Sathish Bhadravati Shivachandra

Immunosuppression in broiler chicks fed aflatoxin and inoculated with fowl adenovirus serotype-4 (FAV-4) associated with hydropericardium syndrome

A total of 240 unvaccinated day-old broiler chicks, which had been found to be negative for antibodies against FAV-4, were divided into four groups of 60 chicks each. Group A was fed aflatoxin at 1 ppm from 7 days to 7 weeks of age. Group V was infected intra-abdominally at 14 days of age with 0.2 ml of FAV-4, having a titre of 105.5 TCID50 per 0.2 ml. The combined group AV was given the aflatoxin and infected with FAV-4. The fourth group C served as the control. More pronounced clinical signs, a higher mortality rate (56.7%), and reductions in body weight gain and in the organ to body weight ratios of the bursa and spleen were recorded in group AV. A significant (p<0.01) reduction in the HI antibody titre following vaccination against Newcastle disease, and of skin thickness in the delayed hypersensitivity test following sensitization with DNCB, indicated an additive immunosuppressive effect from aflatoxin and FAV-4 on the humoral and cell-mediated immune responses in group AV compared to groups A and V. Microscopically, marked depletion and degeneration of lymphocytes in the thymus, bursa, spleen and caecal tonsils were observed in group AV up to 5 weeks PI.

Prevalent serotypes of Pasteurella multocida isolated from different animal and avian species in India.

Identification and estimation of the prevalence ofPasteurella multocida organisms in different animal and avian species in India during November 2000 to July 2003 was carried out. Out of 418 samples collected from different outbreaks suspected to be caused byP. multocida, a total of 206 bacterial cultures were identified asP. multocida on the basis of cultural, morphological and biochemical characteristics. All the 206 cultures were isolated from different domestic animal species (cattle, buffalo, sheep, goat, pig and rabbit), avian species (chicken, duck, quail, turkey, goose) and wild animals such as leopard and deer. Serotyping ofP. multocida cultures revealed the presence of various serotypes (A:1, A:3, A:1,3, A:4, B:2, D:1and –:1) among the livestock population. P. multocida polymerase chain reaction (PCR) assay applied on different forms of bacterial cultures (bacterial culture lysate, direct bacterial colony and mixed bacterial culture lysate) yielded an amplified product of ∼460 bp specific forP. multocida. The results of PCR assay correlated well with conventional methods of identification. The present investigation revealed the presence of varied serotypes among livestock and PCR assay was found to be useful for rapid, sensitive and specific diagnosis of pasteurellosis in animals and avian species.

A review of hemorrhagic septicemia in cattle and buffalo

Abstract Hemorrhagic septicemia (HS), an acute, fatal and septicemic disease of cattle and buffaloes caused by Pasteurella multocida, is important in tropical regions of the world, especially in African and Asian countries. The prevalence of disease has been well documented with predominant isolation of P. multocida serotypes B:2 and E:2. Conventional methods of identification such as serotyping, biotyping, antibiogram determination and pathogenicity as well as molecular methods (P. multocida-specific polymerase chain reaction (PCR), a serogroup B-specific PCR assay, multiplex capsular typing system and loop-mediated isothermal amplification techniques) and characterization (restriction endonuclease analysis, randomly amplified polymorphic DNA analysis, repetitive extragenic palidromic PCR and enterobacterial repetitive intergenic consensus PCR analysis) are applied in parallel for rapid epidemiological investigations of HS outbreaks. Although several vaccine formulations including alum precipitated, oil adjuvant and multiple emulsion vaccines are commercially available, the quest for suitable broadly protective HS vaccines with long-lasting immunity is on the upsurge. Concurrently, attempts are being made to unravel the mysteries of the pathogen and its virulence factors, pathogenesis and determinants of protective immunity as well as diversity among strains of P. multocida. This review highlights the advances in these various aspects of HS.

