In vitro bactericidal efficacy of equine polymorphonuclear leukocytes against Corynebacterium equi

Polymorphonuclear leukocytes from adult horses were separated from whole blood, using a 2-step Percoll gradient, and were tested for bactericidal function against Corynebacterium equi. Staphylococcus aureus, an organism against which equine neutrophils have proved efficacy, was a positive control. The percentage of uptake after a 15-minute preincubation of the neutrophils and bacteria in the presence of normal horse serum was also calculated. The results indicated that equine neutrophils effectively phagocytosed and killed C equi and S aureus. The percentage of uptake for S aureus (95% +/- 3%) was greater than that for C equi (85% +/- 6%) (P less than 0.001), but the bactericidal efficacy was equivalent. More than 90% of the ingested or attached bacteria were destroyed during the 3-hour incubation period (mean percentage of C equi killed = 96 +/- 2%; mean percentage of S aureus killed = 91 +/- 8%). These results indicated that a failure of bacterial killing by neutrophils is unlikely to be important in the pathogenesis of C equi pneumonia in the horse.     

Pulsed-field gel electrophoresis and distribution of the genes zag and fnz in isolates of Streptococcus equi

Streptococcus equi subsp. equi and subsp. zooepidemicus are important pathogens of the equine respiratory tract. Isolates of both subspecies were examined by pulsed-field gel electrophoresis (PFGE). With the exception of eight isolates, a unique band pattern was displayed for each of the 48 subsp. zooepidemicus isolates tested. A method to distinguish isolates of the genetically very homogeneous subsp. equi has hitherto not been available, although several methods have been tested. By the use of PFGE, 50 isolates of subsp. equi could be divided into eleven groups, each with a unique pulsotype. In addition, the recently characterised genes encoding the cell-wall proteins ZAG and FNZ of S. equi subsp. zooepidemicus strain ZV were shown by Southern blots to be present in all 98 tested isolates, including the type strains of the two subspecies. Binding assays showed that the expression of the two genes clearly differentiate between the two subspecies.     

Bacterial isolates and antimicrobial susceptibilities in equine bacterial ulcerative keratitis (1993--2004)

REASONS FOR PERFORMING STUDY: Bacterial ulcerative keratitis is a common and often vision-threatening problem in horses. Emerging bacterial resistance to commonly used topical antibiotics has been demonstrated. Previous antibiotic use may alter the antimicrobial susceptibility of bacterial isolates. OBJECTIVES: To document aerobic bacterial isolates and associated bacterial susceptibilities from horses with ulcerative keratitis treated at the University of Tennessee between January 1993 and May 2004 and determine whether prior antibiotic therapy affected antimicrobial susceptibility of the isolates. METHODS: Medical records from horses with ulcerative keratitis and positive aerobic bacterial cultures and antimicrobial susceptibility were evaluated. Clinical history regarding antibiotic therapy prior to culture was documented. RESULTS: Fifty-one aerobic bacterial isolates from 43 horses were identified. Streptococcus equi subspecies zooepidemicus was the most commonly isolated organism, accounting for 33.3% of all isolates, followed by Pseudomonas aeruginosa (11.8%), Staphylococcus spp. (11.8%) and Gram-negative nonfermenting rods (7.8 %). No resistance was noted amongst S. equi ssp. zooepidemicus to cephalothin, chloramphenicol or ciprofloxacin. Only 64 % of S. equi ssp. zooepidemicus isolates were sensitive to bacitracin. No resistance was noted among P. aeruginosa to gentamicin, tobramycin or ciprofloxacin. Antibiotic therapy with neomycin-polymixin B-bacitracin prior to presentation and culture was documented in 11/17 horses in which S. equi ssp. zooepidemicus was isolated and in 4/6 horses in which P. aeruginosa was isolated. Three horses received topical corticosteroids prior to culture, of which 2 had polymicrobial infections. CONCLUSIONS: S. equi ssp. zooepidemicus and P. aeruginosa were the most frequently isolated bacterial organisms in equine ulcerative keratitis. No significant trends in aminoglycoside or fluoroquinolone resistance were noted among these organisms. However, the resistance of S. equi ssp. zooepidemicus to bacitracin with common use of this antibiotic suggests that previous antibiotic therapy probably affects antimicrobial resistance. POTENTIAL RELEVANCE: Therapy prior to culture may play an important role in antimicrobial susceptibility of corneal bacterial isolates. Corticosteroid use may increase the risk of polymicrobial infections of corneal ulcers, leading to a worse prognosis. Although significant fluoroquinolone resistance has not been documented in the veterinary literature, these antimicrobials should be reserved for known infected corneal ulcers and not used for prophylaxis. Empirical antibiotic therapy should not only be guided by clinical signs, but also take into consideration previous antimicrobial and corticosteroid therapy.     

