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.

Detection and Analysis of West Nile Virus Structural Protein Genes in Animal or Bird Samples.

West Nile virus (WNV) is an important zoonotic pathogen, which is detected mainly by identification of its RNA using PCR. Genetic differentiation between WNV lineages is usually performed by complete genome sequencing, which is not available in many research and diagnostic laboratories. In this chapter, we describe a protocol for detection and analysis of WNV samples by sequencing the entire region of their structural genes capsid (C), preM/membrane, and envelope. The primary step is the detection of WNV RNA by quantitative PCR of the NS2A gene or the C gene regions. Next, the entire region containing the structural protein genes is amplified by PCR. The primary PCR product is then amplified again in parallel reactions, and these secondary PCR products are sequenced. Finally, bioinformatic analysis enables detection of mutations and classification of the samples of interest. This protocol is designed to be used by any laboratory equipped for endpoint and quantitative PCR. The sequencing can be performed either in-house or outsourced to a third-party service provider. This protocol may therefore be useful for rapid and affordable classification of WNV samples, obviating the need for complete genome sequencing.

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.

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.

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.

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.

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.

Equid Herpesvirus 8 Isolated From an Adult Donkey in Israel.

We report the isolation of Equid herpesvirus 8 from a rescued donkey that suffered severe postcastration complications. Despite intensive treatment, the donkey deteriorated and was euthanized. Postmortem virologic analysis revealed the isolation of a herpesvirus that is closely related to herpesviruses reported from donkeys and horses in Australia, China, and Ireland, causing respiratory disease in donkeys and abortion in mares. To our knowledge, this is the first report of this equid herpesvirus in Israel. The potential significance of this herpesvirus to the equid population in Israel needs further investigation.

High Exposure to Toxoplasma gondii and Neospora Spp. in Donkeys in Israel: Serological Survey and Case Reports.

Toxoplasma gondii and Neospora spp. are closely related cyst-forming coccidian parasites, which infect various animal species and have considerable zoonotic and economic implications, respectively. Both parasites are endemic in Israel and have been reported to infect wild and domestic animals. This study was conceived to evaluate the serologic exposure of donkeys to these parasites. Serum samples were collected from 98 donkeys. Half of them (n = 49) were from animal shelters in Israel, and the rest (n = 49) were working donkeys from the Palestinian Authority. The donkeys were screened for the presence of anti-Toxoplasma and anti-Neospora antibodies by immunofluorescence antibody tests (IFATs). The seroprevalence of T. gondii and Neospora spp. was 94% and 70%, respectively, and 69% of the donkeys were exposed to both parasites. In addition, N. caninum tissue cysts were documented in two donkeys during post-mortem examination. This is the first report of the exposure of donkeys to T. gondii and Neospora spp. in the area. The high prevalence found in this study suggests that donkeys may have a role in the maintenance of these parasites in the area, thus serving as a source of infection for the definitive hosts.

Molecular characterization of the re-emerging West Nile virus in avian species and equids in Israel, 2018, and pathological description of the disease.

BACKGROUND: In this report we describe the molecular and pathological characteristics of West Nile virus (WNV) infection that occurred during the summer and fall of 2018 in avian species and equines. WNV is reported in Israel since the 1950s, with occasional outbreaks leading to significant morbidity and mortality in birds, high infection in horses and humans, and sporadic fatalities in humans. METHODS: Animal and avian carcasses in a suitable condition were examined by post-mortem analysis. Tissue samples were examined for WNV by RT-qPCR and the viral load was quantified. Samples with sufficient material quality were further analyzed by Endpoint PCR and sequencing, which was used for phylogenetic analysis. Tissue samples from positive animals were used for culturing the virus in Vero and C6/36 cells. RESULTS: WNV RNA was detected in one yellow-legged gull (Larus michahellis), two long-eared owls (Asio otus), two domesticated geese (Anser anser), one pheasant (Phasianus colchicus), four hooded crows (Corvus cornix), three horses and one donkey. Pathological and histopathological findings were characteristic of viral infection. Molecular analysis and viral load quantification showed varying degrees of infection, ranging between 70-1.4 × 106 target copies per sample. Phylogenetic analysis of a 906-bp genomic segment showed that all samples belonged to Lineage 1 clade 1a, with the following partition: five samples from 2018 and one sample detected in 2016 were of Cluster 2 Eastern European, two of Cluster 2 Mediterranean and four of Cluster 4. Four of the positive samples was successfully propagated in C6/36 and Vero cell lines for further work. CONCLUSIONS: WNV is constantly circulating in wild and domesticated birds and animals in Israel, necessitating constant surveillance in birds and equines. At least three WNV strains were circulating in the suspected birds and animals examined. Quantitative analysis showed that the viral load varies significantly between different organs and tissues of the infected animals.

