While it is commonly hypothesized that sexual differentiation in the mammalian brain is initiated mainly by gonadal sex steroids, recent evidence has suggested that dopaminergic (DA) neurons within the rodent midbrain have sex differences independent of gonadal secretions. More recently, it has been reported that Sry (the sex-determining region of the Y chromosome) is directly involved in this difference. The possibility of sexual dimorphism in the mouse midbrain needs to be elucidated. In the present study, the midbrain of C3H mice, which is little understood, was investigated histologically and immuno-histoplanimetrically to reveal sexual and developmental differences. The female ventral tegmental area appeared to contain higher immunoreactivity to tyrosine hydroxylase (TH) than that of males at 11 weeks of age, whereas general histological differences between the sexes were not clearly found. The TH-immunoreactive (TH-ir) neurons within the A8, A9, and A10 mesencephalic areas were examined separately. There was the sex difference in the time period when TH-ir cell numbers significantly increased, indicating that the growth rate of midbrain DA nuclei also differs and that the midbrain DA system may trace different processes of sexual maturation between the sexes. These differences between female and male may reflect the direct regulation by Sry or the multiple effects of both Sry and sex steroids. Further experiments are needed to determine which factor forms this difference in the growth pattern in the numbers of TH-ir neurons.
Nitric oxide (NO) has been proposed as a mediator of postnatal mammary gland development. We investigated the immunohistochemical localization of three isoforms of nitric oxide synthase (NOS) enzymes, neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS), in the mouse mammary gland during reproductive cycle. The NOS isoforms detected in normal mouse mammary glands were the constitutive forms of NOS (nNOS and eNOS). nNOS was localized to the alveoli, myoepithelia, lactiferous ducts and blood vessel endothelia, while eNOS was localized in the alveoli, lactiferous ducts and blood vessel endothelia. The strongest immunoreactivity for both constitutive NOS isoforms was observed in pregnant mice. The differential staining intensity of NOS enzymes in the mammary gland led us to conclude that nitric oxide in the mouse mammary gland is mainly synthesized by constitutive NOS isoforms, and suggest that NO has functional roles in post-pubertal growth and differentiation of the mammary gland.
The expression level of phospholipase D (PLD) was examined in the lungs of mice during postnatal development. Immunoprecipitation analysis revealed a moderate basal level of PLD1 in the lungs at postnatal 1 week, with a gradual increase occurring until week 4. The pattern of PLD2 paralleled that of PLD1, although the quantity of PLD2 was markedly less. PLD was immunostained in the most bronchiolar epithelium, some pulmonary macrophages and alveolar cells at postnatal week 1, with increased immunoreactivity in those cells with lung development at postnatal week 4. These findings suggest that the increase of PLD expression is associated with pulmonary alveolarization, possibly by involvement in cell proliferation and differentiation during postnatal mouse lung development.
The genetic diversity of the partial S1 gene involving the hyper variable region for infectious bronchitis (IB) vaccine strains in Japan were compared with those of IB virus isolated from the field in Japan. Field isolates have mainly been classified into three major genotypes, JP-I, JP-II and JP-III, since 2003; however, the 4/91 genotype was detected from recent field isolates in Japan. The virus neutralization (VN) activity with vaccine immunized serum was investigated to evaluate the protective effects of vaccines against Japanese field isolates. In the results of the VN test, antiserum immunized with the GN and C78 (JP-I), TM-86w and Miyazaki (JP-II) and 4/91 (793B) vaccine strains could neutralize a high rate of field isolates of homologous genotype (75% of field isolates of JP-I, 100% of that of JP-II and 100% of that of 793B, respectively). For field isolates of JP-III, even though there are no homologous genotype vaccine strain, some strains of JP-III were neutralized with immune serum from vaccine strains of the heterologous genotype. In this study, a correlation between serological property and genotype was found for JP-I, JP-II and 793B. Our results suggested that an effective vaccine could be predicted in accordance with the genotype of field isolates.
Onychomycosis is a fungal infection of fingernails or toenails caused by several species of fungi and yeasts. A Japanese monkey, Macaca fuscata, displayed severe onychomycosis in his 4 limbs. Diagnosis and etiological agent identification were performed by conventional and DNA-mediated methods. The accumulated findings of this case revealed Trichosporon montevideense, which has long been considered to be a nonpathogenic yeast. Here, we present the first report of an involvement of T. montevideense in an onychomycosis case.
