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ThesisItem Open Access MOLECULAR DETECTION AND CHARACTERIZATION OF CLOSTRIDIUM PERFRINGENS TOXIN GENES RESPONSIBLE FOR NECROTIC ENTERITIS IN POULTRY OF ANDHRA PRADESH(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2008-04) PRAVEEN KUMAR, N; VINOD KUMAR, N(MAJOR); RANI PRAMEELA, D; SUJATHA, KABSTRACT: The present study was carried out to investigate the prevalence of Clostridium perfringens toxin genes responsible for necrotic enteritis in poultry of Andhra Pradesh. A total of 691 faecal samples (396 necrotic enteritis suspected and 295 apparently healthy) comprising of cloacal swabs from live birds and intestinal scrapings from dead birds were collected from different districts of Andhra Pradesh viz., Chittoor, Guntur, Nellore, Krishna, East Godavari and West Godavari. Gross pathological studies of affected birds revealed necrosis of the small intestinal mucosa and submucosa with fibrin deposition resulting in pseudo membrane formation and turkish towel appearance was noticed in the small intestine. Microscopically lumen of intestine with fibrinonecrotic material which forms a visible pseudo membrane composed of cell debris, necrotic/distorted villi, inflammatory cells and clumps of bacteria were observed. The samples were inoculated in to fluid thyoglycollate broth and incubated overnight. DNA extracted from 24 h broth cultures by boiling method were screened by multiplex PCR for simultaneous detection of α, β and β2 toxin genes. Out of 396 (broiler 282 & layer 114) necrotic enteritis suspected samples 337/396 (85.1%) were positive for α toxin gene of which 189/337 (56.08%) were β2 toxin gene positive. Out of 295 (broiler 182 & layer 113) apparently healthy samples 61/295 (20.67%) were positive for α of which 4/61 (6.55%) were β2 positive. None of the sample showed amplification of β toxin gene indicating the absence of C. perfringens type C. As some recent studies focused the involvement of NetB toxin in pathogenesis, therefore, uniplex PCR amplification of NetB gene was done from alpha toxin gene positive samples (C. perfringens type A) yielded no positives for NetB toxin gene. From chi square analysis a significant difference (p<0.01) in the prevalence of toxin genes (cpa & cpb2) between necrotic enteritis suspected and apparently healthy at 99% level of confidence with an increased number of positives from necrotic enteritis suspected group. The present research indicates C. perfringens type A along with β2 toxin gene might be responsible for causing necrotic enteritis in broilers and layers in Andhra Pradesh. The multiple sequence analysis of the amplified partial cpa and cpb2 gene sequences revealed 100% homology between the present isolates, and with selected published sequences from GenBank were found to be 98-99% and 94-99% homology respectively. The phylogenetic analysis of the cpa gene of the present C. perfringens isolate (MG600591) with the selected published sequences of cpa revealed the close segregation in distinct clade with cpa gene of broiler isolate of C. perfringens (GU581186) from Iran. The phylogenetic analysis of three cpb2 sequences of present isolates (MF471364; MF471366; KX001813) segregated into close group of poultry originated sequences with exception of MF471365 which segregated in distinct clade with noporcine originated C. perfringens sequence (AY609173) from USA. Since alpha toxin gene (cpa) is considered as signature toxin gene for C. perfringens, amplification of cpa by PCR is considered as confirmative diagnosis of C. perfringens. Hence, in the present study all the PCR positives for cpa (n=398) were isolated by culturing revealed only 221/398 (55.52%) isolates indicating PCR is more sensitive in detecting C. perfringens when compared with isolation by culturing. In the present study culture supernatant of B. subtilis isolated from healthy intestinal contents of birds successfully inhibited C. perfringens by disk diffusion method indicating its importance as a probiotic in controlling necrotic enteritis in poultry.ThesisItem Open Access CHARACTERIZATION OF Staphylococcus aureus ISOLATES OF ANIMAL ORIGIN(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2003-11) ARUNA, VEDAVALLI; DHANA LAKSHMI, K(MAJOR); JANAKIRAMA SARMA, B; ANJANEYULU, YABSTRACT : The present investigation