BACTERIOLOGICAL STUDIES ON SUB-CLINICAL MASTITIS IN COWS AND BUFFALOES WITH TRAILS FOR ITS TREATMENT

SAYED, S.M.; F. MANSY, MARIUM; EL-BERBAWY, S.M.

doi:10.21608/avmj.2011.174542

BACTERIOLOGICAL STUDIES ON SUB-CLINICAL MASTITIS IN COWS AND BUFFALOES WITH TRAILS FOR ITS TREATMENT

Assiut Veterinary Medical Journal

Article 13, Volume 57, Issue 129, April 2011, Page 1-17 PDF (459.28 K)

Document Type: Research article

DOI: 10.21608/avmj.2011.174542

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Authors

S.M. SAYED¹; MARIUM F. MANSY²; S.M. EL-BERBAWY²

¹Animal Health Research Institute, Assiut Regional Laboratory.

²Animal Health Research Institute, Assiut Regional Laboratory

Abstract

This study is concerned with bacterial causes of subclinical mastitis in baladi cows and buffaloes. A total of 94-baladi cow and 80-buffaloes were examined. All animals rear in a smallholder private cases and hand milked. Screening tests of the milk samples using both field tests (California Mastitis Test and Modified Whiteside Test), revealed that 26(27.74%) & 30(31.9%) baladi cows and 13(16.3%) & 14(17.5%) of buffaloes showed positive results by both tests, respectively. These positive milk samples were examined bacteriologically on general and specific enriched media. The isolated bacteria were 58 isolates from baladi cows and 27 isolates from buffaloes. These isolates resembled two categories: contagious bacteria 14 (24.14%) & 11 (40.74%) and environmental bacteria 44(75.86%) & 16(59.26%) in baladi cows and buffaloes, respectively. The single infection resembled 19.23 and 15.38%, while mixed infection was 80.77 and 84.62% in baladi cows and buffaloes, respectively. The isolated contagious strains were Staph. aureus and Strept. dysgalactiae 6(10.35%) for both species and Strept. agalactiae 2(3.45%) in baladi cows. In buffaloes, Staph. aureus were 11 isolates (40.74%). Concerning to environmental bacteria in baladi cows E. coli were 12(20.69%); Staph. saprophyticus 10(17.24%);Staph. intermedius 9(15.52%);Strept. Pyogenes and Strept. uberis 3(5.17%) for each, whileStaph. epidermidis and Klebsiella pneumoniae 2(3.45%) for both and Enterobacter aerogenes was isolated from a single milk sample (1.72%). In buffaloes, E. coliand Staph. intermedius resembled 3(11.1%) for both, Staph. saprophyticus 5(18.52%), Staph. epidermidis 2(7.41%); Strept. uberis andCitrobacter diversus 1(3.7%)for each. Antimicrobial susceptibility testing revealed that all isolated strains were sensitive to Ciprofloxacin and Ofloxacin with percentage 100% followed by Gentamycin 97.6%, Kanamycin 90.5% and Doxycycline 85.7%. All tested bacterial isolates showed resistance to Cloxacillin, Ampicilin, Amoxicillin and Cefotaxime. Treatment of some subclinical cases carried on three groups (each of three baladi cows and one buffalo), the first and second group treated with intramammary infusion of Gentamicin and kanamycin, respectively and the third group subjected for intramammary treatment with 10% Egyptian fennel honey solution. All milk samples in the 1^st group were negative for CMT&MWST and bacterial culture post treatment, while in 2^nd group two out of four milk samples at 7^th &10^th days post treatment were positive for CMT&MWST and bacterial culture. The third group all milk samples post treatment were positive for CMT & MWST and negative for bacterial culture. It can be concluded that high prevalence of subclinical mastitis caused by Staphylococcus spp. and E. coli, so strict hygienic measures and effective control for pathogens should be applied. Program of teat dipping and intramammary antibiotic treatment at drying off period is thus recommended. In addition to, enhancing the awareness between dairy farmers is the main point for control of subclinical mastitis in dairy animals.

Keywords

Keywords: Subclinical mastitis; bacteriological examination; baladi cows; buffaloes

Full Text

Animal Health Research Institute,

Assiut Regional Laboratory.

