Kerala Agricultural University, Thrissur

The history of agricultural education in Kerala can be traced back to the year 1896 when a scheme was evolved in the erstwhile Travancore State to train a few young men in scientific agriculture at the Demonstration Farm, Karamana, Thiruvananthapuram, presently, the Cropping Systems Research Centre under Kerala Agricultural University. Agriculture was introduced as an optional subject in the middle school classes in the State in 1922 when an Agricultural Middle School was started at Aluva, Ernakulam District. The popularity and usefulness of this school led to the starting of similar institutions at Kottarakkara and Konni in 1928 and 1931 respectively. Agriculture was later introduced as an optional subject for Intermediate Course in 1953. In 1955, the erstwhile Government of Travancore-Cochin started the Agricultural College and Research Institute at Vellayani, Thiruvananthapuram and the College of Veterinary and Animal Sciences at Mannuthy, Thrissur for imparting higher education in agricultural and veterinary sciences, respectively. These institutions were brought under the direct administrative control of the Department of Agriculture and the Department of Animal Husbandry, respectively. With the formation of Kerala State in 1956, these two colleges were affiliated to the University of Kerala. The post-graduate programmes leading to M.Sc. (Ag), M.V.Sc. and Ph.D. degrees were started in 1961, 1962 and 1965 respectively. On the recommendation of the Second National Education Commission (1964-66) headed by Dr. D.S. Kothari, the then Chairman of the University Grants Commission, one Agricultural University in each State was established. The State Agricultural Universities (SAUs) were established in India as an integral part of the National Agricultural Research System to give the much needed impetus to Agriculture Education and Research in the Country. As a result the Kerala Agricultural University (KAU) was established on 24th February 1971 by virtue of the Act 33 of 1971 and started functioning on 1st February 1972. The Kerala Agricultural University is the 15th in the series of the SAUs. In accordance with the provisions of KAU Act of 1971, the Agricultural College and Research Institute at Vellayani, and the College of Veterinary and Animal Sciences, Mannuthy, were brought under the Kerala Agricultural University. In addition, twenty one agricultural and animal husbandry research stations were also transferred to the KAU for taking up research and extension programmes on various crops, animals, birds, etc. During 2011, Kerala Agricultural University was trifurcated into Kerala Veterinary and Animal Sciences University (KVASU), Kerala University of Fisheries and Ocean Studies (KUFOS) and Kerala Agricultural University (KAU). Now the University has seven colleges (four Agriculture, one Agricultural Engineering, one Forestry, one Co-operation Banking & Management), six RARSs, seven KVKs, 15 Research Stations and 16 Research and Extension Units under the faculties of Agriculture, Agricultural Engineering and Forestry. In addition, one Academy on Climate Change Adaptation and one Institute of Agricultural Technology offering M.Sc. (Integrated) Climate Change Adaptation and Diploma in Agricultural Sciences respectively are also functioning in Kerala Agricultural University.

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Open Access
Bacteria associated with respiratory infections in poultry
(Department of Veterinery Microbiology, College of Veterinary and Animal Science, Mannuthy, 2007) Jesto, George; KAU; Krishnan Nair, G
This study was undertaken to isolate and identify of bacteria from respiratory tract of poultry and to study the antibiogram of the isolates. Birds showing respiratory signs were sacrificed, postmortem examination was conducted and samples such as nasal, tracheal and air sac swabs and lungs were collected after taking all sterile precautions. A total of 105 samples were collected by sacrificing birds showing clinical signs. Isolation of causative bacteria was made by culturing on brain heart infusion agar, Mac Conkey agar and blood agar. For identification of isolates all the procedures were followed as described by Barrow and Feltham (1993). A total of 31 bacterial isolates were obtained from samples. A total of 12 Escherichia coli isolates were isolated and identified, 4 Pasteurella multocida isolates and 15 Staphylococcus sp. Isolates were isolated and identified biochemically. Out of 15 Staphylococcus sp. isolated and identified 11 isolates (73.33 per cent) were coagulase negative This result indicate that CoNS were more frequently isolated from staphylococcal infections although they do not possess the virulent coagulase activity. So importance must be given to CoNS also, as given to coagulase positive staphylococci and much study need to be diverted to find the virulence factors and role of them in producing bacterial infections in poultry. Multi drug resistance (resistance to at least three antimicrobials) was found among all E. coli isolates obtained in the study. Hence it may be concluded that the high level of resistance observed among poultry E coli isolates obtained in the study may be due to incorporation of antibiotics in feed as growth promoters. As 100 per cent sensitivity is shown to enrofloxacin and chloramphenicol by P. multocida isolates, these two drugs may be used for treating pasteurellois. Amoxycillin clavulanic acid (Ac) and cephalexin (Cp) was found to be the most effective antibiotic against Staphylococcus sp. in the study. The plasmid DNA content of the seven isolates of E. coli was analysed on agarose gel electrophoresis but correlation between the number of plasmids and antibiotic resistance could not be ascertained in this study. In conclusion, the results of this study provide evidence for significant antimicrobial resistance among bacterial isolates from birds. Long term prospective studies involving isolation, identification and antibiogram from more samples are required to identify novel pathogens causing respiratory disease in birds. Such studies provide data on temporal and spatial difference in antibiotic resistance patterns, which in turn helps the scientific community to design better disease control strategies.
