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Open Access
Immunogenicity of an indigenous isolate of newcastle disease virus and Its usefulness as a vaccine strain
(Department of microbiology, College of veterinary and animal sciences, Mannuthy, 1983) Murugan, M R; KAU; Suloohana, S
The newly isolated mesogenic strain of Newcastle disease virus (NT) from an ailing mynah was studied in detail with particular reference to its biological characteristics, pathogenicity and immunogenicity. The results of various studies were compared with that of Komorov strain, a known mesogenic strain. The titer attained in developing chick embryos, mean death time of inoculated chick embryos at terminal dilutions, neuropathogenicity index in day old chicks and intravenous pathogenicity index were 109.5 /0.2 ml, 87 hours 0.63 and 0.00 respectively for the MT strain. The above values in order were 1010.5/0.2 ML, 76.5 hours, 1.16 and 0.000 for the Komorov strain. The infectivity of MT strain was labile at 560C for 10 minutes and the haemagglutinin was completely lost within five minutes. On the other hand the infectivity and haemagglutinin of K strain were comparatively resistant. Strain MT was pathogenic to day old chicks in which 26.6% mortality was noticed. In recovered chicks sufficient HI antibodies were seen and all of them withstood challenge. Although comparable results were obtained for Komarov strain, it was less pathogenic to day old chicks. Though 23.3% of chicks manifested clinical symptom only 3.3% died and the remaining birds recovered. In three weeks old chicks MT and K strain were found to be nonpathogenic either by S/C or oculonasal route. The inoculated chicks were immune when challenged six weeks later. Even in six weeks old chicks having no base immunity no post-inoculation reactions could be detected. All the chicks showed a rise in antibody titer reaching the peak level by the end of the third week and were resistant to challenge after six weeks. In chicks aged six weeks having a base immunity with strain were also free from any post infection reaction either by I/M or S/C route or inoculation. Chicks in both the groups produced HI antibodies and was always higher in those received infection by I/M route. The peak titers were obtained at the end of the third week and then declined. Though the titers were low by the end of the 6th week all the chicks were resistant to ND when exposed to a virulent virus. 2.9% of the chicks that received K strain by I/M route showed post inoculation reaction and died of ND. The remaining chicks and those in the S/C group behaved the same way as those received NT strain. Though the antibody response of chicken to MT and K were not statistically significant in all the three experiments, MWU test revealed that MT has a significantly higher immunogenic effect than K as the former always had a higher means than the latter. The ability to infect in contact chicks was also investigated. Strain MT was less efficient in this property giving only 25% to 28% transmission. On the other hand K strain revealed significantly higher transmissibility as it could spread to 62.5 to 75% of the inoculated in contact chicks. The mesogenic strain MT is quite safe in chicks of three weeks of age and above. It is also a good immunogen producing HI antibodies which protected the chicks from challenge even after six weeks. However the strain can be recommended as a vaccine strain only after further field trials and its effects on egg production are worked out.
Open Access
Comparison of serological tests for the detection of leptospira antibodies in immunised animals
(Department of microbiology, College of veterinary and animal sciences, Mannuthy, 1980) Ravikumaran Nair, R; KAU; Abdulla, P K
Leptospirosis is a widespread disease of man and animals and is of considerable economic importance besides being a public health problem. The leptospira infection in man and animals may be confirmed either by isolation of the organisms or by detection of specific antibodies in the serum and tissues of infected animals. Isolation of Leptospira is time consuming and beyond the scope of many diagnostic laboratories. In the present study the sensitivity of passive haemagglutination test was compared with the established microscopic agglutination test utilizing rabbit hyperimmune serum as the source of antibody. Leptospira serotypes were grown in Korthof’s medium enriched with 10% haemolysed rabbit serum. By 7 – 10 days satisfactory concentration of the organisms was obtained and was used for MA test. Passive haemagglutination test was carried out employing ethanol extracted antigen from concentrated leptospiral cultures. The PHA test was carried out after determining the optimum dilution of antigen required to sensitize sheep erythrocytes. Hyper immune sera to both serotypes were raised in rabbits by a series of intravenous inoculations. Serum samples for antibody titration was collected at weekly intervals from seven days following the first injection till the 49th day. Antibody titration by MA and PHA tests have shown that all the three animals inoculated with L. autumnalis had a uniform titre of 1:400 on the seventh day whereas the other three animals inoculated with L. pyrogenes showed a low titre of 1:100 by MA test. The PHA titre of both the groups remained the same ie 1:5. The maximum titre of 1:28000 for L. autumnalis was attained on the 21st day and remained unchanged until 35th day. The maximum PHA titre was attained only on 35th day (1:160). The rabbits inoculated with L. pyrogenes showed a maximum titre of 1:3200 by MA and 1:80 by PHA. The results obtained tend to show that PHA titres after reaching the maximum level remained detectable for longer period when compared to MA titres. Erythrocyte sensitizing substance from both the serotypes and the sera samples collected periodically from immunized rabbits were preserved at – 200 C at varying length of time upto three months. There was no deterioration in the stability or potency of ESS or sera on storage.