was carried out to isolate and characterize S. aureus from different animal and poultry species. Characterization was done by examination of the isolates for simultaneous coagulase and manitol fermentation, DNase, hemolysin, dermonecrotoxin production and antibiogram. The isolates were also subjected to finger printing analysis by REP and BOX-PCR. A total of 50 samples were collected from different animal species viz., buffalo, dog, goat, poultry and emu for isolation of S. aureus. The number of isolates from 14 buffalo milk samples,13 from birds, 13 from dogs and 10 from goats were 8, 6, 10 and 7, respectively. Identification of isolates by simultaneous testing of coagulase and mannitol fermentation was less time consuming and a convenient method. Most of the coagulase positive strains showed thermonuclease activity in toluidine blue DNA agar. Out of all the hemolysis positive isolates only one isolate from Emu bird exhibited dermonecrosis in rabbits. The RF for metronidazole was 100% and was more than 50% for ampicillin, penicllin-G, nitrofurantoin, amoxycillin and streptomycin. The RF of 25 – 50% was noticed for kanamycin and erythromycin. For doxycylcline, tetracycline, trimethoprim norfloxacin and cephalexin the RF was less than 25%. Out of 31 DNA samples of S. aureus analyzed by PCR technique, 14 samples produced PCR amplified products in REP and BOX and they were grouped into 6 major clusters in REP and 10 in BOX. There was extensive variability in DNA finger prints of S. aureus indicating differences in the genetic make up of S. aureus strains without any consideration for the animal source.ThesisItem Open Access ISOLATION AND CHARACTERIZATION OF INDIGENOUS INFECTIOUS BURSAL DISEASE VIRUS ISOLATES IN AND AROUND HYDERABAD(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2009-03) SOUMYA, VENGA REDDY; DHANALAKSHMI, K(MAJOR); SATYANARAYANA CHETTY, M; MADHURI, DABSTRACT : The present study was taken up with the objective of isolation and characterization of IBDV isolates. Ten pooled bursal samples (each pool consisting of five samples) were screened for IBD virus by AGID, out of which 5 pooled samples were positive for IBD virus. Chicken embryos were used for isolation of IBD virus by CAM route. Out of five samples two samples, PDP-1 and VH-1 yielded IBD virus after three serial passages in 11 day old chicken embryos. These isolates produced characteristic lesions like dwarfing of embryo, cutaneous edema, hemorrhages on thigh muscles, toes and greenish yellow discolouration of the liver in chicken embryos. These isolates could be adapted to BHK-21 cell cultures only after 5 successive passages as evidenced by stabilization of typical cytopathic effects. At 3rd passage level, CPE started with rounding of cells 72 h, PI. Large numbers of cells showed rounding and clumping, increase in cytoplasmic granularity at 96 h, of PI in 4th passage. In 5th passage, CPE started early i.e., 48 h, of PI and by 72 h, 60% of the cells were involved. There was extensive detachment of cells by 96 h, PI. Cell culture adapted isolates were characterized for physico-chemical properties. They were resistant to chloroform but inhibited by 1.0% formalin for 6 h, 0.1% formalin for 24 h. They were viable even after exposure to 560C for 90 min. They were resistant to acidic pH of 2.0 but completely inactivated at pH 12.0. Immunological characterization of cell culture adapted IBDV isolates by AGID and CIE revealed clear precipitation lines. The polypeptide analysis by SDS-PAGE revealed that two isolates contained identical viral polypeptides with similar molecular weights and band pattern. Both isolates revealed 5 poly peptides, in addition to these two faint bands. The molecular weights of the 5 polypeptides were: (91 KD) VP1, (52 KD) VPx, (42 KD) VP2, (32 KD) and VP3, (27KD) VP4 and molecular weights of two faint bands were 70 KD and 66 KD. In this study, RT-PCR was performed on cell culture fluids and corresponding bursal tissues to detect the virus. RT-PCR was performed using P1 and P2 primers specific to hyper variable region of VP2 gene pertaining to IBDV. These studies resulted in generation of specific amplification of 643 bp from samples tested. Pathogenecity studies with the cell culture adopted field isolates inoculated in 3 week old chicks revealed 50 % mortality with PDP-1, 