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Anaplasma centrale has been used in cattle as a live blood vaccine against the more pathogenic Anaplasma marginale for over 100 years. While A. marginale can be propagated in vitro in tick cell lines, facilitating studies on antigen production, immunisation and vector-pathogen interaction, to date there has been no in vitro culture system for A. centrale. In the present study, 25 cell lines derived from 13 ixodid tick species were inoculated with the Israeli vaccine strain of A. centrale and monitored for at least 12 weeks by microscopic examination of Giemsa-stained cytocentrifuge smears. Infection of 19 tick cell lines was subsequently attempted by transfer of cell-free supernate from vaccine-inoculated tick cells. In two separate experiments, rickettsial inclusions were detected in cultures of the Rhipicephalus appendiculatus cell line RAE25 28-32 days following inoculation with the vaccine. Presence of A. centrale in the RAE25 cells was confirmed by PCR assays targeting the 16S rRNA, groEL and msp4 genes; sequenced PCR products were 100% identical to published sequences of the respective genes in the Israeli vaccine strain of A. centrale. A. centrale was taken through three subcultures in RAE25 cells over a 30 week period. In a single experiment, the Dermacentor variabilis cell line DVE1 was also detectably infected with A. centrale 11 weeks after inoculation with the vaccine. Availability of an in vitro culture system for A. centrale in tick cells opens up the possibility of generating a safer and more ethical vaccine for bovine anaplasmosis., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.

Artemisone was evaluated, in in vitro and in vivo, for control of bovine babesiosis caused by Babesia bigemina and Babesia bovis parasites. In vitro, artemisone reduced parasitemia in a dose-dependent manner: the inhibitory effects increased gradually, reaching a maximum inhibition of 99.6% and 86.4% for B. bigemina and B. bovis, respectively 72 h after initiation of treatment with initial parasitemia of 0.5%. In calves infected with either B. bigemina or B. bovis artemisone treatment was well tolerated and prevented development of acute babesiosis in all animals except for one B. bovis-infected calf. The treatment did not eliminate all blood parasites, and recovered animals carried a persistent low-level infection. Treatment with artemisone may be useful as an alternative drug for preventing the pathology that results from babesiosis, without interfering with acquired immune protection following recovery from an acute babesiosis infection or vaccination., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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., (© 2013 Blackwell Verlag GmbH.)

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., (© 2013 Blackwell Verlag GmbH.)

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.

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.

Susceptible dairy cattle were immunized with attenuated, live calf-derived, in vitro-cultured or biologically cloned Babesia bovis, with non-viable exoantigens, or with recombinant rhoptry-associated protein 1 (rRAP1). Antibody response assessed by the indirect fluorescent assay (IFA) and by the growth inhibition activity in vitro showed that seroconversion correlated with neutralization activity in vitro in all immunized groups, but not with protective immunity in vivo. The protective responses elicited by immunization with completely avirulent biologically cloned live parasites, or by the exoantigens were sufficient for highly susceptible dairy cattle, in which prime immunization with blood-derived attenuated parasites cause clinical babesiosis. Upon challenge with virulent live parasites all immunized calves were solidly protected, but only partial protective immunity was acquired by rRAP1 immunization.

