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 Diyarbak?r 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.     

A comparison of real-time PCR and reverse line blot hybridization in detecting feline haemoplasmas of domestic cats and an analysis of risk factors associated with haemoplasma infections

ABSTRACT: BACKGROUND: Three species of feline haemoplasma are recognised: Mycoplasma haemofelis (Mhf), 'Candidatus Mycoplasma haemominutum' (CMhm) and 'Candidatus Mycoplasma turicencis' (CMt). This study compared a reverse line blot hybridization (RLB) assay for simultaneous detection of Mhf, CMhm with three separate quantitative real-time polymerase chain reaction (qPCR) assays used for diagnosis of Mhf, CMhm and CMt. The RLB and qPCR assays were applied to DNA extracted from blood samples collected from 154 cats from Trinidad and Tobago. RESULTS: CMhm and Mhf DNA were detected using both RLB and qPCR. CMt DNA was detected by qPCR only. Comparing RLB and qPCR for the detection of CMhm DNA, 40 (26.3%) and 48 (31.6%) cats, respectively, were positive. The difference was more marked for Mhf, with RLB detecting a total of only 11 (7.2%) positive cats whereas qPCR detected 41 (27.0%) positive cats. Using qPCR as a gold standard, haemoplasma infected cats were more likely to be retrovirus positive (OR = 5.68, P = 0.02) and older (median age 5.5 years), than non-infected cats. In addition, CMhm positive cats were more likely to be male (OR = 3.4, P = 0.04). CONCLUSIONS: Overall the qPCR was more sensitive than RLB. In addition, age (median 5.5 years) and retrovirus positivity were risk factors for infection with the feline haemoplasmas in this study population. Further studies on feline haemoplasma infections in cats are needed to determine the significance of detecting small amounts of haemoplasma DNA, feline retrovirus infection and other associated risk factors on the clinical manifestation of disease.     

Molecular survey of canine vector-borne diseases in stray dogs in Thailand

Despite the large population of stray dogs in Thailand, there is limited information on the prevalence of canine vector-borne diseases (CVBDs). In this study, a molecular survey was conducted to determine the prevalence of Babesia spp., Ehrlichia canis, Hepatozoon spp., Anaplasma platys and Mycoplasma spp. in dogs in Thailand. Of the 181 dog blood samples tested by PCR, 78/181 (43.1%) were found to be infected with one or more pathogens. The overall prevalence rates of Mycoplasma spp., Hepatozoon spp., Babesia spp., A. platys and E. canis infections were 19.9%, 18.8%, 9.4%, 4.4% and 3.9%, respectively. To the authors' knowledge, this is the first report of Mycoplasma infection in Thailand in dogs. The current findings are important for future surveillance of CVBDs and designing appropriate approaches for diagnosis and control for the diseases in Thailand.     

PCR detection of Anaplasma platys in blood and tissue of dogs during acute phase of experimental infection

Four dogs were experimentally infected with Anaplasma platys to determine changes in real-time TaqMan PCR detection in blood and tissue, microscopically detectable parasitemia, and platelet concentrations during the first 28 days of infection. Buffy-coat blood cells were PCR positive for A. platys DNA at 4 days after inoculation and remained positive in all dogs until day 14. Marked thrombocytopenia and low parasitemia occurred in dogs during that initial period. During 17 and 28 days post-inoculation, the PCR results on buffy-coat blood cells were intermittently negative in each dog with marked thrombocytopenia and no microscopic evidence of parasitemia. Bone marrow and splenic aspirates collected from the A. platys-infected dogs were tested by real-time TaqMan PCR. Two dogs were PCR positive in spleen and marrow at 28 days post-inoculation, when PCR results for buffy-coat blood cells were negative. Spleen and/or bone marrow samples should be considered as additional samples for PCR testing of dogs, particularly when blood samples are PCR negative during the acute phase of A. platys infection.     

