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Detection of Chlamydophila caviae and Streptococcus equi subsp. zooepidemicus in horses with signs of rhinitis and conjunctivitis
Veterinary Microbiology 142 (2010) 440–444
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Detection of Chlamydophila caviae and Streptococcus equi subsp. zooepidemicus in horses with signs of rhinitis and conjunctivitis Wolfgang Gaede a, Karl-Friedrich Reckling a, Anette Schliephake a, Dirk Missal a, Helmut Hotzel b, Konrad Sachse c,* a b c
State Institute for Consumer Protection of Saxony-Anhalt, Dept. for Veterinary Medicine, Stendal, Germany Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Institute of Bacterial Infections and Zoonoses, Jena, Germany Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Institute of Molecular Pathogenesis, Naumburger Str. 96a, 07743 Jena, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 July 2009 Received in revised form 12 October 2009 Accepted 13 October 2009
At a stud farm of Trakehner horses, all 33 foals of a birth cohort developed conjunctivitis and serous to muco-purulent rhinitis, and 7 older horses showed recurrent signs of conjunctivitis. Examination of nasal and conjunctival swabs by bacterial and cell culture, as well as real-time PCR, ArrayTube microarray analysis and DNA sequencing led to the identification of Chlamydophila (C.) caviae (first description in horses) and Streptococcus (S.) equi subsp. zooepidemicus. We presume a synergistic effect associated with these two agents by hypothesising that primary lesions were set by C. caviae and subsequently aggravated by Streptococcus equi subsp. zooepidemicus. Indications supporting this assumption include (i) the conjunctivitis caused by mono-infection with C. caviae, (ii) recurrent clinical symptoms in the affected animals, and (iii) the absence of a sustained clinical effect of antibiotic therapy with trimethoprim–sulfonamide, enrofloxacin and amoxicillin. The detection of C. caviae in horses raises questions about the significance and natural host range of this agent. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Chlamydophila caviae Streptococcus equi subsp. zooepidemicus Conjunctivitis Rhinitis Horse Real-time PCR DNA microarray testing
1. Introduction Infections of horses with chlamydiae have been known for decades, although it is not quite certain whether the horse represents a natural or aberrant host for these obligate intracellular bacteria. Up until the end of the 1990s, a number of papers reported clinical cases ranging from respiratory disease (McChesney et al., 1982; Moorthy and Spradbrow, 1978; Popovici and Hiastru, 1968), to conjunctivitis (Moorthy and Spradbrow, 1978; Pienaar and Schutte, 1975), polyarthritis (Blanco Loizeiler et al., 1976; McChesney et al., 1974; Pienaar and Schutte, 1975), encephalo-hepatitis (Blanco Loizeiler, 1968) and abortion (Blanco Loizeiler et al., 1976; Bocklisch et al., 1991; Dilbeck
* Corresponding author. Tel.: +49 3641 804334; fax: +49 3641 804228. E-mail addresses: konrad.sachse@fli.bund.de, [email protected] (K. Sachse). 0378-1135/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2009.10.011
et al., 1985; Gla´vits et al., 1988; Lehmann and Elze, 1997; Pienaar and Schutte, 1975; Popovici and Hiastru, 1968). These cases were ascribed to Chlamydia psittaci (old classification), albeit diagnostic testing was often confined to serology. An isolate from a horse with serous nasal discharge (Wills et al., 1990) was later reclassified as Chlamydia pneumoniae (Storey et al., 1993). More recently, investigations of equine abortion cases revealed involvement of Chlamydophila (C.) psittaci (Henning et al., 2000; Szeredi et al., 2005), which had been redefined as a species in a revised taxonomic scheme (Everett et al., 1999). In a study on horses with recurrent airway obstruction, Theegarten et al. (2008) identified both C. psittaci and C. abortus in healthy and diseased horses, but signs of acute chlamydial infection combined with inflammation of lung tissue were only observed in clinical cases. In contrast, Mair and Wills (1992) isolated Chlamydia psittaci from 15 out of 300 horses, but found no association between the presence of these bacteria and signs of clinical ocular or respiratory disease.