Non-oxidative antibacterial activity of bovine neutrophil granule proteins towards mastitis pathogens

Acid extracts of bovine neutrophil granules displayed potent antibacterial activity towards a number of mastitis pathogens in vitro. Killing of pathogens by acid extractable granule protein was dependent on incubation time, protein concentration, bacterial cell load, pH and ionic strength. Gram-negative and Gram-positive organisms showed variable sensitivity to granule extract. Strains of Staphylococcus aureus were the most resistant of tested organisms to granule extract. Gram-negative organisms were neither consistently more nor less sensitive than Gram-positive organisms. Maximal killing of Gram-positive pathogens, after 30 minutes exposure to granule extract at 37 degrees C, occurred between pH 7.0 and 8.0. The Gram-negative organism Escherichia coli B117 was more sensitive to neutrophil granule extract at pH 5.0.     

The pathogenic equine streptococci

Streptococci pathogenic for the horse include S. equi (S. equi subsp. equi), S. zooepidemicus (S. equi subsp. zooepidemicus), S. dysgalactiae subsp. equisimilis and S. pneumoniae capsule Type III. S. equi is a clonal descendent or biovar of an ancestral S. zooepidemicus strain with which it shares greater than 98% DNA homology and therefore expresses many of the same proteins and virulence factors. Rapid progress has been made in identification of virulence factors and proteins uniquely expressed by S. equi. Most of these are expressed either on the bacterial surface or are secreted. Notable examples include the antiphagocytic SeM and the secreted pyrogenic superantigens SePE-I and H. The genomic DNA sequence of S. equi will greatly accelerate identification and characterization of additional virulence factors and vaccine targets. Although it is the most frequently isolated opportunist pyogen of the horse, S. zooepidemicus has been the subject of few contemporary research studies. Variation in the protectively immunogenic SzP proteins has, however, been well characterized. Given its opportunist behavior, studies are urgently needed on regulation of virulence factors such as capsule and proteases. Likewise, information is also very limited on virulence factors and associated gene regulation of S. dysgalactiae subspecies equisimilis. It has recently been shown that equine isolates of Streptococcus pneumoniae are clonal, a feature shared with S. equi. All equine isolates express capsule Type III, are genetically similar, and have deletions in the genes for autolysin and pneumolysin. In summary, the evolving picture of the interaction of the equine pathogenic streptococci and their host is that of multiple virulence factors active at different stages of pathogenesis. The inherent complexity of this interaction suggests that discovery of effective combinations of immunogens from potential targets identified in genomic sequence will be laborious.     

Results of bacteriological and cytological examinations of the endometrium of subfertile mares in stud farms in Serbia

Uterine microbiology, antimicrobial susceptibility and endometrial cytology were investigated in a total of 51 mares with fertility problems from 16 different stud farms in Serbia. Uterine cultures were performed after collection with a double guarded uterine swab, and endometrial cytology was evaluated after collection of endometrial cells with a special device (cytology brush). In 21 of 51 mares, at least one bacterial species was isolated from the uterus; the most frequent were Streptococcus equi subsp. zooepidemicus (13 isolates) and E. coli (four isolates). All isolates of Streptococcus equi subsp. zooepidemicus were susceptible to penicillin. Results from endometrial cytology were inconsistent; in 17 animals with positive bacteriological culture, cytology was not altered. It can be concluded that in Serbia, as in many other contries, Streptococcus equi subsp. zooepidemicus is the main cause for equine endometritis. It can be easily diagnosed by uterine culture but endometrial cytology does not always prove the existence of an endometrial infection with this agent.     

