Multi-locus sequence typing of Streptococcus equi subspecies zooepidemicus strains isolated from cats

Abstract

Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) causes outbreaks of fatal respiratory disease in dog shelters and fatal respiratory and neurologic disease in cat shelters. We conducted multi-locus sequence typing analysis on S. zooepidemicus isolates from 5 Canadian and 3 Israeli cats with severe respiratory and neurologic disease, plus 1 isolate from a clinically normal shelter cat. Our aim was to determine if feline outbreaks are clonal and whether there is commonality between feline and canine strains. ST363 was identified as the causative strain of a Canadian outbreak of S. zooepidemicus–linked disease, and is a double-locus variant of ST173, which was isolated from one of the Israeli cats. ST363 was also isolated from the clinically normal cat, indicative of the potential for enzootic infection in shelters. Strains within the ST173 clonal complex were responsible for 2 large canine outbreaks in the United States and the United Kingdom, as well as the death of 1 cat in the United States outbreak. ST215 was isolated from 2 cats in the Israeli outbreak, and is unrelated to the ST173 complex. We conclude that S. zooepidemicus outbreaks in cat shelters are clonal and that strains within the ST173 clonal complex are pathogenic for both dogs and cats.

Multi-locus sequence typing (MLST) is a molecular technique that allows for genetic comparison of bacterial strains and is based on sequencing 7 highly conserved housekeeping genes.¹⁹ An MLST scheme has been developed for the β-hemolytic, Lancefield group C streptococcal bacterium Streptococcus equi subspecies zooepidemicus (S. zooepidemicus).¹⁹ S. zooepidemicus is an equine mucosal commensal responsible for opportunistic disease in a wide host range and in horses in particular.¹⁵ Many S. zooepidemicus isolates have been typed by MLST within the endogenous reservoir population of the equine nasopharynx.⁹,¹⁶ However, only a subset of this population has been associated with equine disease, typically a single clone of S. zooepidemicus that may also be a member of a pathogenic clonal complex.^9,16

In contrast to the horse, S. zooepidemicus is not a commensal in the dog.^6,8,17 Yet, epizootics of fatal necrohemorrhagic pneumonia affecting hundreds of dogs in shelters and kennels have occurred.¹⁷ MLST evaluation of 6 canine disease outbreaks in Britain and the United States revealed clonality and a group of S. zooepidemicus strains that are pathogenic for dogs.¹⁷ Without an endogenous mucosal reservoir population of S. zooepidemicus, it is unclear how the shelter dogs came to be infected.

In 2010, outbreaks of fatal S. zooepidemicus pneumonia, rhinitis, and meningitis were reported in cats residing in Israeli and Canadian cat shelters.^2,3 At that time, outbreaks of acute fatal necrohemorrhagic pneumonia caused byS. zooepidemicus had been reported in hundreds of sheltered and kenneled dogs for 3 decades.^4,7,10,14 In those outbreaks, feline S. zooepidemicus infection was rarely reported: a cat contracted fatal septicemia in 1 shelter, and 3 cats developed severe pneumonia in a second shelter.¹⁷ S. zooepidemicus is not a commensal in the cat.^1,6 How the cat shelter outbreaks in Israel and Canada were initiated is unclear. We report herein on the application of MLST to 9 feline isolates of S. zooepidemicus we compiled from 2008 to 2015 and compare the results with those for pathogenic canine isolates.

We studied 8 isolates of S. zooepidemicus that were cultured after autopsy of 3 mature long-term residents of a large British Columbia (BC) cat shelter (BC shelter 1, cases 1–3; case 1 was reported previously³), a juvenile feral cat found on Vancouver Island (case 9), a 15-wk-old rescue kitten flown from Egypt to Calgary, Alberta (case 8), and 3 cats from a large Israeli cattery reported previously² (cases 5–7; Table 1). Of the unpublished cases, cases 2, 3, and 9 were autopsied at the Animal Health Centre (Abbotsford, BC, Canada). Case 8 was autopsied at the Diagnostic Services Unit in Calgary. Other than routine bacterial culture, ancillary testing was conducted at the discretion of the pathologist based on history, gross findings, and microscopic observations in each cat.

