Seroprevalence and B1 gene Phylogeny of Toxoplasma gondii of Dogs and Cats in Republic of Korea.

The outbreak of human toxoplasmosis can be attributed to ingestion of food contaminated with Toxoplasma gondii. Toxoplasmosis recently increased in domestic and stray dogs and cats. It prompted studies on the zoonotic infectious diseases transmitted via these animals. Sero- and antigen prevalences of T. gondii in dogs and cats were surveyed using ELISA and PCR, and B1 gene phylogeny was analyzed in this study. Toxoplasmosis antibodies were measured on sera of 403 stray cats, 947 stray dogs, 909 domestic cats, and 2,412 domestic dogs collected at nationwide regions, Korea from 2017 to 2019. In addition, whole blood, feces, and tissue samples were also collected from stray cats (1,392), stray dogs (686), domestic cats (3,040), and domestic dogs (1,974), and T. gondii-specific B1 gene PCR was performed. Antibody prevalence of stray cats, stray dogs, domestic cats, and domestic dogs were 14.1%, 5.6%, 2.3%, and 0.04%, respectively. Antigen prevalence of these animals was 0.5%, 0.2%, 0.1%, and 0.4%, respectively. Stray cats revealed the highest infection rate of toxoplasmosis, followed by stray dogs, domestic cats, and domestic dogs. B1 gene positives were 5 of stray cats, and identified to high/moderate pathogenic Type I/III group. These findings enforce that preventive hygienic measure should be strengthened at One Health level in dogs and cats, domestic and stray, to minimize human toxoplasmosis infections.

First report of Bluetongue virus isolation in the Republic of Korea and analysis of the complete coding sequence of the segment 2 gene.

This study investigated the possible presence of the Bluetongue virus (BTV) in the Republic of Korea (ROK). Cell cultures were used to test blood samples collected from abattoirs throughout the country. Testing identified a single BTV isolate, which was characterized as BTV serotype 1 based on a nucleotide sequence analysis of the segment 2 gene. This report therefore indicates that BTV serotype 1 is present in the ROK. The potential importance of BTV in the ROK has been overlooked because cattle are mostly unaffected by the virus and because sheep, the most severely infected hosts, are uncommon in the ROK. However, as recent BTV serotype 8 outbreaks in Europe have demonstrated, certain BTV strains have the potential to cause severe disease in cattle. Additionally, with climate change continuously expanding the regions in which Culicoides vectors are able to survive, there is an increased need to study BTV in the Far East and ROK. To better prepare for future outbreaks of BTV, a sustained and effective level of surveillance for BTV in livestock will need to be established.

Detection and differentiation of Schmallenberg, Akabane and Aino viruses by one-step multiplex reverse-transcriptase quantitative PCR assay.

BACKGROUND: Schmallenberg virus (SBV), Akabane virus (AKAV) and Aino virus (AINV) are members of the Simbu serogroup within the genus Orthobunyavirus, family Bunyaviridae, which can cause reproductive disorders including abortion, stillbirth and congenital malformation in ruminants. Because, the clinical signs are similar, confirmatory diagnosis requires viral detection to differentiate infection between these three viruses. METHODS: In this study, a one-step multiplex reverse-transcriptase quantitative PCR (one-step mRT-qPCR) was developed for the simultaneous detection and differentiation of SBV, AKAV and AINV. RESULTS: The detection limit of the one-step mRT-qPCR for SBV, AKAV and AINV were 2.4 copies (10 (0.6) TCID 50/ml), 96.2 copies (10 (1.5) TCID 50/ml) and 52.3 copies (10 (1.2) TCID 50/ml), respectively. Various field samples such as bovine serum, bovine whole blood, bovine brain, goat serum and Culicoides were analyzed using the one-step mRT-qPCR and compared with previously published RT-qPCRs. The test results of the field samples were identical for the one-step mRT-qPCR and RT-qPCRs, which showed all samples to be negative for SBV, AKAV and AINV, except for one bovine brain sample (1/123) that was positive for AKAV. CONCLUSION: The one-step mRT-qPCR allows for the simultaneous detection of three viral pathogens (SBV, AKAV and AINV) that cause reproductive failure.

Species Diversity and Seasonal Distribution of Culicoides spp. (Diptera: Ceratopogonidae) in Jeju-do, Republic of Korea.

