Systemic toxoplasmosis in a cat under cyclosporine therapy.

Toxoplasma gondii, an obligatory intracellular protozoan parasite infecting warm-blooded animals, can cause toxoplasmosis, a major zoonosis. A male neutered, domestic cat was referred to the Hebrew University Veterinary Teaching Hospital due to dyspnea after long term treatment with cyclosporine for obsessive self-grooming and pruritis. After thorough diagnostics, including non - invasive imaging, broncho-alveolar lavage, blood serology, hematology and biochemistry, and evaluation of the aspirated fluid components, a severe pneumonia and abdominal effusion were detected with observation of free tachyzoites under light microscopy from lavage fluids. PCR and DNA sequencing of broncho-alveolar lavage was positive for T. gondii. Despite aggressive treatment with antibiotics, oxygen supplementation and T. gondii specific antimicrobials, the cat died. It is suggested that potential candidates for cyclosporine be screened for T. gondii antibodies, kept entirely indoors and not fed uncooked meat in order to prevent exposure to T. gondii infection.

Troglostrongylus brevior is the dominant lungworm infecting feral cats in Jerusalem.

Feline lungworms infect the respiratory tract of wild and domestic cats, causing infection often associated with clinical disease. Until recently, Aelurostrongylus abstrusus has been considered the most relevant species of lungworm, while Troglostrongylus brevior was considered of less significance. Fecal samples of feral cats from Jerusalem, Israel, collected over a year, were examined for first stage lungworm larvae (L1) using the Baermann method. Positive samples were morphologically identified, and their species identity was molecularly confirmed. Forty of 400 (10.0%) cats were lungworm-positive, of which 38/40 (95.0%) shed Troglostrongylus brevior and 6/40 (15.0%) shed Aelurostrongylus abstrusus. Four cats (10.0%) had mixed infections with both lungworm species. L1 shedding was associated with clinical respiratory signs in 11 (19.0%) T. brevior shedding cats of a total of 58 cats manifesting respiratory signs, while 23/342 (6.7%) cats without respiratory signs were L1-positive (p = 0.006). Non-respiratory clinical signs were also found to be more prevalent in L1 shedders (p = 0.012). A young kitten ≤ 4 weeks of age shed T. brevior L1 larvae. DNA sequences of both lungworm species using the ribosomal internal transcribed spacer 2 (ITS2) locus were > 99% similar to other sequences deposited in GenBank, suggesting that T. brevior and A. abstrusus ITS2 sequences are both highly conserved. In conclusion, L1 shedding in feral cats from Jerusalem were mostly caused by T. brevior with only a small proportion involving A. abstrusus, different from many studies from other geographical regions.

Urinary incontinence associated with Mesocestoides vogae infection in a dog.

Peritoneal larval cestodiasis caused by Mesocestoides spp. is a rare infection in dogs. A 6-year-old female dog was presented for veterinary care with urinary incontinence which started 1 year earlier. After performing hematology, ultrasound, and computerized tomography, an exploratory laparotomy revealed canine peritoneal larval cestodiasis (CPLC) with the presence of Mesocestoides vogae (syn. Mesocestoides corti) tetrathyridia confirmed by morphological identification and PCR and DNA sequencing. Parasitic cysts were found around the urinary bladder and appeared to inhibit its normal function. An initial treatment with 5 mg/kg praziquantel subcutaneously every 2 weeks for four treatments failed to alleviate the clinical signs, and only treatment with fenbendazole at 100 mg/kg P.O. twice daily for 28 days was associated with the disappearance of ascites and regaining of urinary control. This is the first report of CPLC associated with urinary incontinence in dogs and the first description of this cyclophyllidean cestode in dogs in Israel.

Neospora caninum in crows from Israel.

A cross-sectional Neospora caninum seroprevalence study was performed on free ranging crows (Corvus cornix, Corvus monedula and Corvus splendens) from Israel in order to assess their exposure to this pathogen and evaluate their role as potential hosts or as sentinels of infection. Using the modified agglutination test (MAT) with a cutoff titer of 1:100, 30 out of 183 crows (16.4%) were found to be N. caninum seropositive. Positive results were validated and confirmed by the indirect fluorescent antibody test (IFAT). There was 100% agreement between tests when cut-off titers of 1:50 and 1:100 were applied for the IFAT and MAT, respectively. PCR analysis of brain extracts from all crows resulted in the detection of N. caninum DNA for the first time in crows belonging to two species, C. cornix and C. monedula. The high N. caninum seroprevalence in crows suggests that widespread exposure to infection with N. caninum exists especially in central and northern Israel and that crows may act as suitable markers for disease prevalence in the areas in which they are found.

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.

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.

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.

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.

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.