Se18.9, an anti-phagocytic factor H binding protein of Streptococcus equi

Evasion of phagocytosis is an important virulence determinant of Streptococcus equi (S. equi subsp. equi), the cause of equine strangles and distinguishes it from the closely related but much less virulent S. zooepidemicus (S. equi subsp. zooepidemicus). We describe Se18.9, a novel H factor binding protein secreted by S. equi but not by S. zooepidemicus that reduces deposition of C3 on the bacterial surface and significantly reduces the bactericidal activity of equine neutrophils suspended in normal serum for both S. equi and S. zooepidemicus. Se18.9 is secreted abundantly by actively dividing cells and is also bound to the bacterial surface. Strong serum and mucosal antibody responses are elicited in S. equi infected horses. Although a gene identical to se18.9 was not detected in S. zooepidemicus, sequences encoding proteins of similar size with similar signal peptide sequences were found in 3 of 12 randomly selected strains. Since Se18.9 is unique to S. equi, and immunoreactive with convalescent sera and mucosal IgA, it has potential for immunodiagnosis and for study of mucosal antibody response to S. equi.     

Dissemination of the superantigen encoding genes seeL, seeM, szeL and szeM in Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus

Bacterial superantigens are one of the major virulence factors produced by Streptococcus pyogenes and Staphylococcus aureus. The two novel superantigen encoding genes seeM and seeL were described for S. equi subsp. equi which is known as the causative agent of strangles in equids. In the present study previously characterized S. equi subsp. equi strains and strains of various other animal pathogenic streptococcal species and subspecies were investigated for the presence of the superantigen encoding genes seeM and seeL by polymerase chain reaction. According to these studies seeL and seeM appeared to be a constant characteristic of all investigated S. equi subsp. equi strains. Surprisingly, one S. equi subsp. zooepidemicus strain (S.z. 122) was also positive for both genes. The species identity of this S. equi subsp. zooepidemicus strain could additionally be confirmed by sequencing the 16S rRNA gene and the 16S-23S rDNA intergenic spacer region. The superantigen encoding genes could not be found among additionally investigated S. equi subsp. zooepidemicus strains or among strains of seven other streptococcal species. The seeL and seeM genes of the S. equi subsp. equi strain S.e. CF32 and the genes szeL and szeM of the S. equi subsp. zooepidemicus strain S.z. 122 were cloned and sequenced. A sequence comparison revealed a high degree of sequence homology between seeL, szeL, speL and seeM, szeM and speM, respectively. The superantigenic toxins L and M seemed to be widely distributed virulence factors of S. equi subsp. equi, rare among S. equi subsp. zooepidemicus but did not occur among a number of other animal pathogenic streptococcal species.     

Surveillance programme for important equine infectious respiratory pathogens in the USA

The prevalence and epidemiology of important viral (equine influenza virus [EIV], equine herpesvirus type 1 [EHV-1] and EHV-4) and bacterial (Streptococcus equi subspecies equi) respiratory pathogens shed by horses presented to equine veterinarians with upper respiratory tract signs and/or acute febrile neurological disease were studied. Veterinarians from throughout the USA were enrolled in a surveillance programme and were asked to collect blood and nasal secretions from equine cases with acute infectious upper respiratory tract disease and/or acute onset of neurological disease. A questionnaire was used to collect information pertaining to each case and its clinical signs. Samples were tested by real-time PCR for the presence of EHV-1, EHV-4, EIV and S equi subspecies equi. A total of 761 horses, mules and donkeys were enrolled in the surveillance programme over a 24-month study period. In total, 201 (26.4 per cent) index cases tested PCR-positive for one or more of the four pathogens. The highest detection rate was for EHV-4 (82 cases), followed by EIV (60 cases), S equi subspecies equi (49 cases) and EHV-1 (23 cases). There were 15 horses with double infections and one horse with a triple infection. The detection rate by PCR for the different pathogens varied with season and with the age, breed, sex and use of the animal.     

马腺疫链球菌fneB基因LAMP检测方法的建立及应用

为了建立快速简便的fneB-LAMP检测法用于马腺疫病原马链球菌马亚种(S.equi)的检测,本试验采用恒温水浴进行LAMP扩增,建立可视化fneB-LAMP检测方法.结果显示:对大肠埃希菌、无乳链球菌、金黄色葡萄球、蜡状芽孢杆菌和克雷伯杆菌5种菌与标准S.equi菌株检测,只能够检测出标准阳性菌株;对新疆昭苏县3个马...     