Biting midges belonging to the genus Culicoides (Diptera: Ceratopogonidae) were collected by Mosquito Magnet(®) and black light traps at 5 sites on Jeju-do, Republic of Korea (Korea), from May-November 2013 to determine species diversity and seasonal distribution. A total of 4,267 specimens were collected, of which 99.9% were female. The most common species was Culicoides tainanus (91.8%), followed by C. lungchiensis (7.2%) and C. punctatus (0.6%), while the remaining 4 species accounted for <0.5% of all Culicoides spp. that were collected. High numbers of C. tainanus were collected in May, followed by decreasing numbers through August, and then increasing numbers through November when surveillance was terminated. Peak numbers of C. lungchiensis were collected during September, with low numbers collected from May-August and October-November. The presence of C. lungchiensis in Korea was confirmed by morphological and molecular analyses.

Detection of Rickettsia monacensis from Ixodes nipponensis collected from rodents in Gyeonggi and Gangwon Provinces, Republic of Korea.

A total of 1,305 ticks were collected from wild rodents captured monthly, except July and August, during 2008 at three US-ROK operated military training sites and three US military installations in Gyeonggi and Gangwon Provinces, the Republic of Korea (ROK). Ixodes nipponensis was the most frequently collected tick (n = 1,299, 99.5 %), followed by Ixodes pomerantzevi (n = 6, 0.5 %). The ticks were pooled (1-15/sample) and tested by nested polymerase chain reaction (nPCR) for spotted fever group (SFG) rickettsiae with primer sets targeting the outer membrane protein B (ompB), citrate synthase (gltA), and 17-kDa antigen gene loci. A total of 115/197 (58.4 %) pools were positive by nPCR for the outer membrane protein ompB. Nucleotide sequence analysis of 105/115 (91.3 %) ompB targeted nPCR positive products showed a high degree of similarity to Rickettsia monacensis (99.3-100 %, n = 87) and R. japonica (99.5-100 %, n = 18). From the 87 positive samples demonstrating a high degree of similarity to R. monacensis, 15 were selected and analyzed by nPCR for gltA and the 17-kDa genes. A total of 12/15 pooled samples were positive for by nPCR for gltA, with amplicons demonstrating a high degree of similarity to R. monacensis (99.3-99.7 %). A total of 13/15 pooled samples were positive by nPCR for the 17-kDa gene, with amplicons demonstrating a high degree of similarity to R. monacensis (99.4-100 %). These findings demonstrate that R. monacensis is distributed throughout Gyeonggi and Gangwon Provinces in the ROK. Furthermore, data suggest a relative high prevalence of R. monacensis in the tick, I. nipponensis.

Livestock agroterrorism: the deliberate introduction of a highly infectious animal pathogen.

Agroterrorism refers to attacks with any of a variety of biological or chemical agents against commercial crops or livestock populations, either as targets in their own right or as vehicles to attack humans. An agroterrorism incident would generally involve bioterrorism, and potential agents include pathogens such as viruses, bacteria, or fungi. Within the context of agroterrorism, livestock agroterrorism is described as the intentional introduction of an animal-borne infectious disease with the goal of spreading fear, producing economic losses, and/or threatening social stability. Causing human illness or human casualties is another potential goal of livestock agroterrorism. Livestock agroterrorism is considered to be attractive to terrorists because biological agents that affect livestock or poultry are more readily available and more difficult to monitor than are agents that infect humans. In addition, a terrorist attack on animal husbandry may have huge economic consequences with no human casualties. Therefore, a biological attack that targets the animal husbandry sector should be regarded as both a "high-consequence" event and a grave national security risk. This review addresses the use of biological weapons that may be used to target livestock or poultry rather than agricultural inputs or equipment. It first defines livestock agroterrorism. Then, the common priority disease agents that may be used to target livestock or poultry in an agroterrorist attack and that are attractive to terrorists are outlined.

Toxoplasma gondii and Rickettsia spp. in ticks collected from migratory birds in the Republic of Korea.

