Diagnostic performance of a rapid immunochromatographic test for the simultaneous detection of antibodies to Theileria equi and Babesia caballi in horses and donkeys.

BACKGROUND: Equine piroplasmosis is caused by two tick-borne protozoan parasites, Theileria equi and Babesia caballi,, which are clinically relevant in susceptible horses, donkeys, and mules. Moreover, equine piroplasmosis significantly constrains international trading and equestrian events. Rapidly diagnosing both parasites in carrier animals is essential for implementing effective control measures. Here, a rapid immunochromatographic test for the simultaneous detection of antibodies to T. equi and B. caballi was evaluated using samples from horses and donkeys collected in Greece, Israel, and Italy. The results were compared with an improved competitive enzyme-linked immunosorbent assay (cELISA) for detecting antibodies to both parasites using the same panel of samples. METHODS: Blood samples were collected from 255 horses and donkeys. The panel consisted of 129 horses sampled at four locations in northern Greece, 105 donkeys sampled at four locations in Sicily, and 21 horses sampled at two locations in Israel. The rapid test and the cELISA were performed according to the manufacturer's instructions, and the results were subjected to a statistical analysis to determine the sensitivity and specificity of both tests and their association. RESULTS: The immunochromatographic test provided a result within 15 min and can be performed in the field, detecting both pathogens simultaneously. The overall coincidence rate between the rapid test and the cELISA for detecting antibodies against T. equi was 93% and 92.9% for B. caballi. The rapid test's sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for T. equi were above 91.5%. Sixteen samples were positive for both parasites in the rapid test and eight in the cELISA. Either test had no significant association between T. equi and B. caballi detection. The detection rates of both parasites were significantly higher in Italy than in Greece or Israel and in donkeys than in horses. The agreement for T. equi between the results of both tests was high in Greece (93.8%) and Italy (95.2%) and moderate in Israel (76.2%). For B. caballi, the specificity and NPV of the rapid test were high (94.2% and 98.3%, respectively), although the sensitivity and PPV were moderate (69.2% and 39.1%, respectively) due to the small sample size. However, for B. caballi, the sensitivity was higher with the rapid test. CONCLUSIONS: The rapid test detected T. equi and B. caballi simultaneously in the field, potentially replacing laborious cELISA testing and is recommended for import/export purposes. The test can also be helpful for the differential diagnosis of clinical cases, since seropositivity may rule out equine piroplasmosis since it does not indicate current or active infection.

Persistence of Anti-Rabies Antibody Response in Horses Following Vaccination.

Rabies is a fatal zoonotic disease affecting all mammalian species. It is caused by the rabies virus and is prevalent worldwide. Horses are not commonly infected with rabies but their vaccination is recommended due to the potential zoonotic risk. This study aimed to evaluate the duration of immunity following rabies vaccination in horses. A total of 126 serum samples were collected from 93 horses, vaccinated 6 to 91 months before sampling. Rabies-virus-neutralizing antibody (RVNA) levels were evaluated using the Rabies Fluorescent Focus Inhibition Test (RFFIT). A protective RVNA titer of above 0.5 IU/mL was found in 112 (88.9%) of the samples and 84 (90.3%) of the horses. Antibody titers declined over time (rho = -0.271, p = 0.002); however, there was no significant difference in antibody titers or the prevalence of unprotected horses between the time intervals following vaccination. Purebred horses had lower antibody titers (p = 0.024). The response to booster vaccination was inspected in ten horses, and increased antibody titers were found in eight of them. The results of this study demonstrate the prolonged persistence of protective immunity in horses following rabies vaccination, in some cases, for up to eight years. Therefore, the current annual vaccination strategy should be re-evaluated. A rate of 9.7% of poor responders should be considered from an epidemiological perspective in order to minimize the risk of emergence of the disease.

First Detection of Anti-Besnoitia spp. Antibodies in Equids in Israel and the Palestinian Authority.