37.5 % mortality with VH-1.The dead birds revealed characteristic lesion i.e., haemorrhages in thigh and pectoral muscles, enlarged and haemorrhagic bursae. Histopathological changes were more pronounced in bursa of Fabricius. The changes were characterized by lymphoid necrosis, follicular degeneration and proliferation of connective tissue.ThesisItem Open Access ISOLATION AND MOLECULAR CHARACTERIZATION OF INDIGENOUS GOATPOX VIRUS FROM TELANGANA REGION(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2009-01) VIJAYALAXMI, V; SATYANARAYANA CHETTY, M(MAJOR); Anjaneyulu, YABSTRACT:In view of the increasing incidence of goatpox out breaks the present study has been taken up with a view to isolate goatpox virus from field cases and characterize them. The disease surveillance reports on goatpox were collected for the years 1997-2008 and analysed. The analysis of epidemiological data revealed 176 outbreaks in Andhra Pradesh The outbreaks were recorded more during the years 2000-2003 and 2004-2007.Case fatality rates ranged from 3.7% - 49.4 % during the period. Samples were collected from two outbreaks of goatpox. Both the samples were positive for goatpox by Agar gel immunodiffusion. Attempts were made to isolate the virus by passaging in chicken embryos and then adapted to vero cell lines. Infected embryonating chicken eggs died between 48 h. and 120 h. Post Inoculation (PI) depending on the passage. CAM was edematous, thickened and congested. Slight hemorrhages were observed in 3rd passage. Embryo material after 3rd passage gave positive reaction when tested by Agar gel immunodiffusion with positive serum. EID50/ml (embryo infective dose 50) of the virus isolates ranged from 0.5x102 to 1x102 for the virus from 1st isolate while the virus from 2nd isolate had titres ranging from 0.7x102 to 0.9x102 / ml. Embryo passaged virus was then adapted to vero cells. On first passage cells revealed granulation and shrinkage of cells at 24 h – 48h post infection, syncitia at 72 h - 96 h PI while peeling of monolayer started at 120 h PI. Complete detachment of monolayer was seen in 6 days. Virus from 1st isolate had a titre of 104.9 TCID50 /ml and 2nd isolate had 105.2 TCID50/ml titre. Goatpox virus (GPV) was successfully purified by sucrose density gradient centrifugation. Two bands formed on the gradient, one at the interface of 50 and 60 percent sucrose layer and another at 40 percent sucrose layer. AGID and Immuno Electrophoresis were used to characterize the isolates. They resulted in a single precipitation line with hyperimmune serum. Physicochemical characterization of the viruses with regard to temperature, pH, ether and chloroform were carried out. The viruses were completely inactivated at 60oC in 30 min and 70oC in 10 min. Chloroform, ether and pH 3 were detrimental to goatpox viral isolates. Electron microscopy of the virus isolates revealed brick shaped virus with size of 250 x 290 nm. SDS - Polyacrylamide gel electrophoresis of the isolates revealed a total of 20 bands. Of these, five were major bands with size ranging from 22 to 67kd. Seroprevalance studies on goatpox was studied by employing AGID and Counter Immuno Electrophoresis (CIE). CIE was found more sensitive with positive percentage of 22.5 compared to 13.75 by AGID in a total of 80 serum samples. Polymerase chain reaction was used for detection of specific nucleotide sequences of GPV in samples and cell culture adapted virusesThesisItem Open Access ISOLATION, CHARACTERIZATION AND ANTIBIOGRAM PATTERN OF SALMONELLA AND ESCHERICHIA COLI IN ENTERIC INFECTIONS OF PIGS IN ANDHRA PRADESH(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2008-12) RUPA SUNDARI, A; SATYANARAYANA CHETTY, M(MAJOR); SREENIVASULU, D; SRI LATHA, ChABSTRACT: Multiple serovars of Salmonella and serotypes of E. coli are responsible to cause severe infections in pigs leading to morbidity and mortality and economic loss to swine industry. These bacteria also affect human health and have public health impact. Isolation and identification of indigenous Salmonella serovars / E. coli serotypes is of prime importance in order to carryout research on development of vaccines and for the study of molecular epidemiology. Hence, an attempt was made in this direction in the present study. Among 101 faecal samples screened for salmonellosis the notable