Isolation, cloning, and pathologic analysis of Trypanosoma evansi field isolates.

In recent years, the emergence of highly pathogenic Trypanosoma evansi strains in the Philippines has resulted in substantial losses in livestock production. In this study, we isolated T. evansi from infected-water buffaloes in the Philippines and analyzed their virulence using mice and cattle. A total of 10 strains of T. evansi were isolated. Evaluation of the virulence of each strain using mice depicted significant differences among the strains in the prepatent period, the level of parasitemia, and the survival time of the infected animals. In mice infected with the highly pathogenic T. evansi, signs of excessive inflammation such as marked splenomegaly and increase more than 6-fold in the number of leukocytes were observed at 8 days post-infection. To study the virulence of the parasite strains in cattle (which are the common T. evansi hosts in Philippines), cattle were infected with the T. evansi isolates that showed high and low virulence in mice. The rate of parasite growth and the length of the prepatent periods were found to be similar to those observed in mice for the respective strains. The cattle infected with the highly pathogenic strain developed anemia and a marked decrease in leukocyte counts. To determine the cause of the pathological changes, we analyzed the expression levels of inflammatory cytokines and observed up-regulation of tumor necrosis factor-α in anemic infected cattle. Our findings suggest that the epidemic of T. evansi in the Philippines is characterized by T. evansi strains with varying virulences from low to very high pathogenicity in cattle.

Development and application of a quantitative real-time PCR for the diagnosis of Surra in water buffaloes.

Trypanosoma evansi (T. evansi) causes the disease called Surra in domestic animals, which is of great economic importance in South Asian countries. In order to improve the diagnosis of Surra, we endeavored to develop a real-time PCR assay for the detection and quantification of parasites in water buffaloes using specific primers for the T. evansi Rode Trypanozoon antigen type (RoTat) 1.2 Variable Surface Glycoprotein (VSG) gene, which is a known diverse DNA region in trypanosomes. The quantitative detection limit of the assay was 10(2) trypanosomes per mL of blood, and the identity of the amplicon was confirmed in all assays by melting curve analysis. To evaluate the clinical applicability of this procedure, detection and estimation of parasitemia in blood samples obtained from water buffaloes and horses were conducted. T. evansi was detected in 17/607 (2.8%) blood samples, with parasitemia levels ranging from >10(1) to 10(7) parasites per mL of blood. Interestingly, out of the 17 PCR positive animals, 3 had previously received trypanocidal treatment and 1 had abortion history. These data indicate that real-time PCR for the estimation of putative parasitemia levels is a quantitatively and objectively applicable technique for clinical diagnosis of Surra, and could help to understand disease stage and risk of transmission of T. evansi.

A survey of abortifacient infectious agents in livestock in Luzon, the Philippines, with emphasis on the situation in a cattle herd with abortion problems.

In the Philippines, insufficient consideration has been given to the implementation of systematic control measures against major abortifacient infectious agents in livestock. To elucidate the epidemiology of abortifacient infectious agents in livestock, the prevalence of four abortifacient agents was assessed. Initially, a total of 96 cattle including 17 cows with history of abortion were examined in a herd in Luzon at the request of the farm owner. Six (35.3%) of the 17 aborting cows were found to be serologically positive for Neospora caninum (N. caninum), whereas the seroprevalence in non-aborting cows was 15.9% (10/63). Four of the 6 serologically positive aborting cows were also RT-PCR-positive for bovine viral diarrhea virus (BVDV). Two (12.5%) of the 16 bulls examined were also found to be infected with BVDV, suggesting a putative risk factor of transmission via semen. Based on sequence analysis, the isolates detected belong to BVDV type 1b group. Furthermore, an epidemiological survey of abortifacient infectious agents was conducted with various species of livestock from herds located in Luzon. Out of the 105 water buffalo samples collected, 4 (3.8%) were indicated positive to N. caninum, 2 (1.9%) to Toxoplasma gondii (T. gondii) and 2 (1.9%) to Trypanosoma evansi (T. evansi). The overall seroprevalence of N. caninum in goat and sheep were 23.6% (21/89) and 26.3% (10/38), respectively. BVDV was not detected in these herds. The findings of this exploratory study indicate a relationship between infection and bovine abortion and that a lager study is required to statistically confirm this relationship.