Abstract:
The objective of this study was to evaluate the existence of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus as probable agents associated with naturally occurring infection of the equine upper respiratory disease in Mashhad area. Nasal swabs samples from thirty horses with upper respiratory tract infections were collected. The bacteria isolated and identified were Streptococcus equi subsp. equi (1 isolate), Streptococcus equi subsp. zooepidemicus (25 isolates), Pasteurella sp. (11 isolates), Staphylococcus sp. (17 isolates), Bacillus sp. (4 isolates), Pseudomonas sp. (4 isolates), Proteus sp. (1 isolate), Neisseria sp. (1 isolate) and E. coli (1 isolate). All 25 isolates of Streptococcus equi subsp. zooepidemicus and the isolate of Streptococcus equi subsp. equi were characterized by biochemical tests and molecular techniques. For molecular identification of the subspecies S. equi and S. zooepidemicus two genomic region SeM and sodA were amplified. This study is the first report of molecular identification of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus in Iran.
INTRODUCTION
Streptococcus equi subsp. equi is worldwide known as the causative agent of strangles, a contagious inflammatory disease of the Upper Respiratory Tract (UPR) and associated lymph nodes of equids. The outcome is only rarely fatal due to complications (Sweeney et al., 2005). Strangles is a common infection among the horse population. It is a notifiable disease based on laboratory confirmation or clinical symptoms. Speed of diagnosis is of great importance to prevent spreading of the disease, as morbidity is high. There is a great demand by clinicians and horse owners for earlier laboratory confirmation than is provided with conventional cultivation. S. equi subsp. zooepidemicus is also an important pathogen of equines being associated with respiratory tract infections of foals and with uterine infections in mares (Radostits et al., 2007). S. equi subsp. zooepidemicus as a member of Lance field`s serogroup C is regarded as archetypal species of the closely related species S. equi subsp. equi (Chanter et al., 1997; Harrington et al., 2002; Timoney, 2004). Streptococcus species identification is customarily based on biochemical typing schemes, including Lancefield grouping (Quinn et al., 1994).
So far there has not been any report of molecular identification of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus from horses with upper respiratory tract infections in Iran. Therefore, the present study was designed to investigate the different methods for identification of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus and on the basis of this knowledge devise a Polymerase Chain Reaction (PCR) system that can quickly detect S. equi and S. zooepidemicus and distinguish between them.
MATERIALS AND METHODS
Clinical investigations: Thirty horses aged 20 months to 22 years, located at 5 racing clubs or referred to the clinic of the Ferdowsi School of Veterinary Medicine were enrolled in the study between January 1st, 2006 and June 1st, 2007. The group of animals investigated was mainly composed of thoroughbred, turkomanbred and mixed breed horses.
Clinician diagnosed respiratory tract disease was by detection of abnormal clinical signs related to the respiratory tract such as fever, cough, nasal discharge and the enlargement of lymph node, change in respiratory rate, abnormal auscultation of the trachea and thoracic cavity and loss of appetite.
Bacteriological examination: Samples for bacteriological examination were collected from nasal cavities of horses using sterile swabs (NS) taken from distant part of the nasal cavity after careful cleaning of the nares and anterior part of nasal mucosa with disinfectant. All samples were collected in duplicated and processed for bacterial isolation as soon as possible, usually within 0.5-4 h after collection. At least two cultures were made for each specimen. Samples were streaked into trypticase soy agar with 5% (v/v) ox blood. All cultures were incubated in 5% (v/v) CO2 at 37°C for 24-48 h. Typical β-haemolytic streptococci-like colonies were detected on blood agar and identified by characteristic colony morphology, Gram staining and biochemical tests including catalase. Isolates identified as S. equi fermented salicin and sucrose but not sorbitol, lactose, raffinose, inulin, trehalose, glycerine, or mannitol. The isolates hydrolysed starch but not aesculin. Isolates identified as S. zooepidemicus gave the same biochemical results but fermented sorbitol and lactose (Quinn et al., 1994).
Polymerase chain reaction: The DNA extraction was performed according to the method described previously (Alber et al., 2004), with some modifications. For this three colonies of the strain to be investigated were suspended in 50 mL TE buffer (10 mmol L-1 Tris-HCl, 1 mmol L-1 EDTA, pH 8.0) containing 1 mL mutanolysin (5 U mL-1) and incubated for 60 min at 37°C in a water bath. The suspension was subsequently treated with 1 mL proteinase K for 120 min at 56°C. After boiling for 15 min at 100°C the suspension was centrifuged (10, 000 g, 5 min) and the supernatant cooled before use.
Three separate PCR mixtures, largely based on that previously described (Timoney and Artiushin, 1997; Newton et al., 2000; Alber et al., 2004), were used but with modification. Three versions of PCR were used in ongoing attempts to improve sensitivity and reliability with further nested set of primers. The primers for these PCRs are shown in Table 1. In brief, PCR-1 was conducted by mixing 2 μL of extracted sample with 3 μL of 10x PCR buffer, 0.9 μL (50 mmol L-1) magnesium chloride, 0.6 μL (10 mmol L-1) dNTP mix, 0.6 μL each first round primer at 25 pmol μL-1, 0.5 μL AmpliTaq, 21.8 μL water. During the first round of the reaction, the mixture was heated at 95°C for 1 min followed by 35 cycles of 94°C for 1 min, 58°C for 1.5 min, 72°C for 1 min followed by 10 min at 72°C.
PCR-2 was conducted in the same way as PCR-1 with the exception of the primers. Also, PCR-3 mediated identification based on the superoxide dismutase A encoding gene (sodA) was conducted in the same way as PCR-1 but annealing temperature was 70°C.
