation as a strategy to increase conjugated linoleic acid in dairy products

101

쓼

LINA COSSIGNANI, FRANCESCA BLASI Department of Pharmaceutical Sciences, Section of Food Science and Nutrition University of Perugia, Via San Costanzo, 06126 Perugia, Italy

SUMMARY Conjugated linoleic acids (CLA) are a group of non-methylene interrupted octadecadienoic acids, naturally present in different foods, among which foods derived from ruminants, as milk and dairy products. The rumenic acid (9cis, 11trans-CLA) is the predominant isomer in milk fat, followed by 10trans, 12cis-CLA and other isomers in traces. In order to characterize the profile of CLA isomers found in dairy products, the silver-ion high performance liquid chromatography (Ag+-HPLC) equipped with multi-columns provides satisfactory resolution. To evaluate the fatty acid (FA) composition of dairy products, the high resolution gas chromatography (HRGC) analysis of derivatized FA, as fatty acid methyl esters, is generally used. Actually, there is always more interest of researchers in increasing the content of CLA in foods, including milk and dairy products. In fact, a lot of studies, carried out on animals and humans, have suggested that CLA may have important human health benefits, among which antiatherogenic, antiadipogenic, and antidiabetic properties. Among the strategies to increase CLA content during the manufacturing of dairy products, fermentation is one of these. CLA content in fermented dairy products is extremely variable (generally from 3.4 to 8.8 mg/g fat) and strongly linked to the strain type, to the aerobic or anaerobic conditions and to other parameters, as pH, time and temperature of growth culture. Strains of Bifidobacterium, Enterococcus, Lactobacillus, Lactococcus, Propionibacterium and Streptococcus can be considered potential CLA-producers. The manipulation of the feed and dietary regimen of the animals is an alternative strategy to increase CLA content. Linseed, soybean, olive, canola, sunflower and fish oils, added to feed, can improve the unsaturated FA contents, including CLA, in milk and dairy products. Alternatively, interest in the enzymatic modification of FA composition of dairy products in order to produce healthier fats, is increasing. In this review, an overview of the main strategies to increase CLA content in dairy products, with particular focusing on fermentation, is reported.

KEY WORDS CLA, nutraceutical, dairy products, milk quality, fermentation.

CHEMISTRY OF CLA Conjugated linoleic acids (CLA) are a mixture of positional (from 7,9- to 11,13-) and geometric (cis,cis; cis,trans; trans,cis; or trans,trans) isomers of linoleic acid (9cis, 12cis-18:2); differently from linoleic acid, the double bonds in CLA are adjacent, without intermediate methylene groups. Among this type of fatty acids (FA), the 9cis, 11trans isomer was identified in ruminant milk fat for the first time by Parodi (1977), and then named “rumenic acid”. Later, it was confirmed that 9cis, 11trans-CLA is the predominant isomer in milk fat (75-90% of the total CLA), followed by 10trans, 12cis-CLA; trace amounts of other isomers (9trans, 11trans; 7trans, 9cis) have been also found1. Figure 1 shows the two main CLA isomers detected in dairy products.

CLA CONTENT OF FERMENTED DAIRY PRODUCTS Linoleic acid is the key precursor of rumenic acid, which can be synthesized by biohydrogenation of linoleic acid, car-

Corresponding Author: Francesca Blasi (francesca.blasi@unipg.it).

Figure 1 - The predominant isomers in milk fat: 9cis, 11trans-CLA and 10trans, 12cis-CLA.

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102

Fermentation as a strategy to increase conjugated linoleic acid in dairy products

Table 1 - Content (mg/g of fat) of CLA and starter cultures of fermented milks. FERMENTED MILK

CLA

Microorganism Lb. lactis

1.9a-9.2a

Lb. buchneri

4.3a

Lb. reuteri

4.8a

Lb. helveticus

5.4a

Lb. brevis

1.7b-10.5b

Lb. viridescens

1.8b-5.7b

Lb. casei

1.13c-6.3a

Lb. plantarum

0.51c-4.9a

B. animalis ssp. lactis

15.5d-18.5d

B. bifidum

0.46c-4.2e

B. breve

2.3e-4.4e

B. pseudolongum ssp. pseudolongum

2.7e-4.2e

a Pandit et al., 2012; b Puniya et al., 2008; c Kuhl et al., 2016; d Florence et al., 2009; e Gorissen et al., 2012.

ried out by ruminal and lactic acid bacteria. The concentration of CLA in cow milk normally ranges between 2.0 and 6.4 mg/g of fat, and is mainly affected by the lactation stage, rumen microflora, and dietary regime2. Milk and dairy products are the main source of CLA in the human diet, providing about 70% of the total CLA daily intake, which varies between 70 and 430 mg/day3. Table 1 and 2 show the content, expressed as mg/g of fat, of CLA (total isomers) in fermented milks and yogurts. It can be noted that the CLA content in fermented dairy products is extremely variable (in fermented milk from 1.9 to 18.5 mg/g of fat; in yogurt from 2.1 to 20.8 mg/g of fat) and strongly linked to the strain type and to the conditions of growth culture. In fermented dairy products, the concentration of CLA varies, more commonly, between 3.4 and 8.8 mg/g fat.

STRATEGIES TO INCREASE CLA CONTENT IN DAIRY PRODUCTS Strategies to increase CLA content are based on the modification of the technological processes used for the manufacturing of dairy products4. Fermentation is one of the main

approaches employed to increase CLA content in a natural manner; it is strain-dependent, because of the different linoleate isomerase activity of the species. Recently, the main bacteria species used for the production of functional dairy products have been summarized4. In fact, in addition to the rumen bacteria, strains of Bifidobacterium, Enterococcus, Lactobacillus, Lactococcus, Propionibacterium, Streptococcus can be considered as potential CLA-producers, because of the presence of the linoleate isomerase enzyme. Numerous studies have shown that strains of various bacteria can be used as starter cultures for the development of functional fermented milk and yogurt, having higher contents of CLA5-12. Generally, a vegetable oil, a rich source of linoleic acid, is supplemented to the incubation mixture as substrate for improving CLA production. Different types of oils have been used to this aim (sunflower, hydrolyzed soy oil, grape seed oil, castor oil) and different concentration tested. For example, Puniya et al.5 used sunflower oil at the concentration of 0.25, 0.5 and 1.0% v/v, while Vieira et al.9 at 1.7% v/v. In addition, the optimal conditions of pH, time and temperature for the production of CLA are very important parameters; values of pH-optimum from 6.5 to 9.5, and temperature from 10 Â°C to 34 Â°C are reported7. The fermentation time ranges from 12 to 70 hours. Some authors reported that the concentration of the 9-cis, 11-trans isomer was about three times higher when a mixed rumen bacterial strain was incubated under aerobic conditions rather than under anaerobic conditions. It must be also emphasized that an inhibitory effect of LA on the growth of strains has been reported, suggesting that the conversion of LA to CLA isomers ca be related to a detoxification mechanism. Alternatively, thermal and high pressure processing has been also used13. Other strategies, employed to increase CLA content, are based on the manipulation of the feed and dietary regimen of the animals. A lot of researches have shown that the addition to the feed of linoleic acid-rich supplements, such as linseed, soybean, olive, canola, sunflower and fish oils14-16, can slightly increase both the CLA content and the concentration of unsaturated fatty acids in milk and dairy products. Recently, Ianni et al.17 reported that cows fed with a diet supplemented with dried grape pomace produced milk with an increased concentration of rumenic acid. Recently, there has been an increasing interest in the modification of fatty acid composition of foods, including dairy products. It is possible to produce healthier fats, for example structured lipids with CLA esterified in sn-2 position or medium chain triacylglycerols containing CLA, with nutritional advantages due to their real bioavailability18-20.

