The effect of a live Neospora caninum tachyzoite vaccine in naturally infected pregnant dairy cows.

Neosporosis, caused by the intracellular protozoan Neospora caninum, is a major cause of abortion and reproductive failure in cattle worldwide. The principal route of transmission of neosporosis is via in utero infection of the offspring. There is no effective prophylactic treatment or vaccine available against bovine neosporosis. A N. caninum NcIs491 isolate was examined for its ability to immunize and reduce abortions in naturally infected dairy cows under field conditions. N. caninum-seropositive pregnant dams were inoculated with 10(8) live tachyzoites during mid-term pregnancy. A total of 520 N. caninum seropositive dams were included in this study, of these, 146 were immunized and 374 cows served as a non-vaccinated control group. A significantly lower incidence of abortion was observed in vaccinated compared to non-vaccinated cows, 16 and 26% respectively (P=0.01), with a vaccine efficacy of 39%. However, the number of seropositive offspring remained similar in both groups. Overall, this field trial suggests that vaccination with live N. caninum tachyzoites should be considered as an effective measure to reduce abortions caused by neosporosis in naturally infected cows.

Genetic polymorphism of Babesia bovis merozoite surface antigens-2 (MSA-2) isolates from bovine blood and Rhipicephalus annulatus ticks in Israel.

This study demonstrated the genetic diversity among MSA-2c, MSA-2a1 and MSA-2b proteins of Babesia bovis isolates obtained from bovine blood and Rhipicephalus annulatus tick samples. The least identities that were observed among the deduced amino acid sequences of MSA-2c, MSA-2a1 and MSA-2b were 55, 63, and 71%, respectively. During the study four B. bovis calves, aged about 1 month, were found to be infected with virulent field strains and developed babesiosis. Probably, the calves had received insufficient antibodies, or the antibodies raised against the vaccine strain did not cross-protect against virulent field isolates. The complete msa-2 locus from the Israeli B. bovis vaccine strain and two field isolates were characterized. Similarly to the Australian strains and isolates, the msa-2 loci of the examined Israeli strain and isolates had only two msa-2 genes - msa-2c and msa-2a/b - located between msa-2c and orfB. Several of the examined samples, contained different MSA-2 genotypes concurrently. No obvious geographical relationships among isolates from various regions of Israel were established. Moreover, in the phylogenetic analyses, the Israeli deduced MSA-2 amino acid sequences of the three examined genes were clustered together with sequences derived from other countries, proving that the msa-2 gene sequences of B. bovis shared the same genetic characters worldwide. The present study clearly showed that the MSA-2 proteins of B. bovis isolates from Israel were genetically distinct from the vaccine strains. Thus, further research will be needed in order to understand the genetic diversity mechanisms of B. bovis, and the immunological responses of the infected animals.

Dynamics of Besnoitia besnoiti infection in cattle.

Bovine besnoitiosis is caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti. This disease progresses in two sequential phases: a febrile acute phase with oedemas and respiratory disorders, and a chronic phase characterized by the presence of subcutaneous tissue cysts and skin lesions. Serious consequences of the infection are poor body condition, sterility in bulls and eventual death. The role of host/parasite-dependent factors, which play a major role in the pathogenesis of the disease, is not yet fully elucidated. Isolate/strain virulence, parasite stage, dose and the route of parasite inoculation were studied under different experimental conditions, which make it difficult to compare the results. Data on host-dependent factors obtained from naturally infected cattle showed that (i) the seroprevalence of infection is similar in both sexes; (ii) seropositivity increases with age; (iii) both beef and dairy cattle are susceptible to the infection; and (iv) the cell-mediated immune response is likely to play a major role because a T cell response has been observed around several tissue cysts. Whether colostral antibodies are protective and to what extent the humoral immune response might reflect the disease/protection status require further research. Thus, a well-established experimental bovine model could help to clarify these important questions. The dynamics of B. besnoiti infection in cattle and available knowledge on relevant factors in the pathogenesis of the infection are reviewed in the present work.

Comparative analysis of mitochondrial markers from four species of Rhipicephalus (Acari: Ixodidae).