Table 1.

Detailed results obtained in the current study or in prior published studies as indicated.

Case	Origin	Date	Diagnosis	Bacterial culture and PCR	ST	PCR for FHV	PCR for FCV
1	BC shelter 1	2008	Rhinitis, meningitis	Sez*	363	+*	−*
2	BC shelter 1	2010	Rhinitis, meningitis	Sez	363	ND	ND
3	BC shelter 1	2011	Rhinitis, bronchopneumonia	Sez	363	ND	ND
4	BC shelter 2	2011	Elective surgery, tonsil swab	Sez, Pm, Bb	363	ND	ND
5	Israel	2007	Rhinitis, pneumonia, meningoencephalitis	Sez†	173	ND†	ND†
6	Israel	2007	Rhinitis, pneumonia	Sez, Mg†	215	ND†	ND†
7	Israel	2007	Rhinitis, meningoencephalitis	Sez†	215	ND†	ND†
8	Egypt/Calgary	2014	Rhinitis, pneumonia, conjunctivitis	Sez, Cf‡, Bb‡, Mf‡	358	−‡	−‡
9	Vancouver Island	2015	Otitis media/interna, rhinitis, subdural abscess	Sez	175v§	ND	ND

Bb = Bordetella bronchiseptica; Cf = Chlamydia felis; FCV = feline calicivirus; FHV = feline herpesvirus; Mf = Mycoplasma felis; Mg = Mycoplasma gateae; ND = not done; Pm = Pasteurella multocida; Sez = Streptococcus equi subspecies zooepidemicus; ST = sequence type.

^*Results from Britton and Davies.³

^†Results from Blum et al.²

^‡Results obtained by use of the feline respiratory disease RT-PCR panel (IDEXX Reference Laboratories, Calgary, Alberta, Canada).

^§Isolate had 6 genes identical to ST175 and absence of a yqiL value.

In case 8, lung tissue was submitted for a commercial feline respiratory disease reverse-transcription (RT-)PCR panel (IDEXX Reference Laboratories, Calgary, Alberta, Canada). The PCR test was negative for feline calicivirus (FCV), feline herpesvirus 1 (FHV), and H1N1 influenza virus, and positive for Chlamydia felis, Bordetella bronchiseptica, and Mycoplasma felis. Upon microscopic examination of tissues, no evidence for viral, chlamydial, or mycoplasmal disease was detected. B. bronchiseptica and S. zooepidemicus were cultured from the lung. Pasteurella multocida and B. bronchiseptica were isolated from the tonsil of case 4, a clinically normal cat from BC shelter 2 that was undergoing elective surgery. Following case workup, the tonsillar and autopsy S. zooepidemicus isolates from the Canadian cats were cryopreserved at −80°C for future study.

For MLST analysis, thawed isolates from the 6 Canadian cats and reconstituted lyophilized cultures from the 3 Israeli cats were inoculated onto Columbia blood agar with 5% sheep blood (Oxoid, Nepean, ON, Canada) and MacConkey agar (Oxoid) and incubated at 35°C ± 2°C in 5–10% CO₂ and aerobic conditions, respectively. Agar plates were observed at 24 and 48 h for bacterial growth, and bacterial isolates were subcultured to obtain a pure isolate for identification purposes. Bacterial identifications were performed based on colony morphology, growth characteristics, Gram stain, biochemical testing, and molecular techniques. All isolates of β-hemolytic streptococci were speciated using the API 20S kit (bioMérieux, St. Laurent, QC, Canada).