Bacteriological studies on sub-clinical mastitis in cows and buffaloes with trails for its treatment

(With 5 Tables)

S.M. Sayed; Marium F. Mansy

and S.M. El-Berbawy

(Received at 15/3/2011)

دراسات بکتريولوجية علي إلتهاب الضرع الخفي في الأبقار والجاموس

مع محاولات لعلاجه

سيد محمد سيد, مريم فؤاد منسي , سعد محروس البرباوى

أجريت هذه الدراسة لمعرفة البکتريا المسببة لإلتهاب الضرع الخفي في الأبقار البلدية والجاموس, حيث تم فحص 94 بقرة بلدية و80 جاموسة. کانت جميعها ملکية خاصة وفي مجموعات صغيرة وتحلب يدويا. أسفرت نتائج الفحص باستخدام اختباري الکاليفورنيا ماستيتس(CMT) والويتسيد المعدل (MWST) عن ايجابية الفحص لعدد 26(27.74%) و30(31.9%) من الأبقار البلدية وعن عدد 13(16.3%) و14(17.5%) من الجاموس لکلا الاختبارين علي الترتيب. وقد خضعت هذه العينات الإيجابية للاختبارات الحقلية للفحص البکتريولوجى وتم زرعها على المستنبتات البکترية العامة والخاصة لزيادة فرص العزل وتم عزل 58 عترة من الأبقار البلدية و27 عترة من الجاموس تمثل مجموعتين من المسببات البکترية کانت علي النحو التالي البکتريا الوبائية 14عترة (24.14%) و11عترة(40.74%) وبکتريا التلوث البيئي 44عترة(75.86%) و16عترة (59.26%) من الأبقار البلدية والجاموس , علي التوالي. وکانت العدوى المنفردة تمثل 19.23 و15.38% أما العدوى المختلطة کانت تمثل 80.77 و84.62% من العترات المعزولة في الأبقار البلدية والجاموس. تمثلت البکتريا الوبائية في عزل ميکروب المکور العنقودي الذهبي بنسبة 40.74% في الجاموس , أما في الأبقار البلدية تم عزل المکور العنقودي الذهبي والسبحي ديسجلاکتيا 10.35% لکل منهما والمکور السبحي أجالاکتيا 3.45%. أما بکتريا التلوث البيئي في الأبقار البلدية فقد تمثلت في عزل الميکروب القولوني 20.69% والمکور العنقودي سابروفيتکس 17.24% والمکور العنقودي انترميديس 15.52% والمکور السبحي بيوجين والمکور السبحي أوبرس 5.17% لکل منهما والمکور العنقودي أبيدرميدس والکلبسيلا نومني 3.45% لکل منهما وکذلک تم عزل ميکروب أنتيروباکتر أيروجينز بنسبة 1.72%. أما في الجاموس تمثلت بکتريا التلوث البيئي قي عزل الميکروب القولوني والمکور العنقودي انترميديس 11.1% لکل منهما والمکور العنقودي سابروفيتکس 18.52% والمکور العنقودي أبيدرميدس 7.41% والمکور السبحي أوبرس والستروباکتر دايفرسيس 3.7% لکل منهما. وبإجراء اختبار الحساسية لأهم هذه العترات کل على حدة ضد 15 من المضادات الحيوية المختلفة أسفرت النتائج عن حساسية جميع العترات المعزولة للسيبروفلوکساسين وأفلوکساسين بنسبة 100% والجنتاميسين 97.6% والکاناميسين 90.5% ودوکسي سيکيلين 85.7%, کما أنها أظهرت مقاومة لکل من کلوکسيلين وأمبيسيلين أموکسيسيلين وسيفوتکسيم. وبعلاج بعض الحيوانات المصابة بالتهاب الضرع الخفي في ثلاث مجموعات (کل مجموعة 3 أبقار بلدية وجاموسة واحدة)؛ خضعت المجموعة الأولي والثانية لحقن الضرع باستخدام جنتاميسين وکاناميسين علي التوالي, أما المجموعة الثالثة تم حقن الضرع بمحلول 10% عسل الشمر المصري. بعد العلاج کانت جميع عينات اللبن في المجموعة الأولي سلبية لاختباري الکاليفورنيا ماستيتس والويتسيد المعدل وکذلک للعزل البکتيري. أما المجموعة الثانية کانت عينتان من أربع عينات من اللبن بعد العلاج في اليوم السابع والعاشر ايجابية لاختباري الکاليفورنيا ماستيتس والويتسيد المعدل وکذلک للعزل البکتيري. بينما کانت جميع عينات اللبن بعد العلاج في المجموعة الثالثة ايجابية لاختباري الکاليفورنيا ماستيتس والويتسيد المعدل وسلبية للعزل البکتيري. ويمکن الخلاصة إلي أن المسبب الرئيسي لالتهاب الضرع الخفي يرجع إلي الميکروب المکور العنقودي والميکروب القولوني؛ وللسيطرة علي هذا المرض يجب أتباع الوسائل الصحية وکذلک رفع الوعي الصحي بين مربي الحيوانات الحلابة.