W. Gaede et al. / Veterinary Microbiology 142 (2010) 440–444
While S. equi subsp. zooepidemicus is considered to be part of the normal upper respiratory tract flora (Clark et al., 2008; Timoney, 2004), the agent was the most frequently isolated pathogen in clinical respiratory disease of young horses, typically associated with pharyngitis and purulent pneumonia (Clark et al., 2008; Rolle and Mayr, 2007). Chapman et al. (2000) and Clark et al. (2008) detected this bacterium at high rates in trachea samples. It seems that imbalances and disturbances in the interaction between pathogen and host defence, which can be triggered by viral co-infections, high summer temperatures, tissue injury or transport stress, are a prerequisite for the occurrence of clinical manifestations (Oikawa et al., 1994; Timoney, 2004). In the present report, we describe an outbreak of conjunctivitis and rhinitis at a horse farm where mixed infection with chlamydiae and S. equi subsp. zooepidemicus was diagnosed. Rather unexpectedly, the chlamydial agent involved was identified as C. caviae. 2. Materials and methods 2.1. Case history In the period from April 27 to the end of June 2006, each newborn foal of the 7 Trakehner mares of a stud farm of 40 horses in the state of Saxony-Anhalt developed conjunctivitis, rhinitis and cough. The symptoms started at the age of 2–3 weeks. After about 4 days, the signs of rhinitis changed from serous to mucous-purulent in most cases. The body temperature was 38.6–38.8 8C during the acute period. One foal died from pneumonia at the age of 8 weeks in July. In addition, diarrhoea was also observed in several foals beginning 1–2 days after the first respiratory symptoms and before antibiotic treatment. All horses of the farm periodically showed signs of conjunctivitis. The initial antibiotic therapy (1st to 8th week) included two intramuscular applications of penicillin/streptomycin (given two times on two consecutive days) before the foals were sent to pasture. Subsequently, a trimethoprim/ sulfonamide combination was given orally on five consecutive days, which stopped purulent inflammations, but recurrent symptoms were observed a few days later (repeated every other week by the owner). Amoxicillin was also given once in between (week 6). In the second treatment cycle (week 9), Baytril1(enrofloxacin) was orally administered for 5 days. Although, in the third cycle (week 10), the administration period of trimethoprim/sulfonamide was extended to 10 days, clinical symptoms were recurring. Finally, another Baytril treatment for 5 days was conducted in week 14. As the described manifestations of the disease recurred at the end of August of the same year, i.e. 14 days after the end of antibiotic therapy, the owner agreed to sampling of all diseased foals and laboratory testing of the specimens. 2.2. Samples and initial processing Conjunctival and nasal swabs were taken for standard bacteriological, virological and DNA testing, as well as isolation of chlamydiae from 6 foals that survived the first
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infection. The procedure for detection of bacterial agents comprised growth on blood agar (Oxoid, Wesel, Germany, Cat.-Nb. PB 5177 A) under aerobic and micro-aerobic conditions with 12% CO2 at 37 8C, as well as selective Gassner agar (Oxoid, Cat.-Nr. PO 5021 A), selective Pasteurella agar (Oxoid, Cat.-Nr. PB 5175 A) and selective Mycoplasma agar (Oxoid, Cat.-Nr. CM 0401, including supplement Cat.-Nr. SR 0059C). For isolation of viruses, several cell cultures were inoculated and cultivated for three passages. PCR testing was conducted for Influenza A Virus (according to Spackman et al., 2002), Equine Herpesvirus Types 1 and 4 (Benetka et al., 2002), Equine Arteritis Virus (De Vries et al., 1990) and Mycoplasma spp. (Van Kuppeveld et al., 1992). For simultaneous isolation of bacterial DNA, viral DNA and RNA from swabs, the High Pure Viral Nucleic Acid Isolation Kit (Roche Diagnostics, Mannheim, Germany) was used according to the instructions of the manufacturer. From culture fluids, DNA was extracted using the High Pure PCR Template Preparation Kit (Roche). 2.3. Real-time PCR DNA extracts of swab samples were examined using a Chlamydiaceae family-specific assay targeting the 23S rRNA gene (23S-rtPCR) based on the protocol published previously (Ehricht et al., 2006), but extended by inclusion of an internal amplification control (Pantchev et al., 2009). 2.4. ArrayTube (AT) DNA microarray testing Species identification of chlamydial agents involved was performed using the ArrayTube1 DNA microarray assay as described previously (Sachse et al., 2005; Borel et al., 2008). 2.5. DNA sequencing To verify the 23S-rtPCR and AT test data, sequencing of a discriminatory region of the 16S rRNA gene (Hotzel et al., 2005) and the whole chlamydial ompA gene (Sachse et al., 2008) was conducted as described previously. The sequences have been deposited at GenBank accession numbers GQ332574 and GQ332575, respectively. 2.6. Culture of chlamydial agents To isolate chlamydial agents, Buffalo Green Monkey (BGM) cells and yolk sacs of embryonated chicken eggs were inoculated with sample material and cultured for several passages according to standard procedures (Sachse et al., 2003; Tang et al., 1957). Replication of chlamydiae was monitored by real-time PCR of culture aliquots. 3. Results The results of PCR testing and cultural bacteriological examination are shown in Table 1. In 5 of 6 tested foals, the real-time PCR assay specific for the family Chlamydiaceae was positive. The highest titres of chlamydial agents were detected at the beginning of sampling, i.e. 14 days after the
W. Gaede et al. / Veterinary Microbiology 142 (2010) 440–444
442
Table 1 Results of DNA assays and bacteriological examination. 23S-rtPCR Chlamydiaceae (Ct)
ArrayTube assay
24.7 27.4
C. caviaea,b C. caviaea,b
n.d. n.d.