serovars Salmonella Derby, S. Typhimurium and S. Typhisuis surfaced as major incriminating agents in pigs while the E. coli serotypes identified were O1, O2, O5, O8, O10, O12, O22, O24, O60, O88, O89, O92, O123, O138, O154 and O159. The bacterial aetiology was confirmed with standard procedures and protocols of Bergey’s manual of determinative bacteriology, ninth edition. The haemagglutination pattern of Salmonella and E. coli exhibited agglutination of erythrocytes viz., cattle, sheep, goat, chicken, rabbit, guinea pig and mice indicating the importance of fimbriae in colonization of organisms in intestine and production of toxins. Anti-sensi disk patterns revealed that the drugs of choice were chloramphenicol, co-trimoxazole and gentamicin for Salmonella infection while gentamicin, amoxycillin and chloramphenicol were the suitable drugs for chemotherapy of E. coli infection. The in vivo pathogenicity studies in mice revealed that S. Typhimurium was most pathogenic followed by S. Derby and S. Typhisuis whereas the pathogenicity studies carried out in chicks with E. coli serotypes viz., O1, O2, O8, O10, O22, O88, O92, O123, O138, O138 and O159 produced characteristic lesions and mortality in affected chicks. An innovative study on effect of Salmonella serovars viz., S. Typhimurium and S. Derby and E. coli serotypes O1, O24, O60 O89, O92, O123, O138 and O159 on germination of seeds were found to be toxic causing the inhibition of germination. Cefotaxime extract of Salmonella serovars on SDS-PAGE yielded 11 polypeptide bands ranging in molecular weight from 14 to 95 kDa while fimbrial extract of E. coli revealed a single band of 18 kDa.ThesisItem Open Access STUDIES ON THE ANTIGENIC RELATIONSHIP BETWEEN BTV-2, 9 & 15 IDENTIFIED IN ANDHRA PRADESH(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2008-11) DEEPTHI, B; SREENIVASULU, D(MAJOR); SATYANARAYANA CHETTY, M; ESWARA PRASAD, PABSTRACT : The presence of multiple serotypes of the midge-borne bluetongue virus and lack of effective vaccine are the major impediments in controlling bluetongue in sheep. Attempts are being made to develop the vaccine employing the available serotypes to control the disease in the state. Hence, it is essential to identify the antigenic relationship among the serotypes to identify the candidate vaccine strains to be incorporated in the preparation of vaccine. Further, interference phenomenon was also reported among the serotypes. To understand the antigenic relationship between BTV-2, 9 and 15 serotypes, the viruses were propagated in BHK21 cell lines which showed 90% of the CPE by 72 hrs. Purification of the BTV serotypes by polyethylene glycol precipitation revealed a single light scattering zone at 50-60% sucrose interface. Presence of BTV in the purified samples was confirmed by RNA extraction and observed the segmented nature of the nucleic acid. The purified virus samples of the BTV serotypes 2, 9 and 15 were used to raise hyper immune serum in rabbits. Neutralizing antibodies for the BTV serotypes were detected by day 21 PI and the titres persisted up to day 60 PI, the period under study. Maximum titres induced against BTV-2, 9 and 15 were 1:320, 1:256 and 1:640 respectively. Reciprocal cross neutralization test was employed to find out the R% values between BTV-2, 9 and 15 which indicated the extent of antigenic relationship between the serotypes. R% value between BTV-2 and BTV-9 was recorded as 2.8. R% value of 3.53 and 2.8 were observed between BTV-2 & 15 and BTV-9 & 15 respectively. The R% values recorded in the present study revealed a weak antigenic relationship between the BTV serotypes. The extent of antigenic relationship between the BTV serotypes was also determined by multiple sequence alignment of the nucleotide and amino acid sequences of the reference BTV serotypes 2, 9 and 15. The sequence analysis of the VP2 gene revealed a homology of 47-53% and 29-41% at the nucleotide and amino acid levels respectively. R% values obtained using reciprocal cross neutralization test with the BTV-2, 9 and 15 serotypes isolated in native sheep of Andhra Pradesh and the genomic analysis of the reference serotypes of BTV-2, 9 and 15 revealed very weak antigenic relationship and were highly divergent.ThesisItem Open Access STANDARDIZATION OF RT-PCR FOR TYPING OF BTV-2, 9 & 