Summary

Keywords: Subclinical mastitis, bacteriological examination, baladi cows, buffaloes.

Introduction

Mastitis is incriminated as one of the critical problems of the dairy animals leads to losses during the lactation period. These losses are primary due to lower milk yield, reduced milk quality and higher costs of treatment and control (Palanivel et al., 2008). The prevalence of subclinical mastitis in dairy herds is often surprising to producers, moreover, subclinically infected udder quarters can develop clinical mastitis and the rate of new infections can be high (Zdunczyk et al., 2003).

Subclinical mastitis is the most serious form, as both infected udder and milk show no obvious clinical abnormalities, whereas, several causative organisms are discharged with milk for long time during which the causative organism acts as invisible potential source of spreading infection in the herd without the farmer being aware of it, so the infection becomes difficult to eradicate. This may cause sever harm from the epizootiological and epidemiological as well as economic points of view (Salem et al., 1993).

Mastitis pathogens of dairy animals are numerous, but the majority of udder infections caused by pathogens of two categories including, contagious bacteria as Staphylococcus aureus, Streptococcus agalactiae and Streptococcus dysgalactiae, and environmental bacteria as E. coli, Strept. uberis and other Staphylococcus sp. (Bramley et al., 1996; El-Balkemy et al., 1997 and Anwer et al., 2003). Staph. aureus seemed to bethe predominant organisms causing subclinical mastitis (Kader et al., 2002 and Ali et al., 2008). It may predispose the herd to infection by coliforms or other pathogens (Ibtisam et al., 1993).

In Egypt, buffaloes are the first animal for milk yield and the majority of these buffaloes are private ownership (Metry, 1996). The greatest problem in the treatment and control of mastitis is emergence of drug resistance pathogens (Jha et al., 1994). The pattern of drug resistance continues to change in a particular area depending upon various epidemiological factors and indiscriminate use of antibiotics (Choudhury and Narayan, 1984).

The intramammary fennel honey infusion was applied in subclinical mastitic cows resulted in significant decline of total bacterial count and highly significant increase in milk yield (Abd El-Hafeez et al., 2005).

Due to economic and public health importance of subclinical mastitis, the present study was aimed to detect subclinical mastitis and determine the predominant contagious and environmental bacteria causing it in baladi cows and buffaloes using the most available media for isolation. Determination of antibiogram of the most prevalent bacterial isolates and trials for treatment of some affected cases was the second aim.

Materials and Methods

Ninety-four baladi cows and eighty buffaloes with clinically sound udder and secreting apparently normal milk were included in this study. All animals related to small-scale herds (1-4 animals). They reared in private cases under the farmers hand and hand milked twice daily. The animals ages ranged from 4-6 years and at different stages of lactation. They housed in a barn with dust ground and their feed consists of green fodder, hay and concentrates.

Quarter milk samples (366 from baladi cows and 314 from buffaloes) were examined at once, where 10 and 6 quarters were blind non lactating, respectively, using the field tests, (California Mastitis Test, CMT, using Delaval Mastitis Test, 3804101, Poland, Schalm et al., 1971 and modified Whiteside Test, MWST, Murphy and Hanson, 1941).

For bacteriological examination, ten ml of fresh milk samples from 30 baladi cows and 14 buffalos which showed sub-clinical mastitis positive reaction (individual sampling), as a pooled milk samples were of the four quarters in a sterile screw capped vials, were collected aseptically. Milk samples centrifuged at 3000 rpm for 20 minutes, then a loopfull from milk sediment streaked onto Azid Blood Agar plate and a loopfull was inoculated into each of nutrient broth (Himedia Lab. Limited, India) MacConkey broth (Biomark Lab. India) and modified EC-medium (Diffco No.7197405) with Novobiocin 2%. The previously inoculated tubes were incubated at 37˚c for 24 hours. From the incubated tubes, a loopfull was streaked onto the surface of each of the nutrient agar, blood agar with 5% sheep blood, Mannitol salt agar (BBL), MacConkey agar (Biomark Lab. India) and Sorbitol MacConkey agar plates (Diffco). The inoculated plates were incubated overnight aerobically at 37˚c.