Table 2 - Content (mg/g of fat) of CLA and starter cultures of yogurts. YOGURT

CLA

S. thermophilus + L. delbrueckii ssp. bulgaricus

2.1a-16.5b

S. thermophilus + L. delbrueckii ssp. bulgaricus + B. bifidum

2.7c-4.2c

S. thermophilus + L. delbrueckii ssp. bulgaricus + B. breve

2.3c-4.4c

S. thermophilus + L. delbrueckii ssp. bulgaricus + B. pseudolongum ssp. pseudolongum

3.0c-4.4c

S. thermophilus + L. delbrueckii ssp. bulgaricus + L. acidophilus L10

4.7d-11.0d

S. thermophilus + L. delbrueckii ssp. bulgaricus + B. animalis

3.6a-9.6d

S. thermophilus + L. delbrueckii ssp. bulgaricus + L. acidophilus

3.3a-20.8e

Akalin et al., 2007; b Florence et al., 2009; c Gorissen et al., 2012; d Santo et al., 2012; e Dave et al., 2002.

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L. Cossignani et al. Large Animal Review 2019; 25: 101-104

BIOLOGICAL ACTIVITIES AND POTENTIAL ADVERSE EFFECTS OF CLA CLA can be considered a multifunctional natural nutraceutical from the rumen, since it shows numerous potential health properties. For this reason, in the last two decades, there was an explosion of interest into deepening the knowledge on biological activity of dairy and fermented products. A lot of recent papers, carried out on animals and humans, confirmed that CLA isomers possess antiatherogenic21, antiadipogenic22, anticancerogenic23, antinflammatory24 and antidiabetic25 properties. Moreover, CLA isomers have effect on weight loss26. As regards fermented dairy products, milk fermented with Lactobacillus casei NCDC19 decreases epididymal fat mass and adipocyte size in obese mice27. In any case, it must be underlined that generally the bioactivity is linked to a specific isomer, and that these properties can be additive, antagonistic, or independent. According to experimental trials on animals, a 70-kg individual would benefit from a daily intake of 3.5 g of CLA2. Human studies on CLA supplementation in doses of up to 7 g/day have shown no adverse effects. Some studies focused on genotoxicity/antigenotoxicity of CLA by using test in vitro as comet test28,29. It has been reported that CLA possess antigenotoxic properties, evaluated using HepG2 cells, mainly acting as desmutagenic agents, and that qualitative/quantitative effects strongly depend on CLA isomers. The main CLA isomers (9cis, 11trans and 10trans, 12cis) of fermented dairy products were effective against 4-nitroquinoline N-oxide-induced DNA damage. At the same time, Aydin et al.30 suggested that CLA (200 mg/kg/day) and whey protein (5 mg/kg/day) can improve the antioxidant defenses of rats, against acrolein-induced toxicity. The potential adverse effects associated with CLA intake, such as its role in oxidative stress, glucose metabolism, milk fat depression, and liver functions, have been reported3,31.

ANALYSIS OF CLA IN FERMENTED DAIRY PRODUCTS In recent years, there has been an increasing interest in analytical methods, reliable and precise, useful for identification and quantification of CLA isomers in dairy products, nutraceuticals, and nutritional supplements. The technique of silver ion (or “argentation”) chromatography was often used for CLA analysis, in particular the silver-ion high performance liquid chromatography (Ag+-HPLC) equipped with single column32 or multi-columns33. This last approach led to a more satisfactory chromatographic resolution of the CLA isomers found in dairy products. However, a combination of high resolution gas chromatography (HRGC) and Ag+HPLC was found to be necessary to resolve all CLA isomers34. Because of the multiplicity of geometrical and positional CLA isomers, complete separation and accurate analysis of complex mixtures are needed. To evaluate the FA composition of fermented dairy products, the HRGC analysis of derivatized FA has been generally used35. Lipases have been widely used to separate the two most common CLA isomers (9cis, 11trans-CLA and 10trans, 12cisCLA)36. The single most comprehensive method for CLA

103

analysis is the high resolution NMR spectroscopy37, which permitted the identification and quantification of all the positional (7,9- to 11,13-) and geometrical CLA isomers. Since dairy products are subjected to heat treatments to elongate their shelf-life, it is important to evaluate the oxidative modifications of CLA38 and the productions of volatile compounds39,40 during thermal processes. Another interesting aspect is the determination of the position esterified by CLA in the glycerol backbone, important aspect for physiological/nutritional reasons41 and for detection of adulterations42-44.

CONCLUSIONS Despite the interesting biological activities of CLA isomers, dietary CLA intake is relatively low to promote the desired physiological effects. Therefore, the improved production of CLA by bacteria in foods is an important technological challenge. In this perspective, more research is needed in order to exploit the potential of milk fermentation as a valid approach for manufacturing dairy products with high CLA content. In this context, the optimization and standardization of fermentation conditions are necessary to obtain reproducible concentrations of CLA isomers in functional foods and nutraceuticals. Moreover, the development of simple and fast analytical methods is desirable for quality control procedures.

