ECHO-MORPHOMETRIC MEASUREMENTS OF UDDER AND TEATS IN SHEEP 33

ECHO-MORPHOMETRIC MEASUREMENTS OF UDDER AND TEATS IN SHEEP 33 Relationships between somatic cell count and certain udder and teat echo-morphometric measurements in mastitis caused by Staphylococcus aureus in Awassi sheep Z. BANI ISMAIL*, M. ALEKISH, O. AL-SHEYAB Faculty of Veterinary Medicine, Jordan University of Science and Technology, Irbid 22110 Jordan *Corresponding author: zuhair72@just.edu.jo SUMMARY The objectives of this study were to describe the echo structure of the udder and teats in normal and in mastitic lactating Awassi sheep and to report possible relationship between high somatic cell count in milk and certain echo-morphometric measurements in Staphylococcus aureus mastitis. B-mode ultrasonography was used to examine the udders and teats of 5 normal, 5 subclinical and 7 clinical mastitis affected sheep. The echomorphometric measurements of the udder and teats revealed a significant difference in gland cistern width (GCW), gland cistern area (GCA) and gland wall thickness (GWT) in clinical mastitis in comparison to those in subclinical mastitis and normal udders. In normal udders, the mean ± SD of GCW, GCA and GWT were 6.3±1.5, 16±2.5 and 0.6±0.5 respectively. In subclinical and clinical mastitis however, these values were 7±2.5 and 4.4±0.8, 19±2.9 and 13±1.5, 0.7±0.1 and 1.3±0.4 respectively. Spearman correlation coefficient analysis revealed a significant association between somatic cell linear scores (LnSCC) and teat wall thickness in sheep affected with clinical mastitis. In this study the normal ultrasonographic appearance of the udder and teats in Awassi sheep was described for the first time. Mastitis significantly affected gland wall thickness, gland cistern width and gland cistern area. These data could be useful in determining the degree of udder tissue involvement and may give insights on the prognosis of affected ewes. Keywords: Awassi sheep, Mastitis, Staphylococcus aureus, Somatic cell count, Ultrasonography RESUME Relations entre le nombre de cellules somatiques et certaines mesures morphométriques du pis et trayons au cours de mammites causées par Staphylococcus aureus chez la brebis Awassi Les objectifs de cette étude étaient de décrire par échographie la structure de la mamelle et des trayons chez la brebis Awassi en lactation afin d évaluer un possible rapport entre le nombre de cellules somatiques dans le lait et certains mesures morphométriques. L échographie en mode B a été utilisée pour examiner les mamelles et les trayons de mamelles de brebis saines, souffrant de mamittes sub-cliniques et souffrant de mammites cliniques. L analyse morphométrique des mesures de la mamelle et des trayons a révélé une importante différence de largeur de la citerne (GCW), de la taille de la citerne (GCA) et de l épaisseur de la paroi (GWT) chez les animaux atteints de mammite clinique par rapport aux témoins et animaux atteints de mammite subclinique. Pour les pis témoins, la moyenne ± écart-type de GCW, GCA et GWT étaient respectivement de 6,3 ± 1,5, 16 ± 2,5 et 0,6 ± 0,5. Lors de mammite subclinique ces valeurs étaient de 7 ± 2,5 et 4,4 ± 0,8, 19 ± 2,9. Elles étaient de 13 ± 1,5, 0,7 ± 0,1 et 1,3 ± 0,4 lors de mammites cliniques. L analyse des coefficients de corrèlation de Spearman a révélé une association significative entre taux de cellules somatiques linéaires (CETC) et épaisseur de la paroi du trayon chez les brebis atteintes de mamittes cliniques. Ces données pourraient être utiles pour déterminer le degré de l implication du tissu mammaire et évaluer un pronostic lors de mamittes chez la brebis. Mots-clés : brebis, Awassi, mammite, Staphylococcus aureus, nombre de cellules somatiques, échographie, trayon, morphométrie Introduction Staphylococcus aureus (S. aureus) is the most commonly isolated microorganism from clinical and subclinical mastitis in ruminants [1, 2, 14]. Cure rates of S. aureus mastitis are low and infected animals remain a major source of the microorganism in the environment causing major economical impact [1, 2, 14]. Inside the udder, S. aureus invades deep and forms many microabscesses that lead to permanent tissue damage [9]. Therefore, early and accurate diagnosis of mastitis is prudent to successful treatment. Moreover, the detection of structural changes at the tissue level in the affected udder can be clinically important for selecting the most appropriate treatment as well as predicting an accurate prognosis. Conventional diagnostic techniques such as somatic cell count (SCC), California Mastitis Test (CMT), and bacterial cultures remain the gold standard for detecting mastitis including clinical and subclinical cases, however, these techniques are unable to detect changes in the tissue level or to predict the prognosis in individual cases [8, 10, 13]. The structure of the udder and teats of ruminants have been previously described in normal and in mastitis cases [4, 6]. More recently, ultrasonography has been used for obtaining important udder and teat morphometric measurements [3, 5, 15]. Ultrasonographic measurements of the udder and teats were used to develop a set of desirable traits that are associated with higher milk productivity and used in genetic selection of sheep [15]. Furthermore, another set of ultrasonographic measurements were used to evaluate the effect of the milking process on udder and teat structures

