Transstadial and intrastadial experimental transmission of Ehrlichia canis by male Rhipicephalus sanguineus

The acquisition and transmission of rickettsial pathogens by different tick developmental stages has important epidemiological implications. The purpose of this study was to determine if male Rhipicephalus sanguineus can experimentally acquire and transmit Ehrlichia canis in the absence of female ticks. Two trials were performed where nymphal and male R. sanguineus were simultaneously acquisition fed on the same infected donor hosts, and transstadially or intrastadially exposed male ticks were fed on separate pathogen-free dogs as a test for transmission. A single-step p30-based PCR assay was used to test canine and tick hosts for E. canis infections before and after tick feeding. E. canis was detected after either intrastadial or transstadial passage in male ticks, the organism remained detectable in both tick groups after transmission feeding, and both tick groups transmitted the rickettsia to susceptible dogs. Infection of dogs via tick feeding resulted in milder clinical signs and lower antibody titers than intravenous inoculation of carrier blood, but further investigation is needed to understand the mechanisms responsible for this observation. These results demonstrate that male R. sanguineus can take multiple feedings, and that they can both acquire and transmit E. canis in the absence of female ticks. This tick development stage could be important in transmission of E. canis, and perhaps related pathogens, between vertebrate hosts under natural and experimental conditions.     

Antibody-ELISA for Trypanosoma evansi: Application in a serological survey of dairy cattle, Thailand, and validation of a locally produced antigen

Trypanosoma evansi is generally considered a mild pathogen in bovines. However, in Asia, acute and chronic signs have been observed in cattle, with high levels of parasitaemia, abortion and death. Investigations in Asian cattle are needed to better understand this epidemiological situation. To generate comparable data at a regional level, development and standardization of an antibody-enzyme linked immunosorbent assay for T. evansi (ELISA/T. evansi) was initiated and applied in an epidemiological survey carried out in dairy cattle in Thailand. A batch of 1979 samples was collected from dairy farms located throughout the country's four regions. Soluble T. evansi antigens initially produced in France were also produced in Thailand for comparison and technology transfer. Screening of 500 samples allowed us to identify reference samples and to determine the cut-off value of the ELISA. Seropositive animals – some of them confirmed by PCR – were found in the four regions, in 12 out of 13 provinces, in 22 out of 31 districts, in 56 farms out of 222 (25%, 95%CI ± 6%) and in 163 animals out of 1979 (8.2, 95%CI ± 1.2%). Estimated seroprevalence in 35 farms ranged between 1% and 30%, and in 21 farms it was >30%. Approximately 25% of survey cattle were exposed to the infection, in various situations. A sub-sample of 160 sera was tested on both antigens. Wilcoxon's (Z = 1.24; p = 0.22) and McNemars's tests (CHI2 = 3.55; p = 0.09) did not show any significant differences, showing that the locally produced antigen is suitable for further evaluation in the surrounding countries. Use of this standardized serological method will broaden knowledge of the prevalence and impact of the disease at the regional level in South-East Asia. Further validation of this ELISA will be necessary in other host species such as buffalo, horse and pig.     

Host surveys, ixodid tick biology and transmission scenarios as related to the tick-borne pathogen, Ehrlichia canis

The ehrlichioses have been subject to increasing interest from veterinary and public health perspectives, but experimental studies of these diseases and their etiologic agents can be challenging. Ehrlichia canis, the primary etiologic agent of canine monocytic ehrlichiosis, is relatively well characterized and offers unique advantages and opportunities to study interactions between a monocytotropic pathogen and both its vertebrate and invertebrate hosts. Historically, advances in tick-borne disease control strategies have typically followed explication of tick-pathogen-vertebrate interactions, thus it is reasonable to expect novel, more sustainable approaches to control of these diseases as the transmission of their associated infections are investigated at the molecular through ecological levels. Better understanding of the interactions between E. canis and its canine and tick hosts would also elucidate similar interactions for other Ehrlichia species as well as the potential roles of canine sentinels, reservoirs and models of tick-borne zoonoses. This article summarizes natural exposure studies and experimental investigations of E. canis in the context of what is understood about biological vectors of tick-borne Anaplasmataceae.     

