Large Animal Review 5 - 2021 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

05/21

Bimonthly, Year 27, Number 5, October 2021

LAR Large Animal Review

ISSN: 1124-4593

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

ORIGINAL ARTICLES BOVINE • Clinical follow-up in fattening bulls affected by Bovine Respiratory Disease (BRD) after pharmacological treatment with Tulathromycin and Ketoprofen: clinical score and ultrasonographic evaluations • Effect of Yucca Schidigera inclusion in milk replacer for veal calves on health status, antimicrobial use and growth performance

OVINE • Investigation of the gene carriage rates for Staphylococcus aureus, mecA, vanA and nuc genes in the nasal and milk specimens from the sheep caretakers with sheep • Effects of rosemary distillation residues substitution to oat-hay on diet digestibility, metabolic profile and growth performance of Barbarine lamb ewes

CAPRINE • Histo-morphological alterations in testis of goat (Capra hircus) induced by atrazine in vitro: evaluation of ameliorating effect of vitamin

EQUINE • Serum trace element levels in Equine Herpesvirus 1 infected horses

CASE REPORTS CAPRINE • Traumatic lateral hysterocele complicated with intestinal adhesions in a pregnant goat

SOCIETÀ ITALIANA VETERINARI PER ANIMALI DA REDDITO ASSOCIAZIONE FEDERATA ANMVI

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INDEX

Anno 27, numero 5, Ottobre 2021

Rivista indicizzata su: CAB ABSTRACTS e GLOBAL HEALTH IMPACT FACTOR (2021): 0.417

BOVINE Clinical follow-up in fattening bulls affected by Bovine Respiratory Disease (BRD) after pharmacological treatment with Tulathromycin and Ketoprofen: clinical score and ultrasonographic evaluations ENRICO FIORE, ANASTASIA LISUZZO, ANDREA BELTRAME, BARBARA CONTIERO, ELIANA SCHIAVON, ELISA MAZZOTTA

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

Effect of Yucca Schidigera inclusion in milk replacer for veal calves on health status, antimicrobial use and growth performance

Managing Editor: Matteo Gianesella

OVINE Investigation of the gene carriage rates for Staphylococcus aureus, mecA, vanA and nuc genes in the nasal and milk specimens from the sheep caretakers with sheep

Technical Editor: Enrico Fiore 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.

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

SILVIA GROSSI, RICCARDO COMPIANI, GIANLUCA BALDI, CARLO ANGELO SGOIFO ROSSI

ÖMER AKGÜL, GÜLHAN BORA, HÜSEYIN GÜDÜCÜOĞLU

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Effects of rosemary distillation residues substitution to oat-hay on diet digestibility, metabolic profile and growth performance of Barbarine lamb ewes YAGOUBI YATHREB, SMETI SAMIR, MEKKI ILYES, MAHOUACHI MOKHTAR, ATTI NAZIHA

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CAPRINE

Histo-morphological alterations in testis of goat (Capra hircus) induced by atrazine in vitro: evaluation of ameliorating effect of vitamin RAYEES AHMAD BHAT, SONAL GUPTA, CONCETTA SAOCA, CLAUDIA GIANNETTO, GIUSEPPE PICCIONE, FRANCESCO FAZIO

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Direttore Responsabile Sabina Pizzamiglio Stampa Press Point - Via Cagnola, 35 20081 Abbiategrasso (MI) - Tel. 02/9462323 Spedizione Poste Italiane SPA - Spedizione in A.P. D.L. 353/2003 (Conv. in L. 27/02/2004 N. 46) Art. 1, Comma 1, DCB Piacenza

EQUINE Serum trace element levels in Equine Herpesvirus 1 infected horses MARIA LUISA MARENZONI, ONUR İ SKEFLI, FATMA ATES ALKAN, LUCA AVELLINI, ÇAGˇLA PARKAN YARAMIS, M. ERMAN OR

Concessionaria esclusiva per la pubblicità E.V. Soc. Cons. a r.l. Palazzo Trecchi - 26100 Cremona Ufficio Pubblicità: Paola Orioli Tel. 0372/403539 - E-mail: info@sivarnet.it Prezzo di copertina: € 10,00. La rivista è inviata a tutti i veterinari interessati ai settori degli animali da reddito con il versamento di € 52,00 per l’Italia; € 62,00 per l’Estero. Servizio abbonamenti: Tel. 0372/403507. Ai Soci SIVAR in regola con il pagamento della quota associativa, la rivista è inviata gratuitamente in quanto la quota è comprensiva dell’abbonamento alla rivista stessa.

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CASE REPORTS

CAPRINE

Traumatic lateral hysterocele complicated with intestinal adhesions in a pregnant goat KHAN SHARUN, KALLEMUCHIKAL MANIKANDAN MANJUSHA, ROHIT KUMAR, ABHISHEK CHANDRA SAXENA, PRAKASH KINJAVDEKAR, 291 UJJWAL KUMAR DE, ABHIJIT MOTIRAM PAWDE, AMARPAL

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E. Fiore et al. Large Animal Review 2021; 27: 243-249

Clinical follow-up in fattening bulls affected by Bovine Respiratory Disease (BRD) after pharmacological treatment with Tulathromycin and Ketoprofen: clinical score and ultrasonographic evaluations

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ENRICO FIORE1, ANASTASIA LISUZZO1*, ANDREA BELTRAME2, BARBARA CONTIERO1, ELIANA SCHIAVON3, ELISA MAZZOTTA1 1

Department of Animal Medicine, Production and Health, University of Padua, Viale dell’Università 16, 35020 Legnaro, Italy 2 Veterinarian free Practitioner 3 Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), Viale dell’Università 10, 35020 Legnaro, Italy

SUMMARY Bovine respiratory disease (BRD) is recognized as one of the most important disease in beef industry because it may negatively influence animal welfare and farm economy. An early diagnosis is necessary to improve prognosis and outcome, as well to provide for specific management treatment for the affected animals. The aim of this study is to assess the feasibility of the ultrasonographic technique as diagnostic method for BRD in fattening bulls after pharmacological treatment with tulathromycin and ketoprofen. Sixty Limousine fattening bulls of 10.23 ± 1.37 months’ age belonging to a single farm were enrolled in this study. Animals were divided into two groups according to ultrasonographic score (US score) of lungs: Group C or control group (US score < 3; 29 animals), and Group D or disease group (US score ≥ 3; 31 animals). Both groups received a clinical examination and a TUS (thoracic ultrasonography) at time 0 (T0; day of restocking) and time 7 (T7; +21 days). Furthermore, group D received additional clinical examinations and TUS at time 1 (T1; day of diagnosis and treatment), time 2 (T2; +1 day), time 3 (T3; +2 days), time 4 (T4; +3 days), time 5 (T5; +7 days), and time 6 (T6; +14 days). Statistical differences between groups were evaluated by parametric and not-parametric test. The lung lesions such as total areas of hepatization and fluid alveolograms were measured and analyzed with mixed regression models over time. The two groups showed a significant difference both in US score and clinical symptoms (respiratory score, nasal and ocular discharges and rectal temperature) when comparing T1 of group D with T0 of group C. Over the time, the treatment of group D was effective in reducing the US score and rectal temperature after 2 and 1 days, respectively, while ocular discharges was reduced after 2 days. The ultrasound evaluation of the general health status showed an improvement after 7 days from treatment. However, a reduction of total areas of hepatization and fluid alveolograms were evidenced in 3 days. In conclusion, the pulmonary ultrasound has been evidenced as a feasible and sensitive tool both for BRD early diagnosis and treatment effectiveness evaluation in fattening bulls.

KEY WORDS Ultrasonography; Lung’s lesions; Bovine Respiratory Disease; Tulathromycin; Fattening bulls.

INTRODUCTION Bovine respiratory disease (BRD) is a syndrome involving infectious agents, host immune response and environmental factors. Stressors as transport or dietary changes may predispose animals to primary infection by viral or bacterial pathogens1,2. Among viral agents that mostly contribute to BRD there are Bovine Herpesvirus type 1 (BHV-1), Parainfluenza-3 virus (PI3), Bovine Viral Diarrhea Virus (BVDV), Bovine Adenovirus and Bovine Respiratory Syncytial Virus (BRSV); whereas among bacterial agents there are Mannheimia haemolytica, Mycoplasma bovis, Pasteurella multocida, and Histophilus somni3. Clinical observations of systemic and respiratory signs are often used for the diagnosis of BRD. However, the combination

Corresponding Author: Anastasia Lisuzzo (anastasia.lisuzzo@phd.unipd.it).

of these parameters present a low sensitivity and specificity, near to 60%, with a consequent treatment of healthy subjects or missed detection of sick animals4,5. Actually, BRD may be present as subclinical form, with lung lesions and without clinical signs6. Furthermore, BRD is recognize as one of the most important disease in beef industry because it may negatively influences the growth, long-term productivity and increases the costs due to treatment, mortality, and decreased feed efficiency4,7,8. An early diagnosis is necessary to improve prognosis and outcome of BRD, such as to facilitate specific management and correct treatment of animals5,9. Ultrasonography is a non-invasive, cost-effective, practical and rapid method (only 20-30 seconds/time procedure in optimal condition) to diagnose BRD in field9-11. In fact, bacterial and viral agents of BRD may result in non-aerated lung lobules that allow the ultrasound investigation by the replacement of reverberation artifacts to hypoechoic and anechoic structures12-14. Compared to diagnosis based on clinical ob-

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servation, lung ultrasonography shows a greater sensitivity, between 80 to 94%, and specificity, between 94 to 100%, according to different conditions and techniques10. These findings are important to perform an early diagnosis in order to reduce animal suffering and to avoid unnecessary antimicrobial treatments to avoid the antimicrobial resistance15. The aim of this study is to assess the feasibility of the ultrasonographic technique as diagnostic method for BRD in fattening bulls after pharmacological treatment with tulathromycin and ketoprofen.

MATERIAL AND METHODS Animals and clinical examination This study was conducted in February 2021 in a single beeffattening herd (Isola della Scala, Verona, Italy). A single stock of sixty Limousine fattening bulls was enrolled in this study with an average age of 10.23 ± 1.37 months. Animals came from France and were kept on outdoor dirt-floor pens. A total mixed ration (TMR) was provided once a day and water was ad libitum. All animals were weighed and vaccinated against bovine Parainfluenza-3 Virus (PI3), Bovine Viral Diarrhea Virus (BVDV), Bovine Herpes Virus-1 (BHV-1) and Bovine Respiratory Syncytial Virus (BRSV) (Cattle Master 4; Zoetis Italia S.r.l., Rome, Italy); against Clostridium Spp. (Bravoxin 10; MSD Animal Health Italia S.r.l., Rome, Italy); and against Pasteurella haemolytica (Rispoval Pasteurella; Zoetis Italia S.r.l., Rome, Italy) on the day of restocking. Furthermore, an antiparasitic treatment with moxidectin (Cydectin 0.5% Pour on; Zoetis Italia S.r.l., Rome, Italy) was performed on the same day. All animals were evaluated after 21 days, and an average daily gain (ADG) was calculated. A vaccination booster for PI3, BVDV, BHV-1, BRSV and Clostridium Spp. was performed on the same day. All animals were singularly conducted in a cattle crush (Taurus MC Deluxe Gate; Te Pari, Oamaru, New Zeland) where received a clinical examination by veterinarian of Veterinary Teaching Hospital - University of Padua. The respiratory score (R score)16 based on cough, nasal and ocular discharges, and rectal temperature was conferred on a scale of 0 to 3 for each parameter. Animals were considered sick when the total score was greater or equal to 5, or if at least two parameters were reported over than 2. Due to the different cut-offs for identifying sick animals, percentage of diseased animals based on R score (DA%) were calculated.

Thoracic Ultrasonography (TUS) evaluation A thoracic area from 10th to 3rd intercostal space (ICS) was shaved before performing the thoracic ultrasonography (TUS) and ethyl alcohol (90%) was used as a transducing agent. TUS evaluation was performed with portable ultrasound scanner (MyLabOneTM, Esaote S.p.a., Genova, Italy) equipped with a multi-frequency convex probe (SC3421, Esaote S.p.a., Genova, Italy; 2.5-6.6 MHz). Six lung areas were investigated: between 10th and 7th ICS for the caudal lung; between 6th and 5th ICS for the middle lung; and between 4th and 3rd ICS for the cranial lung of both sides. The firsts two ICS were not evaluated due to the heavy forelimb muscles of post-weaned beef calves as described by Pravettoni et al. (2021). All scans were performed with constant ul-

trasound settings frequency of 4.3 MHz, 15 cm depth acoustics window, 100% gray scale gain, and time-gain compensation was in a neutral position. The ultrasonographic score (US score) on a 6-point scale10 was established during TUS. According to it, a score of 0 was equal to a normal aerated lung; 1 indicated diffuse comet-tail artifacts without consolidation; 2 indicated lobular consolidation; 3 indicated lobar consolidation; 4 lobar consolidations of two lobes; and 5 indicated lobar consolidation of three or more lobes. An US score greater or equal to 3 was consistent with bacterial bronchopneumonia. Images were saved in a digital imaging and communications in medicine (DICOM) format and used for a post-sampling quantitative assessment (MyLabDeskTM, Esaote S.p.a., Genova, Italy) of lungs’ lesions. The lung lesions such as hepatizations and fluid alveolograms were measured in cm2 for the six lung’s areas. The total lung consolidation represents the sum the US score for each area.

Experimental study design and laboratory analysis Animals were divided into two groups according to ultrasonographic score: group C and group D. The group C or control group had an US score lesser than 3 and enrolled 29 fattening bulls. Group D or disease group had an US score greater or equal to 3 and enrolled 31 fattening bulls. These animals were considered affected by bovine respiratory disease (BRD)10 and received a single subcutaneous (SC) injection of a long-acting macrolide and non-steroidal anti-inflammatory (FANS) at the day of diagnosis (tulathromycin and ketoprofen, 2,5 mg/kg + 3 mg/kg, Draxxin plus, Zoetis Italia S.r.l., Rome, Italy). Nasal swabs were collected from group D animals before drug treatment (35 cm swab length, and agarized blister pack; Medical Wire & Equipment Co Ltd, Corsham, United Kingdom). The nasal swabs were gathered in a cold box at 4 °C and delivered refrigerated laboratory of Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe; Legnaro, Padua, Italy) within 1 hour. The nasal swabs were analyzed for bacterial culture and Minimum Inhibitory Concentration (MIC) evaluation. Group C and group D received clinical examination and TUS at time 0 (T0; day of restocking) and time 7 (T7; after 21 days). Furthermore, group D received additional clinical examinations and TUS at time 1 (T1; day of diagnosis and treatment), time 2 (T2; after 1 day), time 3 (T3; after 2 days), time 4 (T4; after 3 days), time 5 (T5; after 7 days), and time 6 (T6; after 14 days). No one animal of group C showed clinical signs suggestive for BRD or presented an US score greater or equal to 3 during the study. Moreover, 15 of the 60 beef fattening bulls presented a TUS indicative for BRD at the day of restocking, despite it was not possible the T0 evaluations for these animals.

Data elaboration In order to perform the statistical analysis, data were converted to numeric scale. Specifically, nasal and ocular discharges relevance was pointed as follow: absent = 0; unilateral = 1; bilateral = 2; and abundant bilateral = 3. Whereas lung TUS evaluations outlined a lesion scores: absence of lesions = 0; comet tail = 1; hepatization = 2; fluid alveolograms = 3; comet tail and hepatization = 4; comet tail and fluid alveolograms = 5; hepatization and fluid alveolograms = 6; comet tail, hepatization and fluid alveolograms = 7. The global lesion score was obtained through the sum of lesion score of each area.

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Statistical analysis

RESULTS

Statistical analysis was performed using R software ver. 4.0.317 implemented with “rcmdr” package and with S.A.S system software (version 9.4; SAS Institute Inc., Cary, North Caroline, USA). In general, a p-value ≤ 0.05 was accepted as significant. The data distribution was assessed by Shapiro-Wilk normality test before all statistical analysis. Statistical differences between the two groups were evaluated according to data distribution: two unpaired sample Wilcoxon test for not normally distributed data (US score, R score, nasal and ocular discharges) and t-test for normally distributed data (rectal temperature). The DA% was evaluated with z-test for two samples, whereas ADG was evaluated with one-way ANOVA test. A linear mixed models (LMM) was used to analyze differences of weight. The model considered as fixed factors group, time, and their interaction. Animal was considered as random and repeated effect. The follow-up of group D during time was performed with Kruskal-Wallis test for not normally distributed data (US score, R score, nasal and ocular discharges, global lesion score, lesion score, hepatization’s areas and fluid alveolograms’ areas). The DA% was evaluated with k proportions chi-squares test. An LMM was used for rectal temperature. The model included the fixed factor of time, whereas animals was considered as repeated and random effect. The same model implemented with rectal temperature as covariate was used to evaluate the total area of hepatization. Instead, the total area of fluid alveolograms was evaluated with a generalized linear mixed model zero-inflated (GLIMMIX) with Poisson distribution which considered the same factors as previous model. For all mixed models, post-hoc pairwise comparison among least squares means were performed using Bonferroni correction.

The two groups did not show differences in weight at T0 and T7 (p-value = 0.398), and in ADG (1.72 kg/day in group C compared to 1.90 kg/day in group D; SEM = 0.15; p-value = 0.389). Furthermore, a significant time effect was evidenced (p-value < 0.0001) with a greater weight at T7 (407 kg for group C and 398 Kg for group D, SEM =6.52) compared to T0 (371 kg for group C and 358 kg for group D, SEM=6.42). No significant difference in US score between groups at T0 (median value of 2 with interquartile range (IQR) of 2-2 in both groups in clinical findings were reported (Figure 1); p-value = 0.054). Whereas, comparing group D at T1 to group C at T0, a significant difference was evidenced (median value of 2 with IQR of 2-2 for group C and 3 with IQR of 3-4 for group D; pvalue < 0.001). Alternatively, US score at T7 did not show difference between groups (median value of 2 with interquartile range (IQR) of 2-2 in both groups; p-value = 1.000). R score did not report significant difference at T0 (p-value = 0.219), whereas a significant difference was showed between group D at T1 and group C at T0 (median value of 3 with IQR of 2.75-5 for group C and 5 with IQR of 4.5-6 for group D; pvalue < 0.001), and between the two groups at T7 (median value 3 with IQR of 3-5 for group C and 5 with IQR of 3.75-5 for group D; p-value = 0.006). The DA% was significantly different between groups at T0 (42.86% for group C and 0% for group D; p-value = 0.036), between group D at T1 versus group C at T0 (42.86% for group C and 83.87% for group D; p-value = 0.001), and between groups at T7 (40.00% for group C and 75.00% for group D; p-value = 0.019). Cough was never observed in animals of both groups during the study. Nasal discharge was not significant at T0 (p-value = 0.105), while it was significantly different between group D at T1 and group C at

Figure 1 - Box plot of clinical examination [ultrasonographic score (US score), respiratory score (R score), ocular and nasal discharges, and rectal temperature] of both groups (group D = orange, and group C = blue) at T0 and T7, and between T1 of group D and T0 of group C (T0/T1). Asterisk (*) indicates significant differences between groups within parameter and time points.

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T0 (median value 0 with IQR of 0-2 for group C and 2 with IQR of 2-2 for group D; p-value < 0.001). However, this parameter was not significant at T7 (p-value = 0.097). Ocular discharge was not significant at T0 (p-value = 0.907), while it differed significantly between group D at T1 and group C at T0 (median value 0 with IQR of 0-0 for group C and 0 with IQR of 0-1 for group D; p-value = 0.039). However, a not significant difference was evidenced at T7 (p-value = 0.065). Temperature assessment by rectal measurement was not significantly different between groups at T0 or T7 (p-value = 0.973 and 0.090, respectively), whereas a significant difference was present between group D at T1 and group C at T0 (39.45±0.46 °C in group C and 40.07±0.08 °C in group D; p-value < 0.001). In group D a significant difference was observed in all the examined clinical parameters over time (US score, p-value < 0.001; R score, p-value < 0.001; DA%, p-value < 0.001; nasal and ocular discharges, p-value < 0.001 and p-value = 0.003, respectively; and rectal temperature, p-value < 0.001; Figure 2). US score at T0 was significantly different from T1 (median value of 2 with IQR of 2-2 versus 3 with IQR of 3-4), whereas T1 varied significantly from T3 (median value of 2 with IQR of 2-2.25), T4, T5, T6, and T7 (median value of 2 with IQR of 22 for T4, T5, T6, and T7). R score at T0 was significantly different from T1 (median value of 3 with IQR of 2-3 versus 5 with IQR of 4.5-6), T2 (median value of 5 with IQR of 4-5), T3 (median value of 5 with IQR of 4-5), T4 (median value of 5 with IQR of 4-5), and T7 (median value of 5 with IQR of 3.75-5.25), whereas T1 differed from T5 (median value of 4 with IQR of 2.25-5) and T6 (median value of 3.5 with IQR of 3-5). DA% at T0 (0%) was significantly variated from all other time points (T1: 83.87%, T2: 92.86%, T3: 100%, T4: 87.10%, T5: 63.33%, T6: 46.15%, and T7: 75.00%), whereas no significant difference were evidenced between T1 and the other time points. Nasal

discharge at T0 were significantly different from T1, T2, T3, and T4 (median value of 0 with IQR of 0-0 at T0 versus 2 with IQR of 2-2 at T1, T2, T3, and T4), whereas T1 did not show a significant variation within the other time points. Ocular discharge at T0 differed significantly from T1 (median value of 0 with IQR of 0-0 versus 0 with IQR of 0-1), whereas T1 differed from T3, T4, T5, and T6 (median value of 0 with IQR of 0-0 for T3, T4, T5, and T6). Rectal temperature showed a significant changes between T0 and T1 (39.37±0.12 °C versus 40.07±0.08 °C), nevertheless T1 differed significantly from T2, T3, T4, T5, and T6. The TUS evaluation in group D during clinical follow-up evidenced not significant changes about the lesion scores among the lung caudal areas of (p-value = 0.165 and 0.622 for left and right caudal areas, respectively) and cranial lung areas (p-value = 0.073 and 0.454 for left and right cranial areas, respectively); conversely, significant variations in lesion scores among the lung middle areas (p-value < 0.001 and 0.001 for left and right middle areas, respectively) were reported. Particularly, TUS examination of the lung middle left area at T0 differed significantly from T1 (median value of 0 with IQR of 0-1 versus 3 with IQR of 0.75-6), whereas T1 varied significantly from T5, T6, and T7 (median value of 0 with IQR of 0-0 for T5 and T6, and IQR of 0-1 for T7). Furthermore, the TUS of the lung middle right area at T0 differed from T1 (middle value of 2 with IQR of 1-2 versus 2 with IQR of 0-4.5), while T1 differed significantly from T5, T6, and T7 (median value of 0 with IQR of 0-0.75 for T5, IQR of 0-0 for T6, and IQR of 0-0.5 for T7). The clinical follow-up showed a significant positive trend of the global lesion score (p-value < 0.001) (Figure 3), with a difference of T5, T6 and T7 (median value of 4 with IQR of 4-8 for T5 and T6, and IQR of 2-8 for T7) from T1 (median value of 12.5 with IQR of 10-16).

Figure 2 - Box plot of clinical examination [ultrasonographic score (US score), respiratory score (R score), ocular and nasal discharges, and rectal temperature] of group D during clinical follow-up over time. One asterisk (*) indicates significant differences of time points from T0 within parameter, while two asterisk (**) indicates significant differences of time points from T1.

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Figure 3 - Box plot of global lesion score during clinical follow-up of group D over time. One asterisk (*) indicates significant differences of time points from T0 within parameter, while two asterisk (**) indicates significant differences of time points from T1.

