A.A. Kumar

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.

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.

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.

Antibiotic Sensitivity Patterns among Indian Strains of Avian Pasteurella multocida

An investigation was carried out to study the antibiotic sensitivity of avian strains ofPasteurella multocida and to select an effective antimicrobial agent for control of avian pasteurellosis in India. A total of 123 strains ofP. multocida recently isolated from different avian species (chicken, duck, turkey, quail, and goose), from different regions of India were subjected to antibiotic sensitivity tests using 20 different antibiotics. Absolute resistance was observed against sulfadiazine. The studies indicated that the strains were most sensitive to chloramphenicol (73.98%), followed by enrofloxacin (71.54%), lincomycin (64.23%), norfloxacin (61.79%) and doxycycline-HCl (56.91%). The majority of the strains were found to exhibit intermediate sensitivity. Chloramphenicol was selected and suggested for treatment. Antibiogram studies also revealed the emergence of multidrug-resistant strains ofP. multocida among Indian poultry.

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.

REP-PCR analysis of Pasteurella multocida isolates from wild and domestic animals in India.

Repetitive extragenic palindromic sequence-based PCR (REP-PCR) was used to characterize 67 field isolates of Pasteurella multocida originating from different animal species and geographical regions of India. REP-PCR was found to be rapid and reproducible (three repeats were done). These isolates yielded different 23 profiles which were clustered into eight groups. The discrimination index was moderate (D value 0.83). Somatic and antigenic typing of the isolates did not reveal any correlation with REP-PCR profiles. There was no host-specific, type-specific, region-specific or pathenogenicity-specific pattern. The REP profiles of isolates obtained from wild animals were similar to those obtained from domestic animals. Two common bands were present in all the isolates irrespective of somatic or antigenic types. The results were not comparable with earlier findings, which had shown high discrimination index and correlation with disease presentation.

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.

Differentiation of avian Pasteurella multocida strains by single-primer PCR.

Pasteurella multocida, a Gram-negative coccobacillus and the causative agent of fowl cholera, is known to affect a wide range of domestic and wild birds, causing high mortality. The disease may result from strains of P. multocida belonging to several capsular (A, B, D, E and F) and somatic serotypes (1–16) (Rimler and Glisson, 1997). Generally, diagnosis of the disease in natural outbreaks largely depends on conventional methodologies comprising bacterial isolation and identification by serotyping and biochemical characterization, which reveal the presence of variable serogroups/types in different geographical regions (Rhoades and Rimler, 1987). However, it has been observed that conventional characterization is not sensitive enough to identify and differentiate each strain involved in natural outbreaks (Snipes et al., 1990; Wilson et al., 1993; Biswas et al., 2004; Shivachandra et al., 2005). Alternatively, DNA-based methods have been applied more recently for rapid identification and differentiation of avian strains of P. multocida originating from different regions (Blackall and Miflin, 2000; Townsend et al., 2001). Although, a number of typing techniques are currently available with varying stringencies and discriminatory powers, a simple and rapid method would greatly enhance the preliminary differentiation of strains (Olive and Bean, 1999). One such methods is randomly amplified polymorphic DNA-PCR (RAPDPCR). Single-primer PCR or RAPD-PCR analysis uses oligonucleotide primers that amplify certain sections of the genome by PCR to produce identifiable banding patterns that are useful in strain differentiation (Blackall and Miflin, 2000). RAPD-PCR assay for P. multocida was developed to provide a fast and resource-efficient method of classifying individual field strains with high specificity (Welsh and McClelland, 1990; Williams et al., 1990; Chaslus-Dancla et al., 1996; Huber et al., 2002).

Endocrine dysfunction in chronic severe ehrlichiosis with or without babesiosis in dogs.

Emaciation and general weakness are common clinical signs in many chronic diseases, probably owing to interplay between anorexia, increased energy demands of an ailing animal and the changing endocrine environment (Ziegler et al., 1994). The adrenal glands, the thyroid, and the pancreas are of prime importance in energy metabolism and in the adaptation of mammals to their environment. Canine monocytic ehrlichiosis caused by Ehrlichia canis is a tick-borne rickettisial disease, manifested by a variety of clinical signs and mortality in the chronic severe form. Deficiencies in the adrenal and thyroid glands have opposing effects of catabolism and anabolism, respectively. It seems that the progressive wasting in ehrlichiosis may involve a multiplicity of factors. Ehrlichiosis (with or without babesiosis), being a catabolic disease, may be responsible for disturbed thyroid metabolism and altered secretion of the endocrine pancreas and adrenals. In recent years, endocrine disruption has been reported in clinical trypanosomosis (Varshney and Varshney, 1999) and babesiosis in dogs (Varshney et al., 2003). However, no literature reports are available on the endocrine status of dogs suffering from chronic severe ehrlichiosis with or without babesiosis. The present study was carried out to determine whether thyroid functions, cortisol secretion from the adrenals or insulin secretion from the pancreas are affected during chronic severe canine ehrlichiosis with or without babesiosis.