Molecular detection of Cryptosporidium spp. infections in water buffaloes from northeast Thailand

The objectives of this study were to determine the individual and herd-level prevalence and genotype of Cryptosporidium and to identify putative risk factors associated with Cryptosporidium spp. infections in water buffaloes in northeast Thailand. Fecal samples from 600 water buffaloes of 287 farms in six provinces were collected and tested using DMSO-modified acid-fast staining and polymerase chain reaction. The overall prevalence of Cryptosporidium infections in buffaloes was 5.7 and 8.7 % among individual animals and herds, respectively. The provinces with highest infected Cryptosporidium were located in the Sakon Nakhon Basin in the northern part of the region. In addition, higher herd prevalence was observed among farms with more than five buffaloes (30 %) than those with five or less animals (16.2 %). Thirty (88.2 %) of the 34 Cryptosporidium-positive samples were Cryptosporidium parvum and four (11.8 %) were Cryptosporidium ryanae.     

The effect of the DNA preparation method on the sensitivity of PCR for the detection of Trypanosoma evansi in rodents and implications for epidemiological surveillance efforts

Trypanosoma evansi is responsible for the most largely distributed animal trypanosomosis, affecting a wide range of wild and domestic animals. Its surveillance requires the implementation of standardized and reliable diagnostic tools. Although the development of polymerase chain reaction (PCR) tools has greatly improved our diagnostic capacity, factors affecting their sensitivity need to be acknowledged and accounted for in the interpretation of results. The targeted gene and the primer design have already been shown to greatly affect the sensitivity of a PCR, and the best-performing sets of primers have been previously identified. However, the sensitivity of the PCR is also largely influenced by the DNA extraction or sample preparation method. In this paper, we selected 6 DNA extraction or blood sample preparation methods representative of what would be used in a budget-constrained setting: phenol–chloroform, Chelex^®, Flexigen (Qiagen^®) kit, Genekam^® kit and two original protocols using sodium hydroxide. We studied the effects of the preparation method on the detection limit of the subsequent PCR. Our results show that the extraction method strongly affects the PCR sensitivity. The classical phenol–chloroform extraction method allowed for the PCR with the lowest detection limit. Some combinations of extraction method and primer set had detection limits that were not compatible with their use as a reliable diagnostic test, and would severely reduce the performance of a surveillance program. Therefore, we encourage laboratories to carefully select their sample preparation and PCR protocols, depending on the aimed sensitivity, cost, safety, time requirement and objectives.     

Risk factors of Neospora caninum infection in dogs and cats in dairy farms in Western Thailand

The objectives of this study were to investigate the risk factors of Neospora caninum infection in dogs and cats found in dairy herds in western Thailand. A case-control study was conducted in dairy herds in three western provinces including Nakhon Pathom, Ratchaburi and Kanchanaburi. Blood samples of pets from 14 positive dairy herds and 26 herds from negative neighbouring farms which were randomly selected, in total blood samples from dogs and cats from 40 herds were collected and examined for antibodies against N. caninum infections using competitive ELISA and indirect fluorescent antibody test (IFAT). No seropositive cats were found. Of the 38 positive dogs, four (10.5%) were seropositive to N. caninum, which was higher than the proportion of seropositive in the negative herd population (one of 76, 1.3%). The higher proportion of seropositive farm dogs as compared with neighbouring dogs tended to be significant (P = 0.055).     

Molecular and serological prevalence of Babesia bovis and Babesia bigemina in water buffaloes in the northeast region of Thailand