Infection of cattle with Neospora caninum protozoa, the causative agent of bovine protozoal abortion, results in robust cellular and humoral immune responses, particularly CD4(+) T-lymphocyte activation and gamma interferon (IFN-gamma) secretion. In the present study, N. caninum SRS2 (NcSRS2) T-lymphocyte-epitope-bearing subunits were incorporated into DNA and peptide preparations to assess CD4(+) cell proliferation and IFN-gamma T-lymphocyte-secretion immune responses in cattle with predetermined major histocompatibility complex (MHC) genotypes. In order to optimize dendritic-cell processing, NcSRS2 DNA vaccine was delivered with granulocyte macrophage-colony-stimulating factor and Flt3 ligand adjuvant. The synthesized NcSRS2 peptides were coupled with a palmitic acid molecule (lipopeptide) and delivered with Freund's adjuvant. Cattle vaccinated with NcSRS2 DNA vaccine alone did not induce T-lymphocyte activation or IFN-gamma secretion, whereas subsequent booster inoculation with NcSRS2-lipopeptides induced robust NcSRS2-specific immune responses. Compared to the response in control animals, NcSRS2-lipopeptide-immunized cattle had significantly increased NcSRS2-specific T-lymphocyte proliferation, numbers of IFN-gamma-secreting peripheral blood mononuclear cells, and immunoglobulin G1 (IgG1) and IgG2a antibody levels. The findings show that N. caninum NcSRS2 subunits bearing T-lymphocyte epitopes induced cell-mediated immune responses similar to the protective immune responses previously described against live parasite infection, namely T-lymphocyte activation and IFN-gamma secretion. The findings support the investigation of NcSRS2 immunogens for protection against N. caninum-induced fetal infection and abortion in cattle.

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.

Overwhelming evidence has accumulated of the effectiveness of immunization with live attenuated vaccines to control tick-borne diseases of livestock. Despite several disadvantages, vaccination with live attenuated organisms against tropical theileriosis, babesiosis and possibly heartwater constitutes one of the most cost-effective intervention strategies. Although great advances have been made through genomics and proteomics research, this has not yet translated into effective non-living vaccines. As a result, there is a continuing necessity to use available live vaccines in tick and tick-borne disease-control strategies adapted to conditions prevailing in many parts of the world.

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.

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.

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.

Cryptosporidium parvum is an apicomplexan parasite that infects humans and ruminants. C. parvum isolated from cattle in northeastern Turkey and in Israel was genotyped using multiple polymorphic genetic markers, and the two populations were compared to assess the effect of cattle husbandry on the parasite's population structure. Dairy herds in Israel are permanently confined with essentially no opportunity for direct herd-to-herd transmission, whereas in Turkey there are more opportunities for transmission as animals range over wider areas and are frequently traded. A total of 76 C. parvum isolates from 16 locations in Israel and seven farms in the Kars region in northeastern Turkey were genotyped using 16 mini- and microsatellite markers. Significantly, in both countries distinct multilocus genotypes confined to individual farms were detected. The number of genotypes per farm was higher and mixed isolates were more frequent in Turkey than in Israel. As expected from the presence of distinct multilocus genotypes in individual herds, linkage disequilibrium among loci was detected in Israel. Together, these observations show that genetically distinct populations of C. parvum can emerge within a group of hosts in a relatively short time. This may explain the frequent detection of host-specific genotypes with unknown taxonomic status in surface water and the existence of geographically restricted C. hominis genotypes in humans.

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.

Two separate groups of Bos taurus bulls, one of 106 and the second of 27 animals, imported to Israel from areas free of Babesia bovis and Babesia bigemina, were vaccinated against babesiosis with a bivalent live attenuated vaccine. In light of the fact that routine vaccination is recommended at the weaning age, these bulls--of highly susceptible breeds--were kept under close surveillance to prevent losses that might be caused by severe clinical reactions to their vaccination at the age of 16-18 months. Seven days after vaccination, about one-third of the 106 bulls in the first group developed clinical signs of B. bigemina infection, which peaked at day 9, and then diminished from day 11, when the patent period known for B. bovis infection was observed. Because of the severe clinical responses a total of 36% of the bulls required babesicidal treatment. Despite the treatment Babesia were not sterilized: 33 and 68% of the animals remained PCR positive for B. bigemina and B. bovis, respectively. To mitigate the severe responses to vaccination, the 27 bulls of the second group were vaccinated in two-steps: they were inoculated initially with avirulent culture-derived parasites and then vaccinated with the conventional donor-derived vaccine a month later. None of the bulls in the latter group developed clinical babesiosis, all were serologically positive to B. bigemina, and 67% showed seroconversion to B. bovis. In light of the experience described here, it is suggested that sensitive older cattle be vaccinated against babesiosis by priming them with avirulent in vitro-cultured parasites and then inoculating them with the conventional donor-derived vaccines.

