Serological detection of Rickettsia spp. and evaluation of blood parameters in pet dogs and cats from Bangkok and neighboring provinces.

Rickettsiosis is caused by Orientia spp. and Rickettsia spp., arthropod-borne zoonotic intracellular bacteria. The close relationships between pet dogs, cats and owners increase the risk of rickettsial transmission, with limited studies on the seroprevalence in pets. This study investigated the prevalence of rickettsia exposure among dogs and cats in Bangkok and neighboring provinces. The samples from 367 dogs and 187 cats used in this study were leftover serum samples from routine laboratory testing stored at the Veterinary Teaching Hospital. In-house Enzyme-linked immunosorbent assay (ELISA) tests included IgG against the scrub typhus group (STG), typhus group (TG), and spotted fever group (SFG). The seroprevalence in pet dogs was 30.25% (111/367), including 21.53% for STG, 4.36% for TG, and 1.09% for SFG. Co-seroprevalence consisted of 2.72% for STG and TG, 0.27% for STG and SFG, and 0.27% for pangroup infection. The prevalence in cats was 62.56% (117/187), including 28.34% for STG, 4.28% for TG, and 6.42% for STG. Co-seroprevalence in cats consisted of STG and TG (4.28%), STG and SFG (5.35%), TG and SFG (3.21%), and three-group infection (10.69%). No significant difference in seroprevalence for the three serogroups was observed in any of the 64 districts sampled. The mean hematocrit level significantly decreased in seropositive dogs (P<0.05). Seropositive dogs and cats were detected in significantly greater numbers of anemia cases than nonanemia cases (P<0.05) (odds ratio: 7.93, 0.44, p = 0.00, p = 0.01). A significantly higher number of seropositive cats had decreased hemoglobin levels (P<0.05) (odds ratio: 3.63, p = 0.00). The seropositive samples significantly differed among older cats (P<0.05). These high exposures in pet dogs and cats could constitute important relationship dynamics between companion animals and rickettsial vectors. Significantly decreased hematocrit and hemoglobin levels indicated anemia in the exposed dogs and cats. The study findings will raise awareness of this neglected disease among pet owners and veterinary hospital personnel and aid in future public health preventative planning.

Bacterial DNA and osteoarthritis in dogs with patellar luxation and cranial cruciate ligament rupture.

Background and Aim: The association between bacterial DNA in stifle joints, including those with cranial cruciate ligament rupture (CCLR) and medial patellar luxation (MPL), and osteoarthritis in dogs remains elusive. This study investigated the potential association between the detection of bacterial DNA and osteoarthritis in dogs using a broad-range polymerase chain reaction technique targeting the 16S ribosomal RNA gene. Materials and Methods: Synovial fluid (35 samples) and knee tissue samples (32 samples) were obtained from 35 dogs diagnosed with CCLR (n = 20; 11 males and nine females) or MPL (n = 15; five males and 10 females) who underwent a surgical operation between October 2014 and April 2015. Results: Dogs with CCLR had a higher average osteoarthritis score than those with MPL (2.0 ± 0.9 vs. 0.5 ± 0.9; p = 0.005). Bacterial DNA was detected in the stifle joints of 60.71% of dogs with MPL. Pelomonas spp. (25.00%), Halomonas spp. (17.86%), and 5 other species (17.86%) were the most frequently identified bacteria. Bacterial DNA was detected in 41.03% of dogs with CCLR. Pelomonas spp. (15.38%), Sphingomonas spp. (10.26%), Halomonas spp. (5.13%), and 4 other species (10.26%) were the most frequently identified bacteria. No significant difference was observed in the prevalence of bacterial DNA obtained from tissue samples (46.88%) or joint fluid samples (51.43%). The presence of bacterial DNA was not associated with the type of knee injury (MPL or CCLR; p = 1.000). There was a higher prevalence of bacterial DNA in samples from dogs with moderate-to-severe osteoarthritis (94.44%) than in those with minimal osteoarthritis (41.18%), and a significant association between the presence of bacterial DNA and moderate-to-severe osteoarthritis was identified (p < 0.01). Conclusion: Dogs with moderate-to-severe osteoarthritis were more likely to have bacterial DNA in their stifle joints than those with no or minimal osteoarthritis. These findings provide valuable insight into the potential role of bacterial DNA in joint tissue or joint fluid and the development of osteoarthritis in dogs.

Systematic evaluation of TaqMan real-time polymerase chain reaction assays targeting the dsb and gltA loci of Ehrlichia canis in recombinant plasmids and naturally infected dogs.

