PATHOGENESIS AND BACTERIOLOGICAL ETIOLOGY PROFILE OF SUBCLINICAL MASTITIS OF SHE-CAMELS INFLUENCED IN ECONOMIC LOSSES IN AL-TAIF GOVERNORATE (SHORT TITLE: SUBCLINICAL MASTITIS IN SHE-CAMELS AT TAIF)

Pathogenesis and Bacteriological Etiology Profile of Subclinical Mastitis of She-camels Influenced in Economic Losses in Al-Taif Governorate (Short title: Subclinical mastitis in she-camels at Taif)

Sherifa M. Sabra^* and Eman M. Sharaf^*

* Dept. of Microbiology, Science Collage, TaifUniversity, KSA. 

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                                           Abstract

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Keywords: CMT, SCC, CNS, E.coli.

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الهيئة المرضية والأسباب البکتيريولوجية لالتهاب الضرع الخفي للنوق مؤدية لخسائر اقتصادية

بمنطقة الطائف

شريفة مصطفي صبره , إيمان محمود شرف

التهاب الضرع الخفي هو مرض للمرضعات من الحيوانات يؤدي إلى فقدان إنتاج الحليب، والجودة، وإحراز تقدم في بعض الأحيان إلى التهاب الضرع, فضلا عن قد يؤدي إلي جفاف الضرع. تم جمع مجموعه 160 عينة من الحليب من أرباع النوق لعدد 40 ناقة, من قطعان في منطقة الطائف يظهروا بصحة جيدة بدون أي علامات مرضية في مرحلة الرضاعة. تم إجراء اختبار کاليفورنيا وعد الخلايا الجسدية بالإضافة إلي العزل والتصنيف البکتيريولوجي للعينات بوجود خمسة من النوق کعينات ضابطة. أظهرت النتائج أن معدل ايجابية التهاب الضرع الخفي 23,8%, أکثر ايجابية لاختبار کاليفورنيا انحصرت بين معدلي (+1 - +3), عدد الخلايا الجسدية بين (2X⁴10 – 4,2X ⁵¹⁰) خلية/ملل. العزلات الأکثر في النسبة المکورات العنقودية السالبة للتجلط 19,4%, تليها المکورات العنقودية الذهبية 14,4%, ثم العصويات القولونية 5,6%, Strept.dysagalactiae 3,8%, Strept.agalactiae 2,5%, وأخيرا کلابسيلا الرئوية 1,9%. قد حصلت دقة الاختبارات المستخدمة على أهداف موضوعية مع نتائج العزلات الميکروبية وتحديد الهوية. کشف الهدف من التهاب الضرع الخفي في النوق في الطائف کمصدر للحليب ومنتجاته خصيصا للعائلة. نتائج الدراسة الحالية تبين النسبة المئوية حوالي ربع عينات الحليب المختبرة، التي تشير إلى أن هناک خسائر في الحليب بمقدار الربع. ويجب أن تسيطر علي التهاب الضرع الخفي من خلال رفع مستوى البيئة المراعي، واختبار للحليب وکذلک لتشخيص المرض في وقت مبکر مع اتخاذ جميع الاحتياطات لتقليل والوقاية من الإصابة.

Introduction

Subclinical mastitis is a form of mastitis characterized by change in milk composition with no signs of gross inflammation or milk abnormalities. Changes in milk composition can be detected. Chief pathogens causing subclinical mastitis include Strept.agalactiae Staph.aureus. Strept.dysagalactiae. The organisms live inside the udder and survives only for a short time outside the mammary gland. It spreads primarily during milking, contaminated hands and materials. The Staph.aureus lives inside and outside the udder on the teat skin. The microorganism can cause subclinical mastitis and spreads by the same way as Strept.agalactiae. Yeast can also cause mastitis, overuse of antibiotics and poor sanitation contribute to yeast mastitis was recorded by Macdonald Campus of McGill University, 2012.

