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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 MOLECULAR CHARACTERIZATION OF LISTERIA MONOCYTOGENES ISOLATES OBTAINED FROM DIFFERENT ANIMAL SPECIES(AAU, Anand, 2009) MATHAKIYA, RAFIYUDDIN A.; ROY, ASHISHListeria 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 pouhry products are the main sources of human listeriosis cases and outbreaks. In human, it causes septicaemia, meningitis, encephalitis, abortion and death in some cases while 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 late-term abortions or septicemia in neonates. The present study was undertaken to characterize all twenty-eight L. monocytogenes field isolates obtained from different animal species which were maintained at the Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand. The characterization was done on the basis of phenotypic character (CAMP test) and with an aid of new molecular techniques like PCR, PCR-RFLP. The quantitation by CFU counting and lowest detection limit by PCR was also performed to know the sensitivity level of PCR. Phenotypic characteration by CAMP test on all the 28 field isolates revealed positive reaction for twenty-three isolates with characteristics enhancement of haemolytic zone with S. aureus on Sheep Blood Agar (SBA) and five isolates (LM 01, LM 03, LM 05, LM 15 and LM 22) showed weak haemolytic zone. The template DNA was prepared from 28 field isolates and reference strain Listeria monocytogenes 4b (MTCC 1143) by boiling method as per standard protocol. L. monocytogenes inlA, inlB, plcB and m/J genes specific primers (inlA F: 5'-CCT AGC AGO TCT AAC CGC AC -3' and inlA R: 5'-TCG CTA ATT TGG TTA TGC CC-3'); (MB F: 5'-AAA GCA CGA TTT CAT GGG AG -3' and inlB R: 5'-ACA TAG CCT TGT TTG GTC GG-3'); (plcB F: 5'-GGG AAA TTT GAC ACA GCG TT -3' mdplcB R: 5'-ATT TTC GGG TAG TCC GCT TT-3') and (m//F: 5'-TGT AAC CCC GCT TAC ACA GTT-3' and inlJR: 5'-TTA CGG CTG GAT TGT CTG TG-3') were used to amplify virulence associated genes and virulent strain specific gene region. The PCR amplification carried out for the detection of virulence associated genes inlA (lmo0433), inlB (lmo0434), and plcB (lmo0205) gave a posifive result in all the 28 field isolates of of L. monocytogenes as well as all the reference strain Listeria monocytogenes 4b (MTCC 1143). The desired amplified product of approximately 255 bp, 146 bp and 261 bp was generated from virulence associated genes inlA, MB, and plcB, respectively, from all the 28 isolates of L. monocytogenes and the reference strain using the primer pairs for inlA, inlB, and plcB genes. Similarly, PCR amplification of inlJ (lmo282J) gene was carried out for the detection of virulent strain among all the 28 field isolates of L. monocytogenes. The desired amplified product of approximately 611 bp was generated from all the 28 isolates of L. monocytogenes and the reference strain Listeria monocytogenes 4b (MTCC 1143) using the primer pairs for m/J gene. PCR-RFLP analysis, which is commonly used for genotyping, was carried out for ten field isolates (LM 07, LM 13, LM 14, LM 20, LM 23, LM 24, LM 25, LM 26, LM 27 and LM 28) and reference strain Listeria monocytogenes 4b (MTCC 1143) having intense band in PCR product of inlA and inlJ genes was carried out for both inlA and inlJ genes by using four restriction endonucleases Rsal (Fast Digest), HindIII (Fast Digest) and DpnI, AluI, respectively. Digestion of 255 bp PCR products of inlA gene with Rsal yielded two fi-agments of approximately 151 bp and 104 bp, with Hindlll resulted in two fragments of 149 bp and 106 bp. On 2% agarose gel electrophoresis, all the 10 field isolates as well as the reference strain yielded similar RFLP profiles with each of the RE used. Similarly, Digesfion of 611 bp PCR products of inlJ gene with Alul yielded four fragments of approximately 274 bp, 161 bp, 150 bp and 26 bp digested. On 4% agarose gel electrophoresis, all the 10 field isolates as well as the reference strain yielded similar RFLP profiles with Alul while none of the samples was digested with DpnI. The quantitation by CFU counting and lowest detection limit by PCR was performed by ten fold serial dilution of the culture in BHI broth. For the sensitivity tests, the plate