Detection of Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli in Brazilian mastitic milk goats by multiplex-PCR

ABSTRACT: This study evalueted the prevalence of Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli in milk samples from 257 goats (513 half-udders) and ten bulk tanks, from ten dairy goat farms of São Paulo State, Brazil, by multiplex-PCR. The samples were screened by microbiological culture (gold-standard), and tested by different multiplex-PCR protocols for the detection of each bacterium. A total of 178 half-udders resulted positive by microbiological culture, with coagulase-negative staphylococci (70%), S. aureus (13.5%), S. intermedius (7.9%), and Enterobacteriaceae (4%) the prevalent pathogens. In other way, multiplex-PCR detected 173 pathogens in 151/523 (28.9%; CI95% 25.2-32.9%) milk samples 144/513 (28.1%) half-udders and 7/10 (70%) bulk tanks, with E. coli (86/162, 51.9%) and S. aureus (50/162, 30.9%) the prevalent ones in half-udders, and S. aureus (6/10, 60%) and E. coli (4/5, 36.4%) in bulk tanks. Multiplex-PCR showed a high performance for the detection of three bacteria at a time in mastitic goat milk direct from half-udders or bulk tanks. Thus, this multiplex-PCR protocol proved to be an adequate tool for the identification of the most common mastitis pathogens, independent of their phenotypic characteristics in the diagnosis of clinical mastitis in goats, allowing a continuous and better vigilance and monitoring the herd, being included in quality programs.

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Perlakuan Celup Puting setelah Pemerahan terhadap Keberadaan Bakteri Patogen, Staphylococcus aureus, Streptococcus agalactiae, dan E. coli pada Sapi Perah Penderita Mastitis Subklinis di Peternakan KUNAK Bogor

Jurnal Sain Veteriner ◽

10.22146/jsv.29293 ◽

2017 ◽

Vol 35 (1) ◽

pp. 63

Author(s):

Herwin Pisestyani ◽

Etih Sudarnika ◽

Rachmi Ramadhanita ◽

Abdul Zahid Ilyas ◽

Chaerul Basri ◽

...

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Streptococcus Agalactiae ◽

Pathogenic Bacteria ◽

Subclinical Mastitis ◽

Lactation Period ◽

E Coli ◽

Milk Samples ◽

Agar Media ◽

Teat Dipping

Mastitis is udder inflammation that can be caused by the entry of pathogenic bacteria through the teat. One of the treatments that can prevent bacteria infection is teat dipping. This research was aimed to identify the effectof teat dipping after milking to the presence of pathogenic bacteria that cause subclinical mastitis (Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli) and the presence of pathogenic bacteria that cause subclinicalmastitis by quarter of udder. Milk samples were taken from normal lactation period of dairy cows, clinically healthy, and positive subclinical mastitis status when the intervention was applied. Sixty seven samples of quarters from 18dairy cows were identified to the presence of pathogenic bacteria that cause subclinical mastitis before teat dipping and at 1st, 2nd, and 3rd months after teat dipping. Identification of the presence of pathogenic bacteria was done byculturing samples on selective agar media. The presence of common pathogenic bacteria, Staphylococcus aureus, and Escherichia coli decreased significantly after teat dipping, while Streptococcus agalactiae and other pathogenicbacteria did not decrease significantly. The presence of pathogenic bacteria based on the quarter of udder did not show the significance difference. Application of teat dipping after milking was able to decrease the presence ofpathogenic bacteria that cause subclinical mastitis.

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Absence of Bacterial Adherence in the Establishment of Experimental Mastitis in Mice

Veterinary Pathology ◽

10.1177/030098587801500609 ◽

1978 ◽

Vol 15 (6) ◽

pp. 770-775 ◽

Cited By ~ 16

Author(s):

J. C. Anderson

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Mammary Gland ◽

Significant Role ◽

Streptococcus Agalactiae ◽

Staphylococcus Epidermidis ◽

Alveolar Epithelium ◽

Bacterial Adherence ◽

E Coli ◽

Suckling Mice

The possibility of adherence of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli or Streptococcus agalactiae to the epithelium of the mammary gland was investigated by inoculating them into this gland of mice. S. aureus, S. epidermidis and E. coli did not adhere to alveolar epithelium in suckling or non-suckling mice. S. agalactiae adhered to alveolar epithelium in non-suckling mice but adhesion was not sufficiently strong to withstand suckling. Bacterial adherence probably does not play a significant role in the establishment of mastitis by these organisms.

