Department of Pathology and Infectious Diseases, Royal Veterinary College (RVC), University of London, Hawkshead Lane, North Mymms AL9 7TA, UK
Correspondence
Victoria J. Chalker
vicki.chalker{at}hpa.org.uk;
vix.chalker{at}ntlworld.com
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ABSTRACT |
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Present address: Health Protection Agency, 61 Colindale Avenue, London NW9 5HT, UK.
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INTRODUCTION |
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In dogs, mycoplasmas are thought to be part of the normal bacterial flora in the upper respiratory tract (Rosendal, 1982), but there are conflicting reports about the presence of mycoplasmas in the lower respiratory tract of healthy dogs. Randolph et al. (1993)
found that the lungs of up to 27 % of healthy dogs were colonized, whereas other authors have failed to detect mycoplasmas in the lower respiratory tract of healthy dogs (Rosendal, 1982
). The role of individual Mycoplasma sp. in respiratory infections of dogs is not well understood, but they are thought to colonize the lungs during pneumonia (Rosendal, 1982
). Of the species listed above, M. bovigenitalium, M. canis, M. cynos, M. edwardii, M. feliminutum, M. gateae and M. spumans have been isolated from dogs with respiratory disease (Armstrong et al., 1972
; Rosendal, 1978
). Pneumonia in dogs has been reproduced by experimental endobronchial inoculation with an isolate of M. cynos that had been isolated from a dog with pneumonia, and also by exposure of non-infected dogs to dogs infected with M. cynos (Rosendal, 1972
; Rosendal & Vinther, 1977
). In addition, several cases have been described in which mycoplasmas have been isolated in pure culture from dogs with respiratory disease, although typing to the species level has not been performed (Randolph et al., 1993
; Chandler & Lappin, 2002
).
Canine infectious respiratory disease (CIRD, kennel cough) is a complex disease involving a variety of pathogens that, when present concurrently, may act synergistically to enhance the severity of the disease (Appel & Binn, 1987). The infectious agents traditionally associated with CIRD are canine adenovirus, canine parainfluenza virus and the bacterium Bordetella bronchiseptica. Recently, however, Streptococcus equi subspecies zooepidemicus (Chalker et al., 2003a
) and canine respiratory coronavirus (CRCoV; Erles et al., 2003
) have both been found to be associated with CIRD in dogs. Outbreaks of CIRD are more common and severe in dogs that are housed communally in training institutions and rehoming centres. The majority of studies examining CIRD have not included mycoplasmas as potential causative agents, and the few that have determined whether mycoplasmas were present did not identify the individual species, limiting their value. Techniques currently used to identify canine mycoplasmas include growth inhibition and immunofluorescence tests which require specific antisera, and are therefore limited to specialized laboratories. The identification of species is further complicated by the fact that the colonies of several canine mycoplasmas are morphologically identical, and infections with a mixture of species are common. Because of this, several colonies from each case must be identified to the species level to increase confidence in the presence or absence of individual species. All these factors have contributed to the lack of testing for mycoplasmas, and the extent to which mycoplasmas are involved in respiratory infections in dogs is unknown.
The principal aim of this study was to develop molecular-based diagnostic tests for the most common canine mycoplasmas in order to determine which species of mycoplasmas are present in healthy dogs and those with CIRD.
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METHODS |
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The additional dog population, (B), included in the study comprised 153 dogs visiting a training centre. Dogs entered the centre periodically for a few months of training, prior to being housed in family homes. All training dogs were less than 2 years of age, in excellent condition, and had complete clinical histories. Dogs were housed in groups of two or fewer per kennel, but shared runs and air space with other dogs. All dogs were regularly vaccinated against all the agents listed for population (A). Unlike population (A), in which CIRD was endemic, outbreaks of CIRD occurred only sporadically in the training kennel and were of reduced severity. Dogs in the training kennel were monitored over a 12 month period from 2001 to 2002, and clinical respiratory disease graded as above. Tonsillar swabs were taken from a proportion of dogs in the kennel each month, and from dogs during an outbreak of CIRD. A total of 153 dogs were swabbed in the following clinical categories: (1) n=110, (2) n=27, (3) n=16, (4) n=0, (5) n=0.
