Molecular detection of virulence genes associated with pathogenicity of Gram positive isolates obtained from respiratory tract of apparently healthy as well as sick goats

Aim: To know the prevalence of gram positive isolates and their virulence genes obtained from respiratory tract of apparently healthy as well as sick goats. Material and Methods: Nasal swabs and tissue samples were screened for the presence of microbial pathogens by cultural isolation, biochemical confirmation. Further, these isolates were subjected to the virulence gene detection by PCR. Results: Out of 144 isolates, 57 isolates of gram positive pathogens were obtained and confirmed as Staphylococcus spp. (43 isolates) and Streptococcus spp. (14 isolates) on the basis of Gram staining, morphology, cultural identification and biochemical characters. Five isolates (11.6%) were found to be positive for Coagulase gene; 11 isolates (25.6%) were found to be positive for clfA gene; and 14 isolates (32.6%) were found to be positive for Spa gene. Conclusion: The presence of these genes confirmed the pathogenic potential of gram positive pathogens and their association with clinical manifestations in respiratory tract infections of goats.


Introduction
range and are associated with various clinical manifestations in small ruminants, viz., pneumonia, Staphylococci and Streptococci are group of mastitis and pyogenic infections.bacteria that cause a wide spectrum of clinical clfA gene codes for clumping factor, helps in manifestations, such as pneumonia, wound infections, infection process by facilitating bacterial binding via septicemia, endocarditis, mastitis and metritis in solubilised or immobilized fibrinogen.Because animals.It is responsible for both nosocomial and fibrinogen plays significant role in platelet thrombus community-acquired infections [1].These species formation, it is likely that clfA may be involved in occur as commensals on skin and mucous membranes; bacterial platelet interactions.Therefore, it is some may act as opportunistic pathogens causing implicated as virulence factor [3]. spa gene codes for pyogenic infections.They are gram positive cocci (1 the IgG binding region of the protein A and is well µm) that tend to occur in irregular clusters resembling known for the binding ability for its immunoglobulin bunches of grapes.They are facultative anaerobes, Fc region.spa has affinity with solubilised or fermentative and catalase positive.They are nonimmobilized von Willebrand factor (vWF) and motile, oxidase-negative and do not form spores. identified as a novel Staphylococcal adhesin [4].coa Streptococcus species having similar characteristics gene codes for enzyme coagulase and coagulase as Staphylococcus species but are catalase negative production is important phenotypic determinant of S. and found in chain of different lengths; acts as aureus which is associated with virulence [5].In commensals on the mucosae of the upper respiratory addition to these, it produces several other tract and lower urogenital tract [2].Many species are extracellular virulence factors which affect the host saprophytic including those of veterinary importance.cell metabolism.These genes are associated with the These both are major pathogens having wide host pathogens: Primarily the isolates were characterized protein and is thought to play a role in epithelial cell by KOH, Catalase, Oxidase and O-F tests [2].adherence and invasion, and resistance to Following primary biochemical tests, the isolates were phagocytosis [6].These genes are associated with the characterized by various secondary biochemical tests virulence of Streptococcus spp.
[7].Secondary biochemical tests include Coagulase In the present study, the animals were screened Test, t-DNAse production, Voges-Proskeur (VP) test, for the respiratory tract infections caused by CAMP reaction, Lancefield grouping and Carbohydrate Staphylococcus spp.and Streptococcus spp.by fermentation test.cultural and molecular techniques.Along with their primary and secondary biochemical characteristics, DNA Extraction: The template DNA from colony the isolates were also examined for the presence of was prepared with minor modifications [8].Briefly, various virulence associated genes by PCR to study from the culture plate, bacterial colonies were picked their possible role in pathogenicity.
and suspended in 100 µl milli-Q water.The samples were boiled for 15 min, cell debris was removed by

Materials and Methods
centrifugation at 5000 rpm and the supernatant was Sample collection: During the present study, a total collected and used as a template DNA. of 102 nasal swabs were collected from apparently Polymerase chain reaction (PCR) for amplification healthy and sick goats from organized farms and 96 of virulence associated genes: PCR was carried tissue samples were collected from post-mortem out with template DNA (3 µl), forward and reverse animals and animals slaughtered in the local meat primers (1 µl), 12.5 µl of master mix (2x) and 7.5 µl market and transported to the laboratory on ice.All the DNAse Free Water in a total volume of 25 µl.The samples were processed and screened for possible DNA was amplified using the specific cycling microbial pathogens.conditions (Table 2).PCR products were separated in Cultural isolation of the organisms: Bacterial 1.5% agarose gel for 90 min at 80 V, stained with isolation was done following standard technique by ethidium bromide (1%) [added @ 0.5 µl/100ml] and inoculating tissue samples and nasal swabs primarily detected by UV transillumination (wavelength 312 on blood agar and plates were incubated for 24-48 hrs nm) [9].Amplified genes were identified on the basis o of fragment size shown in Table 1 and the cyclic at 37 C.After incubation, the nature of growth and cultural conditions for PCR are explained in table 2. characters of colonies were studied.Preliminary morphological identification was done based on