A novel sandwich enzyme-linked immunosorbent assay (ELISA) was established to determine the serum insulin concentrations in domestic cats. By using a solid-phase mouse anti-bovine insulin monoclonal antibody and a peroxidase-conjugated guinea pig anti-rat insulin polyclonal antibody, feline serum insulin concentrations in the range of 0.1 to 3.6 ng/ml could be measured. The intraassay CV and interassay CV were less than 6% and less than 10%, respectively. The present insulin assay will strongly help studies on feline diabetes mellitus.
Dendritic cells (DCs) are professional antigen presenting cells (APCs) that possess an extraordinary capacity to stimulate naïve T cells and initiate a primary immune response. To develop a DC-based immunotherapy for feline immunodeficiency virus (FIV) infection, we carried out a study to characterize DCs from FIV-infected cats and compared the observations with those obtained from healthy controls. DCs were derived from adherent peripheral blood mononuclear cells that had been cultivated with recombinant feline interleukin 4, granulocyte macrophage colony-stimulating factor and heat-inactivated autologous plasma. Various parameters, such as cell morphology, surface phenotype, endocytosis and mixed leukocyte reaction (MLR), were analyzed to characterize feline DCs. Monocyte-derived DCs from FIV-infected cats as well as those from healthy controls showed a dendritic appearance and expressed an APC-like phenotype (CD1c+, CD80+ and MHC class II+). However, the expression level of CD1a was variable in the DCs derived from FIV-infected cats, although this was not the case in the DCs derived from the healthy controls. DCs from the FIV-infected cats retained the ability to take up dextran via the mannose receptor and also showed an apparent MLR, indicating that these cells could be useful in immunotherapy. In this study, monocytes obtained from FIV-infected cats could differentiate into functional DCs, suggesting that they might be used in a DC-based immunotherapy against FIV infection.
The aim of this experiment was to evaluate the immunomodulating activities of inactivated Propionibacterium granulosum cell walls and E. coli lipopolysaccharide (PG/LPS) on porcine immunity. Piglets were intramuscularly administered PG/LPS (1 ml/10 kg body weight) once or twice. The function of natural killer cells, lymphocytes and neutrophils and the adjuvant effect on antibody induction by attenuated classical swine fever virus (CSFV) and inactivated Mycoplasma hyopneumoniae vaccination were evaluated. The results showed that the cytotoxicity of natural killer cells and proliferation of lymphocytes in response to mitogen stimulation were significantly enhanced (P<0.05) in those pigs receiving PG/LPS injection compared with the controls. However, there was no significant effect on the phagocytic activity of neutrophils (P>0.05). PG/LPS also displayed adjuvant effects with CSFV and Mycoplasma hyopneumoniae vaccines. Moreover, pigs receiving two injections of PG/LPS showed a 20.8% growth enhancement compared with untreated pigs. Thus, PG/LPS caused positive immunoregulation of porcine innate immune system effectors, non-specific activation of lymphocytes and antibody production.
We compared the relative resistance and soluble variant surface glycoprotein (VSG)-specific responses in (C57BL/6 × BALB/c)-F1 (B6B-F1) and C3H mice during infection with Trypanosoma brucei brucei, the hemoprotozoan parasite causing a debilitating disease in man and livestock. We demonstrated that C3H mice are relatively more trypanosusceptible, as evidenced by their reduced ability to control parasitemia and shorter survival time, than B6B-F1 mice. Quantitative differences in the pattern of cytokine and antibody (Ab) production were observed between the 2 mouse strains following infection with T. b. brucei. Thus, although both mouse strains recorded detectable levels of IFN-γ, TNF-α, NO and IL-10 in plasma and lymph nodes, as well as plasma IgM, IgG1, IgG2a, IgG2b and IgG3 Abs against VSG, the susceptible C3H mice only exhibited trace levels of Abs of all isotypes and yet produced elevated levels of IFN-γ, TNF-α and NO, compared to the relatively trypanotolerant B6B-F1 mice. In aggregate, these data strongly suggest that trypanosome-infected C3H mice have an immunological defect, manifested not only by suppression at the B cell clonal level, but also at the level of protective T cell and macrophage phenotypes.