culture method was used to confirm the number of CFU and PCR was performed from each diluted culture and it showed the amplification up to 2 x10 to power 1 CFU/ml of broth using primer pair of inlA gene. This result indicates the lowest detection of Z. monocytogenes organism was 2 x10 to power 1 CFU/ml by PCR. Thus, in order to assess the analytical sensitivity, meaning the lowest number of L. monocytogenes detectable by PCR is a low-cost and rapid procedure that can be appropriated for the detection in real time of low L. monocytogenes levels in naturally contaminated food and is suitable to implement in the food industry.ThesisItem Open Access PREVALENCE AND MOLECULAR CHARACTERIZATION OF CANINE PARVOVIRUS FROM DIARRHOEIC DOGS(AAU, Anand, 2009) DHIRAJLAL, UMRETIYA SANDIP; JHALA, M. K.Canine parvovirus (CPV), a member of the genus Parvovirus, family Parvoviridae is a non-enveloped, single-stranded DNA virus of approximately 5 kb genome. The virus was first described in 1978, with the original isolate being termed CPV type 2 (CPV-2). In 1979, a variant CPV strain designated CPV type 2a (CPV-2a) became widespread, followed by further antigenic variants designated CPV type 2b (CPV-2b) and 2c (CPV-2c), which emerged in 1984 and 2000, respectively. In due course, CPV-2a, CPV-2b and 2c replaced CPV-2. The vaccines presently in use are CPV-2 strain specific. Hence, the objective of the present study was to detect and genotype CPV at different locations of Gujarat state by Polymerase chain reaction (PCR), using primer sequences derived from the conserved VP2 region of the genome, and to subsequent Restriction fragment length polymorphism (RFLP) analysis of the PCR product. The RFLP analysis employed the REs Hphl and Mboll in order to differentiate the CPV-2 antigenic variants. A few epidemiological factors like breed, age, sex, season and vaccination status affecting the infection were also studied. Haemagglutination (HA) and Haemagglutination inhibition (HI) tests were also employed so as to compare their sensitivity with PCR. From three different districts of Gujarat state, a total of 55 faecal samples were collected from dogs showing signs of diarrhea by sterile rectal swabs during December 2008 to April 2009. The samples were clarified and processed by HA-HI and PCR (using M1/M2 primers) assays for CPV detection. PCR detected the virus in 20 (36.36%) samples. Significantly more cases (40.91%) were seen in dogs below six months of age than dogs above six months of age (28.57%). Local breed showed maximum incidence (54.54%) followed by Doberman (42.85%), Labrador (41.66%), Alsatian (37.5%), Great Dane (33.33%) and Pomeranian (18.18%). Maximum number of cases (45.00%) was recorded in winter season i.e. during the months of December to February than in summer months (13.33%). Male dogs revealed higher incidence (41.05%) than female dogs (25.00%). CPV-unvaccinated dogs showed higher incidence (42.86%) of CPV than the CPV-vaccinated dogs (29.63%). HA and HI tests using pig erythrocytes yielded 17 (30.91%) samples positive for CPV with 85.00% sensitivity, 100%) specificity and 94.54 % overall agreement relative to PCR results. PCR amplification with primer pair M1/M2 yielded product of -252 bp in all 20 positive samples similar to that of vaccine (Puppy DP, Intervet) strain and the specificity of PCR amplicons was confirmed by RFLP using RE Fast Digest Dral. Nine representative PCR positive samples (representing six breeds of dog) were processed further for genotyping study. Primer pair Pabs/Pabas amplified all selected nine positive faecal samples with the product of 682 bp, but the vaccine strain could not be amplified suggesting that the strain present in faecal samples was not CPV-2 and was a variant. The specificity of PCR amplicons was confirmed by RFLP using RE Fast Digest Hinji. PCR amplification with primer pair Pbs/Pbas yielded the product (428 bp) specific to primers used suggesting that the strain in faecal samples was not CPV-2a but some other variant strain. Primer pair Hfor/Hrev used earlier in one study could not amplify any of the nine CPV positive faecal samples, but the vaccine strain was amplified, while primer pair Hfor/Pbas could amplify all nine CPV positive samples and vaccine