The study of aerobic bacterial flora of the upper respiratory tract of equines from Jammu and Kashmir region of India

Aim: To isolate aerobic bacterial micro flora residing in the upper respiratory tract of equines used by the pilgrims and tourists in Jammu & Kashmir. Materials and Methods: 88 apparently healthy equines and 53 equines with respiratory tract diseases were used in this study. Swab samples were collected from the upper respiratory tract of equines. Isolation and identification of the bacteria was conducted under aerobic conditions. Each of the sample processed yielded at least one type of bacteria species. Results: A total of 321 bacterial isolates were recovered from both groups of equines. The majority of the isolates were Gram positive (84.11%) and the rest were Gram negative (15.88%). Bacterial isolates identified in order of the magnitude were Streptococcus equi subsp. zooepidemicus (17.44%), Micrococcus spp. (9.96%), Corynebacterium (9.65%), Staphylococcus intermedius (9.65%), Staphylococcus aureus (8.72%), Bacillus spp. (7.16%), Streptococcus pneumonia (5.60%), Staphylococcus chromogens (5.60%), Streptococcus equismilis (5.29%), Pseudomonas aeruginosa (5.29%), Rhodococcus equi (3.73%), Escherichia coli (3.73%), Klebsiella pneumoniae (3.42%), Proteus vulgaris (3.42%), and Streptococcus equi subsp. equi (1.24%). Conclusion: The present study reveals the predominance of Gram positive bacteria in both healthy and diseased animals. Bacteria were recovered at a higher rate from diseased equines than from apparently healthy animals. Streptococcus equi subsp. zooepidemicus was mainly found to be associated with respiratory tract infections.


Introduction
bacteria are generally considered as secondary invaders of upper respiratory tract in equines.The most common India has about 20 million equids maintained opportunistic bacteria associated with respiratory infections under different farming systems and employed for include Streptococcus equi subsp.zooepidemicus, various purposes, out of which Jammu & Kashmir Actinobacillus equuli, Bordetella bronchiseptica, (J&K) has approximately 5.3 million equines [1].In Echerichia coli, Pasteurella spp.and Pseudomonas particular, equines in this region are used by Gujjars aeurogenosa [4].and Bakarwals to earn their livelihood primarily Therefore this study was designed to explore and through tourism.Due to their ability to survive on differentiate the bacterial flora of the diseased and lesser quantity of food and due to their ability to apparently healthy animals in the upper respiratory conserve more energy, equines can be used in harsh tract.Moreover, our study will yield highly relevant environments and for difficult working conditions [2] data regarding important zoonotic bacterial pathogens such as a visit to the important religious shrines in J&K; that can be contracted from equines.Furthermore, our Mata Vaishno Devi Shrine and Holy Amarnath Cave.
data will be helpful to determine and assess the Besides, equines are widely used by tourists for horse prevalence of bacterial species in upper respiratory riding and trekking.Equines are vulnerable to many tract of equines in the northern temperate environment diseases and among them respiratory problems are the of J&K, India.most severe health issues observed in working equines and have a high prevalence rate of 30% [3].So far most