References 1. Christie, W.W., X. Han. (2012). Lipid Analysis: isolation, separation, identification and lipidomic analysis, 4th ed., An Oil Press Title. WP, Woodhead Publishing Limited, Cambridge, UK. 2. Hur S.J., Kim H.S., Bahk Y.Y., Park Y. (2017). Overview of conjugated linoleic acid formation and accumulation in animal products. Livest Sci, 195: 105-111. 3. Polidori P., Vincenzetti S., Pucciarelli S., Polzonetti V. (2018) CLAs in animal source foods: healthy benefits for consumers. In: Mérillon JM., Ramawat K. (eds), 1-33, Bioactive Molecules in Food. Reference Series in Phytochemistry. Springer, Cham. 4. Kuhl G.C., De Dea Lindner J. (2016). Biohydrogenation of linoleic acid by lactic acid bacteria for the production of functional cultured dairy products: a review. Foods, 5: 13. 5. Puniya A.K., Chaitanya S., Tyagi A.K., Singh S.D.K. (2008) Conjugated linoleic acid producing potential of lactobacilli isolated from the rumen of cattle. J Ind Microb Biotech, 35: 1223-1228. https://doi.org/ 10.1007/s10295-008-0429-3. 6. Florence A.C.R., da Silva R.C., Santo A.P.D., Gioielli L.A., Tamime A.Y., de Oliveira M.N. (2009). Increased CLA content in organic milk fermented by bifidobacteria or yoghurt cultures. Dairy Sci Tech, 89: 541553. https://doi.org/10.1051/dst/2009030. 7. Gorissen L., Raes K., De Smet S., De Vuyst L., Leroy F. (2012). Microbial production of conjugated linoleic and linolenic acids in fermented foods: Technological bottlenecks. Eur J Lipid Sci Tech, 114: 486-91. https://doi.org/10.1002/ejlt.201100239. 8. Pandit A., Anand S., Kalscheur K., Hassan A. (2012). Production of conjugated linoleic acid by lactic acid bacteria in milk without any additional substrate. Int J Dairy Tech, 65: 603-608. https://doi.org/10. 1111/j.1471-0307.2012.00870.x 9. Vieira C.P., Cabral C.C., da Costa Lima B.R.C., Paschoalin V.M. F., Leandro K.C., Conte-Junior C.A. (2017). Lactococcus lactis ssp. cremoris MRS47, a potential probiotic strain isolated from kefir grains, increases cis-9, trans-11-CLA and PUFA contents in fermented milk. J Funct Foods, 31: 172-178. https://doi.org/10.1016/j.jff.2017.01.047. 10. Akalin A.S., Tokusoglu O., Gonc S., Aycan S. (2007). Occurrence of conjugated linoleic acid in probiotic yoghurts supplemented with fructooligosaccharide. Int Dairy J, 17: 1089-1095. https://doi.org/10.1016/j. idairyj.2007.02.005. 11. Santo A.P.D., Cartolano N.S., Silva T.F., Soares F., Gioielli L.A., Perego

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28. Blasi F., Dominici L., Moretti M., Villarini M., Maurelli S., Simonetti M.S., Damiani P., Cossignani L. (2012). In vitro genotoxicity/antigenotoxicity testing of some conjugated linoleic acid isomers using comet assay. Eur J Lipid Sci Tech, 114: 1016-1024. https://doi.org/10.1002/ ejlt.201200064. 29. Lombardi G., Vannini S., Blasi F., Marcotullio M.C., Dominici L., Villarini M., Cossignani L., Moretti M. (2015). In vitro safety/protection assessment of resveratrol and pterostilbene in a human hepatoma cell line (HepG2). Nat Prod Comm, 10: 1403-1408. https://www.ncbi.nlm. nih.gov/pubmed/26434128. 30. Aydın B., ekero lu Z.A., ekero lu V. (2018). Acrolein-induced oxidative stress and genotoxicity in rats: protective effects of whey protein and conjugated linoleic acid. Drug Chem Tox, 41: 225-231. https://doi.org/10.1080/01480545.2017.1354872. 31. 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 highyielding dairy cow. J Dairy Res, 83: 438-441. https://doi.org/10.1017/ S0022029916000509. 32. Cossignani L., Giua L., Lombardi G., Simonetti M.S., Damiani P., Blasi F. (2013). Analysis of CLA isomer distribution in nutritional supplements by single column Ag+-HPLC. J Am Oil Chem Soc, 90: 327-335. https://doi.org/10.1007/s11746-012-2176-x. 33. Rodríguez-Castañedas J. L., Peña-Egido M. J., García-Marino M., García-Moreno C. (2011) Quantitative determination of conjugated linoleic acid isomers by silver ion HPLC in ewe milk fat. J Food Comp Anal, 24: 1004-1008. https://doi.org/10.1016/j.jfca.2010.10.009. 34. Blasi F., Giua L., Lombardi G., Codini M., Simonetti M.S., Damiani P., Cossignani L. (2011). Improved HRGC separation of cis,trans CLA isomers as Diels-Alder adducts of alkyl esters. J Chrom Sci, 49: 379-383. https://doi.org/10.1093/chromsci/49.5.379. 35. Cossignani L., Giua L., Urbani E., Simonetti M.S., Blasi F. (2014). Fatty acid composition and CLA content in goat milk and cheese samples from Umbrian market. Eur Food Res Tech, 239: 905-911. https://doi.org/10.1007/s00217-014-2287-8. 36. Giua L., Cossignani L., Simonetti M.S., Lombardi G., Blasi F. (2012). Candida rugosa lipase selectivity towards trans,cis- and cis,trans- conjugated linoleic acid isomers. Eur Food Res Tech, 235: 53-59. https://doi.org/10.1007/s00217-012-1731-x. 37. Alexandri E., Ahmed R., Siddiqui H., Choudhary M.I., Tsiafoulis C.G., Gerothanassis I.P. (2017). High resolution NMR spectroscopy as a structural and analytical tool for unsaturated lipids in solution. Molecules, 22: 1663. https://doi.org/10.3390/molecules22101663. 38. Giua L., Blasi F., Simonetti M.S., Cossignani L. (2013). Oxidative modifications of conjugated and unconjugated linoleic acid during heating, Food Chem, 140: 680-685. https://doi.org/10.1016/j.foodchem. 2012.09.067. 39. Cossignani, L., Giua, L., Simonetti, M.S., Blasi F. (2014). Volatile compounds as indicators of conjugated and unconjugated linoleic acid thermal oxidation. Eur J Lip Sci Tech, 116:407-412. http://doi.org/10.1002/ ejlt.201300205. 40. Urbani E., Blasi F., Chiesi C., Maurizi A., Cossignani L. (2015). Characterization of volatile fraction of saffron from central Italy (Cascia, Umbria). Int J Food Prop, 18: 2223-2230. https://doi.org/10.1080/ 10942912.2014.968787. 41. Cossignani L., Blasi F., Bosi A., D’Arco G., Maurelli S., Simonetti M.S., Damiani P. (2011). Detection of cow milk in donkey milk by chemometric procedures on triacylglycerol stereospecific analysis results. J Dairy Res, 78:335-342. https://doi.org/10.1017/S0022029911000495. 42. D’Arco G., Blasi F., Cossignani L., Di Giacomo F., Ciavardelli D., Ventura F., Scipioni S., Simonetti M.S., Damiani P. (2012). Composition of meat and offal from weaned and fattened rabbits and results of stereospecific analysis of triacylglycerols and phosphatidylcholines. J Sci Food Agric, 92:952-959. https://doi.org/10.1002/jsfa.4676. 43. Blasi F., Lombardi G., Damiani P., Simonetti M.S., Giua L., Cossignani L. (2013). Triacylglycerol stereospecific analysis and linear discriminant analysis for milk speciation. J Dairy Res, 80:144-151. https://doi.org/10.1017/S0022029912000635. 44. Blasi F., Montesano D., De Angelis M., Maurizi A., Ventura F., Cossignani L., Simonetti M. S., Damiani P. (2008). Results of stereospecific analysis of triacylglycerol fraction from donkey, cow, ewe, goat and buffalo milk. J. Food Comp. Anal. 21:1-7. https://doi.org/10.1016/j.jfca.2007.06.005.

