Molecular characterization of the Israeli B. bigemina vaccine strain and field isolates.

The present study demonstrated the genetic character of the Israeli Babesia bigemina vaccine strain and field isolates, based on rap-1a and rap-1c gene sequences. The RAP-1a of blood-derived Israeli B. bigemina field isolates shared 100% amino acid sequence identity. However, comparison of RAP-1c from various Israeli B. bigemina field isolates revealed that the total sequence identity among the field isolates ranged from 98.2 to 100%. High identity was observed when RAP-1a sequences from the Israeli vaccine strain and field isolates were compared with RAP-1a from Egypt, Syria, Mexico and South Africa, while, the Israeli RAP-1c sequences showed the highest identity to the Mexican isolate JG-29 and to the PR isolate from Puerto-Rico. Based on sequence variations between the rap-1a of the vaccine strain and that of the field isolate, and between the rap-1c of the vaccine strain and that of the field isolates, nPCR-RFLP procedures were developed that enable, for the first time differentiation between the Israeli B. bigemina vaccine strain and field-infection isolates. These assays could serve as fast and sensitive methods for detection and differentiation between Israeli B. bigemina vaccine strains and field isolates, as well as for epidemiological investigations.

Differentiation between Israeli B. bovis vaccine strain and field isolates.

The present study demonstrated for the first time the ability to distinguish between the Israeli Babesia bovis vaccine strain and field isolates. The existence of an additional EcoRI restriction site in the rhoptry-associated protein-1 (rap-1) gene, which is unique to the Israeli vaccine strain, and the abolition of one of the HaeIII restriction sites in the rap-1 gene of the vaccine strain enabled distinction between the Israeli B. bovis vaccine strain and field isolates, and this was the basis for polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) development. ClustalW sequence alignment of RAP-1-deduced amino acids of the Israeli B. bovis strains and of field isolates showed that the total sequence identity among the RAP-1 amino acid sequences ranged from 97.5% to 100%. However, comparison between amino acids of RAP-1 of the Israeli vaccine strain and of field isolates, on the one hand, and B. bovis strains from Argentina, Mexico, Brazil, and USA, on the other hand, revealed 90% identity. The PCR-RFLP assay offered the great advantage of being able to distinguish between vaccine and field isolates in mixtures and provide new insight into the molecular epidemiology of B. bovis infections in Israel.

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.

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.

Experimental transmission of field Anaplasma marginale and the A. centrale vaccine strain by Hyalomma excavatum, Rhipicephalus sanguineus and Rhipicephalus (Boophilus) annulatus ticks.

The cattle rickettsia Anaplasma marginale is distributed worldwide and is transmitted by about 20 tick species, but only Rhipicephalus simus, a strictly African tick species, has been shown to transmit the vaccine strain of A. centrale. The aim of the present study was to examine transmission of field strains of A. marginale and of the vaccine strain of A. centrale by three tick species -Hyalomma excavatum, Rhipicephalus sanguineus and Rhipicephalus (Boophilus) annulatus - to susceptible calves. Two genetically distinct Israeli field strains of A. marginale, tailed and non-tailed (AmIsT and AmIsNT, respectively), were efficiently transmitted by R. sanguineus, whereas H. excavatum transmitted only the tailed isolate, and R. (Boophilus) annulatus did not transmit A. marginale. None of the three tick species transmitted A. centrale. By means of msp1a primers in PCR assays, amplicons of similar sizes were obtained from either A. marginale-infected calves that were used for acquisition feeding, from R. sanguineus fed on the infected calves, or from calves to which anaplasmosis had been successfully transmitted by these ticks. Although an A. centrale-specific fragment was amplified from salivary glands of R. sanguineus, no transmission to susceptible cattle occurred during 3 months of observation, and anaplasmosis was not induced in splenectomized calves that were subinoculated with blood from calves on which R. sanguineus had fed.

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.

Concomitant infection of cattle with the vaccine strain Anaplasma marginale ss centrale and field strains of A. marginale.

