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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
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
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.
Open Access
Nucleic acid and protein profile of Pasteurella multocida of avian origin
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2001) Rajalakshmi Radabai, S; KAU; Krishnan Nair, G
Four avian field isolates (one from quail and three from ducks) suggestive of Pasteurella sp. were compared with the avian Pasteurella multocida reference strain (LKO) for their biochemical properties, drug resistance patterns, whole cell and outer membrane protein profiles and restriction enzyme digestion pattern of chromosomal DNA. Biochemical characterisation revealed two biotypes among the four field isolates; namely P. multocida subsp. septica (quail isolate) and P. multocida subsp. multocida (duck isolates). The study of antibiogram using 14 chemotherapeutic agents revealed the presence of multiple drug resistance in all the four field isolates and reference strain. Whole cell protein profiles of the four field isolates and reference strain revealed 7 to 11 bands of molecular weights ranging from 197.4 kDa to 2.5 kDa. Polypeptide bands in the molecular weight range of 36 to 38 kDa (which were attributable to the protein-H), 29 to 32 kDa and 10 to 11 kDa were common to all the isolates of P. multocida. The major outer membrane proteins (OMPs) in the molecular weight range of 32 to 33 kDa and 27 kDa region were common to all the four field isolates and reference strain. II Unique protein bands in each of the isolate were indicative of variant forms of the same organism. Restriction enzyme analysis of chromosomal DNA yielded different restriction fragments in each of the isolates which were not easily distinguishable. Hence, of the tests used, phenotypic characters, biotype, antibiogram and protein profile were found to be more effective in differentiating the isolates rather than restriction enzyme digestion patterns of genomic DNA.
Open Access
Seroprevalence of HydroPericardium Syndrome in Broiler chickens
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2000) Binu, T V; KAU; Koshy John
In the present study, seroprevalence of hydropericardium syndrome (HPS) in broiler chickens in Kerala was evaluated. The results of agar gel precipitation test (AGPT), counter imrnunoelectrophoresis (eIE) and indirect enzyme linked imrnunosorbent assay (ELISA) were compared in detecting HPS antibodies. A total of 350 sera samples were collected from different parts of Kerala. In the first phase, 50 sera samples were screened by AGPT, eIE and indirect ELISA. Three samples were found positive by AGPT and eIE. Seven samples were found to be posi ti ve by indirect ELISA. Based on the comparison, another 300 samples were screened using indirect ELISA and 17 samples were found to be positive. A total of 24 samples were found positive for HPS antibodies out of 350 samples screened by indirect ELISA. The seroprevalence of 6.86 percentage was evaluated in broiler chickens in Kerala using indirect ELISA. The syndrome was experimentally reproduced in three-week-old broiler subcutaneous inoculation viz. , chicks. intramuscular, Various routes of and intraperitoneal routes were tried. The lesions were more pronounced when the birds were inoculated by subcutaneous route. Chick mortality was high on the fourth day. Electron microscopy of the infected liver homogenate revealed hexagonal virus particles of about 75 nm in size. These are suggestive of adenovirus. Histopathology of necropsied birds showed degeneration and necrosis of myocardial fibres, necrosis of hepatocytes and focal areas of coagulation in liver. Basophilic intranuclear inclusion bodies were demonstrated in hepatocytes. Vacuolar degeneration of kidney tubules, lymphoid depletion of bursa, vascular sclerosis of spleen and congestion and oedema of lungs were also seen.
Open Access
Isolation and characterization of Chlamydia psittaci with emphasis on protein profile
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2001) Binu, K Mani; KAU; Mini, M
For isolation of Chlamydia psittaci 46 clinical cases associated with abortions in livestock from livestock farms and Veterinary hospitals in and around Thrissur were screened. Impression smear staining of the clinical materials USin9 Giemsa, Modified Ziehl Neelsen and Gimenez revealed two positive cases. Both were from aborted foetuses, one from bovine (M-121) and the other from caprine (M-430). Chick embryo inoculation by YS route was used for isolation of the organism. The organisms could be isolated only from the two cases positive by preliminary screening, as confirmed by staining of YS impression smears. The mortality rate in CE was more in case of M-430 compared to the other two isolates (M-121 and reference isolate, P-156). All the three isolates produced patchy haemorrhagic lesions in YS and skin of embryoS. An overall isolation percentage of 4.3 was obtained in this study. All the three isolates were resistant to sodium sulphadiazine. Pathogenicity studies indicated that, all the three isolates were of moderate virulence for mice and guinea pigs. Based on AGPT, the local isolates were confirmed as Chlamydia psittaci. All the three isolates caused rounding and swelling of fibroblast cells and syncytia formation at 24h PI in Mc Coy cell line. By 48 to 72 h PI, these effects became more prominent with detachment of cells from adjacent cells and from the glass surface. Purified EBs were prepared from infected Mc Coy cell line by Urografin gradient centrifugation. After confirming the presence of sufficient amount of protein by Biuret method, they were used for SDS-PAGE. A total of 12 polypeptide bands were obtained for both the bovine isolates while the caprine isolate gave 10 bands. Molecular weights of 148 kDa and 135 kDa were present only in P-156 and 152 kDa and 137 kDa were unique for M-121. Bands at 152 kDa, 148 kDa, 137 kDa, 135 kDa, 32 kDa, 18 kDa, 15 kDa and 7 kDa were lacking in M-430. This isolate was having unique bands of 155 kDa, 19 kDa, 12.2 kDa and 6.4 kDa.