The suspected colonies were identified: morphologically, by Gram's stain and biochemically confirmed according to Quinn et al. (1994), using catalase activity, coagulase test as well as Novobiocin (5 mcg) and polymixin-ß sulphate (300 U) sensitivity tests for identification of Staphylococcus spp. Identification of Streptococci spp. was done by catalase test, haemolytic activity, sodium hippurate hydrolysis, aesculin hydrolysis on blood agar with 0.1% aesculin, growth in 6.5% Nacl broth, growth on MacConkey agar, Sorbitol & lactose fermentation and Bacitracin, 0.04 unit susceptibility. Enterobacteriaceidentified biochemically by conventional IMVIC (Indole, Methyl red, Voges-proskauer and citrate utilization) test, motility, triple sugar iron agar (TSI) inoculation and sugar fermentation (Sorbitol, raffinose & cellobiose), according to Quinn et al. (1994).

Antibiogram ofthe recovered isolates was adapted using antimicrobial susceptibility testing by disc diffusion standard technique according to Quinn, et al. (1994). The isolated strains were tested against 15 antibiotics (Ampicilin 10 µg, Amoxicillin 25 µg, Cefotaxime 30 µg, Cephalexin 30 µg, Ciprofloxacin 5 µg, Cloxacillin 1 µg, Doxycycline 30 µg, Gentamicin 10 µg, kanamycin 30 µg, Neomycin 30 µg, Novobiocin 30 µg, Ofloxacin 5 µg, Oxytetracycline 30 µg, Trimethoprim 5 µg, Spiramycin 100 µg), ( Bioanalyse-Turkey).

Treatment trials: Selected baladi cows and buffaloes with subclinical mastitis were allocated into three groups (each of three cows and one buffalo). The first group administered Gentamicin 100 mg/ quarter (B.V.Co., France), the second was administered Kanamycin 100000 I.U./ quarter (Univet, Ireland). All drugs were administered twice daily after milking for three consecutive days and the third group was subjected for intramammary treatment with Egyptian fennel honey. Unprocessed honey diluted with sterile normal saline solution to achieve 10% honey solution then, filtered under complete aseptic conditions using sterilized filter papers to remove any debris, wax, or large particles, Al-Waili (2003). Ten ml of 10% honey solution intramammary/ quarter infused daily for three successive doses guarded with intra-muscular administration of antihistaminic (Abd El-Hafeez et al., 2005). The milk samples were collected before treatment and at the 4^th & 7^th and 10^thdays post treatment. Each samples was subjected to CMT & MWST and for bacterial culture.

Results

Detailed obtained results were illustrated in Tables (1-5).

In this study, the outcome of treatment in first group all milk samples after treatment were negative for CMT & MWST and for bacterial culture. In the second group all milk samples at 4^th day post treatment were negative for CMT & MWST and for bacterial culture, but two out of four milk samples were positive CMT & MWST and also for bacterial culture at 7^th &10^th days post treatment. In the third group, all milk samples after treatment were positive for CMT & MWST and negative for bacterial culture.

Table 1: Incidence of subclinical mastitis in examined baladi cows and buffaloes by both tests.

Positive for bacterial culture		Modified Whiteside test				California mastitis test				No. of animals	Types of animals
%	No.	Negative		Positive		Negative		Positive
%	No.	%	No.	%	No.	%	No.	%.	No.
27.7	26	68.1	64	31.9	30	72.3	68	27.7	26	94	Cows
16.3	13	82.5	66	17.5	14	83.3	67	16.3	13	80	Buffaloes

Table 2: Incidence of subclinical mastitis quarters in the examined baladi cows and buffaloes

Types of animals	Examined quarter	One quarter		Two quarters		Three quarters		four quarters		Total
Types of animals	Examined quarter	No.	%	No.	%	No.	%	No.	%	No.	%
Cows	366	10	2.73	7	1.91	7	1.91	6	1.64	30	8.20
Buffaloes	314	7	2.23	4	1.27	3	0.96	0	0	14	4.46

Table 3: The frequency percentage of the single and mixed infection in positive milk samples of the examined baladi cows and buffaloes by using bacteriological examination.