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IL PORTALE DEL VETERINARIO

DI FIDUCIA

Il portale del Veterinario di Fiducia è una piattaforma multimediale rivolta a Medici Veterinari che svolgono attività clinica e manageriale negli allevamenti italiani. È gestito dalla SIVAR (Società Italiana Veterinari per Animali da Reddito - Federata ANMVI) che ne è anche proprietaria. 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

DDD

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

CODICE EGO

➜

➜ DATABASE EPIDEMIOLOGICO SANITARIO Raccoglie dati sanitari ed epidemiologici di cui dispone solo il veterinario d’azienda

AREA LIBERA DEL PORTALE Alcune sezioni e funzioni (es. notizie e materiali didattici sono di libero accesso e non richiedono l’inserimento di credenziali (né password né username EGO). AREA RISERVATA DEL PORTALE Alcune sezioni e funzioni sono accessibili solo utilizzando il proprio Codice Ego (username e password) dopo averne richiesto l’attivazione alla casella: vetdifiducia@anmvi.it oppure info@sivarnet.it. Sono in area riservata le seguenti funzionalità: – DES (Database Epidemiologico Sanitario) – DDD (Database per il Monitoraggio degli Antibiotici) – forum di discussione – alcuni materiali didattci

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ISCRIVITI ALLA SIVAR PER IL 2019 E RICHIEDI L’ABILITAZIONE AI SERVIZI DEL PORTALE VETERINARIO DI FIDUCIA: AVRAI ACCESSO A DES, DDD E FORUM INVIO RICHIESTA A info@sivarnet.it o vetdifiducia@anmvi.it

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S. Reiichiro et al. Large Animal Review 2019; 25: 107-110

Evaluation of marsupialization combined with long-term administration of antibacterial agents in calves with omphalophlebitis and secondary liver abscess

107

SATO REIICHIRO1, SHINOZUKA YASUNORI1, ONDA KEN1, OCHIAI HIDEHARU1, YAMADA KAZUTAKA1 1

School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuou-ku, Sagamihara, Kanagawa 252-5201, Japan

SUMMARY Umbilical vein marsupialization was performed on three calves with abscesses of the umbilical vein and liver as revealed by ultrasonography. After surgery, the umbilical vein was washed with a warm physiological saline containing a susceptible antibacterial agent, and a systemic antimicrobial agent was administered for 14 days. There were no postoperative complications in any of the three calves, and each returned to production.

KEY WORDS Antibacterial agent, calf, liver abscess, marsupialization, omphalophlebitis.

INTRODUCTION

MATERIAL AND METHODS

Bacterial infections of the umbilical region of calves develop soon after birth due to insanitary birthing environments, failure of passive immunity, or incomplete disinfection of the umbilical cord1. Infectious diseases of the umbilical region consist of umbilical cord inflammation outside the abdominal cavity, omphalophlebitis, umbilical arteritis, and urachal infection. Pathological progression eventually leads to abscess formation. Resultant bacteremia caused by these diseases may lead to complications (i.e. polyarthritis, pneumonia), and subsequently, reduce the productivity of calves. The umbilical arteries are transected and involute into the abdominal cavity during parturition; however, the umbilical vein and urachus lack elastic fibers and do not involute easily, facilitating the establishment of infections. Of the 322 cases of umbilical cord diseases, Yanmaz et al.2 reported omphalophlebitis to be among the most intractable, inducing the highest mortality among calves. A delay in the diagnosis and treatment of the umbilical vein infection leads to umbilical vein abscesses requiring drainage through marsupialization of umbilical vein (MUV), as abscesses reaching the porta hepatis are difficult to remove. Presently, there is no effective treatment method for managing the clinical progression of the disease and the eventual spread of the abscesses to the porta hepatis, because the removal of abscesses from within the liver parenchyma is challenging. The present study reports on the satisfactory outcome of MUV, and long-term, post-surgical administration of antibacterial agents on three calves in which liver abscesses, secondary to omphalophlebitis, were observed.

Three calves with omphalophlebitis involving the liver were treated surgically by marsupialization of the umbilical remnant. The medical records were reviewed to identify the breed, sex, age, bacterium isolated from the abscess, post-operative management including the duration and type of antibacterial agent administered, and the day from surgery to discharge (Table 1). Written informed consent was obtained from the respective owner for publication of this case report and accompanying images.

Corresponding Author: Sato Reiichiro (r-sato@azabu-u.ac.jp).

RESULTS Case 1 Holstein, female, 4 months old; body weight (BW), 134.5 kg. The calf exhibited swelling of the umbilical region at approximately 1 month of age. As the owner observed no other abnormalities, no action was taken, and breeding was continued. At 4 months of age, the owner observed abscess drainage from the umbilical region and requested a diagnosis. The following observations were registered from the initial examination of the calf: a temperature of 39.3°C, heart rate of 102 bpm, respiratory rate of 66 bpm, satisfactory movement of the rumen, and no complaints with appetite or activity. The umbilical region was distended to 13x7x7 cm with abscess drainage observed from the tip. Fever was present and tenderness was found on palpation. A blood test showed an increase in leukocytes (13,700/µl; reference range, 4,900-12,000/µl). An ultrasound (3.5-MHz convex probe; MyLab One VET, Esaote, Maastricht, Netherlands) revealed a tubular structure linking the umbilical region to the liver (Figure 1A, 1B). Moreover, multiple high echogenicity spots (0.5-1 cm diameter) were imaged in a number of locations in the liver parenchyma. No abnormalities were observed in the

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Evaluation of marsupialization combined with long-term administration of antibacterial agents in calves

Table 1 - Patient characteristics (breed, age, bacteria, therapeutic procedures). Therapeutic

Case No.

Breed

Sex

Age (month)

BW (kg)

Holstein

female

134.5

2 3

Japanese Black Brown Swiss

male female

1 1

50.0 53.0

Isolated Bacterium

Antibacterial agent

T. pyogenes

P. mirabilis

T. pyogenes

P. mirabilis

T. pyogenes

Period of Antibacterial agent administration (day)

Irrigation period (day)

Days from surgery to discharge (day)

Figure 1 - Ultrasound image of the cranial abdomen of case 1. (A) Scanner placement transverse plane immediately cranial to the umbilicus. The lumen of the vein has an area of relatively high echogenicity (arrow). (B) The infected umbilical vein (*) penetrating the liver parenchyma (L).