MLST was performed on confirmed S. zooepidemicus isolates using the internal fragments of genes for carbamate kinase (arc), ribonucleoside-diphosphate reductase (nrdE), prolyl-tRNA synthetase (proS), signal peptidase I (spi), thymidylate kinase (tdk), triosephosphate isomerase (tpi), and acetyl-CoA acetyltransferase (yqiL)¹⁹ on the 9 S. zooepidemicus isolates. The fragments were amplified in volumes of 25 µL (Illustra puReTaq Ready-To-Go PCR beads, GE Healthcare Biosciences, Piscataway, NJ) using 50 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. The PCR products were filtered (Amicon Ultra-0.5 centrifugal filters, Millipore, Burlington, ON, Canada), a PCR was performed (BigDye Terminator v.3.1 cycle sequencing kit, Applied Biosystems, Thermo Fisher Scientific, Waltham, MA), and the final products were purified (BigDye XTerminator, Applied Biosystems) prior to sequencing (3130 Genetic Analyser, Applied Biosystems). The sequences determined for both strands were compared with sequence types (STs) in the S. zooepidemicus database (PubMLST, https://goo.gl/3BJ1KD) for ST allocation. Sequences not in the database were submitted for ST assignment.

All 9 S. zooepidemicus isolates were gram-positive cocci that were β-hemolytic on blood agar and fermented sorbitol and lactose but not trehalose. Seven of the 9 isolates exhibited mucoid colony morphology, whereas 2 were non-mucoid. MLST analysis results are presented in Table 1. A clonal infection was identified in cases 1–3 in which ST363, a novel ST that has not been previously reported, to our knowledge, was identified in all 3 cats. ST363 is a double-locus variant of ST173, the strain also identified in case 5. ST173 is the founding member of a pathogenic clonal complex that includes ST18 and ST316.^17,19

S. zooepidemicus ST173 was responsible for the large 2007 clonal outbreak of canine necrohemorrhagic pneumonia in a Nevada shelter.¹⁷ One cat in the Nevada outbreak was fatally infected systemically with ST316, a single-locus variant (SLV) of ST173.¹⁷ ST18, another SLV of ST173, was responsible for the 2008 outbreak of fatal pneumonia in greyhounds in the United Kingdom.¹⁹ Half (4 of 8) of the autopsied cats in our study were infected with strains of S. zooepidemicus within the ST173 clonal complex. Clearly, strains within the ST173 clonal complex have virulence for both dogs and cats.

Clonal infection in cats and dogs can also occur independently of strains in the ST173 complex. In a shelter outbreak in Pennsylvania, large numbers of dogs and 3 pneumonic cats were infected with ST315.¹⁷ Similarly, isolates from Israeli cases 6 and 7 were allocated to ST215, a strain unrelated to either ST315 or the ST173 complex. ST215 has not been reported in association with either feline or canine disease and was originally isolated from an equine tracheal wash (https://goo.gl/y7nmTc).

Sporadic infection with S. zooepidemicus was also observed in our study. The isolate from the Vancouver Island feral cat could not be assigned to an ST because the yqiL allele could not be amplified. The other 6 alleles matched ST175 (hereafter, isolate ST175v). Neither ST175v nor the case 8 isolate (ST358) had any molecular relationship to published canine strains or to the strains identified from the cat shelters. ST175 is a strain isolated from a horse with pneumonia (https://goo.gl/bBe2ZT), whereas ST358 is a newly recognized ST with no published information regarding clonal relationships. Sporadic disease has been identified in dogs. S. zooepidemicus strains isolated from 4 pet dogs diagnosed with rhinitis, dermatitis, pleural effusion, and hemorrhagic pneumonia, respectively, were similarly disparate and distinct from clones associated with fatal pneumonia in canine shelter outbreaks.⁵

How S. zooepidemicus initially infects a dog or cat and then enters a shelter environment is unclear. The original source of the bacterium in the canine outbreaks was never identified.¹⁷ Increased incidence of oropharyngeal S. zooepidemicus colonization was demonstrated in dogs residing on horse farms,¹ and S. zooepidemicus was isolated from the nasal passages of a dog with lymphoplasmacytic rhinitis, resident of a stud farm,¹¹ suggesting that horses may be an important source of initial infection. In our study, 3 of the 9 cats were infected with strains linked to STs (ST215 and ST175) of S. zooepidemicus related to equine respiratory disease. Interspecies transmission of pathogenic strains can thus occur and may be one mechanism by which dogs and cats become infected.

