Haemoparasites of free-roaming dogs associated with several remote Aboriginal communities in Australia

Background Tick-borne haemoparasites Babesia vogeli and Anaplasma platys are common among the free-roaming canine populations associated with Aboriginal communities in Australia, whilst the prevalence of haemoplasmas, which are also suspected to be tick-borne, remained unexplored. The aim of this study was to determine the prevalence of haemoplasma infection in these populations, and to identify any correlation with other haemoparasites. Blood was collected from 39 dogs associated with four Aboriginal communities and screened for infection using PCR and serology. DNA was purified and PCR analyses for piroplasms, Anaplasmataceae family bacteria and haemoplasmas performed. Serum was analysed using a commercial haemoparasite ELISA. Prevalence of infection was compared between communities. Results Seventeen dogs (44%) were infected (PCR positive) with Mycoplasma haemocanis, eight (21%) with ‘Candidatus Mycoplasma haematoparvum’, 20 (51%) with A. platys, and 17 (44%) with B. vogeli. Two dogs were infected with a novel haemoplasma as determined by DNA amplification and sequencing. Two dogs (5%) were serologically positive for Dirofilaria immitis antigens, one (3%) was positive for Ehrlichia canis antibodies and nine (24nbsp;%) were positive for A. platys antibodies. Co-infections were frequent. Haemoplasma prevalence was highest (73%, 16/22) in Central Australia and lowest (22%, 2/9) in Western Australia (p = 0.017). In contrast, B. vogeli prevalence was low in Central Australia (18%, 4/22) but higher (78%, 7/9) in Western Australia (p = 0.003). Conclusions This is the first time haemoplasma infections, including a novel species, have been molecularly documented in Australian dogs. The wide regional variation in prevalence of some of the haemoparasite infections detected in this study warrants further investigation.


Background
Dogs associated with the Aboriginal communities in the remote regions of Australia are considered to be freeroaming as they are owned but not confined in any manner. Amongst this canine population, infestation with the brown dog tick (Rhipicephalus sanguineus) is endemic and infections with Anaplasma platys and Babesia vogeli, tick-borne haemoparasites associated with R. sanguineus, are widespread [1]. The canine haemoplasmas Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum' are also believed to have R. sanguineus-mediated transmission [2], with free-roaming behaviour suspected of being a significant risk factor for the relatively high haemoplasma prevalence (20%) in Tanzanian dogs [3]. A recent PCR-based study of a canine hospital population in Sydney, New South Wales did not identify any haemoplasma infections (N. Hetzle, personal communication), although the blood was collected from non free-roaming owned dogs in a large city where the brown dog tick is very rarely found.
The aim of this study was to determine the prevalence of canine haemoplasma infections in free-roaming dogs from several remote Aboriginal communities (see Figure 1), Ti Tree (Nturiya and Pmara) in the Northern Territory (Central Australia), Tiwi Islands (off the coast of the Northern Territory), Goodooga (north-western New South Wales) and Bidyadanga (Western Australia), using realtime quantitative PCR analysis. A further aim was to determine if such infections were associated with other haemoparasites.

Population
Of the 39 dogs sampled, 20 were male and 19 were female ( Table 1). Most of the dogs were entire. The majority of dogs were adults (n = 25), with equal numbers (n = 7) of puppies and juvenile dogs.

PCR Results
PCR results were available for all samples for all assays. DNA was successfully purified and amplified from all canine blood samples as determined by the presence of adequate levels of canine internal control gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH; threshold cycle: 18.2 to 23.3).
See Table 1    amplification of target DNA from the remaining two positive samples resulted in a failure to sequence). All dogs positive for M. haemocanis and/or 'Ca. M. haematoparvum' were also positive by the panhaemoplasma assay, but two additional dogs were positive only on the pan-haemoplasma assay (threshold cycle: 36.7 & 37.9), corresponding to approximately one to ten haemoplasmas per reaction (equivalent to 5 μl DNA template); one from Nturiya (Ti Tree) and one from Bidyadanga. Of these dogs, one was negative for all other haemoparasites, and the other was positive for A. platys and B. vogeli. Sequencing of a 600 bp 16 S rRNA gene fragment amplified from these discordant samples showed that they were both infected with the same novel haemoplasma species [EMBL:HE577612] (Figure 2: phylogenetic tree). The novel haemoplasma had highest identity (82.8 to 84.9%) to the haemofelis group of haemoplasmas [4], closely followed by the haemominutum group of haemoplasmas (79.1 to 83.7%) and the nonhaemotropic mycoplasmas Mycoplasma fastidiosum and Mycoplasma cavipharyngis (82.3 and 82.5% respectively). The 23 bp deletion common to haemofelis group haemoplasmas was not present in the novel haemoplasma gene sequence. Attempts to amplify a larger 16 S rRNA gene fragment and a ribonuclease P ribosomal gene fragment from these two samples were unsuccessful.

