Out of 165 samples processed, 88 (53.33%) Staphylococci isolates were obtained using conventional method. The sample details and percent positivity is as per Table 1. Predominance of
Staphylococcus in such types of canine affection dogs was in consonance with findings from west Bengal
(Dey et al., 2016) and Jammu
(Vanni et al., 2009), where 57.6% and 62.58% of cases were attributed to this bacterium, respectively. However, higher involvement has also been reported as 86.5
(Katarzyna et al., 2013), 86.5
(Ruzauskas et al., 2015), 94
(Melter et al., 2017) per cent cases, respectively. Whereas other reports
(Tarazi et al., 2015; Reddy et al., 2016; Habibullah et al., 2017 and
Yadav et al., 2018) stated lesser involvement of bacteria, principally because these studies were targeted to
S. aureus only.
Skin is considered primary site of
Staphylococcus colonization, so, maximum cases were reported either as pyoderma or abscess/ wound. Even higher involvement than to present findings have been reported, in Jammu (68.55%) and Chennai (59 .0%) by
(Beigh et al., (2013) and
Chitra et al., (2018), respectively. The lower incidence can be attributed to hot and dry environment in this region which is less favourable for growth of organisms. Though, a lower isolation rate (16%) was observed from healthy and diseased dogs in south China
(Feng et al., 2012). A similar incidence of otitis infection
(Penna et al., 2009; Hamed and Shahram, 2012) and nasal infection
(Katarzyna et al., 2013) were reported and substantiate findings of present work. Likewise, 58.8% incidence of ocular disease in dogs were attributed to same pathogen by
Varges et al., (2009). These infections are likely to get its origin from bacterial population of skin and externally communicating mucous membranes.
Though the species of Staphylococcus involved in canine skin and associated membrane affection can be differentiated on biochemical testing but now- a- days PCR becomes method of choice (
Sasaki et al., 2010;
Tarazi et al., 2015). A simultaneous attempt was made to identify these isolates with PCR, using 16s
rDNA based primers. All these 88 tentatively positive isolates yielded 228 bp fragment which confirmed them as
Staphylococcus spp
. Further, 46/88(52.27%) isolates were confirmed as
S. aureus through PCR using
sa442 gene primer.
Though,
Kusumaningruma et al., (2016) observed the 16S rDNA primer as
Staphylococcus aureus specific, however, in our study,
Staphylococcus spp. other than
Staphylococcus aureus, also showed positive result with this primer. So, 16S rDNA primer and 442 bp primer should be employed consecutively for genus and species level identification.
(Martineau et al., 1998).
During the characterization, 51/88 (57.95%) isolates were found positive for coagulase production and designated as Coagulase positive
Staphylococcus spp., whereas remaining 37 were considered as Coagulase negative Staphylococcus (CoNS). Surprisingly, five isolates which were detected positive in tube coagulase test, were negative in PCR based on sa442 gene. These were most likely to be
S. pseudintermedius, which showed very similar biochemical profile to
S. aureus and widely involved in canine affection
(Markey et al., 2013). As in phenotypic coagulase test, 51 isolates were found positive and only 46 identified as
Staphylococcus aureus. Previously also, a similar rate of 51.4% and 56.25% of coagulase positive staphylococcus have been reported by
Penna et al., (2009) and
Dilek et al., (2010), respectively. However, in contrast to the present finding, a higher
i.e., 72%
(Mubita et al., 1995) and 87.2%
(Bean et al., 2016) and lower isolation rate of 28.07% (Silva, 2001), 35.4%
(Ghosh et al., 2003) and 20.5% (Arzu
et al., 2009) of coagulase positive
Staphylococcus have also been reported, respectively.
Therefore, these isolates were further screened by
coa gene specific PCR. Out of 46 isolates of
S. aureus, 42 (91.30%) isolates were found positive for
coa gene, in which single amplicons of variable size were detected. Amplicon size of 600, 680, 710 and 850 bp were detected among 11, 9, 12 and 10 isolates, respectively. Only one isolate which was confirmed as
S. aureus did not reveal any band indicating negative for
coa gene, two isolates detected negative in tube coagulase test were found positive in
coa gene based PCR.
Combining the genotypic and phenotypic results, out of 46 PCR positive isolates of
Staphylococcus aureus, 42 and 04 isolates were confirmed as coagulase positive and negative, respectively by
coa gene base PCR. On the other hand, out of five tube coagulase positive but Sa442 gene negative isolates, 4 isolates were found negative for
coa gene PCR. This way out, 51
Staphylococcus isolates positive for tube coagulase test, 09 detected negative in
coa gene PCR and 04 among them were proven
coa gene negative
Staphylococcus aureus (CoNS) and other 05 might belong to other species. Moreover, during the study, two isolates detected negative in tube coagulase test were found positive in
coa gene based PCR, which corroborated with earlier reports of
Gharib et al., (2013) and
Parth et al., (2016). Thus, the amplification of all the isolates by PCR was found to be an accurate and superior technique.
In earlier studies
(Arzu et al., 2009), for coagulase gene polymorphism by PCR, isolates produced one or more fragment sizes of PCR products
viz., 470, 810, 490, 521, 570, 326, 437, 697, 710 and 1500, respectively. Number of repeats were similar with
Khichar et al., (2012) who employed similar primer for bovine mastitic
Staphylococcus aureus. However, in contrast to this, three amplicons of 600, 680 and 850 bp were recorded
(Sanjiv et al., 2008). The reason for the polymorphism may be deletion or insertional mutations, particularly in 3’ end region of the coagulase gene.
Considering haemolysin production, out of 88 isolates, 12 (13.64%), 45 (51.14%), 28 (31.82%) and 3 (3.41%) showed
alpha,
beta, gamma and
alpha-
beta haemolysin production respectively. Predominance of beta haemolysin producing
Staphylococcus was in accordance with previous reports
(Reddy et al., 2016; Habibullah et al., 2017). Earlier reports of similar frequency
(Bhagat et al., 2017) reported 09.64%
alpha, 56.63%
beta, 32.53%
gamma and 01.21%
alpha-
beta haemolysins. Contrastingly, 33.96 per cent of
alpha, 49.06 per cent of
beta, 07.55 per cent of
gamma and 09.43 per cent of
alpha-
beta haemolysins have though been reported from Gujarat state
(Parth et al., 2016).
In detection of X-region of protein A in
spa gene by PCR, 26 (56.52%) isolates out of 46 isolates of
Staphylococcus aureus were found positive in which single amplicon in variable numbers was detected. Amplicon size of 243, 270, 296 and 306 bp with calculated number of 5, 6, 9 and 6 repeats, respectively were detected among 5 (19.23%), 6 (23.07%), 9 (34.61%) and 6 (23.07%) isolates, respectively indicative of polymorphisms of
spa gene.
Similar to the present findings, product size of 206, 243, 262, 277, 292, 306 and 339 bp with calculated numbers of 7, 8, 9, 10, 10, 11 and 12 repeats have been reported, respectively
(Khichar et al., 2012). An study from Gujarat state
Bhagat et al., (2016) revealed, 32 (58.18%) isolates out of 55 isolates of
Staphylococcus aureus were found positive for X-region of Protein A in
spa gene showing single amplicons of variable sizes
viz., 160, 243, 270, 296 and 306 bp with calculated number of 5, 8, 10, 10 and 11 repeats, respectively. Eighty isolates of
S. aureus from noses of dogs were found positive for X-region of
spa gene showing single amplicons of variable sizes
viz., 290 bp, 310 bp 270 bp, respectively (
Arzu et al.,2009).
