BACTERIOLOGICAL STUDIES ON MASTITIS OF DAIRY ANIMALS WITH SPECIAL REFERENCE TO MYCOPLASMA INFECTION IN MENOFIA AND KALUOBIA GOVERNORATES, EGYPT

Dept. of Mycoplasma,

Animal Health Research Institute, Dokki

Bacteriological studies on mastitis

of dairy animals with special reference to Mycoplasma infection in Menofia

and Kaluobia Governorates, Egypt

(With 4 Tables and 7 Figures)

By

Fadia ABDELHAMEED and Eman M. Sharaf*

* Dept. of Bacteriology, Animal Health Research Insitiute, Shebin-El-Koom

(Received at 11/3/2009)

دراسات بکتريولوجية عن مرض التهاب الضرع في الحيوانات الحلوبة مع اشــارة خاصة لعدوى الميکوبلازما بمحافظتي المنوفية والقليوبية – مصر

فادية عبد الحميد ، ايمان شرف

کان الغرض من اجراء البحث معرفة مدى انتشار مرض التهاب الضرع الظاهري والخفي في بعض مزارع الابقار الفريزيان والجاموس الحلوبة (ذات الاعداد المحدودة) بمحافظتي المنوفية والقليوبية لموسم حليب واحد (نوفمبر 2007 الى نهاية ديسمبر 2008 ) وکذلک معرفة أهم المسببات البکتيرية مع الترکيز على معرفة مدى تواجد عدوى ميکروب الميکوبلازما. واتضح من الفحص الاکلينيکي لضروع الحيوانات المصابة ظاهريا مع نتائج اختبار کاليفورنيا للحيوانات السليمة ظاهريا أن معدل الأنتشار العام لمرضي التهاب الضرع الظاهري والخفي في مزارع الأبقار کان 9.56 % و 27.50 % على التوالي. وفي مزارع الجاموس کان معدل الأنتشار العام 6.00 % و 17.50 % على التوالي مما يشير الى ارتفاع معدل الاصابة في الابقار الفريزيان عن الجاموس. أجملت نتائج الفحص البکتريولوجي للعينات الالبان المجمعة من الابقار والجاموس المصابة بالتهاب الضرع الظاهري والخفي عن مدى سيادة الميکروب العنقودي الذهبي. هذا وقد تبؤت عدوى الميکوبلازما موقعا هاما ضمن الميکروبات المسببة لمرض الالتهاب الضرع الوبائي في الابقار الفريزيان (حيث بلغت نسبة الاصابة 19.70 % من الابقار المصاب اکلينيکيا). هذا بالاضافة الى احتلال ميکروب الميکوبلازما المرتبة الثانية (28.58 %) مباشرة عقب الميکروب العنقودي الذهبي (42.86 %) کسبب مسئول عن حالات التهاب الضرع المستعصي الذي لا يستجب للعلاج في الابقار الفريزيان. ولم يتم عزل الميکوبلازما من الجاموس. وتم عزل 28 عترة من ميکوب الميکوبلازما مصنفة بيوکيميائبا من الابقار المصابة اکلينيکيا وتم تحديد نوع ميکوبلازما الأبقار (Mycoplasma bovis) باستخدام استخدام إختبارات البيولوجيا الجزيئية المتمثلة في اجراء اختبار انزيم البلمرة المتساسل التفاعلي ( PCR ) الذي کان أکثر سلاسة من وسائل التصنيف التقليدية. وقد تم انجاز اختبار الحساسية بالمضادات الحيوية لميکروب العنقودي الذهيي ونوقشت نتائجه. الحالات الايجابية لعدوى الميکوبلازما خاصة ميکوبلازما الابقار – سواء بطرق العزل التقليدية أو باستخدام البيولوجيا الجزيئبة – بجب استبعدها من قطيع الادرار باسرع وقت لتجنب احتمالات انتفال العدوى للحيوانات المجاورة وکذلک لتجنب انتقال العدوى للمواليد الحديثة والعجول. في المزارع المصابة بالميکوبلازما يجب اجراء فحص الميکوبلازما بالتوازي مع الکشف الدوري لالتهاب الضرع مع استبعاد أو التخلص من الحالات الايجابية.