In group D, the hepatization areas concerning both caudal lung regions did not present any significant trend over time (p-value = 0.213 and 0.622, respectively for left and right caudal lung); conversely, a significant positive trend for the middle lung (pvalue < 0.001 and 0.001, respectively for left and right caudal lung). The evaluation of hepatized lungs areas of the cranial regions reported a significant difference in cranial right lung (p-value = 0.024) rather than cranial left lung (p-value = 0.086). Hepatization areas of the middle-left lung showed a significant reduction among T1, T5 and T6 (median value of 2 with IQR of 0-3.68 cm2 versus 0 with IQR 0-0 cm2 in T5 and T6), whereas the same parameter in right lung was sensibly reduced between T1 and T6 (median value of 1.64 with IQR of 0-4.79 cm2 versus 0 with IQR 0-0 cm2). In addition, cranial right lung evaluation showed evidenced significant reduction between T1 and T5 (median value of 4.31 with IQR of 2.32-4.61 cm2 versus 2.11 with IQR 1.30-3.09 cm2). Finally, total hepatization areas (Figure 4) reported a significant decrease between T1 (10.99±0.76 cm2) and T4, T5, T6 and T7 (6.72±0.65, 5.16±0.66, 4.64±0.70 and 5.02±0.75 cm2, respectively) (p-value < 0.001). The fluid alveolograms areas of both caudal lungs’ regions (pvalue = 0.500 and 0.525, respectively for left and right caudal lung) and cranial right lung (p-value = 0.267) were not significantly changed over time. Conversely, the middle lung areas reported significant variations (p-value < 0.001 and 0.001, respectively for left and right caudal lung), as though; the cranial left lung regions (p-value = 0.029). Fluid alveolograms areas of the middle-left lung showed a difference between T0 and T1 (median value of 0 with IQR of 0-0 cm2 versus 0 with IQR of 0-0.27 cm2) and between T1 and other time points (median value of 0 with IQR of 0-0 cm2); in right lung, this ultrasonographic parameter showed a similar trend in T1 (median value of 0 and IQR of 0-0.05 cm2). Cranial left lung areas reported differences among T0 and T1, and T2 (median value of 0 with IQR 0-0 cm2 versus 0 with IQR 0-0.57 cm2 for T1 and IQR of 0-0.64 cm2 for T2), and between T1 and T3, T4, T5, T6 and T7 (median value of 0 with IQR 0-0.08 cm2 for T3, IQR 0-0.10 cm2 for T4, IQR 0-0 cm2 for T5, T6 and T7). Furthermore, the total fluid alveolograms areas presented significant differences among T1 (1.73±0.44 cm2) and T4, T5, T6, T7 (0.26±0.13, 0.31±0.14, 0.18±0.12, 0.21±0.14 cm2) (p-value = 0.004) (Figure 5).

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Figure 4 - Box plot of total area of hepatization in group D during clinical follow-up over time. One asterisk (*) indicates significant differences of time points from T0 within parameter, while two asterisk (**) indicates significant differences of time points from T1.

Figure 5 - Box plot of total area of fluid alveolograms in group D during clinical follow-up over time. One asterisk (*) indicates significant differences of time points from T0 within parameter, while two asterisk (**) indicates significant differences of time points from T1.

DISCUSSION Animal’s transport for restocking represents an important stressor which may predispose beef cattle to infectious diseases. BRD is usually suspected when the animals show respiratory and ocular signs, besides the subclinical presentations are frequently not detected. Nevertheless, an early diagnosis is essential to avoid economic losses and preserve animal welfare8,18,19. Animals affected by BRD often report lower ADG values compared to healthy10,20. The administration of tulathromycin have been reported to improve the ADG in BRD affected animals comparing to the control group21,22. This finding may be associate to a manipulation of ruminal fermentation that may positively influence animals’ productivity3,23,24. Our results report no statistical differences between healthy and sick animals both in ADG or in body weight during the study period. These findings may suggest that an early treatment with tulathromycin could decrease the possibility of body weight reduction during the firsts 21 days of restocking. R score showed greater value in group D at T1 compared to group C at T0, but no differences were evidenced between

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Clinical follow-up in fattening bulls affected by Bovine Respiratory Disease (BRD) after pharmacological treatment

groups at T0. The greater R score in group D at T1 may be attributed to higher incidence of nasal discharge, hyperthermia, and ocular discharges in this group. The evaluation of these parameters may be difficult because of the clinical presentation can be inconstant in terms of severity and persistency, particularly during the viral replication (hyperthermia followed by nasal and ocular discharges)2,6,25. DA% showed greater value in group D at T1 compared to group C at T0, besides the control group reported a greater value compared to group D and C at T0. This finding may be associated to the detection of hyperthermia and the absence of cough. These parameters may be influenced by the stress for the animals during the operators handlings5,19. According to the literature, in our study 40% of sick animals were detected in group C (R score), confirming that clinical diagnosis may lack in specificity26. Macrolides are often use to treat BRD due to their bacteriostatic capacity, accumulation on leucocytes and bronchial secretions, and for their greater concentration in lung tissue than plasma3,27. Particularly, tulathromycin has been reported effective both in targeting pathogens and in modulating the inflammatory response such as avoid uncontrolled self-perpetuating inflammation during BRD28. In addition, tulathromycin shows a higher clinical efficacy compared to other antimicrobial, as previously reported Nutsch et al. (2005) and Torres et al. (2013). Animals in group D showed significant greater US score, R score, DA%, nasal discharge and rectal temperature, whereas ocular discharge values at T1 (day of diagnosis and treatment) compared to T0 (day of restocking). These findings confirm the BRD typical presentation characterized by respiratory and systemic signs6. During the clinical follow-up after the treatment with tulathromycin and ketoprofen, the US score showed an improvement after 2 days (T3) and until the end of the study. The improvement of ultrasound lung scores may indicate the healing of the lung tissue30. R score improved after 7 days (T5) and until 14 days (T6). However, among the parameters considered for R score, nasal discharge did not show a significant recovery, while ocular discharge and rectal temperature improved after 2 days (T3) and 1 day (T2), respectively, and until 14 days (T6). Nevertheless, DA% did not improve after the treatment. Finally, US score, nasal and ocular discharges, or body temperature assessments reported no differences between group D and C at the end of the study period. Conversely, R score and DA% were statistically higher in group D. TUS can be used to evaluate the severity of lung lesions represented as different degrees of non-aerated lung tissue (consolidation) due to inflammation and exudate deposits14,31. In group D, the global lesion score and the pulmonary middle areas score of both lungs showed a significant improvement after 7 days from treatment (T5) and until the end of the study. These findings suggest that general health status of lungs and single health status of middle areas improved after 7 days from treatment since the lesion score derives from the presence and the type of lesions on the investigated areas, while the global lesion score derives from the sum of the single lesion score. The areas of hepatization and fluid alveolograms were evaluated to support the analysis of lung health status after treatment. In the present study, hepatization areas of middle left lung and cranial right lung improved after 7 days (T5), whereas they were improved after 14 days (T6) in the middle right lung. However,

the total hepatization areas showed a significant improvement after only 3 days from treatment (T4), with a reduction of about 6.00 cm2 after 7 days (T5) and until the end of the study. However, some consolidated areas were still present after 21 days. The fluid alveolograms areas were improved after 1 days (T2), in the middle area of both lungs, and after 2 days (T3), in the cranial left lung until the end of the study. Moreover, the total area of fluid alveolograms improved after 3 days (T4) with a reduction of about 1.50 cm2 after 7 days until the end of the study. According to the literature, the present study suggest that the combination of tulathromycin and ketoprofen can be successfully used in animals affected by BRD to treat acute lung lesions, particularly effective for the most sensitive pulmonic areas (cranial and middle lung of both sides)14,21,30. Moreover, the antimicrobics administration seems to be more efficient during early stage of the disease, in order to prevent the onset of chronic and severe pulmonary lesions.

CONCLUSION The use of pulmonary ultrasound was recognized to be a sensitive diagnostic method to assess respiratory diseases and the response to treatment in fattening bulls. Moreover, tulathromycin and ketoprofen treatment was effective in reducing the ultrasound score and rectal temperature after 2 and 1 days, respectively. Despite nasal discharge was not improved after the treatment, the ocular discharge was reduced after 2 days from the medical protocol. This study confirmed the cranial and middle areas of both lung sides are the most affected during BRD. The medical treatment has been reported efficient in reducing lung lesion after 3 days. Some areas of lung hepatization remained at the end of the study, confirming that the treatment is more effective in acute conditions of the disease. Acknowledgments: The authors would like to thank the veterinarian Pietro di Salvo from Zoetis S.r.l. company and workers of the farm Cooperativa Agricola Zootecnica “La Torre” (Isola della Scala, Verona, Italy) for their help during the study. Author Contributors: Conceptualization, E.F., A.L., and E.M.; Methodology, E.F., A.L., and E.M.; Software, B.C.; Formal analysis, A.L., B.C., and E.S.; Investigation, E.F., A.L., and A.B.; Resources, E.F., and E.S.; Data curation, E.F., and A.L.; Writingoriginal draft preparation, E.F., and A.L.; Writing-review and editing, E.F., A.L., E.M., and E.S.; Visualization, A.L.; Supervision, E.F.; Project administration, E.F.; Funding acquisition, E.F. All authors have read and agreed to the published version of the manuscript. Funding: The study was supported by Zoetis S.r.l. regarding the costs of drug and ultrasound evaluations on the farm. Ethical approval: No approval from Ethics Committee was required. No invasive medical procedures were executed to perform the study. The study was performed with the consent of the animals’ owner during the routinary clinical activity of the Veterinary Teaching Hospital, University of Padua. Animal care and procedures are in accordance with the Guide for the Care and Use of Laboratory Animals and Directive 2010/63/EU for animal experiments (National law: D.L. 26/2014). Data Availability Statement: The data will be available by sending an email to the corresponding author. Conflict of Interest: The authors declare no conflict of interest.

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17. Team R Development Core A Language and Environment for Statistical Computing. R Found. Stat. Comput. 2018, 2, https://www.R-project.org. 18. Buckham Sporer, K.R.; Weber, P.S.D.; Burton, J.L.; Earley, B.; Crowe, M.A. Transportation of young beef bulls alters circulating physiological parameters that may be effective biomarkers of stress. J. Anim. Sci. 2008, 86, 1325-1334, doi:10.2527/jas.2007-0762. 19. Timsit, E.; Dendukuri, N.; Schiller, I.; Buczinski, S. Diagnostic accuracy of clinical illness for bovine respiratory disease (BRD) diagnosis in beef cattle placed in feedlots: A systematic literature review and hierarchical Bayesian latent-class meta-analysis. Prev. Vet. Med. 2016, 135, 67-73, doi:10.1016/j.prevetmed.2016.11.006. 20. Kiser, J.N.; Lawrence, T.E.; Neupane, M.; Seabury, C.M.; Taylor, J.F.; Womack, J.E.; Neibergs, H.L. Rapid communication: Subclinical bovine respiratory disease - loci and pathogens associated with lung lesions in feedlot cattle. J. Anim. Sci. 2017, 95, 2726-2731, doi:10.2527/jas2017.1548. 21. Godinho, K.S.; Wolf, R.M.L.G.; Sherington, J.; Rowan, T.G.; Sunderland, S.J.; Evans, N.A. Efficacy of tulathromycin in the treatment and prevention of natural outbreaks of bovine respiratory disease in European cattle. Vet. Ther. 2005, 6, 122-135. 22. Nutsch, R.G.; Skogerboe, T.L.; Rooney, K.A.; Weigel, D.J.; Gajewski, K.; Lechtenberg, K.F. Comparative efficacy of tulathromycin, tilmicosin, and florfenicol in the treatment of bovine respiratory disease in stocker cattle. Vet. Ther. 2005, 6, 167-179. 23. Armato, L.; Gianesella, M.; Fiore, E.; Arfuso, F.; Rizzo, M.; Zumbo, A.; Giudice, E.; Piccione, G.; Morgante, M. Effect of live yeast & yeast cell wall Saccharomyces cerevisiae diet supplementation on faeces chemical composition and growth performance in growing and finishing beef steers. Large Anim. Rev. 2016, 22, 203-210. 24. Armato, L.; Gianesella, M.; Morgante, M.; Fiore, E.; Rizzo, M.; Giudice, E.; Piccione, G. Rumen volatile fatty acids × dietary supplementation with live yeast and yeast cell wall in feedlot beef cattle. Acta Agric. Scand. A Anim. Sci. 2016, 66, 119-124, doi:10.1080/09064702.2016.1272628. 25. Blakebrough-Hall, C.; Dona, A.; D’occhio, M.J.; McMeniman, J.; González, L.A. Diagnosis of Bovine Respiratory Disease in feedlot cattle using blood 1H NMR metabolomics. Sci. Rep. 2020, 10, 1-12, doi:10.1038/s41598-01956809-w. 26. White, B.J.; Renter, D.G. Bayesian estimation of the performance of using clinical observations and harvest lung lesions for diagnosing bovine respiratory disease in post-weaned beef calves. J. Vet. Diagnostic Investig. 2009, 21, 446-453, doi:10.1177/104063870902100405. 27. Mzyk, D.A.; Bublitz, C.M.; Martinez, M.N.; Davis, J.L.; Baynes, R.E.; Smith, G.W. Impact of bovine respiratory disease on the pharmacokinetics of danofloxacin and tulathromycin in different ages of calves. PLoS One 2019, 14, 1-23, doi:10.1371/journal.pone.0218864. 28. Fischer, C.D.; Beatty, J.K.; Duquette, S.C.; Morck, D.W.; Lucas, M.J.; Buret, A.G. Direct and Indirect anti-inflammatory effects of tulathromycin in bovine macrophages: Inhibition of CXCL-8 secretion, induction of apoptosis, and promotion of efferocytosis. Antimicrob. Agents Chemother. 2013, 57, 1385-1393, doi:10.1128/AAC.01598-12. 29. Torres, S.; Thomson, D.U.; Bello, N.M.; Nosky, B.J.; Reinhardt, C.D. Field study of the comparative efficacy of gamithromycin and tulathromycin for the treatment of undifferentiated bovine respiratory disease complex in beef feedlot calves. Am. J. Vet. Res. 2013, 74, 847-853, doi:10.2460/ ajvr.74.6.847. 30. Hussein, H.A.; Binici, C.; Staufenbiel, R. Comparative evaluation of ultrasonography with clinical respiratory score in diagnosis and prognosis of respiratory diseases in weaned dairy buffalo and cattle calves. J. Anim. Sci. Technol. 2018, 60, 1-11. 31. Rademacher, R.D.; Buczinski, S.; Tripp, H.M.; Edmonds, M.D.; Johnson, E.G. Systematic thoracic ultrasonography in acute bovine respiratory disease of feedlot steers: impact of lung consolidation on diagnosis and prognosis in a case-control study. Bov. Pract. 2014, 48, 1-10.

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Effect of Yucca Schidigera inclusion in milk replacer for veal calves on health status, antimicrobial use and growth performance

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SILVIA GROSSI*1, RICCARDO COMPIANI1, GIANLUCA BALDI1, CARLO ANGELO SGOIFO ROSSI1 1

University of Milan, Faculty of Veterinary Medicine, Department of Veterinary Science for Health, Animal Production and Food Safety, Milan 20133, Italy

SUMMARY Antimicrobial resistance is a global health problem. White veal calves face many challenges during the first period of life which, in combination with their immature physiological systems, may explain their high susceptibility to infections and increased use of antibiotics, mainly in the form of mass preventive treatments. The use of antibiotic alternatives, such as natural extracts, to improve calf immune function is gaining interest in rearing white veal calves. This study evaluates the effect Yucca Schidigera inclusion in veal calf ’s milk replacer on immune functionality and growth performances. The trial involved 1015 male Friesian calves divided in two groups: control (CON) and treatment (TREAT), differing for the inclusion of Yucca Schidigera extracts. The zootechnical performances were evaluated: mortality, morbidity and slaughtering performances such as carcass weight, incidence of underweight carcasses, average daily gain and meat colour. At slaughterhouse, pulmonary score evaluation was performed to understand the severity and incidence of respiratory diseases. In terms of immune functionality and health status, the number, type and days of treatments were analysed. Also, on 30 calves per group, blood samples were taken to evaluate the serum antioxidant capacity and the haemoglobin level at d0 and d90. In terms of zootechnical performances, no statistically significant differences were found. The incidence of pulmonary lesions was comparable in the two groups. No statistically significant differences were found also in terms of haemoglobin levels and oxidative stress. ROMs and OXY levels were similar between groups (284.92 in TREAT vs 365.23 in CON for ROMs and 237.90 HClO/mL in TREAT vs 228.45 HClO/mL in CON for OXY). In terms of antibiotic use, the control group received both more mass (17 in TREAT vs 21 in CON) and individual treatments (140 calves treated in TREAT vs 300 in CON), with an average increase of the days on treatment per animal (44.85 in TREAT and 57.52 in CON). The inclusion of Yucca Schidigera allowed a reduction of the antibiotics use, but did not affects growth performance and carcass characteristics.

KEY WORDS Antimicrobial Resistance; Veal Calves; Natural Extract; Yucca Schidigera.

INTRODUCTION Antimicrobial resistance is a global health concern. Globally, 700,000 people each year die from infection with antibioticresistant organisms (AROs), one third in children aged under 5 years1 and it is projected to cause 10 million deaths per year by the year 2050. Also, infection caused by antibiotic resistant microorganism represent an increasingly important cost item in a state’s health care budget, that is expected to reach 100 trillion of dollars in 20502. The mechanisms besides the acquisition of resistance characteristics by microbes such as bacteria, is complex, and still unclear3. It is generally accepted that resistant bacteria are a result of selection pressure4. Suspected principal foci of selection

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

pressure include misuse and abuse of antimicrobials in both human medicine and food-producing animals farming for treatment or prevention (in the form of prophylaxis or metaphylaxis) of disease2,5. In a meta-analysis of data from 901 studies from 2000 to 2018, the proportion of antibiotics with resistance higher than 50% increased from 0.15 to 0.41 in chickens, 0.13 to 0.34 in pigs, and 0.12 and 0.23 in cattle. The highest resistance rates were observed among antibiotics used most commonly to avoid the onset of pathologies that can worsened animal productivity and welfare (tetracyclines, sulphonamides etc.)6. Among the different zootechnical species there are many differences in terms of use of antibiotics, considering the specific intrinsic characteristics of both the animals reared and the management systems. Globally, swine and poultry farming accounted for the highest use of antibiotics, while cattle farming is in third position in terms of milligrams per population correction units (PCU) (172 mg/PCU, 148 mg/PCU and 45 mg/PCU respectively)6. Also, the physiological stage of life of

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the animal reared can determine a different use of antibiotics. Young animals are more susceptible to diseases than adults and also their ability to cope is impaired, because of the low immune functionality and low antibody production. Consequently, the quantities and number of antimicrobials treatments are higher in young animals7,8,9. In swine farming, 80% of the total treatments are administered before the tenth week of life, and the most common are oral mass treatments7. Also, in beef cattle farming the use of antibiotics increase during the earlier stages of life. A clear example is the situation in white veal calves farming. The incidence of treatments, especially mass treatments, is much higher than in the fattening of adult beef cattle, and 12% to 13% of the total treatment are mass treatment10. White veal calves face many challenges during the first period of life, including birth, transportation, mixing procedures, inappropriate management conditions and new housing environments11. Furthermore, all these challenges occur at an age at which the calf is immature and several physiological systems are still developing and not completely functional yet, such as the gastrointestinal tract (GIT), the thermoregulatory and the acquired immune systems12. The combination of the indicated challenges and the immature physiological systems of the calves may explain the high susceptibility of calves to infections, and the higher use of antibiotics especially for enteric diseases and Bovine Respiratory Disease (BRD). While the first can sharply increase the mortality rate in the first 180 days of life, BRD is the main cause of production losses and welfare issues, with an economical loss of about 35 to 180 €. Traditionally, to prevent those heavy losses, farmers use metaphylactic mass treatments at the arrival and also others when the season or the specific situation of a batch are critical8,9. This situation must change because of antimicrobial resistance. In fact, in white veal farms the incidence of resistant bacteria is between highest in the zootechnical sector, specifically in terms of Methicillin-Resistant Staphylococcus Aureus (LA-MRSA)13. Alternative solutions to antibiotics have to be found, that can ameliorate the natural immune response of the calves, allowing to reduce the antibiotic use without an impairment in animal welfare and productivity standards. In the last few years, natural products such as oil and extracts, are increasingly being studied in animal nutrition, because of their specific functional properties. Those products contain a pool of different active compounds, such as flavonoids, glucosinolates and isoprene derivatives, that have had an antibiotics-like and antioxidant-like action in different studies, done either in vivo or in vitro14. The extract from Yucca Schidigera has been proven to be rich in functional compounds such as ellagic acid, quercetin, tannins and cinnamic acids, saponins and other phenolic compounds, such as yuccaols15. The main biological activities are related to yuccaols and saponins16,17. The Yucca Schidigera extracts have shown positive results in zootechnical applications in food-producing animals, monogastric and ruminants, both in terms of production performances and immune functionality18,19,20. In terms of cattle farming, the inclusion of Yucca Schidigera extracts has led to a better immune functionality in vivo21 (Mowat et al., 1999), mainly due to a better development of the immune response and a higher proliferation of the immune cells22 and to a modulatory activity on the inflammatory reaction, that limits its negative effects through an action on specific transcription factors23.

Furthermore, Yucca’s saponins can have a modulating action on gut microflora. The combination of those factors can lead to better growth performance, especially in the early stages of life and in stressful conditions, as highlighted by de Sousa et al. (2019)20. Furthermore, saponins from Yucca Schidigera have shown a potential antimicrobial effect against Escherichia coli, mainly due to a lytic action on the bacterial membrane24, and a potential anticoccidial effect in calves, reducing the excretion of oocysts25. The purpose of this study was to evaluate the potential effect of Yucca Schidigera extracts in the milk replacer of white veal calves on health status, immune functionality, antibiotic use and zootechnical performances, with the overall aim to reduce the use of antibiotics without welfare and productivity issues.

MATERIALS AND METHODS Animals, housing and experimental groups The study took place in a veal calves fattening facility, located in the Piemonte region. For the purpose of the study, calves were housed in two different barns, filled completely in one day each (17th February and 8th March). The trial started at 17th of February 2019 and end at 2th October 2019, following the entire fattening period. As the legislation reports, for the first 8 weeks of ages, all the calves were housed in single cage. For the other 8 months, calves were housed in group pens, with six calves each26. In the trial, 1015 male calves were involved, mostly HolsteinFriesian and some crossbreeds. In order to avoid the bias due to the batch, at the arrival, the animals were blocked by body weight, and assigned to the two experimental groups (Table 1), following the output of a numerical randomized procedure of Excel. In order to avoid a possible influence of the housing environment, both barns were divided into control and treatment. The two experimental group was: i) Control (CON), milk replacer without any nutraceutical inclusions; ii) Treatment (TREAT), milk replacer with the inclusion of two different nutraceutical supports, based on Yucca Schidigera extracts (Table 2). The first (MiniMix START 0-60) was specially formulated to promote the immune function and antioxidant status of the calves, while the second (MiniMix PROGRESS CB) to counteract the digestive disorders. The first nutraceutical support was administered for the first 60 days of fattening (15 g/head/day), while the second one only in case of digestive issues, for only 5-7 days (20 g/head/day) from the 40th day of life. The feeding plan and the characteristics of the milk replacers used were the same for the two experimental group. During the hole fattening period three different milk replacer were used, to better satisfy the specific nutritional requirements in the different physiological phases, accordingly to the NRC (2001)27. The Yucca Schidigera extracts was mixed directly into the milk replacer. Milk was offered in two equal meals daily at 09:00 and 17:00. All the calves were bucket-fed the milk-replacer diet. During the hole fattening period all the calves received the same type and amounts of solid feed. All the calves had free access to the water. In terms of sanitary management protocol, at restocking both the groups receive oxytetracycline for eleven days as a preventive mass treatment. Calves from the control group received also colistin, as another mass treatment at restocking, for five

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Table 1 - Experimental groups: CON vs TREAT. CON

TREAT

Characteristics n of calves

490

525

Average arrival weight, kg

47.8

47.6

24.1

22.8

Average arrival age, d

Restocking sanitary protocol d0 to d5

Mass feed treatment: Oxytetracycline + Colistin

Mass feed treatment: Oxytetracycline

Nutraceutical support d0 to d60

15 g/head/day of MiniMix Start 0-60

In case of digestive disorders

20 g/head/day of MiniMix Progress CB

Table 2 - Characteristics of the two nutraceutical premixes.