34 BANI ISMAIL (Z.) AND A. MIRZAEI (A.) and to predict mastitis by a process of correlation between milk SCC and certain ultrasonographic measurements [11]. In Awassi sheep, the echographic characterization of the udder and teats in normal ewes and ewes affected with clinical, subclinical and chronic mastitis caused by S. aureus have not been reported previously. Hence, the objectives of this study were to describe the normal echo structure of the udder and teats in normal and in mastitic lactating Awassi sheep and to report possible relationship between high somatic cell count in milk and certain echo-morphometric measurements in S. aureus mastitis. Materials and methods ANIMALS All experimental protocols performed in this study were reviewed and approved by the Jordan University of Science and technology Animal Care and Use Committee (JUST- ACUC). This study was performed using 12 lactating sheep affected with mastitis and 5 randomly selected normal lactating sheep in similar stages of lactation as a control group. All sheep were about 2-5 years of age and weighing approximately 50 to 70 kg. All sheep were of Awassi breed and were in their first month of lactation. Sheep included in the study were from different local flocks. All sheep were milked manually. The 12 sheep with udder or milk abnormalities were presented to the Veterinary Health Center for evaluation of udder or milk secretion abnormalities but were healthy otherwise according to owners claims. At presentation, all sheep were subjected to a complete physical examination including attitude, heart rate, respiration rate, rectal temperature and rumen motility patterns. The udder and teat were examined by palpation to detect any abnormal signs such as hotness, swelling, or masses. Milk was examined grossly to evaluate its nature such as watery, bloody or presence of clots. Individual milk samples were then collected aseptically from each udder half and placed separately in sterile plain tubes and sent to the laboratory immediately for microbial culture. Another individual milk sample was collected from each udder half and sent to the laboratory for the determination of somatic cell count. MILK MICROBIAL CULTURE For microbial culture, 1ml of each milk sample was spread on MacConkey blood agar and incubated at 37Cofor 18 to 24 hours. Bacteria from positive culture plates were then identified based on colony morphology, gram staining characteristics and hemolytic features. Further bacterial identification was performed using coagulase test followed by fermentation of sugars (maltose, mannitol and trehalose) [8]. Only milk samples that revealed pure culture of S. aureus were included in the study. SOMATIC CELL COUNT DETERMINATION Somatic cell count was performed manually as described previously [12]. Briefly, milk samples were shaken via vortex. 0.01 ml of milk was smeared on one centimeter square on a glass slide, dried, and fixed then stained with Newman-Lambert stain (methylene blue, tetrachlorethane, glacial acetic acid and ethanol) to count the somatic cells. Thirty fields were counted under light microscope at 100X oil immersion lens. The following equation was then used to determine the total somatic cells per ml: Somatic cells/ml= microscopic factor (MF) * (WBCs/field) SCC were converted to natural log and analyzed statistically. UDDER AND TEAT ULTRASONOGRAPHY All udders and teats of the selected sheep in the study were subjected to an ultrasonographic examination. A B-mode ultrasonography machine (Sonostar, China) was used in this study. The direct contact technique (transcutaneous echography) was used for examination of the gland and teat structures using 5 to 7.5 MHz frequency sector transducers [3, 13]. All ewes were imaged at least 2 hours after the last milking to make sure that the udder is full of milk. Excessive hair was clipped and an acoustic contact gel was applied to the skin. To image the gland cistern and parenchyma, the transducer was placed on the lateral and caudal surfaces of each udder half [5] and images were produced in longitudinal and transverse sections. To image the teat, the teat was filled by milk by squeezing the udder half by hand making sure that equal pressure is applied each time. Images were obtained in longitudinal and transverse sections. The following measurements were made for each ewe: teat cistern width and area, teat wall thickness, udder cistern width and area, udder wall thickness, and glandular parenchyma. STATISTICAL ANALYSIS Data were analyzed using SPSS statistical package (IBM, SPSS version 20, 2011). Descriptive statistics were performed using frequency as well as means and standard deviation (SD) to describe the numeric data. Student t-test and one way ANOVA for independent samples were used to compare means of the normally distributed numeric variables. Correlations of SCC and ultrasonographic udder and teat parameters were analyzed using Spearman s rank correlation coefficient for numeric variables. The SCC values were converted to natural log to achieve normality.

