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.

Neospora caninum: Chronic and congenital infection in consecutive pregnancies of mice.

Neospora caninum, the causative agent of bovine neosporosis is the major cause of abortion in cattle worldwide. The principal route of transmission is via in utero infection of the offspring. Congenitally-infected dams remain persistently infected for life and might undergo abortions in consecutive pregnancies. In the present study, the effect of N. caninum in chronic and congenital infection was examined. CD1 mice were infected intra-peritoneally with live tachyzoites of the NcIs491 isolate, while non-infected mice served as a control. There were no clinical signs of infection observed following inoculation, but high titers of specific anti- N. caninum antibodies were detected. A month after infection, when chronic-infection was established, mice were mated. Fertility, litter size and mortality rate were monitored within two generations of four consecutive pregnancies. During a nine months period of the study all females maintained high level of antibodies, while the non- infected control mice remained seronegative. There was no difference in the fertility rate of the dams, or in the litter size of infected and control mice. Mortality of offspring of the first and second generations of the infected dams was observed within the two first weeks of life. The vertical transmission was analyzed by PCR assay of offspring brains. PCR positive results were found in all 13 litters of the first generation tested during four consecutive pregnancies. The rate of vertical transmission slightly decreased in successive pregnancies being 74.2%, 59.5%, 48.1% and 40% for the first to fourth pregnancies respectively. In the second generation 21 out of 28 litters were found positive and the overall rate of vertical transmission was 28.5%. In chronically and congenitally infected dams N. caninum infection was maintained during all successive pregnancies for about 9 months. The results show that CD-1 outbred mice are a suitable model for studying chronic and congenital neosporosis.

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.

Neospora caninum: in vivo and in vitro treatment with artemisone.

Neosporosis caused by Neospora caninum has global economic, clinical, and epidemiological impacts, mainly in the cattle industry. Currently, there is no useful drug for treatment of neosporosis. This publication is the first to describe the significant benefits that artemisone has on Neospora infections both in vitro and in vivo. Artemisone is a new semi-synthetic 10-alkylamino artemisinin that is superior to other artemisinin derivatives in terms of its significantly higher antimalarial activity, its tolerance in vivo, lack of detectable neurotoxic potential, improved in vivo pharmacokinetics and metabolic stability. Low micromolar concentrations of artemisone inhibited in vitro Neospora development. Prophylactic and post-infection treatment profoundly reduced the number of infected cells and parasites per cell. In the in vivo gerbil model, a non-toxic dose prevented typical cerebral symptoms, in most animals. There were no signs of clinical symptoms and brain PCR was negative. Most treated gerbils produced high specific antibody titer and were protected against a challenge. Overall, artemisone could be considered as a future drug for neosporosis.

Genetic diversity of anaplasma species major surface proteins and implications for anaplasmosis serodiagnosis and vaccine development.

The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes several pathogens of veterinary and human medical importance. An understanding of the diversity of Anaplasma major surface proteins (MSPs), including those MSPs that modulate infection, development of persistent infections, and transmission of pathogens by ticks, is derived in part, by characterization and phylogenetic analyses of geographic strains. Information concerning the genetic diversity of Anaplasma spp. MSPs will likely influence the development of serodiagnostic assays and vaccine strategies for the control of anaplasmosis.

Expression of Anaplasma marginale major surface protein 2 operon-associated proteins during mammalian and arthropod infection.

The antigenically variant major surface protein 2 (MSP2) of Anaplasma marginale is expressed from a 3.5-kb operon that contains, in a 5'-to-3' direction, four open reading frames, opag3, opag2, opag1, and msp2. This operon structure was shown to be conserved among genotypically and phenotypically distinct A. marginale, A. ovis, and A. centrale strains. The individual OpAG amino acid sequences are highly conserved among A. marginale strains, with identities ranging from 95 to 99%. OpAG2 and OpAG3 were expressed by all examined A. marginale strains during the acute rickettsemia in the mammalian host and, like MSP2, localize to the bacterial surface. OpAG2 and OpAG3 were also expressed in an infected Ixodes scapularis tick cell line. In contrast, the same A. marginale strains expressed only OpAG2 in two different Dermacentor spp. during transmission feeding. OpAG1 expression was not detected in the infected mammalian host, the infected tick cell line, or within infected Dermacentor ticks. The differential expression of outer membrane proteins from within an operon is a novel finding in tick-transmitted bacteria, and the regulation of expression may be broadly applicable to understanding how the pathogen adapts to the mammalian host-tick vector transition.

Pan-Mediterranean comparison for the molecular detection of Theileria annulata.

Seven laboratories decided to compare their molecular diagnostic techniques to identify Mediterranean theileriosis caused by Theileria annulata. Each laboratory used either PCR or PCR and reverse line blot hybridization (RLB) to identify T. annulata. Five laboratories sent their own samples to laboratory 4 to be recoded and passed on to at least two other laboratories. A total of 120 blood samples were analyzed during this study, generating 540 results. Laboratory 1 sent only T. annulata-infected samples (positive control batch), and all the laboratories testing this batch found 100% infection. Laboratory 2 sent only negative samples from a Mediterranean area where T. annulata was unknown, and two laboratories out of three found a few positive samples in these negative samples. For the remaining samples, detection performance was variable. Agreement between laboratories ranged from 21.4 to 91.3%. The overall mismatch between laboratories was around 30% by whatever technique used. This paper describes the methodological parameters that could explain the variation of results.

Expression of major surface protein 2 variants with conserved T-cell epitopes in Anaplasma centrale vaccinates.

Major surface protein 2 (MSP-2), identified as a protection-inducing immunogen against Anaplasma marginale challenge, is an immunodominant outer membrane protein with orthologues in all examined Anaplasma species. Although immunization with live Anaplasma centrale has long been used to induce protection against acute disease upon challenge with virulent A. marginale, its MSP-2 structure and whether MSP-2 variants are generated during persistence of the vaccine strain was unknown. In this study, we showed that the A. centrale vaccine strain persisted for a minimum of 4 years postvaccination and generated sequential MSP-2 variants. Comparison of amino acid sequences encoded by A. centrale msp-2 transcripts from the initial postimmunization period and from sequential time points during persistence of the vaccine strain revealed a central hypervariable domain flanked by conserved amino and carboxy-terminal regions. This structure corresponded to that shown in A. marginale MSP-2, where the central hypervariable region encodes variant B-cell epitopes in the extracellular domain and the flanking transmembrane domains are rich in CD4(+)-T-cell epitopes. Importantly, at least four CD4(+)-T-cell epitopes are conserved between the two species, a finding consistent with A. marginale challenge triggering a recall response of CD4(+) T cells induced by A. centrale vaccination. The genomic arrangement is conserved between A. centrale and A. marginale with multiple msp-2 pseudogenes and a single operon-linked expression site for the full-length msp-2. This conservation of both genomic structure for generating MSP-2 variants and the CD4(+)-T-cell epitopes between these two genetically distinct Anaplasma species indicates that they present a similar repertoire of MSP-2 epitopes to the immune system and that this similarity may be responsible for all or part of the A. centrale vaccine efficacy.