The diagnostic significance of the plasma concentration of N-terminal pro-brain natriuretic peptide (NT-proBNP) was evaluated in 72 dogs with mitral valve insufficiency and 36 control dogs. In the controls, the plasma NT-proBNP concentration was 163.9 ± 114.7 (SD) pmol/l. The values in those with International Small Animal Cardiac Health Council (ISACHC) functional classification of heart failure class Ia, Ib, II and IIIa mitral valve insufficiency were 302.8 ± 257.1 (n=21), 634.2 ± 642.5 (n=23), 1,277.9 ± 756.2 (n=18) and 1,908.9 ± 538.8 (n=10) pmol/l, respectively; those in the class Ib or severer groups were significantly higher than that in the controls. In dogs in which the intensity of cardiac murmurs was Levine 3, 4, 5 and 6, plasma NT-proBNP concentrations were 647.6 ± 577.3 (n=27), 1,184.7 ± 841.0 (n=18), 1,532.4 ± 784.2 (n=10) and 1,461.8 ± 932.2 (n=4) pmol/l, respectively, and were significantly higher than that in the controls. The plasma NT-proBNP concentration was significantly correlated with the cardiac size (VHS) and LA/Ao (r=0.611, n=89, p<0.01; and r=0.705, n=91, p<0.01, respectively). When dogs with ISACHC class II or IIIa were regarded as heart failure, the cut-off value was 713.5 pmol/l, and the sensitivity and specificity were 0.913 and 0.857, respectively. These findings could indicate that plasma NT-proBNP concentration was significantly associated with the severity of heart failure due to mitral valve insufficiency in dogs. Further investigation is required to determine factors other than heart failure affecting plasma NT-proBNP concentration.
Echocardiographic values need to be established for each age and breed to be applied to equine medicine. The present research aims to describe echocardiographic measurements in Spanish fillies and colts of different age, to compare these values with those previously described for other equine breeds and to determine whether there is an influence of gender on echocardiographic measurements. Seventy unsedated and healthy Spanish foals of both sexes (39 females and 31 males) were divided into five age groups: A (22-60 days; n=15), B (61-90 days; n=13), C (91-180 days; n=18), D (181-270 days; n=12) and E (271-394 days; n=12). The following echocardiographic measurements were made: left ventricular internal dimensions, left ventricular free wall thickness and interventricular septum thickness at systole and at diastole. Mitral and tricuspid valve thickness was also determined. Opening velocity of the mitral valve in early diastole (DE slope), pattern of transmitral flow (E/A ratio) and echocardiographic mitral valve E point-septal separation (EPSS) were calculated from specific points in the anterior leaflet of the mitral valve. Aorta diameters were measured at the level of the valve leaflets, at the sino-tubular junction, at the valve level and at the sinus of Valsalva. Performance indices of the left ventricle were obtained. Most of the echocardiographic parameters increased with age, being the increases more marked up to 180 days of age. Fraction shortening, ejection fraction, stroke volume index, fractional septum and fractional wall thickening did not differ between groups. Some differences were found between fillies and colts in the different age groups, which could have been linked to the different body weight.
Paramylon is a β-(1-3)-D-glucan isolated from Euglena gracilis Z. This study was designed to evaluate the protective effects of paramylon on liver injury induced by carbon tetrachloride (CCl4) in rats. Wistar stain male rats were orally administered paramylon (500, 1,000 and 2,000 mg/kg body weight) before treatment with a single intraperitoneal dose of 50% CCl4 (2 ml/kg body weight). The rats were sacrificed 24 hr later, and blood samples were collected for assay of serum biochemical parameters. The livers were excised to evaluate the activity of antioxidant enzymes. Histopathological examination of the livers was also performed. The results showed that the treatment of paramylon prevented elevation of the serum levels of hepatic enzyme markers and inhibited fatty degeneration and hepatic necrosis induced by CCl4. Pre-administration of paramylon reduced the liver apoptotic index. The treatment of paramylon recovered reductions of activity of hepatic superoxide dismutase, catalase and glutathione peroxidase induced by CCl4. These results demonstrate that paramylon exhibits protective action on acute hepatic injury induced by CCl4 via an antioxidative mechanism. To the best of our knowledge, this is the first report of a hepatoprotective effect based on the antioxidative action of paramylon.
