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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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ThesisItem Open 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.ThesisItem Open Access ISOLATION, IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF BACTERIAL PATHOGENS FROM RESPIRATORY TRACT OF APPARENTLY HEALTHY AS WELL AS SICK GOATS(AAU, Anand, 2011) AHER, TUSHAR KISAN; ROY, ASHISHA number of factors are responsible for economic losses to the goat industry; among them the pneumonia due to various bacterial species imposes serious constraints on goat production all over the world because of high mortalities. The major health problem of small ruminants is pneumonia/pleuropneumonia, which may be caused by Mycoplasma and Pasteurella species alone or in conjunction with other microbes. Pneumonia in small ruminants constitutes a serious setback to the growth in this group of animals with resultant economic losses in many parts of the world. Thus, the present study was undertaken with a view to know preponderance of this bacterial spp. in relation to respiratory tract infections in apparently healthy and sick goats. The objectives were isolation, identification, nucleic acid based detection of virulence associated and toxigenic potentials and in vitro antibiotic sensitivity patterns of the isolates from respiratory tract infections of apparently healthy as well as sick goats. In the present investigation, total 102 nasal swab samples and 96 tissue samples were collected from apparently healthy as well as sick goats. Bacterial isolation was done following standard technique by inoculating tissue sample and nasal swab sample primarily on blood agar and plates incubated for 24-48 hrs at 37°C. After incubation, the nature of growth and cultural characters of colonies were studied. Preliminary morphological identification was based on Gram's staining. Specific identification and biochemical characterization of the isolates was done as per the standard techniques. In this study, ten different types of bacteria were isolated. It includes Mycoplasma spp. (0.7%), P. inultocida (0.7%), Staphylococcus spp. (29.9%), Micrococcus spp. (4.2%), Streptococcus spp. (9.7%), Bacillus spp. (19.4%), E. coli (18.8%), Proteus spp. (4.9%), Klebsiella spp. (5.6%) and P. aeruginosa (6.3%). The most prevalent species of bacteria found was Staphylococcus spp. Gram positive organisms were more prevalent in apparently healthy goats (46.5%) than sick goats (11.8%). Gram negative organisms were more prevalent in sick goats (24.3%) than apparently healthy goats (16.7%). From 102 nasal swab samples- 68 isolates, 32 lung samples- 31 isolates, 32 trachea samples- 26 isolates, 32 tonsil samples- 19 isolates were obtained. Out of which. Gram positive bacteria were 91 (63.2%), whereas Gram negative bacteria was 52 (36.1%) and a single isolate was identified as Mycoplasma spp. (0.7%). From 102 nasal swabs, total 68 isolates were obtained and there were total nine different types of bacteria isolated, viz.. Mycoplasma spp. (1.5%), P. multocida (1.5%), Staphylococcus spp. (38.2%). Micrococcus spp. (8.82%), Streptococcus spp. (7.4%), Bacillus spp. (33.8%), E. coli (5.9%), Klebsiella spp. (1.5%) and P. aeruginosa (1.5%). The most pre\alent bacterial species found in nasal swab were Staphylococcus spp. From. 32 lung samples, total 31 isolates were obtained and there were total seven different types of bacteria isolated, viz., Staphylococcus spp. (19.4 %). Streptococcus spp. (12.9%), Bacillus spp. (6.5%), E. coli (35.5%), Proteus spp. (6.5%), Klebsiella spp., (9.7%) and P. aeruginosa (9.7%). The most prevalent bacterial species found in lung was E. coli. From 32 tracheal samples, total 26 isolates were obtained and there are total six different types of bacterial species were isolated. It involves Staphylococcus spp. (30.8 %), Streptococcus spp. (7.7%)), Bacillus spp. (11.5%), E. coli (34.6%), Proteus spp. {1.1%) and P. aeruginosa (1.1%). The most prevalent bacterial species found in trachea was E. coli. From 32 tonsillar samples, total 19 isolates were obtained and there are total six different types of bacterial species were isolated. It involves Staphylococcus spp. (15.8 %), Streptococcus spp. (15.8%), E. coli (15.8%o), Proteus spp. (15.8%), Klebsiella spp., (21.1%) and P. aeruginosa (15.8%)). The most prevalent bacterial species found in tonsil was Klebsiella spp. Molecular characterization of the isolates by PCR based method was applied for specific detection as well as detection of virulence associated and toxigenic genes.ThesisItem Open Access MOLECULAR CHARACTERIZATION OF CAPRINE MYCOPLASMA ISOLATES(AAU, Anand, 2010) PATEL, AMITKUMAR HIMMATBHAI; ROY, ASHISHMycoplasmas belongs to the class Mollicutes and are among the smallest freeliving microorganisms capable of auto replication. Mycoplasmas are highly fastidious bacteria, difficult to culture and slow growing. Many