Samples.
Dogs from population (A) were necropsied within 4 h of euthanasia. Bronchial lavage (BL) and tracheal samples were taken, sampled immediately and then stored at 70 °C. The method of BL sampling has been described previously (Chalker et al., 2003a), but in brief ensures that the sample is not contaminated by micro-organisms higher up the respiratory tract. Tracheal tissue samples were stored in Hanks' balanced salt solution at 70 °C, and BL samples were stored in Hanks' balanced salt solution with the addition of 15 % mycoplasma-free, heat-inactivated horse serum. Tonsillar swabs were not stored after use. Tissue samples were fixed in 10 % formol saline and embedded in paraffin, and stored at ambient temperature until use.
Mycoplasma isolation and growth conditions.
Mycoplasmas were routinely cultured at 37 °C in 95 % nitrogen and 5 % CO2 on Ureaplasma medium (Mycoplasma Experience; Windsor et al., 1975) and Mycoplasma medium (ME media, Mycoplasma Experience; Hannan et al., 1997
), and in 5 % CO2 and 95 % air at 37 °C in Ureaplasma and Mycoplasma broth (Mycoplasma Experience). Tracheal samples and tonsillar swabs were spread onto agar and then added to liquid media, whereas 0·05 ml BL was plated onto solid medium and 0·1 ml added to broth. All broth cultures were incubated for at least 7 days, and agar cultures for at least 28 days. Cultures were examined daily for the first 7 days and weekly thereafter.
Mycoplasmas were isolated from the air of the kennel of population (A) by leaving a Mycoplasma solid medium agar plate open for 1 h on top of the kennel, prior to incubation as above. Mycoplasmas from positive samples were purified, and individual clones stored at 70 °C prior to identification to species level. Due to the morphological similarity of canine mycoplasma colonies, several colonies from each sample were taken for further analysis. All mycoplasma type strains were obtained from the National Collection of Type Cultures (NCTC), Colindale, London (A. laidlawii PG8 NCTC10116, M. arginini G230 NCTC10129, M. cynos H381 NCTC10142, M. edwardii NCTC10132, M. felis CO NCTC10160, M. feliminutum PG15 NCTC10159, M. gateae C5 NCTC10161, M. maculosum PG15 NCTC10168, M. molare H542 NCTC10144, M. opalescens MHS408 NCTC10149, M. spumans PG13 NCTC10169, Mycoplasma sp. strain VJC358 NCTC11743), except for Mycoplasma sp. strain HRC 689, which was obtained from the University of Florida, and U. canigenitalium AE39, which was kindly donated by Mycoplasma Experience.
Mycoplasma identification.
Due to the high similarity of the 16S rRNA genes of canine Mycoplasma species (Chalker & Brownlie, 2004), PCR tests were developed for the species listed in Table 1
to species-specific regions identified in the 16S/23S rRNA intergenic spacer region. Primers and amplification parameters are also listed in Table 1
. All PCRs commenced with an initial denaturation at 95 °C for 5 min and were followed by the specific annealing step listed in Table 1
and then by final extension at 72 °C for 5 min. PCR reactions (50 µl) included 5·0 µl 10x magnesium-free buffer (0·1 M Tris/HCl, 0·5 M KCl, pH 8·3), 1·5 mM MgCl2 (Promega), 0·5 µl (0·5 Units) Taq DNA polymerase (Promega), 0·2 mM PCR nucleotide mix (Promega), 0·025 µg forward primer (Myc1; 5'-CACCGCCCGTCACACCA-3'), 0·025 µg reverse primer (see Table 1
), and
1 µg mycoplasma DNA (isolate or positive control type-strain) or 1 µl water (negative control). DNA was extracted from mid-exponential-phase cultures of mycoplasmas (520 ml) using the DNeasy tissue kit (QIAGEN), according to the manufacturer's instructions.