Open Access
Development and evaluation of outer membrane protein vaccine against duck pasteurellosis
(Department of Veterinary Microbiology, College of Veterinary and Animal Science, Mannuthy, 2007) Ranjini, A R; KAU; Krishnan, Nair G
A research work was undertaken to prepare effective vaccines against P. multocida grown under different conditions and their immunopotency was assessed in one month old ducklings. The purity of the P. multocida A:l strain (DP1) was confirmed as per standard procedure. Pathogenicity of the isolate was assessed in six to eight weeks old mice. The isolate killed the mice with in 8 h intra peritoneally and within 24 h when injected subcutaneously. Whole cell protein of P. multocida (DP1) was extracted and they were subjected to discontinuous system of SDS- PAGE which revealed 20 to 26 visible protein bands of molecular weight ranging from 102 to 19 kDa. After growing P .multocida in iron sufficient and iron restricted medium its OMPs were extracted and they were analysed by SDS PAGE. In iron sufficient medium, 10 protein bands of MW which ranging from 91.84 to 19.02 kDa were revealed. In addition to the above a protein band with MW of 97.8 kDa were detected in iron restricted medium.The protein concentration was estimated by Modified Lowry method,and it was found to be 4mg/ml. The Median Lethal Dose (LDso) of P. multocida when determined in one, month old ducklings was found to be 10 -7.13 which contained 13 cells. Oil adjuvanted formalin inactivated vaccines were prepared from DPl grown under three different conditions viz., TSB, BHIB and BHIB supplemented with 100 u M 2,2' dipyridyl. Sterility, safety and potency test of the vaccines were done as per standard procedures. A total of 120 one month old ducklings were divided in to four groups with 30 birds in each group. The first three groups were vaccinated with ordinary bacterin, OMP vaccine prepared under iron sufficient condition and OMP vaccine prepared under iron restricted condition respectively and the fourth group served as control.The birds were vaccinated with 0.5millilitre of vaccine intramuscularly. The blood was collected from all the ducks on day 0,7, 14,21,28,45 and 60 day PV. Passive haemagglutination was done and the increase in antibody titre was observed from day 7 PV onwards for groups I,.Il and In. The highest antibody titre was obtained at 14th day PV and 21 st day PV for aMP vaccine prepared under iron restricted condition. All the vaccine groups had shown a significant difference from the control group at all stages of study. On homologous challenging OMP vaccine under iron restricted media on 42nd day PV afforded 60 per cent protection. On 60th day PV afforded 50 per cent protection. Though the aMP vaccine prepared under iron restricted condition was found to provide high antibody titre, the protection percentage afforded by it in comparison with ordinary bacterin and aMP vaccine prepared under iron sufficient media was low. Hence, the aMP vaccine prepared under iron restricted condition need to be subjected to further investigation.
