Detection of and subsp. in horses with signs of rhinitis and conjunctivitis

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Detection of and subsp. in horses with signs of rhinitis and conjunctivitis

Wolfgang Gaede, Karl-Friedrich Reckling, Anette Schliephake, Dirk Missal, Helmut Hotzel, Konrad Sachse

To cite this version:

Wolfgang Gaede, Karl-Friedrich Reckling, Anette Schliephake, Dirk Missal, Helmut Hotzel, et al..

Detection of and subsp. in horses with signs of rhinitis and conjunctivitis. Veterinary Microbiology,

Elsevier, 2010, 142 (3-4), pp.440. �10.1016/j.vetmic.2009.10.011�. �hal-00587283�

Accepted Manuscript

Title: Detection of Chlamydophila caviae and Streptococcus equi subsp. zooepidemicus in horses with signs of rhinitis and conjunctivitis

Authors: Wolfgang Gaede, Karl-Friedrich Reckling, Anette Schliephake, Dirk Missal, Helmut Hotzel, Konrad Sachse

PII: S0378-1135(09)00531-8

DOI: doi:10.1016/j.vetmic.2009.10.011

Reference: VETMIC 4639

To appear in: VETMIC Received date: 1-7-2009 Revised date: 12-10-2009 Accepted date: 13-10-2009

Please cite this article as: Gaede, W., Reckling, K.-F., Schliephake, A., Missal, D., Hotzel, H., Sachse, K., Detection of Chlamydophila caviae and Streptococcus equi subsp. zooepidemicus in horses with signs of rhinitis and conjunctivitis, Veterinary Microbiology (2008), doi:10.1016/j.vetmic.2009.10.011

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Accepted Manuscript

S HORT C OMMUNICATION 1

2

Detection of Chlamydophila caviae and Streptococcus equi subsp. zooepi-

3

demicus in horses with signs of rhinitis and conjunctivitis

4 5 6

Wolfgang Gaede ^a , Karl-Friedrich Reckling ^a , Anette Schliephake ^a , Dirk Missal ^a , Helmut 7

Hotzel ^b , and Konrad Sachse ^c * 8

10 9

a State Institute for Consumer Protection of Saxony-Anhalt, Dept. for Veterinary Medicine, 11

Stendal, Germany 12

b Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Institute of Bacte- 13

rial Infections and Zoonoses, Jena, Germany 14

c Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Institute of Mo- 15

lecular Pathogenesis, Jena, Germany 16

17 18 19

*Corresponding author:

20

Tel. +49-3641-804334, fax +49-3641-804228, e-mail: konrad.sachse@fli.bund.de 21

22 23 24 25

Abstract 26

At a stud farm of Trakehner horses, all 33 foals of a birth cohort developed conjunctivitis and 27

serous to muco-purulent rhinitis, and 7 older horses showed recurrent signs of conjunctivitis.

28

Examination of nasal and conjunctival swabs by bacterial and cell culture, as well as real-time 29

PCR, ArrayTube microarray analysis and DNA sequencing led to the identification of Chla- 30

mydophila (C.) caviae (first description in horses) and Streptococcus (S.) equi subsp. zooepi- 31

demicus. We presume a synergistic effect associated with these two agents by hypothesising 32

that primary lesions were set by C. caviae and subsequently aggravated by Streptococcus equi 33

subsp. zooepidemicus. Indications supporting this assumption include i) the conjunctivitis 34

caused by mono-infection with C. caviae, ii) recurrent clinical symptoms in the affected ani- 35

mals, and iii) the absence of a sustained clinical effect of antibiotic therapy with 36

trimethoprim-sulfonamide, enrofloxacin and amoxicillin. The detection of C. caviae in horses 37

raises questions about the significance and natural host range of this agent.