A field isolate of Theileria annulata (Uzbek strain) was obtained from calves infected by Hyalomma anatolicum ticks collected from an endemic region in Uzbekistan. Schizont-infected bovine cells that had been established and propagated in cell culture were examined for attenuation both in vivo, by inoculating cells from various passages into calves, and in vitro for metalloproteinase activity. During serial subcultivation a gradual reduction in virulence and in enzyme activity in cells infected with the Uzbek strain were observed. Complete attenuation of the Uzbek isolate was obtained at about passage 80, and only traces of proteolysis were detected in gelatin substrate gels. In contrast, there was no direct correlation between virulence and enzyme levels in an Israeli strain. While schizonts of the Israeli strain were completely attenuated at passage 80, proteolysis in the substrate gels was detected up to passage 197. Solid immunity was observed in calves immunized with attenuated T. annulata schizonts of the Uzbek strain upon challenge with the homologous H. excavatum sporozoites. For a strain to be used for vaccine production, it appears that animal inoculation still remains the most reliable method to assess the degree of attenuation and protection.

Friesian cattle were immunized with two inoculations of anti-tick Bm86 (Tick-GARD) vaccine and were challenged 30 or 90 d later with Boophilus annulatus larvae derived from 1.2 g of eggs. No nymphs or adult ticks were found on the immunized cattle during four weeks after challenge. Repeated infestations (2 to 4) with larvae on three other calves during a period of 160 and 390 d after the immunization did not result in development of nymphal and adult stages. In control, non-immunized cattle infested with corresponding batches of larvae 1380 to 4653 replete adult female ticks were collected. Larvae issued from Babesia bovis-infected female ticks transmitted the infection to Bm86-immunized cattle, but the progeny of B. bigemina-infected females did not. Since B. bigemina is transmitted exclusively by nymphal stages of Bo. annulatus these results support the observation that immunity induced by Bm86 affects the larval stage of this tick.

The persistence of Babesia bovis and B. bigemina infection in Friesian cows, following vaccination with attenuated live vaccines, was assessed by subinoculation of blood into splenectomized calves. Subinoculation tests showed that B. bigemina persisted in two out of 20 cows vaccinated 10 and 46 months previously, and that B. bovis persisted in 11 out of 22 cows vaccinated 10 to 47 months previously. Antibody was detected in five B. bigemina - and 15B. bovis -vaccinated cows. Parasites of both species persisted among the serologically negative cows, whereas blood obtained from serologically positive cows failed to transmit infection. It is concluded that in the absence of reinfection Friesian cattle may spontaneously eliminate B. bigemina and B. bovis infection after various periods following vaccination.

Bovine anaplasmosis caused by the intraerythrocytic rickettsia Anaplasma marginale is the most prevalent tick-borne disease of cattle worldwide. The most efficient method to control anaplasmosis is by vaccination using live Anaplasma centrale, a closely related species or subspecies of low pathogenicity that is capable of inducing significant protection against the more virulent A. marginale. In the present study, we applied PCR assays to detect and discriminate field infection with A. marginale from A. centrale persistently infected vaccinates. Direct and one-stage nested PCR were based on A. centrale mbp58 specific sequence, with the assay sensitivity level of 0.00001% for nested PCR performed in a single amplification step. Size polymorphism in the A. marginale msp1 alpha gene among strains was used to design a PCR capable of discriminating between the Israel T and NT strains of A. marginale and the encoded MSP1a size polymorphism was confirmed by immunoprecipitation. The detection of A. centrale in 72% of vaccinated field-grazing cattle clearly indicated that the majority of vaccinated cattle remain carriers. A. marginalewas detected in 64% of these vaccinated cattle, demonstrating that, as expected, natural transmission occurs within the endemic region. The lack of severe A. marginaleoutbreaks in this region, despite ongoing transmission, is consistent with protection being provided by widespread vaccination with A. centrale.