strain yielding expected product (915 bp) specific to the primer pair used. Digestion of PCR product amplified by primer pair Hfor/Pbas with RE Hphl differentiated field strain of CPV from vaccine strain i.e. CPV-2. PCR products amplified by primer pairs Pbs/Pbas and Hfor/Pbas were digested by RE MboII, which detects Glu-426 mutation at nt 4062-4064 and hence, all nine CPV positive faecal samples and recognized as CPV-2c.ThesisItem Open Access “ISOLATION, IDENTIFICATION, MOLECULAR CHARACTERIZATION OF BRUCELLA FROM REPRODUCTIVE DISORDERS OF ANIMALS AND SERODETECTION OF BRUCELLA ANTIBODIES(Anand Agricultural University, 2008) SANJAY GHODASARA; Dr. Ashish RoyBrucellosis is a major bacterial zoonosis of global importance. The causative organism is Gram-negative facultative intracellular pathogens that may affect a range of different mammals including man, cattle, sheep, goats, swine, rodents and marine mammals. Brucellosis is caused by different species of Brucella and is cause significant economically losses to live stock industries. The disease is manifested by reproductive disorder, viz., infertility, retained placenta, abortions and endometritis in livestock species, hence it leads to loss in productivity of animals and consequently to economic lossesThesisItem Open Access “ISOLATION OF PPR VIRUS, DETECTION BY ELISA AND DIFFERENT GENE BASED RT-PCR”(Anand Agricultural University, 2008) Pooja Choudhary; Dr. M.K. JhalaPeste des petits ruminants (PPR) is a severe viral disease of goats and sheep with high morbidity and sometimes high mortality characterized by fever, erosive stomatitis, conjunctivitis, gastroenteritis and pneumonia. PPR is caused by Peste des petits ruminants virus (PPRV), a Paramyxovirus of the Morbillivirus genus. The disease causes severe economic losses to small ruminant husbandry and is presently considered as one of the major threats to about 200 million small ruminant population of the country. The present study was aimed to detect PPRV in clinical samples using s-ELISA and to derive estimates of overall, locationwise and specieswise incidence of PPRV. The study also involved the isolation of PPR virus from clinical samples and assessment of F, N and H gene targets for detection of PPRV by RT-PCR fromThesisItem Open Access GENE CLONING AND SEQUENCE ANALYSIS OF CHICKEN INFECTIOUS ANAEMIA (CIA) VIRUS(Anand Agricultural University, Anand, 2008) CHANNABASAYYA HIREMATH; Dr. M. K. JhalaChicken infectious anemia virus (CIAV) is an economically important and contagious avian pathogen with a worldwide distribution, causing chicken infectious anemia (CIA) in chickens. CIAV is a non-enveloped, icosahedral virus having a single stranded circular DNA genome and belongs to genus Gyrovirus, within the family Circoviridae. In the present study, the suspected CIA incidences were confirmed by PCR applied to different affected tissues collected from clinically affected birds from poultry flocks in and around Anand district of Gujarat. To study the extent of genetic variability of the field CIAV, the nucleotide sequences of VP1, VP2 and VP3 genes of CIAV were determined directly from the cloned PCR products, without isolating the virus in cell culture to obviate the potential genetic nucleotide changes that can possibly occur during cell culture passage. The gene sequences and the deduced aminoacid sequences of VP1, VP2 and VP3 of CIAV were compared with the other Indian and foreign sequences available in GeneBank and phylogenetic analysis based on nucleotide and deduced aminoacid sequences of VP1 was carried out to access the genetic distance of CIAV circulating in the field.ThesisItem Open Access ISOLATION, IDENTIFICATION, BIOCHEMICAL CHARACTERIZATION, ANTIBIOGRAM PATTERN AND MOLECULAR CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS FROM CLINICAL AND SUBCLINICAL MASTITIC MILK(Anand Agricultural University, Anand, 2008) BHANDERI BHARAT BABUBHAI; Dr. Ashish RoyStaphylococci are most significant among pathogen causing wide spectrum of diseases in both human and animal beings. Mastitis a disease of cows & buffaloes involved in severe economic losses to farmers & livestock owners world wide. The disease primarily leading to inflammatory conditions of udder is manifested as acute, sub