Besnoitia is a tissue cyst forming coccidia, which affects multiple host species worldwide. Equine besnoitiosis is characterized mainly by generalized skin lesions and cysts in the scleral conjunctiva. Recent reports revealed exposure to Besnoitia in equines in Europe and the United States. However, the exposure to Besnoitia spp. in the Israeli equine population was never investigated. The aim of this study was to evaluate the seroprevalence and associated risk factors for besnoitiosis in equids in Israel. A cross-sectional serosurvey was performed using serum samples of apparently healthy horses (n = 347), donkeys (n = 98), and mules (n = 6), and exposure to Besnoitia spp. was determined by an immunofluorescent antibody test (IFAT). Anti-Besnoitia spp. antibodies were detected in 17.7% equids, 6.9% horses, 33.3% mules, and 55.1% donkeys. The seroprevalence in donkeys was significantly higher than in horses (p < 0.001). A significant association between the geographic location and seropositivity was found both in horses and donkeys, which was significantly higher (p = 0.004) in horses sampled in southern Israel, and donkeys sampled in Israel versus the Palestinian Authority (p < 0.001). This is the first serosurvey of Besnoitia infection in equines in Israel, and the results are consistent with reports from Europe. The clinical significance of equine besnoitiosis should be further investigated.

High infection rates of Toxoplasma gondii in cattle, sheep and pigs from Israel.

Toxoplasma gondii is an obligate intracellular parasite belonging to the phylum Apicomplexa, which causes the zoonotic disease toxoplasmosis. T. gondii infects almost all warm blood animals. Generally, infected animals are asymptomatic and remain infected for life. Infection of humans occurs by consumption of infected undercooked meat or contaminated vegetables, fruit and water. The aim of this study was to evaluate the prevalence and seroprevalence of T. gondii in livestock in Israel. For the serological screening we investigated the presence of antibodies against T. gondii in sera using the indirect fluorescent antibody test (IFAT). Molecular screening was preformed using conventional PCR and nested PCR for the detection of T. gondii DNA in tissue samples. Serum samples of 249 cattle and 138 sheep were collected from farms. This serological survey showed high seroprevalence with seropositivity of 29 % in cattle and 33 % in sheep. In addition, 526 paired sera and tissue samples from cattle, sheep and pigs were obtained in slaughterhouses. The serological prevalence of T. gondii in healthy animals intended for human consumption was 29.4 % in cattle, 26.1 % in sheep and 8.1 % in pigs. The molecular detection of T. gondii in the tissue samples was 7.5 % in cattle, 7.3 % in sheep and 6.3 % in pigs. Considering the combined positive results from both serological and molecular assays, exposure to or infection with the parasite was present in 26.2 % of the samples (33.8 % in cattle, 30.3 % in sheep, 12.5 % in pigs). The prevalence of T. gondii in pigs was significantly lower compared to that of cattle and sheep (P < 0.001). All PCR positive samples from animals and 12 human positive samples were genotyped using a restriction fragment length polymorphism (PCR-RFLP) method. The results showed the existence of atypical genotypes in the majority of the samples and no correlation between animals and human samples could be determined from this study. Widespread exposure to T. gondii in Israel with the presence of parasite DNA in meat from cattle, sheep and pigs meant for human consumption was found.

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.

Neospora spp. Seroprevalence and Risk Factors for Seropositivity in Apparently Healthy Horses and Pregnant Mares.

Equine Neospora infection has been linked to neurological disorders and infertility in horses. This study looked into the risk factors for infection and the exposure to Neospora spp. in horses. The study was performed in two independent populations in Israel. The first consisted of apparently healthy horses, and the second consisted of mares examined during pregnancy and after parturition. Sera samples collected from horses and mares were tested for Neospora exposure by the indirect fluorescent antibody test (IFAT). The study revealed seroprevalence of 24% in apparently healthy horses and 66.4% and 48.6% in mares during gestation and after parturition, respectively. Among the investigated risk factors, older age (p = 0.026) and housing in both stalls and paddocks (p = 0.033) in apparently healthy horses, and Arabian breeds (p = 0.005) in pregnant mares, were found to be significantly associated with Neospora spp. seropositivity in univariable, but not multivariable, statistical analysis. This study revealed high exposure of equines to Neospora parasites, especially mares. Horse farm management, in combination with active surveillance, including serological testing and follow up, could help reduce the spread of the parasite among horses in endemic areas.