of the research remained focused on viral rather than
Study area: The present study was carried out in areas bacterial causes of respiratory tract infections because of Jammu and Kashmir (J&K) in which equine rearing is highly prevalent.The study was conducted from September, 2010 to August, 2011.Samples were collected from five regions of J&K viz.R.S. Pura, Katra, Tangmarg, The study of aerobic bacterial flora of the upper respiratory tract of equines from Jammu and Kashmir region of India coccus intermedius and S. aureus which were isolated Samples: Samples were collected from both clinically as pure cultures in samples collected from nasopharynx, affected animals and apparently healthy animals.A the majority of cultures on plates revealed mixed total of 141 nasal swab samples were collected from infections with bacteria.A total of 321 isolates were five regions of J&K.The swab samples were collected obtained from the 141 nasal swabs, of which 270 from both nostrils after cleaning the anterior part of (84.11%) were Gram-positive and 51 (15.88%) were nostrils of equines using manually made 15 inch long Gram-negative.The isolation rate of Gram positive sterile swabs.bacteria was significantly higher (P < 0.01) than Gram Bacteriological isolation was done as soon as negative bacteria, a finding that is in good agreement possible.Brain heart infusion (BHI) broth was used for with previous report [6].The data regarding the the primary enrichment of bacterial isolates.The bacterial isolates recovered from nasopharynx of growth in BHI was streaked onto sheep blood agar with equines with respiratory disease and apparently healthy 5% (v/v) defibrinated sheep blood.All cultures were equines are shown in Table -2.incubated aerobically at 37°C for 48 hrs.Representative Major proportions of bacterial isolates recovered colonies from culture positive plates were subcultured from both apparently healthy and sick equines were on the 5% sheep blood agar for isolation of pure Gram-positive.In the present study, detailed investigacolonies.Colony morphology, colour, size, elevation, tion was carried out to explore the resident flora of status of haemolysis were recorded.Pure colonies were bacteria inhabiting the upper respiratory tract of transferred to blood agar slants for further biochemical apparently healthy equines and equines with upper tests.These culture isolates were subjected to Gram's respiratory tract diseases in order to determine the staining procedure, and tests for primary and secondary associated risks to the persons handling them, especially biochemical tests for identification of culture [5].
the owners, tourists and pilgrims.It was found that Characterisation of Streptococcal isolates was done equines in this part of India are a reservoir of some using Histrep identification kit (Himedia, Mumbai, zoonotically important bacterial pathogens like India) and Histrep Latex agglutination kit (Himedia, Rhodococcus equi and S. zooepidemicus and pose Mumbai, India) and the results were interpreted as per threat to tourists and handlers to contract the infection the manufacturer's instructions.
[7-8].In this study, the quality and quantity of bacterial