umbilical arteries or the urachus. Collectively, these results presented a diagnosis of omphalophlebitis and secondary hepatic abscesses. Aerobic and anaerobic culturing of the aseptic samples from the abscess leakage were conducted. Drug susceptibility tests (BD Sensi-Disc, Becton Dickinson Japan, Tokyo, Japan) were performed on the isolated bacteria using penicillin (PC), ampicillin (ABPC), cefazolin (CZ), oxytetracycline (OTC), kanamycin (KM) and enrofloxacin (ERFX). Trueperella pyogenes (T. pyogenes) and Proteus mirabilis (P. mirabilis) were isolated from the abscess samples and were found to be susceptible to CZ, KM, and ERFX. Additionally, T. pyogenes showed susceptibility to PC and OTC; and P. mirabilis, to ABPC. Judging complete removal of the umbilical vein abscess to be problematic, MUV was implemented on the 3rd day following admission. The cow was fasted for 24 h prior to the surgery, and intramuscular cefazolin sodium was administered (5 mg/kg. Cefazolin-Chu; Fujita Pharmaceutical, Tokyo, Japan) to prevent perioperative infection. The calf was guided to a recumbent, dorsal position before general anesthesia was administered. Following closure of the umbilical opening with purse-string sutures, a local anesthetic (Kyoritsu Seiyaku Corporation, Tokyo, Japan) with procaine hydrochloride was applied to the surgical site. The skin was incised in a fusiform shape around the umbilical opening, and following the separation of the umbilical region and the subcutaneous tissue, a sterilized surgical glove

was placed over the umbilical region. Nylon suture (USP-3) was used to ligature the exposed umbilical cord and glove and, thereby, prevent leakage from the umbilical opening. An incision was made of the skin, 5 cm toward the cranial side from the umbilical region and, 1.5 cm paramedially on the right side, and approximately 10 cm towards the cranial side. The swollen umbilical vein, proceeding from the abdominal cavity, was made to adhere to the peritoneum and the greater omentum, and subsequently, connected to the liver. After circular excision of the umbilical stump, the freed umbilical vein was tugged to the right of the paramedian site while retaining the cranial end of the incisional site. Following closure of the umbilical region (median plane) with synthetic, absorbent sutures; the swollen umbilical vein was clipped in place near the porta hepatis using intestinal clamps. A warm, physiological saline solution, containing 2 g of dissolved cefazolin sodium, was then slowly and repeatedly infused in, and suctioned out from the umbilical vein, held outside the abdominal cavity, until the solution became clear. The clips near the porta hepatis were then released. The small, paramedian opening was sutured shut from the caudal section, and the freed umbilical vein was fixed in place by suturing it to the tunica muscularis at the cranial tip of the opening. The skin incision was ligatured with intradermal, buried sutures using synthetic, absorbent thread. Procaine penicillin G (5,000 IU/kg, Kyoritsu Seiyaku Inc., Tokyo, Japan) and kanamycin (10 mg/kg, Ryusan Kanamycin-

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Chu 100, Kyoritsu Inc., Tokyo, Japan) were administered for 14 days, following the surgery. On the 5th day post-surgery, physiological saline solution was slowly infused in and suctioned out from the umbilical vein opening without applying pressure, and irrigation was performed until the solution appeared clear. Two weeks after the surgery, the umbilical vein was removed, and abdominal closure was performed. The calf was released from care 30 days following the initial surgery and delivered her first calf 25 months post-surgery. At 32 months, she continued to undergo breeding.

Case 2 Japanese black cattle, male, 1 month old; BW, 50 kg. The calf was examined 25 days after birth due to insufficient appetite and abscess drainage from the umbilical region. The initial examination showed a temperature of 39.2°C, heart rate of 102 bpm, respiratory rate of 22 bpm, and sluggish activity. The umbilical region was distended to 5x4x4 cm. Fever and tenderness were observed on palpation, with induration in one area. Deep palpation of the calf in the standing position detected an egg-sized swelling from the umbilical cord in the right cranial direction. A blood test evinced a mild increase in leukocytes (12,700/µl; reference range, 4,900-12,000/µl). An ultrasound revealed a tubular structure linking the umbilical region to the liver. Moreover, large, unequal echogenicity spots of 0.5 cm diameters were identified at a number of locations in the liver parenchyma. No abnormalities were observed in the umbilical arteries or the urachus. Collectively, these results presented a diagnosis of omphalophlebitis and a secondary hepatic abscess. Aerobic and anaerobic culturing of the abscess drainage were conducted; as in Case 1, T. pyogenes and P. mirabilis were isolated. A drug susceptibility test showed that both bacteria were susceptible to ABPC, CZ, KM and ERFX. Additionally, T. pyogenes showed susceptibility to PC. Judging complete extraction of the umbilical vein abscess to be problematic, MUV was performed on the 14th day after admission. The same methods for pre-surgical management and surgical operations, employed in Case 1, were applied to this case. The swollen umbilical vein proceeding out of the abdominal cavity was made to adhere to the peritoneum and the greater omentum and was then connected to the liver (Figure 2). Procaine penicillin G (5.000 IU/kg) and kanamycin (10 mg/kg) were administered for 14 days postsurgery. As in Case 1, saline solution with dissolved cefazolin was used for 14 days until it appeared clear after the irrigation of the umbilical vein opening. Two weeks post-surgery, the umbilical vein was removed and abdominal closure was performed. On the 25th day following the surgery, the calf was released from care and was slaughtered for meat at the age of 30 months.

Figure 2 - Intraoperative appearance of the infected umbilical vein (*) penetrating the liver (arrow) of case 2. Cr, cranial; Cd, caudal; R, right side; L, left side.

(800 mg/100 ml, reference range, 400-700 mg/100 ml). An ultrasound revealed a tubular structure linking the umbilical region to the liver. Moreover, low echogenicity nodules (1 cm diameter) were scattered in the liver parenchyma. A contrastenhanced computed tomography (CT; BrightSpeed16, GE Healthcare, Port Washington, NY, U.S.A.) was performed before surgery. Iohexol (Omnipaque; Daiichi-Sankyo Co., Ltd., Tokyo, Japan) was used as a contrast agent. Multiple unenhanced nodules, suggestive of intra-hepatic abscesses, were observed (Figure 3). No abnormalities were seen in the umbilical arteries or the urachus. From the ultrasound and CT examination, a diagnosis of multiple hepatic abscesses issuing from the umbilical vein was made. Aerobic and anaerobic culturing of the abscess drainage were

Case 3 Brown Swiss, female, 1 month old; BW, 53 kg. The calf was examined 14 days post-partum due to anorexia, umbilical region swelling, and abscess drainage. Initial examination showed a temperature of 39.7°C, heart rate of 130 bpm, respiratory rate of 48 bpm, and sluggish activity. The umbilical region was distended to 5x4x4 cm. Fever and tenderness were observed upon palpation, with induration in one area. A blood test showed an increase in leukocytes (15,400/µl; reference range, 4,900-12,000/µl) and hyperfibrinogenemia

Figure 3 - Contrast enhanced computed tomography showed multiple hepatic abscess of case 3. Arrows show the multiple hepatic abscess.

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Evaluation of marsupialization combined with long-term administration of antibacterial agents in calves

performed as in Case 1, and T. pyogenes was isolated. A drug susceptibility test showed sensitivity of T. pyogenes to PC, ABPC, CZ, KM, OTC, and ERFX. Judging complete extraction of the umbilical vein abscesses to be problematic, MUV was implemented on the 4th day after admission. The same pre-surgical management and surgical operations used in Case 1 were applied to this case. The swollen umbilical vein proceeding from outside the abdominal cavity was made to adhere to the peritoneum and the greater omentum, and was subsequently, connected to the liver. Procaine penicillin G (5.000 IU/kg) was administered for 14 days post-surgery. To irrigate the umbilical vein opening, as in Case 1, saline solution containing cefazolin was used for 5 days until it appeared clear. At 13 days post-surgery, the umbilical vein was removed, and abdominal closure was performed. On the 23rd day following the surgery, the calf was released from care. The cow continued breeding at 8 months of age.