Theileria orientalis is one of the benign species of Theileria that is widely distributed in Japan and is sometimes responsible for serious economic losses in the livestock industry. In the present study, we surveyed the current status of T. orientalis infection in grazing cattle in the eastern areas of Hokkaido (Taiki, Otofuke, Shintoku, and Shin-Hidaka districts) using molecular methods, as well as traditional methods, of diagnosis. The genes encoding the major piroplasm surface protein (MPSP) and p23 of T. orientalis were identified using highly detectable polymerase chain reaction (PCR). Results of the MPSP-PCR assay indicated that grazing cattle in these districts, after about 1.5 months pasturage, showed high rates of infection, ranging from 10.0-64.8%. Although the main MPSP and p23 genotypes detected were the Ikeda- or Chitose-types, an MPSP gene closely relating to that found in Okinawa prefecture, and a p23 gene closely relating to the Australian (Warwick) Buffeli-type gene, were found in the cattle in Shintoku and Shin-Hidaka districts. The present survey indicated that there were at least five types of T. orientalis classified by their MPSP genes in Hokkaido, Japan, and that T. orientalis infection rates are still high in this region.
Plants are attractive vaccine production and oral delivery systems. Cereals are excellent candidate for edible vaccines, which can express and store high levels of proteins for extended periods of time without degradation. In this study, we produced a 14-kDa protective surface antigen of Ascaris suum L3 larvae and its fusion chimera with a mucosal carrier molecule cholera toxin B subunit (CTB) in rice (Oryza sativa L.) under the control of the endosperm-specific glutelin-B promoter. We found that the recombinant protein expression levels reached 1.5 μg per seed, a comparably high amount as compared to previously reported transgenic rice expression experiments. Potentials of transgenic rice plants as a source of oral vaccines against swine roundworm are discussed.
This retrospective study examined 8 cases of pathologically diagnosed insulinoma. Surgical intervention was performed in all cases, once insulinoma was suspected based on unique clinical signs, hypoglycemia, and/or findings on ultrasonography. At the same time, insulin and glucose concentrations were measured before surgery for clinical diagnosis. Although all cases displayed typical clinical signs and hypoglycemia, insulin concentrations varied with 3 of 8 cases showing levels within the reference interval. In addition, to confirm the reliability of measured values, we submitted serum samples from 4 cases to two commercial veterinary laboratories. Results differed considerably between laboratories, with no apparent correlations between the two. In Laboratory A, 3 of 4 cases were above the reference interval, and 1 case was in the middle of the reference interval. Conversely, in Laboratory B, 3 of 4 cases were above the reference interval, and 1 case was below the reference interval. Split decisions regarding the diagnosis of insulinoma were seen for 2 of the 4 cases.
An eosinophilic substance is usually observed in the mouse nasal septum, and its volume increases with age. In contrast to descriptions in textbooks defining the eosinophilic substance as amyloid, our previous report revealed that the observed eosinophilic substance is not amyloid, but consisted of collagen and an amorphous material. Furthermore, it was suggested that the amorphous material was produced by the clear hematoxylin and eosin (HE)-stained nasal gland epithelial cells. In this study, we investigated the deposition process of the amorphous material produced by nasal gland epithelial cells in the interstitium morphologically. In most cases, the amorphous materials in the clear HE-stained nasal gland epithelial cells accumulated at the basal portion. Collagen fibers surrounding the nasal glands partially disappeared, whereas the amorphous material in contact with the rough endoplasmic reticulum of the nasal gland epithelial cells continued to the amorphous material in the interstitium. These findings suggested that the amorphous material produced by the clear HE-stained nasal gland epithelial cells migrated to the interstitium through the partial opening of the basement membrane.
A 4-year-old, male, dachshund was referred to a certain local veterinary hospital because of a soft and fluctuant swelling in the left upper cervical region. The swelling was surgically removed and appeared to be filled with bloody mucus. Grossly, the swelling was identified as salivary mucocele and showed small multifocal whitish ossified tissue on its surface. Microscopically, the wall of salivary mucocele appeared as granulation tissue surrounding mucin, which was composed of loose edematous and vascularized connective tissue containing chronic inflammatory cells such as lymphocytes, plasma cells and macrophages. Characteristically, present case had ossifying components formed by metaplastic spindle cells in the wall of salivary mucocele. Therefore, the present case was diagnosed as salivary mucocele with osseous metaplasia in a dog.
