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Anand Agricultural University, Anand

Anand Agricultural University (AAU) was established in 2004 at Anand with the support of the Government of Gujarat, Act No.(Guj 5 of 2004) dated April 29, 2004. Caved out of the erstwhile Gujarat Agricultural University (GAU), the dream institution of Sardar Vallabhbhai Patel and Dr. K. M. Munshi, the AAU was set up to provide support to the farming community in three facets namely education, research and extension activities in Agriculture, Horticulture Engineering, product Processing and Home Science. At present there seven Colleges, seventeen Research Centers and six Extension Education Institute working in nine districts of Gujarat namely Ahmedabad, Anand, Dahod, Kheda, Panchmahal, Vadodara, Mahisagar, Botad and Chhotaudepur AAU's activities have expanded to span newer commodity sectors such as soil health card, bio-diesel, medicinal plants apart from the mandatory ones like rice, maize, tobacco, vegetable crops, fruit crops, forage crops, animal breeding, nutrition and dairy products etc. the core of AAU's operating philosophy however, continues to create the partnership between the rural people and committed academic as the basic for sustainable rural development. In pursuing its various programmes AAU's overall mission is to promote sustainable growth and economic independence in rural society. AAU aims to do this through education, research and extension education. Thus, AAU works towards the empowerment of the farmers.

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Author: BHANDERI, BHARAT BABUBHAI × End date: 2011 × Start date: 2011 × Type: Thesis ×
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    ThesisItemOpen Access
    PHENOTYPIC AND GENOTYPIC IDENTIFICATION AND METAGENOMIC ANALYSIS OF SUBCLINICAL MASTITIC PATHOGENS IN COWS
    (AAU, Anand, 2011) BHANDERI, BHARAT BABUBHAI; Jhala, M. K.
    Subclinical mastitis occurs with no visible changes in the appearance of the milk and/or the udder, but milk production decreases which leads to economic losses to the farmers and dairy industry. There are many microbial pathogens involved in causing subclinical mastitis in cows. The present study was undertaken to know incidences of subclinical mastitis in organized farms using Somatic Cell Count (SCC) and bacteriological examination (International Dairy Federation-IDF guidelines), California Mastitis Test (CMT) and impregnated pH strip test followed by characterization and PCR based detection of important mastitic pathogens. Metagenomic analysis of subclinical mastitis milk was also done to determine the complex microbial diversity in udder environment during subclinical mastitis. A total of 349 quarters of 89 lactating cows comprising 31 Triple cross (TP) (Kankrej x Jersey x Holstein Friesian), 29 Kankrej, 17 Gir and 12 Holstein Friesian (HF) affiliated with Anand Agricultural University, Anand were screened for subclinical mastitis. Overall 52.8 per cent (47/89) cows were found to be positive for subclinical mastitis infection in one or more quarters. The highest incidence of subclinical mastitis was found in Triple cross cows (74.19%), followed by Gir cows (58.82%), HF cows (50%) and Kankrej cows (27.58%). Overall quarter wise incidence for subclinical mastitis was found to be 30.66 per cent (107/349). The highest incidence was found in Gir cows (38.80%) followed by Triple cross cows (38.08), HF cows (33.33%) and Kankrej cows (15.04%). The highest incidence of subclinical mastitis was found in fore left quarter (28.03%), followed by hind left quarter (27.1%), fore right quarter (24.29%) and hind right quarter (20.56%). Of the 47/107 cows/quarters positive for subclinical mastitis, 39/47 (82.97%) cows and 82/107 (76.63%) quarters were also positive by CMT and 27/47 (57.44%) cows and 56/107 (52.33%) quarters were positive by impregnated pH strip test. Cultural isolation ft'om 107 subclinically positive quarter milk samples yielded 126 bacterial isolates. Staphylococci was the most predominant bacterial species accounting for 53.97 per cent (68/126) of all the isolates, followed by 21.43 per cent (27/126) CAMP (Christie-Atkins-Munch-Peterson) test positive Str. agalactiae, 18.25 per cent (23/126) Micrococci, 4.77 per cent (6/126) E. coli and 1.58 per cent (2/126) Bacillus species. Out of 68 Staphylococci isolates, 38 (55.89%) isolates showed fermentation on Mannitol Salt Agar (MSA), whereas 30 (44.11%) isolates were mannitol non fermentive. Of the total 30 S. aureus identified by PCR, 21 (70%) were mannitol fermentive and 9 (30%) mannitol non fermentive. Thirty one (45.58%)) Staphylococci were found to be positive for pigment production, whereas 37 (54.42%) isolates produced white colonies on