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Growth and Death of Selected Microorganisms in Ultrafiltered Milk

Journal of Food Protection ◽

10.4315/0362-028x-49.3.233 ◽

1986 ◽

Vol 49 (3) ◽

pp. 233-235 ◽

Cited By ~ 6

Author(s):

PATRICIA HAGGERTY ◽

NORMAN N. POTTER

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Skim Milk ◽

Laboratory Scale ◽

The Other ◽

Streptococcus Faecalis ◽

E Coli ◽

Milk Samples ◽

D Values ◽

D Value

Studies were made to compare the growth and death of Staphylococcus aureus, Streptococcus faecalis and Escherichia coli in skim milk concentrated by ultrafiltration to that in unconcentrated skim milk. Skim milk was volume concentrated to 2× in laboratory-scale stirred UF cells. Behavior of the organisms was analyzed in four inoculated milk samples: 2× retentate, 1× water-diluted retentate, milk equivalent (retentate plus permeate) and unconcentrated skim milk. Growth of each organism and of total aerobes did not vary in the four milk samples at either 7 or 13°C. For S. faecalis and E. coli, D-values for samples heated to 62.7°C did not significantly differ in the four milk samples (p>0.01). The D-value of S. aureus in water-diluted retentate was slightly but significantly lower than those in the other three milk samples (p<0.01), possibly due to the lowered lactose level in this sample.

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Public health risks of Escherichia coli and Staphylococcus aureus in raw bovine milk sold in informal markets in Egypt

The Journal of Infection in Developing Countries ◽

10.3855/jidc.9509 ◽

2018 ◽

Vol 12 (07) ◽

pp. 533-541 ◽

Cited By ~ 2

Author(s):

Walid Elmonir ◽

Etab Abo-Remela ◽

Azza Sobeih

Keyword(s):

Public Health ◽

Escherichia Coli ◽

Staphylococcus Aureus ◽

Health Risks ◽

Raw Milk ◽

Informal Markets ◽

E Coli ◽

Milk Samples ◽

Public Health Risks ◽

And Staphylococcus Aureus

Introduction: Milk is an important food in Egypt and most of it is sold as raw milk in informal markets. Methodology: This study was conducted to investigate the public health risks of Escherichia coli and Staphylococcus aureus in milk sold in informal markets in Egypt. A total of 121 milk samples were analyzed for occurrence, virulence genes and antibiotic resistance of E. coli and S. aureus. Results: A total of 35/121 (28.9%) of milk samples were contaminated with 16/121 (13.2%) E. coli, 22/121 (18.2%) S. aureus, and 3/121 (2.5%) both isolates. Shiga-toxin producing E. coli (STEC), Enterotoxigenic E. coli (ETEC) and Enterotoxigenic S. aureus were detected in 5/121 (4.1%), 2/121 (1.7%) and 8/121 (6.6%) of the examined milk samples, respectively. Multiple drug resistances (MDRs) were showed by 14/16 (87.5%) and 21/22 (95.5%) of E. coli and S. aureus isolates, respectively. E. coli isolates showed high resistance for cephalothin (87.5%), ampicillin (68.8%) and tetracycline (68.8%), but were sensitive for gentamicin and chloramphenicol. Resistance phenotypes of E. coli were diverse; however, STEC isolates were significantly associated with co-resistance to cephalothin, ampicillin and tetracycline (P< 0.05). Two (9.1%) of S. aureus isolates were methicillin-resistant (MRSA) but sensitive to gentamicin (GS-MRSA). Five (22.7%) of S. aureus isolates were gentamicin-resistant methicillin-sensitive S. aureus (GR-MSSA). S. aureus isolates also showed high resistance for ampicillin (100%), tetracycline (90.1%) and sulfamethoxazole-trimethoprim (90.1%). Conclusion: These findings highlighted the potential public health hazards of E. coli and S. aureus pathogens in raw milk sold in informal markets in Egypt.

