Neospora-related abortions in sheep in Israel - A serological diagnostic challenge in an endemic area.

Abortions in sheep flocks is a common multifactorial problem, which affects animal productivity and welfare. Israel is endemic to several infectious abortifacient pathogens, with neosporosis identified as a prominent cause. High seroprevalence of N. caninum is present in the general sheep population, making complicate to associate it as the causative agent of abortion in sheep. Here we describe two investigations of abortion storms in sheep flocks with high seroprevalence of neosporosis. In flock A, higher anti-Neospora antibody titers were demonstrated in aborting versus non-aborting ewes, suggesting that it may be the cause of abortions. In flock B, several infectious abortifacients were identified, but only the seroprevalence of border disease differed statistically between aborting and non-aborting ewes. These reports highlight the difficulty of diagnosing the cause of abortion in a multifactorial situation, and confirm the necessity to assess paired samples from aborting and non-aborting ewes, for reliable interpretation of the results.

The involvement of protozoan parasites in sheep abortions - A ten-year review of diagnostic results.

Abortions in sheep flocks affect animal health and lead to significant losses in productivity, with severe economic consequences. In recent years, the role of protozoan parasites as the cause of ovine abortions has been significant. Here, the diagnosis of infectious causes of abortions in sheep in Israel in the last decade is reviewed, focusing on parasitic pathogens. Analysis of the serological data of sheep sera (including aborted fetuses) submitted for diagnoses between 2010 and 2019 revealed overall seroprevalence of 67.4 % and 46.7 % for Neospora spp. and Toxoplasma gondii respectively, with high rates of co-exposure (32.4 %). The seroprevalence of T. gondii was higher in aborting ewes than in pre-sale examinations (48.2 % and 28.9 %, respectively (P < 0.001)). The seroprevalence of Neospora spp. was significantly higher than the seroprevalence of Toxoplasma (P < 0.001), and was similar in samples from aborting ewes and in samples from pre-sale examinations. In addition, the presence of anti-Neospora spp. antibodies was the most prominent finding diagnosed in aborted fetuses (22.9 % of aborted fetuses, significantly higher than any other organism, P < 0.001). The results of this study demonstrate that in endemic areas the seroprevalence of N. caninum in sheep may be high, and should be considered as an important cause of abortions. However, since the seroprevalence is high even in non-aborting ewes, in order to determine the causative agent of abortion in endemic flocks, a comprehensive epidemiological investigation is warranted.

Serological and Molecular Prevalence of Babesia caballi in Apparently Healthy Horses in Israel.

Babesia caballi is a tick-borne hemoparasite of equines and one of the causative agents of equine piroplasmosis, which poses a great concern for the equine industry regarding animal welfare and international horse movement. The parasite is endemic in Israel; however, its seroprevalence in the area was never evaluated due to antigenic heterogenicity in the gene used in the commercially available kit. Blood samples were collected from 257 horses at 19 farms throughout the country and screened for the presence of anti-B. caballi antibodies via an indirect immunofluorescent antibody test (IFAT) and for the presence of parasite DNA by nested PCR. The seroprevalence of B. caballi was 69.6% and its molecular prevalence was 9.7%. The geographical area, horse's sex, breed, housing, exposure to ticks, and specifically to Hyalomma marginatum, and co-infection with Theileria equi were found to be significantly associated with serologic exposure in univariable analysis, while the geographical area and horses' sex remained significant in the multivariable analysis. The results of this study demonstrate a high level of exposure to B. caballi and identify important risk factors for infection. The difference between the serological and molecular prevalence, probably related to parasite clearance, is also highlighted.

The Effect of Vaccination with Neospora caninum Live-Frozen Tachyzoites on Abortion Rates of Naturally Infected Pregnant Cows.

Neosporosis is a major cause of abortions in cattle worldwide. Recently a live attenuated vaccine showing promising results in preventing abortions, when administered at mid-pregnancy to seropositive cows, was developed. In this study, vaccination with 2 × 108 live frozen N. caninum tachyzoites (NcIs491) was used to immunize naturally infected seropositive pregnant dairy dams. The study was performed under field conditions in four herds, and a follow-up of three subsequent pregnancies was analyzed. A total of 1136 cows were serologically examined. Total seroprevalence was 41.4%, with 25.1% of the cows having titers of 1:800 or higher. Abortion rates were significantly higher in cows with high antibody titers (≥1:800) for two consecutive pregnancies. Vaccination was administered to 114 out of 285 cows with antibody titers higher than 1:800. Immunization resulted in lower abortion rates at three of the farms. Vaccine efficacy ranged from -19.8% to 75% at different farms, with overall efficacy of 28.4% in all four farms and overall efficacy of 58.2% in the three farms with positive results. Our results showed different vaccine efficacy in studied farms, suggesting that frozen live vaccination may generally be an effective method to control neosporosis in cattle.

Infection dynamics of Theileria equi in carrier horses is associated with management and tick exposure.

The tick-borne equine hemoparasite, Theileria equi, is endemic in many parts of the world where prevalence may be high, and most infected horses are apparently healthy but serve as life-long carriers. To determine the factors that affect T. equi dynamics, we followed parasitic loads in apparently healthy horses at four time points during one year. A total of 1094 blood samples were collected from 395 horses, along with ticks and demographic and clinical data. Infection and load of T. equi were tested by PCR and qPCR, and for the spring dataset, infection was also tested serologically by IFAT (n = 268). Theileria equi was molecularly detected in 64.8 % of the horses. The agreement between molecular and serological results was 79.8 % (K > 0.674) and positively correlated with parasitic load. Infection was associated with pale mucus membranes, lower packed cell volume and higher total solids (all P < 0.001), although these changes had only minor clinical importance. While parasitic loads in qPCR-positive samples (n = 561) were generally low (mean = 7.9-10-4% parasitized erythrocytes), younger horses showed higher loads, possibly suggesting development of immunity. Infection and parasitic load were associated with housing management and tick exposure, illustrating different patterns of exposure. Endemic stability is suggested in pasture farms with constant exposure to ticks, where parasite prevalence was high (96 %) and associated with T. equi 18S rRNA genotype D, low parasitemia and high antibody titers. Endemic instability can be suggested in case were horses are kept in paddocks (prevalence = 49 %) with intermittent exposure to ticks, where infection was associated with high parasitemia when ticks were present. A steady state is suggested in stabled horses (prevalence = 46 %), with no exposure to ticks; where infection was associated with genotype A, low parasitemia and low antibody titers. The ability to identify different risk groups within endemic areas may improve the administration of suitable treatment and control practices in an effort to reduce the risk of clinical disease.

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

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

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

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

Bovine immune response to inoculation with Neospora caninum surface antigen SRS2 lipopeptides mimics immune response to infection with live parasites.

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.