Mitochondrial sequences of four mitochondrial markers: 12S rRNA, 16S rRNA, cytochrome c oxidase subunit I (COX1) and cytochrome b (CytB) from four Rhipicephalus species were analyzed to establish genetic relationships and enable molecular identification. Field-collected samples from the species Rhipicephalus annulatus, Rhipicephalus bursa, Rhipicephalus sanguineus and Rhipicephalus turanicus were amplified by PCR and compared with GenBank™ annotated sequences. PCR products were obtained using primers that were designed to amplify orthologous sequences from different tick species and genera. The average intra-species sequence identity was 98.5-99.5%, while the average inter-species identity was 86.5-89.6%, reflecting a ≈ 10% decrease in the identity, when different species are compared. The "closest" two species, in terms of sequence identity, were R. sanguineus and R. turanicus, while the "least close" ones were R. annulatus and R. sanguineus. Molecular identification of each species was accomplished by a combined restriction analysis of 12S, COX1 and CytB markers, obviating the need for field sample sequencing. The restriction mapping data suggest that by using several markers, each with a unique digestion pattern, the identity of a given sample could be determined at the species level. It is anticipated that with the accumulation of more information on additional species and markers, molecular identification will become a standard approach for tick classification, complementing morphological taxonomy.

First detection of Ixodes ricinus on beef cattle in Israel.

This is the first report of the presence of Ixodes ricinus on beef cattle in Israel. Up to now, in the Middle East this tick was considered to be confined to Turkey and northern Iran. In the present study, tick samples collected from field-grazing beef cattle in western Galilee (northern Israel) were first examined morphologically for species-specific taxonomical features and then by molecular characterization. Ticks identified morphologically as I. ricinus were then examined by PCR with four different molecular markers: 12S rRNA, 16S rRNA, COX1 and cytochrome B. The PCR products were sequenced and compared with annotated I. ricinus sequences in GenBank™ and the analyzed sequences from the collected samples shared 98-99% identity with reported I. ricinus sequences. In contrast, sequences from the collected ticks shared identity of 91% or less with annotated sequences from other Ixodes species. Multiple alignments and neighbor-joining analyses performed for each of the four markers reinforced the results obtained from pairwise alignments. These findings demonstrated for the first time the presence in Israel of the tick species I. ricinus - with results confirmed by a combination of morphological examination and molecular analyses.

Cross-protection between geographically distinct Anaplasma marginale isolates appears to be constrained by limited antibody responses.

The rickettsia Anaplasma marginale causes the haemolytic disease bovine anaplasmosis, an economic problem in tropical and subtropical areas worldwide. The closely related but less pathogenic Anaplasma centrale is commonly used as a live vaccine to prevent anaplasmosis, but it can only be produced from infected blood. UFMG1 is a low pathogenic Brazilian strain of A. marginale, which has been shown to protect cattle against a high pathogenic Brazilian isolate. As UFMG1 can be grown in tick cells, the strain was proposed as a possible cell culture-derived vaccine. We have evaluated whether UFMG1 could protect cattle against a geographically distant heterologous strain, using A. centrale vaccination as a standard for comparison. Trial calves were infected with UFMG1, A. centrale or PBS. UFMG1-infected animals were more symptomatic than those infected with A. centrale, but none required treatment. All calves were then challenged with the Israeli A. marginale Gonen strain (one of the most prevalent strain in Israel). The A. centrale group had the mildest symptoms, while UFMG1 and control groups both had a more severe response. Nevertheless, the challenge did not cause life-threatening disease in any group. Animals infected with A. centrale had a significantly higher IgG response than UFMG1, when measured in an ELISA against initial bodies from their homologous strain or Gonen. The level of cross-reactivity of the response to initial infection correlated significantly with reduced symptoms after challenge. In conclusion, UFMG1 had limited effect in preventing disease by the geographically distant heterologous Gonen strain. While the low pathogenicity of the Gonen strain in this trial makes it impossible to conclusively state that UFMG1 would have given no protective effect against more serious disease, the comparatively low IgG response to UFMG1 suggests it would not have been as effective as A. centrale.

Evidence for extensive genetic diversity and substructuring of the Babesia bovis metapopulation.