species are important veterinary pathogens causing respiratory infection, mastitis, conjunctivitis, arthritis, and occasionally abortion. M mycoides subsp. capri (Mmc), M. capricolum subsp. capricolum (Mcc) and M. capricolum subsp. capripneumoniae (Mccp) are some of the most virulent species that have been associated with caprine respiratory disease which all are belongs to a group of very closely related mycoplasmas known as the "M mycoides cluster," which also includes 2 other pathogens of ruminants: M. mycoides subsp. mycoides SC (MmmSC), and the bovine group 7. The present work was carried out with the objective of characterization of ninteen isolates of M. mycoides subsp. capri and eleven isolates of M. capricolum subsp. capricolum (which are being maintained at Department of Veterinary Microbiology, College of Veterinary Science and A. H., Anand were isolated from healthy and infected goats of Gujarat state). Preliminary confirmation of the mycoplasma isolates by slide agglutination test, molecular characterization of the mycoplasma isolates by PCR and PCR- RFLP profiling, in vitro detection of virulence associated genes and characterization of membrane protein by one-dimensional SDS-PAGE of mycoplasma isolates was carried out during the present work. Revival of the mycoplasma isolates was done on Modified Hank's Balanced salt solution liquid ( MBHS-L) medium & Modified Hank's Balanced salt solution agar ( MBHS-A) medium. For preliminary confirmation of mycoplasma isolates slide agglutination test were employed. Identification of Mycoplasma isolates by PCR was carried out using mycoplasma genus specific primer pairs viz., (GPO-l(F)/ MGSO(R)), which yielded an amplified products of 715 bp for all the (30) isolates of mycoplasma. Identification of mycoplasma isolates was also carried out by PCR using M. mycoides subsp. capri and M. capricolum subsp. capricolum specific primers (CAP-21(F)/ CAP-21(R)), which yielded an amplified products of 1225 bp for all the (30) isolates of mycoplasma and also further restriction enzyme Asel and Sspl were used for characterization of the amplified products.. Detection of virulence associated LppA gene in M. mycoides subsp. capri was carried out by PCR using LppA gene specific primers (MMMLC2(F) and MMMLCl(R)). No amplified product could be detected in all the isolates (19) of M. mycoides subsp. capri. Detection of virulence associated LppA gene in M. mycoides subsp. capricolum was carried out by PCR using LppA gene specific primers (MCCPLl(F) and MCCPLl(R)), which yielded as expected 1356 bp long sequence of the LppA gene for all the isolates (11) of M mycoides subsp. capricolum. Detection of virulence associated GlpO gene was carried out by PCR using GlpO gene specific primers (GlpO(F) and GlpO(R)) of the M. mycoides subsp. capri, yielded as expected 1189 bp long sequence of the GlpO gene for all the isolates (19) of M. mycoides subsp. capri. Further digestion of the 1189 bp amplicons with the enzyme HpaI resulted in 2 fragments, 652 bp and 537 bp, while with the enzyme EcoAll resulted in 2 fragments, 826 bp and 363 bp, and with the enzyme Bpil resulted in 2 fragments, 941 bp and 248 bp. GlpO enzyme may play a central role in the virulence of M mycoides subsp. capri by the fact that the H2O2 and reactive oxygen species (ROS) act as powerful mediators for cell injury directly by inflammatory processes or by inducing pro-inflammatory genes. Characterization of the membrane protein was carried out by one-dimensional SDS-PAGE, yielded 6 band pattern for M. mycoides subsp. capri and 5 band pattern for M. mycoides subsp. capricolum. A band with 62 kDa is considered to be the lipoprotein A for M. mycoides subsp. capri and band with 58 kDa is considered to be the lipoprotein A for M. capricolum subsp. capricolum. The lipoprotein is reported to have role in pathogenesis and immune response against the mycoplasmal agents.ThesisItem Open Access “MOLECULAR GROUPING AND PATHOGENIC ANALYSIS OF LISTERIA MONOCYTOGENES BY CLONING AND SEQUENCING OF INLJ GENE”(Anand Agricultural University, Anand, 2010) Pragnesh B. Madariya; Dr. I. H. KalyaniListeria monocytogenes, a facultative intracellular pathogen, is responsible for severe food-borne infections in humans and can also cause invasive disease in many different animal species, including farm ruminants cattle, sheep, and goats. Several animal-derived L. monocytogenes-contaminated food products, including raw milk, pasteurized milk, chocolate milk, butter, soft cheeses, processed meat and poultry products are the main sources of human listeriosis cases and outbreaks. In ruminants it primarily causes encephalitis and uterine infections. The encephalitic form of animal listeriosis is characterized by neurological signs, including circling, excessive salivation, unilateral facial paralysis and uterine infections are characterized by lateterm abortions or septicemia in neonates. The present study was undertaken to