Components

MiniMix START 0-60

MiniMix PROGRESS CB

Yucca Schidigera extracts and dehydrated yeast culture of Saccharomyces cerevisiae

20,00

11,00

Analytical values, % DM Crude protein Crude fats

1,50

0,90

Ash

11,00

23,00

Cellulose

23,00

12,50

0,75

1,80

Methionine

0,30

0,15

Doses (g/head/day)

days, as reported in Table 1. After restocking the standard sanitary management protocols didn’t differ between the two groups.

Parameters Indicators of the immune functionality were analysed in combination with data about health status, antimicrobials use and zootechnical parameters, observed both at the farm level and at slaughterhouse.

Production performances, health status and antibiotic use The animals were monitored on a daily basis by the farm’s veterinary staff. Mortality and morbidity were recorded daily with also the relative causes. The veterinary treatments administered in both groups were recorded on the farm register, divided in individual and mass treatments. The quantities of antibiotics administered were calculated, based not only on the number of animals involved, but also on the duration of each intervention (days on treatment). At slaughterhouse, the carcass weight, fattening and conformation score and colour indicators were recorded. The average daily gain (ADG, kg/head/day of weight) was then calculated. Furthermore, during the post mortem inspection, the pulmonary scores were done, according to Leruste et al. (2012)28. Both the left and right lungs were examined (cranial and caudal lobe for the left lung and cranial, caudal and intermediate lobe for the right lung). The presence of lung lesions was eval-

uated, in terms of both number and also severity and extension, with a 0 to 3 evaluation score, reported in Table 3.

Evaluation of oxidative stress levels (TEST ROMs and OXY) On 30 calves per group, at d0 and d90, blood samples were taken to determine the antioxidant status. Blood samples were collected from the jugular vein using tubes with EDTA just before morning feeding, and the tubes were immediately placed on ice. The collected samples were centrifuged at 2500 × g for 15 min at 4 °C, and thereafter plasma samples were stored at -20 °C until analysis. Serum was analysed for serum antioxidant status, through the oxygen reactive metabolites (ROMs) test, and for antioxidant capacity through the OXY-adsorbent test. The ROMs test was done according to Iorio et al. (2003)29. The ROMs test allows to determine the blood concentration of the hydroperoxides (ROOH), substances belonging to the wide class of the so-called reactive oxygen metabolites (Reactive Oxygen Metabolites, ROMs), markers of the tissue damage generated by the peroxidation of lipids, amino acids, proteins, and nucleic acids. In this test, hydroperoxides, after reacting with a suitably buffered chromogen (N, N-diethyl-para-phenyl diamin), develop a coloured derivative, which is detected photometrically. The concentration of hydroperoxides, directly proportional to the intensity of the colour, it is expressed in Carratelli Units (1 U CARR= 0.08 mg hydrogen peroxide / dL). The OXY-adsorbent test was done according to Iorio et al.

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Table 3 - Lungs evaluation. Score*

Images

Healthy lungs, light pink-orange colour

Notes

Lungs with a small lesion grey-red coloured

Lungs with one big or several small lesions grey-red coloured, with total surface smaller than a lobe

Lungs with lesion greyred coloured, with a total surface of at least a lobe and/or presence of abscesses

*Evaluation scale based on Leruste et al., (2012)

(2003)29. This test evaluates the capacity of the serum samples to cope with a massive oxidant attack, inducted in vitro by a hypochlorous acid solution. Unreacted HClO radicals further react with the chromogen solution of N, N-diethyl-p-phenylendiamine and form a colored complex, which is measured at 505 nm. The results of the test are expressed as mol HClO/mL. The highest are the results, the better is the defence ability of the plasma.

to analysis of variance, using the General Linear Model procedure of SAS.

RESULTS Zootechnical, health and productive parameters The zootechnical and productive data, registered at the farm level and at slaughterhouse, are summarized in Table 4. For these parameters, no statistically significant differences were found. Mortality and incidence of lighter carcasses, weighing less than

Statistical analysis Statistical analyses were performed using SAS 9.3 (2010; SAS Institute Inc., Cary, NC, USA). The data obtained were subjected Table 4 - Zootechnical performances. N°

Mortality, % (n)

ADG, kg/d

Carcass Weight, kg

Losses (<110 kg), %

Color L,

Control

490

2.86 (14)

1.270

170.23

1.26

41.71

10.01

4.26

Treatment

525

4.76 (25)

1.273

169.17

2.00

42.14

9.77

4.21

Table 5 - Results of the pulmonary score evaluation. Score 1

Pulmonary Score, % (n) Score 2 Score 3

Score 4

Control

43.61 (181)

19.04 (79)

23.61 (98)

13.73 (57)

Treatment

46.88 (210)

15.40 (69)

23.88 (107)

13.84 (62)

Table 6 - Evaluation of the oxidative stress at day 0 and 90 and evaluation of the haemoglobin level during the hole fattening period. day

Control

Treatment

ROMs, U/Carr

d0 d90

259.00 365.23

258.69 284.91

OXY, µmol HClO/mL

d0 d90

316.37 228.45

303.81 237.90

Hb, g/dL

d0 d90

9.29 8.92

9.14 8.92

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S. Grossi et al. Large Animal Review 2021; 27: 251-257

255

Table 7 - Individual treatment. Stable B1 Stable C1 C

Total calves per group

265

N° calves treated individually

152

Weighed Total (n° calves* days of treatment) C T

253

225

272

490

525

148

931

425

19.046 38.8

20.761 39.5

Total days of treatment Average treatment days for each calf

110 kg, were similar between groups (respectively, 2.86% vs 4.76% in TREAT and 1.26% vs 2% in CON). No differences were also found for ADG, final carcass weight and colorimetric characteristics. The results of the pulmonary inspection are summarized in Table 5. Also, for pulmonary score there were no statistically significant differences between the two groups. The higher number of severe lesions (score 2 and 3) highlighted in the TREAT group was only a function of the greater numerosity of calves in this group (525 in TREAT vs 490 in CON). In fact, in percentage terms, the distribution of observations in the 4 different classes was similar between the two groups.

Evaluation of oxidative stress levels (TEST ROMs and OXY) In Table 6 are reported the data obtained from the ROMs and OXY analysis at d0 and d90. No statistically significant differences were found also for those parameters. Indeed, the inclusion of the two nutraceutical nu-

trients did not affect the antioxidant capacity of calves. In both groups at d90 the level of ROMs (284 U/Carr in TREAT vs 365 U/Carr in CON), and OXY (237.90 HClO/mL in TREAT vs 228.45 HClO/mL in CON) were similar.

Antimicrobials use: type of treatment and days on treatment In Table 7 and 8 are reported all the treatments done in both stable and in both groups, divided for “individual” (Table 7) or “mass” treatment (Table 8). Also, the number of calves treated and the days on treatment are reported, to better quantify the use of antibiotics in both groups. As visible in Table 7, the number of calves treated individually in the CON group was higher (152 and 148 in CON vs 45 e 95 in TREAT), but because calves treated in TREAT group undergone to a longer treatment period, the total days on individual treatment didn’t differ between the two groups. As visible in Table 8, calves from the CON group received two more mass treatment in each barn, one (colistine) as a part of

Table 8 - Mass treatment, total treatment and days on treatment per group. Stable B1 Stable C1 C Total calves per group Mass treatment 15-25/02 15-19/02 20-25/02 22/02 02-04/03 08-15/03 08-11/03 11-15/03 16/03 16-20/03 27-30/03 05-08/04 24-28/04 11-16/05 26-30/05 09-13/05 18-23/06 09-13/07 02-06/08 26-31/08 N° calves treated individually

265

253

Oxytetracicline Colistine Sulf. + Trimethoprim Tulathromycin Tildipirosin Oxytetracicline Doxicicline Amoxicillin Amoxicillin Oxytetracicline Oxytetracicline Amoxicillin 152

Weighed Total (n° calves* days of treatment) C T

225 -

272 -

Oxytetracicline Colistine Sulf. + Trimethoprim Tulathromycin Tildipirosin Oxytetracicline Doxicicline Amoxicillin Amoxicillin Oxytetracicline Oxytetracicline Amoxicillin 148

Total days on treatment Average days on treatment for each calf

490

525

27.255 2.915 1.325 1.590 265 795 1.800 900 1.125 225 1.325 900 900 1.325 1.350 1.325 1.125 2.940 2.450 1.325 1.350

23.119 2.783 253 2.176 272 1.265 1.088 1.088 1.265 1.632 1.265 1.360 3.150 2.625 1.265 1.632

931

425

28.186 57,52

23.544 44,85

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Effect of Yucca Schidigera inclusion in milk replacer for veal calves on health status

the standard sanitary protocol and one as a result of an impairment in the health status. CON calves resulted to be treated four more times than the TREAT group. Also, the total days on treatment was affected, even when the individual and mass treatment were considered together, resulting in higher total and average days on treatment in the CON group (Table 8).

DISCUSSION The spread of antimicrobial resistance and its implication in human health has led the zootechnical producers to rethink about their standard management and sanitary practices, to find out how to reduce this phenomenon. The white veal calf farming is also deeply involved in this process. Traditionally, a high use of orally administered mass treatments is done, especially for preventive purposes. Those type of treatments are considered the main cause of the growth of strains of resistant bacteria. This research was conducted to verify the effect of an inclusion of Yucca Schidigera extracts on growth, health, immune function of white veal calves and on the average use of antibiotics, specifically mass treatments. In bibliography it was proved that Yucca Schidigera can have many important biological implications, especially because of its content in saponins and resveratrol. Besides having a strong immunostimulatory capacity, it also promotes digestive function, both at ruminal and intestinal levels18. Considering the antioxidant status, as visible in Table 5, it seems that the administration of Yucca Schidigera extract did not significantly alleviate the oxidative stress, even if calves from the TREAT group showed a slightly lower values for ROM analyses and, on the other hand, slightly higher values for µmols of HClO/mL, factors indicating a better reaction of the antioxidant systems. However, other studies that involved Yucca Schidigera in animal nutrition and in in vitro tests, showed positive effects on the oxidative status of treated animals. Resveratrols and other specific polyphenols, called “yuccaols”, have been shown to have a remarkable antioxidant effect, often even superior to the reference antioxidants, both in vivo and in vitro tests (Test Teac)15,17. Unfortunately, the difference found in this study was only a numerical difference and it wasn’t statistically significative. Several studies have also shown a potential stimulating effect of saponins on immune cells and on the production of antibodies22. Neither there was a positive effect of yucca administration in terms of pulmonary score.

CONCLUSIONS Considering the result obtained in this trial, it can be concluded that the inclusion of a nutritional support based on natural extracts as Yucca Schidigera, is not sufficient to obtain a real and significant reduction in the consumption of antibiotics. The continuous spread of antimicrobial resistance pushes the zootechnical producers and technicians to study and search new strategies to reduce the use of antibiotics, without at the same time affecting animal welfare and productivity. Natural extracts and essential oils surely will have many applications in the future, because of their multiple biological functions. But, as underlined in this research, this is not enough to obtain a real im-

provement in antibiotic reduction. To achieve this result is necessary to rethink about the hole farming process, not only the nutritional integration. In fact, there are many different aspects that can impact on both animal immune function and pathogens circulation in white veal calves farming. Creating an “integrated management system” that consider all those aspects that can affect animal strengthens and welfare can be the only way to effectively reduce the use of antibiotics. All those factors can be summarized in the more general concept of “animal welfare”. In fact, a lower animal welfare can cause both a higher spread of pathogens and a stronger incidence of disease. It could be explained considering the effect of stress on immune function and production parameters. A lower welfare exposes the animals to a higher stress levels, that can have a detrimental effect on the immune system, lowering their ability to cope with pathogens. On one hand, it is necessary to individuate and limit all the risk factors that can lead to a higher microbial circulation, or to a less immune functionality. On the other side, it is important to find out how animal welfare and strengthens may be improved and act on those aspects.

Acknowledgment First author Silvia Grossi received Galloway Scotbeef award for Graduates at the University of Milan for € 3.000 for the realization of the experimental master’s thesis related to the livestock sector of beef cattle.

References 1.

World Health Organization (2019). New Report Calls for Urgent Action to Avert Antimicrobial Resistance Crisis. World Health Organization website. https://www.who.int/newsroom/ detail/29-04-2019-newreport-calls-for-urgentaction to avert-antimicrobial-resistance-crisis. 2. O’Neil J. (2016), AMR Review, Review on Antimicrobial Resistance: tacking drug-resistant infection globally: final report and recommendations. 3. Woolhouse M., Ward M., van Bunnik B., Farrar J. (2015) Antimicrobial resistance in humans, livestock and the wider environment. Phil. Trans. R. Soc. B 370: 20140083; http://dx.doi.org/10.1098/rstb.2014.0083. 4. Van den Bogaard A.E., Stobberingh E.E. (2000). Epidemiology of resistance to antibiotics. Links between animals and humans. Int J Antimicrob Agents.14(4):327-35. doi: 10.1016/s0924-8579(00)00145-x. PMID: 10794955. 5. Fish D.N., Ohlinger M.J. (2006). Antimicrobial resistance: factors and outcomes. Critical Care Clinics 22,291±311. https://doi.org/10.1016/ j.ccc.2006.02.006 PMID: 16678001. 6. Van Boeckel T.P., Brower C., Gilbert M., Grenfell B.T., Levin S.A, Robinson T.P., Teillant, A., Laxminarayan R. (2015). Global trends in antimicrobial use in food animals. PNAS first published March 19, 2015; https://doi.org/10.1073/pnas.1503141112. 7. Callens B., Persoon D., Maes D., Laanen M., Postma M., Boyen F., Haesebrouck F., Butaye P., Catry B., Dewulf J. (2012). Prophylactic and metaphylactic antimicrobial use in Belgian fattening pig herds. Preventive Veterinary Medicine, 106:53-62. 8. Pardon B., De Bleecker K., Hostens M., Callens J., Dewulf J., Deprez P. (2012a). Longitudinal study on morbility and mortality in white veal calves in Belgium. BMC Veterinary Research, 8(1):26. 9. Pardon B., Catry B., Dewulf J., Persoons D., Hostens M., De Bleecker K., Deprez P. (2012b). Prospective study on quantitative and qualitative antimicrobial and anti-inflammatory drug use in white veal calves. J. Antimicrob. Chemother., 67(4):1027-1038. 10. Pardon B., Hostens M., Duchateau L., Dewulf J., De Bleecker K., Deprez P. (2013). Impact of respiratory disease, diarrhea, otitis and arthritis on mortality and carcass traits in white veal calves. BMC Veterinary Research, 9:79. 11. Hulbert L.E., Moisa S.J. (2016). Stress, immunity, and the management of calves. J Dairy Sci., 99:3199–216. doi: 10.3168/jds.2015-10198.

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S. Grossi et al. Large Animal Review 2021; 27: 251-257 12. Marcato F., van den Brand H., Kemp B., van Reenen K. (2018). Evaluating Potential Biomarkers of Health and Performance in Veal Calves. Front. Vet. Sci. 5:133. doi: 10.3389/fvets.2018.0013. 13. Graveland H., Wagenaar J.A., Heesterbeek H. et al. (2010). Methicillin resistant Staphylococcus aureus ST398 in veal calf farming: human MRSA carriage related with animal antimicrobial usage and farm hygiene. PLoS ONE, 5: e10990. 14. Rhodes, M.J.C., Price, K.R. (1997). Identification and analysis of plant phenolic antioxidants. Eur. J. Cancer prev., 6:5188. 15. Oleszek W., Sitek M., Stochmal A., Piacente S., Pizza C., Cheeke P. (2001a). Steroidal saponins of Yucca Schidigera Roetzl. J. Agric. Food Chem., 49:4392-4396. 16. Qu L., Wang J., Yao X., Huang P., Wang Y., Yu H., Han L., Zhang Y., Wang T. (2018). Spirostane-Type Saponins obtained from Yucca Schidigera. Molecules, 23:167. 17. Piacente S., Montoro P., Oleszek W., Pizza C. (2004). Yucca Schidigera Bark: Phenolic Constituents and Antioxidant Activity. J. Nat. Prod., 67:882-885. 18. Cheeke P.R., Piacente S. Oleszek W. (2006). Anti-infiammatory and anti-arthritic effetcs of Yucca Schidigera: a review. Journal of Inflammation, 3:6. 19. De Oliveira C.A.C., Perez A.C., Merino G., Prieto J.G., Alvarez A.I. (2001). Protective effects of Panax ginseng on muscle injury and inflammation after eccentric exercise. Comparative Biochemistry and Physiology, 130(C):369-377. 20. Sun D.S., Shi B.L, Tong M.M., Yan S.M. (2018). Improved performance and immunological responses as a result of dietary Yucca Schidigera extract supplementation in broilers, Italian Journal of Animal Science, 17:2, 511-517, DOI: 10.1080/1828051X.2017.1358593. 21. De Sousa O.A., Cooke R.F., Brandão A.P., Schubach K.M., Schumaher T.F., Bohnert D.W., Marques R.S. (2019). Productive and physiological responses of feeder cattle supplemented with Yucca Schidigera extract

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during feedlot receiving. J. Anim. Sci. 2019.97:208–219 doi: 10.1093/jas/sky412. Mowat A.M., Smith R.E., Donachie A.M., Furrie E., Grdic D., Lycke N. (1999). Oral vaccination with immune stimulating complexes. Immunology Letters, 65:133-140. Francis G., Kerem Z., Harinder P.S.M., Becker K. (2002). The biological action of saponins in animal system: a review. British Journal of Nutrition, 88:587-605. De Oliveira C.A.C., Perez A.C., Merino G., Prieto J.G., Alvarez A.I. (2001). Protective effects of Panax ginseng on muscle injury and inflammation after eccentric exercise. Comparative Biochemistry and Physiology, 130(C):369-377. Sen S., Makkar H.P.S., Muetzel S., Becker K. (1998). Effect of Quillaja saponaria saponins and Yucca Schidigera plant extract on growth of Escherichia Coli. Lett. Appl. Microbiol., 27:35-38. Rambozzi L., Min A.R.M., Menzano A. (2011). In vivo anticoccidial activity of Yucca Schidigera saponins in naturally infected calves. Journal of Animal and Veterinary Advances. 10. 391-394. 10.3923/ javaa.2011.391.394. Decreto Legislativo n. 126 del 7 luglio 2011. Attuazione della direttiva 2008/119/CE che stabilisce le norme minime per la protezione dei vitelli. Gazzetta Ufficiale n. 180 del 4 agosto 2011. NRC. 2001. Nutrient Requirements of Dairy Cattle. 7th rev. ed. Natl. Acad. Sci. Washington, DC. Leruste H., Brscic M., Heutinck L.F.M., Visser E.K., Wolthuis-Fillerup M., Lindahl I.L., Shalkop W.T., Dougherty R.W., Thompson C.R., Van Atta, G.R., Bickoff E.M., Walter E.D., Livingstone A.G., Guggoloz J., Wilson R.H., Sideman M.B., De Eds F. (1957). Alfalfa saponins. Studies on their chemical, pharmacological, and physiological properties in relation to ruminant bloat. USDA Techincal Bullettin No 1161, Washington, D.C.. Iorio E.L. (2003) ed. d-ROMs test e stress ossidativo. 1a ed. Grosseto: Diacron International, 2003.

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Investigation of the gene carriage rates for Staphylococcus aureus, mecA, vanA and nuc genes in the nasal and milk specimens from the sheep caretakers with sheep

259

ÖMER AKGÜL*1, GÜLHAN BORA1, HÜSEYIN GÜDÜCÜOĞLU2 1

Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Van Yüzüncü Yıl University, VAN, Turkey Department of Medical Microbiology, Faculty of Medicine, Van Yüzüncü Yıl University, VAN, Turkey

SUMMARY Methicillin-resistant S. aureus (MRSA) is an important pathogen that may cause serious infections in the humans and animals. The pathogenity of Staphylococcus aureus emerges associated with the factors such as antibiotic resistance, immune evasion, invasion capability and virulence. By the impairment of the immune system, S. aureus-borne skin and soft tissue infections as well as serious infections such as pneumonia, septicemia and osteomyelitis may develop in the human bodies. S. aureus is one of the most common causes of the intramammary infections (IMI) in the dairy ruminants. The present study aimed to identify the presence of S. aureus, mecA and vanA genes in humans and animals (sheep) in the rural corporations on commercial dairy sheep farms in Eastern Turkey. It was also targeted to evaluate nuc gene positivity of Stapyhylococcus aureus strains isolated from humans and animals. Totally 78 (12.7%) S. aureus strains were isolated and identified from 612 materials taken from the sheep caretakers (nasal swab: 204) and the sheep (204 specimens for nasal swab and 204 specimens for milk each from sheep). S. aureus was distributed in 27 (13.2%; 204), 16 (7.8%; 204) and 35 (17.2%; 204) of the nasal swab specimens taken from the sheep caretakers, the sheep and sheep’s milk specimens, respectively. Antibiotic susceptibility testing of 78 S. aureus isolates performed by Vitek2 device revealed that the highest antibiotic resistance was against benzylpenicillin. Gene analysis for 12 MRSA strains isolated in the specimens of the sheep caretakers and sheep was performed by single-Polymerase Chain Reactions (sPCR) for detection of mecA and vanA genes. Twelve MRSA isolates were found positive for mecA gene carriage. On the other hand, 78 S. aureus isolates were not found to carry vanA gene. All of the 12 MRSA and 66 Methicilline-susceptible S. aureus (MSSA) isolates were found positive for nuc gene carriage. It was concluded that MRSA strains isolated from the sheep caretakers and sheep had impact on the public health and created at risk for food chain.

KEY WORDS mecA, vanA, nuc, human, sheep.