35.5 37.6 36 No Ct
C. caviae No signal No signal No signal
Aerobic sporeforming bacteria
Nasal swab
31.6
C. caviaea
16
Nasal swab
32.4
C. caviaea
2
20
Nasal swab
34.8
C. caviae
2
20
Nasal swab
34.2
C. caviae
3
19
Nasal swab
28.2
C. caviaea
4
18
Nasal swab
29.3
C. caviaea
5 5 5 3 6
10 10 10 20 10
Nasal swab Conjunctival Conjunctival Conjunctival Conjunctival
32.3 32.9 30.9 36.9 No Ct
n.d. n.d. C. caviae n.d. n.d.
S. equi ssp. zooepidemicus (as dominant bacteria) S. equi ssp. zooepidemicus (as dominant bacteria) S. equi ssp. zooepidemicus (nearly pure culture) S. equi ssp. zooepidemicus (nearly pure culture) S. equi ssp. zooepidemicus (nearly pure culture) S. equi ssp. zooepidemicus (nearly pure culture) n.d. n.d. n.d. n.d. n.d.
Age at time of sampling (weeks)
Sample type
1 2
14 18
Nasal swab Nasal swab
1 2 3 4
16 20 20 19
Conjunctival Conjunctival Conjunctival Conjunctival
1
16
1
Time of sampling (weeks after the end of antibiotic treatment)
Foal [8_TD$IF]no.
2
4
4
5
swab swab swab swab
swab swab swab swab
Bacteriological examination Main agent
Additional findings
few non-specific aerobic bacteria
Acinetobacter lwofii
Non-haemolytic Cocci Very few Pantoea agglomerans
Very few Enterobacter cloacae
n.d., not done. a Confirmed by ompA sequencing (GenBank acc. no. GQ332575). b Additionally confirmed by 16S rRNA gene sequencing (GenBank acc. no. GQ332574).
Fig. 1. DNA microarray testing of equine samples using the ArrayTube1 assay. Results are shown for a nasal swab of foal no. 1 at 14 days after the end of antibiotic treatment (A and B) and the first passage of cell culture of the same sample (C and D). (A and C) Images of the stained microarrays. (B and D) Bargraph showing specific hybridisation signals for Chlamydiaceae consensus probes (1), Chlamydophila genus-specific probes (2) and C. caviae speciesspecific probes (3), whereas the signals for the rest of the chlamydia species-specific probes (4) are absent or represent non-specific cross-hybridisation. (Signal 5 in (D) represents a cross-hybridisation with one of the three probes for Waddlia chondrophila.)