Fecal Microbiome Features Associated with Extended-Spectrum ß-Lactamase-Producing Enterobacterales Carriage in Dairy Heifers.

Extended-spectrum ß-lactamases (ESBLs) are a growing public health threat, and one key human exposure point is through livestock and the food supply. Understanding microbiome factors associated with fecal ESBL carriage can help detect and ideally assist with controlling and preventing ESBL dissemination among livestock. The objective of this study was to investigate the diversity and composition of the heifer fecal microbiota in ESBL-producing Enterobacterales (ESBL-PE) carriers and noncarriers. A total of 59 fecal samples were collected from replacement heifers between 12 and 18 months old from eight dairy farms in central Israel. Genomic DNA was extracted, and 16S rRNA amplicon sequencing was performed (Illumina short reads), focusing on a comparison between 33 ESBL-PE carriers (55.9%) and 26 (44.1%) noncarriers. Samples were analyzed and compared using QIIME2 (DADA2 pipeline and taxonomic assignment with SILVA database) and associated R packages for alpha and beta diversity and taxonomic abundances. Alpha diversity (Shannon diversity) and beta diversity (unweighted UniFrac) showed no significant difference between ESBL-PE carriers and noncarriers. Heifers from farms feeding calves with pooled colostrum had higher ESBL-PE carriage rates than heifers from farms feeding with individual mother colostrum (p < 0.001). Taxonomical abundance analysis revealed that the most common bacterial phyla were Bacteroidetes (44%) and Firmicutes (38%). There was no significant difference in taxonomic composition between ESBL-PE carriers and noncarriers at the phylum and genus levels. However, LEfSe biomarker discovery analysis identified several genera which were significantly different between carriers and noncarriers. For example, Prevotellacaea, Bacteroides, Rikenellaceae, and uncultured Bacteroidales were more abundant in ESBL carriers than noncarriers. Some aspects of microbiota composition differ between ESBL carriers and noncarriers in dairy heifers, specifically the abundance of certain genera. Feeding with pooled colostrum may play a role in that assembly. These could potentially serve as markers of ESBL-PE carriage. However, further research is needed to determine whether these observed differences have a significant impact on colonization with ESBL-PE.

Virus Infection in Equine.

The relationship between men and horses has significantly evolved over the last century [...].

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.

Equine Encephalosis Virus.

Equine encephalosis (EE) is an arthropod-borne, noncontagious, febrile disease of horses. It is caused by EE virus (EEV), an Orbivirus of the Reoviridae family transmitted by Culicoides. Within the EEV serogroup, seven serotypes (EEV-1-7) have been identified to date. This virus was first isolated from a horse in South Africa in 1967 and until 2008 was believed to be restricted to southern Africa. In 2008-2009, isolation of EEV in an outbreak reported from Israel demonstrated the emergence of this pathogen into new niches. Indeed, testing in retrospect sera samples revealed that EEV had already been circulating outside of South Africa since 2001. Although EEV normally does not cause severe clinical disease, it should be considered important since it may indicate the possible spread of other related, much more pathogenic viruses, such as African horse sickness virus (AHSV). The spread of EEV from South Africa to central Africa, the Middle East and India is an example of the possible emergence of new pathogens in new niches, as was seen in the case of West Nile virus, and should be a reminder not to limit the differential list when facing a possible outbreak or a cluster of clinical cases. This review summarizes current knowledge regarding EEV structure, pathogenesis, clinical significance, and epidemiology.

West Nile Virus in Common Wild Avian Species in Israel.