Susceptibility of equine bacterial isolates to antimicrobial agents

In vitro antimicrobic susceptibility patterns of commonly isolated aerobic gram-positive and gram-negative bacterial pathogens of equine origin were determined, using the agar-plate dilution method. All organisms were recent clinical isolates and included Corynebacterium (Rhodococcus) equi, Corynebacterium pseudotuberculosis, (coagulase positive) Staphylococcus sp, Streptococcus equi, Streptococcus zooepidemicus, Actinobacillus sp, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella. In vitro susceptibility levels were outlined for 14 antimicrobics as follows: amikacin less than or equal to 4.0 micrograms/ml, ampicillin less than or equal to 1.0 microgram/ml, amoxicillin less than or equal to 1.0 microgram/ml, cefadroxil less than or equal to 8.0 micrograms/ml, chloramphenicol less than or equal to 8.0 micrograms/ml, erythromycin less than or equal to 1.0 microgram/ml, gentamicin less than or equal to 2.0 micrograms/ml, kanamycin less than or equal to 4.0 micrograms/ml, penicillin less than or equal to 1.0 microgram/ml, tetracycline less than or equal to 1.0 microgram/ml, sulfadimethoxine less than or equal to 10.0 micrograms/ml, ormetoprim/sulfadimethoxine less than or equal to 0.5/9.5 micrograms/ml, sulfadiazine less than or equal to 10.0 micrograms/ml, and trimethoprim/sulfadiazine less than or equal to 0.5/9.5 micrograms/ml.     

Detection of Bacteria in Equine Synovial Fluid by Use of the Polymerase Chain Reaction

Equine synovial fluid aliquots were inoculated with Salmonella enteritidis, Escherichia coli, Actinobacillus equuli, Staphylococcus aureus , and Streptococcus zooepidemicus to obtain approximate concentrations of 1000, 100, 10, and 1 colony forming U/mL. Synovial fluid aliquots were also inoculated with an unquantitated inoculum of Bacteroides fragilis and Clostridium perfringens. Inoculated synovial fluid was incubated in trypticase-soy broth or Columbia broth for approximately 12 hours. Then aliquots were removed for DNA extraction and polymerase chain reaction (PCR) analysis for detection of a 531 base-pair segment of bacterial DNA corresponding to a region of the 16S ribosomal gene. Duplicate samples of inoculated synovial fluid were prepared for microbial culture. Bacteria were detected in all samples inoculated with bacteria but not in control synovial fluid samples. Under experimental conditions there was no difference between microbial culture and PCR analyses for detection of bacteria. Experimentally, PCR was able to detect bacteria in synovial fluid within 24 hours of inoculation.     

The bactericidal effect of N-acetyl-beta-D-glucosaminidase on bacteria.

The bactericidal activity of N-acetyl-beta-D-glucosaminidase (NAGase) on some of the potential bacterial pathogens of the cow was determined. NAGase treatment significantly decreased the mean log10 number of Actinomyces pyogenes (P less than 0.01) and Staphylococcus aureus (P less than 0.05) after 2 and 4 hours of incubation at 37 degrees C. Similarly NAGase treatment significantly (P less than 0.05) reduced the mean log10 number of Streptococcus agalactiae and Pseudomonas aeruginosa after 4 hours incubation at 37 degrees C. NAGase did not reduce the numbers of Escherichia coli or Enterobacter aerogenes after either 2 or 4 hours incubation. Since NAGase and presumably other lysosomal enzymes are free on normal mucosal surfaces such as the uterus it is suggested that this direct bactericidal activity may be an important component of the normal defense mechanisms.     