Summary

Nine small-scale dairy farms include 982 animals (Friesian cows, 565 and buffaloes, 417) at Kaluobia and Menofia Governorates were clinically inspected for detection of udder and/or milk abnormalities, and the apparently healthy animals were subclinically tested by California mastitis test to indicate the prevalence of clinical and subclinical mastitis during one lactation season; Nov., 2007 – Dec., 2008). The isolated bacterial pathogens were identified by conventional culturing procedures whereas the isolated Mycoplasma was molecularly identified using Polymerase Chain Reaction (PCR). The current results indicated that overall prevalence of clinical and subclinical mastitis in cows' farms was 9.56 % and 27.61 %, respectively, and in buffaloes' farms was 6.00 % and 17.50 %, respectively. The high susceptible rate (P < 0.01) of cows to mastitis than buffaloes was discussed. Staphylococcus aureus was the predominant mastitis pathogen of clinical and subclinical mastitis in dairy cows and buffaloes. Mycoplasma could not be isolated from buffaloes. In Friesian cows, Mycoplasma occupied a considerable level of the isolated contagious mastitis pathogen from the clinically mastitic cases (19.70 %) and it occupied the second grade (28.57 %) following Staphylococcus aureus (42.86 %) as etiologic agent responsible for incurable mastitis of cows. Twenty-eight isolates of Mycoplasma were biochemically determined and 17 of them were identified as Mycoplasma bovis by PCR, which is easier and more flexible tool than conventional culturing procedures. In-vitro antibiotic-disc-susceptibility to Staphylococcus aureus was achieved. The culturally and/or molecularly (PCR) positive cases of Mycoplasma mastitis, in particular Mycoplasma bovis mastitis, should culled as early out the dairy herd to avoid the lateral transmissions of infection to neighboring unaffected animals, and to avoid the transmission of infection to neonates and calves. Mycoplasmal examination, in the infected farm, should regularly do in paralleling with routine detection of mastitis until elimination by segregate/cull the infected cases.

Key words: Subclinical, clinical & incurable mastitis in cows & buffaloes, bacteria, Mycoplasma, PCR.

Introduction

Mastitis is continuously implicated as one of the serious tribulations of the dairy cows and buffaloes inducing severe economic losses during the lactation season. These losses are primarily due to lower milk yield, reduced milk quality, and higher costs of treatment and control (Palanivel et al., 2008 and Nicholas et al., 2009). Microorganisms causing mastitis of dairy animals are numerous, but mastitis pathogens of dairy ruminants can be categorized as environmental (Escherichia coli and other Coliforms) or contagious (Mycoplasma species; streptococcus agalactiae and Staphylococcus aureus) depending on their primary reservoir (environment versus infected mammary gland) (Edmonson and Bramely, 2004 and Reddy et al., 2007). Mastitis caused by contagious pathogens appears to be more prevalence than the environmental mastitis (Riekerink et al., 2006 and Zaitoun, 2008).

On the other side, Mycoplasma mastitis is incriminated as a major enemy facing the dairy industry (Nicholas et al., 2009). Contagious mastitis caused by Mycoplasma of dairy cows and buffaloes is a worldwide disease (Riekerink et al., 2006 and Nicholas et al., 2008) and firstly reported in Egypt by El-Ebeedy et al. (1985) and Zaitoun (1990), respectively. The later authors emphasized that Mycoplasma mastitis coming Egypt through importation of infected Friesian cows. During the past 20 years, several reports on various forms of Mycoplasma mastitis in dairy cows and buffaloes on farms and in individual (private) cases at different localities of Delta-region and of Upper Egypt's Governorates were subsequently published (Eissa, 1986; Ahmed and Sabry, 1989; Zaitoun, 1990; Zaitoun et al., 1991; El-Shabiny, and Abou-El-Makarem, 1994). This may obviously refers to the spread of Mycoplasma infection throughout the Egyptian farms and it may become inherent or endemic in some areas. Various types of Mycoplasmas were isolated from dairy Friesian cows and buffaloes with mastitis. These Mycoplasmas included Mycoplasma bovis, Mycoplasma bovigenitalium and Mycoplasma arginini. Mycoplasma bovis appears to be the most important etiologic agent of Mycoplasma mastitis (Nicholas et al., 2008).   

