Quantitative analysis of Babesia ovis infection in sheep and ticks.

A quantitative PCR, based on the gene encoding Babesia ovis Surface Protein D (BoSPD) was developed and applied to investigate the presence of Babesia ovis (B. ovis) in its principal vector, the tick Rhipicephalus bursa (R. bursa), and in the ovine host. Quantification of B. ovis in experimentally-infected lambs showed a sharp increase in parasitemia 10-11 days in blood-inoculated and adult tick-infested lambs, and 24 days in a larvae-infested lamb. A gradual decrease of parasitemia was observed in the following months, with parasites detectable 6-12 months post-infection. Examination of the parasite load in adult R. bursa during the post-molting period using the quantitative PCR assay revealed a low parasite load during days 2-7 post-molting, followed by a sharp increase, until day 11, which corresponded to the completion of the pre-feeding period. The assay was then used to detect B. ovis in naturally-infected sheep and ticks. Examination of samples from 8 sheep and 2 goats from infected flocks detected B. ovis in both goats and in 7 out of the 8 sheep. Additionally, B. ovis was detected in 9 tick pools (5 ticks in each pool) and two individual ticks removed from sheep in infected flocks.

Transmission of Babesia ovis by different Rhipicephalus bursa developmental stages and infected blood injection.

In this report, the transmission efficacy of Babesia ovis, the principal causative agent of ovine babesiosis, was studied by infestation of lambs with different Rhipicephalus bursa stages or by injection of infected blood. Infected blood injection induced acute babesiosis in splenectomized lambs, while only mild clinical signs were observed in intact animals. Both splenectomized and intact lambs developed high antibody titer, detectable for at least 180 days post infection. Infestation of splenectomized and intact lambs with infected tick larvae did not induce clinical babesiosis or specific serum response in any of the examined animals. Similarly, infestation of one splenectomized lamb with partially-fed infected R. bursa males did not induce any clinical response or seroconversion. Nymph infestation caused a mild clinical response followed by specific seroconversion, in one out of five lambs. All animals infested with infected unfed adults (males and females) showed mild-to-severe clinical signs 8 to 12 days post infestation. The acute phase was followed by a marked seroconversion. Our results indicate that the principal transmission of B. ovis is performed by adult R. bursa ticks, and that the host reaction can last as long as 6 months following the acute infection.

Molecular detection of Babesia ovis in sheep and ticks using the gene encoding B. ovis surface protein D (BoSPD).

The gene encoding Babesia ovis surface protein D (BoSPD) was cloned from B. ovis cDNA library. This gene encodes a polypeptide chain of 155 amino acids, including a predicted 22 amino acid signal peptide. Sequence analysis of the BoSPD suggested that it is a surface protein with no known domains. BLAST analysis followed by multiple alignments showed four orthologs from other Apicomplexan species and suggested that BoSPD is specific for B. ovis. BoSPD-based PCR was then developed to specifically detect B. ovis in experimentally-infected sheep and Rhipicephalus bursa ticks, as well as in field samples. The PCR enabled detection of B. ovis at a calculated parasitemia of 0.0016% and was shown to be specific for B. ovis. Moreover, the BoSPD PCR allowed detection of prolonged subclinical infection in experimentally-infected lambs and in dissected organs of experimentally-infected ticks. Finally, the PCR was used to detect parasitemia in blood samples from naturally-infected sheep and in R. bursa ticks collected from sheep in an infected flock. These results suggest that the BoSPD gene sequence can be used as a specific and sensitive marker, allowing detection of subclinical parasitemia in sheep and in ticks. Based on its predicted properties, BoSPD may be considered as a candidate for anti-B. ovis vaccine development or a target for anti-B.ovis treatment.

Neosporosis in naturally infected pregnant dairy cattle.

Neosporosis caused by caused by the apicomplexan parasite Neospora caninum is one of the major causes of infectious abortion in bovines worldwide. A long-term prospective study was performed in a dairy herd endemic for N. caninum in order to analyze the impact of neosporosis on the proportion of aborting cows. A total of 1078 pregnant cows were tested for presence of antibodies and the proportion of abortions was calculated. The overall seroprevalence of N. caninum found in the herd was 35.5%. The percentage of abortions in seropositive cows was 3 times higher than in their seronegative counterparts (21.6 and 7.3%, respectively). No statistically significant association was found between the antibody level of positive during pregnancy and the proportion of aborting cows. However, 41.2% of the dams with antibody titers of 1:12,800 aborted. The risk of abortion for such dams was 2.7 times higher than for other seropositive cows which had lower titers of antibodies (p=0.0072). In the follow-up examinations of the seropositive cows during several pregnancies, the overall percent of abortions observed was significantly higher than in seronegative individuals (49.3 and 16.9%, respectively; p<0.0001). Moreover, the proportion of repetitive abortion observed was 5 to 1 (17.4 and 3.5%) in seropositive and seronegative dams, respectively (p<0.001). The rate of vertical transmission in positive dams was 61.0% and it appeared to be directly associated with antibody levels: the higher the titer in the dams during pregnancy, the higher the percentage of sero-positivity in their calves. Increased proportion of abortions was observed in seropositive cows both in summer and winter in comparison with spring and autumn. It was found that in seropositive cows, an increased number of pregnancies, which was directly related to the age of the dam, has been associated with an increased number of abortions.