Open Access
Evaluation of pathogenicity and antigenic relationship of salmonella isolates from poultry
(Department of Veterinery microbiology, College of Veterinary and Animal Science, Mannuthy, 2007) Sunil, G; KAU; Koshy, John (Guide)
In the present study the pathogenicity and the antigenic relationship of three local isolates of S. gallinarum was compared with that of a reference/known strain. The antigenic homogeneity/heterogeneity of these isolates was studied using SDS PAGE and AGID. All the four isolates when subjected to antibiogram revealed similar pattern. The only difference was with isolate X 4 which was susceptible to co-trimoxazole while all the other three were resistant. Except for the reference/known strain BGL, all the other three isolates were found pathogenic to mice. All the four isolates were pathogenic to day-old chicks. Isolates QS 1 and X 4 were most pathogenic. Major clinical signs were somnolence, weakness, inappetance and whitish diarrhea and were more prominent in IM inoculated group. All chicks that died during the experimental period gave positive Salmonella isolation from liver, spleen, heart blood, lungs, yolk and ceca. At the end of experimental period survived birds were sacrificed and they gave positive isolation only from ceca. All the four isolates were pathogenic to layers. The clinical signs observed were listlessness, inappetance, ruffled feathers, shrunken comb, greenish-yellow diarrhea with gradual weight loss and was more prominent in IV challenged group, followed by those inoculated by oral and IC routes. The most pathogenic strain was QS 1. Cloacal swabs examined for Salmonella revealed intermittent shedding in all groups. The highest re-isolation was obtained from liver. The egg production was affected in all test groups and was most severely affected in IV group. No significant difference was noticed between different routes of inoculation and among different isolates in re-isolation from egg. The re-isolates were confirmed as S. gallinarum by standard biochemical reactions. The gross and histopathological changes observed in experimental infection with all the isolates were the same. There was enlargement and necrosis of liver and spleen, with congestion of heart and lungs. Day-old chicks had omphalitis. Histopathologically there was congestion, necrosis and infiltration of inflammatory cells into the internal organs, including submucosa of intestine. Sodium dodecyl sulphate - polyacrylamide gel electrophoresis analysis of the OMPs of all four isolates revealed 13 different polypeptide bands. These bands were of similar molecular weight in all the four isolates, indicating antigenic homogeneity. Agar gel immunodiffusion carried out using hyper immune serum raised against isolates BGL and QS 1 revealed lines of identity between the four isolates, indicating antigenic homogeneity by serology.
Open Access
Polymerase chain reaction for the detection of canine parvovirus in faeces of dogs
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2004) Josemi, Mathew; KAU; Mini, M
A study was undertaken to compare the ability of haemagglutination (HA), polymerase chain reaction (PCR) and seminested PCR for the diagnosis of canine parvovirus (CPV) infection by detecting CPV from faecal samples of clinically suspected dogs. Characterization of vaccine strain and field strains of CPV was performed by restriction enzyme analysis (REA) in this study. Attempts were made to isolate CPV from faecal samples in MDCK cell line. One hundred and twentysix faecal samples were collected from dogs suspected for CPV infection and 40 faecal samples were also collected from normal healthy non-vaccinated as well as vaccinated dogs after 15 days of vaccination, that were brought to veterinary hospitals attached to KAU. All the samples were screened by HA, PCR and seminested PCR to detect CPV. No significant difference in HA titre could be appreciated on comparing the titre using PBS, pH 7.2 and PBS – BSA as diluents. The haemagglutination reaction by CPV was found to be favoured by a slightly acidic pH in the range of 4.0 to 6.0. Chloroform treatment of faecal samples had no influence on HA titres above 5 log2. Among 126 faecal samples screened, 43.65 per cent, 61.11 per cent and 73.81 per cent were tested positive for CPV infection by HA, PCR and seminested PCR respectively. Therefore, seminested PCR was found to be a more sensitive and specific method over HA and PCR for the early diagnosis of CPV infection. All the faecal samples from healthy non-vaccinated and vaccinated dogs after 15 days of vaccination were tested negative by HA, PCR and seminested PCR. Restriction enzyme analysis using HinfI, Rsa I and Sau 961 revealed no difference in the fragment length patterns between CPV vaccine strain and field strain. Attempts to isolate CPV from faecal samples in MDCK cell line were found unsuccessful. The occurrence of CPV infection among non-vaccinated dogs was found to be higher than that of vaccinated dogs. Vaccine failure was also observed after MLV vaccination. The distribution of CPV infection was highest among dogs between two to four months of age. Breed-wise distribution of CPV infection showed highest distribution in German shepherd dogs when compared to other breeds. Most of the cases of CPV were noticed during February and July in the year 2003 and during March to June in the year 2004.