INTRODUCTION Staphylococcus aureus is an important pathogen that plays an important role in the infections that develop in the humans and animals1. The pathogenity of S. aureus emerges associated with the factors such as antibiotic resistance, immune evasion, invasion capability and virulence2. S. aureus have been reported to be colonized in the skin or nasopharyngeal regions of the healthy subjects at a rate of approximately 25-30%3. By the impairment of the immune system, S. aureus-borne skin and soft tissue infections as well as serious infections such as pneumonia, septicemia and osteomyelitis may develop in the human bodies4. S. aureus is one of the most common causes of the intramammary infections (IMI) in the dairy ruminants. The rates of clinical and subclinical mastitis infection in the sheep were identified as 5-11% and 0.22-2.06% in the sheep, respectively5, 6 . Although, mammary is considered as an essential source of contamination with S. aureus for milk and dairy farm envi-

Corresponding Author: Ömer Akgül (o.akgul@yyu.edu.tr)

ronment, other body regions (nasal cavity) may also play an important role7. Methicilline-resistant Staphylococcus aureus is defined as an important infectious agent for the hospital-acquired infections (Hospital-acquired MRSA; HA-MRSA) and an important pathogen in the community (Community-acquired MRSA; CAMRSA)8. Different CA-MRSA clones (Livestock-associated MRSA; LA-MRSA) that created serious concerns in the humans exposed in the dairy environments have been identified in the different countries of the world9. It has been shown that LAMRSA particularly infects dairy employers10 and veterinary physicians11 due to close contact. Several studies have reported the presence of MRSA in the regions of the Mediterranean countries (Greece, Spain and Italy) that sheep and goat milk as well as commercial dairy products are commonly used12, 13. The assessment of the risk size created by mecA and vanA gene carriage of S. aureus strains on public health is also considered to be important. It is particularly targeted to provide contribution to the scientific world on the fact that which treatment options should be developed in either human and veterinary medicine against particularly livestock-associated S. aureus strains by revealing their antibiotic resistance profiles14. The resistance profiles of S. aureus species against antibiotics

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Investigation of the gene carriage rates for Staphylococcus aureus, mecA, vanA and nuc genes in the nasal

create great concerns. Beta-lactams, macrolides and tetracyclines are commonly used in the treatment of S. aureus-associated infections15. However, the frequency of the multidrug resistant MRSA strains that show aminoglycoside and macrolide-lincosamide-streptogramin resistance reached very high rates16. The identification of medically important MRSA plays an important role in struggling with these resistance phenomena. MRSA emerges due to synthesis of PBP2a, PBP2LGA or PBP2 as the specific molecules determined by mecA gene15, 17, 18. Vancomycin is one of the primarily preferred antibiotics in the treatment of the MRSA infections3. However, the presence of Vancomycin Intermediate and Vancomycin Resistant S. aureus (VISA ve VRSA) clinical isolates posed serious public health concerns in the recent twenty years3, 19. Thermostable deoxyribonuclease (DNase) is a specific thermostable DNAse that breaks down DNA encoded by nuc gene. The nuc gene is a specific target of the PCR-based methods for the identification of S. aureus20. The nuc gene is one of the most commonly used indicators for differentiation of S. aureus from other Staphylococcus species and determination of its prevalence21. However, several PCR studies have reported that false MRSA-associated identification results due to the presence of S. aureus specific nuc gene were also obtained22. The present study aimed to identify presence and rate of S. aureus carriage detected in dairy sheep farms (nasal swab and milk specimens) and sheep caretakers (nasal swab specimens) in the Eastern Anatolia Region (Van Province) of Turkey. In this region, it is aimed to reveal the antibiotic resistance profile seen in S. aureus isolates isolated from humans and animals. Especially, it was aimed to reveal the prevalence of methicillin and vancomycin resistance S. aureus in sheep caretakers and dairy sheep farms. In addition, it was aimed to evaluate the risk posed by people associated with dairy sheep farms on public health.

nical mastitis (38; 18.6%) and subclinical mastitis (166; 81.4%). In the flocks detected with clinical mastitis the milk specimens from the sheep with healthy appearance and positive CMT test result were evaluated. The isolation, identification and antibiotic susceptibility tests were performed in the Bacteriology Laboratory of The Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Van Yüzüncü Yıl University and the Bacteriology Laboratory of The Department of Medical Microbiology, Van Yüzüncü Yıl University Dursun Odabas Medical Center. Swabs were used in obtaining nasal specimens from humans and sheep. Nasal swab specimens were directly taken into 5 ml Mueller-Hinton Broth. The milk specimens were also taken from the sheep simultaneously with obtaining nasal swab specimens. Nasal swab specimens were incubated at 37 °C for 18-24 hours in the incubator. The incubated swab and milk specimens were directly inoculated onto Baird Parker agar (including Egg Yolk Tellurite supplement) and incubated at 37 °C for 48 hours. The black colonies with a surrounding 2-5 mm clear field were assessed as suspected S. aureus. The catalase (H2O2) and Gram positive strains with coccus morphology that indicated β-hemolysis on blood agar were stored at -20 °C in the 10% Nutrient Broth for species identification and subsequently molecular characterization. Species identification and antibiotic susceptibility tests were performed in the Bacteriology Laboratory of The Department of Medical Microbiology, Van Yüzüncü Yıl University. Initially, the strains stored at -20 °C in the 10% Nutrient Broth were inoculated onto 7% human blood TSA II and incubated at 37 °C for 24-48 hours. The pure colonies that grew in the 7% human blood TSA were used to prepare 0.5 McFarland bacterial suspension. Its identification and antibiogram test were performed in the Gram positive panels using The BD Phoenix automated microbiology system (Becton Dickinson, USA). S. aureus isolates were stored at -20 °C in the 10% Nutrient Broth for molecular characterization.

MATERIALS AND METHODS Molecular Characterization Results of S. aureus Isolates

Isolation, Identification and Antibiogram Test for S. aureus Totally 612 specimens were collected from the 204 different sheep caretaker and family members working in the care of sheeps (nasal swab specimens), both nasal swabs (n= 204) and milk specimens (n= 204) from the same of sheeps with mastitis out of 17 rural corporations (an average of 100 to 500 head sheeps) on commercial dairy sheep farms in Eastern Turkey. According to the anamnesis, sheep herds with previously experienced mastitis problems were included in the study. The milk specimens of the sheep were obtained by identification of cli-

The molecular characterization procedure of the isolated and identified S. aureus species by sPCR was carried out in Molecular Biology Laboratory of the Department of Pharmaceutical Microbiology, Faculty of Pharmacy. For this purpose, nuc gene (279bp), mecA gene (310bp) and vanA gene (1032bp) carriage rates of S. aureus isolates were analyzed. The reference primers of the DNA amplicons analyzed in this study were presented in Table 1. DNA extraction was carried out in accordance with the procedure described in the G-spinTM Total DNA Extraction Mini Kit (Intronbio, KOREA). The identification of S. aureus nuc gene was performed ac-

Table 1 - Reference primers used in the study. Target

nuc

mecA

vanA

Primer

Sequence (5’-3’)

Product size (bp)

Reference

Forward

GCGATT GAT GGT GAT ACG GTT

279

[20]

Reverse

AGC CAA GCC TTG ACG AAC TAA AGC 310

[20]

1032

[22]

Forward

CCA ATT CCACAT TGT TTC GGT CAT A

Reverse

GTA GAA ATG ACT GAA CGT CCG ATA A

Forward

ATG AAT AGA ATA AAA GTT GC

Reverse

TCA CCC CTT TAA CGC TAA TA

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Ö. Akgül; Large Animal Review 2021; 27: 259-268

cording to the method reported by Sahebnasagh et al.21. PCR amplification mixture contained 5µl template DNA, 2 µl PCR Buffer (10X), 1 µl MgCl2 (50 mM), 4 µl dNTPs (1 mM), 1 µl nuc1 and nuc2 primers (10 Pmol), 0.5 µl Taq DNA polymerase (5 U/µl) and 10.5 µl double distilled water to obtain a 25 µl final solution. PCR temperature cycling conditions were performed for totally 30 cycles at 94 °C for 5 min at initial denaturation stage, following 94 °C for 1 min, 50 °C for 1 min, 72 °C for 2 min and 72 °C for 10 min at the final stage. Then, 5 µl PCR products were run on the 1.7% agarose gel electrophoresis including ethidium bromide (0.5 µg/ml) in the 1X TBE (Tris-HCL, Boric acid, EDTA) buffer at 80 V for 120 min. The bands were analyzed by Gel Logic 2200 Imaging System as the gel imaging system. S. aureus ATCC 29213 and PCR water were used as positive and negative controls, respectly. The identification of S. aureus mecA gene was performed according to the method reported by Sahebnasagh et al.21. PCR amplification mixture contained 5 µl template DNA, 2µl PCR Buffer (10X), 1 µl MgCl2 (50 mM), 4 µl dNTPs (1 mM), 2 µl mecA1 and mecA2 primers (10 Pmol), 1µl Taq DNA polymerase (5 U/µl) and 8 µl double distilled water to obtain a 25 µl final solution. PCR temperature cycling conditions were performed for totally 30 cycles at 94 °C for 5 min at initial denaturation stage, following 94 °C for 30 sec, 55 °C for 1 min, 72 °C for 30 sec and 72 °C for 5 min at the final stage. Then, 5 µl PCR products were run on the 2% agarose gel electrophoresis including ethidium bromide (0.5 µg/ml) in the 1X TBE (TrisHCL, Boric acid, EDTA) buffer at 80 V for 120 min. The bands were analyzed by Gel Logic 2200 Imaging System as the gel imaging system. S. aureus ATCC 25923 and PCR water were used as positive and negative controls, respectly. The identification of S. aureus vanA gene was performed according to the method reported by Saadat et al.23. PCR amplification mixture contained 2 µl template DNA, 2µl PCR Buffer (10X), 1 µl MgCl2 (50 mM), 4 µl dNTPs (1 mM), 4 µl Forward and Reverse primers (10 Pmol), 0.25 µl Taq DNA polymerase (5 U/µl) and 2.75 µl double distilled water to obtain a 20 µl final solution. PCR temperature cycling conditions were performed for totally 30 cycles at 98 °C for 2 min at initial denaturation stage, following 98 °C for 10 sec, 50 °C for 1 min, 72 °C for 90 sec and 72 °C for 10 min at the final stage. Then, 5 µl PCR products were run on the 1.5% agarose gel electrophoresis including ethidium bromide (0.5 µg/ml) in the 1X TBE (Tris-HCL, Boric acid, EDTA) buffer at 100 V for 100 min. The bands were analyzed by Gel Logic 2200 Imaging System as the gel imaging system. Vancomycin-resistant Enterococcus ATCC 51299 and PCR water were used as positive and negative controls, respectly.

Statistical Analysis Descriptive statistics for the studied variables (characteristics)

261

were presented as count and percent. Proportions were compared with Z test or Fisher’s Exact test for two proportions. Statistical significance levels was considered as 5% and MINITAB for windows (ver: 14) statistical program was used for all statistical computations.

Ethics Committee Approval The human part of the study was carried out due to Approval of Clinical Researches Ethics Committee of Van Yüzüncü Yıl University Medical Faculty, Dated on 16th February 2018 and Decision Number: 2018/19. Animal part of the study was performed due to the Approval of Animal Experiments Local Ethics Committee of Van Yüzüncü Yıl University (decision number: 2018/01).

RESULTS Isolation, Identification and Antibiogram Results of S. aureus Totally 78 (12.7%) S. aureus strains were isolated from 612 materials taken from the sheep caretakers and sheep. S. aureus was identified in 27 (13.2%) of 204 nasal swab specimens taken from the sheep caretakers. Nasal swabs belonging to sheep caretakers from 17 different farms showed positivity in 6 (35.3%) different dairy sheep farms and 35 [17.2%; 11 (64.7%) out of 17 dairy sheep farms are positive] of the nasal swab specimens (n=204) and milk specimens (n=204) from the sheep, respectively. S. aureus was isolated in 5 [2.5%; 2 (11.8%) out of 17 dairy sheep farms are positive] of the 38 milk specimens from the sheep with clinical mastitis. On the other hand, S. aureus was identified in 30 [14.7%; 11 (64.7%) out of 17 dairy sheep farms are positive] of the 166 milk specimens from the sheep with subclinical mastitis (Table 2). S. aureus was found positive in both nasal and milk samples of three sheep with subclinical mastitis in a farm. MRSA colonization determined in both nasal and milk samples of a sheep. In addition, nasal MRSA presence was detected in one of the sheep caretakers at this farm. The antibiotic resistance profiles of the MRSA isolates obtained in the specimens of the sheep caretakers and sheeps were presented in Table 3. The 4 MRSA strains obtained from the sheep caretakers were found to manifest the highest antibiotic resistance against cefoxitin, benzylpenicillin, ampicillin, oxacillin and clindamycin (100%). These MRSA isolates were found to be susceptible to quinopristin/ dalfopristin, tigecycline and rifampin. It was exhibited that 2 MRSA strains isolated from the nasal specimens of the sheep demonstrated the highest antibiotic resistance against cefoxitin, benzylpenicillin, ampicillin, oxacillin, erythromycin and clindamycin (100%). It was identified that one MRSA isolated from the milk specimens of the sheep with clinical mastitis indicated the highest

Table 2 - The distribution of S. aureus and MRSA isolates in the specimens taken from the sheep caretakers and sheep. Bacteria

S. aureus MRSA

Nasal swap of sheep caretakers (n=204) 1

Nasal swap of sheep (n=204) 2

Sheep milk (Clinical Mastitis) (n=38) 3

Sheep milk Sheep milk (Subclinical (Mastitis) Mastitis) (N=204) (n=166) 4 5

p(1-2)

p(3-4)

p(3-5)

p(4-5)

p(1-5)

p(2-5)

27 (13,2%)

16 (7,8%)

5 (13.2%)

30 (18.1%)

35 (17.2%)

0,075

0,431

0,511

0,818

0,269

0,004

4 (2%)

2 (1%)

1 (2,6%)

5 (3%)

6 (2.9%)

0,410

0,896

0,914

0,968

0,522

0,152

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Investigation of the gene carriage rates for Staphylococcus aureus, mecA, vanA and nuc genes in the nasal

Table 3 - Antibiotic resistance profiles of MRSA isolates. Nasal S. aureus of the sheep caretaker (n=4)

Antibiotics

Nasal S. aureus of sheep (n=2)

S. aureus of the sheep milk with Clinical mastitis (n=1)

S. aureus of the sheep milk with subclinical mastitis (n=5)

Cefoxitin Screening

Benzylpenicillin

Ampicillin

Oxacillin

Gentamycin

Ciprofloxacin

Levofloxacin

Moxifloxacin

Inducible Clindamycin resistance

Erythromycin

Clindamycin

Q/D*

Linezolid

Vancomycin

Tetracycline

Tigecycline

Nitrofurantoin

Rifampin

SXT*

*Q/D: Quinupristin/ Dalfopristin; SXT: Sulfamethoxazole/Trimethoprim

antibiotic resistance to cefoxitin, benzylpenicillin, ampicillin, oxacillin, gentamycin, erythromycin and clindamycin. This MRSA isolate was found to be susceptible to the other antibiotics. The 5 MRSA isolates of the sheep with subclinical mastitis indicated the highest antibiotic susceptible to tigecycline, nitrofurantoin and rifampin. The antibiotic resistance profiles of the S. aureus isolates obtained in the specimens of the sheep caretakers and sheep were presented in Table 4, respectively. The 27 S. aureus strains obtained from the sheep caretakers were found to manifest the highest antibiotic resistance against benzylpenicillin (17; 63%), ampicillin (13; 48%) and erythromycin (13; 48%). All the S. aureus isolates were found to be susceptible to vancomycin. It was exhibited that 16 S. aureus strains isolated from the nasal specimens of the sheep demonstrated the highest antibiotic resistance against benzylpenicillin (14; 87.5%) and ampicillin (11; 69%). All the S. aureus isolates were found be susceptible to tetracycline, tigecycline and rifampin. It was identified that 5 S. aureus isolates isolated from the milk specimens of the sheep with clinical mastitis indicated the highest antibiotic resistance to benzylpenicillin (5; 100%). It was also determined that 5 S. aureus isolates were highly susceptible to the other antibiotics. The 30 S. aureus isolates of the sheep with subclinical mastitis indicated the highest antibiotic resistance to benzylpenicillin (26; 87%) and gentamicin (19; 63%). All the 30 S. aureus isolates were detected to be susceptible to tigecycline and rifampin. It was detected that 12 S. aureus [15.4%; 9 (52.9%) out of 17 dairy sheep farms are positive isolates] obtained in the specimens of the sheep caretakers and sheep were phenotypically

MRSA. Of these 12 S. aureus isolates; 4 [33.3%; (4/ 17, 23.5%)], 2 [16.7%; (2/17, 11.8%)], and 6 [50%; (3/17, 17.6%)] were distributed in the nasal swab specimens of the sheep caretakers, nasal swabs and milk specimens of the sheeps, respectively.

Molecular Characterization Results of the S. aureus Isolates Gene analysis for phenotypically 12 MRSA and 66 Methicillin-susceptible S. aureus (MSSA) strains isolated in the specimens of the sheep caretakers and sheep was performed by single-Polymerase Chain Reactions (sPCR) for detection of mecA and vanA genes. Twelve of the 78 S. aureus isolates were found positive for mecA gene carriage (Figure 1). No mecA gene was detected in 66 MSSA isolates. It was also revealed that 78 S. aureus isolates were negative for vanA gene carriage. All the 12 MRSA and 66 Methicilline-susceptible S. aureus (MSSA) isolates were found positive for nuc gene carriage (Figure 2).

The Statistical Analysis Results As a result of statistical analysis, a significant difference was found between sheep nasal samples and S. aureus isolated from mastitis, but no difference was found between MRSA rates (Table 2). No statistically significant difference was detected between the rates of S. aureus and MRSA isolated from the nasal swab specimens of the sheep caretakers and sheep. No difference was found between the sheep with clinical and subclinical mastitis with respect to carriage of S. aureus and MRSA strains. It has been revealed that humans and animals carry the same risk

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Table 4 - Antibiotic resistance profiles of S. aureus isolates. Nasal S. aureus of the sheep caretaker (n=27)

Antibiotics

Nasal S. aureus of sheep (n=16)

S. aureus of the sheep milk with Clinical mastitis (n=5)

S. aureus of the sheep milk with subclinical mastitis (n=30)

Cefoxitin Screening

Benzylpenicillin

Ampicillin

Oxacillin

Gentamycin

Ciprofloxacin

Levofloxacin

Moxifloxacin

Inducible Clindamycin resistance

Erythromycin

Clindamycin

Q/D*

Linezolid

Vancomycin

Tetracycline

Tigecycline

Nitrofurantoin

Rifampin

SXT*

*Q/D: Quinupristin/Dalfopristin; SXT: Sulfamethoxazole/Trimethoprim

for exposure to S. aureus and MRSA in the sheep farms in Van Province.

DISCUSSION S. aureus may also cause severe invasive diseases (osteomyelitis and septic endocarditis), soft tissue and skin infections as well as it is a member of normal flora (nasal region) in humans24. Several studies have addressed livestock-associated nasal carriage of S.aureus25, 26. It has been exhibited in a study carried out in Latium (Italy) that 5 MRSA out of 12 S. aureus strains were isolated from the 14 swab specimens (nasal, oral and skin) taken from the sheep caretakers, between 2013 and 201525. Carfora et al.26 have determined three MRSA strains in the nasal specimen from the farm owners and from the hands and nasal of milker in Rome. In the present study, 4 MRSA out of 27 S. aureus strains were detected in 204 nasal swab specimens from the sheep caretakers. The rate of nasal S. aureus and MRSA was lower compared with the other studies26, 27. This difference was considered resulting from S. aureus colonization in the exposed environment, racial features, geographical residence and differences between the isolation methods. Staphylococcus species are isolated from various body regions (nasal and mammary region) of the healthy ruminants. Mastitis, impetigo, abscess, vaginal infections, abort, osteomyelitis and rhinosinusitis may develop due to Staphylococcus28. Researchers have reported that nasal region is the most available site for the initial colonization of S. aureus in the small ru-

minants (sheep) and functions as the primary source in the infections7. The nasal swab specimens (n=204) of the sheep from the 17 different sheep farms of our regions indicated 16 [7.8%; 4 (23.5%) out of 17 dairy sheep farms are positive] S. aureus strains. A study carried out in Algeria has reported that 45 (%36.6) S. aureus strains were isolated in the 123 milk specimens obtained from the animals with clinical mastitis29. Vasileiou et al.28 have reported 26% subclinical mastitis caused by S. aureus in the sheep flocks. Compared with the other researches, the isolation rate of S. aureus from subclinical mastitis was lower in our region 28, 30. While the rate of clinical mastitis is reported to be less than 5% in average sheep every year in the USA, clinical mastitis was detected in sheep at the same rate in our Region 31, 32. This result was considered to be associated with S. aureus colonization in the sheep population, milking techniques (mechanical milking or hands) and stockyard environment (compliance to sheep capacity). Mechanical milking was performed in only two of the 17 different sheep farms in our region. S. aureus was found to be more prevalent in hand milked sheep farms. There are differences between the protocols for treatment of MSSA and MRSA strains in the infections caused by S.aureus in the humans and animals. After development of methicillin resistance in the course of time, semi-synthetic penicillins such as oxacillin, nafcillin and cloxacillin have been introduced to clinical practice as the altenative treatment option33. Beside that, vancomycin is only one of the few antibiotics that MRSA strains are susceptible. Trimethoprim sulfamethoxazole (SXT), cip-

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Investigation of the gene carriage rates for Staphylococcus aureus, mecA, vanA and nuc genes in the nasal

Figure 1 - PCR image of mecA gene of MRSA isolates isolated from the human and sheep specimens.

*1-4: nasal MRSA of the sheep caretaker; 5-6: Nasal MRSA of sheep; 7 Milk MRSA with clinical mastitis; 8-12: Milk MRSA with subclinical mastitis; NC: Negative Control; PC: Positive Control, S. aureus ATCC 25923; M: 100 bp DNA Marker.

rofloxacin, clindamycin, doxycycline and rifampin are the oldest antibiotics used in the treatment of particularly resistant S. aureus isolates34. However, it is known that different antibiotic resistance profiles developed by S. aureus strains significantly increased due to irregular and excessive use of the antibiotics in humans and animals. Ahmadi et al.35 have reported in their study that 55 MRSA isolates isolated from human nasal specimens indicated susceptibility to mupirocin, gentamicin and fusidic acid. It has been stated in a study carried out in Italy that 3 MRSA isolates were isolated in the nasal swab specimens from the sheep caretakers. All these isolates were shown to be resistant to amoxicillin-clavulanic acid, cefotaxime, doxycycline, erythromycin, amoxicilline, penicilline and tetracycline. However, all MRSA isolates were detected to be susceptible to vancomycin8. Cong et al.3 have noted that human VRSA strains demonstrate high antimicrobial susceptibility. It was determined in our study that 27 S. aureus strains obtained from the sheep caretakers manifested the highest antibiotic resistance to benzylpenicillin (17; 63%), ampicillin (13; 48%) and erythromycin (13; 48%). All the S. aureus isolates were found to be

susceptible to vancomycin and rifampin. Compared with other studies, differences between the antibiotic resistance rates were considered to be associated with the exposed environment and S. aureus colonization. Different studies have evaluated the antibiotic resistance rates of S. aureus isolates isolated from the nasal swabs of the sheep worldwide36, 37. Abdel-Moein and Zaher36 have suggested that all the isolates isolated from the nasal specimens of the sheep were resistant to penicilline, oxacilline ve cefoxitin. It has been determined in Tunisia that S. aureus strains isolated from the nasal specimens of the sheep exerted resistance to penicillin (8.8%), ciprofloxacin (4.4%), tobramycin (%2.2%) and tetracycline (2.2%)37. El-Deeb et al.38 have demonstrated that antibiotic resistance rate of MRSA strains isolated from nasal specimens of the healthy animals in Saudi Arabia against penicillin, oxacillin and cefoxitin was 100%. It was exhibited in the present study that 16 S. aureus strains isolated from the nasal specimens of the sheep manifested the highest antibiotic resistance to benzylpenicillin (14; 87.5%) and ampicillin (11; 69%). All the S. aureus isolates were found be susceptible to vancomy-

Figure 2 - PCR image of nuc gene of S. aureus isolates isolated from the human and sheep specimens.

*1-4: nasal MRSA of the sheep caretaker; 5-6: Nasal MRSA of sheep; 7 Milk MRSA with clinical mastitis; 8-12: Milk MRSA with subclinical mastitis; 13-14; Nasal S. aureus of the sheep caretaker; 15-16: Milk S. aureus of the sheep with mastitis; NC: Negative Control; PC: Positive Control, S. aureus ATCC 29213; M: 100 bp DNA Marker.