ECHO-MORPHOMETRIC MEASUREMENTS OF UDDER AND TEATS IN SHEEP 35 Results and discussion The udders and teats of 17 ewes (12 affected with mastitis and 5 normal) in their first month of lactation were imaged. All animals classified as normal revealed normal vital signs. The udder and teats were apparently normal by palpation. The average of somatic cell count in the milk of normal sheep was 216 x10 3 cells/ ml. No bacteria were isolated from all normal sheep. Twelve ewes were classified as having mastitis (7 clinical and 5 subclinical mastitis). Milk microbial cultures revealed colonies of pure S. aureus. In sheep affected by clinical mastitis, the udder and milk secretion were apparently abnormal but no systemic signs were observed. Abnormal udders were swollen and hot and the secretion was watery with clots present. The average somatic cell count in this group was 16 x 10 6 cells/ml (minimum value 3.6 x10 3 cells/ ml and maximum value 44 x 10 6 cells/ml). In the subclinical group, the general health of the ewes was within normal limits and there were no abnormal changes detectable upon the examination of the udder and milk secretion. The average somatic cell count in this group was 4 x 10 6 cells/ml (minimum value 6.8 x10 3 cells/ ml and maximum value 10 x 10 6 cells/ ml). In this study, good quality images were obtained for the gland and teats using 5 to 7.5 MHz sector probes using the direct contact method. In normal udders, the two udder halves appeared to be completely separated by a thick hyperechogenic septum with one gland cistern and teat cistern and one teat canal for each udder half. The teat canal in Awassi sheep could be visualized as a tiny hypoechogenic line leading to a wider homogenous hypoechogenic to an anechogenic space representing the teat cistern (Figure 1A). Similar to previous findings in other ruminants, different layers of teat wall could be appreciated. The normal teat wall has been described as a 3 layered structure [4, 6]. The outer layer appeared as a thin hyperechoic line representing the skin, the middle thicker homogenous layer with less echogenicity representing muscular and connective tissues and the inner layer appeared as a hyperechogenic line representing the mucosa. As described previously, the gland cistern in normal sheep appeared continuous with the teat cistern [4, 6]. The gland cistern appeared as a large anechoic space with few echogenic particles (Figure 2A). The venous ring of Furstenberg appeared as echogenic figure-like projections extending into the gland cistern [4, 6]. Figure 1: Ultrasonographic image of the teat in Awassi sheep (Transverse section, 6.6 MHz sector probe). A: Normal udder; B: Subclinical mastitis. Notice the smaller teat cistern area and thickened teat wall in subclinical mastitis. C: Clinical mastitis. Notice significant thickening and increased echogenicity of the teat wall. Figure 2: Ultrasonographic image of the parenchyma and gland cistern in sheep (Transverse section, 6.6 MHz sector probe). A: Normal udder; B: Subclinical mastitis. Notice that difference in echogenicity of the gland cistern between normal udder and udders with subclinical mastitis. C: Clinical mastitis. The parenchyma is hyperechoic and heterogeneous and the gland cistern appears as heterogeneous and hyperechoic structure in clinical mastitis.