Migratory birds disperse ticks and associated tick-borne pathogens along their migratory routes. Four selected pathogens of medical importance (Coxiella burnetii, Rickettsia spp., Francisella tularensis, and Toxoplasma gondii) were targeted for detection in 804 ticks (365 pools) collected from migratory birds at Hong and Heuksan Islands in the Republic of Korea (ROK) from 2010 to 2011 and 2016. Toxoplasma gondii and Rickettsia spp., were detected in 1/365 (0.27%) and 34/365 (9.32%) pools of ticks, respectively. T. gondii and five rickettsial species were recorded in ticks collected from migratory birds for the first time in ROK. The five rickettsial species (R. monacensis, Candidatus Rickettsia longicornii, R. japonica, R. raoultii, and R. tamurae) were identified using sequence and phylogenetic analysis using ompA and gltA gene fragments. Rickettsia spp. are important pathogens that cause rickettsiosis in humans, with cases recorded in the ROK. These results provide important evidence for the potential role of migratory birds in the introduction and dispersal of T. gondii and Rickettsia spp. along their migratory routes and raise awareness of potential transmission of zoonotic tick-borne pathogens associated with migratory birds in the ROK.

Simultaneous detection of Rift Valley Fever, bluetongue, rinderpest, and Peste des petits ruminants viruses by a single-tube multiplex reverse transcriptase-PCR assay using a dual-priming oligonucleotide system.

The aim of this study was to develop a highly sensitive and specific one-step multiplex reverse transcriptase PCR assay for the simultaneous and differential detection of Rift Valley Fever virus (RVFV), bluetongue virus (BTV), rinderpest virus (RPV), and Peste des petits ruminants virus (PPRV). These viruses cause mucosal lesions in cattle, sheep, and goats, and they are difficult to differentiate from one another based solely on their clinical presentation in suspected disease cases. In this study, we developed a multiplex reverse transcriptase PCR to detect these viruses using a novel dual-priming oligonucleotide (DPO). The DPO contains two separate priming regions joined by a polydeoxyinosine linker, which blocks extension of nonspecifically primed templates and consistently allows high PCR specificity even under less-than-optimal PCR conditions. A total of 19 DPO primers were designed to detect and discriminate between RVFV, BTV, RPV, and PPRV by the generation of 205-, 440-, 115-, and 243-bp cDNA products, respectively. The multiplex reverse transcriptase PCR described here enables the early diagnosis of these four viruses and may also be useful as part of a testing regime for cattle, sheep, or goats exhibiting similar clinical signs, including mucosal lesions.

Molecular Detection and Phylogeny of Tick-Borne Pathogens in Ticks Collected from Dogs in the Republic of Korea.

Ticks are important vectors of various pathogens that result in clinical illnesses in humans and domestic and wild animals. Information regarding tick infestations and pathogens transmitted by ticks is important for the identification and prevention of disease. This study was a large-scale investigation of ticks collected from dogs and their associated environments in the Republic of Korea (ROK). It included detecting six prevalent tick-borne pathogens (Anaplasma spp., A. platys, Borrelia spp., Babesia gibsoni, Ehrlichia canis, and E. chaffeensis). A total of 2293 ticks (1110 pools) were collected. Haemaphysalis longicornis (98.60%) was the most frequently collected tick species, followed by Ixodes nipponensis (0.96%) and H. flava (0.44%). Anaplasma spp. (24/1110 tick pools; 2.16%) and Borrelia spp. (4/1110 tick pools; 0.36%) were detected. The phylogenetic analyses using 16S rRNA genes revealed that the Anaplasma spp. detected in this study were closely associated with A. phagocytophilum reported in humans and rodents in the ROK. Borrelia spp. showed phylogenetic relationships with B. theileri and B. miyamotoi in ticks and humans in Mali and Russia. These results demonstrate the importance of tick-borne disease surveillance and control in dogs in the ROK.

Molecular Detection and Phylogenetic Analysis of Tick-Borne Pathogens in Ticks Collected from Horses in the Republic of Korea.

The horse industry has grown rapidly as a leisure industry in the Republic of Korea (ROK) in parallel with an increased demand for equestrian activities. As a result, there has been an increase in horse breeding and equestrian population and potential exposure to ticks and their associated pathogens. To provide a better understanding of the potential disease risks of veterinary and medical importance, a study was conducted to determine the geographical distribution and diversity of ticks collected from horses and vegetation associated with horse racetracks/ranches throughout the ROK. This included a survey of five associated common pathogens, Anaplasma phagocytophilum, Ehrlichia chaffeensis, Borrelia spp., Babesia caballi, and Theileria equi. A total 9220 ticks were collected from horses and associated pastures. Ticks were identified to species, stage of development, and sex. Two species of ticks, Haemaphysalis longicornis (99.9%) and Ixodes nipponensis (0.1%) were identified. Two of the target pathogens, A. phagocytophilum and Borrelia spp., were detected in 5/1409 tick pools (0.35%) and 4/1409 pools (0.28%) of H. longicornis, respectively, both of which are zoonotic pathogens of medical importance. The results of 16S rRNA phylogenetic analysis of A. phagocytophilum showed a close relationship to strains distributed in China, USA, Germany, Italy, Turkey, and Poland. Borrelia spp. showed a close relationship, based on 16S rRNA gene, to the strains reported from the USA (B. burgdorferi and B. americana) and Japan (B. tanukii and B. garinii). These results provide information about the potential risks of veterinary and medical importance and the development of mitigation strategies for disease prevention.