DISCUSSION Umbilical cord inflammation arising outside the abdominal cavity3-5, omphalophlebitis, umbilical arteritis, and urachal inflammation caused by diffusion of the infection within the abdominal cavity, generally comprise the infectious diseases of the umbilical region affecting newborn calves. Of the 322 cases of umbilical cord disease, Yanmaz et al.2 reported omphalophlebitis as one of the most intractable diseases, and thus, induces the highest mortality among calves. Omphalophlebitis progresses to septicemia, bacterial infectious diseases, and formation of hepatic abscesses, becoming an economically damaging disease6. Surgical removal of the umbilical vein is viable for cases of umbilical vein abscess without the involvement of the liver parenchyma. However, for abscesses penetrating the liver parenchyma, MUV is performed on account of the difficulty of partially removing the liver3,6-9. When conducting MUV, two methods8,10,11 allow for the appropriate drainage4,10,12,13 of abscesses within the umbilical vein. Edwards et al.10 and Marchionatti8 have devised a new type of incision at the region where veins enters the liver (right, cranial abdominal wall), and have reported a method of fixing the umbilical vein thereto. This positioning allows the vein to exit the abdominal cavity directly ventral to the liver. Although the risk of introducing infected tissue and infecting the abdominal cavity is likely, covering the tip of the umbilical vein with a vinyl or a surgical glove can mitigate the risk. Steiner et al.8 have reported a method of extending the umbilical vein, several centimeters from the xiphoid process, and fixing it to the cranial side of an incised wound on the caudal umbilical cord. Both methods require frequent drainage of the infection from within the umbilical vein. However, applying pressure when cleaning the umbilical vein increases the risk of further intra-hepatic progression of infection, and pressurized cleansing worsens the pathology and may induce septicemic shock and consequent death8. The three cases herein presented used the Edwards et al.10 and Marchionatti11 methods to drain the liver directly to the area outside the abdominal cavity. To prevent the expulsion of umbilical vein abscesses into the liver, the hepatic opening was clipped close with intestinal clamps prior to cleansing the umbilical vein with a warm, physiological saline solution, repeatedly infused and suctioned out without applying pressure.

In addition, while inflammatory bacteria and bacterial liver abscesses can be treated by abscess drainage and the administration of an appropriate antibacterial agent, intractable bacterial liver abscesses accompanying chronic granulomatous disease can reportedly be treated with a satisfactory prognosis by combining percutaneous trans-hepatic abscess drainage with long-term administration of antibacterial agents14.

CONCLUSIONS In the three cases herein reported, the performance of MUV and pre-surgical bacterial investigations, and post-surgical, long-term administration of antibacterial agents can be considered to enable post-surgical recovery of productivity, free of secondary diseases. However, owing to the considerable risk of a superinfection and the emergence of resistant bacteria when administering antibacterial agents, the inflammatory bacteria must be treated, antibacterial agents with corresponding susceptibility be used, and monitoring for the possibility of resistant bacteria be maintained. This research suggests that MUV and long-term administration of antibacterial agents can be used to effectively treat omphalophlebitis and the secondary hepatic abscesses.

ACKNOWLEDGEMENTS The authors thank the staff and student assistants at Azabu University for taking care of the heifer during hospitalization. This work was partially supported by a research project grant awarded by the Azabu University Research Services Division.

References 1. Divers T.J., Peek S.F. (2018). Urinary Tract Diseases. In: Rebhun’s Diseases of Dairy Cattle, 3rd ed., 548-552, Elsevier, St. Louis, MO. 2. Yanmaz L.E., Dogan E., Okumus Z., Kaya M., Hayirli A. (2017). Estimation of outcome of umbilical diseases based on clinical examination: A retrospective study involving 322 calves. Isr J Vet Med, 72: 40-44. 3. Trent A.M., Smith D.F. (1984). Surgical management of umbilical masses with associated umbilical cord remnant infections in calves. J Am Vet Med Assoc, 15: 1531-1534. 4. Bohy A., Moissonnier P. (1990). Umbilical disease in Charolais calves: a retrospective study of 115 surgically treated cases. Point Vét, 22: 542-551. 5. Geishauser T., Gründer H.D. (1992). Nabelentzündung beim Kalb- Ein Rückblick auf 104 Faelle. Tieraerzl Umschau, 47: 304-320. 6. Baxter G.M. (1989). Umbilical masses in calves: Diagnosis, treatment and complications. Compend Contin Educ Pract Vet, 11: 503-515. 7. Parker J.E., Gaughan E.M. (1988). Partial hepatic resection for treatment of a single liver abscess in a dairy heifer. Vet Surg, 17: 87-89. 8. Steiner A., Lisher C.J., Oertle C. (1993). Marsupialization of umbilical vein abscesses with involvement of the liver in 13 calves. Vet Surg, 22: 184-189. 9. Rings D.M. (1995). Umbilical hernias, umbilical abscesses, and urachal fistulas. Surgical considerations. Vet Clin North Am Food Anim Pract, 11: 137-148. 10. Edwards R.B. 3rd, Fubini S.L. (1995). A one-stage marsupialization procedure for management of infected umbilical vein remnants in calves and foals. Vet Surg, 24: 32-35. 11. Marchionatti E., Nichols S., Babkine M., Fecteau G., Francoz D., Lardé H., Desrochers A. (2015). Surgical management of omphalophlebitis and long term outcome in calves: 39 Cases (2008-2013). Vet Surg, 45:194-200. 12. Becker M., Kaegi B., Waxenberger M. (1985). Bovines Pericard eine Bioprothese für den Verschluss von Bauchdeckendefecten beim Kalb. Schweiz Arch Tierheilk, 127: 379-383. 13. Sen T.B., Paul M.K. (1989). Further studies on the use of nylon mosquito net mesh in hernioplastie in bovine. Indian J Anim Health, 28: 65-66. 14. Johnston R.B., Newman S.L. (1977). Chronic granulomatous disease. Pediatr Clin North Am, 24:365-376.