Background and Aim: Because of the diversity of local genotypes of Ehrlichia canis, genes targeted by TaqMan real-time polymerase chain reaction (RT-PCR) assays should be systematically evaluated. This study evaluated the amplification efficiency, linearity, precision, and sensitivity of two TaqMan RT-PCR assays targeting the dsb and gltA loci of E. canis in recombinant plasmids and naturally infected dogs. Materials and Methods: Thirty blood samples were collected from dogs showing clinical signs of canine monocytic ehrlichiosis at the Kasetsart University Veterinary Teaching Hospital, Bangkok, Thailand. The dsb and gltA genes were amplified by conventional PCRs (cPCRs) on the blood samples and were then sequenced. Meanwhile, RT-PCR was used to detect dsb and gltA genes in 10-fold dilutions of the recombinant plasmids. Results: Both dsb and gltA were amplified with a high degree of linearity (R2 ≥0.975 and 0.993, respectively) in all dilutions, although the mean percentage of relative standard deviation of gltA was lower, but the difference was non-significant. The detection limits of RT-PCR and cPCR were 10-7 and 10-6, respectively, for both loci. RT-PCR targeting dsb (22/30; 73.3%) and gltA (15/30; 50%) yielded a number of positive results that did not differ significantly (p=0.06). The RT-PCR positive results of the dsb gene (22/30) differed significantly from that of cPCR (11/30) (p=0.004). In contrast, the RT-PCR positive results of the gltA gene (15/30) did not differ significantly from that of cPCR (12/30) (p=0.43). The mean Ct value (30.2) based on dsb RT-PCR of 22 positive cases was higher than that of gltA RT-PCR (Ct=27.4) on 15 positive cases. The Ct values from dsb RT-PCR were >30 in all seven discordant samples that were not detected by the gltA RT-PCR. Conclusion: RT-PCR targeting the dsb gene was more sensitive for detecting E. canis in naturally infected dogs. This study suggested that TaqMan RT-PCR of the dsb gene should be selected for E. canis research in this region.

Detection of antibodies against three zoonotic Bartonella spp. and cross-reactivity among species and Coxiella burnetii in dogs and cats from Central Thailand.

Dogs and cats are important reservoir hosts of bacterial zoonotic pathogens, especially the Proteobacteria, Bartonella spp., and Coxiella burnetii. Bartonella spp. and C. burnetii are Gram-negative intracellular bacteria causing cat-scratch disease and query fever, respectively. Despite these two pathogens being dangerous, studies of their seroprevalence and cross-reactivity are limited in Thailand. The objectives of this study were to detect the seroprevalence of three zoonotic species of Bartonella and to evaluate cross-reactivity among Bartonella spp. and with C. burnetii. In total, 570 dog and cat serum samples were detected for antibodies against Bartonella spp. and C. burnetii using an indirect immunofluorescence antibody (IFA) test. At titer ≥ 1:64, tested serum that had a fluorescent intensity score ≥ 2 was interpreted as positive. Additionally, possible factors related to the seroprevalence were analyzed consisting of sex, breed, age, residing area, and ectoparasite control. Overall, the seroprevalence levels of Bartonella spp. and C. burnetii were 13.16% and 1.23%, respectively. All antigens of Bartonella were reacted to sera (1.23-7.72%), furthermore, both phases of C. burnetii were revealed in sera (0.35-1.05%). Interestingly, there was a poor agreement of cross-reactivity among Bartonella spp. at 5.56-8.70%, while cross-reactivity between Bartonella spp. and C. burnetii also showed poor agreement (2.80%). It is suggested that dogs and cats are important reservoirs of Bartonella spp., even in animals with ectoparasite control. The Bartonella seroprevalence was high in pure-breed animals with ectoparasite control, reflecting that Bartonella spp. infections can occur in owned, well-cared-for, and asymptomatic dogs and cats.

Detection of zoonotic Bartonella species in ticks and fleas parasitizing free-ranging cats and dogs residing in temples of Bangkok, Thailand.