Molecular Detection and Phylogenetic Analysis of Anaplasma and Borrelia Species in Ticks Collected from Migratory Birds at Heuksan, Hong, and Nan Islands, Republic of Korea.

The extended distribution and potential introduction of exotic ticks and associated tick-borne pathogens along the northern and southern routes of migratory birds pose zoonotic tick-borne disease risks to wild and domestic animals and incidentally to humans. A knowledge of bird migratory patterns, species of attached ticks, and associated pathogens during their migrations to and from their feeding and nesting grounds is central to understanding associated tick-borne disease risks. Tick-borne disease surveillance was conducted from 2010 to 2011 and 2016 at Hong-do (do = island), Heuksan-do, and Nan-do, major stopovers for migratory birds in Republic of Korea (ROK), as part of the Migratory Birds Research Center bird-banding program for studying bird migration patterns in the ROK. A total of 877 ticks belonging to three genera and nine species were collected, Ixodes turdus (576, 65.7%), Haemaphysalis flava (134, 15.3%), H. longicornis (91, 10.4%), I. nipponensis (56, 6.4%), H. formosensis (7, 0.8%), H. ornithophila (6, 0.7%), H. phasiana (5, 0.6%), H. concinna (1, 0.1%), and Amblyomma testudinarium (1, 0.1%) were collected from 274 birds belonging to 20 genera and 41 species. A total of 15/380 pools (3.95%) were positive for Borrelia species (14 pools of I. turdus and 1 pool of H. flava), while only 1/380 pools (0.26%) was positive for Anaplasma phagocytophilum (1 pool of I. nipponensis). Our findings support the role of migratory birds as possible vectors for the introduction of tick-borne pathogens, which requires continuous monitoring for the potential introduction of ticks and their associated tick-borne pathogens.

Fast duplex one-step reverse transcriptase PCR for rapid differential detection of West Nile and Japanese encephalitis viruses.

The aim of this study was to develop a highly sensitive and specific one-step duplex reverse transcriptase PCR (RT-PCR) assay for the simultaneous and differential detection of West Nile (WNV) and Japanese encephalitis (JEV) viruses. The bioinformatic analysis of published sequences of WNV and JEV revealed conserved regions not targeted by previously reported primers. A total of 13 primers were designed based on these regions to detect all of the WNV and JEV lineages and to discriminate between the two viruses by the generation of 482- and 241-bp cDNA products, respectively. The results indicate that single-tube duplex PCR using these primers is a useful technique for the detection and differentiation of WNV and JEV in plasma or brain tissue. The novel duplex RT-PCR described in this study enables the early diagnosis of these two encephalitic flaviviruses. In addition, this technique may be useful as part of a testing regimen for human patients, horses, and other susceptible animal species, as it is rapid (less than 3.5 h from RNA extraction), sensitive, and specific, and it may enable the differential diagnosis of clinical samples.

Prevalence of antibodies against Anaplasma spp., Borrelia burgdorferi sensu lato, Babesia gibsoni, and Ehrlichia spp. in dogs in the Republic of Korea.

