Knowledge, awareness and perception about equine glanders among veterinarians and medical professionals in India.

Glanders is a highly infectious and notifiable disease of equines that occurs due to Burkholderia mallei. In India, glanders re-emerged in 2006 and thereafter regular outbreaks have been reported in various states (n = 14). Frequent and prolonged contact with equids with glanders may transmit B. mallei infection to humans. This study was designed to learn more about the Knowledge, Awareness and Perception (KAP) of veterinarians, para veterinarians, and physicians about equine glanders, which will help in enhancing the nation-wide glanders eradication programme. A total of 165 respondent's from 11 Indian states and one union territory were surveyed. Most of the respondents (n = 160) were from equine glanders affected or endemic states. Knowledge gap analysis revealed that 40.3 and 22% of the participants were not aware of government regulations and the transmission of glanders, respectively. These are major concerns given the wide spread occurrence of disease in the country. Awareness test on glanders revealed that 65(39.4%) participants would collect biological samples for laboratory confirmation, 67(40.6%) would inform the concerned authorities and 106 (64.2%) replied that they would eliminate the glanders infected equines. Analysis of perception towards equine glanders showed that majority of the participants (n = 113, 68.4%) observed that equine keepers were reluctant to disclose the clinical symptoms of B. mallei infection. Furthermore, non-co-operation and unwillingness by superiors (33.9%), financial (31%), administrative (28.4%), and technical limitations (27.8%) were major constraints under the perception analysis. This study reveals that veterinarians need to be educated on governmental policies and guidelines on equine glanders with regular training and awareness programs. Intersectoral co-ordination to investigate human glanders is also needed.

Detection of Mycobacterium avium Subspecies paratuberculosis (MAP) Microorganisms Using Antigenic MAP Cell Envelope Proteins.

Cell envelope proteins from Mycobacterium avium subspecies paratuberculosis (MAP) that are antigenically distinct from closely related mycobacterial species are potentially useful for Johne's Disease (JD) diagnosis. We evaluated the potential of ELISAs, based on six antigenically distinct recombinant MAP cell envelope proteins (SdhA, FadE25_2, FadE3_2, Mkl, DesA2, and hypothetical protein MAP1233) as well as an extract of MAP total cell envelope proteins, to detect antibodies against MAP in the sera of infected cattle. The sensitivity (Se) and specificity (Sp) of an ELISA based on MAP total cell envelope proteins, when analyzing 153 bovine serum samples, was 75 and 96%, respectively. Analysis of the same samples, using a commercial serum ELISA resulted in a Se of 56% and Sp of 99%. Results of ELISA analysis using plates coated with recombinant cell envelope proteins ranged from a highest Se of 94% and a lowest Sp of 79% for Sdh A to a lowest Se of 67% and a highest Sp of 95% for hypothetical protein MAP1233. Using polyclonal antibodies to MAP total cell envelope proteins, immunohistochemical analysis of intestinal and lymph node tissues from JD-positive cattle detected MAP organisms whereas antibodies to recombinant proteins did not. Finally, polyclonal antibodies to MAP total cell envelope protein and to recombinant SdhA, FadE25_2, and DesA2 proteins immunomagnetically separated MAP microorganisms spiked in PBS. These results suggest that antigenically distinct MAP cell envelope proteins and antibodies to these proteins may have potential to detect MAP infection in dairy cattle.

An update on Mycobacterium avium subspecies paratuberculosis antigens and their role in the diagnosis of Johne's disease.

Mycobacterium avium subsp. paratuberculosis (MAP) is responsible for Johne's disease (JD) or paratuberculosis. Diagnosis of MAP infection by measuring host cell-mediated and humoral immune responses has been a major focus in MAP research. For this purpose, several MAP antigens such as secreted protein, cell envelope protein, cell-mediated immune and lipoprotein antigens have been identified and tested to measure their diagnostic utility with varying degree of success. Identifying the optimal antigen or antigen combinations for diagnosis of infected animals is hindered by the complex nature of the disease, prolonged subclinical infection, the differential expression of antigens and scarcity of well characterized MAP-specific epitopes making selection of a single MAP antigen very difficult. Thus, multiplexing of antigens with larger scale and longitudinal studies may lead to development of cost-effective next generation serodiagnostics. This mini review focuses on the role of different MAP antigens in the diagnosis of JD.

Identification of antigenic proteins from Mycobacterium avium subspecies paratuberculosis cell envelope by comparative proteomic analysis.

Johne's disease (JD) is a contagious, chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The aim of this study was to identify antigenic proteins from the MAP cell envelope (i.e. cell wall and cytoplasmic membranes) by comparing MAP, M. avium subsp. hominissuis (MAH) and M. smegmatis (MS) cell envelope protein profiles using a proteomic approach. Composite two-dimensional (2D) difference gel electrophoresis images revealed 13 spots present only in the image of the MAP cell envelope proteins. Using serum from MAP-infected cattle, immunoblot analysis of 2D gels revealed that proteins in the 13 spots were antigenic. These proteins were identified by liquid chromatography tandem mass spectrometry as products of the following genes: sdhA, fadE25_2, mkl, citA, gapdh, fadE3_2, moxR1, mmp, purC, mdh, atpG, fbpB and desA2 as well as two proteins without gene names identified as transcriptional regulator (MAP0035) protein and hypothetical protein (MAP1233). Protein functions ranged from energy generation, cell wall biosynthesis, protein maturation, bacterial replication and invasion of epithelial cells, functions considered essential to MAP virulence and intracellular survival. Five MAP cell envelope proteins, i.e. SdhA, FadE25_2, FadE3_2, MAP0035 and DesA2 were recombinantly expressed, three of which, i.e. SdhA, FadE25_2 and DesA2, were of sufficient purity and yield to generate polyclonal antibodies. Immunoblot analysis revealed antibodies reacted specifically to the respective MAP cell envelope proteins with minimal cross-reactivity with MAH and MS cell envelope proteins. Identification and characterization of MAP-specific proteins and antibodies to those proteins may be useful in developing new diagnostic tests for JD diagnosis.

Descriptive epidemiology of equine influenza in India (2008-2009): temporal and spatial trends.

Equine influenza is a contagious viral disease that affects all members of the family Equidae, i.e., horses, donkeys and mules. The authors describe the pattern of equine influenza outbreaks in a number of states of India from July 2008 to June 2009. The disease was first reported in June 2008 in Katra (Jammu and Kashmir) and spread to ten other states within a year. All outbreaks of equine influenza in the various states were confirmed by laboratory investigations (virus isolation and/or serological confirmation based on haemagglutination inhibition [HI] assays of paired samples) before declaring them as equine influenza virus-affected state(s). The virus (H3N8) was reported from various locations in the country including Katra, Mysore (Karnataka), Ahmedabad (Gujarat), Gopeshwar and Uttarkashi (Uttarakhand) and was isolated in 9- to 11-day-old embryonated chicken eggs. The virus was confirmed as H3N8 by HI assays with standard serum and amplification of full-length haemagglutinin and neuraminidase genes by reverse transcriptase-polymerase chain reaction. Serum samples (n = 4 740) of equines from 13 states in India screened by HI revealed 1074 (22.65%) samples as being positive for antibodies to equine influenza virus (H3N8).