Badgar Battsetseg

Babesial Vector Tick Defensin against Babesia sp. Parasites

ABSTRACT Antimicrobial peptides are major components of host innate immunity, a well-conserved, evolutionarily ancient defensive mechanism. Infectious disease-bearing vector ticks are thought to possess specific defense molecules against the transmitted pathogens that have been acquired during their evolution. We found in the tick Haemaphysalis longicornis a novel parasiticidal peptide named longicin that may have evolved from a common ancestral peptide resembling spider and scorpion toxins. H. longicornis is the primary vector for Babesia sp. parasites in Japan. Longicin also displayed bactericidal and fungicidal properties that resemble those of defensin homologues from invertebrates and vertebrates. Longicin showed a remarkable ability to inhibit the proliferation of merozoites, an erythrocyte blood stage of equine Babesia equi, by killing the parasites. Longicin was localized at the surface of the Babesia sp. parasites, as demonstrated by confocal microscopic analysis. In an in vivo experiment, longicin induced significant reduction of parasitemia in animals infected with the zoonotic and murine B. microti. Moreover, RNA interference data demonstrated that endogenous longicin is able to directly kill the canine B. gibsoni, thus indicating that it may play a role in regulating the vectorial capacity in the vector tick H. longicornis. Theoretically, longicin may serve as a model for the development of chemotherapeutic compounds against tick-borne disease organisms.

Detection of natural infection of Boophilus microplus with Babesia equi and Babesia caballi in Brazilian horses using nested polymerase chain reaction

The potential role of Boophilus microplus as a natural tick vector of Babesia equi and Babesia caballi in Brazilian horses was assessed using nested polymerase chain reaction (PCR)-based marker assay. B. equi merozoite-specific 218bp gene fragment was detected in almost 96% of horse blood samples, and 45.3-62.5% of females, eggs, larvae, and nymphs of B. microplus collected from 47 horses at Campo Grande in the State of Matto Grosso, Brazil. Except for the partially-fed female ticks, the B. caballi-specific 430bp gene fragment was amplified from horse blood samples, and all developmental stages. Parasite DNA from both species was detected in horse blood samples and B. microplus, with the preponderance of B. equi DNA. No DNA samples were positive solely for B. caballi parasite. Only 32% of the Giemsa-stained thin blood smears were positive for Babesia parasites, as against detection of B. equi parasite DNA in 95.7% of the blood samples by nested PCR. We have obtained molecular evidence that strengthens earlier experimental and ultrastructural studies in Brazil incriminating B. microplus as a natural vector of B. equi, and possibly of B. caballi. The detection of B. equi and B. caballi DNA in eggs and larvae of B. microplus is likewise suggestive of the possibility of both transovarial and transstadial parasite transmission in this tick vector.

Babesia parasites develop and are transmitted by the non-vector soft tick Ornithodoros moubata (Acari: Argasidae).

Ornithodoros moubata ticks were fed on blood infected with Babesia equi. However, the parasites were quickly cleared as evidenced by the disappearance of B. equi-specific ribosomal RNA from the ticks. We hypothesized that if the Babesia parasite can escape midgut-associated barriers a non-vector tick can become infected with Babesia. To test this hypothesis, B. equi parasite-infected blood from in vitro culture was injected into the haemocoel of ticks. B. equi-specific rRNA was surprisingly detected 45 days after injection even in the eggs. Babesia-free dogs were infested with O. moubata ticks that were infected by inoculation with B. gibsoni-infected red blood cells. Parasitaemia and antibody production against Bg-TRAP of B. gibsoni increased gradually. These results indicate that O. moubata may be a useful vector model for Babesia parasites and also a very important tool for studies on tick immunity against Babesia parasites and tick-Babesia interactions.

Detection of Babesia caballi and Babesia equi in Dermacentor nuttalli adult ticks.