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Determinación de la cinética, pruebas de crecimiento y efecto de inhibición in vitro de Lactobacillus casei en Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae y Escherichia coli

Revista de la Facultad de Medicina Veterinaria y de Zootecnia ◽

10.15446/rfmvz.v62n2.51992 ◽

2015 ◽

Vol 62 (2) ◽

Author(s):

Henry Jurado-Gámez ◽

Manuel Gúzman-Insuasty

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Streptococcus Agalactiae ◽

Staphylococcus Epidermidis ◽

Lactobacillus Casei ◽

E Coli

Se determinó la cinética, pruebas de crecimiento y el efecto de inhibición in vitro de Lactobacillus casei sobre Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae y Escherichia coli. Se usaron cepas de casa comercial y cepas aisladas en la Vereda La Victoria, Corregimiento de Catambuco al suroccidente del municipio de Pasto, Nariño, Colombia. Se evaluó el efecto de los antibióticos Dicloxacilina, Cefepima, Cefalotina, Ciprofloxacina, Gentamicina, Penicilina, Trimetropim Sulfa y Ampicilina. Se evaluó la inhibición producida por L. casei y su sobrenadante sobre las bacterias patógenas. El crecimiento de la bacteria láctica se evaluó con tres niveles de pH (2,5, 4,5 y 7), tres concentraciones de sales biliares (0,5, 1 y 2%) y dos de bilis bovina (1 y 1,2%), y dos temperaturas (38 y 45°C). Igualmente se determinó la cinética de crecimiento y las variables pH, azúcar total, proteína y ácido láctico. Mediante HPLC se determinaron los péptidos y los ácidos orgánicos presentes en el sobrenadante. L. casei mostró susceptibilidad a la Ciprofloxacina y Ampicilina, mientras que S. aureus mostró susceptibilidad y resistencia a todos los antibióticos para la cepa comercial y aislada respectivamente, el mismo comportamiento se presentó con S. epidermidis. Las cepas de S. agalactiae y E. coli aisladas y comerciales mostraron susceptibilidad a los antibióticos. La cepa láctica mostró un efecto de inhibición de S. aureus, S. epidermidis y S. agalactiae, pero no fue efectiva con E. coli, igual comportamiento se observó con el uso del sobrenadante de la bacteria láctica. Se encontró crecimiento de 1 x 1010 y 5,1 x 107 UFC/ml para 1 y 1,2 % de bilis bovina; 2,3 x 107, 1 x 109 y 3 x 108 UFC/ml para 0,5, 1 y 2 % de sales biliares respectivamente; 1,1 x 1011, 2,0 x 1010 y 1,0 x 1010 UFC/ml para pH de 2,5, 4,5 y 7 respectivamente. La fase exponencial se encontró a 16:48 horas con un crecimiento de 3 x 1010 UFC/ml. La variables pH, azúcar, acidez y proteína durante la fase exponencial fueron de 4,94, 0,88 mg/l, 2,89 mg/l y 1,9 mg/l, respectivamente. La prueba de HPLC para péptidos mostró la presencia de una cadena VAL-TIR-VAL y para ácidos orgánicos se encontró una producción de 83,46% de ácido láctico. L. casei mostró buenas características probiótica que permitirían su aplicación en ensayos in vivo para el control de microorganismos causantes de mastitis subclínica en vacas.

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Baseline composition and microbial quality assessment of raw milk from endemic small ruminants and Maghrebi camels in the oasis area of Tunisia

10.21203/rs.3.rs-1214012/v1 ◽

2022 ◽

Author(s):

