Review of clinical aspects, epidemiology and diagnosis of haemotropic Mycoplasma ovis in small ruminants: current status and future perspectives in tropics focusing on Malaysia.

Mycoplasma ovis (formerly Eperythrozoon ovis) is an epierythrocytic parasitic bacterium of small ruminants known as haemotropic mycoplasma, which is transmitted mechanically by biting flies and contaminated instruments. Acute mycoplasmosis causes severe haemolytic anaemia and mortality in young animals. At the same time, chronic disease may produce mild anaemia and varying degrees of morbidity depending on several factors, including age, reproductive status, the plane of nutrition, immunological status and the presence of concurrent infection. Haemotropic Mycoplasma ovis is currently recognised as an emerging zoonotic pathogen which is widely distributed in the sheep and goat producing areas of tropics and subtropics, where the disease is nearly endemic. Human infection has been reported in pregnant women, immunocompromised patients and people exposed to animals and arthropods. The current diagnosis of haemoplasma relies on microscopic evaluation of Giemsa-stained blood smear and PCR. Although there are few published reports on the incidence of haemotropic Mycoplasma ovis infection of small ruminants in Malaysia, information on its prevalence, risk factors, severity and economic impacts is grossly inadequate. Therefore, a large-scale survey of small ruminant flocks is necessary to elucidate the current seroprevalence status and molecular characteristics of haemotropic M. ovis infection in Malaysia using ELISA and PCR sequencing technologies. In the future, surveillance programs, including vector forecast, quarantine, monitoring by periodic surveys and public enlightenment, will limit the internal and transboundary spread of M. ovis, enhance control efforts and mitigate production losses in Malaysia.

Molecular survey of haemoplasmas in shelter dogs and associations with Rhipicephalus sanguineus sensu lato.

This study aimed to assess the occurrence of canine haemoplasma infection in domestic dogs and its possible trans-stadial transmission by Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) in shelter dogs in Diyarbakir Province in southeast Turkey. Blood samples (n = 282) collected from domestic dogs were analysed by polymerase chain reaction (PCR) for the presence of canine haemoplasma. Fully engorged nymphs (n = 204) were removed from dogs that were positive for canine haemoplasma by PCR and maintained in an incubator at 28 °C for moulting. Unfed ticks (n = 2185) comprising 2100 nymphs and 85 adults collected from the grounds of the same shelter were also screened. Of 282 dogs, 108 [38.3%, 95% confidence interval (CI) 32.6-44.2] were PCR-positive for canine haemoplasmas. Mycoplasma haemocanis (Mhc) infection (26.2%, 95% CI 21.2-31.8) was observed in a significantly higher number of dogs than was Candidatus Mycoplasma haematoparvum (CMhp) infection (6.7%, 95% CI 4.1-10.3). Co-infections were seen in 15 (5.3%, 95% CI 3.0-8.6) dogs. None of the tick specimens examined were found to be positive for haemoplasma. Partial sequences of the 16S ribosomal RNA (rRNA) gene shared 99-100% identity with the corresponding published sequences for Mhc and CMhp. The present results revealed no trans-stadial transmission of canine haemoplasma species by R. sanguineus s.l. in field conditions.

Evaluating the association between blood genotype or phenotype and haemoplasma infection in UK and Italian cats.

BACKGROUND: In humans, blood groups are associated with varying prevalence of infections. The aim of this study was to determine if associations exist between the feline AB blood group system and haemoplasma infection. METHODS: Data from two studies were combined. In the first study, DNA samples from 131 haemoplasma-infected and 132 haemoplasma-uninfected UK cats underwent pyrosequencing to determine their blood genotype as AA, Ab or bb. In the second study, blood samples from 160 Italian cats of known blood phenotype A, B or AB underwent PCR testing for feline haemoplasma species DNA. RESULTS: Haemoplasma infection was demonstrated in cats of all phenotypes and genotypes. A significantly higher number of Ab genotype cats tested positive for overall haemoplasma infection status (p = 0.04) and for Mycoplasma haemofelis infection (p = 0.03). LIMITATIONS: Haemoplasma-infected Italian cats were few, possibly increasing the chance of type II error, and the presence of purebred cats in the sample population may have had a confounding effect. CONCLUSIONS: Feline haemoplasmas do not appear to preferentially use either blood type A or B antigens as attachment sites for erythrocyte colonisation. Further investigations in a larger number of haemoplasma-infected cats of known blood phenotype are warranted to explain the association between genotype Ab and haemoplasma infection.