A case of peripheral acanthomatous ameloblasoma of the angulus orisa in a 6-year-old female rabbit was described. The tumor composed of confluent follicles of stellate reticulum with peripheral cuboidal to reverse polarized columnar cells with nuclear palisading in the submucosa of the angulus orisa. The follicular cell elements stained positive for pan-cytokeratin. Additionally, there were multifocal keratinizing foci of the stellate reticulum with some mineralization. Calcified product such as enamel or dentin was not formed. Acanthomatous ameloblasoma in the submucosa of the angulus orisa has not been described and it may develop from an ectopic rest of odontogenic tissue. After excision, neither local recurrence nor metastasis has been observed.
In situ hybridization and immunohistochemistry with different types of antibody (monoclonal vs. polyclonal, natural vs. synthetic) was compared to detect porcine circovirus 2 (PCV2) in formalin-fixed, paraffin-embedded tissues from pigs with experimentally and naturally occurring postweaning multisystemic wasting syndrome. PCV2 DNA and antigen was detected in tissues from both experimentally and naturally infected pigs by in situ hybridization and immunohistochemistry, respectively. Statistical evaluation revealed that more PCV2 positive signals were significantly detected in both experimentally and naturally infected pigs by in situ hybridization compared with immunohistochemistry (P<0.05). The results of this study demonstrated that in situ hybridization proved more sensitive than immunohistochemistry for the detection of PCV2 in formalin-fixed, paraffin-embedded lymph node tissues.
Static and dynamic allodynia occurred in a rat model of neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve. Static allodynia was detected within 1 day after the CCI surgery, and persisted for 28 days. Dynamic allodynia displayed a slower course of development with a late onset, and statistically significant changes were achieved between 14 and 28 days after the surgery. Mexiletine at 10 and 30 mg/kg, s.c. produced a significant and dose-dependent inhibition of CCI-induced static and dynamic allodynia on day 14 post-surgery. Pregabalin, used as a reference drug, also significantly inhibited both static and dynamic allodynia at 30 and 60 mg/kg, p.o. These findings rationalize the clinical use of mexiletine for treatment of neuropathic pain.
We conducted a spatial analysis of low pathogenic H5N2 avian influenza (AI) outbreaks, that affected 41 chicken farms in Japan in 2005. A statistically significant (p=0.001) cluster of AI-positive farms was identified in the central part of Ibaraki Prefecture. Inside the AI cluster, the density was high for both chicken farms and chicken population, the proportion of layer finisher type farms was high and the farm size was large. We considered it important to take precautions for AI outbreaks in densely chicken-populated areas and to implement appropriate movement control around the affected farms to prevent transmission among farms located within small distances in the case of AI outbreaks. Spatial scan statistics are applicable in veterinary epidemiology to detection of high risk areas for animal diseases.
The purposes of this study were to determine the optimal dose and delay time for lymphography by injection of Iohexol into popliteal lymph nodes and to assess images of computed tomography by the established protocol. Three different doses (30, 60 and 90 mgI/kg) of water-soluble iodinated contrast medium were injected into 15 popliteal lymph nodes of 10 adult beagles, and fluoroscopy was performed. Filling and duration of contrast media and the number of visible ducts from popliteal lymph nodes to the thoracic duct and its branches were recorded. CT lymphography was performed, and the number of visible thoracic ducts was compared with that found by radiographic lymphography. Radiographs obtained between 130 and 800 seconds after injection of contrast medium provided a detailed view of the thoracic duct. The dose of 60 mgI/kg was determined to enable quality diagnostic imaging without extranodal leakage in radiographic lymphography. There was no significant difference in the number of thoracic ducts between the two modalities at each anatomic location. However, CT lymphography provided images of the thoracic duct with better spatial resolution and without superimposition of surrounding tissue. The present study provides an adequate delay time and injection for identification of the canine thoracic duct, and therefore, this technique could be applied to diagnosis of disease associated with chest lymphatic drainage.