The most predominant bacterial isolates were Micrococcus spp., Staph.aureus, Strept.spp. and Coryne. spp. followed by eight other flora (Barbour et al., 1985). She-camels are able to produce milk in valuable quantity (Faye, 2005). However, (Camelus dromedarius) could be affected by udder infection as mastitis like diary animals, a complex disease occurring worldwide among dairy animals, which lowering milk quality, with heavy economic losses largely due to subclinical mastitis which needs special fast diagnosis (Matofari et al., 2003). Evidence indicates that subclinical mastitis causes suffering of she-camels, reduce milk yield, alters milk properties, impairs preservation and processing and is a public health concern for consumers of camel milk (Tibary and Anouassi, 2000). Very little medical information is known about subclinical mastitis concerning their etiology and occurrence in she-camels (Camelidae) (Kalla et al., 2008). Cases of subclinical mastitis in she-camels have recently been reported in Saudi Arabia, Egypt, and Somalia (Barbour et al., 1985; Mostafa et al., 1987; Abdurahman et al., 1991). The early clinical and laboratory diagnosis for subclinical mastitis and treatment greatly reduces the incidence of clinical mastitis and improve health of she-camels. For monitoring mastitis, a number of tests to detect changes in milk can be routinely used for screening purposes in milking herds. One of the screening procedures, for subclinical mastitis, involves the measurement SCC in milk. SCC is a count of the number of neutrophils and normal udder cells present in milk. An increase in the SCC to more than 5x10⁵ cells/ml is considered to be an indication of udder infection in she-camel (Eberlein, 2007).

SCC, CMT, Adenosine tri-phosphate (ATP), N-acetyl-D-gluco-samimidase (NAGase) and Serum albumin has been used as indirect diagnostic tools for infected and non-infected quarters of the she-camel mammary gland (Abdurahmann, 1996). The reading of test is the leading to the diagnosis, because the basal levels of cells and their physiological variations in the she-camels were still not yet established (Abdurahmann et al., 1992).

Previously it was found that milk of she-camels type (Camelus bactrianus) contains not only leukocytes but also large number of a nuclear cell-like particle, so-called ‘cell fragments’ (Abdurahmann et al., 1992). During the past decade there have been several reports on subclinical mastitis in she-camels (Camelus dromedarius) and a few on (Camelus bactrianus) (Barbour et al., 1985; Obeid, 1983;  Arush et al., 1984; Kospakov, 1976a), but a work has been done to study subclinical mastitis and the udder’s response to bacterial invasion by applied CMT to composite milk samples from the she-camels (Camelus dromedarius) and resulted in the test was useful for screening subclinical infected udders (Barbour et al., 1985; Saber et al., 2010). Quarter milk samples that contained bacteria had significantly higher mean values for SCC, but the mean NAGase levels were not significantly different for the bacteriological negative and positive samples.

There was a low correlation coefficient between the SCC and NAGase in the quarter milk samples from which bacteria were not isolated. The type of bacteria had a significant effect on the SCC but not on the NAGase activity. Quarter samples from which Staph.aureus (coagulase positive) was isolated showed the highest mean SCC and this organism is therefore suspected to be the underlying cause of the subclinical mastitis.

The SCC gave a better indication of the presence of pathogenic microorganisms in milk samples than did NAGase (Guljye et al., 2002). Subclinical mastitis was detected in she-camels milk samples, the causative agents was isolated from 66.7% of the samples. The most prevalent groups were Strept. group D 30%, CNS Staph. 20.1%, Staph.aureus 16%, Strept.dysagalactiae 3.6%, and Strept. agalactiae 1.5%. Other isolatates were Coliforms and Micrococci. Strept. dysagalactiae and Strept.agalactiae had a greater association with subclinical mastitis than the other pathogens. Strept.agalactiae and Staph-aureus were ranked as infectious pathogens while Strept. groupD, Strept.dysagalactiae, CNS, Coliforms and Micrococci were ranked as environmental pathogens (Atofari et al., 2005). Examined milk samples of she-camels, the microbiology identification showed the most predominant pathogens were Micrococcus spp. Staph.aureus, Strept.spp. and Coryne.spp. CMT test gave results with which 60% positive, 21% of she-camels revealed subclinical mastitis (Azmi et al., 2008).  

Subclinical mastitis is prevalent in Saudi camels, and its incidence is influenced by breed, parity, and stage of lactation (Aljumaah et al., 2011). Samples gathered from clinically healthy dromedary camel from Al-Jouf, Saudi Arabia were cultured to detect subclinical udder infection. The milk samples were screened by SCC and CMT. Gram-positive Cocci were the dominant recovered udder pathogen. The mean value of SCC was 125,000 cells/ mm3. Infected quarter had generally higher mean values for SCC and CMT scores. Both SCC and CMT were of value in predicting the infection status of the udder (Saleh and Faye, 2011).