38

*Manuscript

Accepted Manuscript

39 40

Keywords 41

Chlamydophila caviae, Streptococcus equi subsp. zooepidemicus, conjunctivitis, rhinitis, 42

horse, real-time PCR, DNA microarray testing 43

44

1. Introduction 45

Infections of horses with chlamydiae have been known for decades, although it is not quite 46

certain whether the horse represents a natural or aberrant host for these obligate intracellular 47

bacteria. Up until the end of the 1990s, a number of papers reported clinical cases ranging 48

from respiratory disease (McChesney et al., 1982; Moorthy and Spradbrow, 1978; Popovici 49

and Hiastru, 1968), to conjunctivitis (Moorthy and Spradbrow, 1978; Pienaar and Schutte, 50

1975), polyarthritis (Blanco Loizeiler et al., 1976; McChesney et al., 1974; Pienaar and 51

Schutte, 1975), encephalo-hepatitis (Blanco Loizeiler, 1968) and abortion (Blanco Loizeiler et 52

al., 1976; Bocklisch et al., 1991; Dilbeck et al., 1985; Glávits et al., 1988, Lehmann and Elze, 53

1997; Pienaar and Schutte, 1975; Popovici and Hiastru, 1968). These cases were ascribed to 54

Chlamydia psittaci (old classification), albeit diagnostic testing was often confined to serol- 55

ogy. An isolate from a horse with serous nasal discharge (Wills et al., 1990) was later reclas- 56

sified as Chlamydia pneumoniae (Storey et al., 1993). More recently, investigations of equine 57

abortion cases revealed involvement of Chlamydophila (C.) psittaci (Henning et al. 2000;

58

Szeredi et al. 2005), which had been redefined as a species in a revised taxonomic scheme 59

(Everett et al., 1999). In a study on horses with recurrent airway obstruction, Theegarten et al.

60

(2008) identified both C. psittaci and C. abortus in healthy and diseased horses, but signs of 61

acute chlamydial infection combined with inflammation of lung tissue were only observed in 62

clinical cases. In contrast, Mair and Wills (1992) isolated Chlamydia psittaci from 15 out of 63

300 horses, but found no association between the presence of these bacteria and signs of 64

clinical ocular or respiratory disease.

65

Accepted Manuscript

While S. equi subsp. zooepidemicus is considered to be part of the normal upper respiratory 66

tract flora (Clark et al., 2008, Timoney, 2004), the agent was the most frequently isolated 67

pathogen in clinical respiratory disease of young horses, typically associated with pharyngitis 68

and purulent pneumonia (Clark et al., 2008, Rolle and Mayr, 2007). Chapman et al. (2000) 69

and Clark et al. (2008) detected this bacterium at high rates in trachea samples. It seems that 70

imbalances and disturbances in the interaction between pathogen and host defence, which can 71

be triggered by viral co-infections, high summer temperatures, tissue injury or transport 72

stress, are a prerequisite for the occurrence of clinical manifestations (Oikawa et al., 1994;

73

Timoney, 2004).

74

In the present report, we describe an outbreak of conjunctivitis and rhinitis at a horse farm 75

where mixed infection with chlamydiae and S. equi subsp. zooepidemicus was diagnosed.

76

Rather unexpectedly, the chlamydial agent involved was identified as C. caviae.

77 78

2. Materials and methods 79

2.1. Case history 80

In the period from April 27 to the end of June 2006, each newborn foal of the 7 Trakehner 81

mares of a stud farm of 40 horses in the state of Saxony-Anhalt developed conjunctivitis, 82

rhinitis and cough. The symptoms started at the age of 2-3 weeks. After about 4 days, the 83

signs of rhinitis changed from serous to mucous-purulent in most cases. The body temperature 84

was 38.6 – 38.8 °C during the acute period. One foal died from pneumonia at the age of 8 85

weeks in July. In addition, diarrhoea was also observed in several foals beginning 1-2 days 86

after the first respiratory symptoms and before antibiotic treatment. All horses of the farm 87

periodically showed signs of conjunctivitis.

88

The initial antibiotic therapy (1st to 8th week) included two intramuscular applications of 89

penicillin/streptomycin (given two times on two consecutive days) before the foals were sent 90

to pasture. Subsequently, a trimethoprim/sulfonamide combination was given orally on five

91

Accepted Manuscript

consecutive days, which stopped purulent inflammations, but recurrent symptoms were ob- 92

served a few days later (repeated every other week by the owner). Amoxicillin was also given 93

once in between (week 6). In the second treatment cycle (week 9), Baytril ^® (enrofloxacin) was 94

orally administered for five days. Although, in the third cycle (week 10), the administration 95

period of trimethoprim/sulfonamide was extended to 10 days, clinical symptoms were recur- 96

ring. Finally, another Baytril treatment for five days was conducted in week 14.