Long-Term Spatiotemporal Stability and Dynamic Changes in the Haemoparasite Community of Bank Voles (Myodes glareolus) in NE Poland

Long-term field studies on parasite communities are rare but provide a powerful insight into the ecological and evolutionary processes shaping host–parasite interactions. The aim of our study was to identify the principal factors regulating long-term trends in the haemoparasite communities of bank voles, and to this end, we sampled three semi-isolated populations of bank voles (n?=?880) in 1999, 2002, 2006 and 2010 in the Mazury lake district region of NE Poland. Overall, 90.8 % of the bank voles harboured at least one of the species of haemoparasites studied. Whilst overall prevalence (all species combined) did not vary significantly between the surveys, different temporal changes were detected among voles in each of the three sites. In voles from Urwita?t, prevalence increased consistently with successive surveys, whereas in Ta?ty, the peak years were 2002 and 2006, and in Pilchy, prevalence oscillated without a clear pattern. Across the study, bank voles harboured a mean of 1.75?±?0.034 haemoparasite species, and species richness remained stable with no significant between-year fluctuations or trends. However, each of the five constituent species/genera showed a different pattern of spatio-temporal changes. The overall prevalence of Babesia microti was 4.9 %, but this varied significantly between years peaking in 2006 and declining again by 2010. For Bartonella spp., overall prevalence was 38.7 %, and this varied with year of study, but the temporal pattern of changes differed among the three sites. The overall prevalence of Haemobartonella (Mycoplasma) was 68.3 % with an increase in prevalence with year of study in all three sites. Hepatozoon erhardovae had an overall prevalence of 46.8 % but showed a marked reduction with each successive year of the study, and this was consistent in all three sites. The overall prevalence of Trypanosoma evotomys was 15.4 % varying significantly between sites, but showing temporal stability. While overall prevalence of all haemoparasites combined and species richness remained stable over the period of study, among the five haemoparasites, the pattern of spatiotemporal changes in prevalence and abundance of infections differed depending on parasite species. For some genera, host age was shown to play an important role, but a significant effect of host sex was found only for Haemobartonella spp.     

Anaplasma platys: an improved PCR for its detection in dogs

This study compares two PCR assays for the detection of Anaplasma platys in dog blood using primers based on the A. platys 16S rRNA gene. The first approach utilized a "standard" PCR protocol composed of a "single-step" direct amplification using an Ehrlichia genus-specific primer set. The second assay being a "nested" PCR screen that first involved a universal bacterial primer set that amplified the majority of the 16S rRNA gene, followed by the nested round of PCR using an A. platys-specific primer set. Of the 22 dogs sampled, 10 were found to contain A. platys DNA using both protocols, and an additional two dogs were found positive using the nested technique. An extract of A. platys positive genomic DNA was serially diluted and comparison of sensitivities determined between the nested PCR, and a direct assay using A. platys-specific primers. The nested protocol demonstrated an increased sensitivity by at least 2 orders of magnitude when compared to the direct assay alone. Our results indicated that the nested PCR assay with its increased sensitivity would be useful for experimental research investigations as well as offer the potential for use as a routine test in diagnostic pathology.     

Prevalence and Genetic Characterization of Toxoplasma gondii in Pet Dogs in Central China

The prevalence and genotype of Toxoplasma gondii infection in dogs in Henan Province, Central China was investigated. A total of 125 blood samples were collected from pet dogs during April to June 2013, and all samples were examined by indirect hemagglutination antibody test (IHA) and nested PCR. The overall T. gondii prevalence in pet dogs was 24.0% (30/125), with 20.8% (26/125) in IHA and 10.4% (13/125) in PCR, respectively. No statistical associations were found between animal gender and age and the prevalence of T. gondii infection. Thirteen positive DNA samples were genotyped using 11 PCR-RFLP markers, including SAG1, (3’+5’) SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico. Of these, only 2 samples were genotyped with complete data for all loci, and a novel genotype (type III at SAG3 and GRA6 loci, and type I at other loci) was identified. This is the first report of genetic characterization of T. gondii infection in dogs in China.     