Neospora caninum is a coccidian parasite identified as a major cause of abortion in cattle. A combined infection of N. caninum with another taxonomically related parasite of cattle, Besnoitia besnoiti can occur in geographical areas endemic for both species. Both infections are routinely diagnosed serologically, and incorrect diagnosis could occur if immunological cross-reactivity exists between the two parasites. To investigate the possible degree of cross-reactivity, we compared results obtained with two serological techniques, immunofluorescent antibody test (IFA), and Western blot analysis on known positive and negative sera. The test sera were derived from naturally infected cattle and from experimentally infected Mongolian gerbils. In IFA of bovine sera, no cross-reactvity was detected at the commonly used serum dilution cutoffs of 1:200 for N. caninum and 1:256 for B. besnoiti. However, at 1:64 dilution of both cattle and gerbil sera, anti-N. caninum sera reacted with B. besnoiti antigen in some individual samples. Anti-B. besnoiti serum did not react with N. caninum antigen at any dilution. This low level one directional cross-reactivity was confirmed by Western blot analysis. B. besnoiti antigen showed two immunoreactive bands when probed with anti-N. caninum serum, while no bands appeared when N. caninum antigen was probed with B. besnoiti antiserum. Immunization and challenge experiments in the highly susceptible Mongolian gerbil (Meriones unguiculatus) showed essentially no cross-protection between N. caninum and B. besnoiti.

An important objective in vaccination strategies is to activate lymphocytes with particular effector functions. Cellular immunity and the type I cytokine IFN-gamma have been implicated in protective immunity to heartwater. Furthermore, low molecular weight proteins of Cowdria ruminantium have been shown to induce peripheral blood mononuclear cells to proliferate. To determine which lymphocyte subset responds when stimulated with fractionated C. ruminantium proteins, specific short-term lymphocyte cultures were established from cattle immunized with the Welgevonden isolate. Four cattle were immunized, two by infection and treatment and two with inactivated organisms. Cell surface phenotypic analysis of the cultures indicated that CD4+ lymphocytes were enriched over time. This coincided with increased antigen-specific proliferation and IFN-gamma production. Proteins of molecular weights 13-18kDa induced the CD4+-enriched T-cell cultures, derived from each of the animals, to proliferate and produce IFN-gamma. Although the two groups of cattle were immunized differently, their lymphocytes responded similarly. These results extend previous findings by identifying the responder cells as being predominantly IFN-gamma producing CD4+ lymphocytes. This cytokine has been implicated in immunity to the parasite. The low molecular weight proteins that induced CD4+ lymphocytes to proliferate and produce IFN-gamma are therefore likely to be important in protection against heartwater and may have a role in vaccine development.

The major economically important tick-borne diseases of cattle are theileriosis, babesiosis, anaplasmosis, and cowdriosis. Culture-derived attenuated schizonts of Theileria annulata have proved to be safe for all types of cattle and they protect against tick-borne theileriosis. T. parva was also successfully grown in vitro; however, inoculation of cattle with allogeneic schizont-infected cells resulted in rejection and destruction of the parasites together with the host cells. The number of schizont-infected cells needed for immunization is greater than for T. annulata theileriosis. Culture-propagated Babesia bovis and B. bigemina were used for large scale vaccination in the field. An avirulent population of Babesia spp. was obtained by in vitro cloning; inoculation of cattle did not induce clinical babesiosis, but produced specific antibodies. Culture-derived exoantigens of Babesia spp. proved to be completely safe for cattle, however, they conferred less protection than live parasites. Cell-cultured Cowdria ruminantium was highly infective for susceptible animals but, attenuated in vitro, could offer a potential source for vaccination. Anaplasma marginale, successfully grown in tick cell culture, may be developed for vaccines. Factors that should be considered in the developing of vaccines against tick-borne diseases include: the protective immune response to the pathogenic parasite developmental stages, virulence, immunological strain differences, and antigenic variations in cattle and in culture.