From practical point of view, diagnosis of mastitis depends chiefly on the clinical inspection of the grossly affected mammary quarter (s) and thereafter treated by an available antibiotic drug without detection of mastitis pathogen, which consider the cornerstone in mastitis’ treatment and control (Zaitoun, 2008). In general, the selected anti-mastitis drug was usually chosen based on the experience of veterinarians and/or owners. These may attribute to inadequate facilities of the bacteriological examinations in territories, and not all owners accept the bacteriological analyses of the milk of the affected animals to minimize the costs of diagnosis and they irrationally treated the clinically affected cases haphazardly. This may assist in a build-up of bacterial resistance. Therefore, the current work was carried out to elucidate the following items: a) the prevalence rate of mastitis in cows and buffaloes in some private farms at Menoufia and Kalioubia Governorates. b) to indicate the most common mastitis pathogen(s) responsible for intramammary infections of dairy cows and buffaloes.   c) to evaluate the situation of Mycoplasma mastitis, particularly Mycoplasma bovis, among dairy animals population. Antibiotic sensitivity test of the predominant mastitis pathogen(s) was also aimed.

Materials and Methods

Animals:

During the period of investigation (Nov., 2007 to Dec., 2008), a total number of 982 dairy animals were randomly selected and clinically monitored to clear-up any udder and/or milk abnormalities. The apparently healthy cases of the selected animals were subclinically examined using indirect test (California Matitis Test, CMT) (Table 1). The clinically positive cases with signs of udder and/or milk abnormalities were serially enrolled and their therapeutic trials were recorded. Mastitic cases received two consecutive therapeutic trials with adequate therapy without successful results were encountered in our work as incurable mastitis. A composite sample from the clinically affected quarter(s) per each case was collected in screw-capped bottle and sent to the laboratory with minimal of delay, and subjected to bacteriological and mycoplasmal examinations. On the other hand, the subclinically positive quarter(s) of each case were sampled and subjected to the same examinations of the clinically positive samples. To minimize the costs of laboratory examinations, a composite sample from each clinically and/or subclinically positive case was collected and examined.

Samples and Culturing procedures:

I – Bacteriological examinations:

The affected quarter was washed, disinfected, and the first strips were rejected. Approximately 25 ml of intramammary secretion was collected in sterile screw-capped bottle. The collected sample was thoroughly vortexed and divided into two halves. The first half was prepared for bacteriological examinations. The sample aerobically incubated overnight at 37^oC, and thereafter centrifuged and the sediment was streaked onto 10% sheep's blood agar (Gibco) and onto MacConkey agar (Oxoid) plates. The cultured plates were aerobically incubated to  24 – 48 hours at 37^oC. The suspected colonies were picked-up, purified, morphologically identified and biochemically analyzed according to the criteria illustrated by Watts (1990).

II- Mycoplasmal examinations:

The second half of each collected sample was thoroughly mixed in vortex and 0.2 ml was drawn and poured into screw capped bijou bottles containing 1.8 ml of modified Hayflick's broth medium supplemented with inhibitors, fresh yeast extract and horse serum, and incubated at 37^oC (Stalheim, 1990). The incubated bottle was thereafter platted onto agar media and incubated in 5 % Co₂ environment. Two-days post incubation, the cultured plates were examined and agar-blocks of the positive plate with characteristic shape of Mycoplasma colonies were taken and recultured, purified and subjected to digitonin sensitivity test (Stalheim, 1990). The digtonin sensitive strains were identified biochemically viz., glucose fermentation, arginine hydrolysis and film and spots tests (Table 2). The glucose negatve, arginine negative Mycoplasma with production of film and spot was selected to molecular testing using polymerase chain reaction (PCR).  

