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Ehrlichia canis is the etiologic agent of canine monocytic ehrlichiosis (CME) and is a useful model for zoonotic tick-borne pathogens, many of which infect dogs. The purpose of this study was to evaluate rifampin and doxycycline regimens for clearance of E. canis infections in addition to alleviation of CME. Beagles were infected with E. canis by intravenous inoculation with carrier blood and treated with either rifampin or doxycycline after the acute phase of CME. Improved hematological values demonstrated that both treatments effectively relieved signs of the disease. Peripheral blood from all dogs became PCR negative after antibiotic treatment, suggesting that these infections were eliminated and that rifampin is an effective alternative chemotherapeutic agent for treatment of CME.

Tick burdens cause direct damage to hosts and transmit several disease agents, the majority of which are secreted into feeding lesions through tick salivary glands. Reduced incidence of naturally transmitted tick-borne diseases was recently observed among cattle immunized with tick salivary gland extracts (TSGE). The aim of this work was to compare the ultrastructure of salivary glands from Rhipicephalus (Boophilus) microplus removed from cattle that were immunized with TSGE to those from control cattle injected with saline. Partially fed female ticks that infested these cattle were collected, and their salivary glands were removed and compared with light microscopy. More extensive salivary gland damage was observed in ticks collected from cattle immunized with TSGE than those from saline controls. Salivary glands from immunized cattle had extensive necrosis in agranular c and f cells; partial necrosis in granular b, c, and e cells; and slight necrosis in a and d cells. These results indicated that host immunization with TSGE causes detrimental effects to female tick salivary glands and correlated with the reduced incidence of naturally transmitted tick-borne diseases observed among these cattle.

The tropical cattle tick, Rhipicephalus (Boophilus) microplus, is an important ectoparasite of livestock in Thailand that causes economic losses due to the direct effects of tick feeding and by the pathogens they transmit. Intensive acaricide use has several drawbacks, which spurred efforts to develop anti-tick vaccines. Vaccines targeting concealed antigens localized in the tick midgut result in reduced tick fecundity, but molecules localized in the tick salivary glands, which could play a role in pathogen transmission, remain largely unexplored for R. microplus. Calreticulin (CRT) is a protein found in tick salivary glands and saliva, and CRT might facilitate tick feeding and pathogen transmission through anti-thrombotic and complement-inhibition activities. This then suggests that CRT should be evaluated as a vaccine candidate antigen to control cattle ticks in Thailand. The objective of this work was to clone, sequence, and analyze cDNA encoding CRT from salivary glands of R. microplus indigenous to Thailand. Nucleotide sequence analysis showed an open reading frame of 1233 bp. Comparison of the amino acid sequence showed 65-99% identities to other known CRTs from Oryctolagus cuniculus, Rattus norvegicus, Homo sapiens, Bos taurus, R. sanguineus, and R. microplus. The N- and P-domains of CRT were the most conserved, whereas the C-domain was high acid and more variable. CRT primary sequences were most conserved among mammals. Further investigations are warranted to determine whether immunization with Thai B. microplus CRT can affect tick performances and experimental pathogen transmission.

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.

An immunocompetent animal disease model based on infection with Ehrlichia chaffeensis would facilitate research toward understanding mechanisms responsible for the broad range of clinical signs associated with human monocytic ehrlichiosis (HME). The adaptability of this model for the experimental feeding of tick species and stages and for testing therapies comparable to those for human diseases are additional advantages of large animal models. Herein, we summarize pathology reports for calves that developed fatal disease after experimental inoculation with E. chaffeensis. Elevated liver enzyme levels and lung pathology among these calves corroborated earlier reports of severe HME. Thus, an experimental disease model based on infection of outbred immunocompetent hosts with E. chaffeensis could be within our grasp for the first time.

A rapidly increasing stray animal population in Bangkok has caused concern regarding transmission of vector-borne and zoonotic diseases. The purpose of this study was to determine if stray animals in Bangkok are a potential reservoir of Hepatozoon, a genus of tick-borne parasites that has received little attention in Thailand. Blood samples were collected from stray companion animals near monasteries in 42 Bangkok metropolitan districts. Both dogs and cats were sampled from 26 districts, dogs alone from 4 districts and cats alone from 12 districts. Samples were collected from a total of 308 dogs and 300 cats. Light microscopy and an 18 S rRNA gene-based PCR assay were used to test these samples for evidence of Hepatozoon infection. Gamonts were observed in blood smears for 2.6% of dogs and 0.7% of cats by microscopy. The PCR assay detected Hepatozoon in buffy coats from 11.4% of dogs and 32.3% of cats tested. The prevalence of infection was the same between male and female dogs or cats, and PCR-positive dogs and cats were found in 36.6% and 36.8% of the districts surveyed, respectively. There was an association between the percentages of PCR-positive dogs and cats in districts where both host species were sampled. Sequences of representative amplicons were closest to those reported for H. canis. These results represent the first molecular confirmation that H. canis is indigenous to Thailand. The unexpectedly high prevalence of Hepatozoon among stray cats indicates that their role in the epizootiology of hepatozoonosis should be investigated.