characterizeThesisItem Open Access MOLECULAR CHARACTERIZATION OF CAPRINE MYCOPLASMA ISOLATES(Anand Agricultural University, Anand, 2010) PATEL AMITKUMAR HIMMATBHAI; Dr. Ashish RoyMycoplasmas belongs to the class Mollicutes and are among the smallest freeliving microorganisms capable of auto replication. Mycoplasmas are highly fastidious bacteria, difficult to culture and slow growing. Many species are important veterinary pathogens causing respiratory infection, mastitis, conjunctivitis, arthritis, and occasionally abortion. M. mycoides subsp. capri (Mmc), M. capricolum subsp. capricolum (Mcc) andThesisItem Open Access Cloning, Sequencing and Sequence analysis of ompH gene of bovine Pasteurella multocida(Anand Agricultural University, Anand, 2010) SHAH ANKITABEN KANAIYALAL; Dr. M. K. JhalaP. multocida is non-motile, small gram-negative coccobacilli of the family Pasteurellaceae and is a commensal of the upper respiratory tract of many animal species. Haemorrhagic septicaemia (HS) is the most economically important disease caused by Pasteurella multocida. The organism causes a wide range of diseases in domestic animals throughout the world. In India, HS has been reported to be the most fatal of five important epizootic diseases affecting buffaloes and cattle caused by serotype B:2. Outer membrane protein (OMP) of gram negative bacteria plays an important role in disease process as it acts at an interface between the host and pathogen. Protein H (OmpH) is the major OMP in the envelope of P. multocida, is protective and has a potential as a vaccine candidate. The present work was undertaken to characterizeThesisItem Open Access PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS FROM BOVINE MASTITIC ORIGIN FOR THEIR VIRULENCE ASSOCIATED CHARACTERS AND ANTIBIOGRAM PATTERN(Anand Agricultural University, Anand, 2011) Dipti T. Raval; Dr. Ashish RoyMastitis is a disease primarily leading to inflammatory conditions of udder is manifested as acute, sub acute or chronic form, which results in heavy economic losses for the dairy industry. Among various pathogens involve in the disease S. aureus has been reported to be a prominent causal agent. The objectives of the present study were cultural & biochemical characterization, detection of antibiogram pattern & molecular identification of S. aureus isolates. Cultural based detection of virulence associated character and molecular detection of virulence associated gene of Staphylococcus aureus from clinical and subclinical cases of bovine mastitis in and around Anand city of Gujarat were also studied. In the present investigation, a total of 40 isolates of S. aureus were included from theThesisItem Open Access “ISOLATION, IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF BACTERIAL PATHOGENS FROM RESPIRATORY TRACT OF APPARENTLY HEALTHY AS WELL AS SICK GOATS”(Anand Agricultural University, Anand, 2011) TUSHAR KISAN AHER; Dr. Ashish RoyA number of factors are responsible for economic losses to the goat industry; among them the pneumonia due to various bacterial species imposes serious constraints on goat production all over the world because of high mortalities. The major health problem of small ruminants is pneumonia/pleuropneumonia, which may be caused by Mycoplasma and Pasteurella species alone or in conjunction with other microbes. Pneumonia in small ruminants constitutes a serious setback to the growth in this group of animals with resultant economic losses in many parts of the world. Thus, the present study was undertaken with a view to know preponderance of this bacterial spp. in relation to respiratory tract infections in apparently healthy and sick goats. The objectives were isolation, identification, nucleic acid based detection of virulence associated and toxigenic potentials and in vitro antibiotic sensitivity patterns of the isolates from respiratory tract infections of apparently healthy as well as sick goatsThesisItem Open Access ANTIBIOTIC RESISTANCE AND VIRULENCE GENES IN STREPTOCOCCUS AGALACTIAE ISOLATED FROM BOVINE SUBCLINICAL MASTITIS CASES(Anand Agricultural University, Anand, 2011) BEENU JAIN; Dr. M. K. JhalaMastitis is the most important and expensive disease of dairy industry. It results in severe economic losses from reduced milk production, treatment cost, increased labor, milk withheld following treatment and premature culling (Miller et al., 1993). This disease is characterized by physical, chemical and bacteriological changes in the milk and pathological changes in the glandular tissue of the udder. The most important changes in the milk include discoloration, presence of clots and presence of large number of leukocytes. The US National Mastitis Council estimated an overall worldwide annual loss of 2 billion dollar due to mastitis (Anonymous, 2005). The annual economic losses due to mastitis in India have been estimated to be 7165 crores (Basal and Gupta, 2010). Generally mastitis occurs in two forms i.e., clinical or