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cin, tetracycline, tigecycline and rifampin. It was concluded that performing screening tests widely is important since nasal S. aureus strains isolated from the sheep exhibited different antibiotic resistance rates. It has been reported that MSSA strains isolated from sheep milk specimens have shown high tetracycline resistance rates in Turkey39. We have monitored in our study that 5 S. aureus isolates isolated from milk of the sheep with clinical mastitis indicated the highest antibiotic resistance to benzylpenicillin (5; 100%). It was detected that those 5 S. aureus isolates demonstrated high susceptibility to other antibiotics. On the other side, 30 S. aureus isolates isolated from the milk of the sheep with subclinical mastitis presented the highest antibiotic resistance to benzylpenicillin (26; 87%) and gentamicin (19; 63%). All those 30 S. aureus isolates were susceptible to vancomycin, tigecycline, nitrofurantoin and rifampin. It was concluded that antibiotics (particularly penicillin) used for the infections developing in the sheep and exposed resistant S. aureus colonization in the environment played an important role in the increased antibiotic resistance in our region. Methicillin has initially emerged as a great therapeutic medication invention in 19573. It was reported that prevalence of methicillin resistance raised over 40% in the invasive infections and asymptomatic colonized patients in the continent Europe. It is estimated that 30% and 1.5% of the population was colonized with MSSA and MRSA in the USA, respectively40. Macori et al.25 have declared that 5 (41.7%) of 12 S. aureus isolates isolated from the owners and the employers of the sheep farms. Carfora et al.26 have announced that they isolated 2 MRSA isolates in the nasal swab specimens from sheep caretakers. In the present study, 4 (2%) MRSA strains were isolated from the nasal specimens of the 204 sheep caretakers in 17 different sheep farms in our region. Nasal MRSA was not found very prevalent in the sheep caretakers. It was concluded that risk for contamination of MRSA with humans who live in the rural or urban area through sheep caretakers was very low. A study has analyzed the public health risk for MRSA infection resulting from the contact between animals and humans36, 40 . It was reported in a study carried out in Egypt that 2 (3.8%) MRSA isolates were isolated from nasal specimens of 52 sheep36. Researches have demonstrated that MRSA strains were common in the milk specimens of the sheep with mastitis although methicillin was not used in the treatment8. In our study, 2% MRSA (8/408) was detected in the nasal and milk specimens taken from the sheep. The discussed studies have used mecA gene as the target site for the identification of MRSA. In also our study, all the MRSA isolates were mecA positive. It was determined that impact of MRSA was low in the sheep in our region. While MRSA strains isolated from sheep with mastitis are resistant to many antibiotics, MSSA strains show sensitivity to many antibiotics41. In our region, it was predicted that the risk of MRSA will be low in antibiotic applications for the treatment of mastitis infections in sheep. Increased rates of vancomycin resistance is encountered in S. aureus strains. Since vancomycin-resistant S. aureus (VRSA) was initially reported in 2002, 52 isolates have been declared worldwide3. In our study, vancomycin resistance was enountered in none of the 78 S. aureus strains isolated in the specimens of the sheep caretakers and sheep. It was determined that humans carry no risk for VRSA through sheep farms in our region. However, it is concluded that taking the prevalence of vancomycin resistant Enterococcus (VRE) into account before ad-

267

ministration of vancomycin for the human and animal infections. Hirotaki et al.42 have stated that multiplex PCR method targeting nuc gene is 100% sensitive and 100% specific in the diagnosis of Staphylococcus species isolated from humans and various animals. It was noted in a study from Egypt that 2 MRSA strains isolated in the nasal specimens of 52 sheep were positive for carriage of nuc gene36. In our study, nuc gene analysis of 78 S. aureus strains was performed by sPCR ile yapıldı. Gene analysis result indicated that all the 12 MRSA and 66 MSSA isolates were positive for the carriage of nuc gene. We have determined that our study method was 100% sensitive in the identification of MRSA and MSSA isolates. However, nuc gene region encoded a weak DNA band in the MRSA under the same PCR conditions.

CONCLUSIONS As a consequence, S. aureus was found to have a low colonization prevalence between the sheep caretakers and sheep in our high-capacity rural corporations dealing with sheep breed. It was monitored that isolated and identified S. aureus strains indicated a low MRSA and no VRSA prevalence. It has been suggested that monitoring potential risk of MRSA strains would be important even though they were encountered with low levels. MRSA and MSSA strains were found to present a high antibiotic resistance to penicillin in particularly conventional treatment protocols. Therefore, it was determined that administration of the antibiotics in the treatment of S. aureus-associated infections in either humans and animals after performing antibiotic susceptibility tests would be critical. We have concluded that our findings obtained in our study would contribute to the antibiotic treatment protocol in the treatment of the hospital-, community and animal-acquired infections caused by S. aureus in our region.

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YAGOUBI YATHREB1*, SMETI SAMIR1, MEKKI ILYES1, MAHOUACHI MOKHTAR2, ATTI NAZIHA1 1 2

University of Carthage, INRA-Tunisia, Laboratoire des Productions Animales et Fourragères, 2049 Ariana, Tunisia University of Jendouba, ESAK, Le Kef, Tunisia

SUMMARY Introduction - The Rosemary is an aromatic and medicinal plant widely available in the Mediterranean area of which industry distillation generates voluminous residues that could be an alternative animals’ feed in harsh conditions. Aim - This study aimed to evaluate the effect of hay substitution by rosemary distillation residues (RR) in crude form (CRR) or as pellets (PRR) on lamb’s performance, digestibility and nitrogen retention. The metabolic profile and the rumen fermentation parameters were also determined. Materials and methods - 24 ewe-lambs (average body weight: 24 + 1.3 kg) were divided into 3 groups receiving 500 g of concentrate plus 600 g of oat hay for control group (C), 600 g of crude RR for CRR group and 600 g of RR enriched pellets in PRR group. Results and Discussion - Dry matter (DM) intake was similar for hay, CRR and PRR. The DM and crude protein digestibility was not affected by the dietary treatment. The nitrogen balance was positive for all regimens. The rumen ammonia concentration was higher for C and PRR groups, while the volatile fatty acids concentration was similar among groups. The lamb’s growth was similar among groups. The cost of gain was reduced (P<0.05) for lambs fed RR diets. The glycaemia and uraemia were higher (p<0.001) for PRR. Conclusion - In conclusion, the RR can be used in lamb’s feeding at reduced cost without altering lamb’s growth and rumen functionality.

KEY WORDS Rosemary residues; Lamb’s growth; Metabolic profile; Rumen fermentation; Nutrient digestion.

INTRODUCTION Sheep and goat feeding in semi-arid areas is based on natural resources, rangeland and stubble. The biodiversity loss and climate change with water shortage in these regions resulted in land degradation and uncertain availability of such resources1. Consequently, the livestock use common concentrates and other feeds often imported and expensive. In the SouthernMediterranean region, the hay and straw represent the main basal diets for ruminant nutrition; however, for some part of this region, the availability of hay is a greater problem than the availability of concentrate. The transport of hay, fibrous and voluminous, is more expensive than that of concentrate. In order to surmount the feed availability and cost problem, several alternative feed resources, in particular agro-industrial by-products and shrubs are used in goat, sheep and cattle feeding2, 3. In last decades, the distillation of forest plants to produce es-

Corresponding Author: Yagoubi Yathreb (yagoubiyathreb@hotmail.fr)

sential oils became an important industry and generated a great quantity of by-products. The Rosemary is the most forest plant exploited in artisanal or modern distillation industry in the Mediterranean area. In Tunisia, it covers large areas estimated to 346000 ha. The annual collected amount of rosemary is 15600 tons leaving 5460 tons of distilled rosemary residues (RR) whereas the potential of annual RR is around 9000 tons4. This important quantity of RR can be valued to partially overcome the problem of animal-feed shortage. However, they can be considered as low quality roughage but the adaptation of some easy feed technologies such multi-nutritional blocks or pellets lead to obtain effective mix balanced in proteins, fibre and minerals that can enhance animal’s productivity5. The use of rosemary essential oil and the RR as additive for sheep and goats was extensive, but ha d only concerned their antioxidant properties and their effects on meat quality 5, 6. Nevertheless, to our knowledge, the information about the use of RR as basal diet is very scarce. Therefore, the aim of this work was to study the effects of its substitution, in crude state (CRR) or pellet form (PRR), to oat hay on nutrient digestibility, ruminal fermentation, growth and haematological parameters of Barbarine lambs.

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MATERIAL AND METHODS

tal daily amount of urine was conserved at -4 °C after homogenization to determine the Nitrogen content.

All procedures employed in this study meet ethical guidelines and adhere to Tunisian legal requirements (The Livestock Law No. 2005-95 of 18 October 2005, Chapter II; Section 1).

Experimental diets The fresh RR were collected, after essential oil extraction, in the forest in the semi-arid region of the country. They were transferred in plastic bags and dried under ambient conditions for one week. Then, the RR was included in the diet as crude (CRR) or pellets (PRR) forms. To make the pellets, the RR (60%) were ground and mixed with wheat bran (32%) and soybeans (8%) to obtain enhanced roughage. The oat hay was considered as control feed compared to diets containing RR. The chemical composition and feed costs of all experimental foods were given in Table 1.

Experimental design and feeding management Twenty-four Barbarine ewe-lambs [24+1.3 kg of body weight (BW)] were housed in individual pens and randomly assigned to one of three diets. They averagely received 600 g of basal diets: oat hay, crude RR or pellets RR for Control, CRR and PRR group, respectively. Lambs of all groups were complemented by 500 g of local concentrate (80% of barley, 17.5% of soybean meal and 2.5 of mineral and vitamin supplement) and had free access to water. Animals were allowed 15 days as adaptation to experimental conditions where roughages amounts were progressively increased from 300 to 600 g for CRR and PRR and from 400 to 600 g for oat hay, and then the growing trial lasted 67 days. The refusals were removed and weighed daily before the morning feeding. The lambs were weekly weighed to calculate the average daily gain (ADG).

Digestibility and metabolism A metabolism trial was performed at the end of the growing trial. Six lambs from each dietary treatment were transferred to metabolism cages for 5 days of adaptation and 5 days of measurement of nutrient digestibility and nitrogen (N) balance. The feed intake and refusals and faecal output were daily recorded and sampled for further analysis prior to morning feeding. Representative samples were used for dry matter (DM) determination. At the end of the collection period, all faeces samples for each lamb were mixed and homogenized; representative samples were stored for DM, Ash, fibre and CP analyses. The urine was daily collected from each animal in a plastic basin containing 50 ml of 10% H2SO4 solution. Then 10% of the to-

Rumen fermentation Samples of rumen fluid were collected from 6 animals from each group using a stomach tube before the morning feeding (0h), at 2, 5 and 8 hours post feeding. Ruminal pH was immediately measured using a portable pH meter (330i SET; WTW; Germany) after calibration with two buffers (7.00 and 4.01). For ammonia nitrogen analysis, the ruminal liquid was filtered through 4 layers of sterile gaze strips and a part of the filtered liquid was conserved with concentrated H2SO4. The NH3-N was determined according to7 method. For volatile fatty acid (VFA) analysis, another part of ruminal liquid was fixed by a solution containing 0.25 ml H3PO4+0.25 ml Hg Cl2 and frozen. The VFA were analysed using a Gas Chromatography Bruker Scion 460.The separation process was carried out with acapillary column (30 m x 0.25 mm D.I x 0.25 µm film thickness). The injector temperature was at 220°C while the detector temperature at 230 °C. VFA were then identified by comparison of retention times with the patterns.

Haematological parameters Blood samples were collected, at morning prior feeding, into heparinised vacutainers and immediately centrifuged at 3000 g for 15 minutes. Serum samples were transferred into Eppendorf tubes and frozen until analyses. Then, glucose, triglycerides, cholesterol, urea and creatinine concentrations were measured by specific Kits supplied by Phase Biomaghreb (Tunisia) and the absorbance reading was performed by a spectrophotometer (Rayto: RT-1904 C, Germany).

Chemical analyses The DM of oat hay, concentrate, CRR, PRR, feed refusals and faeces was determined by drying at 75 °C until constant weight and then milled through 1 mm screen. Ash was determined at 550 °C for 6 hours; CP content was determined by the Kjeldahl method on distributed feed, refusals, faeces and urine. Acid detergent fiber (ADF), neutral detergent fiber (NDF) and lignin were determined according to8 method using an ANKOM220 fiber analyzer (ANKOM Technology Corp, Macedon, NY, USA). The total phenolic compounds (TPC) were extracted and determined as described by9 using the Folin-Ciocalteu’s assay.

Calculations and statistical analysis The digestibility of the nutrients was measured as the difference between the amount of nutrient ingested minus the

Table 1 - Chemical composition (g/kg DM) and cost of experimental feeds (Tunisian Dinar, TND).

Dry Matter (g/kg)

Concentrate

Oat hay

Pellets rosemary residues

Crude rosemary residues

923

860

928

849

Crude Protein (g/kg)

119

116

NDF (g/kg)

342

667

386

385

ADF (g/kg)

353

175

273

Lignin (g/kg)

6.5

160

TPC (mg tannic acid/gDM)

2.6

8.1

33.8

47.3

Feed cost/ton (TND)

580

400

300

100

NDF: Neutral Detergent Fiber; ADF: Acid Detergent Fiber; TPC: Total Phenolic Compounds.

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amount of nutrient excreted in the feces, expressed as a percentage of the nutrient ingested: 100 x (intake - excreted)/intake. The feed conversion rate was calculated as the ratio of dry matter intake (DMI) to ADG (g DMI / g BW gain). The feed cost/kg gain (TND) was calculated as the ratio of lambs’ total ration cost to the whole weight gain during the experiment. The PROC GLM of SAS (2004)10 was applied. For nutrient intake, lamb’s growth, daily feed cost, nutrient digestibility, nitrogen balance, haematological parameters and the rumen fermentation parameters, a one-way analysis of variance for diet effects was used. The significance was declared at p < 0.05. The differences between groups was compared by the Duncan’s test. The following contrasts were used: *Ct1: The RR presence effect [C vs. CRR + PRR] *Ct2: The administration form of RR effect [CRR vs. PRR]

RESULTS Feed chemical composition The oat hay presented the lowest, the pelleted RR had the highest CP content (50 vs. 116 g/kg DM), while the crude RR had an intermediate value (73 g/kg DM). The NDF and ADF contents were higher for oat hay than both forms of RR; however the RR diets had the highest content of lignin and total phenolic compounds (33.8 and 47.3 vs. 8.1 mg tannic acid/g DM) for PRR, CRR and C, respectively) (Table 1).

Metabolism trial: Iintake, digestibility and nitrogen balance For all groups, the distributed amount of concentrate was entirely consumed. However, there was some refusal in roughages for all groups and the DM intake averaged 459, 455 and 494 g/day for C, CRR and PRR, respectively (p>0.05). The total DM intake was similar for all lambs (p>0.05), while the CP intake was higher for PRR than for both other groups. The nutrient digestibility for C and PRR groups was similar however; it was significantly lower in CRR diet (Table 2). The CP digestibility was unaffected (P>0.05). The RR consumption in both forms (CRR and PRR) increased significantly the N intake (p<0.05; Table 2). The faecal nitrogen (FN) was higher for CRR than C, whereas PRR was intermediate. The urinary nitrogen (UN) was higher for the PRR group compared with both the other groups (C and CRR), but it was low for all groups. Given the high N intake and low FN, the highest retained nitrogen value was presented by PRR (7.66 vs. 6.05 and 4.1 g/d for CRR and C, respectively). The nitrogen efficiency (RN/NI) was higher for lambs receiving PRR (44%) than both other groups (37 and 39% for C and CRR, respectively).

Figure 1 - Ruminal pH evolution.

and 4, respectively. The intake of RR decreased the proportion of acetic acid and increased the propionic acid in the total VFA for CRR and PRR compared to C diet (p<0.05). However, the butyric acid proportion was similar among diets (p>0.05).

Growth trial: feed intake and growth performance During the growth trial, the whole offered amount of concentrate was consumed by all lambs, while the intake of different roughages (oat hay, CRR and PRR) increased progressively especially for CRR. The total dry matter intake was similar for all groups (Table 3). The Final BW, average daily gain (ADG) and feed conversion ratio (FCR; DM intake / ADG) did not differ among groups (p>0.05). The daily feed cost was reduced (p<0.05) for CRR and PRR compared to control group (Table 3). It was 0.47, 0.30 and 0.39 Tunisian Dinars for C, CRR and PRR, respectively.

Hematological parameters The levels of glucose, triglycerides, cholesterol and urea were affected by the dietary treatment (p<0.05). Glucose and urea concentrations increased for PRR treatment compared to other diets. The lowest triglyceride and cholesterol levels were recorded for CRR group; however, no diet’ effect was observed on the creatinine level (Table 3).

DISCUSSION The CRR and PRR roughages had higher CP and lower NDF and ADF than oat hay, which could improve the efficiency of rumen activity11. Nevertheless, both forms contain high level

Ruminal fermentation The mean ruminal pH was higher (6.35) for C than both RR diets (5.95; Table 2). Before the morning feeding, average pH was comparable among diets (p>0.05, Figure 1); two hours after feeding, it decreased (p<0.005) for all groups; then at 8 hours post feeding, the pH value increased slightly for all groups. The ammonia concentration was lower for CRR than the rest of groups (p<0.05). It increased two hours post feeding for all groups (Figure 2). Total VFA were unaffected by the regimens (p>0.05). The evolutions of total and individual VFA are presented in Figures 3

273

Figure 2 - Ammonia-Nitrogen concentration evolution.

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Table 2 - Nutrient intake (g/day), nutrient digestibility (g/kg), nitrogen balance (g/day) and ruminal fermentation parameters of Barbarine ewelambs. C

CRR

PRR

SEM

Ct1

Ct2

Nutrient Intake Dry Matter (DM)

919

915

954

26.29

0.78

0.77

0.53

Crude Protein (CP)

76b

87b

112a

2.51

0.001

636a

523b

633a

0.94

0.001

0.07

0.001

Nutrient Digestibility

Organic Matter

668

568

683

0.95

0.001

0.05

0.001

NDF

595a

465b

566a

1.13

0.001

0.004

0.002

541

460

601

2.66

0.12

0.85

0.04

Nitrogen balance Nitrogen Intake (NI)

11.2b

15.5a

17.4a

1.54

0.001

0.06

Faecal Nitrogen (FN)

5.7b

8.35a

7.04ab

0.41

0.07

0.04

0.21

Urinary Nitrogen (UN)

1.4b

1.1b

2.7a

0.20

0.01

0.26

0.008

Retained nitrogen (RN)

4.1c

6.05b

7.66a

0.25

0.001

0.37ab

0.39b

0.44a

0.03

0.07

0.39

0.03

RN/NI

Ruminal fermentation parameters Ruminal pH

6.35a

5.95b

0.03

0.001

1.0

NH3-N (mg/100 ml)

13.4a

9.5b

12.1a

0.43

0.007

0.01

0.02

TVFA (mol/100 ml)

62.7

70.8

59.5

2.74

0.25

0.67

0.11

Acetic acid (%TVFA)

58.3a

54.1ab

49.0b

1.12

0.01

0.08

Propionic acid (%TVFA)

21.9b

29.9a

31.9a

0.95

0.001

0.40

Butyric acid (%TVFA)

14.2

13.5

14.2

0.96

0.94

0.85

0.77

NDF: Neutral Detergent Fiber; NH3-N: ammonia nitrogen; TVFA: Total Volatile fatty acid; Ct1: C vs. CRR+PRR; Ct2: CRR vs. PRR; a, b, c: Means in the same row with different superscripts differ (P<0.05). SEM: Standard error of means.

of lignin and TPC, which could compromise the efficiency of these roughages. The CP digestibility was lower for CRR (rich in TPC) than PRR diet; the last was enriched in soya and wheat bran, so it contained more protein. An evident effect of protein supplementation on CP digestibility in case of low quality forages was shown12. Generally, the use of CRR to substitute oat hay altered the nutrient digestibility for this group, however the PRR consumption that is the enhanced roughage (PRR) presented similar nutrient digestibility as the control roughage (hay). Probably the richness of these pellets on soya and wheat bran and the administration form (pellets vs. Crude form) was at the origin of this variance of nutrient digestibility. However, the administration of rosemary essential oil or its equivalent in leaves did not alter the DM digestibility13. In the same context, the RR intake did not alter the OM and CP digestibility until the rate of 70% of hay substitution for Queue Fine de l’Ouest lambs14. In the current study, the low digestibility in CRR group could be the consequence of their richness on lignin, which may result in a bound of their proteins and lead to a negative effect on nitrogen digestibility15. The RR consumption in both forms increased the nitrogen intake as previously found for the lambs from Queue Fine de l’Ouest breed at the substitution rates of 30 and 70% to oat hay14. The higher faecal N output for CRR and PRR compared to C group resulted from the higher N intake in the first given the high correlation between N intake and faecal N. In addition, it could be explained by the fact that some of the RR were not digested and therefore excreted in faeces in relationship with the presence of the condensed tannins in the RR, which could complex proteins making them unprofitable to the microflo-

ra and therefore the host animal and could be excreted in faeces15. This hypothesis is particularly valuable for CRR group having the highest FN loss, for which RR with a high TPC content were intake in crude form (Table 1). The urinary N loss was feeble for all animals, but higher for PRR than other diets. This increase as well as the increase of the uraemia for this group resulted from the surplus in N availability. The relationship between high availability of nitrogen and high urinary N was established in previous studies13. The N balance was the highest for the PRR and lowest for control group; so it was proportional to the roughage content of N, a logical result given the same amount of concentrate11. The richness of rosemary pellets in CP resulted in higher nitrogen balance, which was positive for all groups; this is curious for the control group given the low CP content in the oat hay (50 g/kg DM). Furthermore,

Figure 3 - Total volatile fatty acid concentration evolution.

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275

Table 3 - Dry matter intake (DMI), growth, economic parameters and ewe-lamb’s hematological parameters. C

CRR

PRR

SEM

Ct1

Ct2

Roughage DMI (g/d)

469

392

427

6.5

0.17

0.09

0.39

Total DMI (g/d)

910

833

868

16.5

0.17

0.09

0.39

Total DMI (g/kg w0.75)

1.65

0.27

0.08

0.39

Final Body Weight (kg)

30.7

29.9

31.4

0.41

0.35

0.94

0.15

Average Daily Gain (g)

4.53

0.61

0.76

0.34

Feed Conversion Ratio

10.7

10.03

9.3

0.58

0.87

0.61

0.89

Cost of daily feed (TND)

0.47a

0.30c

0.39b

0.001

Feed cost/kg gain (TND)

5.78a

3.87b

4.71ab

0.29

0.03

0.01

0.23

Haematological parameters (mmol/l) Glucose

2.96b

2.78b

3.37a

0.06

0.007

0.42

0.002

Triglycerides

0.23a

0.18b

0.22ab

0.006

0.03

0.08

Cholesterol

1.55

1.10

1.50

0.05

0.005

0.04

0.006

Urea

7.93b

6.36c

11.92a

0.29

0.001

0.06

0.001

106

119

115

7.37

0.76

0.48

0.86

Creatinine

SEM: Standard error of means; Ct1: C vs. CRR+ PRR; Ct2: CRR vs. PRR; TND: Tunisian Dinar. a, b, c : Means in the same row with different superscripts differ (P<0.05).

Figure 4 - Individual volatile fatty acid concentration evolution.