nutrient agar. Forty eight (70.58%) isolates were found positive for coagulase reaction, whereas 20 (29.41%) were negative. Thirty one (45.58%)) isolates exhibited P haemolysin production, 4 (5.89%) a haemolysin and 33 (48.53%)) isolates were non-haemolytic on 5 per cent Sheep blood agar. Phage typing at National Staphylococci Phage typing Centre, Maulana Azad Medical College, New Delhi, using five phage group sets of International Basic Set of 23 phages revealed maximum number of the Staphylococci isolates lysed by group II 14 (82.35%), followed by groups III, Not alloted (NA), I and V with 12 (70.58%), 9 (52.94%), 5 (29.23%) and, 2 (11.76%) respectively. Maximum 11 (64.7%) isolates were lysed with phage number 47 with strong reaction, followed by 10 (58.82%)) isolates with phage numbers 42E and 81, while less effective phage numbers were 71 and 94, which lysed only one strain (5.89% each) and phage number 95 not giving strong reaction with any of the isolates. The methicillin and oxacillin antibiotic sensitivity pattern by disc diffusion method revealed that, all the 68 (100%)) Staphylococci isolates were sensitive. Serotyping of six E. coli isolates (at National Salmonella and Escherichia Centre, Kasauli, Himachal Pradesh for 'O' antigen) resulted in identifying 014, O20, 045, 055 and 0112 serotypes, while one isolate was untypeable (UT). Out of 68 Staphylococci isolates tested for identification of 5. aureus by PCR, 30 isolates were identified as S. aureus by obtaining amplification product of 1318bp using S. aureus specific primer for 23S rRNA. Out of 30 PCR positive S. aureus, 18 (60%)) were positive and rest were negative for coagulase test. All the 27 Streptococci isolates were identified as Str. agalactiae by amplifying 586bp product using Str. agalactiae specific primer for the 16S rRNA while, none were amplified for Str. dysgalactiae (401bp) and Str. uteris (94bp) based on primers specific for the 16S rRNA and 23 S rRNA respectively. All the six E. coli isolates yielded 232bp amplified product using E. coli specific primer targeting DNA sequence coding for the 23 S rRNA. Metagenomic analysis (using GS FLX 454 Life Sciences) of DNA of subclinical mastitis milk sample of TP, Kankrej and Gir cows yielded an out put of 274190 bp, 17,727 bp, 42,548 bp and 1,960, 170, 301 contigs respectively. Average fragment length obtained were 139.89, 104.28 and 141.36 bp for TP, Kankrej and Gir cows respectively. The longest sequence length was 560, 327 and 454 bp, while shortest sequence length was 40, 40, and 41 bp for TP, Kankrej and Gir cows respectively. A total of 54 (2.76%), 39 (22.94%) and 12 (3.99%) sequences for TP, Kankrej and Gir cows respectively could be matched to proteins in SEED subsystems of MG-RAST (Meta Genome Rapid Annotation with Subsystem Technology) (using an e-value cut-off of le-5). Metagenomic analysis of the three breeds identified bacterial organisms belonging to phyla (5), class (8), Subclass / order (15), Family (19), Genus (23) and species (28); of these, 19 genera and 26 species, many of which were fastidious/anaerobic organisms, were identified additionally than the cultural methods. Out of five genera Staphylococcus, Streptococcus, Micrococcus, Bacillus and Escherichia detected in the subclinical mastitis milk samples of TP, Gir and Kankrej breeds by culture based methods, four genera Staphylococcus, Streptococcus, Bacillus and Escherichia were also identified in the corresponding pyrosequencing data, while Micrococcus identified by culture based methods was not found in the pyrosequencing data. In pyrosequencing, over all 28 bacterial species were identified from all the three breeds of cows viz. Leifsonia xyli, Propionibacterium acnes, Streptomyces coelicolor, Chlamydophila abortus, Staphylococcus aureus, Staphylococcus epidermidis, Lactobacillus acidophilus, Streptococcus mitis, Burkholderia cenocepacia, Burkholderia cepacia, Ralstonia solanacearum, Nitrosomonas europaea, Pseudoalteromonas atlantica. Salmonella Dublin, Serratia marcescens, Azotobacter vinelandii, Pseudomonas aeruginosa, Pseudomonas mendocina, Stenotrophomonas maltophilia. Bacillus subtilis, Lactobacillus delbrueckii. Aster yellows witches'-broom phytoplasma, Pannbaculum lavamentivorans, Thermosipho melanesiensis, Aeromonas hydrophila, Escherichia coli, Shigella hoydii and Pseudomonas fluorescens. Of these, except S. aureus and E. coli, all were additionally identified than the culture based method but, Str. agalactiae identified by cultural method was not found in the pyrosequencing data. The role of lesser known or less frequently involved organisms as identified by metagenomic analysis may be further explored in future so as to understand the complete etiopathology of subclinical mastitis in cows.