Theileria annulata, the cause of tropical theileriosis is propagated in cattle with stage-to-stage transmission by Hyalomma ticks. Three stages in the life cycle of the parasite--tick-derived sporozoites, intramononuclear schizonts, and erythrocytic merozoites--infect cattle. When cattle are inoculated with schizont-infected cells, the parasite is transferred from the donor cell to the recipient. The main pathological damage in cattle is induced by the schizont stage. Each development stage of T. annulata elicits a specific immune response. Schizont-infected lymphoid cells can be grown indefinitely in culture and prolonged cultivation results in loss of virulence. Blood-derived schizonts induce stronger immunity than culture-derived schizonts, which suggests that restrictions on the parasite population or antigenic variation occur during prolonged cultivation. The duration of immunity following sporozoite or schizont infections has not yet been determined, but does not appear to be lifelong. The attenuated, culture-derived anti-theileria vaccine proved to be safe and effective in prevention of field theileriosis in large enzootic areas.

Protective immunity against the ehrlichial pathogen Anaplasma marginale has been hypothesized to require induction of immunoglobulin G2 (IgG2) antibody against outer membrane protein epitopes and coordinated activation of macrophages for phagocytosis and killing. In the present study, cell-mediated immune responses, including induction of IgG isotype switching, were characterized in calves immunized with purified outer membranes of the Florida strain of A. marginale. Importantly, these calves were subsequently shown to be protected upon experimental challenge with the Florida strain, and calves which developed the highest IgG2 titers were completely protected against infection. Peripheral blood mononuclear cells (PBMC) obtained after immunization proliferated strongly in response to both whole A. marginale homogenates and purified outer membranes, and this responsiveness persisted until the time of challenge. Responding cells were shown to be CD4(+) T cells, and CD4(+) T-cell lines cultured for 2 to 4 weeks also proliferated specifically in response to A. marginale and produced high titers of gamma interferon. The helper T-cell response included recognition of conserved epitopes, as PBMC proliferation was stimulated by the homologous Florida strain, four genetically distinct A. marginale strains, and Anaplasma ovis. The outer membrane proteins stimulating the PBMC responses in protected calves included major surface proteins (MSPs) MSP-1, MSP-2, and MSP-3, which were previously shown to induce partial protection against infection. These studies demonstrate, for the first time, potent helper T-cell responses in cattle protectively immunized with outer membranes against A. marginale challenge and identify three MSPs that are recognized by immune T cells. These experiments provide the basis for subsequent identification of the helper T-cell epitopes on MSP-1, MSP-2, and MSP-3 that are needed to evoke anamnestic antibody and effector T-cell responses elicited by protein or nucleic acid immunization.