In order to enable monitoring of the reproductive status of the female giant panda after observation of estrus behavior, we developed an enzyme immunoassay (EIA) system for urinary pregnanediol-3-glucuronide (PdG), a progesterone metabolite, using commercial reagents and examined the changes in the urinary concentration of PdG in a female giant panda that showed pseudopregnancy and suspicious pseudopregnancy in 6 consecutive years. The developed EIA system had good reproducibility (intra- and interassay CVs 6.1% and 16.3%, respectively), good parallelism between the standard curve and the dose response curve of serial diluted samples and positive correlation (r=0.836) with the data for PdG in the same samples measured by gas chromatography. Urinary PdG in the female panda showed two phases of increase. The first elevation was observed immediately after estrus with the levels of PdG below 100 ng/Crmg, while the second phase was characterized by a drastic elevation above 100 ng/Crmg until the level began to decrease at the end of pseudopregnancy or suspicious pseudopregnancy. The length of the second phase had wider range than that of the first phase. In the present study, a new EIA assay system for urinary PdG in the female giant panda was developed, and we found that the length of the second phase is unstable in the pseudopregnant and suspicious pseudopregnant giant panda, in contrast with the unstable length of the first phase caused by delayed implantation in the pregnant giant panda.
To provide up-to-date information on the incidence and risk factors of canine testicular tumors, 476 pathological reports of male canine tumors from two institutes of veterinary medicine in Taiwan over a period of 12 years were reviewed. The detection rate of testicular tumors was 16.8% (80/476) in male canine tumors, and 94.1% (80/85) in male genital tumors. Ninety-six testicular tumors from 80 dogs were identified in this study, including 33 (34.4%) seminomas (SEMs), 25 (26%) interstitial cell tumors (ICTs), 22 (22.9%) mixed germ cell-stromal cell tumors (MGCSCTs), and 16 (16.6%) Sertoli cell tumors (SCTs). Of the 96 testicular tumors, 52 (54.2%) tumors developed from cryptorchid testes in 45 dogs (56.3%), and 44 (45.8%) tumors developed from scrotal testes in 35 dogs (44.7%). Cryptorchidism was significantly associated with development of MGCSCTs, SCTs, and SEMs, but not ICTs (p<0.01). The detection rate of testicular tumors in the dog younger than 10-years-old was significantly associated with cryptorchidism (p<0.01). Except for mixed breed dogs, the Maltese breed had high detection rate of testicular tumors, and may have high risk in cryptorchidism in this study. In conclusion, our results indicate that cryptorchidism alone or in addition to age will significantly affect the incidence and type of canine testicular tumors, and a high detection rate of testicular tumors in Maltese dogs is documented for the first time.
To prevent encephalomyelitis caused by Akabane virus, as observed in 2006, vaccination of calves is one of the most effective prophylactic measures. For vaccination of calves, the duration of the maternal antibodies need to be considered because these antibodies are an obstacle to the effectiveness of the vaccine. In order to estimate the age of antibody decay in calves and to find factors influencing the duration of passive immunity, we conducted survival analysis using data from nationwide sentinel surveillance for Akabane disease. The accelerated failure time model based on the presence of interval censored data was used. The best fit model with a log-logistic distribution indicated that the maternal antibodies of beef calves last 1.11 times longer (95% confidence interval [CI]=1.06-1.16) than those of dairy calves. Calves in the western part of Japan and on Kyushu island, Japan, maintained the maternal antibodies 1.17 times (95% CI=1.11-1.23 and 1.10-1.24, respectively) longer than those in the eastern part of Japan. The ages at which beef calves in the eastern part of Japan, western part of Japan and Kyushu loose the antibodies, with 90% probability, were estimated to be 4.1, 4.8 and 4.8 months, respectively, while the ages were 3.7, 4.3 and 4.3 months for dairy calves in the same regions. The duration of maternal immunity to Akabane virus was different for different types of cattle and among different regions. These differences need to be taken into account when a vaccination strategy is adopted for preventing epizootic encephalomyelitis in the future.
A group of horses immunized with inactivated Japanese encephalitis (JE) vaccine (JE-Immune Group) and a group of non-immunized horses (Non-Immune Group) were infected with West Nile virus (WNV). After WNV infection, neutralizing (Nt) antibody (Ab) titers to WNV were higher than those to JE virus (JEV) in the Non-Immune Group, but the NtAb titers to JEV were higher than those to WNV during most of the post-challenge observation period in the JE-Immune Group. Immunoglobulin M (IgM) Abs to WNV tested positive in the Non-Immune Group but negative in the JE-Immune Group, except for in one horse. These results suggest that diagnosis of WNV infection in JE-immunized horses requires serological tests for NtAb and IgM titers to both WNV and JEV.