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Severe outbreak of pasteurellosis in sows: a case description

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GIUSEPPE MARRUCHELLA1, CRISTINA ESMERALDA DI FRANCESCO1, ABIGAIL ROSE TRACHTMAN1, FRANCESCO MOSCA1, ANDREA DI PROVVIDO1,2, CARLA SEBASTIANI3, PIETRO GIORGIO TISCAR1, CHIARA MAGISTRALI3 1

Università degli Studi di Teramo, Facoltà di Medicina Veterinaria, località Piano d’Accio, 64100 Teramo, Italia 2 Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise, Campo Boario, 64100 Teramo, Italia 3 Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, via Salvemini 1, 06126 Perugia, Italia

SUMMARY Sow mortality represents a relevant issue in the modern pig industry and can negatively impact the profitability of farming. Sow mortality rate should range between 3-6% on a yearly basis, but higher rates are quite commonly reported. Disease conditions of the locomotor, digestive, reproductive and urinary systems are most frequently observed in dead/euthanized sows. On the other hand, respiratory diseases are rare in this category of animals, the most severe episodes resulting from the introduction of a “new” infectious agent into the breeding stock, thus in absence of an adequate level of herd immunity. We describe herein the main clinical, pathological and microbiological findings observed during a severe outbreak of respiratory disease, which occurred in a breeding pig herd in Central Italy. The respiratory syndrome affected a very high percentage of pregnant and lactating sows (around 30%), both primiparous and multiparous. Within two weeks, the mortality rate reached 15%. At necropsy, serous-fibrinous pleuritis, along with large foci of acute bronchopneumonia, affecting the cranioventral parts of both lungs, represented the most relevant pathological findings in animals under study. In depth bacteriological investigations identified a capsular type D and hgbB-positive Pasteurella multocida strain as the sole causative agent. Pneumonic pasteurellosis usually affects grower/finishing pigs and represents the final stage of the so-called porcine respiratory disease complex. As a matter of fact, Pasteurella multocida rarely acts as the primary cause of porcine pneumonia, rather being considered an opportunist which complicates primary infections caused by other bacteria and/or viruses. The co-factors which likely contributed to determine such a severe respiratory syndrome in sows are largely unknown. Speculatively, we hypothesize that resulted from the introduction of a “new” strain of P. multocida, which infected immunologically naïve sows. The poor level of biosecurity adopted, as well as the kinetics of this outbreak, argue in favour of such a scenario.

KEY WORDS Sow, respiratory syndrome, Pasteurella multocida, capsular serogroup, virulence factors.

INTRODUCTION In the modern pig industry, sow mortality represents a relevant issue, which can negatively impact the profitability of farming1. Ideally, the sow mortality rate should range between 3-6% on a yearly basis. However, higher rates are quite commonly reported, usually tied to farm-specific management strategies2. Most sow mortality occurs during pregnancy or within the weaning-to-oestrus interval. It is often challenging to obtain reliable diagnostic data, as necropsy and laboratory investigations are not routinely performed in sows. According to literature, disease conditions of the locomotor, digestive and urogenital systems are most frequently observed in dead/euthanized sows. On the other hand, respiratory diseases are rare in this category of animals, the most severe episodes resulting from the introduction of a “new” infectious agent into the breeding stock, thus in absence of an adequate level of herd immunity2-4.

Corresponding Author: Giuseppe Marruchella (gmarruchella@unite.it).

We report herein the main features of an unusual and particularly severe respiratory syndrome, which recently affected a pig breeding farm in Central Italy.

MATERIALS AND METHODS The present outbreak occurred in a medium-sized pig breeding farm (“site 1”), which remained unused for over two years. In October 2017, one hundred and fifty sows were introduced therein, all coming from the same herd of origin. The sows were routinely vaccinated against Aujeszky’s disease, porcine parvovirus, erysipelas and colibacillosis. Reproductive activity was organized in a three-week batch schedule. Piglets were weaned at 28 days and remained at this site until reaching two months of age. Thereafter, they were transferred to a different farm (“site 2”) and raised to the market weight. The two farms were 5 km away from each other and shared the same personnel and equipment. Fattening pigs coming from other farms were simultaneously reared in site 2, no all in/all out strategy being implemented. In December 2017, a severe respiratory disease occurred in

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Severe outbreak of pasteurellosis in sows: a case description Figure 1 Sow. Abundant discharge of bloody-tinged foamy fluid from the nostrils.

the above breeding stock. Six sows were necropsied for diagnostic purposes. Pulmonary and lymph node samples were routinely processed for histopathological investigations. In depth bacteriological culture tests and biomolecular investigations for Mycoplasma spp.5, Influenza A virus 6, Porcine Reproductive and Respiratory Syndrome virus 7, Porcine Circovirus type 28 and Coronavirus spp.9 were also carried out.

RESULTS The respiratory syndrome affected a high percentage of pregnant and lactating sows (around 30%), both primiparous and multiparous. Anorexia, fever, dyspnoea, coughing, cutaneous and snout cyanosis and agalactia were the most relevant signs and symptoms. Within two weeks, the mortality rate reached 15%. At the same time, the mortality of suckling piglets notably increased, as a result of sow agalactia. At necropsy, abundant discharge of frothy and bloody fluid was observed from the nostrils (Figure 1). Serous-fibrinous pleuritis, along with large foci of acute bronchopneumonia, mainly affecting the cranio-ventral parts of both lungs, represented the most relevant pathological findings (Figure 2). Microscopically, fibrinous pleuritis, fibrinous-purulent and necrotizing broncho-pneumonia were mainly observed. Bacteriological investigations yielded the isolation of Pasteurella multocida (P. multocida) as pure culture from all the lungs under study. In one sow, P. multocida was additionally isolated from the spleen. On the contrary, samples always tested negative for all other pathogens. The antibiotic sensitivity was evaluated by agar diffusion test; all the isolates proved to be sensitive to marbofloxacin, amoxicillin, ceftiofur e florfenicol. One isolate of P. multocida was tested according to Cucco et al.10, in order to identify its capsular serogroup and its major virulence. As a result, P. multocida demonstrated to belong to the capsular serogroup D and to be hgbB-positive. Overall, laboratory investigations identified P. multocida as the sole infectious pathogen responsible for such a severe

Figure 2 - Sow. Multifocal areas of acute pneumonia, showing a lobular pattern. Few petechiae are also present at the level of the visceral pleura and on the outer surface of the pericardium.

respiratory disease. The outbreak was resolved over a twoweek timespan and no further cases were reported in the following months. However, the poor reproductive performances led the farmer to remove the entire breeding stock one year later.

DISCUSSION In pigs, P. multocida rarely plays a role as primary causative agent of pneumonia; conversely, it usually acts as an opportunistic pathogen, worsening infections sustained by viruses or Mycoplasma hyopneumoniae. As a matter of fact, pneumonic pasteurellosis usually represents the end stage of the so-called â&#x20AC;&#x153;porcine respiratory disease complexâ&#x20AC;?, a multifactorial and costly syndrome mainly affecting growing/finishing pigs worldwide11,12. Considering that, the present outbreak of pneumonic pasteurellosis shows some unusual features. First of all, it affect-

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ed a category of animals usually spared by severe respiratory diseases, with unexpected high morbility and mortality rates. Moreover, it was caused by P. multocida as the sole and primary agent. We consider unlikely that this was due to a particular combination of the capsular serogroup with one or more virulence factors. In fact, P. multocida capsular serogroup D/hgbB-positive strains are not uncommon in Italy, nor in the rest of the world10,13. It seems difficult to assign a role to additional predisposing factors. The involvement of lactating sows, along with the mild environmental temperatures, lead us to rule out the climate as a relevant predisposing factor. Likewise, the exposure to mycotoxins was considered improbable, as their levels were within the range of tolerability (data not shown). Speculatively, we hypothesize that the disease resulted from the introduction of a “new” strain of P. multocida, which probably originated from site 2 and then infected immunologically naïve sows. The adoption of a poor level of biosecurity, as well as the kinetics of this outbreak, argue in favour of such a scenario. To conclude, we consider that the present case description implies at least three, as simple as relevant “take home messages”: a) P. multocida can primarily cause severe outbreaks of pneumonia in adult animals, at least under intensive rearing conditions. This further highlights the importance of performing a rational and full diagnostic iter; b) in spite of recent reports14,15, the pathogenic variability of P. multocida strains is still poorly understood and goes well beyond the presence of a single virulence factor. Filling this knowledge gap sounds of particular relevance, due to the compelling restrictions to the use of antimicrobials; c) suitable biosecurity strategies are crucial to properly manage the health of livestock.