Bovine babesiosis is a tick-transmitted hemoprotozoan disease that is mainly caused by Babesia bovis and Babesia bigemina and is characterized by significant morbidity and mortality worldwide. The disease is widespread in the northeastern region of Thailand, where an increasingly large part of the livestock is composed of water buffaloes. The present study was therefore conducted to investigate the epidemiological distribution of B. bovis and B. bigemina in water buffaloes in the northeastern region of Thailand. A total of 305 buffalo blood samples were randomly collected from five provinces and simultaneously analyzed by the nested PCR (nPCR) assay, ELISA, and IFAT techniques. The overall prevalence of B. bovis and B. bigemina was 11.2% and 3.6% by nPCR, 14.7% and 5.9% by ELISA, and 16.8% and 5.6% by IFAT, respectively. The high concordance between the molecular and the serological detection tests revealed the specificity and sensitivity of the diagnostic assays used for the detection of infection as well as the endemic stability status of the parasites in the surveyed areas. Statistically significant differences in the prevalence of the two infections were observed on the basis of age and location but not gender. Our data provide valuable information regarding the epidemiology of B. bovis and B. bigemina infection in water buffaloes in the northeastern region of Thailand which will likely be very beneficial for management and control programs of this disease.     

High genetic diversity in field isolates of Trypanosoma theileri assessed by analysis of cathepsin L-like sequences disclosed multiple and new genotypes infecting cattle in Thailand

In this study, we describe the first survey in Thailand of Trypanosoma theileri, a widespread and prevalent parasite of cattle that is transmitted by tabanid flies. Investigation of 210 bovine blood samples of Thai cattle from six farms by hematocrit centrifuge technique (HCT) revealed 14 samples with trypanosomes morphologically compatible to T. theileri. Additional animals were positive for T. theileri by PCR based on the Cathepsin L-like sequence (TthCATL-PCR) despite negative by HCT, indicating cryptic infections. Results revealed a prevalence of 26 ± 15% (95% CI) of T. theileri infection. Additionally, 12 samples positive for T. theileri were detected in cattle from other 11 farms. From a total of 30 blood samples positive by HCT and/or PCR from 17 farms, seven were characterized to evaluate the genetic polymorphism of T. theileri through sequence analysis of PCR-amplified CATL DNA sequences. All CATL sequences of T. theileri from Thai cattle clustered with sequences of the previously described phylogenetic lineages TthI and TthII, supporting only two major lineages of T. theileri in cattle around the world. However, 11 of the 29 CATL sequences analyzed showed to be different, disclosing an unexpectedly large polymorphic genetic repertoire, with multiple genotypes of T. theileri not previously described in other countries circulating in Thai cattle.     

Seroprevalence of Toxoplasma gondii antibodies in stray cats and dogs in the Bangkok metropolitan area, Thailand

Cats and dogs are the most popular pet animals worldwide. Cats are the natural reservoir of Toxoplasma gondii and excrete the resistant oocyst to environments. On the other hands, dogs play a role in the mechanical transmission of the parasite. Stray cats and dogs in the Bangkok metropolitan area are becoming a public concern because there is a considerable increase in their number annually. These facts indicate the risk of mechanically spreading zoonoses including toxoplasmosis to humans since human acquire the infection from infected mammals, either directly or indirectly. In the present study, the presence of T. gondii antibodies was examined in 592 cats and 427 dogs from October 2001 to September 2002 by using a latex agglutination test. T. gondii antibodies were detected in 65 (11.0%) of the 592 cats and 40 (9.4%) of the 427 dogs. The antibody titers in the positive animals ranged from 1:64 to 1:2048. Seroprevalence was significantly higher in female cats than in male cats. The present study suggested that T. gondii was widespread in the stray animals in the Bangkok metropolitan area; therefore, it is essential to control the number of stray cats and dogs in order to reduce the transmission of toxoplasmosis to animals and humans.     

Seroprevalence of Toxoplasma gondii in captive felids in Thailand

The prevalence of antibodies against Toxoplasma gondii was investigated by commercial latex agglutination test kit (Toxocheck-MT 'Eiken') in captive felids maintained at zoos and a wildlife breeding center in different geographic regions of Thailand. Sera from a total of 136 captive felids of 12 species was obtained between 2002 and 2004. The overall seroprevalence of T. gondii was found in 21 of 136 (15.4%) felids. The titers varied from 1:64 (eight samples) to 1:8192 (one sample). The seroprevalence in different geographic regions were from 0% in the northern area to 23% in the southern area. This study suggested a widespread exposure of captive felids to T. gondii in Thailand and this is the first report of serologic analysis for T. gondii in captive felids in Southeast Asia.