The ability of rhoptry-associated protein 1 (RAP-1) of Babesia bovis and Babesia bigemina to confer partial protective immunity in cattle has stimulated interest in characterizing both B-cell and T-cell epitopes of these proteins. It was previously shown that B. bovis RAP-1 associates with the merozoite surface as well as rhoptries and expresses B-cell epitopes conserved among otherwise antigenically different B. bovis strains. An amino-terminal 307-amino-acid domain of the molecule that is highly conserved in the B. bigemina RAP-1 homolog did not contain cross-reactive B-cell epitopes. The studies reported here demonstrate that B. bovis RAP-1 is strongly immunogenic for T helper (Th) cells from B. bovis-immune cattle and that like B-cell epitopes, Th-cell epitopes are conserved in different B. bovis strains but not in B. bigemina RAP-1. Lymphocytes from cattle immune to challenge with the Mexico strain of B. bovis proliferated against recombinant B. bovis RAP-1 protein derived from the Mexico strain. T-cell lines established by stimulating lymphocytes with recombinant RAP-1 protein responded against B. bovis, but not B. bigemina, merozoites. T-cell lines established by repeated stimulation of lymphocytes with B. bovis membrane antigen proliferated strongly against RAP-1, demonstrating the immunodominant nature of this protein. RAP-1-specific CD4+ T cell clones recognized Mexico, Texas, Australia, and Israel strains of B. bovis but neither B. bigemina merozoites nor recombinant B. bigemina RAP- 1. Analysis of cytokine mRNA in RAP-1-specific Th cell clones revealed strong expression of gamma interferon but little or no expression of interleukin-2 (IL-2), IL-4, or IL-10. Gamma interferon production was confirmed by enzyme-linked imunosorbent assay. These results indicate the potential to use selected B. bovis RAP-1 peptides as immunogens to prime for strong, anamnestic, strain-cross-reactive type 1 immune responses upon exposure to B. bovis.

To provide a biological basis for studies designed to establish the mode of transmission of the veterinary pathogen Besnoitia besnoiti, we compared salient features of this pathogen in cattle with those of Sarcocystis hoarensis in rodents. The cysts and cystozoites of these organisms can readily be distinguished morphologically. In contrast to S. hoarensis, which is well adapted to rodents, B. besnoiti fails to mature in jirds or mice and generally is lethal in jirds. Serological reagents discriminately detect these pathogens. B. besnoiti, therefore, can unambiguously be differentiated from S. hoarensis either by morphological or serological methods or on the basis of experimental comparisons of virulence in laboratory rodents.

Colostral antibodies to B. besnoiti were detected by immunofluorescence in four calves born to two Besnoitia-infected dams, with titres ranging from 1:64 to 1:1024. A specific antibody titre of 1:1024 was found in colostrum collected from one of the dams. Two of the newborn calves, when sampled immediately after birth, were serologically negative to B. besnoiti, but became positive on the next day. In all the calves, antibodies were detectable up to the age of 4 months. Observations concerning passive transfer of antibodies from Besnoitia-infected dams to offspring, and transmission of the infection among infected and non-infected closely kept cows, are discussed.

Babesia bovis merozoite surface antigen 1 (MSA-1) induces antibodies capable of neutralizing merozoites in vitro. Both MSA-1 and the co-expressed MSA-2 are encoded by a polymorphic multigene family and are antigenically variant among strains isolated from widely separated geographic regions. In this study, cross-protective immunity between two B. bovis clones, Mexico Mo-7 and Israel-C, that have antigenically unrelated MSA-1 and MSA-2 surface proteins was assessed. Cattle immunized by infection with either clone were significantly protected against challenge with the uncloned Israel Bbv strain. This indicates that epitopes capable of inducing partial protection are shared among different strains and that immunity is not solely dependent upon MSA-1 or MSA-2. However, cattle immunized with the Israel-C clone, derived from the Israel Bbv strain, were significantly better protected against BbV challenge than were cattle immunized with the Mexico Mo-7 clone bearing antigenically unrelated MSA-1 and MSA-2. The significant difference in immunity induced by the homologous strain versus an antigenically variant strain indicates that epitope variation among strains is relevant to immunity against babesiosis.

Gerbils (Meriones tristrami) were infected subcutaneously or intraperitoneally with live culture-grown Besnoitia besnoiti at doses ranging from 10(1) to 10(7) endozoites. All animals injected subcutaneously survived the infection and were refractory to a lethal challenge dose of 10(7) endozoites given intraperitoneally 6 weeks later. By contrast, gerbils surviving primary intraperitoneal inoculation with 10(2) to 10(6) endozoites showed a variable survival rate to challenge. All gerbils developed antibodies to B. besnoiti regardless of the route of inoculation, except for those given 10(1) endozoites intraperitoneally. There was no statistical difference between the immunofluorescent antibody titres developed in groups vaccinated subcutaneously or intraperitoneally (P = 0.556).