Ticks play an important role in human and veterinary medicine particularly due to their ability to transmit protozoan pathogens. In this study we have demonstrated that polymerase chain reaction (PCR) and nested PCR methods enabled detection of Babesia caballi and Babesia equi in field isolates of Dermacentor nuttalli adult ticks from Mongolia. Primers specific for 218 bp fragment merozoite antigen 1 (EMA-1) gene of B. equi successfully amplified products from all samples of D. nuttalli adult ticks while primers for the 430 bp fragment product from BC48 gene of B. caballi amplified products from seven of the 54 samples. Using PCR and nested PCR methods we have found mixed infections with B. equi and B. caballi in the tick vector. The amplified DNA fragment from D. nuttalli ticks was inserted into the EcoRV site of pBluescript SK and sequenced. The sequence of the 430 bp fragment was completely identical to the nucleotide sequence of the USDA strain of B. caballi. These results suggest that D. nuttalli may play an important role as a vector of both B. caballi and B. equi and also may be important in maintaining endemicity of equine piroplasmosis in Mongolia.

Genetic detection of Babesia bigemina from Mongolian cattle using apical membrane antigen-1 gene-based PCR assay

We developed a new nested PCR (nPCR) assay based on the Babesia bigemina apical membrane antigen-1 (AMA-1) gene sequence for parasite-specific detection. The primers were designed to amplify 738-bp and 211-bp fragments of the AMA-1 gene by primary and nested PCRs, respectively. The assay was proven to be specific for the B. bigemina, whereas the previously established SpeI-AvaI nPCR assay amplified not only the target fragment of B. bigemina but also a homologous one from Babesia ovata. The AMA-1 nPCR assay was also evaluated using field DNA samples extracted from 266 bovine blood samples collected from Mongolia in 2010. In a comparative evaluation, 90 (33.8%) and 25 (9.4%) of the blood samples showed positive reactions for B. bigemina by the SpeI-AvaI nPCR and AMA-1 nPCR assays, respectively. The sequencing analysis of the nPCR products confirmed that the AMA-1 nPCR method had specifically detected the target B. bigemina DNA. However, 4 different kinds of sequences were determined among the SpeI-AvaI nPCR amplicons. Two of them were derived from B. bigemina and B. ovata, while the origins of the others were unknown. In the current study, the presence of B. bigemina was clearly demonstrated among Mongolian cattle populations by the current nPCR assay for the first time. Furthermore, our findings also indicate that the AMA-1 nPCR assay may be a useful diagnostic tool for the specific detection of B. bigemina.

RNA interference of cytosolic leucine aminopeptidase reduces fecundity in the hard tick, Haemaphysalis longicornis

Ticks are effective vectors of pathogens because of their blood feeding and high fecundity. This high fecundity is related to the size of the blood meal. Therefore, knowledge of how blood proteins are degraded and converted to proteins, including yolk protein, is important for the development of ways to inhibit the utilization of blood proteins by ticks. RNA interference (RNAi) is becoming a powerful post-transcriptional gene silencing technique that provides insight into gene function. We constructed a double-stranded RNA (dsRNA) based on a previously cloned Haemaphysalis longicornis leucine aminopeptidase (HlLAP) gene to reevaluate the biological role in tick blood digestion. Gene specific transcriptional, translational, and functional disruptions were achieved by the introduction of dsRNA into the ticks. Significantly delayed onset of egg-laying and reduced egg oviposition resulted from the RNAi for the HlLAP gene. These results suggest that HlLAP actually works as a blood digestive enzyme and affects tick fecundity via unknown mechanisms. The reduction of egg oviposition may be caused by a decrease in nutrients, especially free amino acids generated by HlLAP, from the blood meal. This is the first report of an impact on tick reproduction caused by gene silencing of a blood digestion-related molecule.

Phylogenetic relationships of Mongolian Babesia bovis isolates based on the merozoite surface antigen (MSA)-1, MSA-2b, and MSA-2c genes.

We conducted a molecular epidemiological study on Babesia bovis in Mongolia. Three hundred blood samples collected from cattle grazed in seven different districts were initially screened using a previously established diagnostic polymerase chain reaction (PCR) assay for the detection of B. bovis-specific DNA. Positive samples were then used to amplify and sequence the hyper-variable regions of three B. bovis genes encoding the merozoite surface antigen (MSA)-1, MSA-2b, and MSA-2c. The diagnostic PCR assay detected B. bovis among cattle populations of all districts surveyed (4.4-26.0%). Sequences of each of the three genes were highly homologous among the Mongolian isolates, and found in a single phylogenetic cluster. In particular, a separate branch was formed only by the Mongolian isolates in the MSA-2b gene-based phylogenetic tree. Our findings indicate that effective preventative and control strategies are essential to control B. bovis infection in Mongolian cattle populations, and suggest that a careful approach must be adopted when using immunization techniques.