36 BANI ISMAIL (Z.) AND A. MIRZAEI (A.) Figure 3: A: Ultrasonographic image of the udder parenchyma in subclinical mastitis in sheep (Transverse section, 6.6 MHz sector probe). The parenchyma appears heterogeneous with hyper echoic echo-texture. B: Ultrasonographic view of the parenchyma and gland cistern in clinical mastitis in sheep (Transverse section, 5 MHz sector probe). Heterogeneous echo-texture and hyperechoic parenchyma with hyperechoic cistern are apparent. In normal Awassi sheep, the gland parenchyma appeared as homogenous, mixed echogenicity with circumscribed variably sized round hypoechoic zones representing blood vessels and lactiferous ducts. These results are similar to previous reports in other ruminant species [4, 6]. Abnormal changes in the udder and teats associated with mastitis in dairy cows, sheep and goats were previously described [3 7, 14, 15]. Similarly, in this study, the teat wall in Awassi sheep affected with clinical and subclinical mastitis was thickened and appeared more hyperechoic (Figure 2B, 2C). The gland cistern in subclinical mastitis appeared as heterogeneous and hyperechoic structure while the parenchyma appeared heterogeneous with hyper echoic echo-texture (Figure 2B, 3A). In clinical mastitis, the parenchyma appeared hyperechoic and heterogeneous and the gland cistern appeared as heterogeneous and hyperechoic structure, while the parenchyma in subclinical mastitis appeared heterogeneous with hyper echoic architecture (Figure 2C, 3B). These results were in agreement with previously reported data [3 7, 14, 15]. In this study, there were a significant difference in gland cistern width (GCW), gland cistern area (GCA) and gland wall thickness (GWT) in clinical and subclinical mastitis as compared to normal udders. GCW, GCA, and GWT were Parameter Groups Normal Udder Subclinical Mastitis Clinical Mastitis Somatic cell count Teat cistern width (cm) 1.28 ± 0.50 1.27 ± 0.15 1.33 ± 0.16 Teat cistern area (cm 2 ) 1.30 ± 0.20 1.5 ± 0.12 1.6 ± 0.23 Teat wall thickness (cm) 0.4 ±0.05 0.4 ±0.17 0.5 ±0.17 Gland cistern width (cm) 6.3 ± 1.50 a 7.0 ± 2.5 ab 4.4 ± 1.80 c Gland cistern area (cm 2 ) 16 ±2.50 19 ± 2.90 a 13 ±1.50 b Gland wall thickness (cm) 0.6 ± 0.50 a 0.7 ±0.15 ab 1.3 ± 0.94 c Table I: Means ± SD of different udder and teat ultrasonographic parameters in normal Awassi sheep and sheep with clinical and subclinical mastitis. Different superscript letters in a raw indicate significant difference at P 0.05 Group \LnSCC Echo-Morphometric Measurement TCW TCA TWT GWT GCA GCW Clinical Mastitis -.161 -.238 -.499 * -.124 -.101 -.278 Subclinical Mastitis -.438 -.174 -.096 -.053 -.301 -.174 Normal Sheep -.295 -.252.211 -.611.036.587 Table II: Correlation coefficients between LnSCC and echo-morphometric measurements in normal, clinical and subclinical mastitis in Awassi sheep: teat cistern width (TCW), teat cistern area (TCA), teat wall thickness (TWT), glandular wall thickness (GWT), glandular cistern area (GCA), glandular cistern width (GCW). *Correlation is significant at P 0.05 (2-tailed)

ECHO-MORPHOMETRIC MEASUREMENTS OF UDDER AND TEATS IN SHEEP 37 significantly decreased in clinical mastitis in comparison to those in subclinical mastitis and normal udders (Table I). In subclinical mastitis, the GCW, GCA, and GWT were significantly increased in comparison to that in normal udders. These results are similar to previously reported data that suggest that in clinical mastitis, the gland cistern decrease in size because of thickened milk secreting tissues and because of deposition of purulent material [4]. The teat and gland wall were also found to be thickened because of cellular infiltration and edema. Table II shows results of the correlation