Investigation of hemotropic Mycoplasma spp. genotypes in client-owned cats in Thailand.

The genetic information for three feline hemoplasmas is limited in Southeast Asia. According to the limited genetic data, this study modified a nested-PCR method targeting the 16S rRNA gene by designing a novel primary forward degenerate primer. Two hundred and thirty-one archived DNA extracts from the blood of client-owned cats with a variety of diseases were used. The modified nested PCR detected feline hemoplasma DNA in 64 of 231 (27.7 %) samples. Sanger DNA sequencing, BLAST, and phylogenetic analyses revealed nine nucleotide sequences of Mycoplasma haemofelis (Mhf) (3.9 %, 9/231), fifty-three nucleotide sequences of Candidatus Mycoplasma haemominutum (CMhm) (22.94 %, 53/231) and two nucleotide sequences of Candidatus Mycoplasma turicensis (CMtc) (0.86 %, 2/231). The phylogenetic analysis demonstrated separate genotypes of 30 DNA sequences of Thai CMhm. In addition, this analysis elucidated distinct genotypes of CMhm in Thai fishing cats (Prionailurus viverrinus). The domestic cat and Thai fishing cat groups were the two major groups separating Thai CMhm genotypes based on the 16S rRNA. One CMhm sequence in Thai fishing cats was also present in domestic cat CMhm genotypes. This result suggests that transmission of CMhm between domestic cats and Thai fishing cats has likely occurred. One Mhf sequence had low genetic identity (82 % similarity). The phylogenetic analysis confirmed that this sequence was still very closely related to Mhf reference sequences. This Mhf-like genotype could be a candidate novel Mhf genotype. This new genetic information for feline hemotropic Mycoplasma provides valuable information for future feline-related clinical studies.

Detection of specific IgM and IgG antibodies in acute canine monocytic ehrlichiosis that recognize recombinant gp36 antigens.

The efficacy of antibody detection tools for all stages of Ehrlichia canis infections and for various genotypes remains unclear. We produced recombinant gp36 (rgp36) antigens from different isolates of Thai E. canis to confirm the immunoreactivities to these recombinant proteins from naturally infected dogs. Sera and blood samples were taken from 21 dogs naturally infected with E. canis and in the clinical stages of acute phase ehrlichiosis. The expression vectors and competent E. coli produced two isolates of rgp36. These two major rgp36s were recognized by the dogs' sera in Western blotting, with both anti-dog IgM and IgG used as secondary antibodies. The two different genotypes of these local recombinant immunoreactive proteins were gp36 subgroup A (isolate 1055) and subgroup B (isolate 533). The Western blot analyses successfully identified both specific IgM and IgG from the dogs' sera. Of all 21 cases, five dogs presented specific IgM, twenty dogs presented specific IgG, and the commercial test used found fifteen seropositive dogs. There were four dogs that presented both specific IgM and IgG. Only one dog presented specific IgM only. This report is the first identification of a specific IgM in dogs in response to acute infections with E. canis. The recombinant gp36 isolates may be useful as potential antigenic material for subsequent serological tests that have a high possibility for differentiating between acute, chronic, primary, and nonprimary infections with E. canis.

Association of Ehrlichia canis, Hemotropic Mycoplasma spp. and Anaplasma platys and severe anemia in dogs in Thailand.

Canine tick-borne bacteria; Ehrlichia canis, hemotropic Mycoplasma spp. and Anaplasma spp., are organisms transmitted by Rhipicephalus sanguineus ticks. However, only a few clinical studies evaluating dogs infected with these organisms and anemia condition have been published. In this study, the potential tick-borne bacteria linked to anemia were investigated in eighty-one blood samples selected from anemic dogs using a broad range nested-PCR of the 16S rRNA gene. Positive results were shown in 12/81 blood specimens (14.81%). Nucleotide sequences from the PCR products were analyzed using BLAST and resulted in identification of Ehrlichia canis (8), Candidatus Mycoplasma haematoparvum (1) and Anaplasma platys (3). Two other PCR assays were used to detect and identify the positive results of these pathogens including a specific PCR for Ehrlichia canis (gp36) and a specific nested-PCR for hemoplasma species (16S rRNA) and the phylogenetic analyses of E. canis and canine hemoplasmas were performed using these two loci. These specific PCRs revealed co-infection of E. canis and Mycoplasma haemocanis in two cases. These two male dogs had presented with jaundice, severe hemolytic anemia, severe thrombocytopenia, leukocytosis, mild azotemia and hepatitis. Ehrlichia canis was detected in a significantly greater number of severe anemia cases (PCV<15%) than moderate or mild anemia cases (PCV 16-29%) (P<0.05) and these severe anemia cases were 7-fold more at risk of having E. canis infections (odds ratio: 7.11, p=0.020). However, no statistical differences were detected between E. canis detection and degrees of thrombocytopenia or leukopenia. From the results of this study, we conclude that the severity of anemia is associated with E. canis infections rather than the severity of thrombocytopenia.