Open Access
Detection of rotavirus in the faeces of diarrhoeic calves by reverse transcriptase- polymerase chain reaction and silver staining
(Department of Veterinary and Microbiology, College of Veterinary and Animal Science, Mannuthy, 2007) Ambily, R; KAU; Koshy John
A study was undertaken to detect the presence of rotavirus in the faeces of diarrhoeic calves by RT-PCR and RNA-PAGE. Agar Gel Immune Diffusion was performed to detect the presence of rotaviral antigens in faecal samples using hyperimmune serum raised in rabbits. The protein profile of BRV was analyzed using SDS-PAGE. Attempts were made to isolate BRV from faecal samples in MDBK cell line. One hundred and twenty four faecal samples of diarrhoeic calves were collected from University Livestock Farm, Mannuthy, Veterinary Hospitals of Kerala Agricultural University, some dairy farms in Thrissur district and also from individual farmers in and around Thrissur. Twenty samples each were collected from adult cattle above one year of age with diarrhoea and normal healthy calves. All these samples were screened for the presence of BRV by RNA-PAGE, RT-PCR and AGID. Among 124 faecal samples collected 29 (23.39 per cent) samples were detected as positive by RNA-PAGE. The clustered arrangement of the 11 segments of the genome showed a 4:2:3:2 migration pattern, typical of group A bovine rotavirus. Reverse Transcriptase – Polymerase Chain Reaction could detect BRV in 35 (28.23 per cent) samples. By using AGID, only 16 (12.90 per cent) samples were found positive. Among the various tests employed, RT-PCR was found to be more sensitive in the diagnosis of BRV infections. All the 20 faecal samples from adult cattle with diarrhoea were tested negative by the three methods. Rotavirus could not be detected in the faeces of healthy calves by any of the tests employed. The protein profile of BRV revealed nine polypeptides having molecular weight in the range of 16.5 to 131 kDa. The age-wise distribution of BRV infection in calves was studied. It was found that the occurrence of infection was most common in zero to two weeks of age (39.39 per cent) followed by two to four weeks of age (37.04 per cent). The faecal samples which were found positive by RNA-PAGE was inoculated into MDBK cell line. But the attempts to isolate BRV were found unsuccessful.
Open Access
Production and evaluation of vaccines employing Pasteurella multocida A:1 grown under different growth conditions
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2007) Raja Gopal, R; KAU; Krishnan Nair, G
A research work was undertaken to prepare effective vaccines against P. multocida grown under different conditions and their immunopotency assessed in one month old ducklings. The purity of the Pasteurella multocida A: 1 strain (DP1) was confirmed as per standard procedures. Pathogenicity of the isolate was assessed in six to eight weeks old mice. The isolate killed the intraperitoneally inoculated mice within eight hours and within 24 h when injected by subcutaneous route. The organism was grown in different media to assess the amount of capsular material produced. The media employed were DSA, DSA supplemented with 10 per cent FBS and DSA supplemented with 10 per cent FBS and 0.5 per cent yeast extract. The capsule enhancement was measured by capsule demonstration using Maneval staining and by characterization of crude capsular extract. The capsules of the organisms grown in capsule enhancement media when demonstrated using Maneval staining were discernibly larger and denser, when compared to the organisms grown in DSA alone. The capsular polysaccharides increased by approximately 1.6 times when the organism was grown in capsule enhancement media containing 10 per cent FBS and by about 2.06 times when grown in media supplemented with FBS and yeast extract. The potential of the organism to form in vitro biofilm was assessed by growing the organism in nutrient restricted conditions. The organism was grown in 0.32 per cent TSB supplemented with an inert substrate called bentonite clay, for the bacteria to attach and colonize. For quantification of biofilm, plate count by spread plate method was employed and the results showed that the biofilm cells reached a peak on the third day of incubation with an average count of 1.54 ×106 CFU/g of bentonite clay while the planktonic cells were found to be maximum on day one post inoculation, with a peak count averaging about 8.10 ×108 CFU/ml of the broth. Maneval staining of late logarithmic phase of three day old biofilm culture revealed heavily capsulated cells of P. multocida attached as large aggregates and appearing as chains of coccobacillary cells. Also they appeared as a