PCR procedure:

Twelve purified strains of the frozen isolated Mycoplasmas were selected based on the biochemical profile [glucose fermentation (-ve), arginine hydrolysis (-ve) and film and spot (+ve)] and thawed, cultured into PPLO broth medium, incubated for 48 hrs at 37°C. The incubated cultured broths were tested by a Mycoplasma bovis-specific PCR according to the protocol illustrated by Lunini et al. (2006) and Radaelli et al. (2008). Briefly, five milliliters of the cultured broth was harvested for 30 min at 14000 xg and the pellet was primarily washed with Mycoplasma wash solution to remove all adhering medium-additives from the Mycoplasma-cell (Rodwell and Whitecomb, 1983). Thereafter the pellet was resuspended in 300 µl of distilled water treated with 0.1 % of diethylpyrocarbonate and the DNA was extracted from 100 µl using the DNeasy Tissue Kit (QIAamp^®DNA Mini, Qiagen^®). Specific primers were designed for PCR (MYCBV-Fw – 5'-TAT CGG TGA CCC TTT TGC AC-3'; MYCBV-Rw – 5'-TTC CAC TTC CTG ACT CAC CA-3'). A PCR with these primers resulted in the production of a fragment of 348 bp of the oppD (oligopeptide permease D) gene of Mycoplasma bovis. A final volume of 20 µl was used containing 0.2 µM of each primer, 200 µM of dNTPs, 2 units of Taq polymerase (Roche Diagnostics), 1 x 1.5 mM of MgCl₂ and 5 µl of the extracted DNA. The following amplification procedure was used: 95°C for 5' , 36 cycles at 95°C for 1' 59°C for 30" and 72 °C for 30 " and 72 for 7' . Ten microliters of the amplified product were analyzed by electrophoresis in a 1.8 % agarose gel (Agarose, MP, Multi purpose agarose, Boehringer Manheim). Unfortunately, a reference strain of Mycoplasma bovis could not be obtained in our hand and consequently PCR products were sequenced with an automatic capillary system (Applied-Biosystem) to confirm the specificity of the reaction.

On the other hand, specific primers of Mycoplasma bovigenitalium (Mbg-Fw – 5'-CGT AGA TGC CGC ATG GCA TTT ACG G-3';  Mbg-Rw -  5'-CAT TCA ATA TAG TGG CAT TTC CTA C-3') was also incorporated in molecular detection of Mycoplasma bovigenitalium of the examined milk according to protocol illustrated by Kobayashi et al. (1998).

Antibiotic sensitivity test:

Ten isolates of the isolated Staphylococcus aureus strains were randomly selected and in-vitro tested to different members of antibiotics commonly used in field; penicillin (10 IU), ampicillin (10 µg) cloxacilline (5 µg), cephalothin (30 µg), oxytetracycline (30 µg), and enrofloxacin (5 µg). The technical procedure and interpretation of the applied test were carried out as described by Ortez (2005).   

Statistical analyses:

The obtained data of clinical and subclinical mastitis of the examined dairy cows and buffaloes collected, pooled and statistically analyzed. The prevalence of mastitis calculated mathematically by conventional procedure. The significance difference of the prevalence of clinical and subclinical mastitis of the examined cows and buffaloes was carried out using CHI square analysis according to the outlines illustrated by Lewis (1966).

RESULTS

Prevalence of clinical and subclinical mastitis:

Prevalence (% affected) of clinical and subclinical mastitis of the examined dairy cows and buffaloes in different farms was tabulated on Table 1, and the overall prevalence was mathematically calculated (Table 1). On the other hand, the prevalence rate of incurable cases of mastitis in both species was listed on Table 1.

