Nem Singh

Cytokine profiles, apoptosis and pathology of experimental Pasteurella multocida serotype A1 infection in mice

Mice were experimentally infected with Pasteurella multocida serotype A1 to study the cytokine profiles, host cell apoptosis and sequential pathology at different hours of post-infection. Infected mice were dull, anorectic and depressed. A transient leukocytopenia followed by progressive leukocytosis was observed in the course of infection. Serum cytokine profiles showed significantly (P<0.01) higher amount of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and mouse KC) in the infected mice when compared to control mice. The circulating lymphocytes were apoptotic on annexin V staining. Apoptotic nuclei were detected in splenocytes, hepatocytes and infiltrating leukocytes of the lungs on TUNEL staining. The lungs were grossly congested and hemorrhagic, and showed infiltration with polymorphonuclear cells at early and mononuclear cells in the late hours of infection. Alveolar epithelia, inter-alveolar septa and capillary endothelium of the lungs showed ultrastructural changes. Liver had degenerative changes in histological and ultrathin sections.

Pathology of naturally occurring paratuberculosis in water buffaloes (Bubalus bubalis).

Gross and histologic lesions of paratuberculosis were studied in water buffaloes. Small intestines and associated mesenteric lymph nodes of 405 water buffaloes were examined. Of these, 20 animals having visible changes of intestinal thickening, mucosal corrugations, and enlargement of mesenteric lymph nodes exhibited histologic alteration characteristics of mild to moderate granulomatous inflammation. The histologic lesions observed in these animals were classified into 3 grades on the basis of type of cellular infiltration, granuloma formation, and presence of acid-fast bacilli. Grade-1 lesions observed in 8 animals were marked by the presence of scattered epithelioid macrophages amid large number of lymphocytes in the intestinal villi and in the paracortical regions of the associated mesenteric lymph nodes. Another 8 animals classified under grade-2 revealed microgranulomas, infiltration with a larger number of epithelioid macrophages besides lymphocytes in the intestinal villi, and granulomas in the mesenteric lymph nodes. Grade-3 lesions observed in 4 animals were characterized by the presence of epithelioid granulomas and giant cells in the intestines and the mesenteric lymph nodes. The Ziehl-Neelsens stained tissue sections revealed acid-fast bacilli in grade-3 and −2 animals and acid-fast granular debris in grade-1 animals. Among these 20 buffaloes, 14 (70%) were positive in the IS900 specific polymerase chain reaction and 6 (30%) were positive in the bacterial culture.

Comparative study of experimental Foot-and-Mouth Disease in cattle ( Bos indicus ) and buffaloes ( Bubalis bubalus )

Foot-and-mouth disease (FMD) is one of the most contagious diseases affecting wide range of host species with variable severity and decreased productivity. The present study was undertaken to compare the clinical and leucocytic changes in indigenous Indian cattle and buffaloes experimentally infected with FMD virus (FMDV) Asia 1. A mild type of disease was observed in the cattle, more so in buffaloes infected with FMDV. Difference in terms of type, site and healing of lesion was observed between cattle and buffaloes. Foot lesions were more common than tongue in buffaloes, which were mainly evident in bulb of the heel in contrast to interdigital foot lesions in cattle. Further, FMDV infection induced a transient moderate leucopenia with lymphopenia in both cattle and buffaloes, but monocyte levels diverged. Relationship between the raised body temperature, leucocytic changes and lesion development was observed. Microscopic changes were observed in the keratinized epithelium of tongue and foot. The findings of the present study indicated the need to investigate the early leucocytic changes in cattle and buffaloes in depth for better understanding of the disease process.

Mechanisms of Mycobacterium avium subsp. paratuberculosis induced apoptosis and necrosis in bovine macrophages

The interaction between Mycobacterium avium subsp. paratuberculosis (Map) and macrophages is a complex process to maximize the chances of their respective survival. Previous studies have shown that Map induces cell death in macrophages, but the mechanism is not known. In the present study, we investigated the mechanism by which Map induces cell death in bovine macrophages using the fluorescent and electron microscopic techniques. The macrophages infected with an equal number of Map (i.e., multiplicity of infection, MOI=1) showed no changes of cell death, but those macrophages infected at MOI=10 showed the morphological changes consistent with apoptosis. Strikingly, the macrophages infected by Map at MOI=50 showed the changes of apoptosis and necrosis. The Map-induced apoptosis was a caspase-dependent mechanism at MOI=10 while it was caspase- and nitric oxide-independent at MOI=50. The results of the present study suggest that the mitochondrial damage following Map infection initiates the cell death processes in macrophages.

Immune response and viral persistence in Indian buffaloes (Bubalus bubalis) infected with foot-and-mouth disease virus serotype Asia 1.

ABSTRACT Despite their potential role in the spread of foot-and-mouth disease (FMD), the immune response and viral persistence in FMD virus (FMDV)-infected Indian buffaloes (Bubalus bubalis) have been unexplored. We found similar kinetics of neutralizing antibody responses in the sera and secretory fluids of buffaloes following experimental FMDV Asia 1 infection, but the lymphocyte-proliferative response in infected buffaloes was of low magnitude. Despite inducing a significant systemic and secretory immune response, viral persistence seems to be a common outcome in buffaloes following FMDV Asia 1 infection, which is associated with a weak cellular immune response.