Serology
Serological results were available for all dogs, except for an A. platys antibody result for the only dog (from Pmara, Ti Tree) that was positive for E. canis antibodies. Nine dogs (23.7%) were positive for A. platys antibodies ( Table 1). Both dogs that were serologically positive for D. immitis antigen were from Nturiya. No dogs were positive for B. burgdorferi antibodies.  haemoplasma: three with M. haemocanis alone; one with 'Ca. M. haematoparvum' alone; one co-infected with M. haemocanis and 'Ca. M. haematoparvum'; and a further dog with the novel haemoplasma. Three dogs infected with M. haemocanis, one of which was co-infected with 'Ca. M. haematoparvum' , were seropositive for A. platys. One dog co-infected with M. haemocanis and 'Ca. M. haematoparvum' was also seropositive for D. immitis antigen.

Co-infections & concurrent seropositive results
Of the 20 dogs infected with A. platys as determined by qPCR, 13 were cPCR-positive for B. vogeli, six were seropositive for A. platys antibodies (one dog had an unknown result), one was seropositive for E. canis antibodies, and one was seropositive for D. immitis antigen.
Of the 17 dogs infected with B. vogeli as determined by cPCR, five were seropositive for A. platys antibodies (one unknown), one was serpositive for E. canis antibodies, and none were seropositive for D. immitis antigen.
The dog that was seropositive for E. canis antibodies was also PCR positive for A. platys, B. vogeli, M. haemocanis and 'Ca. M. haematoparvum'.

Comparisons between aboriginal communities
Due to the low numbers of dogs from each community, only haemoparasite prevalences from Central Australia (Ti Tree) and Western Australia (Bidyadanga) could be compared. Haemoplasma prevalence was highest (16/22; 73%) in Central Australia and lowest (2/9; 22%) in Western Australia. The difference in haemoplasma prevalence between these two sites was statistically significant (p = 0.017; Fisher's exact test). In contrast, B. vogeli prevalence was low in Central Australia (4/22; 18%) but higher (6/9; 66%) in Western Australia. The difference in B. vogeli prevalence between these two sites was statistically significant (p = 0.003; Fisher's exact test). There was a trend towards a statistically significant difference in A. platys prevalence between Central Australia (8/22; 36%) and Western Australia (7/9; 78%; p = 0.054; Fisher's exact test).