Molecular survey of Bartonella spp. and haemoplasmas in American minks (Neovison vison).

The aim of this study was to perform a molecular survey and characterize Bartonella spp. and haemotropic Mycoplasma (haemoplasmas) in invasive American minks (Neovison vison) from Southern Chile. Additionally, we addressed risk factors for positivity in both groups of agents. Blood and/or tissue samples from 246 minks were analysed by qPCR targeting the nuoG gene for Bartonella spp. and conventional (c)PCR for 16S rRNA for haemotropic Mycoplasma spp. nuoG qPCR-positive Bartonella spp. samples were submitted to cPCR assays (ITS, ribC, gltA, rpoB, pap-31 and ftsZ genes) to perform phylogenetic inferences. Haemotropic Mycoplasma spp. 16S-positive samples were further amplified by cPCR targeting RNaseP gene (160-210 bp) and by two overlapping 16S rRNA cPCR assays to amplify a larger portion of the gene (1,200bp) for phylogenetics. Bartonella DNA was detected in 8.9% of minks (22/246). Out of 22 nuoG qPCR-positive samples, one and two showed positive results in cPCR assays based on ITS and ribC, respectively. Consistent sequencing results were obtained for only one ITS sample (464 bp sequence), which shared 99.6% identity with B. clarridgeiae. Two per cent of minks (5/246) were positive for 16S rRNA haemotropic Mycoplasma-cPCR assay. Two concatenated sequences of 16S rRNA (1,176 and 1,230 bp) were obtained: one sample shared 97.87% identity with haemotropic Mycoplasma sp. from a wild rodent, and the other 96.49% identity with 'Candidatus Mycoplasma haematoparvum' from a dog. All BLAST results were supported by phylogenetic analysis. One haemoplasma RNase P sequence shared 94.86% identity with Mycoplasma haemofelis from a cat. No risk factors for PCR positivity were identified. In a nutshell, Bartonella clarridgeiae and a potentially novel haemoplasma closely related to haemoplasmas previously reported in rodents, dogs, domestic and wild cats were described for the first time in American minks.

Novel High-Throughput Multiplex qPCRs for the Detection of Canine Vector-Borne Pathogens in the Asia-Pacific.

The Asia-Pacific hosts a large diversity of canine vector-borne pathogens (VBPs) with some of the most common and most pathogenic, generating significant mortality as well as a spectrum of health impacts on local dog populations. The VBPs Anaplasma platys, Babesia gibsoni, Babesia vogeli, Ehrlichia canis, Hepatozoon canis and haemotropic Mycoplasma spp. are all endemic throughout the region, with many exhibiting shifting geographical distributions that warrant urgent attention. Moreover, many of these species cause similar clinical signs when parasitising canine hosts, whilst knowledge of the exact pathogen is critical to ensure treatment is effective. This is complicated by frequent coinfection that can exacerbate pathology. Here, we describe the development, optimisation and validation of two novel quadruplex Taq-Man based real-time PCRs (qPCRs) for the specific and sensitive detection of the aforementioned VBPs. To ensure accurate evaluation of diagnostic performance, results of our qPCRs were evaluated on field samples from Thai dogs and compared with both conventional PCR (cPCR) results and next-generation sequencing (NGS) metabarcoding. Our qPCRs were found to be more sensitive at detecting canine VBP than cPCR and generated results similar to those achieved by NGS. These qPCRs will provide a valuable high-throughput diagnostic tool available to epidemiologists, researchers and clinicians for the diagnosis of key canine VBPs in the Asia-Pacific and further afield.

Assessing the natural circulation of canine vector-borne pathogens in foxes, ticks and fleas in protected areas of Argentine Patagonia with negligible dog participation.