Molecular Variability among Strains of Pasteurella multocida Isolated from an Outbreak of Haemorrhagic Septicaemia in India

The applicability of conventional and molecular methods for rapid detection and differentiation ofPasteurella multocida serogroup B isolates involved in an outbreak of haemorrhagic septicaemia affecting Indian buffaloes was studied. Five isolates were obtained and were subjected to phenotypic and genotypic characterization. None of the five isolates could be differentiated on the basis of cultural, biochemical, pathogenicity and antimicrobial sensitivity patterns. Polymerase chain reaction (PCR)-based techniques were found to be specific and sensitive for rapid detection and differentiation of isolates. Repetitive extragenic palindromic (REP-) PCR, enterobacterial repetitive intergenic consensus (ERIC-) PCR and single-primer PCR differentiated all the five isolates into different profiles. All the isolates involved in the outbreak were found to have a genetic profile different from standardP. multocida strain (P52). However, three isolates had similar profiles, whereas each of the remaining two had a different profile. The study indicates the involvement of multiple strains ofP. multocida in a single outbreak of haemorrhagic septicaemia in buffaloes. The results also indicate that molecular methods of detection and typing are superior to conventional methods for rapid epidemiological investigations of haemorrhagic septicaemia.

Immunogenicity of highly conserved recombinant VacJ outer membrane lipoprotein of Pasteurella multocida.

Bacterial lipoproteins are emerging targets for inducing protective immunity against many infectious diseases. VacJ is a highly conserved and widely distributed outer membrane lipoprotein of Pasteurella multocida strains, which are known to affect a wide range of domestic as well as wild animals and birds. In the present study, the gene encoding for mature VacJ outer membrane lipoprotein of P. multocida serogroup B:2 strain P52 was cloned and over-expressed in Escherichia coli as a fusion protein. The purified recombinant VacJ protein (∼44kDa) was used for immunizing mice (6/group) along with adjuvants (FCA and alum) in two experiments. Immunization of mice with rVacJ (30μg and 75μg/mice) elicited humoral immune response with significant (P<0.01) rise in antigen-specific titers of IgG and its subtypes (IgG1 and IgG2a). No protection was noticed in mice immunized with rVacJ (30μg) along with FCA followed by challenge with 100 LD50 of the homologous strain. On the contrary, higher rVacJ dose (75μg) along with FCA and alum provided 66.7% and 50% protection respectively, at reduced challenge dose (8 LD50). The study indicated that a lipidated recombinant VacJ lipoprotein with suitable adjuvants could potentially act as candidate antigen for vaccine development against pasteurellosis in livestock.

Expression and purification of recombinant type IV fimbrial subunit protein of Pasteurella multocida serogroup B:2 in Escherichia coli

Pasteurella multocida serogroup B:2, a causative agent of haemorrhagic secpticaemia (HS) in cattle and buffalo especially in tropical regions of Asia and African countries, is known to possess a type IV fimbriae (pili) as one of the virulent factors. In the present study, ptfA gene encoding for type IV fimbrial subunit of P. multocida serogroup B:2 (strain p52), an Indian HS vaccine strain, has been cloned and over-expressed in recombinant Escherichia coli. The recombinant type IV fimbrial subunit protein (∼31 kDa) including N-terminus histidine tag was purified under denaturing condition and confirmed by western blotting. A homology model of HS causing P. multocida serogroup B:2 fimbrial subunit has also been discussed. The study indicated the potential possibilities to use the recombinant fimbrial protein in developing HS subunit vaccine along with suitable adjuvant.

Comparative sequence analysis of poxvirus A32 gene encoded ATPase protein and carboxyl terminal heterogeneity of Indian orf viruses

Thirteen orf virus (ORFV) isolates from natural outbreaks in sheep and goats belonging to different geographical regions of India were analysed on the basis of ORF108 (a homologue of poxviral A32 gene), which is known to encode for ATPase and involved in virion DNA packaging. Comparative sequence analysis of ATPase proteins revealed highly conserved N-terminal region with five different motifs [Walker A, Walker B, A32L specific motifs (III and IV) and a novel AYDG (motif-V)] among all poxviruses and divergent carboxyl terminus with either single or double RGD sequences among all Indian ORFV isolates. A homology model and secondary structure predictions of N-terminal region of ORFV A32 revealed that most of the poxviruses including ORFV ATPase protein belong to a distinct clade of the HerA/FtsK super family of DNA packaging proteins. Despite differences in host cell specificity and poxvirus infections among animals, DNA packaging motor domain of poxviruses presumed to share remarkable similarities as indicated by the presence of conserved ATPase motifs in the present investigation. The study also indicated the circulation of heterogeneous strains of ORFV in India and possibilities of differentiation of ORFV strains based on C-terminal heterogeneity.