References 1. Jensen T.B., Toft N., Bonde M.K., Kongsted A.G, Kristensena A.R., Sørensen J.T. (2012). Herd and sow-related risk factors for mortality in sows in group-housed systems. Prev Vet Med, 103: 31-37.

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2. Muirhead M.R., Alexander T.J. (2013). Managing Health in the gestation/Dry Period. In: Managing Pig Health, Eds Muirhead M.R., Alexander T.J., 2nd Ed., 227-262. Sheffield, 5m Publishing. 3. D’Allaire S., Drolet R., Chagnon M. (1991). The causes of sow mortality: A retrospective study. Can Vet J, 32: 241-243. 4. Sanz M., Roberts J.D., Perfumo C.J., Alvarez R.M., Donovan T., Almond G.W. (2007). Assessment of sow mortality in a large herd. J Swine Health Prod, 15: 30-36. 5. van Kuppeveld F.J., Johansson K.E., Galama J.M., Kissing J., Bölske G., van der Logt J.T., Melchers W.J. (1994). Detection of mycoplasma contamination in cell cultures by a mycoplasma group-specific PCR. Appl Environ Microbiol, 60: 149-152. 6. Anonymous. (2015). Influenza A virus of swine. In: Manual of diagnostic tests and vaccines for terrestrial animals Ed. Anonymous, 2.8.7, 1-14, OIE Paris. 7. Guarino H., Goyal S.M., Murtaugh M.P., Morrison R.B., Kapur V. (1999). Detection of porcine reproductive and respiratory syndrome virus by reverse transcription-polymerase chain reaction using different regions of the viral genome. J Vet Diagn Invest, 11: 27-33. 8. Ellis J., Krakowka S., Lairmore M., Haines D., Bratanich A., Clark E., Allan G., Konoby C., Hassard L., Meehan B., Martin K., Harding J., Kennedy S., McNeilly F. (1999). Reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic piglets. J Vet Diagn Invest, 11: 3-14. 9. Escutenaire S., Mohamed N., Isaksson M., Thorén P., Klingeborn B., Belák S., Berg M., Blomberg J. (2007). SYBR Green real-time reverse transcription-polymerase chain reaction assay for the generic detection of coronaviruses. Arch Virol, 152: 41-58. 10. Cucco L., Massacci F.R., Sebastiani C., Mangili P., Bano L., Cocchi M., Luppi A., Ortenzi R., Pezzotti G., Magistrali C.F. (2016). Molecular characterization and antimicrobial susceptibility of Pasteurella multocida strains isolated from hosts affected by various diseases in Italy. Vet It, 53: 21-27. 11. Register K.B., Brockmeier S.L., de Jong M.F., Pijoan C. (2012). Pasteurellosis. In: Diseases of Swine, Eds Zimmerman J.J., Karriker L.A., Ramirez A., Schwartz K.J., Stevenson G.W., 10th ed., 798-809, WileyBlackwell, Oxford. 12. Pors S.E., Hansen M.S., Bisgaard M., Jensen H.E. (2011). Occurrence and associated lesions of Pasteurella multocida in porcine bronchopneumonia. Vet Microbiol, 150: 160-6. 13. Davies R.L., MacCorquodale R., Baillie S., Caffrey B. (2003). Characterization and comparison of Pasteurella multocida strains associated with porcine pneumonia and atrophic rhinitis. J Med Microbiol, 52 (Pt 1): 59-67. 14. Oliveira Filho J.X., ZanellaMorés M.A., Rebellato R., Kich J.D., Cantão M.E., Klein C.S., Carvalho Guedes R.M., Coldebella A., Santos Neves de Barcellos D.E., Morés N. (2018). Pathogenic variability among Pasteurella multocida type A isolates from Brazilian pig farm s. BMC Vet Res, 14: 244. 15. De Lorenzi G., Barbieri G., Gherpelli Y., Pangallo G., Magistrali C.F., Gibelli L., Bonilauri P (2019). Focolaio di pleuropolmonite fibrinonecrotico-emorragica in un’azienda da ingrasso. Pages 223-228 in Proc. Meeting Annuale SIPAS, Rezzato, Brescia, Italia.

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FORMATO Tutti i contributi (review, articoli originali, case reports), presentati in un unico file, devono presentare la seguente struttura: Lingua - Inglese o Italiano. Titolo - Il titolo del manoscritto deve essere breve ed esplicativo. Nel caso di lavori in italiano il titolo deve essere tradotto anche in lingua inglese. Sotto il titolo vanno riportati cognomi e nomi dei singoli autori (ad esempio, Smith Tom), a seguire vanno indicate le affiliazioni degli stessi, numerate in ordine progressivo. Il corresponding author deve essere identificato con un simbolo accanto al nome (esempio, Smith Tom*) ed iI nome completo, l’indirizzo postale, il numero di telefono e l’indirizzo e-mail devono apparire sotto le righe di affiliazione sempre sulla pagina del titolo (esempio, * Corresponding author: Smith Tom, University of…). Abstract - Sulla seconda pagina del manoscritto deve essere inserito un abstract (in inglese) con una lunghezza compresa da un minimo di 300 ad un massimo di 500 parole. L’abstract deve contenere in modo conciso e chiaro lo scopo del lavoro, i risultati e le conclusioni degli autori. Bibliografia, figure e tabelle non devono essere incluse nell’abstract.

Rivista ufficiale SIVAR Parole Chiave - Di seguito all’abstract, sempre in lingua inglese, devono essere riportate le parole chiave (key words), da un minimo di 3 ad un massimo di 5, separate tra di loro dal punto e virgola “;”. Corpo del Testo - I manoscritti degli articoli originali devono presentare il seguente schema: introduzione, materiali e metodi, risultati, discussione, conclusioni, ringraziamenti e bibliografia. Nel caso delle review non è previsto uno schema guida, ma l’argomento deve essere trattato in modo completo e suddiviso in capitoli per renderlo il più chiaro possibile. 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

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

R E D D I TO

Istruzioni LAR 2019 italiano_inglese 06/03/19 14:43 Pagina 2

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

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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