Results and Discussion
Gram's staining.Cultural characteristics of the isolates After morphological, cultural and primary were further studied on Potassium Tellurite Agar,   [13,14,15].Lower prevalence of this species production and 5 isolates were positive for Coagulase was also detected in previous studies which were from production.In the present study, the prevalence of pneumonic lung of goats [16,17].Low prevalence of  Staphylococcus spp. was also detected from different revealed an amplified 650 bp product and 2 isolates anatomical sites of respiratory tract of goats [18].revealed an amplified 600 bp product.All the Based on morphological, cultural and coagulase positive isolates of Staphylococcus spp.by preliminary biochemical identification 14 isolates in vitro test were also found positive for coagulase were confirmed as Streptococcus spp.All the isolates gene by PCR detection.The results of the present were subjected to cultural and secondary biochemical study were in agreement with reports of previous tests for specific identification.Out of 14 isolates, 4 studies who designed these oligos complementary to isolates produced partial hemolysis, while others each end of the sequence of the clfA and spa genes failed to produce hemolysis on sheep blood agar.These which specifically amplified the product sizes of 980 isolates were confirmed as Group-D Streptococci, on the bp and 920 bp, respectively [3,4].PCR of coa genes basis of Lancefield grouping by using Latex was also carried out in the present study using specific Agglutination Test Kit (HiMedia).All the isolates primers resulted in polymorphism (850 bp, 650 bp and revealed negative CAMP reaction.These isolates 600 bp).The findings were in contrast with the reports utilized inulin, trehalose, lactose and glucose, except of previous study who used these oligos complesorbitol.In the present study, the prevalence of mentary to each end of the sequence of the coa gene Streptococcus spp. was found 9.7%.The reports of the which showed polymorphism and specifically present study are in agreement with reports of previous amplified the product sizes of 875, 660, 603, or 547 bp study [10,19].Low prevalence of Streptococcus spp.[5].These virulence factors might contribute for were detected in the previous studies [15,20].mixed respiratory infections in goats.The findings The PCR assay was standardized for detection of were also in contrast with the previous reports in individual virulence associated genes of Staphylococcus which 86 positive samples of Staphylococcus spp., spp.namely clfA gene (codes for clumping factor), spa were collected, out of that 42 specimens (48.8%) gene (codes for the IgG binding region of the protein contained the coa gene, 63 specimens (73.3%)A) and coa gene (codes for enzyme coagulase) using contained the clfA gene and 22 specimens (25.5%) specific primer sequences which gave product sizes of contained spa gene (IgG Binding region) [21].The 980 bp, 920 bp and polymorphism (850 bp, 650 bp and findings were also in contrast with the previous studies 600 bp), respectively.Five isolates (11.6%) were were reported that all the coagulase positive isolates found positive for coagulase gene (Fig. 2); 11 isolates out of collected 92 samples of Staphylococcus aureus, (25.6%) were found positive for clfA gene (Fig. 3); and were found to be harbour coa-gene and IgG binding 14 isolates (32.6%) were found positive for Spa gene region (spa-IgG) whereas, 84 (91.3%) isolates were (Fig. 4).Out of 5 Coagulase positive isolates, 1 isolate positive for clfA gene [22].The reports of present revealed an amplified 850 bp product, 2 isolates study were in contrast with previous study [23].The PCR assay was standardized for detection of

Figure- 3 .
Figure-3.Agarose gel showing amplification product of clfA gene for Streptococcus spp.(approximately 980bp) Lane 3,4,8: Positive field isolates Lane 1,2,5,7: Negative field isolates M: 100 bp-1 Kbp DNA ladder the findings, we need to further investigate the role of 9. Relman, D. A. and Persing, D. H. (1996).Genotypic other factors related to host and pathogen which assist methods for microbial identification, PCR protocols in the progression of disease at physiological and for emerging infectious diseases: A supplement to molecular levels.Diagnostic Molecular Microbiology: Principles and Author's Contribution Applications.ASM Press, Washington, D.C. 3-31.10.Ramchandran, S. and Sharma, G. L. (1969).The work was carried out by TKA under the guidance Observations on the incidence and histopathology of of AR with the technical support of PK.PK revised the pneumonia of sheep and goats in India.Ind. Vet.J. 46: manuscript.All authors read and approved the final 16-29.manuscript.11.Asseged, B., Megra, T. and Sisay, T. (2006).The aerobic bacterial flora of the respiratory passageways

To cite this article:
Aher TK, Roy A, Kumar P (2012) Molecular detection of virulence genes associated with pathogenicity of Gram positive isolates obtained from respiratory tract of apparently healthy as well as sick goats, Vet World, 5(11): 676-681, doi: 10.5455/vetworld.2012.676-681virulence of S. aureus.Nutrient agar and Mannitol salt agar (HiMedia).rib gene codes for surface Rib protein and it has Biochemical identification of Gram positiverole in putative virulence.bca gene codes for alpha-C

Table 1 .
Primers used for amplification of virulence genes of Staphylococcus spp., codes for coagulase; clfA, codes for clumping factor; spa, codes for the IgG binding region of the protein A. coa

Table 2 .
Primers used for amplification of virulence genes of Streptococcus spp.
bca, codes for alpha-C protein; rib, codes for surface Rib protein.biochemical identification, 43 isolates of Staphylococcus Staphylococcus was found to be 29.9%.The were confirmed.Out of theses 43 isolates, 15 isolates prevalence rate of the present study is in agreement showed yellow discoloration on Mannitol salt agar, 25 with reports of different authors [10,11].Higher isolates showing partial hemolysis on Blood agar, 13 prevalence of Staphylococcus spp. was detected by isolates showed golden yellow pigmentation on one author as 73.7% from pneumonic lung of goats Nutrient agar and all isolates produced black colonies [12].Low prevalence of Staphylococcus spp. was on Potassium tellurite agar.All Staphylococcus detected in the previous study from pneumonic lung of isolates were found to be negative for t-DNAse sheep

Table - 3
. Cycling condition for PCR of virulence genes of Staphylococcus spp.

Table 4 .
Cycling condition for PCR of virulence genes of Streptococcus spp.