There is a lack of comprehensive studies on the seroprevalence of tick-borne pathogens in the Republic of Korea. Therefore, the aim of this study was to investigate the seroprevalences of Anaplasma spp. (A. phagocytophilum/A. platys), Borrelia burgdorferi sensu lato, Babesia gibsoni, Ehrlichia spp. (E. canis/E. ewingii), and Ehrlichia chaffeensis in dogs across the Republic of Korea in 2017 and 2018. A total of 2215 serum samples collected from 938 companion dogs, 969 shelter dogs, and 308 military working dogs were examined using commercial enzyme-linked immunosorbent assay (ELISA) and indirect fluorescence immunoassay (IFA) kits. Data collected for each animal, including breed, sex, age, region, season, and dog type, were used for statistical analysis. The overall seroprevalence was highest for Anaplasma spp. (15.1 %), followed by Ehrlichia spp. (10.3 %), B. burgdorferi sensu lato (6.4 %), E. chaffeensis (2.3 %), and B. gibsoni (1.7 %). One hundred and sixty-one dogs had antibodies against two or three different pathogens. The most common combinations were Anaplasma spp. - Ehrlichia spp. (2.1 %), Anaplasma spp. - E. chaffeensis (1.4 %), and Anaplasma spp. - B. burgdorferi sensu lato (1.2 %). Season was significantly associated with the seroprevalences of B. burgdorferi sensu lato and Ehrlichia spp., with dogs presenting the highest percentage of positive results during summer. Anaplasma spp. and B. gibsoni were significantly more prevalent in the northern and southern regions, respectively. The seroprevalences of Anaplasma spp., B. burgdorferi sensu lato, and Ehrlichia spp. were significantly higher in military working dogs, while the seroprevalence of E. chaffeensis was higher in companion dogs. The current findings are important for future surveillance of canine tick-borne pathogens and designing appropriate approaches for the diagnosis and control of these pathogens in the Republic of Korea.

Molecular Detection and Phylogenetic Analysis of Anaplasma spp. in Korean Native Goats from Ulsan Metropolitan City, Korea.

Background: Tick-borne diseases (TBDs) can be fatal to humans as well as to animals causing severe economic losses globally to livestock industries. Many countries conduct regular surveillance of TBDs in livestock. Serological and molecular surveillance of TBDs in livestock and humans was carried out in the Republic of Korea. However, there are not a lot of data on analyses of anaplasmosis in Korean native goats and the correlation with rearing methods and seasons. Methods: In this study, goats in Ulsan city were tested for anaplasmosis by PCR and 16S rRNA sequencing. A total of 452 goat blood samples were collected from 20 farms in 2016. The goat farms in Ulsan city had three different types of rearing methods: conventional, confined, and mixed grazing-confined. Results: Forty-nine of the 452 goats (10.8%) were anaplasmosis positive. Sequence analysis of the PCR products from these 49 goats revealed that 39 of 452 goats (8.6%) were Anaplasma bovis positive, and 10 of 452 goats (2.2%) were infected with Anaplasma capra. The highest outbreaks of anaplasmosis occurred in mixed grazing-confined type of farms (27.1%, 33/122) (χ2 = 60.72, df = 2, p < 0.05), but there was no significant difference in the occurrence of anaplasmosis between spring, summer, and fall seasons. Conclusions: This study was the first detection of A. bovis in Korean native goats and its relationship with rearing methods and seasons. These findings suggested that Korean native goats were highly exposed to Anaplasma spp. during summers when the tick population is the highest and in farms employing mixed grazing-confined rearing methods.

Japanese encephalitis virus in culicine mosquitoes (Diptera: culicidae) of the republic of Korea, 2008-2010.

A total of 150,805 culicine female mosquitoes were captured by Mosquito Magnet, black light, and New Jersey light traps, and at resting collections in the Republic of Korea from 2008 to 2010 as part of the U.S. Forces Korea malaria and Japanese surveillance programs. Mosquitoes were identified and culicine mosquitoes placed in pools of up to 30 mosquitoes each, by species and date of collection, and screened for flaviviruses using a reverse transcription-polymerase chain reaction assay. A total of 98/6,845 (1.4%) pools were positive by reverse transcription-polymerase chain reaction for Japanese encephalitis virus (JEV). A total of 92/2,031 (4.5%) pools of Culex tritaeniorhynchus were positive for JEV and accounted for 93.9% (92/98) of all JEV positive pools. A total of 4/804 (0.5%) and 2/175 (1.1%) pools of C. pipiens and C. bitaeniorhynchus, respectively, were positive for JEV. The JEV maximum likelihood estimations (estimated number of viral RNA positive mosquitoes per 1,000) for C. tritaeniorhynchus, C. bitaeniorhynchus, and C. pipiens were 1.71, 1.02, and 0.36 respectively. JEV is a severe health threat for local populations and of significant concern for nonimmune (unvaccinated) U.S. soldiers, civilians, and family members deployed to the Republic of Korea.

A novel metalloprotease from Gloydius halys venom induces endothelial cell apoptosis through its protease and disintegrin-like domains.