W. Gaede et al. / Veterinary Microbiology 142 (2010) 440–444
end of antibiotic treatment. Furthermore, the concentrations of Chlamydiaceae DNA in nasal swabs were higher than in conjunctival swabs. For foal no. 4, the real-time PCR assay was positive only in the nasal swab. From six nasal swabs of four foals, S. equi subsp. zooepidemicus was successfully cultured and isolated as virtually pure or mixed culture. Antibiograms showed sensitivity of the isolated streptococci to b-lactam antibiotics, tetracyclines, fluoroquinolones and aminoglycosides (data not shown). Viral and mycoplasma testing of samples proved negative. Identification of the chlamydial agent at species level was conducted using the AT DNA microarray assay, because this test allows parallel detection of all 9 currently defined species of Chlamydiaceae. AT testing revealed the presence of C. caviae as the only chlamydial species (Table 1). Typical hybridisation patterns obtained by AT testing of a nasal swab of foal no. 1 at 14 days after the end of antibiotic treatment and the first passage of cell culture of the same sample are shown in Fig. 1. The identity of the chlamydial agent was confirmed by sequencing of the signature sequence of the 16S ribosomal RNA gene (GenBank acc. no. GQ332574) and of the complete ompA gene (GenBank acc. no. GQ332575). PCR products from swab samples of foals 1–4 (cf. Table 1) were used as templates. BLAST analysis of the sequences revealed homologies of 99 and 92%, respectively, to C. caviae type strain GPIC. No sequence differences among individual samples were observed, thus suggesting that only one strain of C. caviae was involved. Despite initial signs of chlamydial growth in BGM cell culture and embryonated chicken eggs we failed to obtain a continuously replicating cultural isolate. 4. Discussion The present study revealed a mixed bacterial infection involving C. caviae and S. equi subsp. zooepidemicus as the presumptive causative agents of the outbreak of respiratory and ocular disease in a stud farm. So far, the list of chlamydial agents identified in horses has been limited to C. pneumoniae, C. psittaci and C. abortus. This report provides the first evidence on the occurrence of C. caviae in horses and its likely involvement in respiratory disease. Interestingly, after completion of the present study, the laboratory of one of the authors (KS) again detected this agent in respiratory samples from an unrelated horse breeding farm (data not shown), which might indicate a wider dissemination. In contrast to the present findings, C. caviae has been considered to possess high host specificity for guinea pigs (Everett, 2000). In any case, the detection of this agent raises new questions since the route of infection remains unclear. As there were no guinea pigs at the farm, nor do these animals roam freely in Central Europe, their possible role as source of infection can be ruled out. As highly sensitive and species-specific diagnostic tools for chlamydiae became available only in the 1990s, it is not quite clear whether the present infection of horses with C. caviae represents a unique event. Moreover, the traditional taxonomy did not feature C. caviae as a separate species, but
443
rather subsumed it under the taxon of Chlamydia psittaci. Thus, studies of chlamydial infections in horses mentioning involvement of Chlamydia psittaci which were conducted before the molecular diagnostic age could unwittingly also have referred to C. caviae. In the present study, we used different DNA-based diagnostic tests targeting three different genomic sites, i.e. 23S rRNA gene (real-time PCR and AT test), ompA gene (sequencing) and 16S rRNA gene (sequencing), all of which identified C. caviae. In the case of the ompA gene sequence, a similarity of 92% to the C. caviae type strain is still within the intra-species variability range of this highly variable membrane protein gene. In view of the presence of C. caviae in nearly all swabs from the group of foals, it appears reasonable to assume a contribution of this agent to the described clinical manifestations. In addition, the fact that only C. caviae was found in the conjunctival swabs indicates an association with the conjunctivitis observed in the same animals, even more so since C. caviae is a known agent of conjunctivitis in guinea pigs. Nevertheless, its role as a primary causative agent cannot be defined unambiguously based on the present data. In the present outbreak, C. caviae probably developed synergistic effects with S. equi subsp. zooepidemicus. As a mucosal commensal in horses (Hirsh et al., 2004), the latter requires precursors (Timoney, 2004). We hypothesise that primary lesions were set by C. caviae and subsequently aggravated by Streptococcus equi subsp. zooepidemicus. Indications supporting this assumption include (i) the conjunctivitis caused by mono-infection with C. caviae, (ii) recurrent clinical symptoms in the affected animals, and (iii) the absence of a sustained clinical effect of antibiotic therapy with trimethoprim–sulfonamide, enrofloxacin and amoxicillin. The latter is usually effective against S. equi subsp. zooepidemicus. The long-lasting antibiotic therapy of the diseased foals is the most likely explanation for our inability to grow the C. caviae strain(s) involved, as chlamydiae are known to pass into a state of persistence upon antibiotic treatment. As a general conclusion from the present findings, practitioners and laboratory diagnosticians should consider the possibility of C. caviae being involved in equine infections. Species-specific tests should be used to enable assessment of the clinical importance and zoonotic potential of the infectious agent involved. Finally, the rather unexpected identification of C. caviae in the present case also indicates that our current understanding of host ranges of Chlamydiaceae spp. is not yet complete. 5. Conflict of interest statement None of the authors (Wolfgang Gaede, Karl-Friedrich Reckling, Anette Schliephake, Dirk Missal, Helmut Hotzel, and Konrad Sachse) has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the paper. Acknowledgements We thank Christine Grajetzki, Simone Bettermann and Karola Zmuda for excellent technical assistance.