Babesia bovis is a tick-transmitted haemoprotozoan and a causative agent of bovine babesiosis, a cattle disease that causes significant economic loss in tropical and subtropical regions. A panel of nineteen micro- and minisatellite markers was used to estimate population genetic parameters of eighteen parasite isolates originating from different continents, countries and geographic regions including North America (Mexico, USA), South America (Argentina, Brazil), the Middle East (Israel) and Australia. For eleven of the eighteen isolates, a unique haplotype was inferred suggesting selection of a single genotype by either in vitro cultivation or amplification in splenectomized calves. Furthermore, a high genetic diversity (H = 0.780) over all marker loci was estimated. Linkage disequilibrium was observed in the total study group but also in sample subgroups from the Americas, Brazil, and Israel and Australia. In contrast, corresponding to their more confined geographic origin, samples from Israel and Argentina were each found to be in equilibrium suggestive of random mating and frequent genetic exchange. The genetic differentiation (F(ST)) of the total study group over all nineteen loci was estimated by analysis of variance (Θ) and Nei's estimation of heterozygosity (G(ST')) as 0.296 and 0.312, respectively. Thus, about 30% of the genetic diversity of the parasite population is associated with genetic differences between parasite isolates sampled from the different geographic regions. The pairwise similarity of multilocus genotypes (MLGs) was assessed and a neighbour-joining dendrogram generated. MLGs were found to cluster according to the country/continent of origin of isolates, but did not distinguish the attenuated from the pathogenic parasite state. The distant geographic origin of the isolates studied allows an initial glimpse into the large extent of genetic diversity and differentiation of the B. bovis population on a global scale.

Genetic diversity of Babesia bovis in virulent and attenuated strains.

The aim of this study was to compare the genetic diversity of the single copy Bv80 gene sequences of Babesia bovis in populations of attenuated and virulent parasites. PCR/ RT-PCR followed by cloning and sequence analyses of 4 attenuated and 4 virulent strains were performed. Multiple fragments in the range of 420 to 744 bp were amplified by PCR or RT-PCR. Cloning of the PCR fragments and sequence analyses revealed the presence of mixed subpopulations in either virulent or attenuated parasites with a total of 19 variants with 12 different sequences that differed in number and type of tandem repeats. High levels of intra- and inter-strain diversity of the Bv80 gene, with the presence of mixed populations of parasites were found in both the virulent field isolates and the attenuated vaccine strains. In addition, during the attenuation process, sequence analyses showed changes in the pattern of the parasite subpopulations. Despite high polymorphism found by sequence analyses, the patterns observed and the number of repeats, order, or motifs found could not discriminate between virulent field isolates and attenuated vaccine strains of the parasite.

Identification of Anaplasma centrale major surface protein-2 pseudogenes.

The present study was aimed to identify msp2 pseudogenes and MSP2 variants in the vaccine Anaplama centrale strain. Five msp2 pseudogenes were identified in the A. centrale genome, and multiple MSP2 variants that emerged during both acute and persistent infection were detected. The pseudogene copies of msp2 were truncated; they contained a central hypervariable region flanked by short portions of the 5' and 3' conserved regions. Alignment of the hypervariable region sequence of the expression site of MSP2 variants with msp2 pseudogenes showed that MSP2 variants are generated by two mechanisms, previously described in Anaplasma marginale: (i) recombination of the whole pseudogene into the single msp2 expression site, and (ii) recombination of small segments of pseudogenes into the expression site by segmental gene conversion. The present study showed that the A. centrale MSP2 variants and the msp2 pseudogene repertoire were different from those reported for A. marginale. Unique MSP2 variants and pseudogenes identified in the vaccine strain allow the A. centrale-vaccinated cattle to be superinfected with the field strains of A. marginale. The knowledge gained in the present study on the mechanisms of antigenic variations in the vaccine strain of A. centrale is a further step in the development of a new generation vaccine against anaplasmosis.

Genetic diversity of major surface protein 1a of Anaplasma marginale in beef cattle.

