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.

Molecular detection of Loxodontofilaria spp. in Asian elephants (Elephas maximus) from elephant training camps in Thailand.

Filarial infection is an important disease in human and animal medicine. Several filarial worms are of importance, especially nematodes in the Onchocercidae. The Asian elephant (Elephas maximus) is an endangered animal and is very important from several socio-economic and ecological aspects in Thailand. Various parasites can be found in elephants; however, data related to filarial infections in elephants is limited. The objective of this study was to detect filaria in the blood of Asian elephants in Thailand, based on a polymerase chain reaction (PCR) technique. Blood samples were collected from 208 Asian elephants and detected for filaria using PCR, targeting the region of the internal transcribed spacer 2 (ITS2), the cytochrome c oxidase subunit 1 (cox1), and the RNA polymerase II large subunit (rbp1). In total, 4.33% (9 out of 208) of the sampled elephants had Loxodontofilaria spp. DNA with 100% query coverage. In addition, the obtained cox1 and rbp1 sequences matched with Loxodontofilaria sp., Onchocerca sp., and Dirofilaria sp. There were no identified risk factors (sex, age, location, and packed cell volume) related to Loxodontofilaria infection in elephants. The analyses of the phylogeny of ITS2 sequences demonstrated that the Loxodotofilaria-positive sequences were closely related to Onchocerca dewittei japonica and Onchocerca dewittei dewittei with 100% query coverage. Notably, the concatenated phylogenetic trees of ITS2 and the cox1 and rbp1 genes were closely similar to Loxodontofilaria sp. To describe in detail the genomic DNA of Loxodontofilaria spp., other genes should be additionally studied using a more discriminatory technique, such as DNA barcoding or whole genome sequencing.

In Vitro Anti-Inflammatory and Regenerative Effects of Autologous Conditioned Serum from Dogs with Osteoarthritis.

Osteoarthritis (OA) is mostly incurable and non-regenerative with long-term complications. Autologous conditioned serum (ACS), which is enriched in Interleukin 1 receptor antagonists (IL-1RA) and growth factors, could be an alternative treatment to accelerate the positive therapeutic effects. ACS is proposed to alleviate inflammation by blocking IL-1 receptors. However, to date, there is no report focusing on the cell-mediated anti-inflammation and regenerative effect caused by ACS, especially the ACS from patients. Therefore, this study aims to investigate the therapeutic potential of ACS generated from dogs with spontaneous OA, focusing on its promising anti-inflammatory and regenerative properties in vitro compared to the matched plasma. We found that ACS prepared from ten OA dogs contained significant concentrations of IL-1RA, vascular endothelial growth factor, and transforming growth factor beta, which are key cytokines in anti-inflammation and angiogenesis. Furthermore, we found that ACS suppressed T cell activity by reducing proliferation of effector T cells and simultaneously expanding numbers of immune suppressive FOXP3+ T cells. Lastly, we showed that ACS enhanced the proliferation of osteocytes and fibroblasts and promoted extracellular matrix gene expression in primary chondrocyte culture. Therefore, these studies indicate that ACS prepared from dogs with OA is active as an immunomodulatory and regenerative strategy for use in OA management.

Molecular Detection of Bartonella Species in Rodents Residing in Urban and Suburban Areas of Central Thailand.

Bartonella spp. are Gram-negative zoonotic bacteria transmitted to humans via various blood-sucking arthropods. Rodents have been identified as reservoir hosts of several zoonotic pathogens, including Bartonella spp. In Thailand, studies of Bartonella spp. in rodents from urban areas are limited; thus, a study in this area is necessary. The objectives of this study were to detect Bartonella spp. in rodents in Thailand and to compare the species' distribution across different areas. In total, 70 blood samples from rodents in urban and suburban areas were tested for Bartonella spp. using a conventional polymerase chain reaction that targeted the citrate synthase (gltA) gene. All Bartonella-positive sequences were analyzed using polymorphism in order to build a phylogenetic tree. Approximately 38% of the rodents studied contained Bartonella DNA. Both Rattus exulans (Pacific rat) and R. tanezumi (Asian house rat) contained Bartonella spp. Four species of Bartonella were detected in blood samples: B. tribocorum, B. phoceensis, B. grahamii, and B. rattimassiliensis. In addition, eight Pacific rats contained the B. kosoyi-B. tribocorum complex. Bartonella phoceensis and B. tribocorum-B. kosoyi complexes were found in a specific habitat (p < 0.05). Interestingly, only seven haplotypes were identified in the sequences analyzed, and only haplotype A was found in both rodent species. Finally, a monitoring program for zoonotic Bartonella infection, especially the B. kosoyi-B. tribocorum complex, B. phoceensis, B. grahamii, and B. rattimassiliensis should be established, especially in high-risk areas.