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Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Detection of Chlamydophila caviae and Streptococcus equi subsp. zooepidemicus in horses with signs of rhinitis and conjunctivitis Wolfgang Gaede a, Karl-Friedrich Reckling a, Anette Schliephake a, Dirk Missal a, Helmut Hotzel b, Konrad Sachse c,* a b c
State Institute for Consumer Protection of Saxony-Anhalt, Dept. for Veterinary Medicine, Stendal, Germany Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Institute of Bacterial Infections and Zoonoses, Jena, Germany Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Institute of Molecular Pathogenesis, Naumburger Str. 96a, 07743 Jena, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 July 2009 Received in revised form 12 October 2009 Accepted 13 October 2009
At a stud farm of Trakehner horses, all 33 foals of a birth cohort developed conjunctivitis and serous to muco-purulent rhinitis, and 7 older horses showed recurrent signs of conjunctivitis. Examination of nasal and conjunctival swabs by bacterial and cell culture, as well as real-time PCR, ArrayTube microarray analysis and DNA sequencing led to the identification of Chlamydophila (C.) caviae (first description in horses) and Streptococcus (S.) equi subsp. zooepidemicus. We presume a synergistic effect associated with these two agents by hypothesising that primary lesions were set by C. caviae and subsequently aggravated by Streptococcus equi subsp. zooepidemicus. Indications supporting this assumption include (i) the conjunctivitis caused by mono-infection with C. caviae, (ii) recurrent clinical symptoms in the affected animals, and (iii) the absence of a sustained clinical effect of antibiotic therapy with trimethoprim–sulfonamide, enrofloxacin and amoxicillin. The detection of C. caviae in horses raises questions about the significance and natural host range of this agent. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Chlamydophila caviae Streptococcus equi subsp. zooepidemicus Conjunctivitis Rhinitis Horse Real-time PCR DNA microarray testing
1. Introduction Infections of horses with chlamydiae have been known for decades, although it is not quite certain whether the horse represents a natural or aberrant host for these obligate intracellular bacteria. Up until the end of the 1990s, a number of papers reported clinical cases ranging from respiratory disease (McChesney et al., 1982; Moorthy and Spradbrow, 1978; Popovici and Hiastru, 1968), to conjunctivitis (Moorthy and Spradbrow, 1978; Pienaar and Schutte, 1975), polyarthritis (Blanco Loizeiler et al., 1976; McChesney et al., 1974; Pienaar and Schutte, 1975), encephalo-hepatitis (Blanco Loizeiler, 1968) and abortion (Blanco Loizeiler et al., 1976; Bocklisch et al., 1991; Dilbeck
* Corresponding author. Tel.: +49 3641 804334; fax: +49 3641 804228. E-mail addresses: konrad.sachse@fli.bund.de, [email protected] (K. Sachse). 0378-1135/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2009.10.011
et al., 1985; Gla´vits et al., 1988; Lehmann and Elze, 1997; Pienaar and Schutte, 1975; Popovici and Hiastru, 1968). These cases were ascribed to Chlamydia psittaci (old classification), albeit diagnostic testing was often confined to serology. An isolate from a horse with serous nasal discharge (Wills et al., 1990) was later reclassified as Chlamydia pneumoniae (Storey et al., 1993). More recently, investigations of equine abortion cases revealed involvement of Chlamydophila (C.) psittaci (Henning et al., 2000; Szeredi et al., 2005), which had been redefined as a species in a revised taxonomic scheme (Everett et al., 1999). In a study on horses with recurrent airway obstruction, Theegarten et al. (2008) identified both C. psittaci and C. abortus in healthy and diseased horses, but signs of acute chlamydial infection combined with inflammation of lung tissue were only observed in clinical cases. In contrast, Mair and Wills (1992) isolated Chlamydia psittaci from 15 out of 300 horses, but found no association between the presence of these bacteria and signs of clinical ocular or respiratory disease.