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Mycoplasma cynos riboprobe construction and in situ hybridization (ISH).
The last 145 bp of the 16S rRNA genes, the entire 16S/23S rRNA spacer region, and the first 36 bp of the 23S rRNA gene of M. cynos were amplified as a single amplicon using the PCR reaction described by Chalker & Brownlie (2004). The 450 bp fragment (sequence AF443606) was ligated into pGemT-EASY, which was used to transform E. coli JM109. Transformants were incubated on selective medium: Luria broth with the addition of 100 µg ampicillin ml1 (Sigma), 50 µg X-Gal ml1 (Sigma) and 0·01 M IPTG (Sigma). Positive transformants were identified by PCR, and the insert was confirmed as M. cynos DNA by sequencing. The plasmid containing the insert was then purified and digested with NotI. A digoxigenin-labelled riboprobe based on this plasmid was then synthesized with a Riboprobe Combination System (Roche), according to the manufacturer's instructions. Probe specificity was determined by dot-blot analysis, using a range of bacterial RNA (M. arginini, M. canis, M. cynos, M. edwardii, M. gateae, M. felis, M. molare, M. maculosum, M. opalescens, M. spumans, A. laidlawii, B. bronchiseptica, Clostridium perfringens, E. coli and S. equi subsp. zooepidemicus) that had been isolated as follows. Bacterial cells were lysed in 1 ml Tri Reagent (Sigma) for 5 min at room temperature, mixed with 0·2 ml chloroform, incubated for 10 min at room temperature, and centrifuged at 10 000 g at 4 °C for 15 min. The upper aqueous phase was added to 0·5 ml 2-propanol, incubated for 10 min at room temperature, and centrifuged at 10 000 g at 4 °C for 10 min. The resulting RNA pellet was washed with 1 ml 75 % ethanol and centrifuged at 10 000 g at 4 °C for 5 min. Finally, the RNA pellet was air-dried, resuspended in 50 µl diethylpyrocarbonate-treated water, and stored at 70 °C prior to use. The probe hybridized to M. cynos RNA and weakly to M. felis RNA and A. laidlawii RNA.
In situ hybridization (ISH) was performed on formalin-fixed, paraffin-embedded 4 µm sections of tracheal tissue on Superfrost Plus slides (BDH). Briefly, slides were incubated at 60 °C for 1 h, dewaxed in xylene, and rehydrated by treatment in 100 % ethanol, 70 % ethanol and finally water. Proteinase K treatment for 20 min at 37 °C was used to expose RNA, and enzyme activity was stopped by immersion in water and then 100 % ethanol, prior to air drying. Just prior to covering, 0·5 µl (50 pg) riboprobe or 0·5 µl control (hybridization buffer alone: 5 ml formamide, 1 ml 50 % dextran sulphate, 1 ml 20x SSC) in 49·5 µl hybridization mix (12·5 µl TES: 0·05 M Tris, pH 8·0, 0·0005 M EDTA, 0·5 M NaCl, 2 µl 10 mg ml1 denatured salmon sperm DNA, 35 µl hybridization buffer) was applied to each slide. The slides were then heated at 80 °C for 10 min to denature the nucleic acid, and the riboprobe was hybridized at 68 °C for 2 h. Slides were then washed at 68 °C in 4x SSC (pH 7·0) for 5 min, twice with 1x SSC for 5 min, and then finally in 0·1x SSC at room temperature for 10 min. Slides were then blocked at room temperature for 30 min and incubated with anti-digoxigenin alkaline phosphatase FAb fragments (1 : 500 in blocking solution) for 30 min. The colour reaction was developed with DIG substrate (Roche) in 1 ml DIG buffer 3, following the manufacturer's instructions, and stopped by rinsing in water. Slides were then counter-stained with picric acid in ethanol (1 : 5) for 1 min, immersed in water and then in acetone for 20 seconds. Slides were then dehydrated in xylene and ethanol prior to mounting with DPX resin. Tracheal sections were deemed positive for M. cynos by the observation of dark-staining clumps along the epithelial surface. ISH was performed on tracheal sections from 50 dogs in population (A).