Zahran Khaldi ◽

Mounir Nafti ◽

Mohamed Tabarek Jilani

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Small Ruminants ◽

Goat Milk ◽

Camel Milk ◽

Microbial Quality ◽

Protein Ratio ◽

E Coli ◽

Milk Samples ◽

Casein Protein

Abstract Characteristics and quality aspects of milk from native ovine queue fine de l’Ouest (QFO) and the local goat population were investigated and compared with those of the local Maghrebi camel. A total of 378 individual milk samples were collected from lactating animals reared in the continental oasis region of Tunisia. Samples were analyzed for physical parameters (pH, density, and acidity), chemical composition (dry matter, fat, protein, lactose, casein, ash, and casein-protein ratio), mineral concentrations (Ca, P, Na, and K) and microbiological features (total mesophilic aerobic bacteria (TMAB), total coliform count (TCC), lactic acid bacteria (LAB), sulphite-reducing Clostridium (CSR), yeast and molds (Y/M), Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Salmonella) according to standard methods. The results obtained for milk characteristics revealed noticeable disparities between the three species. The mean values of pH, density, and acidity in milk collected from sheep appeared higher than those in milk collected from goat species. Compared to the camel populations, sheep species produced milk with similar pH but higher density and acidity. Between camel and goat species, pH and acidity were higher in Negga, while the density was similar. For milk composition, the results showed a remarkable variation among all studied species and an obvious superiority of the ovine species over the caprine and camel populations in all the chemical contents being studied, except for the casein-protein ratio, which is in favor of goat species. The milk of QFO sheep, the richest in casein and protein, was expressed with significantly higher levels of calcium and phosphorus than goat and camel milk. Compared to small ruminants, milk from camels is the richest in Na and K. Additionally, more Ca is present in milk from camels than goats. Goat milk, the poorest type of milk in Ca and Na, contains on average more P than camel milk and more K than sheep's milk. The poor bacteriological quality was that of camel milk for all microbial counts. The microbial quality of goat milk was higher than that of ewe milk based on TMAB, TCC, and E. coli counts, while ovine milk was of better quality, referring to LAB, Y/M, and S. aureus values. No significant differences were found for Staphylococcus aureus and Escherichia coli between the examined species. The obtained results highlighted the complete absence of the two dangerous pathogens Salmonella and CSR in all investigated milk samples. The microbiological examination evidenced that the milk of small ruminant species complies with standard criteria required by Tunisian legislation on the hygiene of milk and dairy products. Regarding camel milk, the microbial analysis revealed poor quality that exceeds standard criteria.

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Subclinical mastitis in dairy cows: somatic cell counts and associated bacteria in Mymensingh, Bangladesh

Journal of the Bangladesh Agricultural University ◽

10.3329/jbau.v15i2.35073 ◽

2017 ◽

Vol 15 (2) ◽

Cited By ~ 1

Author(s):

SMMR Sumon ◽

MA Ehsan ◽

MT Islam

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Dairy Cows ◽

Subclinical Mastitis ◽

Mixed Infections ◽

Coagulase Negative Staphylococci ◽

Somatic Cell Counts ◽

Milk Samples ◽

Cell Counts ◽

Bacillus Spp

Subclinical mastitis is an economically important disease of dairy cows and has a prominent place amongst the factors that limit milk production. This study was undertaken to determine the association of somatic cell counts (SCC) and occurrence of bacteria with SCM in smallholder dairy cows in Mymensingh, Bangladesh. A total number of 240 quarters milk samples from apparently healthy lactating cows were subjected to SCC using NucleoCounter® SCC-100 ™ (Chemo Metec). A quarter was considered SCM positive if the quarter had SCC>100 x 103 cells/ml. All subclinical mastitis positive quarter milk samples were subjected to bacteriological examination and isolates were classified into major, minor, uncommon and mixed pathogens. The overall quarter-level prevalence of subclinical mastitis of dairy cows in Mymensingh district was 25% (95% CI, 19.52% to 30.48%). The most frequently isolated bacterial species were Staphylococcus aureus (18.33%) followed by coagulase-negative staphylococci (10%), Enterobacter spp. (6.67%), Escherichia coli (5%), Bacillus spp. (5%) and Pseudomonas aeruginosa (5%). Different bacterial isolates were associated with 90% cases of subclinical mastitis as mono infections or mixed infections. Mono and mixed infections significantly influenced SCC and were the most prominent factors responsible for increasing SCC. Mean SCC was the highest for Bacillus spp. (713.67 x 103 cells/ml) followed by Enterobacter spp. (395.75 x 103 cells/ml), Escherichia coli (386.00 x 103 cells/ml), Staphylococcus aureus (373.82 x 103 cells/ml), coagulase-negative staphylococci (182.67 x 103 cells/ml) and Pseudomonas aeruginosa (138.67 x 103 cells/ml). Major pathogens induced higher SCC (380.72 x 103cells/ml) than minor and other pathogen groups.J. Bangladesh Agril. Univ. 15(2): 266-271, December 2017

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Determinación de la cinética, pruebas de crecimiento y efecto de inhibición in vitro de Lactobacillus lactis en Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae y Escherichia coli