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While S. equi subsp. zooepidemicus is considered to be part of the normal upper respiratory tract flora (Clark et al., 2008; Timoney, 2004), the agent was the most frequently isolated pathogen in clinical respiratory disease of young horses, typically associated with pharyngitis and purulent pneumonia (Clark et al., 2008; Rolle and Mayr, 2007). Chapman et al. (2000) and Clark et al. (2008) detected this bacterium at high rates in trachea samples. It seems that imbalances and disturbances in the interaction between pathogen and host defence, which can be triggered by viral co-infections, high summer temperatures, tissue injury or transport stress, are a prerequisite for the occurrence of clinical manifestations (Oikawa et al., 1994; Timoney, 2004). In the present report, we describe an outbreak of conjunctivitis and rhinitis at a horse farm where mixed infection with chlamydiae and S. equi subsp. zooepidemicus was diagnosed. Rather unexpectedly, the chlamydial agent involved was identified as C. caviae. 2. Materials and methods 2.1. Case history In the period from April 27 to the end of June 2006, each newborn foal of the 7 Trakehner mares of a stud farm of 40 horses in the state of Saxony-Anhalt developed conjunctivitis, rhinitis and cough. The symptoms started at the age of 2–3 weeks. After about 4 days, the signs of rhinitis changed from serous to mucous-purulent in most cases. The body temperature was 38.6–38.8 8C during the acute period. One foal died from pneumonia at the age of 8 weeks in July. In addition, diarrhoea was also observed in several foals beginning 1–2 days after the first respiratory symptoms and before antibiotic treatment. All horses of the farm periodically showed signs of conjunctivitis. The initial antibiotic therapy (1st to 8th week) included two intramuscular applications of penicillin/streptomycin (given two times on two consecutive days) before the foals were sent to pasture. Subsequently, a trimethoprim/ sulfonamide combination was given orally on five consecutive days, which stopped purulent inflammations, but recurrent symptoms were observed a few days later (repeated every other week by the owner). Amoxicillin was also given once in between (week 6). In the second treatment cycle (week 9), Baytril1(enrofloxacin) was orally administered for 5 days. Although, in the third cycle (week 10), the administration period of trimethoprim/sulfonamide was extended to 10 days, clinical symptoms were recurring. Finally, another Baytril treatment for 5 days was conducted in week 14. As the described manifestations of the disease recurred at the end of August of the same year, i.e. 14 days after the end of antibiotic therapy, the owner agreed to sampling of all diseased foals and laboratory testing of the specimens. 2.2. Samples and initial processing Conjunctival and nasal swabs were taken for standard bacteriological, virological and DNA testing, as well as isolation of chlamydiae from 6 foals that survived the first
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infection. The procedure for detection of bacterial agents comprised growth on blood agar (Oxoid, Wesel, Germany, Cat.-Nb. PB 5177 A) under aerobic and micro-aerobic conditions with 12% CO2 at 37 8C, as well as selective Gassner agar (Oxoid, Cat.-Nr. PO 5021 A), selective Pasteurella agar (Oxoid, Cat.-Nr. PB 5175 A) and selective Mycoplasma agar (Oxoid, Cat.-Nr. CM 0401, including supplement Cat.-Nr. SR 0059C). For isolation of viruses, several cell cultures were inoculated and cultivated for three passages. PCR testing was conducted for Influenza A Virus (according to Spackman et al., 2002), Equine Herpesvirus Types 1 and 4 (Benetka et al., 2002), Equine Arteritis Virus (De Vries et al., 1990) and Mycoplasma spp. (Van Kuppeveld et al., 1992). For simultaneous isolation of bacterial DNA, viral DNA and RNA from swabs, the High Pure Viral Nucleic Acid Isolation Kit (Roche Diagnostics, Mannheim, Germany) was used according to the instructions of the manufacturer. From culture fluids, DNA was extracted using the High Pure PCR Template Preparation Kit (Roche). 2.3. Real-time PCR DNA extracts of swab samples were examined using a Chlamydiaceae family-specific assay targeting the 23S rRNA gene (23S-rtPCR) based on the protocol published previously (Ehricht et al., 2006), but extended by inclusion of an internal amplification control (Pantchev et al., 2009). 2.4. ArrayTube (AT) DNA microarray testing Species identification of chlamydial agents involved was performed using the ArrayTube1 DNA microarray assay as described previously (Sachse et al., 2005; Borel et al., 2008). 2.5. DNA sequencing To verify the 23S-rtPCR and AT test data, sequencing of a discriminatory region of the 16S rRNA gene (Hotzel et al., 2005) and the whole chlamydial ompA gene (Sachse et al., 2008) was conducted as described previously. The sequences have been deposited at GenBank accession numbers GQ332574 and GQ332575, respectively. 2.6. Culture of chlamydial agents To isolate chlamydial agents, Buffalo Green Monkey (BGM) cells and yolk sacs of embryonated chicken eggs were inoculated with sample material and cultured for several passages according to standard procedures (Sachse et al., 2003; Tang et al., 1957). Replication of chlamydiae was monitored by real-time PCR of culture aliquots. 3. Results The results of PCR testing and cultural bacteriological examination are shown in Table 1. In 5 of 6 tested foals, the real-time PCR assay specific for the family Chlamydiaceae was positive. The highest titres of chlamydial agents were detected at the beginning of sampling, i.e. 14 days after the
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Table 1 Results of DNA assays and bacteriological examination. 23S-rtPCR Chlamydiaceae (Ct)
ArrayTube assay
24.7 27.4
C. caviaea,b C. caviaea,b
n.d. n.d.