Open Access
Microbial agents associated with eye infection in chicken
(Department of microbiology, College of veterinary and animal sciences, Mannuthy, 2003) Jaison, George; KAU; Jayaprakasan, V
A study was undertaken to find out the microbial agents associated with the recently reported eye infection of chickens in many parts of Kerala. Samples were collected from 83 birds from different parts of Kerala. The bio-materials employed for the study were the conjunctival swabs and air sac materials. Organisms isolated from different stages of infection include 45 isolates of E. coli, 28 isolates of S. aureus, 11 isolates of B. coagulans, , eight isolates of Pseudomonas aeruginosa, two isolates of S. epidermidis, ten isolates of Alloscheria sp., five isolates of Penicillin sp. and three isolates of Scopulariopsis sp. and three isolates of M gallisepticum. On an average, the rate of isolation of microbial agents per sample at the beginning, mid and advanced stage of infection were 0.5, 1.3 and 2.8 respectively, showing that the number and type of organism isolated increased as the disease advanced except in the case of MG which was isolated only from the beginning stage of infection. No viral agents or e . chlamydia could be isolated through embryonated egg inoculation. The findings of the present study suggest that MG could be the primary agent leading to conjunctivitis while other eubacterial and fungal isolates could be secondary invaders complicating the condition. The antibiogram of the eubacterial isolates suggest that proper treatment with antibiotics like ciproflaxacin/gentamycin at the beginning stage of infection could prevent secondary invaders from complicating the condition.
Open Access
Immunoglobulins in ducks and role of bursa of fabricius in their production
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1990) Krishnan, Nair G; KAU; Sulochana, S
A study was undertaken to determine the immunoglobulin profile of ducks and to delineate the role of bursa in their production. Among the four different methods of bursectonomy employed in the study, Cy was found to produce the maximum reduction in body weight compared to other treatments. Marked reduction in bursal weight was also produced by Cy compared to T and ABS groups, while in SBx group the bursa was absent in toto. Surgical bursactomy resulted in significant reduction in spleen size of both surgically bursactomised uninoculated and inoculated ducklings. Histopathological studies revealed that the bursal development was highly suppressed on treatment with Cy. The ABS and testosterone treatments also elicited suppressive effect on bursa, but to a comparatively milder extent. It was evident that bursa had a role in lymphoproliferative reactions of spleen, as indicated by the maximum suppressive effect on spleen by SBx group. Ammonium sulphate at 33% level was found to be ideal for fractionation of duck serum globulins. Two main elution peaks were obtained on subjecting ammonium sulphate precipitated globulins to sephadex G – 200 chromatography. Concentrated and rerun ascending fractions of first major peak yielded purified IgM while those of the second major peak yielded purified IgG. Comparing the three age groups of antigen inoculation in ducklings bursectomised by different methods, total protein levels lower than the control were observed only in SBxSt (groups 1 and 11) and SBxSt (groups 1 and 111). Bacterial agglutination test revealed that in all four groups of bursectomised ducklings, antibody titres far below those of controls were produced. In SRBC agglutination test, lowered antibody titres than the control were observed in groups 1 to 111 of SBx, Cy and T – treated and groups 11 and 111 of ABS treated birds. Group 1 ABS administered ducklings had identical titres as that of control at days 7 – 21 post – inoculation. Bursectomized uninoculated ducklings revealed higher IgM levels than age – matched controls at many of the weeks under study. In bursectomised ducklings administered SRBC, IgM values higher than control level were obtained in the following cases : in groups 1 and 11 of SBxSR and TSR; groups 11 and 111 of CySR; and groups 1 to 111 of ABSR. S. typhimurium inoculated bursectomised ducklings revealed in the following treatments, higher IgM levels than control; in SBxSt groups 1 and 11; CySt groups 1 to 111; TSt group 1; and in ABSt groups 11 and 111. Quantitation of IgG levels in bursectomised ducklings revealed lower than control levels in SBx, Cy and T groups at 1 – 4 weeks of age, while the levels were higher or lower or identical with that of control from week 5. In ABS group the level was lower at 1 – 2 weeks. In bursectomised ducklings administered SRBC, higher IgG levels than control were obtained in groups 11 and 111 of SBxSR, CySR, TSR and ABSR. In group 1, treated ducklings revealed either lower or identical IgG levels compared to age matched controls. S. typhimurium