Isolation of Coxiella burnetii from serum of patients with acute Q fever.

Worldwide there are few isolate collections of the intracellular bacterium Coxiella burnetii, due to the difficulties associated with working with the organism and the scarcity of suitable samples from which to attempt isolation. Particularly lacking are isolates from acute Q fever patients. The aim of this study was to evaluate whether the serum samples taken from patients with confirmed acute Q fever during the early stage of their disease represented a potential source of viable C. burnetii. Isolation was attempted from 65 of these samples by inoculation of the serum into Vero cell culture and was successful in 36 cases (55%). This high success rate was likely due to extended incubation of up to twelve weeks of the inoculated cultures, allowing the growth of the organism to levels detectable by PCR. Retrospective analysis of the time the sera was stored prior to inoculation into culture demonstrated that C. burnetii remained viable for 224 days in samples stored refrigerated and 371 days in samples stored frozen at -20 °C. These results demonstrate that standard serum samples taken from acute Q fever patients are a valuable source of new isolates of C. burnetii, with no special handling of the specimens required to maintain the organism's viability.

Patterns of Flavivirus Seroprevalence in the Human Population of Northern Laos.

A total of 1,136 samples from 289 households in four provinces in northern Laos were subjected to Japanese encephalitis virus (JEV) and dengue virus hemagglutination inhibition (DENV HI). Overall, antibodies to JEV were detected by HI in 620 (54.6%) of 1,136 people; of which 217 (19.1%) had HI activity against JEV only. Antibodies to DENV4 were detected by HI in 526 (46.3%) of 1,136 people; of which 124 (10.9%) had HI activity against DENV4 only. Antibodies to DENV1-3 were detected by HI in 296 (26.1%), 274 (24.1%), and 283 (24.9) of 1,136 people, respectively; of which 7, 1, and 0, respectively, had HI activity against DENV1-3 only. JEV was the most prevalent Flavivirus in Oudomxay, Luangprabang, and Huaphan provinces and DENV4 was the most prevalent in Xiengkhouang province. Seroprevalence for JEV increased with increasing age and wealth and was higher in villages where rice was cultivated in paddy fields and highest for people of Lao-Tai ethnicity.

Seroepidemiological study of outdoor recreationists' exposure to spotted fever group Rickettsia in Western Australia.

Bushland activity has previously been linked to rickettsial exposure in eastern and central regions of Australia, whereas little is known about the risks in Western Australia. The isolation of Rickettsia gravesii sp. nov. from Amblyomma triguttatum ticks and anecdotal reports of low-grade illness among bush recreationists raised the possibility of rickettsial transmission in the State. This study investigated rickettsial seroprevalence and potential risk of exposure to the spotted fever group rickettsiae in rogainers. Our results showed that rogainers active in the bush had a significantly higher risk of seropositivity (immunofluorescence total antibody titer ≥ 128) for the spotted fever group Rickettsia (odds ratio [OR] = 14.02, 95% confidence interval [CI] = 1.38-142.07) compared with a reference population, the overall seroprevalence in the rogainer group being 23.1%.

The economic impact of pig-associated parasitic zoonosis in Northern Lao PDR.