The presence of PCR products were detected by electrophoresis of 5 μL of sample mixed with 1 μL loading buffer in 2.0% (w/v) agarose gel containing 0.04 mol L-1 Tris-acetate (pH 8.3), 0.001 mol L-1 ethylene diamine tetra acetic acid in GNA 100 apparatus at 110 V for 45 min. Product sizes were determined by comparison with the relative mobilities of the 1 kb DNA standard ladder. Gels were examined on a UV transilluminator after immersion in 0.5 μg mL-1 ethidium bromide for 15 min.
RESULTS AND DISCUSSION
The bacteriological culture results of samples from the nasal swabs are shown in Table 2. From the 30 horses enrolled in this study, 65 isolates were obtained. The bacterial strains were Streptococcus equi subsp. equi (1 isolate), Streptococcus equi subsp. zooepidemicus (25 isolates), Pasteurella sp. (11 isolates), Staphylococcus sp. (17 isolates), Bacillus sp. (4 isolates), Pseudomonas sp. (4 isolates), Proteus sp. (1 isolate), Neisseria sp. (1 isolate) and E. coli (1 isolate). Isolation of most bacterial species from the Upper Respiratory Tract (URT) swabs (NS) has not been shown to be associated with respiratory disease in horses, with the exceptions of S. equi and possibly Pasteurella sp. and S. zooepidemicus are generally considered to be normal URT commensal organisms and considerable care is needed in interpreting their presence in URT swabs taken during clinical disease.
Streptococcus equi subsp. equi was isolated from nasal swabs of five year old horse with submandibular lymphadenitis. She was referred to the Ferdowsi School
| Table 2: | Results of bacterial
isolates obtained from nasal swabs cultures of diseased horses |
| Fig 1: | Typical PCR product
of S. equi subsp. zooepidemicus with size of 235
using the SodA lane (3). Negative reactions of S. equi
subsp. zooepidemicus with the primers of SeM
lanes (1 and 2). Lane M, 100 bp ladder (Gene craft) served as
size marker |
| Fig 2: | Typical PCR products
of S. equi subsp. equi with specific oligonucleotide
primers respectively lanes 1, 2 show the PCR products of S.
equi with sized 677 and 325 bp using SeM primers. Lanes
3, band of 235 bp length amplified using SodA. Lane M,
100 bp ladder (Gene craft) served as size marker |
clinic of Veterinary Medicine with complains of sever dyspnea. However, S. equi subsp. zooepidemicus, the opportunistic pathogen, was isolated from 25 horses according to Timoney (2004) produce disease in situation of virus infection or heat stress and it was therefore of interest to investigate in samples from horses with respiratory disease.
Polymerase chain reaction done on genomic DNA extracted from the bacterial colonies confirmed the bacteriological results (Fig. 1, 2).
The closely related Streptococcal species S. equi subsp. equi and subsp. zooepidemicus were identified by polymerase chain reaction using oligonucleotide primers designed according to species-specific parts of the super oxide dismutase A encoding gene (sodA). Further differentiation of both sub species was be performed by amplification of the gene encoding the protein M which could be isolated from S. equi subsp. equi but not from S. equi subsp. zooepidemicus.
The identification and differentiation of S. equi subsp. equi and subsp. zooepidemicus traditionally relies on phenotypic characteristics including serological properties by use of lancefield group C specific antisera and biochemical properties such as the fermentation of lactose, trehalose and sorbitol.
A positive lactose and sorbitol reaction is typical for S. equi subsp. zooepidemicus.
While negative reaction in these entire tests is typical for S. equi subsp. equi (Lammler and Hahn, 1994; Harrington et al., 2002). However, atypical S. equi subsp. equi that ferment one or both of lactose and trehalose, have been described by Grant et al. (1993).
Resent developments in nucleic acid technology such as PCR have resulted in new method that can be used for identification of bacteria.
PCR-mediated identification base on the M-like protein gene had been described for S. equi subsp. equi (Timoney and Artiushin, 1997; Newton et al., 2000).
However, S. equi subsp. equi possesses genes encoding two M-like proteins one of which is S. equi subsp. equi while the other M-like protein is a homologue to the M-like protein of S. equi subsp. zooepidemicus (Timoney et al., 1997).
In addition, truncated forms of M proteins might occur (Chanter et al., 1997). In the present study species-specific segments of an internal part of superoxide dismutase A encoding gene (sodA) was used for specific identification of both subspecies of S. equi.
The species-specific sequence variation of this gene had already been used for identification of various Gram-positive bacteria also including bacteria of genus Streptococcus (Poyart et al., 1998, 2002; Whatmore et al., 2001). The sodA gene sequences were published previously by to Poyart et al. (1998). However, because of the close relationship of the sodA gene sequences of both subspecies of S. equi, the designed oligonucleotide primers did not allow a differentiation of S. equi subsp. zooepidemicus and S. equi subsp. equi since both belong to the pyogenic group of genus Streptococcus.
A differentiation of both subspecies could be performed by detection of the genes encoding antiphagocytic factor of S. equi subsp. equi protein. The occurrence of the antiphagocytic factor seems to be a constant characteristic of S. equi subsp. equi but not of S. equi subsp. zooepidemicus and could be used for differentiation of both subspecies.
The positive sodA gene reaction of the investigated S. equi strains and the positive M-protein reaction of the S. equi subsp. equi strains could be confirmed by PCR are shown in Fig. 1 and 2.
A major concern in this study is the presence of false negatives due to either sequence mutations or a lack of the toxin mediated identification and differentiation of both subspecies. Larger number of strains of different origin should be reinvestigated.
According to our best knowledge, this study is the first report of molecular detection of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus in Iran.
ACKNOWLEDGMENTS
The authors gratefully acknowledge Ferdowsi University of Mashhad for their financial support and the personnel of Microbiology Department for their technical support.