inoculated bursectomised ducklings had higher IgG levels compared to CSt in all three groups of inoculation. Bile of ducks was found to contain only IgM, as evidenced by immunoelecrophoretic and quantitation studies. The IgM level in bile of bursectomised ducklings was found to be lower than that of the control. Yolk of duck eggs contained both IgM and IgG. Significantly higher lymphocyte count between the control and treated groups under study was detected at 4th (in SBx and T) and 8th weeks (in Cy). At 7th week, SBx group had significantly lower lymphocyte count, compared to control. At the fourth week of age, SBx and T groups had significantly lowered heterophil counts, compared to control. SBx group showed significantly higher count than the control at the 7th week, while at the 8th week, Cy – treated birds had markedly lower count, compared to the control. Eosinophil counts in bursectomised ducklings were higher than in control, while the basophil and monocyte counts in control and treated groups were more or less the same. The results obtained from the present study revealed that, 1. Among the different methods of Bx employed Cy produced maximum reduction in body weight, while SBx resulted in total elimination of bursa and significant reduction of spleen size. 2. Bursa had a role in lymphoproliferative reaction of spleen. Cy – produced maximum suppressive effect in bursa while in spleen SBx caused maximum suppression. 3. Ammonium sulphate (33%) was ideal for separation of duck serum globulins. 4. Sephadex G – 200 gel filtration was suitable for purification of IgM and IgG of ducks. 5. Bursa was concerned only with specific antibody production. 6. Elevated IgM and IgG levels were produced in bursectomised birds by extra – bursal B cells. 7. Bile of ducks contained IgM, the concentration of which was lower than the control in bursectomised birds. 8. Egg yolk of ducks contained both IgM and IgG.
Open Access
Detection of Pasteurella multocida in domestic ruminants by isolation and polymerase chain reaction
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2004) Sunitha Karunakaran; KAU; Krishnannair, G
A study was undertaken to examine the occurrence of P. multocida organisms causing HS in apparently healthy and clinically ill domestic ruminants in a specified area of Thrissur district viz., Ollukkara block, covering one per cent of total ruminant population in the area. Besides the samples collected from Ollukkara block area, those brought to the Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy from suspected cases of HS, were also included in the study. A total of 309 samples comprising of nasal swabs, pharyngeal swabs, lung samples and blood samples were processed for isolation of P. multocida and detection by PCR. Detection of P. multocida by PCR was carried out using species-specific (PM-PCR), type-B specific (HS-B PCR) and nested primers. Pasteurella multocida could not be isolated from any of the clinical samples cultured for isolation from Ollukkara block area. Five isolates could be obtained from the blood samples from Palakkad district. Isolates obtained were characterized as P. multocida using standard bacteriological procedures. A reference strain P52 obtained from IVRI was used for comparison. All the isolates were found to be pathogenic for mice. Based on the fermentation patterns of dulcitol, sorbitol, trehalose and xylose all isolates as well as P52 could be biotyped as P. multocida subsp. multocida. All isolates were uniformly sensitive to ten out of twelve antibiotics tested and uniformly resistant to metronidazole. All isolates were confirmed as type-B P. multocida using species-specific primer pairs, KMT1SP6 and KMT1T7 and HS-B specific primer pairs, KTSP61 and KTT72. Multiplex PCR was used to characterize all the serotype B isolates, which generated two bands of size 460 bp and 590 bp. Only 14 clinical samples including the isolates were positive for P. multocida by PM-PCR. The entire samples tested positive by PM-PCR were confirmed as type-B P. multocida by HS-B specific PCR. The 14 samples found positive by PM-PCR when subjected to nested PCR, gave an amplified product of size 214 bp. Since nested PCR could detect Pasteurella multocida DNA in a high proportion of clinical samples found previously negative by PM-PCR, a definite conclusion could not be arrived about the feasibility of using this PCR assay for enhanced detection of P. multocida as only a random number of negative clinical samples could be screened. All the isolates showed a single REP-PCR profile, indicating a high level of homogeneity among them. Among the five isolates examined, only two (BP2 and BuP1) harboured plasmids. The sequence similarity searches of HS-B PCR amplified product with BLAST network showed that the sequence had 99 per cent identity with Pasteurella multocida unknown protein 1 and protein 2 gene (Accession No AF016260).