Types of animals	Single Infection		Double infection		Triple infection		Total
Types of animals	No.	%	No.	%	No.	%	No.	%
Cows	5/26	19.23	10/26	38.46	11/26	42.31	26/26	100
Buffaloes	2/13	15.38	8/13	61.54	3/13	23.08	13/13	100

Table 4: Frequency distribution of bacterial species recovered from subclinical mastitic milk samples of baladi cows and buffaloes.

Bacterial species

Cows

No. Frequency %

Buffaloes

No. Frequency%

Contagious organisms

- Staph. aureus

- Strept. dysgalactiae

- Strept. agalactiae

Environmental organisms

- E .coli

- Staph. saprophyticus

- Staph .intermedius

- Strept. Pyogenes

- Strept. uberis

- Staph. epidermidis

- Klebsiella pneumoniae

- Enterobacter aerogenes

- Hafnia alvei

- Serratia marcescens

- Citrobacter diversus

N.= 14 24.14

6 10.35

2 3.45

N.= 44 75.86

12 20.69

10 17.24

9 15.52

3 5.17

2 3.45

1 1.72

0 0

No. 11 40.74

11 40.74

0 0

N.=16 59.26

3 11.11

5 18.52

3 11.11

0 0

1 3.70

2 7.41

0 0

1 3.70

0 0

1 3.70

Total

58 100

27 100

Table 5: The percentage of in vitro antimicrobial susceptibility pattern of the most frequent isolates against different antibiotics.

Isolated microorganisms

No. of the tested isolates

Ampicilin(10µg)

Amoxicillin (25µg)

Cefotaxime (3µg)

Cephalexin (30µg)

Ciprofloxacin (5µg)

Cloxacillin (15µg)

Doxycycline (30µg)

Gentamycin (10µg)

Kanamycine (30µg)

Neomycin (30µg)

Novobiocin (30µg)

Ofloxacin (5µg)

Oxytetracycline (30µg)

Trimethoprim (5µg)

Spiramycin (1005µg)

Number and percentage of sensitive strains

Staph. aureus

1/16

(6.3%)

5/16

(31.3%)

0/16

(0%)

4/16

(25%)

16/16

(100%)

2/16

(12.5%)

15/16

(93.8%)

15/16

(93.8%)

14/16

(87.5%)

5/16

(31.3%)

9/16

(56.3%)

16/16

(100%)

8/16

(50%)

10/16

(62.5%)

8/16

(50%)

Strept. Spp.

2/12

(16.7%)

5/12

(41.7%)

3/12

(25%)

1/12

(8.3%)

12/12

(100%)

1/12

(8.3%)

12/12

(100%)

12/12

(100%)

11/12

(91.7%)

10/12

(83.3%)

12/12

(100%)

12/12

(100%)

6/12

(50%)

9/12

(75%)

10/12

(83.3%)

E. coil

1/14

(7.1%)

4/14

(28.6%)

13/14

(92.9%)

13/14

(92.9%)

14/14

(100%)

0/14

(0%)

9/14

(64.3%)

14/14

(100%)

13/14

(92.9%)

12/14

(85.7%)

9/14

(64.3%)

14/14

(100%)

8/14

(57.1%)

11/14

(78.6%)

0/14

(0%)

Total

4/42

(9.5%)

14/42

(33.3%)

16/42

(38.1%)

18/42

(42.9%)

42/42

(100%)

3/42

(7.1%)

36/42

(85.7%)

41/42

(97.6%)

38/42

(90.5%)

27/42

(64.3%)

30/42

(71.4%)

42/42

(100%)

22/42

(52.4%)

30/42

(71.4%)

18/42

(42.9%)

Discussion

Subclinical mastitis means that, although there are no visible or palpable external changes, the infection is present and the inflammation occurs in the udder and apparently healthy milk, but subclinical mastitis leads to undesirable effect on milk constituents and its nutritive value. Economical losses are due loss in milk production, discarding abnormal milk and milk withheld from animals treated with antibiotics, degrading of milk due to higher bacterial or somatic cell count, cost of drugs, veterinary services and increased labor cost. In addition, the problems related to antibiotic residues in milk and its products (Bramely et al., 1996).