Revista de la Facultad de Medicina Veterinaria y de Zootecnia ◽

10.15446/rfmvz.v62n2.51993 ◽

2015 ◽

Vol 62 (2) ◽

Author(s):

Henry Jurado-Gámez ◽

Manuel Gúzman-Insuasty ◽

V. Jarrín-Jarrín

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Streptococcus Agalactiae ◽

Staphylococcus Epidermidis ◽

E Coli ◽

Lactobacillus Lactis

Se determinó la cinética, pruebas de crecimiento y efecto de inhibición in vitro de Lactobacillus lactis sobre las bacterias patógenas Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae y Escherichia coli. Los análisis fueron realizados en el laboratorio de microbiología de la Facultad de Ciencias Pecuarias de la Universidad de Nariño. Las cepas patógenas fueron evaluadas por duplicado, la primera con el uso de cepas de referencia y las segundas, cepas aisladas de la región de Nariño, Colombia. Se determinó la inhibición de los antibióticos Gentamicina, Penicilina, Ciprofloxacina, Dicloxacilina, Cefepime, Cefalotina, Trimetropin Sulfa y Ampicilina mediante la metodología de kirby bauer. Se determinó la inhibición de L. lactis y el sobrenadante sobre todas las bacterias patógenas. Se evaluó el crecimiento de la bacteria láctica a 3 concentraciones de sales biliares (0,5, 1 y 2%) y 2 concentraciones de bilis bovina (1 y 1,2%); 3 pH (2, 4,5 y 7) y 2 temperaturas (38 y 45 °C). Se determinó la fase exponencial de crecimiento de la bacteria láctica y las variables cinéticas pH, azúcar, acidez y proteína. Se analizaron los péptidos presentes en el sobrenadante y la producción de ácidos orgánicos mediante HPLC. Se encontraron los microorganismos S. aureus, S. epidermidis, S. agalactiae y E. coli en leche de vacas con mastitis subclínica en la región de Nariño, Colombia; las cepas fueron aisladas para su análisis. Se encontró susceptibilidad de las cepas de referencia a los anbitióticos evaluados y resistencia de las cepas aisladas. Se observó que únicamente la cepa de referencia de S. aureus fue resistente a la bacteria láctica; ambas cepas de S. agalactiae presentaron resistencia; mientras que S. epidermidis y E. coli mostraron susceptibilidad. Se encontró que el sobrenadante inhibe en mayor proporción a las bacterias patógenas de referencia; mientras que las aisladas posee mayor resistencia. La cepa láctica tuvo un crecimiento de 0 y 5 x 109 UFC/ml a concentraciones de 1 y 1,2%; mientras que con sales biliares se encontró crecimiento únicamente a 0,5% (5 x 108 UFC/ml). La prueba de pH indicó crecimientos de 1,4 x 1012, 6,4 x 1011 y 7,5 x 1011 para pH 2, 4,5 y 7 respectivamente y la temperatura, 2,8 x 1012 y 3,1 x 1012 UFC/ml para 38 y 45°C. Durante la fase exponencial se encontraron valores de: 2 x 1011 UFC/ml a las 14:48 horas; 4,29 de pH, 0,62% de acidez, 2,18 mg/l de azúcar y 0,279 mg/l de proteína. Las pruebas de HPLC indicaron la presencia del péptido VAL-TIRL-VAL y 82,9% de ácido láctico. L. lactis posee características in vitro adecuadas para inhibir cepas patógenas presentes en leche con mastitis subclínica de la región de Nariño, Colombia.

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Antibiogram of Escherichia coli and Staphylococcus aureus isolated from milk sold in Kathmandu district

Nepal Journal of Biotechnology ◽

10.3126/njb.v8i3.30080 ◽

2020 ◽

Vol 8 (3) ◽

pp. 82-86

Author(s):

Shyamala Rai ◽

Barsha Karki ◽

Sujita Humagain ◽

Sandesh Rimal ◽

Sandhya Adhikari ◽

...