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the retained N as proportion of N intake was higher for PRR than C and CRR groups. This is a logical result since the nitrogen retention is primarily a function of N intake and in this case the higher nitrogen intake was presented by PRR group. Nitrogen utilization was subsequently improved by wheat bran and soya bean inclusion in the RR pellets (PRR). The average pH for C group ranged within previously reported values (6.1 to 6.8) for sheep16. A ruminal pH varying between 6.5 and 7 is optimal for microbial digestion of fibre and protein17. The decrease of ruminal pH two hours after feeding confirmed other results18 showing the pH decrease during the five hours after feeding in relationship with concentrate intake and VFA concentration increase. In addition, the decrease in pH could be a consequence of dietary N increasing12. The ammonia-Nitrogen concentration for all diets was within the range of requirements for rumen bacteria19; it is sufficient for an optimum digestion and microbial growth20. The ammonia concentrations increased 2 hours after feeding and then decreased for all groups. This tendency was previously reported12 in relationship with the high microbial activity and a favourable pH to the proliferation of protozoa. For PRR diet, the NH3-N augmentation 2 hours after feeding was spectacular corresponding to 150% ammonia concentration of CRR. This difference resulted from CP content and digestibility difference between both RR diets. Five hours post feeding, ammonia concentration decreased for all lambs in relationship with reduced microbial activity. The VFA are the end products of rumen digestion of carbohydrate that depend on the feed energy and starch quality20. Overall, the total VFA was similar for all diets that could be originated by the similarity among diet intake. The concentration before the morning feeding was the lowest. Two hours post feeding, the total VFA concentrations increased significantly among groups, this could result from the rapid rumen degradability of feed’ starch. Five hours after feeding, the VFA concentration in C and PRR decreased in comparison with CRR, but without significant difference, where VFA remained still stable. The acetic and propionic acids had the same pattern for C and PRR. However, the acetic acid was higher with C diet based in hay containing the highest NDF level, which confirm the acetic acid increase with diet rich in fiber. The lower value of this acid with PRR diet was compensated by an increase in propionic acid, even though in some cases could happen that the increase of acetate occurred at the expense of butyrate and not of propionate20. Nevertheless, generally, the high proportion of acetic acid compared with the propionic acid for all groups suggests that the diets are rich in fiber21. The inclusion of RR in animal diets did not affect feed intake, as reported by some earlier works where the inclusion of rosemary as an additive or as the partial or total replacement to oat hay, did not affect the feed intake13, 14, 22. It was reported that rosemary is an odorous herb which can limit its use in animal feeding at high levels of addition23; however, in the current study, the total substitution of hay by RR as roughage was a success. Therefore, RR was appreciated by the ewe-lambs and its consumption was not affected in both forms. The ADG was low for all diets (< 100g/day). The weakness of growth rate could be due to (i) the lamb’s sex, females had lower growth than males and (ii) the climatic conditions, since the experiment occurred in summer, with high temperatures. The similarity of ADG was obtained despite the higher N retention for PRR group (>7 g/d). This could be due to an unbalanced energy: protein ratio; for

such nitrogen supply more energy should be available. Hence, the CRR, without wheat bran and soya, resulted in similar performance as PRR sparing these ingredients. Our results are consistent with other studies24 using alternative foods (feedblocs, acacia and Atriplex). The use of RR as roughages in the current study decreased the daily feed costs, which respond to the objective of the study confirming previous results on lower cost of by-products diet24. The CRR diet recorded the highest economical feed; this result is pertinent showing the possibility of entire replacement of oat hay, expensive especially in the dry years, by crude rosemary by-products as collected in the forest. Hence, feeding RR to sheep could reduce the feed cost without affecting lamb’s performance. In addition, the feed cost per kg of gain was reduced by 30% and 18.5% for CRR and PRR, respectively compared with C diet. For plasma metabolites, the glucose, triglycerides and creatinine concentrations for all groups were within the ranges reported for small ruminant25, 26. The higher glycaemia with PRR can be related to the the wheat bran presence, rich in starch as a polymer of glucose, can originate this augmentation27. The uraemia was higher for PRR group (11.92 mmol/l) being higher than common values but similar to that resulting from carob intake28. This increase resulted from the excess of N availability for this group; similar conclusions were reported concerning the high level of dietary N and high uraemia12. The cholesterol concentration was low for CRR group. This decrease can be due to the high level of tannins in crude RR; in fact, it was shown that the blood cholesterol decreases with intake of carob rich in tannins28. The triglycerides are lipids that store energy in adipose tissue of the animal. The higher triglycerides concentrations observed for C and PRR groups showed that both diets resulted in similar amount of lipids. The rate of blood’ creatinine may be considered as an index of endogenous protein catabolism29. Besides, the concentration of creatinine in blood can be in connection with the degradation of muscle30, in the current study, it could be concluded that all lambs produced the same muscle quantity, had the same protein degradation and consequently the same creatinine rates. Generally, the haematological parameters were not affected by the inclusion of RR in both forms; these results can approve other earlier results13 showing no supply effect of rosemary leaves.

CONCLUSIONS The results of the current study indicated that distillate rosemary residues can be used as unconventional roughage that can substitute oat hay for growing ewe-lambs with similar growth rates. The pellet’s form, enriched with wheat bran and soya, resulted in high nitrogen retention without consecutive better growth; it is a waste of these ingredients, soya bean in particular. Such RR form should be used with higher energetic diet or formulated without soya. Using RR in both forms reduced the total feed cost without altering animal performance; it could be an economically advantageous practice for farmers.

Acknowledgements We wish to acknowledge (i) Dr Margalida Joy and the staff of CITA in Zaragoza (Spain) where some analyses were performed, (ii) the ESAK staff for their collaboration, (iii) Dr Noura Ben

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Youssef, researcher, and Fatma Boussen, technician, in INRAT for their help in some analysis.

Conflict of interest The authors declare that they have no conflict of interest.

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15. Ben Salem H., Atti N., Priolo A., Nefzaoui A. (2002). Polyethylene glycol in concentrate or feed blocks to deactivate condensed tannins in Acacia Cyanophylla Lindl. foliage. 1. Effects on intake, digestion and growth by Barbarine lambs. Anim Sci, 75: 127-135. 16. Molina-Alcaide E., Yanez Ruiz D., Moumen A., Martin Garcia I., (2003). Chemical composition and Nitrogen availability for goats and sheep of some olive by-products. Small Rum Res, 49: 329-336. 17. Cherdthong A., Wanapat M., Wachirapakorn C. (2011). Effects of ureacalcium mixture in concentrate containing high cassava chip on feed intake, rumen fermentation and performance of lactating dairy cows fed on rice straw. Livest Sci, 136 : 76-84. 18. Ramos S., Molina Alcaide E., Cantal Apiedra-Hijar G., Yanez Ruiz D.R., Tejido M.L., Carro M.D. (2011). Digestibility and ruminal fermentation of diets in forage type and forage to concentrate ration in sheep and goats. Opt Med Série A 99: 41-46. 19. Demeyer D.I., Van-Nevel C.J. (1986). Influence of substrate and microbial interaction on efficiency of rumen microbial growth. Reprod Nutr Dev, 26: 161-169. 20. Doreau M., Michalet-Doreau B., Grimaud P., Atti N., Nozière P. (2003). Consequences of underfeeding on digestion and absorption in sheep. Small Rumin Res, 49: 289-301. 21. Cuvelier C., Cabaraux J.F., Dufrasne I., Istasse L., Hornick J.L. (2005). Production, digestion et absorption des acides gras chez le ruminant. Ann Med Vet, 149: 49-59. 22. Kholif A.E., Matloup O.H., Morsy T.A., Abdo M.M., Abu Elella A.A., Anele U.Y., Swanson K.C. (2017). Rosemary and lemongrass herbs as phytogenic feed additives to improve efficient feed utilization, manipulate rumen fermentation and elevate milk production of Damascus goats. Livest Sci, 204: 39-46. 23. Wanapat M., Cherdthong A., Pakdee P., Wanapat S. (2008). Manipulation of rumen cology by dietary lemongrass (Stapf) powder supplementation. J Anim Sci, 86: 3497-3503. 24. Obeidat B.S., Mahmoud K.Z., Maswadeh J.A., Bsoul E.Y. (2016). Effects of feeding Atriplex halimus L. on growth performance and carcass characteristics of fattening Awassi lambs. Small Rum Res, 137: 65-70. 25. Brugere-picoux J. (1987). Particularités de la biochimie clinique des ruminants. Rec Méd Vét, 163: 1043-1053. 26. Dubreuil P., Arsenault J., Belanger D. (2005). Biochemical reference ranges for groups of ewes of different ages. Vet Res, 14: 636-638. 27. Olafadehan O.A., Adewumi M.K, Okunade S.A. (2014). Effects of feeding tannin-containing forage in varying proportion with concentrate on the voluntary intake, haematological and biochemical indices of goats. Trakia J Sci, 1: 73-81. 28. Noor-Ehsan Gobindram M.N., Bognanno M., Luciano G., Lanza M., Biondi L. (2015). Carob pulp inclusion in lamb diets: effect on intake, performance, feeding behaviour and blood metabolites. Anim Pro Sci, 56: 850-858. 29. Patrick G. H., Hopkins J. A., Ramsey W. S., Gilmore A., 1998. Effects of level of protein and type of molasses on digesta kinetics and blood metabolites in sheep. Small Rum Res, 28: 161-170 30. Reist M., Erdin D.K., Von Euw D., Tschuemperlin D., Leuenberger K.M., Delavaud C., Chilliard H.M., Hammon H.M., Kuenzi N., Blum J.W. (2003) Concentrate feeding strategy in lactating dairy cows: Metabolic and endocrine changes with emphasis on leptin. J Dairy Sci, 86: 1690-1706.

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Histo-morphological alterations in testis of goat (Capra hircus) induced by atrazine in vitro: evaluation of ameliorating effect of vitamin E

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RAYEES AHMAD BHAT1, SONAL GUPTA1, CONCETTA SAOCA2, CLAUDIA GIANNETTO2, GIUSEPPE PICCIONE2, FRANCESCO FAZIO2 1 2

Department of Zoology, Kurukshetra University, Kurukshetra, India Department of Veterinary Sciences, University of Messina, Polo SS Annunziata, 98168 Messina, Italy

SUMMARY The aim of this study was to investigate the histomorphological alterations in goat testis which are induced by atrazine. Atrazine [2-chloro-4 (ethylamino)-6 (isopropylamino) -s-triazine] is an agricultural herbicide that are used to prevent pre and post emergence broadleaf weeds in crops such as maize and sugarcane but it has been proved that it interferes with hormonal activity of the humans and animal at extremely low doses. It has now been recognized as endocrine disruptor and thus affects the reproductive performance by reducing fertility. For this purpose, the testis from slaughtered mature goat (Capra hircus) were collected and the restoration effect of α-tocopherol (100 mol L-1) on degenerative alterations generated by atrazine have been evaluated in vitro on sampled tissue. The seminiferous tubules in the experimental group treated with atrazine at dose levels of 1 nM or 100 nM showed histomorphological changes. Small vacuoles were found in the cytoplasm of spermatogonia after 4 hours of exposure to atrazine at a concentration of 1 nM. Chromolysis were also noticed in the spermatogonia and spermatids. The change detected in seminiferous tubules by 1 nM and 100 nM atrazine concentrations was decreased to some extent in the experimental group treated with 1 nM and 100 nM atrazine doses combined with 0.1 nM Vitamin E (Vitamin E is best known for its antioxidant properties). In seminiferous tubules, vacuolization was considerably decreased, the basal lamina remained intact, and nucleus fragmentation was minimized as compared to tubules treated with single pesticide dosage. The results gathered in the current study suggest that Vitamin E therapy was able to reverse atrazine-induced derangements through the improvement of antioxidant capacity and endocrine function.

KEY WORDS α-tocopherol; atrazine; seminiferous tubules; goats; vacuolization; vacuoles.

INTRODUCTION In the process of development of agriculture, pesticides have become an important tool in livestock farming, cropping, horticulture, forestry, home gardening, homes, hospitals, kitchens, roadsides, recreational and industrial areas (Kent, 1991)1. A vast majority of the population in India is engaged in agriculture and is, therefore, exposed to the pesticides due to their high persistence in the environment. These pesticides can be fungicide, herbicide, weedicide or insecticide. The 6-chloro-s-triazine herbicides, including atrazine, cyanazine, propazine and simazine, which inhibit photosynthesis (the Hill reaction, PSII) in plants, are the most heavily used pesticides in the world (Gammon, 2005)2. Atrazine [2-chloro-4 (ethylamino)-6 (isopropylamino) -s-triazine] is an agricultural herbicide that are used to prevent pre and post emergence broadleaf weeds in crops such as maize and sugarcane but it has been proved that it interferes with hormonal

Corresponding Author: Francesco Fazio (ffazio@unime.it).

activity of the humans and animal at extremely low doses. It has now been recognized as endocrine disruptor and thus affects the reproductive performance by reducing fertility. It can act within the brain to disrupt the cascade of hormonal signal need to initiate ovulation. Atrazine maintains high blood levels of 17 beta-estradiol (E2) and prolactin by suppressing the luteinizing hormone surge throughout the estrous cycle. Increase in testicular weight, degenerative changes in seminiferous tubules and abnormality in reproductive functions in rat have been recorded due to exposure of pesticides (Lee et al., 1978; Mishra et. al., 1993)3,4. Chloropyrifos-methyl, diazinon and profenofos induced decline sperm count associated with increased number of abnormal spermatozoa (Berseth et al., 2009)5. The pesticides used in the crop field have adverse effects on livestock (Hambidge, 1986)6. While most of this special issue is devoted to the testis, which is where most drug and chemically induced toxicity of the male reproductive tract is identified, being able to recognize and understand the potential effects of toxicants on the epididymis is immensely important and an area that is often overlooked (Kempinas and Klinefelter, 2014)7. The epididymis is the duct of male reproductive system where the post-testicular sperm differentiation occurs that provides microenvironment that is pivotal for testicular sperm to mature

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Figure 2 - Microphotograph of goat caput showing intact basal and principle cells and luminal space is filled with speramatozoa in control (without treated) (H&E 400X). Figure 1 - Microphotograph of goat caput showing principle cells and luminal space filled with spermatozoa in control (without treated) stained with Hematoxylin and Eosin at 400X.

Figure 3 - Microphotograph of goat caput after exposure with 1 nM concentration of atrazine for 4 h showing intact seminiferous tubules with spermatogonia having vacuolization in cytoplasm (arrow) (H&E 400X).

Figure 4 - Microphotograph of goat caput treated with 1 nM Atrazine concentration after exposure for 6 hours showing high density of spermatozoa (solid arrow) (H&E 400X).

Figure 5 - Microphotograph of goat caput treated with 100 nM Atrazine concentration after exposure for 4 hours showing increase in luminal diameter (double headed arrow) with scanty spermatozoa (H&E 400X).

Figure 6 - Microphotograph of goat caput treated with 100 nM Atrazine concentration after exposure for 6 hours showing detachment from basal lamina (solid arrow), reduction in thickness of epithelium (dashed double headed arrow), increase in diameter of lumen (H&E 400X).

cells (Kempinas and Klinefelter, 2014)7. Various environmental stresses lead to excessive production of ROS causing progressive oxidative damage and ultimately cell death. Oxidative stress can cause a host of serious health problems, including neurodegenerative disease. Scavenging or

detoxification of excess ROS is achieved by an efficient antioxidative system comprising of the non-enzymatic as well as enzymatic antioxidants (Pallavi et al., 2012)8. Antioxidants counteract free radicals and free radicals cause oxidative stress. Antioxidants have various biological activities, including the abil-

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Figure 8 - Microphotograph of goat caput treated with 100 nM Atrazine concentration with 0.1 nM vitamin E after exposure for 4 hours showing recovery in lumen and high sperm density (H&E 400X). Figure 7 - Microphotograph of goat caput after exposure with 1 nM concentration of atrazine with 0.1 nM Vitamin E after exposure for 4 h showing different cell types in epithelium viz. principal cells (solid arrow), basal cells (dashed arrow) and apical cells (hollow arrow) (H&E 400X).

Figure 9 - Microphotograph of goat caput treated with 1 nM Atrazine concentration and 0.1 nM Vitamin E after exposure of 6 hours showing decrease in luminal diameter and some clear appearance of cells in epithelium (H&E 400X).

Figure 10 - Microphotograph of goat caput after exposure with 100 nM concentration of atrazine and 0.1 nM Vitamin E for 6 hours showing increase in thickness of epithelium (double headed arrow), decrease in luminal diameter along with increased density of spermatozoa (solid arrow) (H&E 400X).

ity to counteract pesticide toxicity. Some experimental studies have demonstrated that antioxidants like vitamin C and E can be used to nullify pesticide toxicity (Yousef et al., 2006)9. Vitamin E is probably best known for its antioxidant properties. It helps to stabilize cell membranes and protect the tissues of the skin, eyes, liver, breast, and testes; as “In particular, Vitamin E, thanks to its antioxidant properties, is of paramount importance in the stabilizing the cell membrane and protecting the lungs from oxidative damage from environmental substances; helps heart and muscle cell respiration by improving function with less oxygen. Vitamin E may improve stamina and endurance and reduce cardiovascular disease. Vitamin E reduces platelet aggregation and platelet adhesiveness to collagen, even more than aspirin (Celestini et al., 2002)10. So, the aim of the study was to determine the histomorphological alterations in goat testis which are induced by atrazine. It is in the light of this information, the present study has been focused on the effect of atrazine and its reversal by Vitamin E in caput of goat (Capra hircus).

MATERIALS AND METHODS Collection of Materials Goat Testis: The testis of mature goat (Capra hircus) was procured from slaughter house near Kurukshetra latitude 29.97oN, 76.84o E. The material was brought to the laboratory in the Department of Zoology, Kurukshetra University, Kurukshetra, in ice cold 0.9% normal saline. Pesticide Atrazine: Atrazine was collected from the pesticide/ fertilizer distributor near the Kurukshetra latitude 29.97oN, 76.84oE and stored in dry conditions. Vitamin E: it was collected from the wholesale pharmaceutical distributor in Kurukshetra and brought to the laboratory in the Department of Zoology, Kurukshetra University, Kurukshetra, and stored at 10 °C.

Tissue Processing The tissue was harvested and processed for histological slide preparation. The caput sections were fixed in the Bouins fix-

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Table 1 - Experimental design of in vitro culture of testis treated with nanomolar concentration of Atrazine and Vitamin E. Control (Group A)

Exposure duration

Treatment (Group B)

Treatment (Group C)

Treatment + Ameliorate (Group D)

Treatment + Ameliorate (Group E)

Group A1

4 Hours

Group B1 (1nM : Atrazine)

Group C1 (100nM : Atrazine)

Group D1 (1nM : Atrazine + 0.1nM : Vitamin E)

Group E1 (100nM : Atrazine + o.1nM : Vitamin E)

Group A2

6 Hours

Group B2 (1nM : Atrazine)

Group C2 (100nM : Atrazine)

Group D2 (1nM : Atrazine + 0.1nM : Vitamin E)

Group E2 (100nM : Atrazine + 0.1nM : Vitamin E)

ative for 24 hours. Then the testis tissues were washed in running tap water for 2 hours. These were then dehydrated in upgrade alcohol series. After proper dehydration the tissue were embedded in paraffin wax at 58-60 °C. Blocks were made, trimmed and ribbons of wax having sections of tissue were cut using microtome at 5-micron thickness. These sections were placed on slides and were stretched to remove any folds in the tissue section. The stretched slides were stained with hematoxylin and eosin.

Staining procedure for Hematoxylin-Eosin Stain Stretched slides were de-waxed in xylene for 15 minutes and were then passed through downgrades of alcohols 100% to 30% for 5 minutes each. After keeping in water for 2-3 minutes, the slides were kept in haematoxylin stain for 2 minutes. The excess of stain was removed by giving dip in acid water. The slides were then placed under slow running tap water for another 23 minutes for stain to develop. Further upgrading of slides was done from 30% to 70% alcohol. Then these were double stained by giving a dip in eosin stain, then processed through 70% to 100% alcohol grades. After placing xylene for 10 minutes, the slides were mounted in DPX.

RESULTS Testicular tissue sections (5 m) stained with haematoxylene and eosin revealed normal histoarchitecture of the seminiferous tubules in control group. In the control group, the epididymis revealed the normal arrangement of different types of tissue (Figure 1 and 2). In the experimental group treated with atrazine with dose level 1 nM or 100 nM revealed histomorphological alterations in the seminiferous tubules. Atrazine of 1 nM concentration after 4 hours of exposure duration revealed small vacuoles in the cytoplasm of spermatogonia (Figure 3). Chromolysis were also noticed in the spermatogonia and spermatids. Pycnosis were also visible in some of the germ cells present in seminiferous tubule. As the exposure duration of atrazine increased up to 6 hours the atretogenic changes in different germ cells and somatic cells were enhanced in a time dependent manner (Figure 4). Large vacuoles of varied sizes and shapes were clearly visible in the seminiferous tubules. Various small vesicles were also noticed in nuclei of various germ cells and somatic cells. Atrazine of 100 nM concentration after 4 hours of exposure duration revealed hyalinization and fragmented nuclei. Basal lamina was also detached from the underlying cells in few portions of seminiferous tubules, were frequently observed. Vac-

uoles were of very large size and dislodging of germ cells in seminiferous tubules was observed (Figure 5). Frequency of fragmented nuclei was enhanced with increased exposure duration of 6 hours of atrazine at dose level of 100 nM concentration. It also induced a decline in diameter of spermatocyte. Chromatin gets condensed and apoptosis in spermatogonial cells were also observed (Figure 6). When experimental group treated with 1 nM and 100 nM atrazine dose along with 0.1 nM Vitamin E, the alteration observed in seminiferous tubules by 1 nM and 100 nM atrazine concentrations were reduced to some extent. Vacuolization reduced greatly, basal lamina remained intact and decrease in fragmentation of nucleus was observed in seminiferous tubules as compared to the tubules treated with only pesticide dose (Figures 7, 8, 9 and 10). Thus we can say that administration of vitamin E was able to revert the atrazine induced derangements through promoting antioxidant capacity and endocrine function.

DISCUSSION Effects of atrazine at different dose level of testicular tissue of goat have been analyzed. The results of present investigation demonstrated that degenerative changes in spermatogonial germ cells were induced by nanomolar concentration of atrazine in vitro in dose and time dependent manner. It also demonstrated the ameliorating effect of Vitamin E against atrazine toxicity on caput epididymis of goat. Light microscopic analyses revealed increased thickness of tunica albuginea, atrophied seminiferous tubules, arrested spermatogenesis, decreased Leydig cells/mm2 of interstitial tissue (2.0 ± 0.7/mm2 in high dose received rats), vasodilatation and thrombosis (Dehkhargani et al., 2011)11. The results of present finding are in agreement with the above findings. DMMP (dimethyl methyl phosphonate) altered reproductive function at all dose levels, while histological abnormalities of the testis were seen only in the high-dose group. Changes in the testes of the high-dose animals were characterized by lack of spermatogenesis or by degeneration, vacuolization, and necrosis of cells in the spermatogenic tubules (Dunnick et al., 1984)12. The results of present finding are in agreement with the above findings. Histological examination revealed significant alteration in the testis including focal mild testicular damages, blood hemorrhage and vascular congestion, hypospermatogenesis, dilation and tubular deformity, cellular vacuolated degeneration (necrosis), aspermatogenesis and tubular destruction and atrophy (Al-Jahdali, 2007)13. Results of present study demonstrated that there was increase in atretic percentage of spermatogonial

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germ cells as the exposure duration increased. This study supports the findings of Al-Jahdali (2007)13. The alteration observed in seminiferous tubules by 1 nM and 100 nM atrazine concentrations was reduced to some extent when treated with 0.1 nM Vitamin E. In seminiferous tubules, vacuolization was greatly reduced, the basal lamina remained intact, and nucleus fragmentation was reduced when compared to tubules treated with only pesticide dose. Similar types of degenerating changes were reported by Sharma et al. (2012)8 upon supplementation of Vitamin E at dose level 100 umol L-1 along with exposure of atrazine. The results demonstrated that atrazine induced atretogenic changes like chromolysis, pycnosis, fragmenataton, hyalinization and condensation were dramatically diminished when the atrazine culture was supplemented with 100 umol L-1 concentrations of vitamin E. Thereby indicating protective effect of Vitamin E on atrazine damage induced potential on the cells. Our results are in agreement with the observations of Mediratta et al. (2008)14 who have reported the sub chronic lindane exposure increased MDA level in serum indicative of an elevation in free oxygen radical generation pesticides. Antioxidant supplementation with ascorbic acid produced significant amelioration up to the control level in morphological degeneration due to pesticides. Ascorbic acid exhibited beneficial effects in detoxifying the effects of atrazine in the spermatogonial parameters in rabbits and other mammals.