The present study was aimed to demonstrate genotypic diversity of Anaplama marginale in infected beef herds grazing within anaplasmosis endemic regions. The genotypic diversity was identified among different herds, within each herd, and also within single animals. The Israeli strains revealed unique characteristics of MSP1a repeats and, in addition to the published repeats, six new tandem repeats designated Is1-5, and Is9 were identified. The superinfections of individual Anaplama centrale vaccinated animals with two genotypically different A. marginale strains were detected. Six out of 43 vaccinated animals in the G herd were each infected with two A. marginale strains carrying two distinct genotypes; in this herd the follow-up during years 2003-2007 demonstrated that several animals carried different msp1a genotypes at different time points. Coinfection with two different genotypes of A. marginale in A. centrale vaccinated cattle was observed in another herd, as well. It appears that A. marginale is composed of a heterogeneous changing bacterial population that evolves in the host or, the genotypic diversity implies high transmission intensity by the vector, or both. Learning how this diversity is generated and identification of distinct A. marginale strains coupled with high sequence variation of MSP1a will aid in understanding Anaplasma transmission and disease development.

Vaccination of cattle against tropical theileriosis in Uzbekistan using autochthonous live vaccine.

In Uzbekistan, 1984 cattle were vaccinated with the TAU-219 autochthonous live Theileria annulata vaccine under field conditions. There were no post-vaccination reactions recorded in calves vaccinated with 10 times the recommended dose, under semi-field conditions. After vaccination 53.9% of the vaccinates developed fever over 39.5 degrees C that lasted for a few days, but none developed clinical theileriosis or required drug treatment during the 3-week follow-up. The numbers of animals in which piroplasms were detected before and after vaccination were similar (7.15% and 7.25%, respectively), indicating previous exposure to T. annulata tick infection. Following vaccination none of the 241 pregnant cows aborted. Milk production during 30-45 days was similar in non-vaccinated and vaccinated cows, at about 5.2-5.9L/day. During 1999-2006 a total of 11,000 field-grazing cattle were safely vaccinated in Uzbekistan.

Isolation of Neospora caninum from dairy zero grazing cattle in Israel.

First Israeli Neospora caninum isolates were obtained from brain tissues of aborted fetuses (NcIs491 and NcIs580) from dairy farms endemic for neosporosis and maintaining cattle on zero grazing. Tissues from different parts of the fetus brains were used to infect Vero cells. Tachyzoites of N. caninum were first observed in cultures from days 30 and 32 after infection. To confirm the identity of the isolated parasites, DNA extracts from brains and cultures were tested by PCR with specific primers based on the Nc5 gene. Specific fragments were amplified by PCR from infected cultures of both fetuses on day 25. Susceptible seronegative gerbils (Meriones tristrami) were inoculated intraperitoneally with 10(3) to 10(5) tenfold dilutions of subculture tachyzoites. The inoculated gerbils developed specific antibodies to N. caninum, with end-point serum dilution of 1:4096 in the IFA assay, whereas no neurological signs or deaths were seen during 4 months of observation.

Prevalence and genetic diversity of Anaplasma marginale strains in cattle in South Africa.

Bovine anaplasmosis, caused by the tick-borne rickettsia Anaplasma marginale, is endemic in South Africa and results in considerable economic loss to the cattle industry. This study was designed to characterize strains of A. marginale at the molecular level from cattle raised in communal and commercial farms in the north-eastern and south-western regions of the Free State Province, South Africa, that varied in rainfall and vegetation. Seroprevalence to A. marginale was determined in 755 cattle by an Anaplasma spp. competitive enzyme-linked immunosorbent assay and ranged from 44% to 98% and was similar in both regions. While Anaplasma centrale was not targeted in this study, A. marginale infections were identified by species-specific msp1alpha polymerase chain reaction in 129 of 215 of the samples studied. Similar genetic diversity of A. marginale strains was found in both the north-eastern and south-western regions. The sequences of 29 A. marginalemsp1alpha amplicons from South African strains revealed considerable genetic diversity providing 14 new repeat sequences. However, 42% of MSP1a repeat sequences were not unique to this region. These results indicated the presence of common genotypes between South African, American and European strains of A. marginale. Cattle movement between different parts of South Africa was suggested by the presence of identical A. marginale MSP1a genotypes in north-eastern and south-western regions of the Free State Province. Control strategies for anaplasmosis in South Africa should therefore be designed to be protective against genetically heterogeneous strains of A. marginale.