Bovine anaplasmosis, caused by Anaplasma marginale, the intraerythrocytic rickettsia, is controlled by vaccination with live Anaplasma marginale ss centrale (A. centrale), a subspecies of relatively low pathogenicity. We have experimentally demonstrated that an animal primarily infected with A. marginale, or with the related vaccine subspecies A. centrale can be infected with the heterologous subspecies, and carries both bacteria. The co-infection was detected in experimentally cross-infected calves for up to 3 months after the last inoculation with the heterologous subspecies. The occurrence of characteristic cyclic rickettsemia of A. centrale and A. marginale was observed by examination of Giemsa-stained blood smears, or by the presence of specific rickettsial DNA confirmed in PCR assays based on specific msp1a and msp4 for A. marginale, and on specifically designed msp3 and msp4 primers for A. centrale. Sequence analysis of msp4-specific fragments for each subspecies revealed the presence of dual infection in both calves on days 30 and 60 after cross-inoculation with the heterologous Anaplasma subspecies. The experimental cross-infection of calves clearly demonstrated that the concept of "infection exclusion" does not apply to Anaplasma infection in cattle; as there was no infection exclusion of A. marginale in A. centrale-infected cattle, and vice versa. The present results confirmed our previous findings that cattle grazing in an anaplasmosis-endemic field were subject to concomitant infection with both the vaccine A. centrale and the field A. marginale strains.

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.

Babesia bigemina: attenuation of an Uzbek isolate for immunization of cattle with live calf- or culture-derived parasites.

The virulence of an Uzbek isolate of Babesia bigemina, obtained from infected Boophilus annulatus ticks from an endemic area in Uzbekistan, was attenuated for immunization of cattle with autochthonous calf- or culture-derived parasites in Uzbekistan. After four "slow passages" in vivo the virulence was reduced, as evidenced by the response of calves inoculated with an experimental live frozen vaccine produced from the following passage. The vaccine was safe and protective against homologous virulent challenge under laboratory conditions. The culture-derived experimental vaccine was produced from cultures initiated after 3 passages in vivo followed by 22 passages in vitro. The cultured parasites did not elicit any clinical sign, but inoculated calves seroconverted following vaccination and were protected against the virulent homologous challenge. Both calf- and culture-derived vaccines were safe for cattle grazing in an endemic area in Uzbekistan. Despite the high polymorphism of B. bigemina, as reported from various geographical regions, the Central Asian strain was attenuated similarly to those that form the basis of the existing live B. bigemina vaccines in other parts of the world.

Application of PCR for detection of Clostridium botulinum type D in bovine samples.

Diagnosis of botulism in cows is obtained by detecting the neurotoxin and/or Clostridium botulinum in the suspected animal. The standard method for detecting the toxin is the mouse bioassay. However, in recent years, the use of mice has become very costly and inconvenient in some facilities, and public pressure has been increasing to find alternatives to live animal bioassays. In this manuscript, we describe the use of the polymerase chain reaction (PCR) procedures in the diagnosis field cases of bovine type D botulism. Bovine samples from clinical cases diagnosed as C. botulinum type D according by clinical symptoms and bioassay resulted in expected PCR product ( approximately 497 bp) similar to the C. botulinum type D NCTC 8265 strain while the gene product was confirmed by sequence data.

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.

Vaccination of older Bos taurus bulls against bovine babesiosis.

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.

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.

Detection of the Anaplasma centralevaccine strain and specific differentiation from Anaplasma marginale in vaccinated and infected cattle.

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.

Molecular characterization of an unassigned Israeli Newcastle disease virus isolate.