We collected blood and/or ectoparasites from 49 South American grey foxes (Lycalopex griseus) and two Andean foxes (L. culpaeus) caught in two National Parks of southern Argentine Patagonia (Bosques Petrificados, BPNP; and Monte León, MLNP) where dogs are nearly absent (density < 0.01 dog/km2). Common ectoparasites were the flea Pulex irritans (88% prevalence) and the tick Amblyomma tigrinum (29%). Conventional PCR and sequencing of 49 blood samples, 299 fleas analysed in 78 pools, and 21 ticks revealed the presence of DNA of the following canine vector-borne pathogens: in grey foxes, Rickettsia sp. (3%), hemoplasmas (8%), including Mycoplasma haemocanis, and Hepatozoon sp. (50%); in P. irritans, Bartonella spp. (72% of flea pools from 76% of foxes), mostly B. vinsonii subsp. berkhoffii but also B. rochalimae, Anaplasmataceae (Wolbachia sp.; 60% and 54%), and M. haemocanis/haemofelis (29% and 18%); and in A. tigrinum, Hepatozoon sp. (33% of ticks in 4 of 7 foxes). No piroplasmid DNA was detected in any sample. Andean foxes were negative for all tested pathogens. Two different Hepatozoon haplotypes were detected: the most prevalent was phylogenetically associated with H. felis, and the other with H. americanum and related sequences. Amblyomma tigrinum and Hepatozoon sp. were more abundant and/or prevalent in BPNP than in colder MLNP, 300 km southwards, perhaps located close to the limit for tick suitability. Bartonella v. berkhoffii was also significantly more prevalent in fleas of foxes in BPNP than in MLNP. This study provides novel information about natural host-pathogen associations in wildlife, markedly extends the distribution area in South America of arthropods and vector-borne pathogens of veterinary and public health interest, and contributes preliminary evidence about the potential role of A. tigrinum and P. irritans as vectors, respectively, for potentially new species of Hepatozoon from Lycalopex spp. and for M. haemocanis that should be further investigated.

Vector-borne diseases in cats in Germany.

Vector-borne diseases (VBDs) are caused by a wide range of pathogens, which are transmitted by a variety of vectors, such as ticks and fleas. As a result of climate changes, more vector-borne diseases are becoming endemic in Germany, not only in dogs, but also in cats. For some of the pathogens prevalence data still need to be investigated in Germany. However, natural infections with Bartonella, Anaplasma, haemotropic Mycoplasma and Borrelia species have already been described in German cats. Clinical relevance of these pathogens is not fully understood, and it is still unknown, why most infected cats stay asymptomatic and which predisposing factors contribute to the development of clinical signs in cats. Moreover, there is a risk of zoonotic transmission for some of the pathogens, e.  g., for some Bartonella spp. infections that are associated with cat scratch disease in humans. Due to the increasing number of VBDs in cats in Germany, preventive measures, such as the use of acaricides and insecticides, should be performed on a regular base in order to reduce the risk of these infections.

Serological and molecular prevalence of canine vector-borne diseases (CVBDs) in Korea.

BACKGROUND: Previous surveys in dogs from Korea indicated that dogs are exposed to a variety of vector- borne pathogens, but perception for a nation-wide canine vector-borne disease (CVBD) occurrence has been missing. We report here results of both serological and molecular prevalence studies for major CVBDs of dogs from all over the South Korean Peninsula except for Jeju Island. RESULTS: Serological survey of 532 outdoor dogs revealed the highest prevalence for Dirofilaria immitis (25.2%), followed by Anaplasma phagocytophilum (15.6%), Ehrlichia canis (4.7%) whereas Borrelia burgdorferi showed the lowest prevalence (1.1%). The number of serologically positive dogs for any of the four pathogens was 216 (40.6%). Concurrent real-time PCR assay of 440 dogs in the study indicated that DNA of "Candidatus M. haematoparvum", Mycoplasma haemocanis, Babesia gibsoni, A. phagocytophilum, and Hepatozoon canis was identified in 190 (43.2%), 168 (38.2%), 23 (5.2%), 10 (2.3%) and 1 (0.2%) dogs, respectively. DNA of Bartonella spp., Ehrlichia spp., Leishmania spp., Rickettsia spp. and Neorickettsia risticii was not identified. Analysis of questionnaires collected from owners of 440 dogs showed that the number of dogs with heartworm preventive medication was 348 (79.1%) among which dogs still positive to D. immitis infection were 60 (17.2%), probably due to the mean months of heartworm preventive medication being only 6.5. The high prevalence rates of both "Ca. M. haematoparvum" and Mycoplasma haemocanis in dogs from Korea indicate that these organisms may be transmitted by vectors other than Rhipicephalus sanguineus because this tick species has rarely been found in Korea. This is the first nationwide survey for canine haemotropic mycoplasma infections in Korea. CONCLUSIONS: This study showed that the risk of exposure to major vector-borne diseases in dogs is quite high throughout all areas of South Korean Peninsula. Since achieving full elimination of many pathogens causing CVBDs from infected animals is often impossible even when they are clinically cured, dogs once exposed to CVBDs can remain as lifetime reservoirs of disease for both other animals and humans in the close vicinity, and should therefore be treated with preventative medications to minimise the risk of pathogen transmission by the competent vectors.