Epidemiologic and phylogenetic analysis of the 2018 West Nile virus (WNV) outbreak in Israel demonstrates human infection of WNV lineage I.

As at 12 November 2018, an outbreak of West Nile virus (WNV) was responsible for 139 WNV infection cases in Israel. Here, we characterise the epidemiology of the outbreak and demonstrate that only WNV lineage I was circulating in mosquitoes and responsible for WNV infection in humans. This suggests that the concurrence of the outbreak in Israel with WNV outbreaks in several European countries is not due to a common, more virulent WNV genotype.

West Nile Virus: Seroprevalence in Animals in Palestine and Israel.

West Nile virus (WNV) epidemiological situation in Israel and Palestine, due to their unique location, draws attention following to the global spread of West Nile fever (WNF). Although much information is available from Israel on clinical cases and prevalence of WNV, clinical cases are rarely reported in Palestine, and prevalence is not known. The objectives of this study were to determine WNV seroprevalence in various domestic animals in Palestine and to reevaluate current seroprevalence, force of infection, and risk factors for WNV exposure in horses in Israel. Sera samples were collected from 717 animals from Palestine and Israel (460 horses, 124 donkeys, 3 mules, 50 goats, 45 sheep, and 35 camels). Two hundred and ten horses were sampled twice. The level of WNV antibodies was determined using commercial Enzyme-linked Immunosorbent Assay (ELISA) Kit. Seroprevalence in equids was 73%. Seroprevalence in Israel (84.6%) was significantly higher than in Palestine (48.6%). Seroprevalence in horses (82.6%) was significantly higher than in donkeys and mules (39.3%). Multivariable statistical analysis showed that geographical area, landscape features (altitude), environmental factors (land surface temperature during the day [LSTD]), species, and age significantly influenced WNV seroprevalence. Fourteen of 95 (14.7%) sheep and goats and 14/35 camels (40%) sampled in Palestine were seropositive for WNV. Of the horses that were sampled twice, 82.8% were seropositive for WNV at the first sampling, and all remained seropositive. Three of the seronegative horses, all from Palestine, converted to positive when resampled (8.5%). The results indicate that domestic animals in Palestine were infected with WNV in the past, and the seroconversion indicates that WNV was circulating in Palestine in the summer of 2014. Control measures to prevent human infection should be implemented in Palestine. Anti WNV antibodies in domestic animals suggest that those species can be used as sentinels for WNV activity in areas where most horses are either seropositive or vaccinated.

Comparison between available serologic tests for detecting antibodies against Anaplasma phagocytophilum and Borrelia burgdorferi in horses in Canada.

To investigate the agreement between available serologic tests for the detection of antibodies against Anaplasma phagocytophilum and Borrelia burgdorferi, 50 serum samples from horses of unknown clinical status and at low risk for infection were tested. In addition to a point-of-care enzyme-linked immunosorbent assay (pocELISA), the evaluated tests included 2 indirect fluorescent antibody tests (IFATs) for antibodies against A. phagocytophilum and an IFAT, an ELISA confirmed with Western blot, and the Lyme multiplex assay for antibodies against B. burgdorferi. For each pair-wise comparison between serologic tests, the difference in the proportion of seropositive results as well as kappa and the prevalence-adjusted, bias-adjusted kappa were calculated. The proportion of seropositive results differed significantly in each pairwise comparison of tests for detection of antibodies against A. phagocytophilum, and between the pocELISA and IFAT as well as between the pocELISA and Lyme multiplex assay for detection of antibodies against B. burgdorferi. Agreement based on kappa varied from poor to fair while agreement was improved when evaluating prevalence-adjusted, bias-adjusted kappa. Lack of agreement may be explained by differences in methodology between the evaluated tests, cross-reactivity or false-positive and false-negative tests. In addition to the limitations of serologic test interpretation in the absence of clinical disease, this data suggest that screening of horses for exposure to tick-borne diseases in nonendemic areas may not be warranted.