Enhanced mucosal immune response in cattle persistently infected with foot-and-mouth disease virus

The mucosal immune response to foot-and-mouth disease virus (FMDV) type Asia 1 was examined in experimentally infected cattle by assaying antibodies by the virus-neutralizing test (VNT) and IgA ELISA in two secretory fluids, oesophageal pharyngeal fluid (OPF) and oro-nasal fluid (ONF). Out of 17 animals infected by the intradermo-lingual route, 12 became persistently infected (carriers), as defined by positive antigen capture RT-PCR reactions for FMDV RNA in OPF samples collected at 28 days or later after exposure. This proportion of carriers (71%) with FMDV Asia 1 is comparable to other serotypes of the virus. When the two groups were examined, the carriers and non-carriers showed no difference in the serum antibody titre until the end of the experiment at 182 days post-infection (DPI). However, despite an initial similarity significantly higher neutralizing antibody titres and FMDV-specific IgA response were detected among the carriers than the non-carriers in both of the secretory fluids. The response was higher and more stable in ONF compared to OPF. Thus, mucosal antibody assays have the potential to be used as a means of differentiating carrier from non-carrier cattle. Furthermore, the findings are consistent with the higher mucosal antibody response in carriers being an effect of persistent infection rather than the cause.

Heterologous Expression of a Gene Encoding a 35 kDa Protein of Mycobacterium avium paratuberculosis in Escherichia coli

The full-length open reading frame coding for a potentially immunogenic 35 kDa protein of Mycobacterium avium paratuberculosis was generated using polymerase chain reaction technology. The gene was inserted in-frame into Escherichia coli expression plasmid pQE32. The resulting recombinant plasmid pPMP35 was transformed into E. coli M15. Analysis of the E. coli induced with isopropyl-β-D-thiogalactopyranoside revealed that the protein accumulated into the cytoplasm as insoluble inclusion bodies. The level of expression of the recombinant 35 kDa protein (P35) was more than 30% of the total protein of E. coli cells. Expression of the recombinant protein was confirmed by immunoblotting. The P35 reacted with a rabbit antiserum raised against a sonicate of M. a. paratuberculosis. The protein was also recognized by serum from a goat with clinical paratuberculosis. Further, a polyclonal antiserum against P35 recognized a 35 kDa band in a membrane fraction of M. a. paratuberculosis. Also, the protein provoked a significant skin reaction in outbred guinea pigs sensitized with M. a. paratuberculosis, as well as in those sensitized with Mycobacterium avium. The results indicate that the 35 kDa protein of M. a. paratuberculosis is a membrane protein, having a role in the cellular immune response.

Pathology of Experimental Infection by Pasteurella multocida Serotype A:1 in Buffalo Calves

Pasteurella multocida serotype A:3 has been mostly implicated in pneumonic pasteurellosis in ruminants. In contrast, our previous studies have reported that both serotypes A:1 and A:3 were responsible for respiratory diseases in cattle and buffaloes. However, the pathology and pathogenesis of P. multocida serotype A:1 (Pm A:1) infection have not been studied in ruminants. In the present study, 12- to 15-week-old buffalo calves (Bubalus bubalis) infected by Pm A:1 had fibrinous and suppurative bronchopneumonia with focal areas of coagulation necrosis typical of pneumonic pasteurellosis. For the first time, this study reports the lung pathology and pathogenecity of Pm A:1 infection in calves.

Experimental evidence for competitive growth advantage of genotype VII over VI: implications for foot-and-mouth disease virus serotype A genotype turnover in nature.

In India, systematic genotype replacement has been observed for serotype A foot-and-mouth disease virus. After a decade of co-circulation of genotypes VI and VII, genotype VII emerged as the single dominant genotype since 2001. To derive possible explanations for such epochal evolution dynamics, in vitro intergenotype growth competition experiments involving both co- and superinfection regimes were conducted. Coinfection of BHK-21 cells demonstrated abrupt loss in the genotype VI viral load with commensurate increase in the load of genotype VII as measured by the genotype differentiating ELISA, RT-PCR and real-time RT-PCR. The superinfection dynamics was shaped by temporal spacing of infection, where the invading genotype VII took more number of passages than coinfection to eventually overtake the resident genotype VI. It was speculated that such superior replicative fitness of genotype VII could have been a possible factor for the ultimate dominance of genotype VII in nature.

The relationship between cellular immune response to foot-and-mouth disease virus Asia 1 and viral persistence in Indian cattle (Bos indicus)

Foot-and-mouth disease (FMD), the most contagious animal disease, is associated with persistent viral infection in ruminants, despite the induction of systemic immune response. The present study was performed to decipher the relation between the persistent FMD virus (FMDV) infection and cellular immune response in Indian cattle (Bosindicus) following experimental inoculation of FMDV Asia 1. Persistent viral infection (carriers) was detected by antigen capture RT-PCR on the oesophageal-pharyngeal fluid. Viral excretion was found to be intermittent and strongly variable among the persistently infected Indian cattle. Lymphocyte proliferative (LP) response, assessed as reactivity of peripheral blood mononuclear cells to FMDV Asia 1 antigen (Ag) was of low magnitude indicating a weak primary cellular immune response following infection. LP response to FMDV Ag was higher among the non-carriers than carriers of FMDV Asia 1. An enhanced LP response was associated with the lack of virus shedding in the OPF. The findings of this study are suggestive of relationship between cellular immune response and virus excretion during persistence of FMDV Asia 1 in infected cattle.