coefficient analysis between LnSCC and different echo morphometric measurements of the udder and teats in normal sheep and sheep affected with clinical and subclinical mastitis. LnSCC was only significantly associated with teat wall thickness in sheep affected with clinical mastitis. These results conform well to previously reported data concerning clinical mastitis [11]. In this study, the echo-texture of the udder and teats in normal Awassi sheep and sheep affected with clinical mastitis caused by S. aureus has been reported for the first time. Clinical mastitis caused by S. aureus appeared to significantly affect certain udder and teat echo-morphometric measurements that can be easily performed in the field and may be adopted as a management tool in the fight against this economically important contagious pathogen especially in intensively managed sheep. Acknowledgements The authors would like to thank the Deanship of Research at Jordan University of Science and Technology for sponsoring this work. We also would like to thank Professor Dr. Shawkat Lafi for performing statistical analysis. Conflict of interest The authors have no conflict of interest to declare. References 1. - ANDERSON D.E., HULL B.L., PUGH D.G.: Sheep and Goat Medicine. WG Saunders, Philadelphia, 2002. 2. - CONTRERAS A., SIERRA D., SÁNCHEZ A., CORRALES J.C., MARCO J.C., PAAPE M.J., GONZALO C.: Mastitis in small ruminants. Small. Rum. Res., 2007, 68, 145-153. 3. - DINC D.A., SENDAĞ S., AYDIN I.: Diagnosis of teat stenosis in dairy cattle by real-time ultrasonography. Vet. Rec., 2000, 147, 270 272. 4. - FASULCOV I. R.: Ultrasonography of the mammary gland of the ruminant: A review. Bulg. J. Vet. Med., 2012, 15, 1 12. 5. - FASULKOV I.R., GEORGIEV P., ANTONOV A., ATANASOV A.: B- mode ultrasonography of mammary gland in goats during lactation period. Bulg. J. Vet. Med., 2010, 13, 245-251. 6. - FASULKOV I., KOLEVA M.: Ultrasound imaging findings in acute mammary gland inflammations in goats. J. Mount. Agri. Balkans, 2011, 14, 210 221. 7. - FLOCK M., KLEIN D. HOFMANN-PARISOT M.: Ultrasonographic findings of pathological teat changes in cattle. Wien Tierärztl Monat, 2004, 91, 184 195. 8. - GONZALO C., ARIZNABARRETA A., CARRIEDO J.A., SAN PRIMITIVO F.: Mammary pathogens and their relationship to somatic cell count and milk yield losses in dairy ewes. J. Dairy Sci., 2002, 85, 1460-1467. 9. - LE MARÉCHAL C., JARDIN J., JAN G., EVEN S., PULIDO C., GUIBERT J.M., HERNANDEZ D., FRANÇOIS P., SCHRENZEL J., DEMON D., MEYER E., BERKOVA N., THIÉRY R., VAUTOR E., LE LOIR Y.: Staphylococcus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis. Vet. Res., 2011, 42, 35. 10. - LEITNER G., CHAFFER M., SHAMAY A., SHAPIRO F., MERIN U., EZRA E., SARAN A., SILANIKOVE N.: Changes in milk composition as affected by subclinical mastitis in sheep. J. Dairy Sci., 2004, 87, 46-52. 11. - MILERSKI M., MARGETIN M., ČAPISTRAK A., APOLEN D., ŠPANIK J., ORAVCOVA M.: Relationships between external and internal udder measurements and the linear scores for udder morphology traits in dairy sheep. Czech J. Anim. Sci., 2006, 51, 383 390. 12. - ALEKISH M.O.: The association between the somatic cell count and isolated microorganisms during subclinical mastitis in heifers in Jordan. Vet. Med., 2015, 60, 71 76. 13. - SCOTT P.: Sheep Medicine. London, Manson Publishing, 2007, 271-278. 14. - WHITE E.: The prevalence of mastitis in small ruminants and the effect of mastitis on small ruminant production. N.M.C. Annual Meeting Proceedings, 2007, 119-127. 15. - WÓJTOWSKI J., SLÓSARZ P., JUNKUSZEW A., MILERSKI M., SZYMANOWSKA A., SZYMANOWSKI M.: Application of ultrasound technique for cistern size measurement in dairy goats. Arch. Tierz., 2006, 4, 382-388.