The interaction of Rhodococcus equi and foal neutrophils in vitro

Polymorphonuclear neutrophil leukocytes (PMNL) from 8 healthy foals (2-14 weeks of age) and 2 foals with bacterial pneumonia were separated from whole blood using a 2 step Percoll gradient. Purified PMNL were tested for bactericidal function against Rhodococcus equi and Staphylococcus aureus in the presence of normal horse serum. The percentage uptake after a 15-min pre-incubation of PMNL and bacteria was also calculated. Ultrastructural examination of the interaction of R. equi and normal foal PMNL was performed after 15 min incubation. Results indicated that foal PMNL effectively phagocytose and destroy R. equi and S. aureus in the presence of normal horse serum. The mean percent uptake for R. equi was 99.3 +/- 0.4% and for S. aureus 99.9 +/- 0.1%. Further, 97.8 +/- 0.1% ingested R. equi and 98.4 +/- 0.1% ingested S. aureus were destroyed in the 15-min incubation period. Over the 3-h incubation, 91.9% of remaining R. equi were killed, but only 49.2 +/- 31.9% of S. aureus (P less than 0.01). Total bactericidal effect of foal PMNL, however, was 99.3 +/- 0.4% against R. equi and 99.9 +/- 0.1% against S. aureus. The percentage uptake and total bactericidal efficacy of neutrophils from sick foals was greater than 95%. Ultrastructural examination of the PMNL-R. equi interaction after 15 min incubation revealed phagocytosis of the bacteria and morphologic changes consistent with neutrophil degranulation. This study suggests that a defect in PMNL bactericidal capability is not likely to be a contributing factor in the pathogenesis of R. equi pneumonia in foals.     

Use of specific sugars to inhibit bacterial adherence to equine endometrium in vitro

OBJECTIVE: To determine whether specific sugars inhibit adhesion of Streptococcus zooepidemicus, Pseudomonas aeruginosa, and Escherichia coli to equine endometrial epithelial cells in vitro. SAMPLE POPULATION: Endometrial biopsy specimens collected during estrus from 7 healthy mares. PROCEDURE: Endometrial specimens on glass slides were incubated for 30 minutes at 4 C with suspensions of S. zooepidemicus, P. aeruginosa, or E. coli in phosphate-buffered saline solution (PBSS) alone or with various concentrations of D-(+)-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-(+)-glucose, galactose, or N-acetyl-neuraminic acid. Inhibition of bacterial adherence was determined by comparing adhesion of bacteria (i.e., percentage of glandular epithelial cells with adherent bacteria) suspended in each sugar solution with that of bacteria suspended in PBSS. RESULTS: Mannose and N-acetyl-D-galactosamine inhibited adhesion of E. coli and P. aeruginosa to epithelial cells, whereas only mannose inhibited adhesion of S. zooepidemicus. The other sugars did not affect bacterial adherence. CONCLUSIONS AND CLINICAL RELEVANCE: Mannose and N-acetyl-D-galactosamine appear to play a role in adhesion of S. zooepidemicus, P. aeruginosa, and E. coli to equine endometrium. In horses with uterine infections, use of sugars to competitively displace bacteria from attachment sites on cells may provide an adjunct to antibiotic treatment.     

Determination of intraspecies variations of the V2 region of the 16S rRNA gene of Streptococcus equi subsp. zooepidemicus

The 16S rRNA gene of 39 S. equi subsp. zooepidemicus strains and two S. equi subsp. equi strains was amplified by polymerase chain reaction and subsequently digested with the restriction enzyme Hinc II. A restriction profile with two fragments with sizes of 1250 bp and 200 bp could be observed for both S. equi subsp. equi strains and for 30 of the 39 S. equi subsp. zooepidemicus strains indicating a sequence variation within the V2 region of the 16S rRNA gene of the remaining nine S. equi subsp. zooepidemicus isolates. A segment of the 16S rRNA gene including the hypervariable V2 region of 11 S. equi subsp. zooepidemicus and two S. equi subsp. equi could be amplified by PCR and sequenced. The sequence of the V2 region of eight S. equi subsp. zooepidemicus strains appeared to be identical or almost identical to the sequence of the two S. equi subsp. equi strains. The sequence of the remaining three S equi subsp. zooepidemicus strains differed significantly from the sequence of S. equi subsp. equi. These differences allowed a division of S. equi subsp. zooepidemicus strains into two 16S rRNA types and might possibly have consequences for the taxonomic position of these phenotypically indistinguishable strains of one subspecies. A molecular typing could additionally be performed by amplification of the gene encoding the 16S-23S rRNA spacer region. A single amplicon of the spacer gene of 1100 bp could be observed for one S. equi subsp. zooepidemicus, an amplicon of 950 bp for two S. equi subsp. equi strains and 10 S. equi subsp. zooepidemicus strains, a amplicon of 780 bp for 27 S. equi subsp. zooepidemicus strains and a single amplicon of 600 bp for one S. equi subsp. zooepidemicus strain. The variations of the V2 region of the 16S rRNA gene and the size variations of the 16S-23S rRNA spacer gene were not related to each other. Both variations could be used for molecular typing of this species, possibly useful in epidemiological aspects.     

