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.

Training need assessment for a master training program in Environmental Health program in Vietnam.

Vietnam is facing a shortage of skilled Environmental health workforce. A Training Needs Assessment was conducted to develop a list of environmental health tasks, a list of core competencies and assess the need for a Master of Environmental Health training program in Vietnam. To answer these questions, a cross-sectional study was conducted in Vietnam in 2017, using both qualitative and quantitative methods. The qualitative study involved a desk review, 29 in-depth interviews, two consultative workshops, and two expert meetings. For the quantitative component, 298 environmental health staff working at different levels completed a structured postal questionnaire. Results showed that different sectors were implementing various environmental health tasks but that there was currently no training program focusing on environmental health in Vietnam. Thirteen core competencies for a Master of Environmental Health were recommended. An urgent need to develop training programs to help building environmental health competencies at the Masters degree level was uniformly expressed. This could be achieved by developing a Master of Public Health with an Environmental Health stream in the short-term and a Master of Environmental Health program in the long-term.

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.

Novel genotypes of Coxiella burnetii identified in isolates from Australian Q fever patients.

Coxiella burnetii, the causative agent of Q fever, was first discovered in Australia in 1937. However, little is known about the strains of C. burnetii present in this country. In this study, six published genotyping methods were applied to 42 isolates from Australian patients with acute (n=39) and chronic (n=3) Q fever. All the isolates contained the plasmid QpRS and lacked the acute disease antigen A (adaA) gene. Two methods of genotyping based on single nucleotide polymorphisms (SNPs) also failed to discriminate between the isolates. However, results from the method based on SNPs within the multi-spacer sequence typing (MST) loci determined a novel MST genotype, with the Australian isolates forming a unique phylogenetic clade. Multi-locus variable number of tandem repeats (VNTR) analysis (MLVA) determined 14 genotypes, all of which were novel compared with those previously identified in strains from other countries. Many of these were single locus variants, differing from each other at just one of the 15 loci tested. Our results show that the Australian isolates exhibit significant diversity from previously characterised strains, but are genetically closely related to each other, supporting a model of evolution by clonal expansion in a geographically isolated location.

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.

Knowledge and Perceptions of Highly Pathogenic Avian Influenza (HPAI) among Poultry Traders in Live Bird Markets in Bali and Lombok, Indonesia.

Highly Pathogenic Avian Influenza (HPAI) has been prevalent in Indonesia since 2003 causing major losses to poultry production and human deaths. Live bird markets are considered high risk areas due to the density of large numbers of mixed poultry species of unknown disease status. Understanding trader knowledge and perceptions of HPAI and biosecurity is critical to reducing transmission risk and controlling the disease. An interview-administered survey was conducted at 17 live bird markets on the islands of Bali and Lombok in 2008 and 2009. A total of 413 live poultry traders were interviewed. Respondents were mostly male (89%) with a mean age of 45 years (range: 19-81). The main source of AI information was TV (78%), although personal communication was also identified to be an important source, particularly among female traders (60%) and respondents from Bali (43%). More than half (58%) of live poultry traders interviewed knew that infected birds can transmit HPAI viruses but were generally unaware that viruses can be introduced to markets by fomites. Cleaning cages and disposing of sick and dead birds were recognized as the most important steps to prevent the spread of disease by respondents. Two thirds (n = 277) of respondents were unwilling to report sudden or suspicious bird deaths to authorities. Bali vendors perceive biosecurity to be of higher importance than Lombok vendors and are more willing to improve biosecurity within markets than traders in Lombok. Collectors and traders selling large numbers (>214) of poultry, or selling both chickens and ducks, have better knowledge of HPAI transmission and prevention than vendors or traders selling smaller quantities or only one species of poultry. Education was strongly associated with better knowledge but did not influence positive reporting behavior. Our study reveals that most live poultry traders have limited knowledge of HPAI transmission and prevention and are generally reluctant to report bird deaths. Greater efforts are needed to engage local government, market managers and traders in education and awareness programs, regulatory measures and incentive mechanisms. Understanding and evaluating the social responses to such an integrated approach could lead to more effective HPAI prevention and control.

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.

Patterns and risks of trichinella infection in humans and pigs in northern Laos.

Several outbreaks of trichinellosis associated with the consumption of raw pork have occurred in Laos since 2004. This cross-sectional study was conducted in four provinces of northern Laos to investigate the seroepidemiology of trichinellosis in the human population and determine the prevalence and species of Trichinella infection in the domestic pig population. Serum samples and questionnaire data were obtained from 1419 individuals. Serum samples were tested for Trichinella antibodies by ELISA using larval excretory-secretory (ES) antigens and a subset of 68 positive samples were tested by western blot. The seroprevalence of Trichinella antibodies was 19.1% (95% confidence interval (CI) = 17.1-21.1%). The risk of having antibodies detected by ELISA using ES antigens increased with age, being of Lao-Tai ethnicity, living in Oudomxay province and being male. Tongue and diaphragm muscle samples were collected from 728 pigs and tested for Trichinella larvae by the artificial digestion method. Trichinella larvae were isolated from 15 pigs (2.1%) of which 13 were identified as T. spiralis by molecular typing; the species of the two remaining isolates could not be determined due to DNA degradation. Trichinella spp. are endemic in the domestic environment of northern Laos and targeted preventative health measures should be initiated to reduce the risk of further outbreaks occurring.

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.

A cross-sectional study of Taenia solium in a multiple taeniid-endemic region reveals competition may be protective.