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 trypanosomes in South East Asia: Attempts to control Trypanosoma lewisi using human and animal trypanocidal drugs.

Beside typical human trypanosomes responsible of sleeping sickness in Africa and Chagas disease in Latin America, there is a growing number of reported atypical human infections due to Trypanosoma evansi, a livestock parasite, or Trypanosoma lewisi, a rat parasite, especially in Asia. Drugs available for the treatment of T. brucei ssp. in humans are obviously of choice for the control of T. evansi because it is derived from T. brucei. However, concerning T. lewisi, there is an urgent need to determine the efficacy of trypanocidal drugs for the treatment in humans. In a recent study, pentamidine and fexinidazole were shown to have the best efficacy against one stock of T. lewisi in rats. In the present study suramin, pentamidine, eflornitine, nifurtimox, benznidazole and fexinidazole, were evaluated at low and high doses, in single day administration to normal rats experimentally infected with a stock of T. lewisi recently isolated in Thailand. Because none of these treatments was efficient, a trial was made with the most promising trypanocide identified in a previous study, fexinidazole 100mg/kg, in 5 daily administrations. Results observed were unclear. To confirm the efficacy of fexinidazole, a mixed infection protocol was set up in cyclophosphamide immunosuppressed rats. Animals were infected successively by T. lewisi and T. evansi, and received 10 daily PO administrations of 200mg/kg fexinidazole. Drastic effects were observed against T. evansi which was cleared from the rat's blood within 24 to 48h; however, the treatment did not affect T. lewisi which remained in high number in the blood until the end of the experiment. This mixed infection/treatment protocol clearly demonstrated the efficacy of fexinidazole against T. evansi and its inefficacy against T. lewisi. Since animal trypanocides were also recently shown to be inefficient, other protocols as well as other T. lewisi stocks should be investigated in further studies.

Zoonotic trypanosomes in South East Asia: Attempts to control Trypanosoma lewisi using veterinary drugs.

A growing number of atypical human infections due to the livestock parasite Trypanosoma evansi, or to the rat parasite Trypanosoma lewisi, are reported in humans in Asia. In some cases, clinical evolutions request treatments, however, so far, there were very few attempts to control T. lewisi using trypanocidal drugs. In a study published elsewhere, the efficacy of human trypanocides is evaluated in laboratory rats, and it concludes that none of them is able to cure rats experimentally infected with T. lewisi. Control of T. lewisi in rat would be a step for identification of drugs against this parasite. In the present study, 4 veterinary drugs: diminazene aceturate, isometamidium chloride, melarsomine hydrochloride and quinapyramine sulfate and chloride, were evaluated at low and high doses, in intra-muscular injections to normal rats experimentally infected with a stock of T. lewisi from Thailand. None of these treatments being efficient, a trial was also made using melarsomine hydrochloride in T. evansi infected rats and in mixed T. lewisi and T. evansi infected rats, in order to demonstrate the efficacy of the drugs under the present protocol. T. evansi was cleared from the rat's blood the day after the treatment, while, T. lewisi remained unaffected until the end of the experiment. These observations clearly demonstrated the efficacy of melarsomine hydrochloride against T. evansi and its inefficacy against T. lewisi. In conclusion none of the veterinary drugs was efficient against this stock of T. lewisi. Other protocols using higher doses or other drugs and T. lewisi stocks should be investigated in further studies. The control of T. lewisi infection in Wistar rats, using veterinary trypanocidal drugs, remains so far unsuccessful.

Evaluation of an Indirect-ELISA Test for Trypanosoma evansi Infection (Surra) in Buffaloes and Its Application to a Serological Survey in Thailand.