Open Access
Prevalence of leptospirosis in animals in and around Thrissur
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2004) Manju Soman; KAU; Jayaprakasan, V
A study was undertaken to assess the prevalence of leptospirosis in animals and man, in and around Thrissur. A total of 501 sera samples, collected from dogs, cattle, pigs, rodents (bandicoots and rats) and human beings were subjected to serologic testing, for detection of Leptospira specific antibodies, by MAT, PHA and indirect IgG ELISA. Isolation of Leptospira was tried from blood of clinically suspected cases of human and canine leptospirosis and from kidney tissues of rodents. The study revealed the presence of antibodies against Leptospira in human beings and all the four species of animals examined by the three tests employed. The prevalence rates detected by MAT in canines, bovines, porcines, murines and human beings were 36.36 per cent, 47 per cent, 23.8 per cent, 21.42 per cent and 54.54 per cent respectively. Out of 30 human blood samples subjected to isolation trials in Fletcher'slEMJH semisolid media with enrichment, Leptospira could be isolated from a single human patient, in Fletcher's semisolid medium with 10 per cent rabbit serum. Of the 35 rodent kidney tissues tried for isolation in Fletcher's semi solid media, leptospires were isolated from three bandicoots and two rats. Indirect ELISA was found to be most sensitive, of the three tests,for rapid screening of the population for leptospirosis, while PHA was found to be a fairly good test for diagnosis of acute leptospirosis. The MAT proved to be the most specific test which could also identify the serogroup identity of the infecting Leptospira. Leptospira pomona and L. australis were detected by MAT as the common serogroups prevalent in man, animals and rodents in this area. The prevalence of common leptospiral serovars in animals and man, indicated that human beings which were the end hosts for Leptospira could have acquired the infection mostly from animals like dogs, cattle and pigs, while the isolation ofleptospires from rodents revealed the carrier status of these animals.
Open Access
Seroprevalence of BlueTongue in Sheep and Goats in Kerala
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2003) Chintu Ravishankar; KAU; Krishnan Nair, G
A study was conducted to assess the seroprevalence of BT in sheep and goats in Kerala, employing AGID, T-ELISA and dot-ELTSA tests. A total of 1010 samples collected from all the 14 districts of the State were screened for the presence of group specific antibodies to BTV. Seropositive samples were obtained from 12 out of the 14 districts. Both sheep and goats were found to harbour antibodies to BTY. Based on the results of the 1- ELISA test, the highest prevalence was observed in Thrissur district (21.10 per cent) and the lowest in Ernakulam district (1.25 per cent). Higher prevalence rates were observed in organized farms. The overall prevalence of the disease in the state was found to be 7.82 per cent. Of the tests employed, AGID test was found to be the least sensitive: The maximum number of positive samples were detected by I-ELISA. To check the validity of the I-ELISA results C-I~:LISA was done and there was good correlation between the results of the two tests as only four samples were detected as false positive in the C-ELISA. Dot-ELISA was found to be a simple, quick and specific test with detection levels comparable to T-ELlSA and could be used in the field conditions. Screening of the cattle population also should be done to get a better picture of the disease in the State as they comprise the largest reservoir of BTV in nature.