In the present study (Table 1), results revealed that subclinical mastitis in baladi cows was 27.7% by C.M.T and 31.9% by M.W.S.T. Among cows, subclinical mastitis ranged from 5.5% (Zahid, 2004), 30.69% (Ghosh et al., 2004); 31.98% (El-Balkemy et al., 1997); 59.05% (Sadek, 2008); up to 69.2% (Awad and Abeer, 2003) and 67% in dairy Friesian cows (Nahed Wahba et al., 2005). In case of buffalo^'s milk samples the incidences of subclinical mastitis was 16.3% and 17.5% by C. M. T. and M. W.S.T., respectively. Similar result 18.59% was obtained by Ghosh et al. (2004). The lower incidences 6.95, 9.59% were obtained by Ahmad et al. (1991) and Saini et al. (1994), respectively, while, Shrirame et al. (1997); El- Balkemy et al. (1997) and Sadek (2008) recorded higher incidences of 20.71%, 42.55 and 33.3%, respectively.

The obtained results showed that the incidence of subclinical mastitis in baladi cows, which reared in small holder private cases, handy milking and obtained good management, lower than the incidence in large scale farms and machinery milking which enhance infection transmission. The sub-clinical mastitis incidence varied widely due to changing in management conditions and different diagnostic tests used (Radostits et al., 2000). The high rates of subclinical mastitis in the different areas were mainly due to poor management and unhygienic milking practices (Shem et al., 2001). The lower incidence of subclinical mastitis in buffaloes may be due to their thick streak canal and perfect closure mechanism of teat sphincter (Ghosh et al., 2004). Since the C.M.T. field test is dependable and reliable perfect test in good agreement with bacteriological results (El-Balkemy et al., 1997). It appeared to agree 100% with bacteriological isolation in the present study and proved its superiority than Modified Whiteside test, which detected false positive results. False positive results of Whiteside test is documented (Nahed Wahba et al., 2005).

The results obtained in Table (2), revealed that, the incidences of subclinical mastitis in the examined baladi cows and buffaloes according to the affected quarter, the one-quarter infection was more than two, three and four quarters infections. These results were similar to that obtained by Saini et al. (1994) and Sadek (2008).

As shown in Table (3), the incidence of mixed and single infection were 80.77and 19.23% in positive baladi cow^'s milk samples for subclinical mastitis, respectively, while buffalo^'s milk samples showed mixed infection incidence higher than single infection (84.62 and 15.38%, respectively). These findings reflect an idea about level of environmental bacterial contamination (Sayed and Abd El-Hafeez, 2009). In addition Staph. aureus may predispose the animals to infection by coliforms or other pathogens (Ibtisam et al., 1993). Meanwhile, El-Khodery and Hoedemaker (2005) and Magda Essa (2007) reported that the mixed bacterial infection was lower than single bacterial infection in subclinical mastitis cases.

The results obtained in Table (4) revealed that the frequency percentage of environmental bacteria in baladi cows and buffaloes was 75.86 and 59.26%, respectively.

The environmental bacteria, which may cause mastitis usually originate from the surrounding environment including air, soil, water, bedding material, faecal matter, milking man and milking utensils (Anwer et al., 2003). The portal of entry into mammary gland for Gram-negative bacteria is the teat canal. Once in the gland, bacteria must utilize available substrates in the mammary secretion to replicate and evade host defenses (El-Mahronki et al., 2006). The obtained results indicated exposure of teat end to the environmental bacteria.

The highest incidence of environmental bacteria in the present study was 20.69 and 11.11% for E. coli in both cows and buffaloes, respectively. E. coli was widely reported to be subclinical mastitis bacteria in cows and buffaloes (Ahmad et al., 1991; Ahmed and Azza, 2001; Kader et al., 2002; Anwer et al., 2003; Awad and Abeer, 2003; Magda Essa, 2007 and Sadek, 2008). Its persistence within the mammary environment was of the recurrent quarter E. coli mastitis and its spread among other quarters and cows during the milking process (Bradley and Green, 2001).

Staph. saprophyticus,Staph. intermedius, Strept. Pyogenes, Strept. Uberis, Staph. epidermidis, Klebsiella pneumoniae, Enterobacter aerogenes, Hafnia alvei, Serratia marcescens and Citrobacter diversus were identified to be as environmental subclinical mastitis pathogens in baladi cows and buffaloes (Mokhbatly et al., 2001 and Kotb, 2006).