meshwork of aggregated and chain forming cells. The capsular polysaccharide estimation of biofilm cells revealed a 3.25 times increase over the planktonic bacteria grown in DSA alone. The biofilm cells on nutrient agar after 24 h at 37°C produced some colony morphotypes characterized by radiating strands from centre to periphery and wavy margin. Median lethal dose (LD50) of P. multocida when determined in one month old ducklings was 23 cells. In the present study, it was unable to arrive at the median lethal dose in six month old ducks as only one duck died after 48 h of virulent challenge. Oil adjuvant formalin inactivated bacterin vaccines were prepared from DP1 grown in TSB, capsule enhancement medium and under biofilm mode and performed the sterility, safety and potency tests of the vaccine employing standard procedures. A total of 160 four week old ducklings were divided into four groups with 40 birds in each group and the first three groups were vaccinated with ordinary bacterin, capsule enhanced bacterin and biofilm vaccine respectively. The fourth group served as control. The birds were vaccinated with 0.5 millilitre of vaccine intramuscularly in the thigh region. Blood was collected from all the ducks pre-vaccination, at weekly intervals upto 28th day post PV and on day 42 PV by cardiac puncture or by jugular venipuncture. Passive haemagglutination using GA-SRBC sensitized with sonicated antigen of DP1 was used to measure the humoral immune response. The IHA titres obtained for biofilm vaccine group on day 14 was very much higher than the other two groups. The antibody titre was observed from day seven onwards for all the groups. All the vaccine groups have shown significant difference from the control group at all the stages of the study. Groups I and II were having no significant difference in their mean titres during the entire study. On homologous challenging, biofilm vaccine gave higher protection rates of 70 and 90 per cent than the 60 and 80 per cent protection rates of ordinary and capsule enhanced bacterins, when challenged with 200 and 100 LD50 doses respectively. Biofilm vaccine was proved to be the best among the three vaccines tried. The capsule enhanced vaccine did not provide any additional advantage over the ordinary bacterin vaccine. Elaborate field trials are to be done before advocating the vaccine for commercial use.
Open Access
Molecular methods based detection of pathogenic mycoplasmas of chicken
(Department of Veterinery Microbiology, College of Veterinary and Animal Sciences, 2006) Dipu, M K; KAU; Jayaprakasan, V
A study was undertaken for the detection of Mycoplasma DNA from the specimens by Polymerase Chain Reaction (PCR), differentiate the three significantly pathogenic mycoplasmas of chicken namely M. gallisepticum, M. synoviae and M. iowae from the other less pathogenic ones based on the result of polymerase chain reaction. Attempts were also carried out to isolate Mycoplasma from clinical samples testing positive by polymerase chain reaction and from a few randomly selected negative samples and to comprehend the different strains of M. gallisepticum, if any, among chicken of various age groups. Out of a total of 225 birds subjected for the study 25 were found positive for the presence of avian Mycoplasma by genus-specific PCR. Thirty samples from these twenty-five birds were positive. Tracheal swabs form all these birds were positive. Fifteen isolates were obtained from these twenty five birds when the tracheal swabs were directly streaked onto BHI agar whereas when these tracheal swabs were collected in BHI broth initially and later subcultured onto BHI agar following a positive result in PCR / colour change of the broth, only 13 of them yielded colonies. Five of the isolates were found to be M. gallisepticum by MG-PCR and these were obtained upon direct inoculation of BHI agar with tracheal swabs from birds. Those samples collected in BHI broth yielded MG colonies only in two cases. Non-MG colonies were found interspersed with MG colonies in agar plates and thus the selective isolation of MG colonies must be performed prior to subculture. The utility of PBS and buffered peptone water supplemented with glycerol as transit media for clinical samples intended for PCR was evidenced. The isolates obtained were successfully lyophilized and stored at –70°C throughout the span of the study. The PCR-RFLP pattern of the obtained MG isolates revealed uniformity among the isolates. None of the samples yielded positive result in MS-PCR and MI-PCR. The sensitivity and usefulness of molecular methods based detection of pathogenic mycoplasmas of chicken over isolation techniques could be appreciated.