97

As the described manifestations of the disease recurred at the end of August of the same year, 98

i.e. 14 days after the end of antibiotic therapy, the owner agreed to sampling of all diseased 99

foals and laboratory testing of the specimens.

100 101

2.2. Samples and initial processing 102

Conjunctival and nasal swabs were taken for standard bacteriological, virological and DNA 103

testing, as well as isolation of chlamydiae from 6 foals that survived the first infection. The 104

procedure for detection of bacterial agents comprised growth on blood agar (Oxoid, Wesel, 105

Germany, Cat.-Nb. PB 5177 A) under aerobic and micro-aerobic conditions with 12% CO 2 at 106

37°C, as well as selective Gassner agar (Oxoid, Cat.-Nr. PO 5021 A), selective Pasteurella 107

agar (Oxoid, Cat.-Nr. PB 5175 A) and selective Mycoplasma agar (Oxoid, Cat.-Nr. CM 0401, 108

including supplement Cat.-Nr. SR 0059 C). For isolation of viruses, several cell cultures were 109

inoculated and cultivated for three passages. PCR testing was conducted for Influenza A Vi- 110

rus (according to Spackman et al. 2002), Equine Herpesvirus Types 1 and 4 (Benetka et al., 111

2002), Equine Arteritis Virus (de Vries et al., 1990) and Mycoplasma spp. (van Kuppeveld et 112

al., 1992).

113

For simultaneous isolation of bacterial DNA, viral DNA and RNA from swabs, the High Pure 114

Viral Nucleic Acid Isolation Kit (Roche Diagnostics, Mannheim, Germany) was used accord- 115

ing to the instructions of the manufacturer. From culture fluids, DNA was extracted using the 116

High Pure PCR Template Preparation Kit (Roche).

117

Accepted Manuscript

118

2.3. Real-time PCR 119

DNA extracts of swab samples were examined using a Chlamydiaceae family-specific assay 120

targeting the 23S rRNA gene (23S-rtPCR) based on the protocol published previously 121

(Ehricht et al., 2006), but extended by inclusion of an internal amplification control (Pantchev 122

et al., 2009).

123 124

2.4. ArrayTube (AT) DNA microarray testing 125

Species identification of chlamydial agents involved was performed using the ArrayTube ^® 126

DNA microarray assay as described previously (Sachse et al., 2005, and Borel et al., 2008).

127 128

2.5. DNA sequencing 129

To verify the 23S-rtPCR and AT test data, sequencing of a discriminatory region of the 16S 130

rRNA gene (Hotzel et al., 2005) and the whole chlamydial ompA gene (Sachse et al., 2008) 131

was conducted as described previously. The sequences have been deposited at GenBank ac- 132

cession numbers GQ332574 and GQ332575, respectively.

133 134

2.6. Culture of chlamydial agents 135

To isolate chlamydial agents, Buffalo Green Monkey (BGM) cells and yolk sacs of embryo- 136

nated chicken eggs were inoculated with sample material and cultured for several passages 137

according to standard procedures (Sachse et al., 2003; Tang et al., 1957). Replication of 138

chlamydiae was monitored by real-time PCR of culture aliquots.

139 140 141

3. Results

142

Accepted Manuscript

The results of PCR testing and cultural bacteriological examination are shown in Table 1. In 5 143

of 6 tested foals, the real-time PCR assay specific for the family Chlamydiaceae was positive.

144

The highest titres of chlamydial agents were detected at the beginning of sampling, i.e. 14 145

days after the end of antibiotic treatment. Furthermore, the concentrations of Chlamydiaceae 146

DNA in nasal swabs were higher than in conjunctival swabs. For foal no. 4, the real-time PCR 147

assay was positive only in the nasal swab. From six nasal swabs of four foals, S. equi subsp.

148

zooepidemicus was successfully cultured and isolated as virtually pure or mixed culture. An- 149

tibiograms showed sensitivity of the isolated streptococci to β-lactam antibiotics, tetracy- 150

clines, fluoroquinolones and aminoglycosides (data not shown). Viral and mycoplasma testing 151

of samples proved negative.