Ecological and evolutionary drivers of haemoplasma infection and bacterial genotype sharing in a Neotropical bat community

Most emerging pathogens can infect multiple species, underlining the importance of understanding the ecological and evolutionary factors that allow some hosts to harbour greater infection prevalence and share pathogens with other species. However, our understanding of pathogen jumps is based primarily around viruses, despite bacteria accounting for the greatest proportion of zoonoses. Because bacterial pathogens in bats (order Chiroptera) can have conservation and human health consequences, studies that examine the ecological and evolutionary drivers of bacterial prevalence and barriers to pathogen sharing are crucially needed. Here were studied haemotropic Mycoplasma spp. (i.e., haemoplasmas) across a species‐rich bat community in Belize over two years. Across 469 bats spanning 33 species, half of individuals and two‐thirds of species were haemoplasma positive. Infection prevalence was higher for males and for species with larger body mass and colony sizes. Haemoplasmas displayed high genetic diversity (21 novel genotypes) and strong host specificity. Evolutionary patterns supported codivergence of bats and bacterial genotypes alongside phylogenetically constrained host shifts. Bat species centrality to the network of shared haemoplasma genotypes was phylogenetically clustered and unrelated to prevalence, further suggesting rare—but detectable—bacterial sharing between species. Our study highlights the importance of using fine phylogenetic scales when assessing host specificity and suggests phylogenetic similarity may play a key role in host shifts not only for viruses but also for bacteria. Such work more broadly contributes to increasing efforts to understand cross‐species transmission and the epidemiological consequences of bacterial pathogens.     

First detection of Ehrlichia platys in dogs and ticks in Okinawa, Japan

We investigated Ehrlichia platys infection of dogs and ticks in Okinawa, Japan. Using E. platys specific primers, E. platys and HE3-R, PCR-positive results were obtained with 32.0% (64/200) of blood samples of dogs and 3.8% (3/77) of ticks. The nucleotide sequences of the amplified DNA fragment from the dogs and the ticks infesting them were identical, and the sequence corresponded to that of the E. platys Gzh981 strain. We concluded that there is a cyclic maintenance of E. platys between dogs and ticks in Okinawa.     

MSG1, a surface-localised protein of Mycoplasma suis is involved in the adhesion to erythrocytes

Mycoplasma suis is a member of the group of uncultivable haemoplasmas which colonise erythrocytes of a wide range of vertebrates. Adhesion to erythrocytes is the crucial step in the unique haemoplasma life cycle. Due to the lack of a cultivation system, no adhesion structures have been identified so far. In order to determine potential adhesion molecules of M. suis, we screened genomic M. suis libraries. The protein MSG1 with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) similarity was identified. The encoding gene msg1 is 1011bp in size. The overall homology of the deduced amino acid sequence to GAPDHs of other pathogenic mycoplasmas ranged from 52.6% to 54.5%. Recombinant MSG1 expressed in Escherichia coli exhibited GAPDH activity. Immunoblot and immunoelectron microscopy analyses using antibodies against rMSG1 verified the membrane and surface localisation of native MSG1 in M. suis. Furthermore, we showed that rMSG1 binds to erythrocyte lysate in a dose-dependent manner. E. coli transformants which express MSG1 on their surface acquire the ability to adhere to porcine erythrocytes. This adhesion could be specifically and significantly inhibited by rMSG1 and antibodies to MSG1. In conclusion, our studies indicate that the membrane-associated MSG1 represents the first putative adhesion protein identified in the group of haemoplasmas.     

Mycoplasma haemocanis infection--a kennel disease?

Mycoplasma haemocanis (formerly Haemobartonella canis) is a red blood cell parasite that causes disease mainly in immunosuppressed and splenectomized dogs. Clinical outbreak of the disease resulted in failure of a large experimental project. We aimed to identify whether M. haemocanis has increased prevalence in kennel-raised dogs. In a prospective study, we compared the prevalence of M. haemocanis in whole blood (anti-coagulated by use of EDTA) collected from pet dogs (University of Illinois, Urbana Champaign, Ill.; n = 60) with that in blood from dogs raised in three distinct kennels in western Europe (WE; n = 23), eastern Europe (EE; n = 20), and North America (NA; n = 20). Screening included antibody testing and microscopy of blood smears. The presence of M. haemocanis was identified using a polymerase chain reaction (PCR) assay for specific DNA of the organism. None of the pet dogs (0%) was test positive for M. haemocanis DNA. Mycoplasma haemocanis was found in dogs tested at all of the kennels. Infection rate in the three kennels was 30, 35, and 87%, respectively (all P < 0.001 versus control, chi2-test). Latent infection with M. haemocanis was not a single observation in kennel-raised dogs. Prevalence may be higher than that in a pet dog population. The potential exists for these latent infections to adversely affect or confound research results.     