Open Access
Characterisation of virus isolates from lesser whistling teals and channa species of fish
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2000) Pradeep, V; KAU; Krishnan Nair, G
Characterisation of virus isolates (T18 and T22) from lesser whistling teals (Dendrocygna javanica) and Channa species of fish (Fe and F12) was carried out to determine the similarities if any between the isolates and to identify the role of waterfowls in dissemination of these viruses. The virus isolates. preserved in the Department of Microbiology were revived by passaging through embryonated chicken! duck eggs through allantoic route. After the third passage, all the isolates were found to produce death of the embryos and the allantoic fluid collected agglutinated one percent chicken RBC. The isolates T18, Fs and F12 produced congestion of the embryo and CAM and the embryos showed sub-occipital and interdigital haemorrhages. Isolate T 22 also produced congestion of the embryo and CAM and the embryos were stunted. Liver of the embryos had yellowish brown patches. The EID50 of isolates were 3.2x10s, 5.6 x 105 , 1.65x 107 and 3.16 x 105 respectively for the isolates T18, T22. Fs and F12. The infectivity and haemagglutinating activity of all the isolates were retained at pH 7.2, but were completely lost at pH 3.2 and 9.0 and also by treatment at 56°C for 30 min. All the isolates were sensitive to chloroform indicating their enveloped nature. Pretreatment of chicken embryo fibroblast cultures with 1 OO~g! ml of luDR did not inhibit the multiplication of any of the isolates indicating they all had RNA genomes. All the isolates were resistant to treatment with brine indicating that they were capable of survlvinp at high salt concentration. The isolates T18 and Fa produced marked CPE in chicken embryo fibroblast culture with rounding and clumping of cells and syncytia formation. Marked cytoplasmic vacuolation was also observed. Inclusion bodies could not be detected either in nucleus or cytoplasm. For isolates T22 and F12, CPE developed later only and was not as prominent as for T18 and Fa. Rounding of cells and their fusion forming syncytia was noticed by 72 h. Cytoplasmic vacuolation though present was much less marked. Inclusion bodies were absent. Large polykaryocytes were produced by the isolates T18, T 22 and F12 in BHK-21 cell line within 24h after inoculation. Between 48-72h large syncytia were formed. Intracytoplasmic inclusions could be observed by 24h after infection, which were quite prominent by 96h. The isolate Fa failed to produce any CPE in BHK-21 cell line. Pathogenecity tests in day old and six-week-old chicken and ducklings showed that all the four isolates were non-pathogenic when given by the oral Isubcutaneous route or by both. Neither clinical signs or mortality could be observed in the birds. Virus isolation was possible from the cloacal swabs of six-week-old chicken for the isolates T18 and T22 up to the 14th and th day respectively. Antigenic relationship between the isolates was tested by gel diffusion and haemagglutination inhibition tests, which showed that the isolate T18 did not have any similarity with any of the other three isolates. The isolate T 22 showed antigenic similarity by both the tests. Fa showed similarity to T18 by HI test but not by immunodiffusion test. Isolate F12 was found to be distinct from the other three by HI test, but showed some similarity with them by immunodiffusion test. By sodium dodecyl sulphate polyacrylamide gel electrophoresis on 10 percent gels, 7-11 bands could be resolved for the different isolates. Three of the bands were common for all the four isolates and were having molecular weights similar to the three major proteins HN, NP and MP of avian paramyxoviruses, suggesting that the isolates belonged to the paramyxovirus group. Monoclonal antibody typing of the isolates T18 and T 22 at the Central Veterinary Laboratory, Surrey, England confirmed that both belonged to the paramyxovirus group with T18 belonging to group C (velogenic) and T 22 to group E (B1 and LaSota) viruses. The isolates Fe and F12 need to be further typed. It was concluded from the study that all the isolates were enveloped RNA viruses with T18 and T 22 being paramyxoviruses belonging to Group I. The properties of the isolates Fs and F12 resembled the paramyxoviruses and from the similarly in protein profile with the other two viruses can also be concluded to be paramyxoviruses.

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