Prevalence and genetic diversity of equine piroplasms in Tov province, Mongolia

Equine piroplasmosis represents a serious problem in horse industry. Although, researchers suggested the possible use of sub-unit vaccines to control equine piroplasmosis, the genetic diversity of vaccine candidate antigens was not properly investigated. In the present study, we screened 250 horses reared in three different districts of Tov province, Mongolia, for Babesia caballi and Theileria equi using ELISA and nested PCR (nPCR) assays. Among these animals, piroplasms were detected in 128 (51.2%) horses by nPCR assays (B. caballi, 42.4%; T. equi, 6.4%; and mixed infections, 2.4%), while 204 (81.6%) were positive by ELISA (B. caballi, 51.6%; T. equi, 19.6%; and mixed infections, 10.4%). Male and middle-aged horses showed higher positive rates than female and younger or older horses. The findings also suggested that a combination of nPCR and ELISA techniques might be useful to detect horses that were chronically or subclinically infected with piroplasms. B. caballi-BC48 and T. equi-EMA-1 gene sequences, in addition to 18S rRNA, were subjected to phylogenetic analyses, and the findings suggested the presence of genetically diverse populations of equine piroplasms in Mongolia. BC48 sequences were separated into four clades in phylogram, and all the Mongolian sequences determined in the present study were found in a single clade. However, a single BC48 sequence previously isolated from a tick in Mongolia formed a separate branch. Similarly, EMA-1 sequences formed four clades, and Mongolian sequences were observed in two different clades, one of which was formed only of Mongolian sequences and is suggested as a new clade. This is the first report that describes the genotypes of equine piroplasms in Mongolia. The findings also emphasized the need for further investigations to study the effect of genetic diversity observed among BC48 as well as EMA-1 sequences on hosts immune responses.

Construction of Neospora caninum stably expressing TgSAG1 and evaluation of its protective effects against Toxoplasma gondii infection in mice.

Toxoplasma gondii and Neospora caninum are closely related apicomplexan parasites. The surface antigen 1 of T. gondii (TgSAG1) is a major immunodominant antigen and, therefore, is considered to be a good candidate for the development of an effective recombinant vaccine against toxoplasmosis. In this study, N. caninum stably expressing the TgSAG1 gene (Nc/TgSAG1) was constructed using pyrimethamine-resistant DHFR-TS and GFP genes as double-selection markers. The expression level, molecular weight, and antigenic property of recombinant TgSAG1 expressed by the Nc/TgSAG1 were similar to those of the native TgSAG1. The mice immunized with Nc/TgSAG1 induced TgSAG1-specific Th1-dominant immune responses and protected the mice from a lethal challenge infection with T. gondii. These results indicate that N. caninum may provide a new tool for the production of a live recombinant vector vaccine against toxoplasmosis in animals. To our knowledge, this is the first report to evaluate the usefulness of N. caninum-based live vaccine.

The first survey of Theileria orientalis infection in Mongolian cattle

In the present study, we have surveyed the presence of a bovine Theileria protozoan, Theileria orientalis, in Mongolian cattle and engorging tick populations from selected provinces and districts in Mongolia. The percentages of infection in the cattle and ticks ranged from 8.8 to 66.6 and from 3.7 to 73.3, respectively, on a per district basis. The genetic diversity of T. orientalis isolates was also studied, based on the protozoan gene encoding a major piroplasm surface protein (MPSP). At least five genotypes (types 1, 3, 5, 7, and N-3) of T. orientalis were found to be circulating among the Mongolian cattle and tick populations. In particular, types 3 and N-3 were common in most of the districts examined, while a strong geographical relationship among the genotypes was not detected in the present study. This is the first epidemiological report describing the presence of T. orientalis infection in Mongolian cattle.