Babesia canis vogeli: a novel PCR for its detection in dogs in Australia

Babesia canis vogeli is known to cause disease in dogs in Australia, and the rapid detection of various subspecies would enable effective treatment and management. A 21 bp oligonucleotide, "Bab-f" was proposed for the production of larger PCR products with high species specificity that would enable effective sequence analyses to yield subspecies identification. The new forward primer when paired with a previously reported "Babesia common" reverse primer generated a 394 bp product which was successfully amplified and provided subspecies differentiation by sequence analyses. Specificity and sensitivity were reported at 100% on a cohort of 55 dogs.     

Babesia gibsoni: Serodiagnosis of infection in dogs by an enzyme-linked immunosorbent assay with recombinant BgTRAP

The thrombospondin-related adhesive protein of Babesia gibsoni (BgTRAP) is known as an immunodominant antigen and is, therefore, considered as a candidate for the development of a diagnostic reagent for canine babesiosis. The recombinant BgTRAP (rBgTRAP) expressed in Escherichia coli was tested in an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to B. gibsoni in dogs. The ELISA with rBgTRAP clearly differentiated between B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. The sera collected from dogs experimentally infected with closely related parasites, B. canis canis, B. canis vogeli, B. canis rossi, and Neospora caninum, showed no cross-reactivity by the ELISA with rBgTRAP. A total of 107 blood samples collected from dogs that had been diagnosed as having babesiosis at veterinary hospitals in Japan were examined for the diagnosis of B. gibsoni infection by the ELISA and PCR. Ninety-six (89.7%) and 89 (83.2%) of the tested samples were positive by the ELISA and PCR, respectively, while 11 (10.3%) and 4 (3.7%) were ELISA+/PCR- and ELISA-/PCR+, respectively. In addition, the sensitivity of the ELISA with rBgTRAP was much higher than that of previously established ELISAs with rBgP50, rBgSA1, and rBgP32. These results indicate that the rBgTRAP is the most promising diagnostic antigen for the detection of an antibody to B. gibsoni in dogs and that the combined ELISA/PCR approach could provide the most reliable diagnosis for clinical sites.     

Serological and molecular studies on Dirofilaria immitis in dogs from Turkey

We estimated the prevalence of Dirofilaria immitis infection in domestic dogs in five Turkish provinces - Sakarya, Kocaeli, Ankara, Elazig and Mersin - using a commercial ELISA kit for detecting circulating antigen and a PCR test for detecting circulating microfilarial DNA. A total of 211 whole-blood and serum samples were collected from dogs of various breeds, ages and life status (owned or stray). Sample population characteristics were recorded and examined for differences in prevalence. Additionally, we collected 15 blood samples from cats (14 owned and 1 stray) from Ankara province and used PCR to detect D. immitis infection. Twenty-seven (12.8%) of 211 dog samples were positive for D. immitis antigen by ELISA. No differences in prevalence were observed by sex (female: 14.4%; male: 10.7%; P>0.05). The prevalence of D. immitis infection varied with age: 11.8% in younger dogs (0.5-2 years) and 17.5% in older dogs (3-5 years). Prevalence between stray dogs (15.2%) and owned dogs (9.3%) did not differ (P>0.05). Prevalence rates were highest in Kocaeli province (18.3%), followed by Ankara (14.8%), Sakarya (12.3%) and Mersin (10.5%) provinces. Prevalence in Elazig province was 0%. No dogs or cats had microfilarial DNA detectable by PCR.     

Very high prevalence of infection with the human T cell leukaemia virus type 1c in remote Australian Aboriginal communities: Results of a large cross-sectional community survey

Infection with the human T cell leukaemia virus type 1 (HTLV-1) subtype C is endemic among Aboriginal people in central Australia. To provide insights into the risk factors for transmission, we conducted the first large-scale, community-based prevalence study in seven remote Aboriginal communities. Residents >2 years old were invited to participate in the study between August 2014 and June 2018. HTLV-1 infection was defined as a positive western blot (WB) test or a positive HTLV-1 PCR. 720 community residents participated in the study (children <15 years, 142; adults, 578). Prevalences for children and adults were 3.5% (5/142) and 36.8% (213/578), respectively, reaching 49.3% (106/215) for those older than 45 years. A wide range of proviral loads were measured for both asymptomatic and symptomatic participants with no difference within groups according to age or gender; however, median PVL was 1.34 log10 higher for symptomatic participants. The adult prevalence of HTLV-1 infection in central Australia is the highest reported worldwide. Sexual contact is likely to be the predominant mode of transmission.     