Keyword(s):

Public Health ◽

Escherichia Coli ◽

Staphylococcus Aureus ◽

Antibiotic Susceptibility ◽

Raw Milk ◽

Microbial Load ◽

Microbial Quality ◽

E Coli ◽

Milk Samples ◽

And Staphylococcus Aureus

The emergence of antibiotic resistance in microorganisms and the presence of such isolates in milk pose a great risk to public health. Therefore, this study aims to determine the antibiotic susceptibility pattern of Escherichia coli and Staphylococcus aureus isolated from milk and assess the microbial quality of milk. For this, a total of 70 milk samples were collected and the total bacterial count (TBC) was determined. E. coli and S. aureus were isolated using their respective selective media while antibiotic susceptibility testing was carried out by Kirby Bauer Disc Diffusion method. The TBC showed that the raw milk samples contained two-fold higher microbial load while the pasteurized milk samples contained four-fold higher microbial loads than the standard guidelines. A total of 62 isolates were identified from culture-positive milk samples of which 32 were E. coli and 30 were S. aureus. A significant correlation was observed between microbial load and the organism isolated (r = 0.339, p<0.01). All S. aureus isolates were susceptible to Chloramphenicol while 40% were resistant to Cefoxitin, indicating the presence of Methicillin resistant S. aureus (MRSA). Also, 12 multidrug resistant (MDR) S. aureus were identified. While for E. coli, all were susceptible to Chloramphenicol but resistant to Ampicillin. Also, 9 MDR E. coli were detected. Higher resistance was observed among isolates from the raw milk samples than the pasteurized milk. It can be concluded that the milk produced by small-scale farms and dairy industries of Kathmandu district are of poor quality. Hence, routine microbial quality assessment and antimicrobial resistance monitoring should be followed to safeguard public health.

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PRIMARY AND SECONDARY ANTIBODY RESPONSE TO COMPOSITE FORMALIN-INACTIVATED STAPHYLOCOCCUS AUREUS, STREPTOCOCCUS AGALACTIAE AND ESCHERICHIA COLI IN RABBITS

Bangladesh Journal of Veterinary Medicine ◽

10.3329/bjvm.v3i2.11404 ◽

2012 ◽

Vol 3 (2) ◽

pp. 178-180

Author(s):

M Avais ◽

G Muhammad ◽

A Shahzad ◽

MQ Bilal ◽

S Hameed

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Antibody Response ◽

Antibody Titre ◽

Streptococcus Agalactiae ◽

Secondary Antibody ◽

E Coli ◽

Significant Difference ◽

Group A ◽

Group B

Twenty buffaloes clinically positive for mastitis were selected for the collection of milk samples. Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli were isolated from milk of mastitic buffaloes according to the procedures recommended by National Mastitis Council Inc., USA. The composite antigen containing S. aureus, Str. Agalactiae and E. coli was then prepared and the concentration of each isolate was adjusted to 1 x 109 cells / ml. To evaluate the antibody response, nine adult healthy rabbits were divided randomly into three groups (A, B and C) consisting of 3 rabbits each. Composite antigen preparation was injected subcutaneously to the rabbits of groups A and B @ 0.2 ml / rabbit while each rabbit of group C inoculated with 0.2 ml normal saline and served as control. The rabbits of group B were given a booster dose at day 15 of the primary injection to see the secondary antibody response. The level of antibodies specific to S. aureus, Str. agalactiae and E. coli were assayed by indirect haemagglutination test (HA) and finally geometric mean titre (GMT) was calculated. At day 0 to 15, it was observed that the antibody titre was almost same in both groups. At day 30 significant difference in antibody titres was observed between group A and B with GMT of 9.8 against S. aureus, and Str. agalactiae in group A whereas in group B GMT was 97.0 against S. aureus and 39.4 against Str. agalactiae which was higher than group A. The GMT against E. coli was 12.1 and 24.3 in group A and B, respectively. At day 45 and 60 there was progressive decrease in antibody titre against Str. agalactiae in group B. The antibody titre against S. aureus first increased at day 45 and then decreased at day 60 whereas the antibody titre against E. coli remained persistent till day 60 in rabbits of group B. Furthermore, the primary antibody response to E. coli of composite antigen was higher (GMT, 12.1 at day 15) compared with S. aureus and Str. agalactiae (GMT, 9.8 at day 15) whereas the secondary antibody response to S. aureus was higher (GMT, 128) compared with Str. agaiactiae and E. coli (GMT, 24.3 at day 45).

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