Bartonellosis is a vector-borne disease caused by intraerythrocytic bacteria known as Bartonella spp. The potential vectors that transmit Bartonella spp. are fleas, ticks, sand flies, and lice. Several Bartonella spp. cause diseases in humans; however, there is few molecular evidence of Bartonella spp. in vectors in Thailand. The objectives of this study were to investigate Bartonella spp. and to evaluate the spatial distribution of Bartonella spp. prevalence in the ectoparasites parasitizing free-ranging cats and dogs in temple clusters of Bangkok, Thailand. In total, 343 ectoparasites were studied to extract their genomic DNA. Species of all specimens were identified using an identification key and conventional polymerase chain reaction (cPCR) was applied to confirm flea and tick species. Extracted DNA samples were processed using primers that targeted the gltA, rpoB, ftsZ, and ribC genes of Bartonella spp. Then, PCR-positive amplicons were sequenced and a phylogenetic tree was constructed. Recorded data were statistically analyzed using descriptive statistics, the chi-square test, Fisher's exact test, and the odds ratio. Area data were analyzed and a prevalence distribution map was plotted. The major parasitizing ticks and fleas in this study were Rhipicephalus sanguineus and Ctenocephalides felis, respectively. Overall, the prevalence of Bartonella spp. in ectoparasites was 7.00%. The gltA amplicons revealed the presence of B. henselae (4.78%) and B. clarridgeiae (4.78%) in C. felis, and B. koehlerae (1.25%) and B. phoceensis (1.25%) in R. sanguineus. Bartonella DNA was encountered in 16/39 (41.03%) districts and 28.57% of the temple clusters. Bang Khun Thian district had the highest positive proportion and Bang Bon district showed co-evidence of different Bartonella species. In addition, the intervening zones were a risk factor of Bartonella (p < 0.05), and the distribution map showed a scattered pattern of Bartonella-positive clusters. Finally, fleas showed to be important vector reservoirs for Bartonella spp., especially zoonotic species, however, experimental studies are needed to prove the Bartonella transmission in ticks.

Molecular Detection of Bartonella spp. and Hematological Evaluation in Domestic Cats and Dogs from Bangkok, Thailand.

(1) Background: Bartonella spp. are Gram-negative, facultative, intracellular bacteria transmitted by hematophagous insects. Several species cause zoonotic diseases such as cat-scratch disease. Bartonella henselae and Bartonella clarridgeiae are the main species found in Thailand, however, there have been few studies on Bartonella spp. In addition, the hematological evaluation of Bartonella-infected animals is limited in Thailand. The aims of this study were prevalence investigation and hematological evaluation of Bartonella-infected dogs and cats residing in Bangkok, Thailand. (2) Methods: In total, 295 dogs and 513 cats were molecularly evaluated to detect Bartonella spp. using PCR with primers targeting the partial gltA, rpoB, ftsZ, ribC, and groEL genes. In total, 651 domestic animals were evaluated for hematological parameters compared between Bartonella-positive and Bartonella-negative animals. (3) Results: Overall, the prevalence of Bartonella spp. was 1.61% which was found only in free-ranging cats (2.83%). Bartonella henselae and B. clarridgeiae were confirmed from a concatenated phylogenetic tree of partial gltA and ribC genes, with 100% bootstrapping replication. For other housekeeping gene sequences, mixed infection was expected from the amplicons of rpoB, ftsZ, and groEL. Importantly, the mean corpuscular volume (MCV) was significantly increased in Bartonella-positive cats. (4) Conclusions: We suggest that B. henselae and B. clarridgeiae are important species and are still circulating in domestic animals, especially cats. The evaluation of blood parameters, especially a raised MCV, should be of concern in Bartonella infection in asymptomatic cats. Additionally, the knowledge of how to prevent Bartonella-related diseases should be promoted with people in at-risk situations.

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.

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.

Molecular cloning, sequence analysis, and immune recognition of Bm95 from Thai strains of Rhipicephalus (Boophilus) microplus.

The efficacy of existing vaccines against Thai strains of Rhipicephalus (Boophilus) microplus remains to be determined, and these vaccine antigens should be compared to homologues derived from local strains. The purpose of this study was to compare Bm95 from Thai R. microplus to the previously reported sequence from Argentinean ticks. mRNA was isolated from R. microplus midgut samples, and cDNA was amplified with Bm95-specific primers. The cDNA and protein sequences of Thai Bm95 were 94 and 92% identical, respectively, to Argentinean Bm95. Although the sequence was similar to Argentinean Bm95, there were 45-amino acid differences among the homologues. Amplicons encoding Bm95 were cloned into pPICZalphaA and expressed in Pichia pastoris strain KM71H. The recombinant plasmid Bm95 (rBm95) was reactive to bovine hyperimmune serum to Thai R. microplus midgut, indicating that the Thai rBm95 was immunogenic. Further work is needed to compare the efficacies of Thai and Argentinean rBm95 for protection of cattle against infestation with Thai R. microplus.