Declarations of Competing Interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the study reported in this paper.

Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

References 1. 2.

6. 7. 8.

CONCLUSIONS 9.

In conclusion, the results gathered in the current study showed that atrazine-organochloride compound, even at low concentration, has damaging effects on goat testicular tissue as fine morphology and severely impaired spermatozoa formation with negative repercussion on the reproductive performance of the goat. According to the findings herein obtained, the use of antioxidants such as Vitamin E could be a represent a strategy useful in ameliorating testicular histological alteration induced by pesticides. This finding will be of great value in assessing the health hazards of pesticides in domestic animals and in providing a baseline study regarding the reproductive toxicity induced by exposure of pesticides in reproductive tissue altering structural and functional integrity and directly affecting the reproductive potential of target and non-target organism. But this kind of toxic effect can be cured by uptake of these antioxidants in diet.

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Kent J. (1991). «Pesticides in agriculture». Gammon D.W., Aldous C.N., Carr W.C., Sanborn J.R., Pfeifer K.F. (2005). A risk assessment of atrazine use in California: human health and ecological aspects. Pest Manag Sci, 61: 331-355. Lee C.C., Russell J.Q. Minor J.L. 1978. Oral toxicity of ferric dimethyl dithio carbamate (Ferbam) and tetra methylthiuram disulphide (thiram) in rodents. J Toxicol Environ Health, 4: 93-106. Mishra V.K., Srivastava M.K., Raizada R.B. (1993). Testicular toxicity of thiram in rat: morphological and biochemical evaluations. J Industr Health, 31: 59-67. Berseth P.A., Harter A.G., Zidan R., Blomqvist A., Araújo C.M., Scheicher R.H., Jena P. (2009). Carbon nanomaterials as catalysts for hydrogen uptake and release in NaAlH4. Nano Letters, 9(4), 1501-1505. Hambidge K.M., Casey C.E., Krebs N.F. (1986). Trace Elements in Human and Animal Nutrition, Mertz W ed, 2: 3-23. Kempinas W.D.G., Klinefelter G.R. (2014). Interpreting histopathology in the epididymis. Spermatogenesis, 4: e979114. Sharma R.K., Fulia A., Chauhan P.K. (2012). Antioxidant attenuation of atrazine induced histopathological changes in testicular tissue of goat in vitro. Toxicol Int, 19: 260. Yousef M.I., Awad T.I., Mohamed E.H. (2006). Deltamethrin-induced oxidative damage and biochemical alterations in rat and its attenuation by Vitamin E. Toxicology, 227: 240-247. Celestini A., Pulcinelli F.M., Pignatelli P., Lenti L., Frati G., Gazzaniga P.P., Violi F. (2002). Vitamin E potentiates the antiplatelet activity of aspirin in collagen-stimulated platelets Haematologica, 87: 420-426. Dehkhargani F.S., Malekinejad H., Shahrooz R., Sarkhanloo R.A. (2011). Detrimental effect of atrazine on testicular tissue and sperm quality: implication for oxidative stress and hormonal alterations. Iran J Toxicol, 5: 426-435. Dunnick J.K., Gupta B.N., Harris M.W., Lamb J.C. (1984). Reproductive toxicity of dimethyl methyl phosphonate (DMMP) in the male Fischer 344 rat. Toxicol Appl Pharmacol, 72: 379-387. Al-Jahdali M.O., Bisher A.S.B. (2007). Testicular histopathological alterations in rats treated with sumithion® NP 25/2.5 EC, insecticide. J Biol Sci, 7: 520-5. Mediratta P.K., Tanwar K., Reeta K.H., Mathur R., Benerjee B.D., Singh S., Sharma K.K. (2008). Attenuation of the effect of lindane on immune responses and oxidative stress by Ocimum sanctum seed oil (OSSO) in rats. Indian J Physiol Pharmacol, 52: 171-7.

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M.L. Marenzoni et al. Large Animal Review 2021; 27: 285-290

Serum trace element levels in Equine Herpesvirus 1 infected horses

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MARIA LUISA MARENZONI1*, ONUR İSKEFLI2, FATMA ATES ALKAN3,4, LUCA AVELLINI1, ÇAǦLA PARKAN YARAMIS5, M. ERMAN OR2 1 2

3 4 5

Perugia University, Faculty of Veterinary Medicine, Perugia, Italy Istanbul University - Cerrahpasa, Faculty of Veterinary Medicine, Department of Internal Medicine, Istanbul, Turkey Istanbul University - Cerrahpasa, Faculty of Medicine, Department of Biophysics, Istanbul, Turkey Beykent University, Faculty of Medicine, Department of Biophysics, Istanbul, Turkey İstanbul University - Cerrahpasa, Vocational School of Veterinary Medicine, Equine and Equine Training Program, Istanbul, Turkey

SUMMARY Equine herpesvirus 1 (EHV-1) is responsible for several syndromes, such as respiratory disease, abortion, fatal viral pneumonia in neonatal foals, and a neurological syndrome, currently referred to as EHV-1 myeloencephalopathy. The analysis of trace element levels in the sera of EHV-1 infected horses could be useful in clarifying the pathogenesis or pathophysiology of these EHVinduced clinical syndromes. Previous studies have shown significant alterations for some serum trace elements (zinc, iron, copper) in EHV-1 infected horses, and this could be justified by the putative role of these elements in many immunological pathways or by their antiviral activity. The aim of the present study was to perform a comparison by retrospective serological study of 52 EHV-1 infected and non-infected horses, both healthy and ill, to establish whether there were possible alterations in serum levels of arsenic, copper, boron, zinc, iron, chromium, magnesium, manganese, selenium, and silicium. Horses were categorized based on the type of syndrome (respiratory disease, abortion, or neurological disease) and the presence of seroconversion (by virus neutralization) and the result of polymerase chain reaction (PCR) for EHV-1. Levels of serum chromium, copper, selenium, boron, and silicium were significantly different among different groups of EHV-1 infected or non-infected horses. Serum chromium levels were higher in infected horses compared to non-infected individuals (p=0.0001). Levels of serum copper (p=0.001), magnesium (p=0.05), selenium (p=0.004), and silicium (p=0.004) were significantly lower in the horses with neurological disease. While levels of serum chromium (p=0.005) were higher, those of boron (p=0.002) were significantly lower in cases of EHV1 abortion. Overall, the present study revealed alterations in the serum levels of some trace elements between EHV-1 non-infected and infected horses, such as those that aborted or developed neurological signs. However, the relationship between the trace elements and the outcomes of the infection could not be established. Further research is needed to enlighten the effects of trace element alterations on the equine herpesvirus-1 infection pathogenesis in horses.

KEY WORDS Serum trace element levels; iron; zinc; selenium; Equine Herpesvirus-1; horse.

INTRODUCTION Equine herpesvirus 1 (EHV-1) is a virus of considerable importance in equine medicine from an epidemiological, clinical, and economic viewpoint1. It is primarily responsible for causing respiratory disease, abortion, fatal viral pneumonitis in neonatal foals, and a neurological syndrome, currently referred to as EHV-1 myeloencephalopathy (EHM). This last syndrome has been associated with EHV-1 strains of different genotypes. Earlier studies had shown that outbreaks of this clini-

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

cal disease were commonly associated with strains of the virus termed as mutant strains, because they had substitution of nucleotide A with G at position 2254. This resulted in an amino acid change of N for D at position 752 within the DNA Polgene2. These strains were also associated with cases of abortion3,4,5. Although numerous studies have been published to identify specific strains with neuropathogenic potential this issue have not been elucidated yet3,6,7,8,9. A recent research employed a case-control design to investigate possible risk factors associated with an extensive series of related outbreaks of EHM and dietary zinc supplementation was found to decrease the risk of this syndrome10. Moreover, the results of an earlier study, that investigated levels of serum copper, zinc, iron, and cobalt in EHV-1 infected horses, found significant alterations in iron,

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copper and zinc levels when compared with those of a non-infected control group11. Previous studies have shown that trace element levels possessed a dynamic mechanism in the course of infectious disease occurrences12,13,14; this may possibly be linked to their putative role in different immunological pathways15,16,17,18,19,20,21. Furthermore, antiviral activity has also been associated with certain trace elements; this could also have potential therapeutic value15,17. The aim of the present study was to perform a retrospective serological study to compare EHV-1 infected and non-infected horses, either clinically ill or healthy, to determine whether there were possible alterations in serum levels of specific trace elements associated with this virus infection.

MATERIALS AND METHODS

1 (either absence of VN antibodies or negative PCR result for virus on blood or nasal swab or cerebrospinal fluid or tissues). In the absence of a diagnosis of EHM, most of these cases were confirmed to have other diseases characterized by neurological signs. Respiratory signs included nasal discharge, cough, with or without fever.

Sample collection Blood samples from clinically affected and healthy horses, were collected into sterile, non-additive Vacutainer tubes (Becton Dickinson, Milan, Italy), and allowed to clot. Serum was drawn off after centrifugation and stored in sterile 2 ml tubes at -20 °C until analysis. Serum samples used to perform measurement of trace elements were collected at time of clinical illness for diagnostic testing or during routine sanitary monitoring of healthy horses.

Study design A retrospective serological study was carried out using sera collected from 52 horses, to compare serum levels of arsenic (As), copper (Cu), boron (B), zinc (Zn), iron (Fe), chromium (Cr), magnesium (Mg), manganese (Mn), selenium (Se), and silicium (Si). The horses comprised those uninfected with EHV1 and those infected with the virus. Each horse in the infected group was categorized based on whether they represented a case of respiratory disease, abortion, or neurological disease. The horses were all adult and infected horses were from 5 different outbreaks that occurred from 2004-2013, involving especially Thoroughbreds, Standardbreds, Warmbloods as previously described5. The horses were considered EHV-1 infected based on the presence of seroconversion (development of antibodies) or a 4-fold or greater increase in antibody levels based on testing paired sera. Blood samples were collected in the acute phase and after 21 days and tested by virus neutralization (VN). Infection was also based on a positive virus result using the polymerase chain reaction (PCR) assay. The groups used for statistical analysis included the following: controls (horses without clinical signs and negative on both VN and PCR tests for evidence of EHV-1 infection); cases of symptomatic or subclinical EHV-1 infection as confirmed on VN or PCR testing (horses with respiratory signs, mares that aborted, neurological cases); EHV-1 negative cases (clinical cases or healthy horses with an EHV-1 negative result); neurological cases (all the cases with neurological signs); non-neurological cases (all clinical cases, both healthy horses and those exhibiting signs other than those of a neurological nature); EHM cases (confirmed neurological cases positive for EHV-1); cases of EHV-1 positive abortion; cases of EHV-1 negative abortion. The same animal could be classified in different categories based on the specific statistical analysis.

Clinical presentation Abortion was defined as fetal loss before 300 days of gestation, diagnosis of pregnancy was determined by ultrasound examination. The presence and nature of acute neurological signs were determined on neurological examination. Cases with acute neurological signs suggestive of EHM and associated with a positive test result for EHV-1 (VN or EHV-1 nucleic acid detection by PCR on blood or nasal swab or cerebrospinal fluid or tissues) were considered EHM cases. EHV-1 negative neurological cases were classified based on a negative result for EHV-

Testing for EHV-1 infection The PCR and VN test were carried out in accordance with the respective test procedures described in the OIE Terrestrial Manual5,22. Serum samples were also tested for VN antibodies to EHV-4 to confirm that seroreactivity to any positive sample was due to EHV-1 and not EHV-4, taking into consideration the cross-reactivity between the two viruses. Cases of EHV-1 abortion were generally diagnosed by PCR examination of the tissues of the fetus. Respiratory, neurological, and subclinical cases of EHV-1 infection were diagnosed by PCR and/or VN testing within outbreaks of EHV-1 infection5. Where possible, strains of EHV-1 were typed as neuropathogenic or non-neuropathogenic according to a method previously described23.

Measurement of trace elements Levels of As, Cu, B, Zn, Fe, Cr, Mg, Mn, Se and Si in serum were analyzed by Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) Thermo iCAP 6000 series at the Trace Element Analysis Laboratory, Biophysics Department, Istanbul University-Cerrahpasa Medical Faculty, Istanbul, Turkey. The wavelengths used for the detection of each element are shown in Table 1. The plasma operating conditions for the ICP-OES system are provided in Table 2. The peristaltic pump speed was

Table 1 - Wavelengths used for analyzed trace elements with ICPOES. These lines were selected based on their sensitivity and freedom from spectral interferences. Elements

Wavelengths (nm)

Arsenic

189.04

Copper

324.75

Boron

249.77

Zinc

206.20

Iron

259.94

Chromium

267.72

Magnesium

285.21

Manganese

257.61

Selenium

196.09

Silicium

251.61

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M.L. Marenzoni et al. Large Animal Review 2021; 27: 285-290 Table 2 - Instrumental conditions of ICP-OES. Parameter

Value

RF incident power

1.3 kW

Plasma argon flow rate

15 L / min

Auxiliary argon flow rate

0.5 L / min

Nebulizer argon flow rate

0.7 L / min

Mist chamber

Stumar-master

Nebulizer

V-groove

100 rpm, and 1.25-mm-i.d. polytetrafluoroethylene tubing was used as transport lines. The standard concentrations used in development of calibration curves were prepared from standard stock solutions of 1000 µg/ml for each analyzed trace element24,25,26. Internal quality controls (inorganic solutions traceable to NIST certified reference material) for each analyte, were obtained from Chem-Lab NV (Belgium). The serum samples from all the groups were analyzed and compared. Each analysis was repeated three times and element levels averaged. Trace element levels were expressed as micrograms per milliliter of serum (µg/mL).

Statistical analysis Considering the limited numbers of animals comprising each of the groups, non-parametric tests were used to investigate possible differences among the groups. The serum levels of each trace element were compared among the different categories of horses, whether based on the presence or absence of clinical signs of EHV-1 infection, and the results of tests used to establish evidence of infection. Statistical association between serum levels of the selected trace elements and whether neuropathogenic or non-neuropathogenic strains of EHV-1 were detected, was also attempted. The Mann-Whitney or Kruskal Wallis test was used for comparison. A p-value ≤ 0.05 was considered significant in conducting the analysis. Data were analyzed by commercial software R, version 2.8.1 (R, Development Core Team 2007).

RESULTS The results with respect to the different categories of horses are summarized in Table 3. Only two cases of neurologic disease met the criteria for EHM. The other neurological cases included horses with ataxia (n= 7), paresis of the hind limbs (n=5), suspicion of encephalitis (n=3), epilepsy (n=1). Because of the limited number of cases, no further analyses were performed on the EHM group. Respiratory signs were observed in 4 cases and

so they were limited to a slight nasal discharge lasting a day or so, the cases were considered subclinical. Levels of serum chromium, copper, selenium, boron, and silicon were found to be significantly different among the five different groups. Trace element levels in the serum of the EHV1 uninfected healthy horses (controls) are provided in Table 4. Medians and minimum-maximum serum trace element levels in EHV-1 infected and non-infected horses are presented in Table 5. On statistical analysis, serum chromium levels were higher in infected horses compared to non-infected animals (p=0.0001). Medians and minimum-maximum of serum trace element levels in horses with or without neurological signs are presented in Table 6. Serum copper (p=0.001), magnesium (p=0.05), selenium (p=0.004) and silicium levels (p=0.004) were found to be significantly lower in the horses displaying neurological signs. Serum trace element levels in EHV-1 abortion positive and negative mares are presented in Table 7. Whereas the levels of serum chromium (p=0.005) were significantly higher, those of boron (p=0.002) were significantly lower in mares positive for EHV1 abortion. Three cases of neuropathogenic strain infection, one case of EHM, two cases of abortion and five cases of infection with nonneuropathogenic strains of EHV-1, all had similar levels of the trace elements under investigation.

DISCUSSION Based on the outcomes of the present study, significant alterations of serum trace element levels were observed in EHV-1 infection. However, there is a need to define whether these alterations were related to EHV-1 infection or to the particular clinical outcome (respiratory, neurological or abortion) recorded in the mixed population of horses used in this study. There may be some relationship between serum trace element levels and the pathophysiology of the mentioned clinical syndromes. In a previous study11, serum copper and zinc levels were lower in cases of EHV-1 infection. By comparison, the findings of the current study indicated that serum copper levels were lower (p=0.001), and serum zinc levels had no significancy among the various groups. Zinc appeared to have a relevant role in EHV-1 and certain other herpesvirus infections such as bovine herpesvirus type 110,11,12. Traub-Dargatz et al. in 201310 found fewer cases of EHM in horses receiving a zinc dietary supplementation. In another study, levels of serum zinc were decreased in EHV-1 infected horses11. However, this finding should be interpreted with caution since the number of cases and controls (9 infected horses and 9 con-

Table 3 - Grouping of horses based on clinical outcome and EHV-1 test results. Clinical category Abortion

EHV-1 test results Positive

Negative

Total

13*

Neurological disease

Healthy

8 (subclinical cases)

16 (controls)

24*

Total

*37 cases were non neurological cases.

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Table 4 - Parameters of the serum trace element levels, expressed in µg/mL, in the control horses. Trace elements (µg/mL)

Controls (n= 16)

Arsenic

0.12 (0.80-0.18)

Copper

1.29 (0.99-2.23

Boron

0.08 (0.03-0.19)

Zinc

0.92 (0.51-1.44)

Iron

2.63 (0.78-4.8)

Chromium

0.01 (0-0.032)

Magnesium

18.3 (10.65-28.65)

Manganese

0.004 (0-0.27)

Selenium

1.005 (0.71-1.29)

Silicium

3.17 (0.15-7.96)

trols) was limited. Moreover, the study classified EHV-1 infected and control horses based on the outcome of VN testing and clinical examination, without providing a description of the observed clinical signs, nor information about the origin of the animals, their possible genetic relationship, feed, environment, or other factors11. Serum chromium levels were found to be significantly high-

er in horses infected with EHV-1 (p=0.0001) and mares that aborted due to EHV-1 (p=0.005). There is a lack of information in the literature about chromium levels in cases of infectious diseases in horses. Based on the findings of the current study, chromium would be worth investigating further to determine if the element might be associated with abortion in mares caused by EHV-1 or whether it was related to the immune response to infection. Chromium has been reported as beneficial for reducing exercise stress27. If preliminary findings are confirmed, chromium supplements could be easily added to the daily ration of horses. However, additional research on this trace element is needed in horses, taking into consideration parameters such as breed, age, nutritional diet, season of the animals under study. Serum magnesium (p=0.05), selenium (p=0.004), and silicium (p=0.004) were found to be significantly lower in neurological cases. It is possible that these elements could be involved in the pathophysiology of neurological disease28. No statistical differences were found in serum iron levels in the present study. This contrasts with the finding of a previous study11, in which an increase in serum iron was reported in EHV1 infected compared to non-infected horses. However, yet a further study20 found transitory differences in serum iron levels only during the acute phase of experimental EHV-1 infection,

Table 5 - Comparison among medians, expressed in µg/mL (minimum-maximum values in brackets) of analyzed serum trace elements in EHV-1 infected and non-infected horses and their corresponding p-values. Trace elements (µg/mL)

EHV-1 infected cases group (n= 16)

EHV-1 non-infected cases group (n=36)

p-value

Arsenic

0.14 (0.03-0.19)

0.13 (0.05-0.22)

0.76

Copper

1.27 (0.50-1.62)

1.20 (0.81-2.23)

0.98

Boron

0.07 (0.02-0.12)

0.97 (0.03-0.47)

0.07

Zinc

0.90 (0.03-1.80)

0.86 (0.51-1.98)

0.82

Iron

2.36 (1.55-8.90)

2.61 (0.78-4.8)

0.76

Chromium

0.02 (0.004-0.04)

0.01 (0.0-0.03)

0.0001

Manganese

0.003 (0.001-0.03)

0.004 (0-0.03)

0.76

Magnesium

18.34 (7.17-24)

17.78 (5.55-28.65)

0.57

Selenium

0.83 (0.3-1.29)

0.97 (0.65-1.29)

0.37

Silicium

1.25 (0.28-3.61)

1.78 (0.09-7.96)

0.53

Table 6 - Comparison among medians, expressed in µg/mL (minimum-maximum values in brackets) of analyzed serum trace elements in horses with and without neurological signs and their corresponding p-values. Trace elements (µg/mL)

Neurological cases group (n=15)

Non-neurological cases group (n=37)

p-value

Arsenic

0.13 (0.05-0.2)

0.14 (0.03-0.22)

0.16

Copper

1.13 (0.5-1.54)

1.29 (0.87-2.23)

0.001

Boron

0.69 (0.03-0.47)

0.81 (0.02-0.32)

0.12

Zinc

0.86 (0.51-1.8)

0.83 (0.03-1.98)

0.56

Iron

2.67 (1.13-8.90)

2.41 (0.78-5.66)

0.99

Chromium

0.007 (0-0.21)

0.01 (0-0.04)

0.43

Manganese

0.003 (0.001-0.02)

0.004 (0-0.03)

0.26

Magnesium

16.66 (5.55-22.95)

18.3 (7.17-28.65)

0.05

Selenium

0.81 (0.3-1.16)

0.98 (0.39-1.29)

0.004

Silicium

0.59 (0.09-2.70)

2.06 (0.15-7.96)

0.004

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Table 7 - Comparison among medians, expressed in µg/mL (minimum-maximum values in brackets) of analyzed serum trace elements in EHV-1 abortion positive and negative mares and their corresponding p-values. Trace elements (µg/mL)

EHV-1 positive aborted mares group (n=6)

EHV-1 negative aborted mares group (n=7)

p-value

Arsenic

0.14 (0.03-0.17)

0.15 (0.12-0.22)

0.63

Copper

1.45 (0.87-1.62)

1.40 (1.08-1.77)

0.84

Boron

0.07 (0.02-0.12)

0.17 (0.09-0.32)

0.002

Zinc

0.89 (0.62-1.8)

0.73 (0.52-1.50)

0.30

Iron

2.17 (1.55-4.05)

1.77 (1.43-3.75)

0.63

Chromium

0.02 (0.06-0.04)

0.001 (0-0.01)

0.005

Manganese

0.002 (0.001-0.03)

0.004 (0.003-0.01)

0.30

Magnesium

17.39 (7.17-18.53)

19.95 (15.15-25.9)

0.53

Selenium

0.79 (0.39-1.2)

1.06 (0.72-1.22)

0.18

Silicium

0.64 (0.28-3.61)

3.88 (0.18-5.77)

0.53

but not in the recovery phase. Moreover, both studies were carried out on a limited number of cases. Further studies are needed to understand the dynamics of iron in EHV-1 infection. Iron is well recognized as a relevant element in various infections11,20,21. In some of them, iron levels remain low beyond the acute phase of the infection, for instance in the case of Streptococcus zooepidemicus infection21. Serum iron levels were previously investigated also in mares that aborted. Iron storage has been reported during pregnancy of horses29. However, no significancy in serum iron levels was observed between the different groups in the present study. Because the number of cases analyzed in particular categories of this study (EHM cases or cases infected with neuropathogenic or non-neuropathogenic strains of EHV-1) was very limited, possible alterations in serum trace element levels may not have been detected. It should be noted that all the studies carried out on trace element levels in EHV-1 infected or non-infected horses11,20,30 involved relatively few animals. However, based on a limited contribution, it could be worthwhile to increase the number of analyses undertaken from the perspective of cumulative analysis (meta-analysis).

References 1.