Surra, caused by Trypanosoma evansi, is a neglected disease due to frequent subclinical evolution, especially in bovines in Asia. However, acute and chronic signs are regularly observed, with significant sanitary and economic impacts. In this study, we evaluated and applied an indirect-ELISA test for the detection of anti-T. evansi immunoglobulin G in buffaloes using antibovine conjugate. Based on buffalo reference sera from the Philippines, a two-graph receiver operating characteristics analysis (TG-ROC) was conducted to define an optimal cut-off value; sensitivity and specificity were estimated at 92.5% and 94.2%, respectively. A cross-sectional serological survey was carried out in the major buffalo breeding areas of Thailand; 892 buffaloes from 8 provinces were sampled in North, Northeastern, and Southern Thailand. Seropositive buffaloes were found in all 8 provinces, on 20.3% of farms for an overall prevalence of 12.2% (95% CI 10.2-14.5%). Nearly one-third of the sampled population was exposed to infection. Broader sampling would be necessary but is not possible in the southern half-wild breeding systems. According to our results, buffaloes may constitute a large and robust reservoir for T. evansi, which is a permanent threat to other livestock such as cattle and horses as well as wild animals such as elephants in Southest Asia.

Is the Oriental House Rat (Rattus tanezumi) a Potential Reservoir for Trypanosoma evansi in Thailand?

Trypanosoma evansi is a protozoan blood parasite and etiologic agent of "surra," a disease affecting a wide range of domestic and wild mammals, some identified as potential reservoirs. Although T. evansi has been detected in several small wild rodent species, their role in the epidemiology of surra is unclear. There is molecular evidence of T. evansi in wild rodents in Asia, but it is not known whether they can carry the parasite for sufficient time to significantly contribute to the epidemiology of surra. We assessed the susceptibility of the Oriental house rat (OHR; Rattus tanezumi) to T. evansi infection. Five adult male OHRs trapped in Bangkhen district, Bangkok, Thailand, and five laboratory Wistar rats (Rattus norvegicus) as positive controls, were experimentally infected with a local strain of T. evansi. The five controls and three of the five OHRs were highly susceptible and rapidly exhibited the high levels of parasitemia usually observed in Wistar rats. They died or were euthanized just prior to expected death. Two OHRs presented fluctuating levels of parasitemia, without obvious clinical signs, throughout 40 d of monitoring. These results highlight the moderate susceptibility of some OHRs and their ability to carry the infection over time. Along with the molecular evidence of T. evansi in captured OHRs (demonstrated elsewhere), our results bring new information on the potential role of OHRs in the complex epidemiology of surra.

Specific primers for PCR amplification of the ITS1 (ribosomal DNA) of Trypanosoma lewisi.

Trypanosoma lewisi is a mild or non-pathogenic parasite of the sub-genus Herpetosoma transmitted by fleas to rats. In a previous study we described pan-trypanosome specific primers TRYP1 which amplify the ITS1 of ribosomal DNA by hybridizing in highly conserved regions of 18S and 5.8S genes. These primers proved to be useful for detecting T. lewisi DNA in laboratory rats, but a recent large scale survey in wild rodents demonstrated a lack of specificity. In the present study, we designed and evaluated mono-specific primers LEW1S and LEW1R, for the detection and identification of T. lewisi by a single-step PCR. These primers were designed inside the highly variable region of the ITS1 sequence of T. lewisi ribosomal DNA. The product size of 220 bp is specific to T. lewisi. The sensitivity limit was estimated between 0.055 and 0.55 pg of DNA per reaction, equivalent to 1-10 organisms per reaction. All the PCR products obtained from 6 different T. lewisi isolates were more than 98% similar with each other and similar to the sequences of T. lewisi already published in Genbank. All DNA of 7 T. lewisi stocks from China gave the specific 220 bp product. We showed that LEW1S and LEW1R primers enabled sensitive detection and identification of T. lewisi infection in laboratory and wild rats. This assay is recommended for monitoring T. lewisi infections in rat colonies or for studying infections in the wild fauna. An absence of cross reaction with human DNA means that these primers can be used to investigate atypical trypanosome infections in humans. Given the risk of T. lewisi infection in human, we believe that these primers will be beneficial for public health diagnosis and rodents investigation programmes.