Isolation of Besnoitia besnoiti from infected cattle in Portugal.

Besnoitia besnoiti, an obligate intracellular protozoan parasite belonging to the phylum apicomplexa, is the causative agent of bovine besnoitiosis. Besnoitiosis is responsible for significant losses in the cattle industry of Africa and Mediterranean countries due to the high morbidity rate, abortion and infertility in males. The acute stage of disease is associated with the proliferative forms (tachyzoites) and is characterized by fever, whimpery, general weakness and swelling of the superficial lymph nodes. During the following chronic stage, a huge number of cysts are formed mainly in the subcutaneous tissues. This process is non-reversible, and chronic besnoitiosis is characterized by hyper-sclerodermia, hyperkeratosis, alopecia and, in bulls, atrophy, sclerosis and focal necrosis that cause irreversible lesions in the testis. In this paper we report on the identification of large cysts in the skin of a cow and a bull in Portugal, which presented loss of hair and enlargement and pachydermis all over the body. The observation of a two-layered cyst wall within the host cell, the encapsulation of the host cell by a large outer cyst wall, and the subcutaneous localization of the cysts within the host, were characteristic for B. besnoiti. The parasites were isolated from the infected animals and successfully propagated in Vero cells without prior passages in laboratory animals. Morphological characterization of B. besnoiti tachyzoites and the amplification of the 149 bp segment from the internal transcribed spacer 1 (ITS1), aided with specific primers, confirmed the identification of B. besnoiti.

Low seroprevalence of antibodies to Neospora caninum in wild canids in Israel.

The role of domestic dogs in the epidemiology of Neospora caninum as well as the relationship between N. caninum infection of farm dogs and cattle were demonstrated, however, evidence is scarce regarding the role of wild canids in domestic animal neosporosis. The present study was undertaken to evaluate the role of wild canids in the epidemiology of bovine neosporosis in Israel by analyzing the prevalence of antibodies to N. caninum in wild canids. Sera samples were collected from 114 free ranging wild golden jackals (Canis aureus), 24 red foxes (Vulpes vulpes) and nine wolves (Canis lupus), which were collected in Israel during the years 1999-2004. Of a total of 147 wild canids tested antibodies to N. caninum were only found in two golden jackals with IFAT titers of 1:50, and in one red fox and one wolf with IFAT titer of 1:400. The low seroprevalence found in this study (2.7%) indicated that wild canids probably do not have an important role in the epidemiology of N. caninum in Israel. However, since the diet of different species of wild canids and even diverse populations of the same canid species vary, it is possible that other results might be obtained from specific wild canids populations, which scavenge in the vicinity of infected bovines.

Molecular and serological detection of A. centrale- and A. marginale-infected cattle grazing within an endemic area.

A reverse line blot hybridization (RLB) one-stage nested PCR (nPCR) for Anaplasma centrale and a nested PCR for Anaplasma marginale were used to detect infected cattle grazing within an endemic region in Israel. A novel set of PCR primers and oligonucleotide probes based on a 16S ribosomal RNA gene was designed for RLB detection of both Anaplasma species, and the performance of the molecular assays compared. The immunofluorescent antibody test (IFA) was used to detect antibodies to both Anaplasma species, whereas, a highly sensitive and specific competitive enzyme-linked immunosorbent assay (cELISA) was used to detect antibodies in A. centrale-vaccinated cattle. The RLB and the nested PCR procedures showed bacteremia with sensitivity of 50 infected erythrocytes per milliliter. Up to 93% of the A. centrale vaccinates carried specific antibodies that were detected by cELISA, and up to 71% of the vaccinated cattle were found to be naturally infected with A. marginale according to the PCR and the RLB assays. Nevertheless, no severe outbreaks of A. marginale infection occurred among vaccinated herds in this endemic region. It appears that both, molecular tools and serology are useful for evaluation of the vaccine efficacy. In the light of wide natural field infection with A. marginale, strong recommendations to continue the A. centrale vaccination program regime will continue until a new generation of non-blood-based vaccine will be developed.

In vitro cultivation of a south African isolate of an Anaplasma sp. in tick cell cultures.