In order to evaluate the contribution of different wild bird species to West Nile virus (WNV) circulation in Israel, during the months preceding the 2018 outbreak that occurred in Israel, we randomly sampled 136 frozen carcasses of a variety of avian species. Visceral and central nervous system (CNS) tissue pools were tested using WNV NS2A RT qPCR assay; of those, 15 (11.03%, 95% CI: 6.31-17.54%) tissue pools were positive. A total of 13 out of 15 WNV RT qPCR positive samples were successfully sequenced. Phylogenetic analysis indicated that all WNV isolates were identified as lineage 1 and all categorized as cluster 2 eastern European. Our results indicated that WNV isolates that circulated within the surveyed wild birds in spring 2018 were closely related to several of the isolates of the previously reported 2018 outbreak in birds in Israel and that the majority of infected birds were of local species.

Establishing Babesia bovis-Free Tick Colony Following Treatment of the Host with Diminazene Aceturate (Berenil).

Babesia bovis is a widely-spread tick-borne hemoparasite of cattle with major economic and animal welfare consequences. Rhipicephalus (Boophilus) annulatus is a one-host tick which transmits bovine babesiosis in the Middle East and Africa. Laboratory rearing of ixodid ticks is essential for the investigation on ticks or tick-borne diseases. Establishing a tick colony in the laboratory usually originates from ticks harvested in the field, which may be naturally infected with various pathogens. This especially applies to carriage of B. bovis as it is highly prevalent in endemic areas and is transmitted transovarially in ticks. Here, we describe the use of diminazene aceturate (Berenil) in order to establish laboratory colonies of Babesia-free R. annulatus, from ticks collected in the field. Ticks collected in the field were kept until oviposition and hatched larvae were introduced to naïve calves, which led to infection of the calves with B. bovis. Calves were then treated with diminazene aceturate several times until the engorged ticks dropped. The eggs and larvae collected from these ticks were parasite-free, as demonstrated both by infection of splenectomized calves and by PCR. This suggested protocol is a useful tool to create parasite-free tick colony and may, theoretically, also be beneficial to reduce parasite circulation in the field, although not recommended, as resistance to diamenizene aceturate might develop.

Seroprevalence and Risk Factors for Exposure to Equine Coronavirus in Apparently Healthy Horses in Israel.

Equine coronavirus (ECoV) infection is the cause of an emerging enteric disease of adult horses. Outbreaks have been reported in the USA, EU and Japan, as well as sporadic cases in the UK and Saudi Arabia. Infection of ECoV in horses in Israel has never been reported, and the risk of exposure is unknown. Importation and exportation of horses from and into Israel may have increased the exposure of horses in Israel to ECoV. While the disease is mostly self-limiting, with or without supportive treatment, severe complications may occur in some animals, and healthy carriers may pose a risk of infection to other horses. This study was set to evaluate the risk of exposure to ECoV of horses in Israel by using a previously validated, S1-based enzyme-linked immunosorbent assay (ELISA). A total of 41 out of 333 horses (12.3%) were seropositive. Exposure to ECoV was detected in 17 of 29 farms (58.6%) and the seroprevalence varied between 0 and 37.5% amongst farms. The only factor found to be significantly associated with ECoV exposure in the multivariable model was the geographical area (p < 0.001). ECoV should be included in the differential diagnosis list of pathogens in cases of adult horses with anorexia, lethargy, fever and gastrointestinal signs in Israel.

Seroprevalence of Leptospira spp. in Horses in Israel.

Leptospirosis has been reported in both humans and animals in Israel but has not been reported in horses. In 2018, an outbreak of Leptospira spp. serogroup Pomona was reported in humans and cattle in Israel. In horses, leptospirosis may cause equine recurrent uveitis (ERU). This report describes the first identification of Leptospira serogroup Pomona as the probable cause of ERU in horses in Israel, followed by an epidemiological investigation of equine exposure in the area. Serologic exposure to Leptospira was determined by microscopic agglutination test (MAT) using eight serovars. In 2017, serovar Pomona was identified in a mare with signs of ERU. Seven of thirteen horses from that farm were seropositive for serogroup Pomona, of which three had signs of ERU. During the same time period, 14/70 horses from three other farms were positive for serogroup Pomona. In 2015, two years prior to this diagnosis, 259 horses from 21 farms were sampled and one horse tested seropositive for serovar Icterohaemorrhagiae. In 2018, one year later, 337 horses were sampled on 29 farms, with none testing seropositive. Although horses are not considered a major host of Leptospira spp., it appears that horses may be infected, and clinically affected, in the course of an outbreak in other species. The identification of leptospirosis in stabled horses may impose a significant zoonotic risk to people.