35.5 37.6 36 No Ct
C. caviae No signal No signal No signal
Aerobic sporeforming bacteria
Nasal swab
31.6
C. caviaea
16
Nasal swab
32.4
C. caviaea
2
20
Nasal swab
34.8
C. caviae
2
20
Nasal swab
34.2
C. caviae
3
19
Nasal swab
28.2
C. caviaea
4
18
Nasal swab
29.3
C. caviaea
5 5 5 3 6
10 10 10 20 10
Nasal swab Conjunctival Conjunctival Conjunctival Conjunctival
32.3 32.9 30.9 36.9 No Ct
n.d. n.d. C. caviae n.d. n.d.
S. equi ssp. zooepidemicus (as dominant bacteria) S. equi ssp. zooepidemicus (as dominant bacteria) S. equi ssp. zooepidemicus (nearly pure culture) S. equi ssp. zooepidemicus (nearly pure culture) S. equi ssp. zooepidemicus (nearly pure culture) S. equi ssp. zooepidemicus (nearly pure culture) n.d. n.d. n.d. n.d. n.d.
Age at time of sampling (weeks)
Sample type
1 2
14 18
Nasal swab Nasal swab
1 2 3 4
16 20 20 19
Conjunctival Conjunctival Conjunctival Conjunctival
1
16
1
Time of sampling (weeks after the end of antibiotic treatment)
Foal [8_TD$IF]no.
2
4
4
5
swab swab swab swab
swab swab swab swab
Bacteriological examination Main agent
Additional findings
few non-specific aerobic bacteria
Acinetobacter lwofii
Non-haemolytic Cocci Very few Pantoea agglomerans
Very few Enterobacter cloacae
n.d., not done. a Confirmed by ompA sequencing (GenBank acc. no. GQ332575). b Additionally confirmed by 16S rRNA gene sequencing (GenBank acc. no. GQ332574).
Fig. 1. DNA microarray testing of equine samples using the ArrayTube1 assay. Results are shown for a nasal swab of foal no. 1 at 14 days after the end of antibiotic treatment (A and B) and the first passage of cell culture of the same sample (C and D). (A and C) Images of the stained microarrays. (B and D) Bargraph showing specific hybridisation signals for Chlamydiaceae consensus probes (1), Chlamydophila genus-specific probes (2) and C. caviae speciesspecific probes (3), whereas the signals for the rest of the chlamydia species-specific probes (4) are absent or represent non-specific cross-hybridisation. (Signal 5 in (D) represents a cross-hybridisation with one of the three probes for Waddlia chondrophila.)