Discussion
Prevalence of the haemoparasites A. platys and B. vogeli in this report was higher (51.3% & 43.6% respectively; 33.3% dual A. platys & B. vogeli infection) than in earlier studies of a similar population of free-roaming dogs associated with Aboriginal communities, where 43% were positive for A. platys and 21% for B. vogeli, including 11% dual infections [1]. The study [1] also reported marked regional variation, with A. platys infection ranging from 39% in north-western New South Wales (including the Goodooga community) to 50% in coastal Arnhem Land in the Northern Territory (near the Tiwi Islands) and B. vogeli infection ranging from 9% in the Northern Territory to 29% in the Tanami desert (approximately half way between the Tiwi Islands and Ti Tree communities).
Canine haemoplasma prevalence outside of Australia has ranged from 0% in the UK [5] to 40% in Sudan [6], with higher prevalences tending to be seen in warmer climates. This compares to a haemoplasma prevalence rate of 54% in the free-roaming canine population described in this study. Mycoplasma haemocanis prevalence has been reported to be up to 40% [7] and 'Ca. M. haematoparvum' prevalence up to 33% [6] with co-infections identified in up to 17% of haemoplasma positive dogs [8], compared to figures of 44%, 21% and 32%, respectively from the present study. It was not possible to determine whether dogs in this study were coinfected with a M. haemocanis or 'Ca. M. haematoparvum' and the novel haemoplasma due to the methodology. In the authors' opinion the less than 85% identity to known Mycoplasma species and the absence of the characteristic deletion common to the most closely related haemofelis group of haemoplasmas are sufficient to be able to describe the haemoplasma identified as being novel [9]. Unfortunately, copy numbers of the novel haemoplasma within the blood of both dogs identified as being infected were too low to enable molecular description sufficient to name the novel species. The significance and the primary host of the novel haemoplasma have yet to be determined.
Canine heartworm has been reported in domestic dogs in Southern Australia and dingoes in the Northern Territories, so the detection of D. immitis antigen was not unexpected [10,11]. It is uncertain and controversial as to whether Lyme borreliosis occurs in Australia [12]. Currently, there is no evidence that B. burgdorferi is responsible for a local syndrome, which has clinical features reminiscent of Lyme disease, so absence of seropositivity in dogs was to be expected. The positive serological result for E. canis could not be confirmed by Anaplasmataceae family cPCR, as the dog was also positive for A. platys by qPCR. In addition, false positive results for E. canis antibody tests have previously been reported in Australia, indicating the presence of crossreacting antibodies [13], raising the suspicion of a false positive result in this case, however, exposure or coinfection with a low copy number of E. canis organisms could not be ruled out.
Inferences from the statistical analysis were limited by the small number of samples obtained in some of the Aboriginal communities, which in turn were limited by a short time-frame for sample collection, and the freeroaming nature of the dogs. It is hoped that future studies involving haematological analyses in conjunction with PCR-based detection of haemoparasites from a larger number of dogs from a single community will elucidate the potential role of canine haemoplasma infection and disease, as both A. platys and B. vogeli have been associated with a reduction in mean platelet numbers [1] while anaemia has been associated only with B. vogeli. The role of haemoplasmas and their mode of transmission in this setting need to be ascertained to determine whether interventions with antimicrobial and / or ectoparasiticidal agents are appropriate. Identification of additional animals infected with a greater copy number of the novel haemoplasma would be required to enable further characterisation and complete molecular description. A much greater number of samples from Tiwi Islands, Goodooga and Bidyadanga would be required to accurately assess the prevalence of D. immitis infection in these populations.

Conclusions
Dogs living in aboriginal communities represent a naturally occurring model of polymicrobial haemoparasitic infection. Similar to A. platys and B. vogeli infection, haemoplasma infection, either with single species or coinfections was common in the free-roaming canine populations associated with Aboriginal communities. The wide variation in regional infection prevalence between some of the haemoparasite infections warrants further investigations to determine the role of vectors in disease transmission and the influence of concurrent haemoparasite infections on susceptibility and pathogenicity, especially in relation to the occurrence of anaemia and thrombocytopenia.

Samples
EDTA-blood and whole blood (clot tube) were collected from 39 free-roaming dogs associated with four Aboriginal communities, between February 2008 and October 2009. Blood was stored at 4°C, and transported to the laboratory within seven days of collection. Serum was harvested from whole blood after centrifugation. EDTA blood and serum were subsequently frozen at −70°C until required.
Most dogs were of mixed breed, and were considered variable hybrids of the dingo (Canis lupus dingo) and the domestic dog (Canis lupus familiaris). Based on dentition they were assigned to three age classes: puppy (< 3 months), juvenile (3 to 12 months) and adult (> 12 months).