We conducted cross-sectional surveys for taeniasis and cysticercosis in humans, pigs, and dogs in four northern provinces of Laos. Human cysticercosis and taeniasis prevalence was 2.2% (95% confidence interval [CI] = 1.4-3.0%) and 8.4% (95% CI = 6.9-9.9%), respectively. Eating uncooked beef, being male, province of residence, age, and ethnicity were significant risk factors for taeniasis and only province of residence was a significant risk factor for cystiercosis. Thirty-five human tapeworms were recovered during the survey and 33 (94.3%) and 2 (5.7%) were identified as Taenia saginata and T. solium, respectively. Maximum-likelihood adjusted prevalence of T. solium and T. hydatigena in pigs was 4.2% (95% CI = 0.5-7.9%) and 55.9% (95% CI = 47.5-64.3%), respectively, and T. hydatigena taeniasis in dogs was 4.8% (95% CI = 0.0-11.3%). Taenia hydatigena and T. saginata were the most prevalent taeniids in the respective pig and human populations and together may suppress T. solium transmission.

Seroprevalence of major bovine-associated zoonotic infectious diseases in the Lao People's Democratic Republic.

Bovine-associated zoonotic infectious diseases pose a significant threat to human health in the Lao People's Democratic Republic (Lao PDR). In all, 905 cattle and buffalo serum samples collected in northern Lao PDR in 2006 were used to determine seroprevalence of five major bovine zoonotic infectious diseases that included Taenia saginata cysticercosis, bovine tuberculosis, Q-fever, bovine brucellosis, and bovine leptospirosis. Five enzyme-linked immunosorbent assays (ELISAs) were used to test for the presence of antibodies to the diseases, except Taenia saginata, for which we tested for the presence of Taenia metacestode circulating antigens. The overall highest prevalence was for T. saginata (46.4%), with lower prevalence for Q-fever (4%), leptospirosis (3%), tuberculosis (1%), and brucellosis (0.2%). Although there were no significant differences in the proportion of seroprevalence between sex and age of the animals sampled, there were significant differences between the provincial distributions. Further studies are required to determine the seroprevalence of these infections in other locations in Lao PDR, as well as other animal species including humans, in order to develop effective prevention and control strategies. This is the first study to investigate the prevalence of bovine zoonotic infectious agents in the Lao PDR. Positivity was demonstrated for all diseases investigated, with the highest prevalence for T. saginata antigen and Coxiella burnetti antibodies. For T. saginata, there were significant differences in the provincial distribution. Approximately 16% seroprevalence of Coxiella burnetti was noted in Xayabuly Province; however, there are no clear reasons why this was the case, and further studies are required to determine risk factors associated with this observation.

Serologic study of pig-associated viral zoonoses in Laos.

We conducted a serologic survey of four high-priority pig-associated viral zoonoses, Japanese encephalitis virus (JEV), hepatitis E virus (HEV), Nipah virus (NiV), and swine influenza virus (SIV), in Laos. We collected blood from pigs at slaughter during May 2008-January 2009 in four northern provinces. Japanese encephalitis virus hemagglutination inhibition seroprevalence was 74.7% (95% confidence interval [CI] = 71.5-77.9%), JEV IgM seroprevalence was 2.3% (95% CI = 1.2-3.2%), and HEV seroprevalence was 21.1% (95% CI = 18.1-24.0%). Antibodies to SIV were detected in 1.8% (95% CI = 0.8-2.8%) of pigs by screening enzyme-linked immunosorbent assay, and only subtype H3N2 was detected by hemagglutination inhibition in two animals with an inconclusive enzyme-linked immunosorbent assay result. No NiV antibody-positive pigs were detected. Our evidence indicates that peak JEV and HEV transmission coincides with the start of the monsoonal wet season and poses the greatest risk for human infection.

Soil-transmitted helminthiasis in Laos: a community-wide cross-sectional study of humans and dogs in a mass drug administration environment.

We conducted a community cross-sectional survey of soil-transmitted helminthiasis in humans and dogs in four provinces in northern Laos. We collected and tested human and dog fecal samples and analyzed results against sociodemographic data. The prevalence of Ascaris lumbricoides, Trichuris trichiura, hookworm, and Strongyloides stercoralis was 26.1% (95% confidence interval [CI] = 23.7-28.4%), 41.5% (95% CI = 38.8-44.1%), 46.3% (95% CI = 43.3-49.0%), and 8.9% (95% CI = 7.4-10.4%), respectively. We observed strong heterogeneity for helminthiasis by ethnicity, province, and wealth status, which coincided with a risk profile demonstrating that Mon-Khmer persons and the poorest households are highly vulnerable. Necator americanus was the dominant hookworm species infecting humans and Ancylostoma ceylanicum was the only Ancylostoma species detected. Hookworm prevalence in village dogs was 94%, and the dominant species was A. ceylanicum. Necator americanus was also detected in dogs. It appears that dogs have a role in human hookworm transmission and warrant further investigation.

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.

Hepatitis E virus is prevalent in the pig population of Lao People's Democratic Republic and evidence exists for homogeneity with Chinese Genotype 4 human isolates.

The objective of this study was to determine the prevalence and genotypic range of Hepatitis E virus (HEV) in the pig population of northern Lao People's Democratic Republic (PDR). We collected 181 faecal samples from indigenous-breed pigs ≤ 6 months of age and the faeces was stored in RNA stabilisation buffer due to cold-chain and transport limitations. Twenty-one (11.6%) pigs had detectable HEV RNA and 43.5% of village pig herds were infected. Based on a 240 base pair-nucleotide sequence flanking the junction of open reading frames 1, 2 and 3 (ORF1, ORF2 and ORF3) the isolates were phylogenetically classified within genotype 4. Phylogenetic analyses revealed distinct genetic groupings of the Lao HEV isolates and two groups clustered with human and pig HEV isolates from China. This was the first study to demonstrate genotype 4 HEV in Lao PDR and indicates pigs are a potential reservoir for human HEV infection.