The parasitic zoonoses human cysticercosis (Taenia solium), taeniasis (other Taenia species) and trichinellosis (Trichinella species) are endemic in the Lao People's Democratic Republic (Lao PDR). This study was designed to quantify the economic burden pig-associated zoonotic disease pose in Lao PDR. In particular, the analysis included estimation of the losses in the pork industry as well as losses due to human illness and lost productivity. A Markov-probability based decision-tree model was chosen to form the basis of the calculations to estimate the economic and public health impacts of taeniasis, trichinellosis and cysticercosis. Two different decision trees were run simultaneously on the model's human cohort. A third decision tree simulated the potential impacts on pig production. The human capital method was used to estimate productivity loss. The results found varied significantly depending on the rate of hospitalisation due to neurocysticerosis. This study is the first systematic estimate of the economic impact of pig-associated zoonotic diseases in Lao PDR that demonstrates the significance of the diseases in that country.

Coxiella burnetii in western barred bandicoots (Perameles bougainville) from Bernier and Dorre Islands in Western Australia.

The aim of this work is to investigate the presence of Coxiella burnetii in Perameles bougainville and their ticks on two islands off Western Australia. Haemaphysalis humerosa, Haemaphysalis ratti, and Haemaphysalis lagostrophi were collected from P. bougainville on Bernier and Dorre Islands from 2005 to 2007; only Amblyomma limbatum was collected from humans over the same interval. One of 13 tick samples and 1 of 12 P. bougainville fecal samples were positive for C. burnetii DNA using quantitative polymerase chain reaction. DNA fragments had >99% similarity to published C. burnetii sequences. Three of 35 P. bougainville sera tested positive for anti-C. burnetii antibodies using enzyme-linked immunosorbent assay. C. burnetii was found in P. bougainville feces and a H. humerosa tick on Dorre Island and Bernier Island, respectively. This is the first reported use of enzyme-linked immunosorbent assay for screening of P. bougainville sera. The risk of zoonotic Q fever infection for human visitors to these islands is considered relatively low, however, appropriate precautions should be taken when handling western barred bandicoots, their feces and their ticks on Bernier and Dorre Islands.

Zoonotic Bartonella species in fleas and blood from red foxes in Australia.

Bartonella are arthropod-borne, fastidious, Gram-negative, and aerobic bacilli distributed by fleas, lice, sand flies, and, possibly, ticks. The zoonotic Bartonella species, Bartonella henselae and Bartonella clarridgeiae, which are the causes of cat scratch disease and endocarditis in humans, have been reported from cats, cat fleas, and humans in Australia. However, to date, there has been no report of B. henselae or B. clarridgeiae in Australian wild animals and their ectoparasites. B. henselae and B. clarridgeiae were detected in fleas (Ctenocephalides felis) from red foxes (Vulpes vulpes), an introduced pest animal species in Australia, and only B. clarridgeiae was detected in blood from one red fox. Phylogenetic analysis of the ribosomal intergenic spacer region revealed that the B. henselae detected in the current study were related to B. henselae strain Houston-1, a major pathogenic strain in humans in Australia, and confirmed the genetic distinctness of B. clarridgeiae. The identification and characterization of Bartonella species in red foxes in the Southwest of Western Australia suggests that red foxes may act as reservoirs of infection for animals and humans in this region.

Diversity of Bartonella species detected in arthropod vectors from animals in Australia.

A variety of Bartonella species were detected in two species of ticks and three species of fleas collected from marsupial hosts; brush-tailed bettong or woylie (Bettongia penicillata) and western barred bandicoots (Perameles bougainville) and from a rodent host; Rattus fuscipes in Western Australia. Bartonella species were detected using nested-PCR of the gltA gene and the 16S-23S ribosomal internal transcribed spacer region (ITS), and species were characterized using DNA sequencing of the 16S rRNA, gltA, rpoB, ftsZ genes and the ITS region. Bartonella rattaustraliani and B. coopersplainsensis were detected in Ixodes spp. ticks and fleas (Stephanocircus pectinipes) respectively collected from rodents. Two novel Bartonella species were detected from marsupials; Candidatus Bartonella woyliei n. sp. was detected in both fleas (Pygiopsylla hilli) and ticks (Ixodes australiensis) collected from woylies and Candidatus Bartonella bandicootii n. sp. was detected in fleas (Pygiopsylla tunneyi) collected from western barred bandicoots. Concatenated phylogenetic analysis of all 5 loci clarified the marsupial cluster of Bartonella species in Australia and confirmed the species status of these two Bartonella species in ticks and fleas from woylies and western barred bandicoots, which are classified as threatened species and are vulnerable to extinction.