A novel hemorrhagic metalloprotease, halysase, isolated from the snake venom of Gloydius halys induces apoptosis in endothelial cells. The purified metalloprotease is a monomeric glycoprotein with an isoelectric point of 4.8. Analysis of the cDNA sequence encoding halysase revealed that the enzyme consists of multifunctional domains including a proprotein domain, a protease domain, a disintegrin-like domain and a cysteine-rich domain. The metalloprotease has a DECD sequence in the disintegrin-like domain instead of the typical RGD sequence. Halysase strongly inhibits proliferation of human umbilical vein endothelial cells in a dose-dependent manner as well as adhesion of the cells to extracellular matrix proteins. The enzyme specifically hydrolyzes not only extracellular matrix proteins such as fibronectin, vitronectin, and type IV collagen, but also integrins alpha1beta1 and alpha5beta1. The apoptosis of endothelial cells induced by halysase is closely associated with activation of caspase-3 and decreased level of Bcl-X(L)/Bax. Apohalysase, which lacks metalloprotease activity, is also able to induce the apoptosis. Several lines of experimental evidence suggest that the protease domain and the disintegrin-like domain of halysase cooperatively contribute to the induction of endothelial cell apoptosis.

Molecular detection and genotyping of Japanese encephalitis virus in mosquitoes during a 2010 outbreak in the Republic of Korea.

Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen, is one of the major causes of viral encephalitis. To reduce the impact of Japanese encephalitis among children in the Republic of Korea (ROK), the government established a mandatory vaccination program in 1967. Through the efforts of this program only 0-7 (mean 2.1) cases of Japanese encephalitis were reported annually in the ROK during the period of 1984-2009. However, in 2010 there was an outbreak of 26 confirmed cases of Japanese encephalitis, including 7 deaths. This represented a >12-fold increase in the number of confirmed cases of Japanese encephalitis in the ROK as compared to the mean number reported over the last 26 years and a 3.7-fold increase over the highest annual number of cases during this same period (7 cases). Surveillance of adult mosquitoes was conducted during the 2010 outbreak of Japanese encephalitis in the ROK. A total of 6,328 culicine mosquitoes belonging to 12 species from 5 genera were collected at 6 survey sites from June through October 2010 and assayed by reverse-transcription polymerase chain reaction (RT-PCR) for the presence of JEV. A total of 34/371 pooled samples tested positive for JEV (29/121 Culex tritaeniorhynchus, 4/64 Cx. pipiens, and 1/26 Cx. bitaeniorhynchus) as confirmed by sequencing of the pre-membrane and envelope protein coding genes. The maximum likelihood estimates of JEV positive individuals per 1,000 culicine vectors for Cx. tritaeniorhynchus, Cx. pipiens, and Cx. bitaeniorhynchus were 11.8, 5.6, and 2.8, respectively. Sequences of the JEV pre-membrane and envelope protein coding genes amplified from the culicine mosquitoes by RT-PCR were compared with those of JEV genotypes I-V. Phylogenetic analyses support the detection of a single genotype (I) among samples collected from the ROK in 2010.

A diagnostic algorithm to serologically differentiate West Nile virus from Japanese encephalitis virus infections and its validation in field surveillance of poultry and horses.

The detection of West Nile virus (WNV) in areas endemic for Japanese encephalitis virus (JEV) is complicated by the extensive serological cross-reactivity between the two viruses. A testing algorithm was developed and employed for the detection of anti-WNV antibody in areas endemic for JEV. Using this differentiation algorithm, a serological survey of poultry (2004 through 2009) and horses (2007 through 2009) was performed. Among 2681 poultry sera, 125 samples were interpreted as being positive for antibodies against JEV, and 14 were suspected to be positive for antibodies against undetermined flaviviruses other than WNV and JEV. Of the 2601 horse sera tested, a total of 1914 (73.6%) were positive to the initial screening test. Of these positive sera, 132 sera (5.1%) had been collected from horses that had been imported from the United States, where WNV is endemic. These horses had WNV vaccination records, and no significant pattern of increasing titer was observed in paired sera tests. Of the remaining 1782 positive sera 1468 sera (56.4%) were also found to contain anti-JEV antibodies, and were interpreted to be JEV-specific antibodies by the differentiation algorithm developed in this study. The remaining 314 horses (12.1%) for which a fourfold difference in neutralizing antibody titer could not be demonstrated, were determined to contain an antibody against an unknown (unidentified or undetermined) flavivirus. No evidence of WNV infections were found during the period of this study.