Seroprevalence of equine granulocytic anaplasmosis and lyme borreliosis in Canada as determined by a point-of-care enzyme-linked immunosorbent assay (ELISA).

Equine granulocytic anaplasmosis (EGA) and Lyme borreliosis (LB) are an emerging concern in Canada. We estimated the seroprevalence of EGA and equine LB by testing 376 convenience serum samples from 3 provinces using a point-of-care SNAP(®) 4Dx(®) ELISA (IDEXX Laboratories, Westbrook, Maine, USA), and investigated the agreement between the point-of-care ELISA and laboratory-based serologic tests. The estimated seroprevalence for EGA was 0.53% overall (0.49% in Saskatchewan, 0.71% in Manitoba), while the estimated seroprevalence for LB was 1.6% overall (0.49% in Saskatchewan, 2.86% in Manitoba). There was limited agreement between the point-of-care ELISA and an indirect fluorescent antibody test for EGA (kappa 0.1, PABAK 0.47) and an ELISA/Western blot combination for LB (kappa 0.23, PABAK 0.71). While the SNAP(®) 4Dx(®) ELISA yielded expected seroprevalence estimates, further evaluation of serologic tests for the purposes of disease exposure recognition may be needed.

Séroprévalence de l'anaplasmose granulocytaire équine et de la borréliose de Lyme au Canada telle que déterminée par un test ELISA hors laboratoire. L'anaplasmose granulocytaire équine (AGE) et la borréliose de Lyme (BL) sont de nouvelles maladies émergentes au Canada. Nous avons estimé la séroprévalence de l'AGE et de la BL équine en testant 376 échantillons sériques de commodité provenant de trois provinces en utilisant un test ELISA SNAPMD 4DxMD hors laboratoire (IDEXX Laboratories, Westbrook, Maine, États-Unis) et nous avons analysé la concordance entre les tests ELISA hors laboratoire et des tests sérologiques faits en laboratoire. Le total des séroprévalences estimées pour l'AGE était de 0,53 % (0,49 % en Saskatchewan, 0,71 % au Manitoba), tandis que le total de la séroprévalence estimée de BL était de 1,6 % (0,49 % en Saskatchewan, 2,86 % au Manitoba). Il y avait une concordance limitée entre le test ELISA hors laboratoire et un test d'immunofluorescence indirecte pour l'AGE (kappa 0,1, PABAK 0,47) et une combinaison de tests ELISA/immunobuvardage pour BL (kappa 0,23, PABAK 0,71). Même si le test ELISA SNAPMD 4DxMD hors laboratoire a donné des estimations de séroprévalence attendues, une nouvelle évaluation des tests sérologiques à des fins de reconnaissance de l'exposition à une maladie peut être requise.(Traduit par Isabelle Vallières).

Passive surveillance for ticks on horses in Saskatchewan.

Passive surveillance of ticks on horses in Saskatchewan revealed that the horses were parasitized by 3 species, Dermacentor albipictus, D. andersoni, and D. variabilis. The nymphs and adults of D. albipictus occurred on horses earlier in the year than did adults of the 2 other species.

Surveillance passive des tiques sur des chevaux en Saskatchewan. Une surveillance passive des tiques chez des chevaux de la Saskatchewan a révélé que les chevaux étaient affectés par des parasites de trois espèces: Dermacentor albipictus, D. andersoni et D. variabilis. Les nymphes et les adultes de D. albipictus se présentaient chez les chevaux plus tôt dans l'année que les adultes des deux autres espèces.(Traduit par Isabelle Vallières).