This paper describes the first successful in vitro cultivation of a South African isolate of an Anaplasma sp., initially thought to be Anaplasma marginale, in the continuous tick cell line IDE8. Blood from a bovine naturally infected with A. marginale kept on the farm Kaalplaas (28 degrees 08' E, 25 degrees 38' S) was collected, frozen, thawed and used as inoculum on confluent IDE8 cell cultures. Twenty days after culture initiation small intracellular colonies were detected in a Cytospin smear prepared from culture supernatant. Cultures were passaged on Day 34. Attempts to infect IRE/CTVM18 cell cultures with the Kaalplaas isolate derived from IDE8 cultures failed, whereas a reference stock of A. marginale from Israel infected IRE/CTVM18 tick cell cultures. Attempts to infect various mammalian cell lines (BA 886, SBE 189, Vero, L 929, MDBK) and bovine erythrocytes, kept under various atmospheric conditions, with tick cell-derived Anaplasma sp. or the Israeli strain of A. marginale failed. Molecular characterization revealed that the blood inoculum used to initiate the culture contained both A. marginale and Anaplasma sp. (Omatienne) whereas the organisms from established cultures were only Anaplasma sp. (Omatjenne).

An enzyme-linked immunosorbent assay for antibodies to Hepatozoon canis.

Canine hepatozoonosis is a tick-borne protozoal disease caused in the Old World and South America by Hepatozoon canis. An enzyme-linked immunosorbent assay (ELISA) using purified H. canis gamont antigen was applied for the detection of antibodies reactive with H. canis. Evaluation of the ELISA with sera from naturally infected parasitemic dogs indicated that it was sensitive (86%), specific (97%), and comparable to the indirect fluorescent antibody test (IFAT) for the detection of H. canis antibodies. A variable degree of serologic cross-reactivity was found between sera from H. americanum-infected dogs and the H. canis antigen. Dogs experimentally infected with H. canis seroconverted 1-4 weeks post-infection (PI). Antibody levels peaked at 7-9 weeks PI and gradually declined thereafter remaining above the cut-off value until the conclusion of the study 7 months PI. The ELISA will be valuable for serological evaluation of dogs suspected of exposure to H. canis and for epidemiological studies.

Molecular conservation of MSP4 and MSP5 in Anaplasma marginale and A. centrale vaccine strain.

Anaplasma centrale msp4 and msp5 genes were cloned and sequenced, and the recombinant proteins were expressed. The identity between Anaplasma marginale and A. centrale MSP4 was 83% in the nucleotide sequences and 91.7% in the encoded protein sequences. A. centrale msp5 nucleotide sequences shared 86.8% identity with A. marginale msp5, and there was 92.9% homology between A. centrale and A. marginale encoded amino acids of the MSP5 protein. Southern blots hybridized with probes derived from the msp4 and msp5 central regions indicate that msp4 and msp5 of A. centrale are encoded by single copy genes. Recombinant MSP4 and MSP5 fusion proteins reacted with anti-A. marginale monoclonal antibodies ANAR76A1 and ANAF16C, respectively, demonstrating the conservation of conformation-sensitive B-cell epitopes between A. centrale and A. marginale. These data demonstrate the structural and antigenic conservation of MSP4 and MSP5 in A. centrale and A. marginale. This conservation is consistent with the cross-protective immunity between A. marginale and A. centrale and supports the development of improved vaccines based upon common outer membrane proteins.

Ultrastructure of Besnoitia besnoiti tissue cysts and bradyzoites.

Besnoitia besnoiti is an economically important tissue cyst-forming apicomplexan of cattle in Africa and Israel. Tissue cysts and bradyzoites of B. besnoiti from the skin of a naturally infected bull were studied by transmission electron microscopy. Tissue cysts enclosed host cell and bradyzoites. Bradyzoites were 6-7.5 x 1.9-2.3 microm in size and contained organelles found in coccidian merozoites including numerous micronemes, rhoptries, amylopectin granules, and a posteriorly located nucleus. Enigmatic bodies, characteristically found in Besnoitia sp. bradyzoites, were not observed. Therefore, enigmatic bodies should be removed as a generic character of the bradyzoites of Besnoitia species.