The objectives of this study were to diagnose the subclinical mastitis of she-camels with references to diagnosis tests SCC, CMT and microbial isolation and identification of milk, which related to reproduction and economic losses in Al-Taif area.  

Materials and Methods

Animals: Forty lactating she-camels (Camelus dromedarius) from the farms present at Taif area, KSA, was screened for detection of subclinical mastitis. She-camels were of (5-15) years ages, within lactation stages (l-15 months) and suckling their calves. They were housed together and fed with ideal food, all the she-camels were free from clinical mastitis during the sampling period.

Sampling procedure: The quarters milk samples (160) were collected from lactating she-camels (n=40). Before collections of samples the udder and the teats were washed and cleaned with 70% alcohol. Discarded the for-milk from each teats.  Tem ml of milk was taken from each quarter under sterile condition in sterile containers. Recorded the animal number, the quarter (Right or left front, Right or left back), date, time, the samples were kept in ice box till reach laboratory. The quarter milk samples were subjected to microbiological isolation, identification, SCC and CMT tests.

Bacteriological examination: Inoculated 0.01 ml of each milk sample separately and were streaked on blood and MacConkey agar plates; were incubated for 24- 48 h at 37°C. The plates were then examined for growth colony morphology. Individual colonies were picked for identification according to the Scandinavian recommendations on examination of quarter milk samples (Klastrup, 1975). For fastidious organism the Muller-Hinton agar was supplemented with  5-7% sheep blood. Confirmed identification by API and Micro-scan.

California mastitis test (CMT): CMT was carried out using the method (Schalm and Noorlander, 1974). An equal volume of CMT reagent and milk was mixed and the reaction was graded 1,2,3,4 or 5, according to the Scandinavian recommendations,corresponding  to 0, trace, 1, 2 and 3 (Klastrup and Schmidt, 1974). The test was performed by a trained technician. The reactions were interpreted as follows: score 1 = no reaction; score 2 = slight slime which tends to disappear with continued swirling; score 3= distinct slime but without gel formation; score 4 = immediate formation of gel which moves as a mass during swirling; score 5 = gel develops a convex surface and adheres to the bottom of the paddle.

Somatic cell count (SCC): The somatic cell counts (cells/ml) for the quarter milk samples were determined using Nucleo Counter SCC-100 (coulter electronic–ChemometecA/s, Denmark)

Statistical analysis: SCC values were transformed into log in order to get homogeneous variance. Mean and standard deviation were calculated for quantitative data. The relationships between SCC and CMT were estimated by the correlation of Spearman. The parity effect, the udder localization effect, the type of bacterial, contamination effect and the level of CMT reaction were estimated by variance analysis on logSCC. For measuring the parity effect, the variable parity was divided into 3 modalities: primiparous (n=1), second parity (n=7), more than second parity (n=11). The types of bacterial contamination were identified by Hierarchical Classification Analysis (HCA) after Multiple Correspondence Analysis (MCA). These analyses were applied to a table of presence/absence of different pathogens agents. The software SPSS, 18 version and XLSTAT (Addinsoft ©) used for data management and statistical analysis.

Results

The results in Table 1 show the majority of she-camels milk samples were identified for subclinical mastitis which total samples were identified 160 resulted in 38 samples (23.8%) positive, were higher than the normal range, identified as subclinical mastitis according to CMT and SCC. Table 1 shows also the explanation of  positive for CMT and SCC as flow first score 1+ = 2X10⁴  which identified in 20 milk samples referred to 12.5%, on the other hand second score 2+ = 3.1X10⁴ showed in 11 milk samples less than first score equal to 6.9%, lastly third score 3+ = 4.2X10⁵ lower than second score and equal to 4.4%. The isolated pathogens types in Table 2 and Figure 1 show the 76 isolates (47.5%) were identified from the milk sample, as well as founding of isolation for more pathogens from once milk sample. Isolated pathogenes divided into Gram-positive included (Staph.aureus, CNS, Strept.agalactiae and Strept.dysagalactiae) and Gram-negative included (E.coli, Klebsiella pneumonia). The predominant isolates were CNS as 19.4%, then Staph.aureus 14.4%, E.coli 5.6%, Strept.dysagalactiae 3.8%, Strept.agalactiae 2.5%, the last Klebsiella pneumonia 1.9%.

Table 1: CMT and SCC from subclinical milk sample

n*= numbers

m* = main

Table 2: Isolated pathogens from milk samples

n*= numbers

Figure 1. Isolated pathogens from milk samples