Genetic characterization of flea-derived Bartonella species from native animals in Australia suggests host-parasite co-evolution.

Fleas are important arthropod vectors for a variety of diseases in veterinary and human medicine, and bacteria belonging to the genus Bartonella are among the organisms most commonly transmitted by these ectoparasites. Recently, a number of novel Bartonella species and novel species candidates have been reported in marsupial fleas in Australia. In the present study the genetic diversity of marsupial fleas was investigated; 10 species of fleas were collected from seven different marsupial and placental mammal hosts in Western Australia including woylies (Bettongia penicillata), western barred bandicoots (Perameles bougainville), mardos (Antechinus flavipes), bush rats (Rattus fuscipes), red foxes (Vulpes vulpes), feral cats (Felis catus) and rabbits (Oryctolagus cuniculus). PCR and sequence analysis of the cytochrome oxidase subunit I (COI) and the 18S rRNA genes from these fleas was performed. Concatenated phylogenetic analysis of the COI and 18S rRNA genes revealed a close genetic relationship between marsupial fleas, with Pygiopsylla hilli from woylies, Pygiopsylla tunneyi from western barred bandicoots and Acanthopsylla jordani from mardos, forming a separate cluster from fleas collected from the placental mammals in the same geographical area. The clustering of Bartonella species with their marsupial flea hosts suggests co-evolution of marsupial hosts, marsupial fleas and Bartonella species in Australia.

Candidatus Bartonella antechini: a novel Bartonella species detected in fleas and ticks from the yellow-footed antechinus (Antechinus flavipes), an Australian marsupial.

Bartonella are fastidious, Gram-negative, aerobic bacilli belonging to the Alphaproteobacteria group. In the last ten years, the discovery of new Bartonella species from a variety of mammalian hosts, arthropod vectors and geographical areas has increased. More than 20 species of Bartonella have been identified, of which approximately thirteen are associated with disease in humans and animals. Recently, four novel species of Bartonella were isolated from mammalian hosts in Australia: Bartonella australis from eastern grey kangaroos (Macropus giganteus) and Bartonella rattaustraliani, Bartonella queenslandensis and Bartonella coopersplainsensis from rodents. Bartonella-like organisms have also been detected from Ixodes tasmani ticks collected from koalas (Phascolarctos cinereus). However, very little is known about Bartonella spp. in other marsupials in Australia. We report the identification of a novel Bartonella species detected from fleas (Acanthopsylla jordani) and ticks (Ixodes antechini) collected from a small carnivorous marsupial, Antechinus flavipes (Mardos or Yellow-footed antechinus) in the southwest of Western Australia. New nested-PCRs targeting the gltA gene and the ribosomal ITS region were developed as part of the present study. DNA sequencing of the 16S rRNA, gltA, ftsZ and rpoB genes and the ribosomal ITS region revealed that this detection is a distinct Bartonella species and is related to B. australis isolated from kangaroos. This is the first report of two different possible arthropod vectors in Australia (ticks and fleas) being infected with the same species of Bartonella. We propose the name Candidatus Bartonella antechini n. sp. for the recently characterized organism.

Evaluation of a Subunit H5 Vaccine and an Inactivated H5N2 Avian Influenza Marker Vaccine in Ducks Challenged with Vietnamese H5N1 Highly Pathogenic Avian Influenza Virus.

The protective efficacy of a subunit avian influenza virus H5 vaccine based on recombinant baculovirus expressed H5 haemagglutinin antigen and an inactivated H5N2 avian influenza vaccine combined with a marker antigen (tetanus toxoid) was compared with commercially available inactivated H5N2 avian influenza vaccine in young ducks. Antibody responses, morbidity, mortality, and virus shedding were evaluated after challenge with a Vietnamese clade 1 H5N1 HPAI virus [A/VN/1203/04 (H5N1)] that was known to cause a high mortality rate in ducks. All three vaccines, administered with water-in-oil adjuvant, provided significant protection and dramatically reduced the duration and titer of virus shedding in the vaccinated challenged ducks compared with unvaccinated controls. The H5 subunit vaccine was shown to provide equivalent protection to the other two vaccines despite the H5 antibody responses in subunit vaccinated ducks being significantly lower prior to challenge. Ducks vaccinated with the H5N2 marker vaccine consistently produced antitetanus toxoid antibody. The two novel vaccines have attributes that would enhance H5N1 avian influenza surveillance and control by vaccination in small scale and village poultry systems.