Open Access
Polymerase chain reaction based screening of bovine mastitis milk to detect leptospira and mycoplasma
(Department of Veterinery Microbiology, College of Veterinary and Animal Science, Mannuthy, 2007) Jibi, M G; KAU; Krishnan Nair, G
In the present study an attempt has been made to detect the Leptospira and Mycoplasma from bovine mastitis milk by PCR. Simultaneously trials were also made to isolate these fastidious organisms from these milk samples. Direct screening of milk sample by PCR could not successfully detect the DNA of either of these organisms. All the previous studies had revealed that inhibitory factors present in the milk might interfere with the amplification of target DNA present in the milk. To overcome this difficulty initially all the milk samples were preenriched in the broth media, subsequently the target DNA was extracted. The genus specific primer A and B were used to detect the leptospires in milk samples. Out of fifty haemagalactic milk samples screened by PCR only one sample was positive for Leptospira. Fletcher’s semisolid media that could support the growth of reference strain of Leptospira pomona was used for the isolation study. But all the attempts to recover the organism from milk sample were unsuccessful. For the detection of Mycoplasma, genus specific primers MGSO and GPO3 were used. Out of the fifty samples screened only one sample was positive for Mycoplasma. But the M.bovis specific primers PpMB 920-1 and PpMB 920-2 failed to amplify the DNA that was found positive in the genus specific PCR. Since several species of Mycoplasma was found to be associated with the mastitis, organisms detected in this study might not be M.bovis. The media used for the isolation of Mycoplasma were BHI broth and BHI agar. Media were standardized with the reference strain and were found to be capable of supporting the growth of organisms. But all the attempts to isolate the Mycoplasma from milk samples were unsuccessful. It was concluded that PCR is a rapid and sensitive technique to detect the fastidious organisms that are difficult to isolate in the ordinary laboratory conditions. But one difficulty encountered with the PCR from milk samples was the extraction of good quality template DNA that contained fewer inhibitors. Since the rapidity and ease of PCR is largely dependent upon the extraction procedure adopted in each bio materials, the protocol followed in this study needs some modifications.
Open Access
Nucleic Acid based detection of salmonellae in poultry
(Department of Veterinary Microbiology, College of Veterinary and Animal Science, Mannuthy, 2005) Muthuramalingam, M; KAU; Mini, M
In the present study detection of salmonellae by Polymerase Chain Reaction in avian bio-materials was carried out. Isolation of salmonellae from avian bio-materials was also done. Differentiation of salmonellae based on molecular methods was carried out. Thirteen isolates from chicken and two from quails were characterized as Salmonella gallinarum using standard bacteriological procedures. With regard to the fermentation of the sugars all isolates fermented dulcitol, maltose, arabinose, trehalose, and mannitol. Variation in fermentation pattern was observed with xylose and sorbitol. All isolates were uniformly sensitive to chloramphenicol, ciprofloxacin, enrofloxacin, and pefloxacin, while all were resistant to tetracycline, furazolidone and cloxacillin. Two isolates were serotyped as Salmonella gallinarum by the National Salmonella and Escherichia Center, Kasauli. Forty-six samples were positive by both genus specific as well as serovar specific PCR. The genus specific and serovar specific PCR were used to confirm the identity of the isolates. Performing PCR on template DNA prepared from RV broth enriched sample was found to be an extremely rapid method for detection of Salmonella. Restriction enzyme analysis of the amplicon from the rfbS gene with enzyme Tfi І of all isolates revealed the expected 235 bp digestion. All the isolates carried plasmids. Two plasmid profiles were observed among the isolates examined. A multiplex PCR for virulence plasmid was carried out. The expected 571 bp level amplification, which is specific for Spv virulence region and 284 bp level amplification, which is specific for genus Salmonella, were obtained in all the isolates. An allele-specific PCR method was developed for serotype-specific detection of S. gallinarum. The expected 187 bp level amplicons were obtained in all the isolates. The sequence of the rfbS gene product has been submitted to the Genbank and has been assigned the accession No AF 442573 ATCC 9184 . The sequence showed 99 per cent identity with Salmonella gallinarum.

Kerala Agricultural University, Thrissur

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