15(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2008-11) HIMAJA, M; SREENIVASULU, D(MAJOR); SATYANARAYANA CHETTY, M; ALAHA SINGARI, NABSTRACT: The present study was taken up to standardize reverse transcription polymerase chain reaction (RT-PCR) for typing of bluetongue virus serotypes 2,9 and 15 isolated from the native sheep of Andhra Pradesh. Bluetongue viruses 2, 9 and 15 were propagated in BHK-21 cell line. Double stranded RNA extracted from the three serotypes resolved into nine bands with similar migration pattern in ethidium bromide stained 1% agarose gel. RT-PCR for serotyping was standardized using primers specific to VP2 gene of BTV-2, 9 and 15 serotypes. cDNA was synthesized by reverse transcribing the RNA using M-MuLV RT enzyme, after denaturing the ds RNA by heating at 95°C for 5 min in the presence of 10% DMSO. The concentration of 1.75 mM MgCl2, annealing temperature of 45°C for BTV-2, 53.8°C for BTV-9 and 61 59.2°C for BTV-15 and 30 cycle amplification were found optimum for amplification of cDNA of BTV-2, 9 and 15 by PCR. RT-PCR resulted in 653 bp product of BTV-2, 1241 bp product of BTV-9 which were defined by specific primers. However non specific amplification at two different sites i.e 700 bp and 1500 bp was noticed for BTV-15. Specificity of RT-PCR was evaluated. BTV-2 and BTV-9 specific primers could amplify only BTV-2 and BTV-9 respectively where as BTV-15 type specific primers amplified not only BTV-15 but also BTV-2 and BTV-9. Primers used for serotype detection of BTV-15 reacted with RNA of BTV-2 and BTV-9 and resulted in non specific amplification. It appears that the BTV-15 specific primers employed in the present study are not suitable for typing of BTV-15. This may be because of variations between the homologous serotypes from different geographical regions. For further studies, amplified PCR products were cloned into PTZ 57 R/T vector (T/A cloning vector) and then transformed to DH5α E coli cells. The recombinant colonies were screened by blue white selection. Positive colonies were confirmed by colony touch PCR and sequence analysis. BLAST search indicated that sequence obtained from BTV-2 PCR product and BTV-9 cloned products were specific to VP2 gene of BTV-2 and BTV-9 respectively. However, 700 bp and 1500bp products of BTV-15 were identical to VP4 gene of BTV-2, 8, 10, 11, 13 & 18 and VP1 gene of BTV-2, 8 & 10 respectively, indicating the non specific amplification of BTV-15. Multiple sequence alignment of BTV-2 nucleotide and amino acid sequences with other BTV-2 isolates from different geographical regions showed homology of 72-91.71% and 88-99% respectively. Phylogenic tree of these isolates indicate that BTV-2/TPT, BTV-2/ china and BTV-2/Taiwan might have evolved from the same evolutionary pathway. Multiple sequence alignment of nucleotide and amino acid sequences of BTV-9/MBN isolate with other BTV-9 isolates from three different geographical regions showed homology of 70-98% and 76-99% respectively. Phylogenic tree 62 of these isolates indicating that BTV-9/ MBN, BTV-9 Afaundau and BTV-9 Mediterranean basin are closely related and formed a group in monophyletic tree. Where as BTV-9 SA reference strain is having divergence of 30-31% from the remaining three isolates and formed a separate group in monophyletic treeThesisItem Open Access CLONING AND SEQUENCING OF HAEMAGGLUTININ- NEURAMINIDASE (HN) GENE OF NEWCASTLE DISEASE VIRUS (NDV)(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2008-11) NAGAVALLI, Y; Satyanarayana Chetty, M(MAJOR); DHANALAKSHMI, K; Anand Kumar, A; Panduranga Rao, PABSTRACT : It is obvious to say that, the poultry farmer is the ultimate looser both in the farm of economy as well as loosing the chicken because of high mortality due to New- castle Disease (ND). Since the virus is having pleurality in antigenecity with broad host range, it became impediment to develop a suitable vaccine for that geographical location. Hence a modest attempt had been made for the sequence information of HN gene of Indian vaccine strain K which would provide a valuable information for understanding molecular epidemiology, distribution, predicting NDV origin, genotyping, phylogenetic relationship, pathogenecity and virulence. Hence the molecular charecterization of K strain was very important with particular reference to HN gene. The