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.

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.

West Nile virus neutralizing antibody prevalence in donkeys from northern Nigeria.

BACKGROUND: This study aimed to evaluate the prevalence of anti-West Nile virus (WNV) neutralizing antibodies in donkeys from two areas in northern Nigeria. METHODS: Serology was determined by a virus neutralization test in samples collected from 205 healthy adult donkeys. RESULTS: Fifty-seven donkeys (27.8%) tested seropositive for WNV. Donkeys from Zaria were 2.6 times more likely to have been exposed to WNV (p<0.001). CONCLUSION: The results of this study demonstrate that this zoonotic pathogen is prevalent in these areas and that measures should be implemented to reduce the risk for both humans and equids.

Detection of Neospora caninum Infection in Aborted Equine Fetuses in Israel.

In horses, Neospora caninum and Neospora hughesi have been associated with fetal loss, and neurological disease, respectively. This study investigated the role of Neospora spp. infection in equine abortion in Israel. The presence of anti-Neospora spp. antibodies was evaluated in 31 aborting mares by indirect fluorescent antibody test (IFAT) and the presence of parasite DNA in their aborted fetuses was evaluated by polymerase chain reaction (PCR), using two target loci (ITS1 and Nc5). The seroprevalence found in aborting mares was 70.9% and the prevalence by DNA detection in the aborted fetuses was 41.9%. Transplacental transmission from positive mares to their fetuses was 45.4% (10/22), while 33.3% (3/9) of fetuses of seronegative mares also tested positive for Neospora. The use of two PCR targets improved the sensitivity of parasite detection, and positive samples were identified by sequence analyses as N. caninum. These finding suggest that N. caninum could be a significant cause of abortion in horses, and that transplacental transmission in horses is an important way of transmission of N.caninum. The results presented here demonstrated the necessity to use several tests concurrently, including serological and molecular assays in order to confirm the involvement of Neospora in mare abortions.

Twenty Years of Equine Piroplasmosis Research: Global Distribution, Molecular Diagnosis, and Phylogeny.

Equine piroplasmosis (EP), caused by the hemoparasites Theileria equi, Theileria haneyi, and Babesia caballi, is an important tick-borne disease of equines that is prevalent in most parts of the world. Infection may affect animal welfare and has economic impacts related to limitations in horse transport between endemic and non-endemic regions, reduced performance of sport horses and treatment costs. Here, we analyzed the epidemiological, serological, and molecular diagnostic data published in the last 20 years, and all DNA sequences submitted to GenBank database, to describe the current global prevalence of these parasites. We demonstrate that EP is endemic in most parts of the world, and that it is spreading into more temperate climates. We emphasize the importance of using DNA sequencing and genotyping to monitor the spread of parasites, and point to the necessity of further studies to improve genotypic characterization of newly recognized parasite species and strains, and their linkage to virulence.

Exposure of Horses in Israel to West Nile Virus and Usutu Virus.

West Nile virus (WNV) and Usutu virus (USUV) are arboviruses transmitted by mosquito vectors. Whereas WNV is endemic in Israel, the Middle East, Europe, and in the Americas, data regarding the prevalence of USUV in the Middle East is limited. While both viruses share similar reservoirs and vectors, exposure of horses in the area to USUV have never been assessed. The aim of this study was to estimate the seroprevalence and co-exposure of WNV and USUV in horses in Israel. A total of 327 serum samples from healthy unvaccinated horses in Israel collected in 2018 were tested for neutralizing antibodies against WNV and USUV. Seroprevalence for neutralizing antibodies against WNV and USUV was 84.1% and 10.8%, respectively. Management and age were significantly associated with WNV and USUV seropositivity. This is the first report describing exposure of horses in Israel to USUV, which indicates that this zoonotic pathogen should be included in the differential diagnosis list of neuroinvasive disease in this country.