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end of antibiotic treatment. Furthermore, the concentrations of Chlamydiaceae DNA in nasal swabs were higher than in conjunctival swabs. For foal no. 4, the real-time PCR assay was positive only in the nasal swab. From six nasal swabs of four foals, S. equi subsp. zooepidemicus was successfully cultured and isolated as virtually pure or mixed culture. Antibiograms showed sensitivity of the isolated streptococci to b-lactam antibiotics, tetracyclines, fluoroquinolones and aminoglycosides (data not shown). Viral and mycoplasma testing of samples proved negative. Identification of the chlamydial agent at species level was conducted using the AT DNA microarray assay, because this test allows parallel detection of all 9 currently defined species of Chlamydiaceae. AT testing revealed the presence of C. caviae as the only chlamydial species (Table 1). Typical hybridisation patterns obtained by AT testing of a nasal swab of foal no. 1 at 14 days after the end of antibiotic treatment and the first passage of cell culture of the same sample are shown in Fig. 1. The identity of the chlamydial agent was confirmed by sequencing of the signature sequence of the 16S ribosomal RNA gene (GenBank acc. no. GQ332574) and of the complete ompA gene (GenBank acc. no. GQ332575). PCR products from swab samples of foals 1–4 (cf. Table 1) were used as templates. BLAST analysis of the sequences revealed homologies of 99 and 92%, respectively, to C. caviae type strain GPIC. No sequence differences among individual samples were observed, thus suggesting that only one strain of C. caviae was involved. Despite initial signs of chlamydial growth in BGM cell culture and embryonated chicken eggs we failed to obtain a continuously replicating cultural isolate. 4. Discussion The present study revealed a mixed bacterial infection involving C. caviae and S. equi subsp. zooepidemicus as the presumptive causative agents of the outbreak of respiratory and ocular disease in a stud farm. So far, the list of chlamydial agents identified in horses has been limited to C. pneumoniae, C. psittaci and C. abortus. This report provides the first evidence on the occurrence of C. caviae in horses and its likely involvement in respiratory disease. Interestingly, after completion of the present study, the laboratory of one of the authors (KS) again detected this agent in respiratory samples from an unrelated horse breeding farm (data not shown), which might indicate a wider dissemination. In contrast to the present findings, C. caviae has been considered to possess high host specificity for guinea pigs (Everett, 2000). In any case, the detection of this agent raises new questions since the route of infection remains unclear. As there were no guinea pigs at the farm, nor do these animals roam freely in Central Europe, their possible role as source of infection can be ruled out. As highly sensitive and species-specific diagnostic tools for chlamydiae became available only in the 1990s, it is not quite clear whether the present infection of horses with C. caviae represents a unique event. Moreover, the traditional taxonomy did not feature C. caviae as a separate species, but
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rather subsumed it under the taxon of Chlamydia psittaci. Thus, studies of chlamydial infections in horses mentioning involvement of Chlamydia psittaci which were conducted before the molecular diagnostic age could unwittingly also have referred to C. caviae. In the present study, we used different DNA-based diagnostic tests targeting three different genomic sites, i.e. 23S rRNA gene (real-time PCR and AT test), ompA gene (sequencing) and 16S rRNA gene (sequencing), all of which identified C. caviae. In the case of the ompA gene sequence, a similarity of 92% to the C. caviae type strain is still within the intra-species variability range of this highly variable membrane protein gene. In view of the presence of C. caviae in nearly all swabs from the group of foals, it appears reasonable to assume a contribution of this agent to the described clinical manifestations. In addition, the fact that only C. caviae was found in the conjunctival swabs indicates an association with the conjunctivitis observed in the same animals, even more so since C. caviae is a known agent of conjunctivitis in guinea pigs. Nevertheless, its role as a primary causative agent cannot be defined unambiguously based on the present data. In the present outbreak, C. caviae probably developed synergistic effects with S. equi subsp. zooepidemicus. As a mucosal commensal in horses (Hirsh et al., 2004), the latter requires precursors (Timoney, 2004). We hypothesise that primary lesions were set by C. caviae and subsequently aggravated by Streptococcus equi subsp. zooepidemicus. Indications supporting this assumption include (i) the conjunctivitis caused by mono-infection with C. caviae, (ii) recurrent clinical symptoms in the affected animals, and (iii) the absence of a sustained clinical effect of antibiotic therapy with trimethoprim–sulfonamide, enrofloxacin and amoxicillin. The latter is usually effective against S. equi subsp. zooepidemicus. The long-lasting antibiotic therapy of the diseased foals is the most likely explanation for our inability to grow the C. caviae strain(s) involved, as chlamydiae are known to pass into a state of persistence upon antibiotic treatment. As a general conclusion from the present findings, practitioners and laboratory diagnosticians should consider the possibility of C. caviae being involved in equine infections. Species-specific tests should be used to enable assessment of the clinical importance and zoonotic potential of the infectious agent involved. Finally, the rather unexpected identification of C. caviae in the present case also indicates that our current understanding of host ranges of Chlamydiaceae spp. is not yet complete. 5. Conflict of interest statement None of the authors (Wolfgang Gaede, Karl-Friedrich Reckling, Anette Schliephake, Dirk Missal, Helmut Hotzel, and Konrad Sachse) has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the paper. Acknowledgements We thank Christine Grajetzki, Simone Bettermann and Karola Zmuda for excellent technical assistance.
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