virus, that was cultivated in chicken embryos was subjected to RNA extraction by Trizol method and the nucleic acid was amplified by employing RT-PCR. The PCR amplicon was cloned into pDrive cloning vector and confirmed the gene size as 1731bp which coded for 577 amino acids . The nucleotide and the deduced amino acid sequences of ten different isolates were multiple aligned with available database (GenBank) using Clustal W to find out the variations between different serotypes. The multiple alignment among these sequences indicated the conserved nature of HN protein with respect to twelve cysteine residues and five glycosylysation sites . Subsequently, the phylogenetic relationship for these sequences was analysed using PHYLIP software. The phylogenetic tree analysis revealed correlation of K strain with other strains with varied percentage of homology.ThesisItem Open Access ISOLATION AND CHARACTERIZATION OF MAREK’S DISEASE VIRUS (MDV) SEROTYPE-1(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2008-01) SHILPA, KALISETTY; DHANALAKSHMI, K(MAJOR); JANAKI RAMA SARMA, B; RAJASEKHAR REDDY, A; RAMAKOTI REDDY, MABSTRACT : Poultry farms in India are experiencing excessive losses due to MD, in spite of vaccination with the serotype 3 (HVT) and serotype 2 (SB1) vaccines. Present study was undertaken with a view to isolate and identify Marek’s disease serotype 1 from the samples collected during field outbreaks; study the pathogenicity of the field isolates in chickens and determine the immunosuppressive effects of MDV field isolate. Three MDV serotype-1 isolates were recovered from flocks in which mortality due to tumors was observed. One each of the viruses was isolated from HVT vaccinated layer breeders (12 weeks), broiler breeders (39 weeks) and unvaccinated commercial broilers (42 days). MDV isolates were identified as serotype 1 by PCR using MDV serotype 1 specific primers. Of these isolates, DM-01 was selected for further characterization of pathogenicity and immunosuppressive potential in HVT vaccinated and unvaccinated white Leghorn chickens. To determine the pathogenicity and immunosuppressive potential of MDV isolate, and in-vivo experiment was conducted using white Leghorn male chicks. One-day old chicks were divided into three groups. Group 1 and 2 received HVT vaccine subcutaneously at neck region on day 1. On day 6th group 2 and 3 were inoculated intraperitoneally with 500 pfu/chick of MDV- field isolate. Presence of virus was detected in blood, spleen and feather follicles from MDV inoculated chickens using MDV PCR. The over all mortality rate was 15.6% in MDV infected, unvaccinated group and 9.4% in HVT vaccinated MDV challenged group. The frequency of MD lesions observed was 56% in MDV challenged unvaccinated group and 33.3% in HVT vaccinated challenged group. The protection index of HVT vaccine against challenge with field isolate of MDV was 40.5% and the virulence rank calculated was 59.5. The body weight was 13% less in unvaccinated challenge group and 10% less in vaccinated challenge group as compared to control group (vaccinated unchallenged). Relative bursal weight was significantly (≤0.05) lower in group 3 (MDV alone) compared to those of HVT + MDV and HVT groups. The effect of MDV on humoral immunity was assessed by measuring antibody levels to ND vaccination and SRBC inoculation. At 14th and 28th day post challenge there was significant reduction in HI antibody titers to ND vaccine and SRBC antigen respectively. Cell mediated immunity was assessed by measuring CBH response to PHA-P and Lymphocyte proliferation index to Con-A. At 42 days post challenge both the CBH response and lymphocyte proliferation index were significantly decreased in both HVT vaccinated MDV challenged group and unvaccinated MDV challenged groups as compared to HVT alone control group. The results of the present study showed that the DM-01 isolate of MDV recovered during this study was able to cause mortality, MD lesions and immunosuppression in chickens. HVT vaccination partially prevented the effects of MDV serotype 1 challenge. Further, based on the results the MDV isolate can be categorized as vvMDV and there is a need to improve the present vaccines by incorporating attenuated serotype 1 strains of MDV to induce better protection and reduce economic losses to poultry farmers. 16