DNA extraction and PCR analysis
DNA was purified from the samples using the QIAmp W DNA Mini Kit (Qiagen Pty Ltd) according to the manufacturer's recommendations, then shipped to the UK on dry ice.
Purified DNA was subjected to an Anaplasmataceae family conventional PCR cPCR and a Babesia / Theileria spp. cPCR. The Anaplasmataceae family cPCR comprised a primer pair (EHR16SD GGTACCYACAGAAGAAG-TCC & EHR16SR TAGCACTCATCGTTTACAGC) [14], which amplifies a 345 base pair (bp) fragment from the 16 S rRNA gene using the following thermal protocol: 95°C for 10 min and 40 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The Babesia/Theileria spp. cPCR comprised a primer pair (BabgenF GAAACTGCGAA-TGGCTCATTA & BabgenR CGGTAGGCCAATACCC-TACCGTC) [15], which amplifies a 250-270 bp fragment from the 18 S rRNA gene using the following thermal protocol: 94°C for 10 min and 40 cycles of 94°C for 30 s, 65°C for 30 s, and 72°C for 45 s. All reactions used 2x HotStarTaq Master Mix (Qiagen, Crawley, UK) with 200 nM each primer pair, 3.0 mM final MgCl 2 and 2 μL DNA template in a total volume of 20 μL, and were performed in a Tetrad thermal cycler (MJ Research, Waltham, MA, USA). Positive and negative control reactions were used in each assay. Amplification products were visualised on an agarose gel following electrophoretic separation.
Purified DNA was also subjected to the following qPCR assays: species-specific qPCRs for each of M. haemocanis and 'Ca. M. haematoparvum' , each duplexed with a GAPDH qPCR [3], pan-haemoplasma qPCRs [16], and an A. platys-specific qPCR (personnel communication: Amplicons from positive cPCR results were purified using the NucleoSpin W Extract II Kit (Macherey-Nagel, ABgene, Epsom, UK), and subjected to DNA sequencing using Applied Biosystems Big-Dye Ver 3.1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer. BLASTn analysis [18] was performed to compare the 16 S rRNA gene (Anaplasmataceae family & haemoplasmas) and 18 S rRNA gene (Babesia/ Theileria spp.) sequences obtained to those in GenBank. A phylogenetic tree including existing haemoplasma species, as well as selected non-haemoplasma Mycoplasma species, was constructed using MacVector version 12 (MacVector, Inc., Cambridge, United Kingdom) for the 16 S rRNA gene using the neighbour-joining program from a distance matrix [19], corrected for nucleotide substitutions by the Kimura two-parameter model [20]. The data set was re-sampled 1000 times to generate bootstrap percentages. The 16 S rRNA gene fragment of the novel haemoplasma species was deposited in the European Molecular Biology Laboratory Nucleotide Database (HE577612).

Serology
Serum samples were tested by enzyme-linked immunosorbent assay (ELISA) for Dirofilaria immitis (heartworm) antigen and Ehrlichia canis, Borrelia burgdorferi, Anaplasma phagocytophilum antibodies using the SNAP 4Dx kit (Idexx Laboratories Pty. Ltd., Rydalmere, NSW 2116, Australia). Due to cross reactivity between A. platys-and A. phagocytophilum-directed antibodies, in conjunction with the absence of A. phagocytophilum from Australia, a positive A. phagocytophilum ELISA result was taken to indicate exposure to A. platys (Personal Communication: Idexx Laboratories Pty. Ltd.).

Statistical analysis
Data were entered into Microsoft W Office Excel W 2007 (Microsoft Corporation) and statistical analyses performed using Statistical Package for the Social Sciences version 18.0 (SPSS Inc., Chicago, IL). Categorical data, i. e. absence or presence of specific infection, gender, age, Aboriginal community (Ti Tree & Bidyadanga only), were compared using the chi-square test, or the Fisher's exact test where sample sizes were small. Too few dogs were available from Tiwi Islands and Goodooga to allow statistical evaluation of area influence on infection. Insufficient numbers were available in each category to enable multivariate analysis. Significance was taken as p ≤ 0.05.

Animal ethics
All procedures were performed with the approval of the Animal Ethics committee of the University of Sydney (N00/11-2006/3/4492).
Authors' contributions GB, RM, SS and ST conceived of the study. GB and RM collected the samples and performed the ELISAs. ENB, DL and CRH carried out the PCR screening assays, DNA sequencing and analysis. ENB, CRH and ST drafted the manuscript. All authors read and approved the final manuscript.