Following introduction of S. zooepidemicus–free dogs to a kennel for >7 d, 20% of the introduced dogs cultured positive for the bacterium on oropharyngeal swabs, indicative of the existence of enzootic S. zooepidemicus infection in the kennel and the potential for transmission.⁵ Recovery of the pathogenic clonal strain ST363 from the tonsil of case 4 in BC shelter 2 suggests that an enzootic reservoir of the bacterium may also exist in cat shelters from which opportunistic disease can arise. Interestingly, in 2009, a cat from BC shelter 2 died from S. zooepidemicus rhinitis and meningitis.³ Unfortunately, that isolate was unavailable for MLST analysis in our study.

Cats in the Israeli outbreak had clinical signs of rhinitis and were positive on culture for S. zooepidemicus prior to development of pneumonia, suggesting that infection was likely initiated in the nasopharynx.² Likewise, all autopsied Canadian cats exhibited rhinitis from which S. zooepidemicus was isolated. The chronic plasmacytic rhinitis observed in cats in the Canadian shelter outbreak suggested that the bacterium was present in the nasopharynx for at least 1 wk prior to death.³ In the Israeli outbreak, purulent rhinitis preceded the peak of mortality by ~10 mo, suggesting that S. zooepidemicus persisted in the cattery for a long period.²

Bacterial pathogenicity can be potentiated by coinfection with other pathogens. Pneumonia is more readily reproduced experimentally in dogs by intranasal inoculation of S. zooepidemicus with canine influenza virus than with S. zooepidemicus alone.⁸ Nevertheless, natural infection by S. zooepidemicus can cause severe outbreaks of canine disease in the absence of coinfections,¹³ indicative of the innate virulence of canine strains. Of the Canadian cats tested for co-pathogens, one was positive on PCR analysis for FHV³ and case 8 was positive for C. felis and M. felis, although neither cat exhibited microscopic evidence of disease caused by these agents. Case 8 was also coinfected with B. bronchiseptica. Ancillary testing was deemed unnecessary on the other 3 Canadian cats given the lack of gross or microscopic evidence for coinfection. A study of cats rescued from 4 large U.S. hoarding incidents found a high prevalence of S. zooepidemicus (55%; 51 of 92) in cats with upper respiratory disease (URD) by PCR analysis of pooled nasopharyngeal and conjunctival samples.¹² Most of these S. zooepidemicus–positive cats were coinfected with FCV (88%) and M. felis (78%).¹² Thus, coinfection has the potential to play a role in feline S. zooepidemicus–linked disease. Whether coinfection significantly contributes to the pathogenesis of S. zooepidemicus disease is unclear, and further studies are needed to determine the role of co-pathogens in cats.

The 55% prevalence of S. zooepidemicus in hoarded cats may reflect increased opportunity for transmission of enzootic infection associated with long-term residence in crowded and unhygienic environments. In the hoarding study, prevalence was determined only for those cats with URD and was not attempted for all 2,374 cats seized. Hence, it is unknown if the high prevalence was correlated only with the presence of URD or was unrelated to disease status.¹² Given the greater sensitivity of PCR testing when compared to bacterial culture, prevalence determined by molecular testing cannot be compared directly with bacterial culture results reported in other feline prevalence studies.^1,6 However, the high rate of molecular detection of S. zooepidemicus in hoarded cats with URD is intriguing and suggests that prevalence may be underestimated by oropharyngeal culture alone.

Cats with pneumonia or meningitis were not reported from the hoarding seizures,¹² suggesting that S. zooepidemicus may not be highly pathogenic for cats, or that insufficient numbers of the bacterium were present to initiate disease, or that the factors that trigger rapid proliferation of S. zooepidemicus and/or immune compromise leading to acute and fulminating disease were absent. Cats residing in shelters are reported to be subject to significant stress, which may render them susceptible to infectious disease via increased cortisol production and consequent immunosuppression.¹⁸ However, the cat-hoarding observations suggest that stress may not be a significant factor for development of fatal S. zooepidemicus disease. More scrutiny is needed to further elucidate how S. zooepidemicus infection is initiated and maintained and how disease is triggered in cats.