acute or chronic form. The disease may have role of single or multiple etiolological agents leading to inflammatory symptoms. Among various pathogens involve in the disease S. aureus has been reported to be a prominent causal agents for mastitis. Thus the present study was undertaken with a view to know preponderance of Staphylococcus aureus in relation to bovine clinical and subclinical mastitis. The objective were isolation, identification, cultural characterization, PCR & cultural based detection of virulence associated character and in vitro antibiotic sensitivity patterns of Staphylococcus aureus from clinical and subclinical cases of bovine mastitis in and around Anand city of Gujarat.ThesisItem Open Access PREVALENCE AND MOLECULAR CHARACTERIZATION OF CANINE PARVOVIRUS FROM DIARRHOEIC DOGS(Anand Agricultural University, Anand, 2009) UMRETIYA SANDIP DHIRAJLAL; Dr. M. K. JhalaCanine parvovirus (CPV), a member of the genus Parvovirus, family Parvoviridae is a non-enveloped, single-stranded DNA virus of approximately 5 kb genome. The virus was first described in 1978, with the original isolate being termed CPV type 2 (CPV-2). In 1979, a variant CPV strain designated CPV type 2a (CPV-2a) became widespread, followed by further antigenic variants designated CPV type 2b (CPV-2b) and 2c (CPV-2c), which emerged in 1984 and 2000, respectively. In due course, CPV-2a, CPV-2b and 2c replaced CPV-2. The vaccines presently in use are CPV-2 strain specific. Hence, the objective of the present study was to detect and genotype CPV at different locations of Gujarat state by Polymerase chain reaction (PCR), using primer sequences derived from the conserved VP2 region of the genome, and to subsequent Restriction fragment length polymorphism (RFLP) analysis of the PCR product. The RFLP analysis employed the REs HphI and MboII in order to differentiate the CPV-2 antigenic variants. A few epidemiological factors like breed, age, sex, season and vaccination status affecting the infection were also studied. Haemagglutination (HA) and Haemagglutination inhibition (HI) tests were also employed so as to compare their sensitivity with PCR.ThesisItem Open Access ELECTROPHEROTYPING, RT-PCR DETECTION AND G-TYPING OF BOVINE ROTAVIRUS FROM DIARRHOEIC CALVES(Anand Agricultural University, Anand, 2009) SINGH TARUNKUMAR C; Dr. M. K. JhalaAmong the infectious diseases of calves, neonatal diarrhoea is a matter of major concern and is caused by multiple etiological agents. Various infectious agents responsible for causing diarrhoea include viruses, bacteria and parasites. Diarrhoea in calves during 5-10 days of age is commonly due to rotavirus and infected calves excrete very large quantities of virus particles in their faeces and become potential source of infection to other animals. Recognition of significant morbidity and mortality caused by rotavirus in neonatal calves has stimulated studies for definitive diagnosis of the disease as well as molecular typing of the circulating viruses with the ultimate goal of developing effective methods of preventive measures.ThesisItem Open Access Seroprevalence, Isolation, Identification and Molecular Characterization of Mycoplasmas from Healthy and Diseased Goats(Anand Agricultural University, Anand, 2009) PANKAJ KUMAR; Dr. Ashish RoyMany species belonging to the class Mollicutes are pathogenic and of great economic concern in livestock production. The major health problem of small ruminants is pneumonia/pleuropneumonia, which may be caused by Mycoplasma species alone or in conjunction with other microbes. Pneumonia in small ruminants constitutes a serious setback to the growth of this group of animals with resultant economic losses in many parts of the world. There is one interesting group of six closely related Mycoplasmas named the Mycoplasma mycoides cluster, consisting of several ruminant pathogens. This group comprises the following species, subspecies, or strains: M. capricolum subsp. capricolum (Mcc, formerly M. capricolum), M. capricolum subsp. capripneumoniae (Mccp, formerly Mycoplasma sp. strain F38), M. mycoides subsp. capri (Mmc), the M .mycoides subsp. mycoides large-colony (Mmm LC), M .mycoides subsp. mycoides small-colony (Mmm SC) types and Mycoplasma sp. strain PG50.