肉桂酸等4种单体对细菌生物膜的影响

通过大肠杆菌和金黄色葡萄球菌BBF的模型,考察肉桂酸、连翘苷、大黄素、亚硫酸穿心莲内酯对BBF形成的影响。在37℃下将细菌培养24h使其形成BBF后加入样品继续培养24h,用结晶紫染色。通过测量吸光度来考察单体对BBF的影响。结果表明,大黄素、连翘苷、肉桂酸、亚硫酸穿心莲内酯（浓度为1．000mg／mL）对金黄色葡萄球菌BBF的抑制率分别为67．26％、10．91％、18．26％、17．05％;对大肠杆菌BBF的抑制率分别为32。99％、36．13％、49．79％、17．05％。由此可见,大黄素对金黄色葡萄球菌BBF的抑制作用较强,肉桂酸对大肠杆菌BBF的抑制作用较强。     

An in vivo model for the study of Bordetella avium adherence to tracheal mucosa in turkeys

An in vivo model was developed for studies characterizing the adherence of Bordetella avium to the tracheal mucosa of turkeys. Three-week-old turkeys were anesthetized, and the cervical part of the trachea was isolated after tracheostomy was done. A hemostat was applied craniad to the tracheostomy site to occlude the tracheal lumen. Isolated tracheal segments were filled with an aqueous bacterial inoculum for 1 minute, and then excess inoculum and the hemostat were removed. After 1 hour, a 1-cm section was excised from each tracheal segment, and adherent viable bacteria were quantified. Modifications of the procedure were evaluated to produce a model that was technically simple to do, economical, and reproducible. To examine the validity of the model, adherence of B avium was compared with that of Escherichia coli and Staphylococcus aureus. Adherence of B avium to tracheal mucosa was 17 times greater than that with E coli and 1,550 times greater than that with S aureus. Colonization of the tracheal mucosa by B avium was demonstrated in tracheal sections obtained 6 hours after filling with bacterial inoculum. Because the ciliary clearance mechanism of the tracheal segments remained functional, washing of the tracheal lumen had no effect on numbers of associated bacteria. An important advantage of this model over in vitro models is the excellent preservation of the tracheal mucosal surface.     

Chronic endometritis in an Asian elephant (Elephas maximus).

A 48-yr-old female Asian elephant with a history of pododermatitis developed recurrent hematuria beginning in 2002. Transrectal ultrasonography and endoscopic examination in 2004 identified the uterus as the source of hematuria and excluded hemorrhagic cystitis. Treatment with Desloreline implants, antibiotics, and homeopathic drugs led to an improved general condition of the elephant. In July 2005, the elephant was suddenly found dead. During necropsy, the severely enlarged uterus contained about 250 L of purulent fluid, and histopathology revealed ulcerative suppurative endometritis with high numbers of Streptococcus equi ssp. zooepidemicus and Escherichia coli identified on aerobic culture. Additional findings at necropsy included: multifocal severe pododermatitis, uterine leiomyoma, and numerous large calcified areas of abdominal fat necrosis. Microbiologic culture of the pododermatitis lesion revealed the presence of Streptococcus agalactiae, Streptococcus equi ssp. zooepidemicus, Staphylococcus sp., Corynebacterium sp., and Entercoccus sp.     

Multiplex polymerase chain reaction for identification and differentiation of Streptococcus equi subsp. zooepidemicus and Streptococcus equi subsp. equi

The closely related streptococcal species Streptococcus equi subsp. zooepidemicus and S. equi subsp. equi were identified by polymerase chain reaction using oligonucleotide primers designed according to species-specific parts of the superoxide dismutase A encoding gene sodA. A further differentiation of both subspecies could be performed by amplification of the genes seeH and seeI encoding the exotoxins SeeH and SeeI, respectively, which could be detected for S. equi subsp. equi but not for S. equi subsp. zooepidemicus. A further simplification of the identification and differentiation of both subspecies was conducted by sodA-seeI multiplex polymerase chain reaction.