152

Identification of the chlamydial agent at species level was conducted using the AT DNA mi- 153

croarray assay, because this test allows parallel detection of all 9 currently defined species of 154

Chlamydiaceae. AT testing revealed the presence of C. caviae as the only chlamydial species 155

(Table 1). Typical hybridisation patterns obtained by AT testing of a nasal swab of foal no.1 156

at 14 days after the end of antibiotic treatment and the first passage of cell culture of the same 157

sample are shown in Fig. 1.

158

The identity of the chlamydial agent was confirmed by sequencing of the signature sequence 159

of the 16S ribosomal RNA gene (GenBank acc. no. GQ332574) and of the complete ompA 160

gene (GenBank acc. no. GQ332575). PCR products from swab samples of foals 1-4 (cf. Table 161

1) were used as templates. BLAST analysis of the sequences revealed homologies of 99 and 162

92%, respectively, to C. caviae type strain GPIC. No sequence differences among individual 163

samples were observed, thus suggesting that only one strain of C. caviae was involved.

164

Despite initial signs of chlamydial growth in BGM cell culture and embryonated chicken eggs 165

we failed to obtain a continuously replicating cultural isolate.

166

167

168

Accepted Manuscript

4. Discussion 169

The present study revealed a mixed bacterial infection involving C. caviae and S. equi subsp.

170

zooepidemicus as the presumptive causative agents of the outbreak of respiratory and ocular 171

disease in a stud farm.

172

So far, the list of chlamydial agents identified in horses has been limited to C. pneumoniae, C.

173

psittaci and C. abortus. This report provides the first evidence on the occurrence of C. caviae 174

in horses and its likely involvement in respiratory disease. Interestingly, after completion of 175

the present study, the laboratory of one of the authors (KS) again detected this agent in respi- 176

ratory samples from an unrelated horse breeding farm (data not shown), which might indicate 177

a wider dissemination. In contrast to the present findings, C. caviae has been considered to 178

possess high host specificity for guinea pigs (Everett, 2000). In any case, the detection of this 179

agent raises new questions since the route of infection remains unclear. As there were no 180

guinea pigs at the farm, nor do these animals roam freely in Central Europe, their possible 181

role as source of infection can be ruled out.

182

As highly sensitive and species-specific diagnostic tools for chlamydiae became available 183

only in the 1990s, it is not quite clear whether the present infection of horses with C. caviae 184

represents a unique event. Moreover, the traditional taxonomy did feature C. caviae as a sepa- 185

rate species, but rather subsumed it under the taxon of Chlamydia psittaci. Thus, studies of 186

chlamydial infections in horses mentioning involvement of Chlamydia psittaci which were 187

conducted before the molecular diagnostic age could unwittingly also have referred to C.

188

caviae. In the present study, we used different DNA-based diagnostic tests targeting three 189

different genomic sites, i.e. 23S rRNA gene (real-time PCR and AT test), ompA gene (se- 190

quencing) and 16S rRNA gene (sequencing), all of which identified C. caviae. In the case of 191

the ompA gene sequence, a similarity of 92 % to the C. caviae type strain is still within the 192

intra-species variability range of this highly variable membrane protein gene.

193

Accepted Manuscript

In view of the presence of C. caviae in nearly all swabs from the group of foals, it appears 194

reasonable to assume a contribution of this agent to the described clinical manifestations. In 195

addition, the fact that only C. caviae was found in the conjunctival swabs indicates an asso- 196

ciation with the conjunctivitis observed in the same animals, even more so since C. caviae is a 197

known agent of conjunctivitis in guinea pigs. Nevertheless, its role as a primary causative 198

agent cannot be defined unambiguously based on the present data.

199

In the present outbreak, C. caviae probably developed synergistic effects with S. equi subsp.

200

zooepidemicus. As a mucosal commensal in horses (Hirsh et al., 2004), the latter requires pre- 201

cursors (Timoney, 2004). We hypothesise that primary lesions were set by C. caviae and sub- 202

sequently aggravated by Streptococcus equi subsp. zooepidemicus. Indications supporting this 203

assumption include i) the conjunctivitis caused by mono-infection with C. caviae, ii) recurrent 204

clinical symptoms in the affected animals, and iii) the absence of a sustained clinical effect of 205

antibiotic therapy with trimethoprim-sulfonamide, enrofloxacin and amoxicillin. The latter is 206

usually effective against S. equi subsp. zooepidemicus.