Statistical analysis.
A significance level of probability of a type 1 error () of 0·05 was assumed for all analyses. The association between the presence of mycoplasmas and CIRD and the association between the presence of M. cynos and CIRD, clinical respiratory score, age and time in the kennel were analysed using a two-tailed Fisher's exact test.
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RESULTS |
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DISCUSSION |
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The association between isolation of M. cynos and the duration of stay in the kennel indicates that dogs were being infected with this bacterium during the first two weeks in the kennel. However, a decline in the isolation of M. cynos was seen in dogs kept longer than 21 days, which may reflect an active immune response to this bacterium. This concurs with the results of a previous study, in which antibodies to M. cynos were detected 45 weeks after infection, corresponding with the elimination of the organism from the lower respiratory tract (Rosendal, 1978). A later investigation noted a greater susceptibility to mycoplasma infection in younger dogs (Randolph et al., 1993
), and our study found a lower prevalence of M. cynos infection in older dogs.
Experimental endobronchial inoculation of dogs with M. cynos has been shown to produce localized pneumonia with destruction and loss of the bronchial epithelial cilia and alveolar infiltration with neutrophils and macrophages (Rosendal & Vinther, 1977), and in our study M. cynos was detected on the tracheal ciliated epithelium by ISH. It is known that M. cynos can persist in the lung for up to 3 weeks following infection (Rosendal & Vinther, 1977
), and that it can also be isolated from conjunctiva (Rosendal, 1973
), tonsils, and even kennel aerosols. The capacity of M. cynos to persist in the environment is unknown, but other Mycoplasma species can survive for weeks to months outside the host, and the environment could therefore be a source of infection (Nagatomo et al., 2001
). Indeed, M. cynos was isolated from kennel aerosols in this study.
It was not possible to identify several mycoplasmas that were isolated in our study because PCR amplification was not achieved with the primers used. This may be due either to the presence of potentially novel Mycoplasma species with sequences insufficiently similar to the primers or to inhibition of the PCR.
In conclusion, M. cynos is associated with CIRD, and younger dogs are more likely to be infected with M. cynos than older dogs. Canine infectious respiratory disease is a complex syndrome, and in the main study population in this investigation B. bronchiseptica, CRCoV, S. equi subsp. zooepidemicus and M. cynos were all associated with CIRD (Chalker et al., 2003a, b
; Erles et al., 2003
). Further work is required to understand the interaction between these organisms during infection and to investigate whether other pathogens may be involved. Rosendal (1982)
stated that natural cases of pneumonia with M. cynos as the sole agent had not been described, and that this bacterium could be a contributory factor. This suggestion agrees with our findings, as M. cynos was more strongly associated with moderate signs of respiratory disease than with milder disease or severe bronchopneumonia. M. cynos was isolated more frequently from the lower respiratory tract than the upper respiratory tract, and bronchial or tracheal lavage samples may be of more use than upper respiratory tract swabs for the detection of M. cynos. The study of canine mycoplasmas has perhaps been neglected due to the difficulties in isolation associated with mixed infections and the difficulty of identification to species level. This is the first study to comprehensively investigate the presence of individual Mycoplasma species in dogs with respiratory disease. The overall importance and distribution of M. cynos, the mechanisms of pathogenicity and nature of the immune response to this pathogen are currently unknown.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 18 October 2003;
revised 26 June 2004;
accepted 6 July 2004.
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