Statistically, CHI-square analysis revealed that there is a significance increase (P < 0.05) in the susceptibility rate of Friesian cows to clinical mastitis (9.56 %) than buffaloes (6.00 %). Concerning the subclinical mastitis, Friesian cows are highly susceptible (P < 0.01) to the subclinical form of mastitis (27.61 %) than buffaloes (17.50 %). There is no significance difference (P > 0.05) in the prevalence rate of incurable mastitis in the examined cows (3.72 %) and buffaloes        (2.64 %).

Mastitis pathogens:

The isolated bacterial pathogens including Mycoplasma infections of the bacteriologically examined samples of dairy cows and buffaloes were illustrated on Table 2. The Table indicated that Staphylococcus aureus followed by Streptococcus agalactiae was the predominant isolates and Mycoplasma infections occupies the third grade of the isolated pathogen. On the other hand, the microbial pathogens isolated from the incurable mastitis cases of cows and buffaloes were summarized on Table 3, which indicates that Staphylococcus aureus and Mycoplasma infections were the predominant etiologic agents responsible for incurability of intramammary infection.

            The Mycoplasmal examinations indicated that 13 (cases of the examined cows with signs of clinical mastitis, n = 59) harbored Mycoplasma either alone (14.81 %) or coupled with Staphylococcus aureus (9.26 %) in their mammary sections and 28 isolates were determined. Of these isolates, 20 strains were digitonin sensitive and encountered as Mycoplasma strains. The biochemical profiles of the isolated Mollicutes were illustrated on Table 4.

PCR Procedure:

All biochemically glucose negative, arginine negative with positive production of film and spot (n = 20) were molecularly tested by PCR. All PCR-tested isolates were negative to Mycoplasma bovigenitalium and 17 isolates gave the specific product of Mycoplasma bovis (Fig. 4). Unfortunately, 3 strains could not be identified.

Frequent distribution of the isolated mastitis pathogens:

The frequent distribution of the isolated bacterial mastitis pathogens and Mycoplasma of the examined dairy cows and buffaloes with clinical and subclinical mastitis were diagrammed in Figures 2, 3, 4, and 5. The microbial pathogens and Mycoplasma isolated from the incurable mastitis cases were figured on Fig. 6 and 7.

Antibiotic sensitivity test:

Seventy percentage of the tested of Staphylococcus aureus strains (n = 10) were sensitive to penicillin and other antibiotics. Meanwhile the reminder were varies in their resistance. Two strains were penicillin resistant but they were sensitive to ampicillin, cloxacilline, cephalothin, oxytetracycline, and enrofloxacin. One strain was resistant to penicillin and oxytetracycline. All tested isolates was highly sensitive to enrofloxacin.

Fig. 1: Eithidium bromide-stained, 1.8 % agarose electrophoresis of PCR product of the tested Mycoplasma isolates

● 1:  DNA ladder, 100 bp

● Arrow: The arrow refers to the specific band of Mycoplasma bovis, 348 bp.

● 2-10: Field isolates of Mycoplasma bovis (Glucose negative, arginine negative, Film & spots positive)

● 11: Arginine positive Glucose negative Mycoplasma (may be Mycoplasma arginini)

● 12: Field isolated strain of Mycoplasma bovis

Table 1: Prevalence of clinical and subclinical mastitis of the examined dairy cows (n = 565) and buffaloes(n = 417) in small private farm during one lactation season

#: Incurable mastitis is cases did not response to two consecutive therapeutic trials with one and/or two types of anti-mastitis udder  infusions.                                                                                                                                                                                                    

$: Number between parentheses is the percentage of infection.

*: Significance increase at the 0.05 level (χ² = 4.11, P < 0.05).

**: Highly significance increase at the 0.01 level (χ² = 12.834, P < 0.01).

@: There is no significance difference in the rate of incurable mastitis in both examined cows and buffaloes (χ² = 0.520, P > 0.05).

N.B: the prevalence (% affected) of mastitis (clinical plus subclinical form) of dairy cows is therefore 37.17 % and of dairy buffaloes is 23.50 %. The incurable mastitis cases were not encountered as clinical form of mastitis in the present work.