207

The long-lasting antibiotic therapy of the diseased foals is the most likely explanation for our 208

inability to grow the C. caviae strain(s) involved, as chlamydiae are known to pass into a state 209

of persistence upon antibiotic treatment.

210

As a general conclusion from the present findings, practitioners and laboratory diagnosticians 211

should consider the possibility of C. caviae being involved in equine infections. Species- 212

specific tests should be used to enable assessment of the clinical importance and zoonotic 213

potential of the infectious agent involved. Finally, the rather unexpected identification of C.

214

caviae in the present case also indicates that our current understanding of host ranges of 215

Chlamydiaceae spp. is not yet complete.

216

217

218

Accepted Manuscript

Conflict of interest statement 219

None of the authors (Wolfgang Gaede, Karl-Friedrich Reckling, Anette Schliephake, Dirk 220

Missal, Helmut Hotzel, and Konrad Sachse) has a financial or personal relationship with other 221

people or organisations that could inappropriately influence or bias the paper entitled "Detec- 222

tion of Chlamydophila caviae and Streptococcus equi subsp. zooepidemicus in horses with 223

signs of rhinitis and conjunctivitis".

224 225

Acknowledgement 226

We thank Christine Grajetzki, Simone Bettermann and Karola Zmuda for excellent technical 227

assistance.

228 229

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331 332 333

Legend to Figure 1 334

DNA microarray testing of equine samples using the ArrayTube ^® assay. Results are shown 335

for a nasal swab of foal no.1 at 14 days after the end of antibiotic treatment (A, B) and the 336

first passage of cell culture of the same sample (C, D). A, C: Images of the stained microar- 337

rays. B, D: Bargraph showing specific hybridisation signals for Chlamydiaceae consensus 338

probes (1), Chlamydophila genus-specific probes (2) and C. caviae species-specific probes 339

(3), whereas the signals for the rest of the chlamydia species-specific probes (4) are absent or 340

represent non-specific cross-hybridisation. (Signal 5 in Fig. D represents a cross-hybridisation 341

with one of the three probes for Waddlia chondrophila.)

342

Accepted Manuscript

Fig. 1

1 2 4 3

1 2 4 3

A

B

C

D

5

Figure

Accepted Manuscript

Table1: Results of DNA assays and bacteriological examination

Time of sampling (weeks after the end of

antibiotic treatment)

Foal No.

Age at time of sampling

(weeks) Sample type

23S-rtPCR Chlamydiaceae

(Ct)

ArrayTube assay

Bacteriological examination

Main agent Additional

findings 2

1 14 nasal swab 24.7 C. caviae

n.d.

2 18 nasal swab 27.4 C. caviae

n.d.

4

1 16

conjunctival swab 35.5 C. caviae aerobic spore-forming bacteria

2 20 conjunctival swab 37.6 no signal

few non- specific aerobic

bacteria

3 20 conjunctival swab 36 no signal

4 19 conjunctival swab no Ct no signal

4

1 16

nasal swab 31.6 C. caviae

S. equi ssp. zooepidemicus (as dominant

bacteria) Acinetobacter

lwofii

1 16

nasal swab 32.4 C. caviae

S. equi ssp. zooepidemicus (as dominant bacteria)

2 20

nasal swab 34.8 C. caviae S. equi ssp. zooepidemicus (nearly pure culture)

non-haemolytic Cocci

2

20

nasal swab 34.2 C. caviae

S. equi ssp. zooepidemicus (nearly pure culture)

very few Pantoea agglomerans

3 19

nasal swab 28.2 C. caviae

S. equi ssp. zooepidemicus (nearly pure culture)

4

18

nasal swab 29.3 C. caviae

S. equi ssp. zooepidemicus (nearly pure culture)

very few Enterobacter

cloacae

5

5 10 nasal swab 32.3 n.d. n.d.

5 10 conjunctival swab 32.9 n.d. n.d.

5 10 conjunctival swab 30.9 C. caviae n.d.

3 20 conjunctival swab 36.9 n.d. n.d.

6 10 conjunctival swab no Ct n.d. n.d.

a) confirmed by ompA sequencing (GenBank acc. no. GQ332575), ^b) additionally confirmed by 16S rRNA gene sequencing (GenBank acc.no.

GQ332574) n.d. not done

Table