Results and Discussion
isolates from both apparently healthy equines and equines with respiratory diseases was compared.The All the samples (88 nasopharyngeal swabs from quality of bacterial isolates recovered was not much [10].This may be due to differences in the range of different except for S. equi subsp.equi which was animals used and different environmental factors in isolated only from diseased equines implying its their study.S. aureus has been isolated from respiratory pathogenic role in respiratory tract diseases in Jammu tracts of healthy and pneumonic cases of domestic and Kashmir.The recovery of the S. equi subsp.equi animals in many other cases as well [20,21].S. aureus was very low as compared to other reports [9] which is the main inhabitant of the mucous membranes in the could be because of that fact that we did employ any upper respiratory tract of animals.It can be involved as invasive methods for sampling of sites like guttural an opportunistic bacterium, following pathologic role pouch which is an ideal site for bacterial recovery from of stress conditions such as viral infections, and other chronically infected animals.Further the organism is causes of infection in immunosuppressed hosts [22].highly sensitive to desiccation and the mucocilliatory Coagulase negative Staphylococcus was the apparatus of horses is very efficient in removal of this commonly recovered bacterial isolate in this study.It pathogen from the site sampled in the study.Also included S. intermedius and S. chromogens.They were Staphylococcus chromogens was isolated only from isolated at a rate of 21.02% from apparently healthy healthy equines.Out of a total 321 bacterial isolates, equines, and 6.29% from equines with respiratory 270 (84.11%) were Gram-positive bacteria while the problems.The isolation rate of these bacteria was other 51 (15.88%) were Gram-negative bacteria which higher in apparently healthy equines as compared to is in accordance with the earlier reports [10].Of 194 the diseased.However, Boguta et al. [17] did not find bacterial isolates from apparently healthy equines, any variation between the two cases.But in study of majority of the bacteria (85.05%) were Gram positive Fernandes et al. [23], coagulase negative Staphylococcus while as rest (14.94%) were Gram-negative.
bacteria were isolated at a much higher rate of 36.6%The higher recovery rate of Gram-positive from upper respiratory tract samples.This could be due bacteria from apparently healthy equines has also been to a smaller sample size in their study.Bacillus spp. was reported by other workers [10,11].Out of 127 bacterial isolated at a rate of 9.79% from apparently healthy isolates from equines with respiratory diseases, 105 equines and 3.14% from equines with respiratory were Gram-positive while only 22 were Gram-problems.Several researchers have isolated Bacillus negative.Predominance of Gram-positive bacteria spp. at different rates of recovery from different species from diseased equines shows their significant role in of animals [24,25].Most of the Bacillus spp.are being opportunistic equine pathogens.However, the saprophytes that are widely distributed in air, soil and recovery rate in diseased equines (2.4%) was slightly water [18].The recovery rate of Bacillus spp. was greater than the rate for apparently healthy (2.2%) lower in diseased equines as compared to study of equines.These results are comparable to previous Sweeny et al. [25].This may be due to the improved findings of Kester et al. [11].The streptococcal isolates method of sample collection in our study.In some were found in both diseased and apparently healthy previous studies, sampling was done with artificially equines, 46.45% and 18.55% respectively.The available cotton swabs which were shorter in length predominant bacterial isolate found in diseased and could only reach anterior portions of the long equines was S. zooepidemicus (29.13%) which is in nostrils of equines which is exposed to open accordance with other reports [12,13].Recovery of this environment, therefore they isolated Bacillus at higher bacterium frequently without any other primary rate than what we found.Contrary to this, we used 15 infection suggests its primary involvement in inch long manually made swab.During our study, we respiratory tract diseases of equines [14] and that it is first cleaned the anterior portions of the nostril with an important upper respiratory tract pathogen causing 70% alcohol and then sampled the posterior portion of serious inflammatory reaction and mucous deposition the nostril. in the respiratory tract [15].However Wood et al. [16] Klebsiella pneumoniae was the dominant isolate have reported it as a secondary bacterial pathogen in among Gram-negative, with a recovery rate of 2.1% horses suffering from primary viral infection.
from apparently healthy equines and 12.7% from Corynebacterium spp. was encountered as a equines with respiratory problems.It was recovered second predominant bacterium among those recovered more frequently from diseased animals (30%) than from apparently healthy equines.They were isolated at apparently healthy ones (6%).K. pneumoniae was a rate of 13.91% from apparently healthy equines, and isolated in study of Savitha [21] at a rate of 5.51% from 3.14% from equines with respiratory tract diseases.
horses with pneumonia.Since K. pneumoniae inhabits These species were also recovered as common isolates the intestinal tracts of animals, fecal contamination of from upper respiratory tract infections in recently the environment may account for the wide distribution weaned foals [17].They are known to cause a variety of of the organism, and contributes to the occurrence of suppurative conditions in equines [18,19].S. aureus opportunistic infections [18].Escherichia coli was was isolated at a rate of 13.3% from apparently healthy isolated as a second dominant bacterium among Gramequines, and 13.3% from equines with respiratory negative bacteria, with the isolation rate of 4.12% from problems.This is lower as compared to Sweeny et al.
apparently healthy equines, and 3.14% from equines study [19] but comparable to study of Dessica et al. with respiratory problems.E. coli has been also isolated from the nasal tract of other animals [26,27].scientific discussion and manuscript writing, FNF R. equi was isolated at a rate of 2.57% from participated in scientific discussion, drafting and apparently healthy animals, and 5.51% from equines revision of manuscript, SAD helped in sampling, SG with respiratory problems.The bacterium is an helped in Lab work of bacterial isolation and opportunistic pathogen and common soil inhabitant identification, GAB helped in lab work, AAZ helped in [18].Severe equine pneumonia, usually found with sampling and AI participated in drafting and revision abscess formation in the lung tissue, is most often of manuscript.All authors read and approved the final associated with R. equi infection [28,29].R. equi is one form of the manuscript. of the most common isolates from equine respiratory

Table - 1
. Area wise details of sample collection