PCR和分离培养同时检测儿童呼吸道感染的支原体

为了解广州市儿童呼吸道支原体感染情况,用一条共同的上游引物,二条特异性的下游引物建立的ＰＣＲ方法能同时扩增ＭＰ的６９１ｂｐ和ＭＧ的４３８ｂｐ粘附因子基因片段,但不会扩增其他支原体和细菌的ＤＮＡ。     

Prevalence of hepatitis A virus infection in Afro-Brazilian isolated communities in Central Brazil

To investigate hepatitis A virus (HAV) infection rates among isolated African-descendant communities in Central Brazil, 947 subjects were interviewed about demographic characteristics in all 12 isolated Afro-descendant communities existing in the state of Mato Grosso do Sul, Central Brazil, between March 2002 and November 2003. Blood samples were collected and sera were tested for HAV antibodies (total and IgM anti-HAV) by enzyme-linked immunosorbent assay. The overall prevalence of HAV infection was 75.6% (95% CI: 72.7-78.3), ranging from 55.4 to 97.3%, depending on the communities studied. The prevalence of anti-HAV increased significantly with age, from 13.8% in the age 0-5 age group to 96.6% in those older than 40 years. The findings point out an intermediate endemicity of HAV infection in some Afro-Brazilian isolated communities in Central Brazil. In addition, the high proportion of susceptible young subjects could be target of future HAV vaccination programs.     

Canine heartworm in the domestic and wild canids of southeastern Nebraska

The prevalence of canine heartworm (Dirofilaria immitis) was examined in the domestic dog, coyote (Canis latrans), and red fox (Vulpes fulva) populations of southeastern Nebraska. Microfilariae were detected in 21.4% (22 of 103) of the domestic dogs. The average age of infection for dogs was 5.8 yr. Nine of the 22 infected dogs also were positive for Dipetalonema reconditum. Thirty-nine of 443 (8.9%) coyotes were found to have adult heartworms. The average number of male and female worms per heart was 3.7 and 3.9, respectively. The mean age of infected coyotes was 3.6 yr. Red foxes had an infection rate of 4.8% (1 of 21). This fox heart had only 1 immature female worm. A mail survey of 6 veterinary clinics was also conducted. Veterinarians reported infection rates of 0% to 18.8% in domestic dogs for their localities.     

The animal reservoir of Tunga penetrans in severely affected communities of north-east Brazil

Tungiasis is a zoonotic ectoparasitosis caused by the sand flea Tunga penetrans L. (Siphonaptera: Tungidae). This disease is hyperendemic in poor communities of north-east Brazil, causing considerable morbidity in affected human populations, but the animal reservoirs have not been investigated previously in Brazil. To assess the prevalence and intensity of T. penetrans infection in domestic and peri-domestic animals, as well as in the human population, we surveyed two typical communities of north-east Brazil: an urban slum and a traditional fishing village. In the slum we examined 849 humans, 121 cats, 82 dogs, 2 pigs, 2 rabbits, 1 monkey and 56 rodents, comprising 34 rats (Rattus rattus L.) and 22 mice (Mus domesticus L). In the fishing village we examined 505 humans, 68 dogs, 37 cats, 7 donkeys, 4 cattle, 3 pigs and 1 monkey. Tungiasis was common among dogs and cats of both communities, with respective prevalence rates of 67.1% (95% CI: 55.8-77.1) and 30.9% (95% CI: 20.2-43.3) in dogs, 49.6% (95% CI: 40.4-58.8) and 32.4% (95% CI: 18.0-49.8) in cats. Slum rats were 41.2% (95% CI: 24.6-59.3) infested, but the other animals were not. Human prevalence rates were 54.4% (95% CI: 51.0-57.8) in the slum and 52.1% (95% CI: 47.6-56.5) in the fishing village. High prevalence rates (range 31-67%) of tungiasis in humans, pets and rats (but apparently not other animals) indicate the need for an eco-epidemiological approach to control of this anthropo-zoonotic problem.