In the present study, as shown in Table (4), the frequency percentage of contagious bacteria causing subclinical mastitis in baladi cows and buffaloes was 24.14 and 40.47%, respectively (Staph. aureus 10.35%; Strept. agalactiae 3.45% and Strept. dysgalactiae 10.34% in cows and Staph. aureus 40.74% in buffaloes). The contagious bacteria are well adopted to survive in the udder and usually establish mild subclinical infection for long duration (El-Khodery and Hoedemaker, 2005 and Abdel-Khalek and El-Sherbini, 2005) and can spread from infected quarters to other quarters (Bramley et al., 1996 and El-Balkemy et al., 1997). Through previous studies (Ahmad et al., 1991; Ahmed and Azza, 2001; Awad and Abeer, 2003 and Sadek, 2008) the highest incidence of subclinical mastitis in cows and buffaloes was due Staphylococci spp. followed by Streptococci spp. Staph. aureus commonly produce long-lasting infections as it developed sophisticated system to evade phagocytosis and intra-cellular killing by neutrophils or macrophages (Vanfurth and Van Zwet, 1986).

The differences in the distribution of isolated microorganisms for cows from a farm to another may be attributed to differences in the management, housing, and species of animals in addition to an inadequate cleansed milk machine and teat trauma(El-Balkemy et al., 1997).

Bacteriological examination of milk is needed not only for confirmatory process but also for drug sensitivity. Identification of the causative organism and sensitivity testing besides culling of untreatable cows are very important for control of sub-clinical mastitis. In the present study, Table (5) shows the prevalent bacteria isolates tested for antibacterial sensitivity pattern. The obtained results revealed the most effective antimicrobial agent all over the study was Ciprofloxacin, Ofloxacin followed by Gentamycin; Kanamycin and Doxycycline with susceptibility 100, 100, 97.6, 90.5 and 85.7%, respectively. While all tested strains resisted Cloxacillin, Ampicilin, Amoxicillin, Cefotaxime. The obtained results coincided to large extent with that of Abd El-Hafeez (2002); Kader et al. (2002); Abdel-Khalek and El-Sherbini(2005); Magda Essa (2007).

Through the present study, treatment of some subclinical cases with antibiotics selected on the basis of in vitro sensitivity test revealed that, in the first group treated with intramammary infusion of Gentamicin, the milk samples were negative for CMT & MWST and negative for bacterial culture post-treatment. In the second group, treated with intramammary infusion of Kanamycin, two milk samples were positive CMT & MWST and also for bacterial culture after 7^th &10^th days post treatment. These results indicated that application of screening tests leads to earlier detection of subclinically infected quarter and aid in the selection of dairy animals for either production or therapy (Sadek, 2008). The third group was subjected for intramammary treatment with 10 ml of 10% solution of Egyptian fennel honey revealed that all milk samples after treatment were positive for CMT & MWST and negative for bacterial culture. The positive reaction in CMT & MWST depends on the concentration of somatic cell count in the milk (Ahmad et al., 1991). Extremely positive reaction of CMT was recorded post intramammary honey infusion, as leukocytes especially lymphocytes were significantly increased in milk post intramammary honey infusion (Abd El-Hafeez et al., 2005). Hydrogen peroxide activity of honey and phytochemical antibacterial component unique to honey are effective inhibiting the growth of mastitis causing species of bacteria at quite low concentrations (Allen and Molan, 1997).

Treatment of subclinical cases using antibiotics for long time may develop bacterial resistance rather than milk withdrawal period, existence of problems associated with yogurt or cheese processing. In addition the failure to treat subclinical mastitis may allow these animals to be reservoir of infection and increase the potential exposure of uninfected animals to contagious pathogens (Ruegg et al., 2008).

It can be concluded that high prevalence of subclinical mastitis caused by Staphylococcus spp. and E. coli, so strict hygienic measures and effective control for pathogens should be applied program of teat dipping and intramammary antibiotic treatment at drying off period. In addition to, enhancing the awareness between dairy farmers is the main point for control of subclinical mastitis in dairy animals. The use of honey in treatment of subclinical mastitis would obviate for all producers the withholding of milk after therapy and any residue that did not end up in the milk would be more acceptable to consumers.

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