Open Access
Characterization of genomic and plasmid DNA of Chlamydia psittaci isolates
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2001) Sreeja, R Nair; KAU; Mini, M
Four isolates of Chlamydia psittaci (M-28, M-430, M-121, P-156) obtained from ruminant abortion were subjected to restriction enzyme analysis and plasmid profiling to assess molecular level differences/homogenity among the isolates. The changes produced in developing chick embryo and the cytopathic effect in Me Coy cell line by the isolates were also investigated in this study. The C. psittaci isolates preserved in the Department of Microbiology were revived by passaging in embryonated chicken eggs through yolk sac route. Six to seven day-old developing chick embryo were used for propagation of the isolates. The isolates, M-28 and M-430 were found to produce death of the embryos within 10 days. The isolates M-121 and P-156 were not able to cause death within 10 days. The embryos appeared stunted with patchy haemorrhages all over the body and hyperemic legs. The yolk sac was thin walled, with deeply injected blood vessels and the yolk was thin and watery. M-28 and M-430 produced more severe lesions of the inoculated embryos compared with the other isolates. The isolates were grown in Me Coy cell line. All isolates revealed marked CPE, characteristic of C. psittaci, with rounding and clumping of cells to form syncytia at 48 h PI and formation of intra cytoplasmic inclusion bodies by 96 h PI. The isolates M-121 and P-156 took more time for the production of CPE compared with that of M-28 and M-430. The isolates were confirmed as C. psittaci by fluorescence antibody technique which revealed, apple green fluorescing chlamydial inclusions in the infected cell line by 96 h PI. The CE as well as cell line propagated isolates were used as a source of elementary bodies for DNA extraction. The purified EBs were prepared by urografin density gradient centrifugation. After obtaining sufficient amount of EBs, it was subjected to DNA extraction. The isolates M-28, M-430, M-121 and P-156 had DNA concentrations of 1710 ug/ml, 1130 Jlgl Iml, 1545 ug/ml and 1475 ug/ml respectively. Restriction enzyme digestion of all the isolates were done separately with Eeo RI, Hae III and Barn HI. Eeo RI cleaved DNA of the isolates M-430 and P-156 into rune fragments, M-28 into eight and M-121 into ten fragments respectively. The molecular size of bands for all isolates had variation in heavier fragments. Common bands were observed at five regions for all isolates. Hae III cleaved the caprine isolates into eight fragments and the bovine isolate M-121 into seven and P-156 into nine fragments. Variation could be observed on the molecular size of the fragments. Bam HI digested all isolates except M-121 into eight fragments. For M-121, seven fragments were obtained. Common bands were noticed at 0.2 and 0.7 kbp region. All enzymes were found to be useful in the differentiation of C. psittaci isolates as these REs produced variation as well as similarity in the restriction fragment sizes. Plasmid profiling revealed the absence of plasmids ill all the four isolates screened. Thus, a combination of phenotypic and genotypic methods could be used as epidemiological tools in the differentiation of C. psittaci strains of mammalian origin.
Open Access
Seroprevalence and Restriction enzyme analysis of Egg Drop Syndrome virus
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2000) Priya, P M; KAU; Krishnan Nair, G
In the present study, seroprevalence of EDS-76 was conducted in five districts of Kerala in duck and' chicken flocks using HI and ELISA. Out of 322 chicken and 281 duck sera samples screened, an overall incidence of 14.91 per cent and 26.69 per cent respectively were recorded. The high proportion of birds showing antibodies to EDS-76 reveals that the infection is widespread in Kerala and may be the major etiological factor associated with drop in egg production in poultry. Among the two serological tests namely, HI and ELISA employed for the detection of EDS-76 viral antibody, HI was found to be simple, sensitive and reliable. It is concluded that HI test could be used for the detection of EDS-76 infection in poultry flocks. Restriction DNA fingerprinting of the three indigenous strains were carried out in conjunction with the reference strain to check for any genetic variation between the strains. Comparison of the DNA fingerprint of all the four strains digested with restriction endonucleases BamHI, EcoRI and HindIII revealed identical banding pattern thereby conforming the genetic similarity of the strains.

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