Cloning and characterization of type 4 fimbrial gene (ptfA) of Pasteurella multocida serogroup B:2 (strain P52).

Pasteurella multocida is known to affect a wide range of domestic as well as wild animal and avian species (Hunt et al., 2000). Among the diseases caused by P. multocida, haemorrahagic septicaemia (HS) caused by Pasteurella multocida B:2 is considered to be an economically important disease in India owing to the high mortality in infected cattle and buffaloes (Singh et al., 1996). Although several control strategies, including use of bacterins, modified live vaccines and subunit vaccines, have been tried to prevent the outbreak of the disease (Verma and Jaiswal, 1998), none of them has been fully effective. A better approach to control the disease could be the development of recombinant subunit vaccine that can target the virulence factors or antigens involved in the pathogenesis of the disease, which need to be studied in detail before any attempt to use them. One could be targeting of molecules involved in facilitating adhesion of the organism to the internal epithelium of the host, since there have been found to be important determinants of virulence for many pathogenic Gram-negative bacteria, allowing them to resist the fluid flow of luminal contents (Brumell and Finlay, 2000). In the past, different surface components of P. multocida—capsule (Finlay and Falkow, 1989; Moxon and Kroll, 1990), outer membrane protein A (Dabo et al., 2003), sialidases or neuraminidases (Mizhan et al., 2000) and type 4 fimbriae (Gloriosso et al., 1982)—have been incriminated in facilitating the adhesion of the organism to the upper respiratory tract mucosa. However, conclusive evidence justifying their role in pathogenesis is lacking. It has been presumed that the mechanisms of pathogenesis of different serogroups of P. multocida are different, as suggested by the fact that P. multocida serogroup A:1, causing fowl cholera, and serogroup D:1, causing atrophic rhinitis, are involved in localized infection, whereas serogroups B:2 and E:2 mainly cause septicaemic forms of the disease. These variabilities in virulence indicate that there exist marked differences in those components that affect the adhesion or colonization of different serogroups of P. multocida. Differences in the surface proteins are a direct reflection of the nucleotide sequence differences in the

Ribotyping of Indian isolates of Pasteurella multocida based on 16S and 23S rRNA genes.

The applicability of ribotyping based on 16S and 23S rRNA was evaluated for molecular epidemiological studies. Forty-eight isolates of Pasteurella multocida isolated from different hosts and geographical locations and one reference isolate were ribotyped. Only four ribotypes were found. All the isolates including reference isolate from wild carnivores had the same ribotype, though they had different serotypes. The isolate from a tiger had one band in addition to the bands present in the major ribotype. The isolates from lions represented two ribotypes; of these ribotypes, one (r2) had an additional band of 3.6 kbp, which was absent in all other ribotypes. The second ribotype (r4) from a lion had one band missing (6 kbp) that was present in the other ribotypes. These isolates were further typed using ERIC-PCR and REP-PCR. With ERIC-PCR and REP-PCR, higher D values of 0.83 and 0.89 were obtained. The current study revealed that ribotyping is not a very efficient typing tool for use in molecular epidemiology for differentiation of isolates.

Detection and characterization of atypical capripoxviruses among small ruminants in India

Recent developments in molecular biology shed light on cross-species transmission of SPPV and GTPV. The present study was planned to characterize the capripoxviruses which were circulating in the field condition among sheep and goats using RPO30 gene-based viral lineage (SPPV/GTPV) differentiating PCR and sequencing of RPO30 and GPCR genes from clinical samples. Out of 58 scabs (35 sheep and 23 goats) screened, 27 sheep and 18 goat scabs were found positive for capripox virus infections. With the exception of one sheep and one goat scabs, all the positive samples yielded amplicon size according to host origin, i.e. SPPV in sheep and GTPV in goats. In the above two exceptional cases, goat scab and sheep scab yielded amplicon size as that of SPPV and GTPV, respectively. Further, sequencing and phylogenetic analyses of complete ORFs of RPO30 and GPCR genes from six sheep and three goat scabs revealed that with the exception of above two samples, all had host-specific signatures and clustered according to their host origin. In case of cross-species infecting samples, sheep scab possessed GTPV-like signatures and goat scab possessed SPPV-like signatures. Our study identifies the circulation of cross-infecting SPPV and GTPV in the field and warrants the development of single-strain vaccine which can protect the animals from both sheeppox and goatpox diseases.