Cryptosporidium species are frequently associated with diarrhea among AIDS patients in Thailand, and dairy herds are a possible source of some of these infections. A cross-sectional study was performed to determine if Cryptosporidium is present among dairy cows in Thailand. Fecal samples were randomly collected from 363 Holstein-Friesian dairy cows from 108 of 860 farms in the Nong Pho region of central Thailand. The average prevalence of Cryptosporidium among dairy cows was 9.4%, according to an assay for Cryptosporidium-specific antigen (CSA) and 0.6% by microscopic examination of acid-fast stained feces. CSA was detected in all host age categories tested, but was most prevalent among calves (15.1%). Overall, 31.5% of farms were contaminated with Cryptosporidium infections. Fifty percent of poorly managed farms had CSA-positive cows, which were more likely to contaminate water and raw milk, while 12.9% of farms with acceptable management practices had CSA-positive cows. There was no association between the detection of Cryptosporidium and other gastrointestinal parasites. These results indicate that Cryptosporidium is enzootic among Thai dairy cattle, and suggest that cattle could have a role in zoonotic cryptosporidiosis in Thailand.

Boophilus microplus is an important vector of bovine disease agents having a major economic impact on cattle production in many tropical and subtropical countries. Components of tick saliva that enable ticks to feed may also facilitate establishment of tick-borne pathogens in the vertebrate host. It has been suggested that acquired resistance against molecules in tick saliva could inhibit parasite transmission, and there is increasing evidence to support this hypothesis. The effect of immune resistance to B. microplus on the incidence of tick-transmitted pathogens was the focus of this experiment. Groups of four dairy cows were injected with antigen extracts of tick salivary glands, midgut, adjuvant only, or PBS, prior to a grazing period in a pasture in Thailand where ticks are abundant and babesiosis is enzootic. These animals were then observed for evidence of babesiosis throughout the rainy season. A reduction in the incidence of clinical babesiosis was observed among cattle immunized with salivary gland preparations compared to nonimmunized controls (P < 0.05). Immunization with midgut or adjuvant only both resulted in a slight reduction in observed disease compared to the same negative control group. B. bigemina was detected in fewer ticks (24.43%) collected from salivary gland-immunized cattle than those collected from the remaining groups (> or =44.57%). These results indicated that immunization with salivary gland antigens could affect pathogen transmission and appears promising for control of tick-borne diseases of cattle.

Boophilus microplus has a major impact on cattle production, and an antitick vaccine would be a valuable tool for control of this important ectoparasite in Thailand. Previous work has shown that immunization of hosts with different tick tissues has different implications regarding tick feeding and fecundity under experimental conditions. The purpose of this study was to assess the effects of immunization of dairy cattle with B. microplus salivary gland or midgut extracts on natural infestations by this tick species. The different antigen extracts (1 mg total protein) or equivalent amounts of adjuvant alone were injected intradermally every two weeks for a total of three times before allowing cattle to graze in a tick-contaminated pasture. Animals were checked daily, and engorged female ticks collected, counted, weighed, and maintained in tick incubators to observe tick performance parameters, including engorged weight, egg mass weight, nonviable eggs, mortality, oviposition period, egg incubation period, and F1 larval weight. After six months, each group was reimmunized with the same antigen and/or adjuvant, and ticks were again collected and evaluated. Immunization of cattle with salivary gland preparations resulted in reductions in mean tick counts and in engorged female weights. Immunization with midgut antigens reduced tick oviposition and reduced egg mass weights. In addition, more ticks recovered from midgut-immunized cows produce nonviable eggs. This investigation indicates that a vaccine based on these antigen preparations could induce a lasting, protective immune response against B. microplus that would be expected to provide a safe nontoxic means of tick control.

A nucleic acid probe complementary to baboon interleukin 10 (IL-10) mRNA was developed for in situ hybridization. Highly conserved IL-10 protein sequences from several mammals were aligned to design oligonucleotide primers flanking a 270-bp sequence of the target cDNA. RNA was isolated from stimulated peripheral blood mononuclear cells (PBMC). IL-10 cDNA was reverse-transcribed from the total PBMC RNA and amplified with the polymerase chain reaction (PCR). Cloning and sequencing of the PCR product confirmed it to be of baboon IL-10 origin, with 97.8% identity to human and 100% identity to macaque mRNA sequences. The baboon IL-10 DNA probe hybridized in Southern blots to a 7.9-Kbp or 8.6-Kbp band after digestion of genomic baboon DNA with Bam H1 or Eco R1, respectively. Preliminary results with an antisense riboprobe derived from this sequence showed the presence of IL-10 mRNA in sections of granulomatous tissues.

The antibody (Ab) responses of dogs immunized with adult tick salivary gland (TSG), midgut (TMG), or repeated infestations of Rhipicephalus sanguineus were monitored to determine if there is an association between Ab production and R. sanguineus performance. Tick-naïve dogs were immunized with TSG or TMG and subjected to two challenge infestations. The control group was infested five times at 21-day intervals. The ELISA technique was used to measure Ab levels in sera from these dogs, which expressed different forms of resistance against R. sanguineus. In dogs immunized with TSG or TMG, similar Ab levels were detected against TMG, TSG, muscle, synganglion, and reproductive organs. However, these sera had different Ab levels against egg mass, unfed larvae, fed larvae, and nymph antigens. Ab levels to muscle, nerve, and reproductive antigens were lower than those observed when TMG or TSG antigens were used. Sera from dogs immunized with TMG or TSG responded to most tick stages or tissue antigens, whereas repeated infestation sera showed the lowest response among the three groups.