Detection of Brachyspira hyodysenteriae, Lawsonia intracellularis and Brachyspira pilosicoli in feral pigs.

Feral pigs are recognized as being a potential reservoir of pathogenic microorganisms that can infect domestic pigs and other species. The aim of this study was to investigate whether feral pigs in Western Australia were colonized by the pathogenic enteric bacteria Lawsonia intracellularis, Brachyspira hyodysenteriae and/or Brachyspira pilosicoli. A total of 222 feral pigs from three study-populations were sampled. DNA was extracted from faeces or colonic contents and subjected to a previously described multiplex PCR for the three pathogenic bacterial species. A subset of 61 samples was cultured for Brachyspira species. A total of 42 (18.9%) of the 222 samples were PCR positive for L. intracellularis, 18 (8.1%) for B. hyodysenteriae and 1 (0.45%) for B. pilosicoli. Four samples were positive for both L. intracellularis and B. hyodysenteriae. Samples positive for the latter two pathogens were found in pigs from all three study-sites. A strongly haemolytic B. hyodysenteriae isolate was recovered from one of the 61 cultured samples. Comparison of a 1250-base pair region of the 16S rRNA gene amplified from DNA extracted from the isolate and five of the B. hyodysenteriae PCR positive faecal samples helped confirm these as being from B. hyodysenteriae. This is the first time that B. hyodysenteriae has been detected in feral pigs. As these animals range over considerable distances, they present a potential source of B. hyodysenteriae for any domesticated pigs with which they may come into contact.

Evaluation of a positive marker of avian influenza vaccination in ducks for use in H5N1 surveillance.

Control measures for H5N1 avian influenza involve increased biosecurity, monitoring, surveillance and vaccination. Subclinical infection in farmed ducks is important for virus persistence. In major duck rearing countries, homologous H5N1 vaccines are being used in ducks, so sero-surveillance using H5- or N1-specific antibody testing cannot identify infected flocks. An alternative is to include a positive marker for vaccination. Testing for an antibody response to the marker would confirm approved vaccine use. Concurrent testing for H5 antibody responses would determine levels adequate for protection or indicate recent infection, with an anamnestic H5 antibody response requiring further virological investigation. In this study, we have evaluated the use of a TT marker in ducks given avian influenza vaccination. Wild or domestic ducks were tested for antibodies against TT and all 463 ducks were negative. High levels of TT-specific antibodies, produced in twice-TT vaccinated Muscovy ducks, persisted out to 19 weeks. There was no interference by inclusion of TT in an inactivated H6N2 vaccine for H6- or TT-seroconversion. Thus TT is a highly suitable exogenous marker for avian influenza vaccination in ducks and allows sero-surveillance in countries using H5N1 vaccination.

Use of tetanus toxoid as a differentiating infected from vaccinated animals (DIVA) strategy for sero-surveillance of avian influenza virus vaccination in poultry.

Strategies for differentiating infected from vaccinated animals (DIVA) require improvement for increased surveillance of avian influenza (AI), where vaccination is employed to control disease. We propose a novel DIVA approach for chickens using tetanus toxoid (TT) as an exogenous marker independent of serotype and relatedness of circulating and vaccine strains. Of 1779 chickens tested from Australia, Hong Kong and China, 100% were seronegative for TT-specific antibodies without vaccination. Tetanus toxoid adjuvanted to mineral oil was immunogenic in chickens. Co-delivery of both TT and inactivated LPAI (H6N2) vaccines in chickens elicited strong TT and influenza-specific antibody responses, which persisted to 53 weeks post-vaccination. Furthermore, immunization with a combined vaccine composed of TT and AI induced high levels of antibodies to both antigens. We conclude that TT is a highly suitable exogenous marker for AI vaccination in chickens allowing simple and effective monitoring of AI vaccination status.

Rickettsia felis in fleas, Western Australia.

This study is the first confirmation of Rickettsia felis in Australia. The organism was identified from 4 species of fleas obtained from dogs and cats in Western Australia, by using polymerase chain reaction amplification and DNA sequencing of the citrate synthase and outer membrane protein A genes.