Detection of Newcastle disease virus (NDV) and avian pathotyping of NDV isolates are extremely important because the appearance of virulent virus has significant economic consequences in terms of vaccination, eradication, and the ability to export poultry products. By using nucleotide and amino acid (aa) homology analysis, we could demonstrate that a NDV broiler isolate is a velogenic virus. This analysis was done after mean death time and intracerebral pathogenicity index tests gave inconsistent results. By establishing a nucleotide sequence dendrogram, we found that the disputed Ber-Tuvia was clearly in the same group as the known Herev-Laet, a velogenic isolate. The difference between Ber-Tuvia 92 and the Herev-Laet velogenic isolate was 6% as opposed to > 16% of the meso- and lentogenic isolates. The Ber-Tuvia isolate contains the Arg/Arg and Lys/Arg aa at positions 112, 113 and 115, 116, respectively, in the fusion protein cleavage aa sequence, which is typical for virulent NDV isolates.

A ribozyme targeted to cleave the polymerase gene sequences of different foot-and-mouth disease virus (FMDV) serotypes.

Vaccinations against foot-and-mouth disease virus (FMDV) has dramatically reduced the number of disease outbreaks. Nevertheless, there are still many outbreaks in different regions around the world. In an effort to find new ways to control the disease, ribozymes able to cleave FMDV were designed and tested. In this work we tested the ability of FRZ4, a ribozyme targeted to the viral polymerase gene, to cleave polymerase sequences of several FMDV. Homology analysis was used to choose target sequences which consist of two conserved GUC which lie 15 bases apart and, their flanking sequences. These were the basis for the FRZ4 ribozyme gene sequence that contains two catalytic domains. We show that polymerase sequences from A, Asia 1, C and two different O1 Israeli isolates could be specifically cleaved by FRZ4. It is suggested that FRZ4 can cleave polymerase gene sequences from any FMDV serotype.

Cleavage of transcripts of foot and mouth disease virus (FMDV), Asia1 serotype, by ribozymes targeted to the VP3 and VP4 genes.

Two ribozyme genes were designed to cut within the VP4 and VP3 sequences of foot and mouth disease virus (FMDV) Asia1 serotype genome. The two genes were synthesized and cloned into pBluescript under the control of the T3 promoter. The ribozyme designed to cut the VP4 gene contained two catalytic sequences targeted to two GUC triplets that are 16 bases apart. The second ribozyme, intended to cut VP3, contained one catalytic sequence. Ribozymes obtained from run-off transcription from both plasmids were able to cleave viral RNA derived from runoff transcripts of plasmids carrying the proper FMDV cDNA inserts. The significance of these findings is discussed.

Detection and subtyping of foot-and-mouth disease virus in infected cattle by polymerase chain reaction and amplified VP1 sequencing.

Fast and accurate detection of foot-and-mouth disease (FMD) outbreaks is needed to limit spread of the disease by proper vaccination. The use of the polymerase chain reaction (PCR) has revolutionized the way in which viral diseases are diagnosed. Sequence analysis of the amplified VP1 sequence can enable the classification of FMD virus detected in the morbid animal. PCR assays were carried out to identify the virus and its serotype in suspect animals from 2 outbreaks of FMD type O virus. Sequence analysis of the amplified VP1 cDNA showed 78% homology with O1K and over 95% homology between the samples. These findings suggest that the 2 outbreaks were due to infection with the same virus serosubtype.

Molecular epidemiology of foot-and-mouth disease (FMD) in Israel in 1994 and in other Middle-Eastern countries in the years 1992-1994.

The reverse transcriptase-polymerase chain reaction (RT-PCR) and direct sequencing were employed in the diagnosis and typing of foot-and-mouth disease virus (FMDV) in samples taken during the 1994 disease outbreak in Israel. Using PCR, virus isolation and serological methods it was shown that the 1994 disease outbreak in Israel and other Middle-Eastern countries was caused by O1 type virus. Direct PCR sequencing of VP1 genes and homology analysis of the virus isolates revealed that there were two distinct outbreaks in Israel. The first originated in Jordan, moved to the West Bank territory and then to the Lower Galilee. The second outbreak, caused by another virus, was responsible for disease outbreaks in South Lebanon, Upper Galilee and the Golan Heights. When viral sequences of isolates from the 1993 outbreaks in Egypt and Lebanon were included in the analysis, they showed a high degree of VP1 sequence homology between themselves, suggesting a common origin.