Calves vaccinated with Anaplasma centrale were treated with 20 mg/kg of long-acting oxytetracycline (OTC/LA) before or simultaneously with vaccination or up to seven months later. Of 40 animals given one or two of OTC/LA from 3 to 13 days before vaccination, 23 become patent after vaccination, with an average prepatent period almost twice as long as that in non-treated vaccinated controls. Upon challenge with 2 x 10(8) A. centrale per dose all 17 previously non-patent calves showed average maximum parasitemias of 2 to 3.8%. Out of 30 calves treated with two to four doses of OTC/LA from one to four weeks after vaccination, 29 remained negative for A. centrale and reacted to challenge infection with average maximum parasitemias of 6.9-7.8%. Five out of 10 calves receiving OTC/LA simultaneously with the vaccination, and all of a separate group of 10 calves treated with a single dose seven days after vaccination, become patent an average of 51.6 and 63.5 d, respectively, after vaccination. Upon challenge, the five previously non-patent calves showed an average of 5.2% maximum parasitemia. In all groups, only rare parasites were seen in previously patent calves after challenge. Thirty calves treated with 2-4 doses of OTC/LA about six months after vaccination showed no or only a few parasites upon challenge. The above results show that treatment with single or multiple doses of OTC/LA a few weeks before or after administration of live A. centrale vaccine can interfere with elaboration of immunity.

Polyclonal bovine IgM-rheumatoid factors (IgM-RFs) were examined in sera of cattle immunized against babesiosis. An enzyme-linked immunosorbent assay (ELISA) was used enabling rapid screening of serum samples. Results obtained indicate a rise of serum IgM-RF levels with age in healthy bovines. However when animals of similar age and pertaining to the same herd were examined, levels of serum IgM-RF exhibited a wide distribution range. Mean values in 60 sera of 2 yrs old clinically healthy heifers originating from a single herd were of 452.60 +/- 201.26 e.u. at 1 in 1,000 serum dilution and of 202.37 +/- 137.86 e.u. at 1 in 4,000 serum dilution. In a herd where repeated vaccination of dams against babesiosis was carried out 3 to 6 weeks before delivery either with live Babesia bovis parasites or soluble antigens of in vitro grown organisms, no significant differences in mean IgM-RF values were found between revaccinated animals and a control group. Nor did the mean values of serum IgM-RF of calves born to the respective groups of dams exhibit significant differences. It thus appears that immunization of healthy cattle against this parasite does not affect mean serum IgM-RF levels.

The use of the ELISA method for the detection of antibodies to B. besnoiti in cattle is described and compared to the IFAT technique. One hundred and twenty-one sera were examined, of which 61 were sera of calves experimentally infected with B. besnoiti, 52 sera from field animals and eight were sera with high titres of antibodies to other parasites. The specificity of both assays correlates but ELISA seemed to be more sensitive. The ELISA technique provides a rapid and reliable method for the screening of B. besnoiti infection in cattle.

Specific precipitable antibodies of both IgG and IgM classes were detected in sera of cattle naturally infected with B. besnoiti. The amount of specific antibodies of the IgG class precipitated by soluble antigen was in the range of 17-50 micrograms/ml serum while that of the IgM class ranged between 5 and 24 micrograms/ml serum. Specific antibodies precipitated by live B. besnoiti parasites were in amounts of 10 to 22 micrograms/ml serum for IgG and 4 to 26 micrograms/ml serum for IgM. Different ratios of IgG/IgM were obtained by the two methods of precipitation. This might indicate that antibodies to B. besnoiti of the IgM class can be precipitated and detected in sera of naturally infected cattle in similar amounts either by live parasites or by solubilized antigen, whereas antibodies of the IgG class can be preferentially detected when solubilized antigen is used for precipitation.