CONCLUSIONS The variations of priorly published studies related with trace element levels should be taken into consideration. More studies of specific systemic diseases might improve our knowledge of the pathogenesis of infectious diseases. Further studies are needed to better understand trace element involvement in EHV1 infections and whether the role(s) of these elements might have potential for prophylactic or therapeutic interventions in this infection.

10.

11.

ACKNOWLEDGEMENTS 12.

This study was funded by Scientific Research Projects Coordination Unit of Istanbul University-Cerrahpasa [Project number: 2017-20035]. The authors thank Prof. Peter J. Timoney for his critical and constructive review of the work.

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Patel J.R., Heldens J. (2005). Equine 1 (EHV-1) and 4 (EHV-4) - epidemiology, disease and immunoprophylaxis: a brief review. Vet J, 170: 1423. Nugent J., Birch-Machin I., Smith K.C., Mumford J.A., Swann Z., Newton J.R., Bowden R.J., Allen G.P., Davis-Poynter N. (2006). Analysis of equid herpesvirus 1 strain variation reveals a point mutation of the DNA polymerase strongly associated with neuropathogenic versus nonneuropathogenic disease outbreaks. J Virol, 80: 4047-4060. Smith K.L., Allen G.P., Branscum A.J., Cook F.R., Vickers M.L., Timoney P.J. Balasuriya U.B.R. (2010). The increased prevalence of neuropathogenic strains of EHV-1 in equine abortions. Vet Microbiol, 141: 5-11. Fritsche A.K., Borchers K. (2011). Detection of neuropathogenic strains of Equid Herpesvirus 1 (EHV-1) associated with abortions in Germany. Vet Microbiol, 147: 176-180. Marenzoni M.L., Bietta A., Lepri E., Casagrande Proietti P., Cordioli P., Canelli E., Stefanetti V., Coletti M., Timoney P.J., Passamonti F. (2013). Role of equine herpesviruses as co-infecting agents in cases of abortion, placental disease and neonatal foal mortality. Vet Res Commun, 37: 311317. Pusterla N., Wilson D.W., Madigan J.E., Ferraro G.L. (2009). Equine herpesvirus-1 myeloencephalopathy: a review of recent developments. Vet J, 180: 279-289. Pronost S., Léon A., Legrand L., Fortier C., Miszczak F., Freymuth F., Fortier G. (2010). Neuropathogenic and non-neuropathogenic variants of equine herpesvirus 1 in France. Vet Microbiol, 145: 329-333. Dunuwille W.M.B., Yousefi Mashouf N., Balasuriya U.B.R., Pusterla N., Bailey E. (2020). Genome-wide association study for host genetic factors associated with equine herpesvirus type-1 induced myeloencephalopathy. Equine Vet J, 52: 794-798. Pusterla N., Hatch K., Crossley B., Wademan C., Barnum S., Flynn K. (2020). Equine herpesvirus-1 genotype did not significantly affect clinical signs and disease outcome in 65 horses diagnosed with equine herpesvirus-1 myeloencephalopathy. Vet J, 255: 105407. Traub-Dargatz J.L., Pelzel-McCluskey A.M., Creekmore L.H., Geiser-Novotny S., Kasari T.R., Wiedenheft A.M., Bush E.J., Bjork K.E. (2013). Casecontrol study of a multistate equine herpesvirus myeloencephalopathy outbreak. J Vet Intern Med, 27: 339-46. Yörük I., Deger Y., Mert H., Mert N. (2007). Serum concentration of copper, zinc, iron, and cobalt and the copper/zinc ratio in horses with equine herpesvirus-1. Biol Trace Elem Res, 118: 38-42. Orr C.L., Hutcheson D.P., Grainger R.B., Cummins J.M., Mock R.E. (1990). Serum copper, zinc, calcium and phosphorus concentrations of calves stressed by bovine respiratory disease and infectious bovine rhinotracheitis. J Anim Sci, 68: 2893-900. Basit S. (2013). Vitamin D in health and disease: a literature review. Br J Biomed Sci, 70: 161-72. Gunville C.F., Mourani P.M., Ginde A.A. (2013). The role of vitamin D

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Serum trace element levels in Equine Herpesvirus 1 infected horses in prevention and treatment of infection. Inflamm Allergy Drug Targets, 12: 239-245. Suttle N.F., Jones D.G. (1989). Recent developments in trace element metabolism and function: trace elements, disease resistance and immune responsiveness in ruminants. J Nutr, 119: 1055-1061. Shankarand A.H., Prasad A.S. (1998). Zinc and immune function: the biological basis of altered resistance to infection. Am J Clin Nutr, 68: 447-463. Erickson K.L., Medina E.A., Hubbard N.E. (2000). Micronutrients and innate immunity. J Infect Dis, 182: 5-10. Failla M.L. (2003). Trace elements and host defense: recent advances and continuing challenges. J Nutr, 133: 1443-1447. Chaturvedi U.C., Shrivastava R., Upreti R.K. (2004). Viral infections and trace elements: a complex interaction. Curr Sci, 87: 1536-1554. Brosnahan M.M., Erb H.N., Perkins G.A., Divers T.J., Borges A.S., Osterrieder N. (2012). Serum iron parameters and acute experimental EHV 1 infection in horses. J Vet Int Med, 26: 1232-1235. Varma K.J., Powers T.E., Powers J.D., Spurlock S.L. (1984). Standardization of an experimental disease model of Streptococcus zooepidemicus in the equine. J Vet Pharmacol Ther, 7: 183-188. OIE (2017). Equine rhinopneumonitis (infection with equid herpesvirus-1 and -4). In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. https://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/3.05.09_EQUINE_RHINO.pdf. Accessed 12 May 2020.

23. Allen G.P. (2007). Development of a real-time polymerase chain reaction assay for rapid diagnosis of neuropathogenic strains of equine herpesvirus1. J Vet Diagn Invest, 9: 69-72. 24. Bursalıoğlu E.O., Alkan F.A., Barutçu Ü.B., Demir M., Karabul Y., Balkan B., Oz E., Içelli O. (2017). Prediction of electron density and trace element concentrations in human blood serum following radioiodine therapy in differentiated thyroid cancer patients. Measurement, 100: 19-25. 25. Arslan H.H., Tarhan D., Cenesiz S., Alkan F.A., Ozcan U., Arslan E.T., Barutcu U.B., Or M.E. (2018). Evaluation of Trace Element Levels and Antioxidant Metabolism in Cattle with Cutaneous Papillomatosis. Acta Sci Vet, 46: 5. 26. Alkan F.A., Karis D., Cakmak G., Ercan A.M. (2018). Analysis of the relationship between hemorheologic parameters, aluminum, manganese, and selenium in smokers. Biol Trace Elem Res, 187: 22-31. 27. Pagan J.D., Jackson S.G., Duren S.E. (1995). The effect of chromium supplementation on the metabolic response to exercise in Thoroughbred horses. Proc. 14th Equine Nutrition and Physiology Symposium, 96-101. 28. Vitale S., Hague D.W., Foss K., de Godoy M.C., Selmic L.E. (2019). Comparison of Serum Trace Nutrient Concentrations in Epileptics Compared to Healthy Dogs. Front Vet Sci, 6: 467. 29. Satué K., Montesinos P. (2013). Plasma biochemistry in pregnant Spanish purebred broodmares. Comp Clin Path, 22: 113-117. 30. Youssef M.A., El-Khodery S.A., Ibrahim H.M. (2012). Antioxidant trace elements in serum of draft horses with acute and chronic lower airway disease.

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Traumatic lateral hysterocele complicated with intestinal adhesions in a pregnant goat

291

KHAN SHARUN1*#, KALLEMUCHIKAL MANIKANDAN MANJUSHA1#, ROHIT KUMAR1, ABHISHEK CHANDRA SAXENA1, PRAKASH KINJAVDEKAR1, UJJWAL KUMAR DE2, ABHIJIT MOTIRAM PAWDE1, AMARPAL1 1 2

Division of Surgery, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India Division of Medicine, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India

SUMMARY Traumatic abdominal hernias are common in small ruminants. Small ruminants, including goat and sheep, are highly susceptible to acquired abdominal hernias. Injuries leading to the weakening of muscles can facilitate the contents to herniate through the abdominal wall. A three-year-old non-descript pluriparous full-term pregnant goat was presented to the Referral Veterinary Polyclinic and Teaching Veterinary Clinical Complex, ICAR-Indian Veterinary Research Institute, Izatnagar, with an exudating wound on the right lateral abdomen over a pronounced swelling. The goat was previously attacked and bitten by a group of stray dogs two months earlier, following which the swelling started to increase in size progressively. General clinical examination identified an extensive swelling on the right lateral abdomen with fluid thrill. Fetal parts and fetal movements were appreciable during palpation. The presence of a live fetus was confirmed within the hernial contents using radiographic and ultrasonic examination. A circular wound was also identified at the center of the swelling that produced a bloody discharge. The goat was subjected to an emergency cesarean section under sedation and regional anesthesia. The uterus was found herniated through the oblique abdominal muscles into the subcutaneous space. Two live fetuses were recovered from the herniated uterus. The herniated contents were replaced into the abdominal cavity after relieving the adhesion between the intestinal loop and subcutaneous tissue. The hernial ring was freshened, and herniorrhaphy was performed using synthetic polyamide suture material (overlapping mattress sutures). Post-operatively the animal was treated with antibiotics and anti-inflammatory drugs. The traumatic lateral hernia was successfully reduced in the goat after performing an emergency caesarean section and herniorrhaphy. When the life of the fetus is at stake, hysterocele should be considered as a severe condition, and an emergency caesarean section has to be performed in cases where fully developed viable fetuses are present in the gravid uterus.

KEY WORDS Traumatic lateral hysterocele, Lateral hernia, Exudating wound, Caesarean section, Surgical management; Goat.

INTRODUCTION A hernia is the protrusion of visceral organs through a normal or abnormal opening in the body1. It is broadly classified into congenital and acquired hernia. Both congenital and acquired hernias are common in domestic animals, especially in small ruminants. The most common cause of acquired hernia is trauma. A typical hernia constitutes a hernial ring or an opening in the muscle due to trauma or congenital defect and a hernial sac beneath the skin. The hernial contents vary according to the anatomical location2. Multiple factors like suture dehiscence, horn goring, trauma, road traffic accidents, and violent contact with blunt objects can contribute to the development of a hernia. Furthermore, increased intra-abdominal pressure, obesity, and excessive stretching of muscles during pregnancy could predispose the animal to hernias3. In ruminants, goats are more prone to injury and subsequent abdominal hernias than cattle due to their small size and docile Corresponding Author: Khan Sharun (sharunkhansk@gmail.com). # Both Khan Sharun and K. M. Manjusha equally contributed to the work and therefore considered first authors.

nature. It has been found that 71.4% of the abdominal hernia cases in small ruminants are due to trauma4. The nature and type of hernial contents depend upon the site of herniation1. The uterus with its contents can get entrapped in the hernial sacs of abdominal, inguinal, and perineal hernias leading to dystocia1,5. Although most hernias can be managed by performing herniorrhaphy, extensive abdominal wall defects should be corrected by performing hernioplasty4. The present paper describes the clinical findings and surgical management of traumatic lateral hysterocele complicated with intestinal adhesions in a three-year-old non-descript pluriparous full-term pregnant goat.

CASE HISTORY AND CLINICAL FINDINGS A three-year-old non-descript pluriparous full-term pregnant goat weighing 20 kg was presented to the Referral Veterinary Polyclinic and Teaching Veterinary Clinical Complex, ICARIndian Veterinary Research Institute, Izatnagar, Bareilly with a swelling on the right lateral abdomen and an exudating wound on the swelling (Figure 1). History revealed that the goat was

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Traumatic lateral hysterocele complicated with intestinal adhesions in a pregnant goat

Figure 1 - (a) Extensive swelling on the right lateral abdomen of the three-year-old non-descript pluriparous full-term pregnant goat. (b) The discharge (with blood) produced from the circular wound present at the center of the swelling.

previously attacked and bitten by a group of stray dogs two months before, which resulted in progressively increasing swelling at the site of the dog bite. On general clinical examination, the goat was found dull and depressed at the time of presentation. A circular wound measuring about 3 cm (diameter) discharging bloody contents was also identified at the center of the swelling (Figure 1b). On palpation of the swelling, fetal parts were easily identified along with the fluid thrill. Furthermore, fetal movements were also detected during the palpation. The cervix was found to be dilated during the per-vaginal examination. Based on the clinical examination, the condition was tentatively diagnosed as hysterocele. Radiological examination identified the presence of a fetal skeleton within the swelling, confirming the diagnosis

Figure 2 - Radiograph showing the presence of a fetal skeleton within the hernial swelling on the right side of the animal (arrow pointing to the cranial aspect of the animal).

(Figure 2). The ultrasonographical examination of the swelling confirmed the presence of two live fetuses with fetal heartbeats measuring 189 beats/minute (Figure 3).

TREATMENT The goat was subjected to an emergency caesarean section under sedation and regional anesthesia. Epidural anesthesia (lumbosacral site) was induced using 2% lignocaine hydrochloride at the dose rate of 1 ml/7 kg body weight (3 ml total dose). The goat was sedated using an intramuscular injection of midazolam at the dose rate of 0.2 mg/kg body weight. Fluid therapy was initiated using normal saline at the dose rate of 10 ml/kg body weight. The right lateral abdomen was prepared for aseptic surgery. The goat was then positioned on the surgical table in the left lateral recumbency. The skin incision was made over the hernial swelling in continuation to the wound. The subcutaneous tissue was carefully dissected to enter the hernial sac. The herniated contents (gravid uterus) were carefully examined to confirm the absence of adhesions with the hernial sac and the abdominal organs. The uterus was found to be herniated through the oblique abdominal muscles into the subcutaneous space. The gravid uterus was exteriorized

Figure 3 - Ultrasonographic examination confirmed that the fetuses were live with a fetal heartbeat of 189 bpm.

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Figure 4 - (a) The gravid uterus was exteriorized through the incision. (b) Adhesion between the intestine and subcutaneous tissue (arrow).

through the skin incision (Figure 4a). Two fully developed live fetuses were recovered through the incision made in the uterus. After the uterine incision closure, the herniated parts of the uterus were replaced into the abdominal cavity. The adhesions between the intestinal loops and the subcutaneous tissue were removed carefully (Figure 4b). The hernial ring was freshened, and herniorrhaphy was performed using synthetic polyamide suture material (overlapping mattress sutures). The subcutaneous tissue and the skin were then sutured in the routine manner using catgut (No. 2 size) and polyamide (No. 2 size) suture material, respectively. Special care was taken to limit the dead space between different layers of tissue. Post-operatively the goat was treated with antibiotic enrofloxacin at the dose rate of 5 mg/kg body weight for seven days and meloxicam at the dose rate of 0.5 mg/kg body weight for three days via the intramuscular route. A single dose of tetanus toxoid (0.5 ml) was also given through the intramuscular route as a preventive measure.

RESULTS The animal made an uneventful recovery following the surgery. Both the kids were alive, healthy without any malformation, and survived the days following the surgery. The goat started taking feed from the next day onwards. Although mild postoperative inflammatory swelling was observed at the surgical site, it got reduced subsequently. The skin sutures were removed on the tenth postoperative day. The traumatic lateral hernia was successfully reduced in the goat after performing an emergency cesarean section and herniorrhaphy.

DISCUSSION Lateral hernias are considered rare in small ruminants (sheep and goats). Both lateral and ventral hernias can occur through the acquired or congenital defects in the abdominal wall other than the natural orifices1. Hernia ventral to stifle skin fold

is termed as the ventral hernia, and the rest in the abdominal wall is known as the lateral hernia. Lateral or ventral abdominal hernias are commonly seen along the costal arch, ventral abdominal wall, high or low in the flank, and between the last few ribs6. The hernia can also be classified as a reducible or irreducible type. In the case of reducible hernias, the hernial contents can be pushed back into the abdominal cavity. In contrast, the contents of irreducible hernias cannot be replaced into the normal anatomical position within the abdominal cavity due to strangulation, incarceration, and adhesion between the hernial contents and the surrounding tissue6. The swelling associated with the hernia may vary in size and shape. Identification of the hernial ring is the most critical part in confirming the diagnosis of a hernia. The hernial mass should be differentially diagnosed from other conditions such as hematoma, cyst, abscess, and tumor. Radiographic evaluation, including both plain and contrast radiography, will help to identify and differentiate the herniated contents in the case of irreducible hernia1,6. The size of the hernial ring varies in diameter depending on the extent of the defect. A peritoneal lining will be absent in the hernial sac that is solely formed between the skin and the subcutaneous tissues. It could contribute to the development of adhesions between the viscera and the hernial sac resulting in complications. A hernia can be diagnosed based on the history, clinical signs, and physical examination1. Treatment is based on the condition of the animal and the type of hernia. Surgical management offers a rapid method for the treatment of hernia. Abdominal hysterocele in goats due to the gravid uterus can result in a prolonged gestation period. However, the ventral abdominal hysterocele is associated with excessive enlargement of the udder due to the peculiar position of the hernia7. Ventral hysterocele (gravid uterus) can occur in goats due to automobile accidents and subsequent tearing of the abdominal muscles8. The goats that are pregnant with multiple fetuses are highly susceptible to abdominal hysterocele, especially during the advanced stages of pregnancy7. This can be attributed to the progressive weakening of the abdominal muscles, ultimately leading to a hernia. Ventral hernia of the gravid uterus can also occur in goats due to sudden blow or trauma to the abdomi-

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Traumatic lateral hysterocele complicated with intestinal adhesions in a pregnant goat

nal wall9. In the present case, the goat was full-term pregnant with two fetuses. The trauma associated with the dog bite might have resulted in the tearing of oblique abdominal muscles that led to the herniation of the gravid uterus. Contrary to the previous reports7,9, both the kids were alive, healthy, and survived the following days. Delay in the diagnosis and treatment of abdominal hysterocele can result in complications. The gravid horn trapped within the hernial sac can get ruptured, affecting the viability of the fetus10. Such unnoticed herniation of the gravid uterus can result in complications such as tissue necrosis, adhesion between the uterus, omentum, small intestine, and abdominal wall, and ruptured hysterocele10. Therefore, hysterocele involving gravid uterus should be immediately subjected to emergency caesarean section if the fetus is full-term and live8,9. Furthermore, utero-peritoneal adhesions can mimic the ventral hysterocele during the clinical examination11,12. Both utero-peritoneal adhesions and hysterocele can cause prolonged fetal retention in small ruminants11-13. However, both conditions require surgical correction by performing caesarean section11,12. Reproductive diseases and disorders in small ruminants, including sheep and goats, are always associated with substantial economic loss14. The extensive management system commonly used for the rearing of goats makes them more susceptible to trauma12,15. The successful management of obstetrical cases in goats mainly depends on the time of presentation and the degree of damage16. Goats are highly susceptible to stray dog attacks as they are mainly reared under an extensive management system. Dog bites have previously been reported to cause enterocutaneous fistula in goats that required extensive surgery (enterectomy and enteroanastomosis) for the correction17. In the present case, intact intestinal loops were found to have adhered to the subcutaneous tissue. The adhesion might be the result of abdominal trauma due to the dog bite.

CONCLUSIONS Hernias, in general, are not considered an emergency condition. However, delayed treatment can lead to complications like adhesions between viscera and subcutaneous tissues due to loss of muscle elasticity. However, when the life of the fetus is at stake, hysterocele should be considered an emergency condition in all animals, irrespective of the cause. In the present case, fetuses were completely developed, and therefore an emergency caesarean section was considered the most suitable surgical intervention for managing lateral hysterocele.

Funding No substantial funding to be stated.

Disclosure statement All authors declare that there exist no commercial or financial relationships that could, in any way, lead to a potential conflict of interest.

References 1. 2.

4. 5. 6.

10.

11.

12.

13. 14.

15.

16.

Acknowledgments The authors are thankful to the Director, ICAR-Indian Veterinary Research Institute, Izatnagar (UP) and Head, Division of Surgery, ICAR-Indian Veterinary Research Institute, Izatnagar (UP) for the facilities provided.

17.

Abdin-Bey, M.R., Ramadan, R.O. (2001). Retrospective study of hernias in goats. Sci J King Faisal Univ, 2(1): 77-88. Gyang, E.O. (1988). Thoracic and Abdominal wall, Herniorraphy in Large Animals. In: Introduction to Large Animal Surgery. Agitab publishers Ltd, Nigeria. 256-266. Blood, D.C., Radostits, O.M., Gay, C.C. (1997). Diseases of respiratory system. In: Textbook of Diseases of Cattle, Sheep, Pigs, Goats and Horses. W. B. Saunders Company Ltd, Philadelphia, 421-422. Al-Sobayil, F.A., Ahmed, A.F. (2007). Surgical treatment for different forms of hernias in sheep and goats. J Vet Sci, 8(2): 185-191. Radhakrishnan, C., Balasubraman, S., Thilagar, S. (1993). Repair of ventral metrocele (gravid) in a goat. Vet Rec, 132(4): 92-93. Tyagi, R.P.S., Singh, J. (1993). Ruminant Surgery: A Textbook of the surgical diseases of Cattle, Buffaloes, Camels, Sheep and Goats. CBS publishers, New Delhi, 484. Parvez, M.A., Sutradhar, B.C., Pallab, M.S., Hasan, T., Khatun, R. (2016). Surgical correction of ventral abdominal hysterocele in gravid uterus of a doe. Res J Vet Pract, 4(4): 76-79. Vijayanand, V., Gokulakrishnan, M., Rajasundaram, R.C., Thirunavukkarasu, P.S. (2009). Ventral hernia [Hysterocele-gravid] in a goat-a case report. Indian J Anim Res, 43(2): 148-150. Kumar, A., Katiyar, R., Gautam, D., Shivaraju, S., Prasad, J.K., Ghosh, S.K. (2018). Hysterocele along with false or secondary extrauterine pregnancy in a goat: A rare case presentation. Theriogenology Insight, 8(1): 45-47. Peker, C., Uçar, E.H., Varoğlu, O., Erdoğan, G. (2019). An uncommon fetal retention case: Ruptured ventral hernia in a sheep. J Hell Vet Medical Soc, 70(4): 1901-1904. Erdogan, G., Ucar, E.H., Peker, C., Kilic, N. (2015). Prolonged co-twin foetal retention in a sheep: caused by hysterocele? Large Anim Rev, 21(2): 87-89. Khan, S., Satheesh, A., Sabarinadh, V.R., Ranjith Mohan, M., Sidhique, S.A., Panikkassery, S. (2018). Surgical management of dystocia due to uterine adhesion in a goat. J Entomol Zool Studies, 6(6): 988-991 Sharun, K., Erdoğan, G. A. (2019). Review: Obstetrical Emergencies in Small Ruminants. Alex J Vet Sci, 62(1): 1-16. Ali, S., Zhao, Z., Zhen, G., Kang, J.Z., Yi, P.Z. (2019). Reproductive problems in small ruminants (Sheep and goats): A substantial economic loss in the world. Large Anim Rev, 25(6): 215-223. Tanko, F.S., Odinya, A.V., Augustine, A., Dupe, R.A.B., Bala, U., Garba, K., Olu, O.S. (2015). An Eight Year Retrospective Study on the Prevalence of Hernias in Large Animals at the Veterinary Teaching Hospital Ahmadu Bello University, Zaria. Anim Vet Sci, 3(4): 125-127. Manjusha, K.M., Sharun, K., Haritha, C.V., Lekshmi, S.A., Faslu, A.T., Rahman, K.S., Sivaprasad, M.S. (2020). Management of Post-Partum Uterine Eversion in a Doe Using Retention Sutures. Int J Livest Res, 10(1), 88-93. Khan, S., Satheesh, A., Sabarinadh, V.R., Ranjith Mohan, M., Sidhique, S.A., Panikkassery, S. (2018). Surgical management of enterocutaneous fistula secondary to dog bite wound in a Malabari goat. J Entomol Zool Studies, 6(6): 927-929.

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