Reactivity of serum samples of dogs and horses tested by use of class-specific recombinant-based enzyme-linked immunosorbent assays for detection of granulocytic ehrlichiosis

OBJECTIVE: To test serum samples of dogs and horses by use of class-specific recombinant-based ELISA for establishing a diagnosis of granulocytic ehrlichiosis attributable to infection with organisms from the Ehrlichia phagocytophila genogroup. SAMPLE POPULATION: Serum samples from 43 client-owned dogs and 131 horses (81 with signs of acute illness and 50 without signs of disease). PROCEDURE: Serum samples were analyzed, using ELISA with a recombinant 44-kd protein antigen for IgM and IgG antibodies to the human granulocytic ehrlichiosis (HGE) agent (NCH-1 strain). Western blot analyses, using infected human promyelocytic leukemia cells, were conducted on 38 serum samples of horses and 11 serum samples of dogs to verify reactivity to the 44-kd peptide. RESULTS: IgM or IgG antibodies to the HGE agent were detected in 5 to 28% of dog serum samples and 5 to 37% of horse serum samples. Thirty-five of 38 (92%) horse serum samples had corresponding results on both tests (2 positive results for 26 samples and 2 negative results for 9 samples), using an ELISA for IgG antibodies or immunoblotting for total immunoglobulins. All 11 serum samples of dogs had positive results for both methods. CONCLUSION AND CLINICAL RELEVANCE: These ELISA with recombinant 44-kd antigen are suitable for detecting IgM or IgG antibodies to the HGE agent in serum samples of dogs and horses. Positive results for serum samples of horses from Connecticut, New York, Virginia, and Georgia indicate that the HGE agent is widely distributed in tick-infested areas of the eastern United States.     

Diagnosis of equine piroplasmosis in Xinjiang province of China by the enzyme-linked immunosorbent assays using recombinant antigens

The prevalence of equine piroplasmosis in Xinjiang province, China, was examined by enzyme-linked immunosorbent assays (ELISAs). A total of 70 serum samples were taken from horses pastured on three farms in western Xinjiang, and examined for diagnosis of equine Babesia equi (B. equi) infection and B. caballi infection by ELISAs using recombinant equi merozoite antigen 1 (EMA-1) and recombinant P48 antigen, respectively. Of the 70 samples, 28 (40.0%) and 17 (24.3%) samples were positive for B. equi infection and B. caballi infection, respectively. In addition, 11 (15.7%) samples were positive for both B. equi and B. caballi infections. These results indicate that equine piroplasmosis is widespread and therefore a cause for serious concern in western Xinjiang. To our knowledge, this is the first report describing a survey on equine piroplasmosis in Xinjiang province, China.     

青海省海西地区马焦虫病的血清学诊断

用ELISA方法调查了青海省海西地区马焦虫病的流行情况。从海西地区的格尔木市和乌兰县的牧户中收集了199份马属动物的血清样品，用马巴贝斯原虫的重组抗原P蚰48（GST-BcSAGlt）蛋白作为ELISA诊断抗原。进行了马巴贝斯原虫的诊断检查。从199份血清样品检出61份马巴贝斯原虫阳性，阳性率为30，65％。结果显示马焦虫病已在该地区广泛传播，并已严重威胁该地区的马属动物。     

Diagnosis of equine piroplasmosis in Brazil by serodiagnostic methods with recombinant antigens.

Serum samples from horses in the States of Sao Paulo and Mato Grosso do Sul, Brazil were examined for diagnosis of equine piroplasmosis by both the latex agglutination test (LAT) and enzyme-linked immunosorbent assay (ELISA) with recombinant antigens. Of the 47 samples analyzed, 38 (81%) and 42 (90%) samples were positive for B. equi infection and B. caballi infection, respectively. In addition, 35 (75%) samples were positive for both B. equi and B. caballi infections. These results indicate that equine piroplasmosis is widespread and therefore a cause for serious concern in the States of Sao Paulo and Mato Grosso do Sul, Brazil.     

Seroprevalence of antibodies against equine arteritis virus in horses residing in the United States and imported horses

OBJECTIVE: To compare seroprevalence of antibodies against equine arteritis virus (EAV) in horses residing in the United States with that of imported horses. DESIGN: Serologic survey. SAMPLE POPULATION: Serum samples from 364 horses on 44 equine operations in California and 226 horses imported from various countries. PROCEDURE: Serum samples were collected from each imported horse and from up to 20 horses on each operation. For resident horses, the number of sampled horses on each operation was determined on the basis of the number of horses on the operation. Samples were tested for antibodies against EAV by use of a serum neutralization test. RESULTS: 1.9% of resident horses and 18.6% of imported horses were seropositive to EAV, including 16.1% of imported stallions. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that the EAV seroprevalence of horses residing in California is considerably lower than that of imported horses, including imported stallions.     

Serologic confirmation of Ehrlichia equi and Borrelia burgdorferi infections in horses from the northeastern United States

OBJECTIVE: To determine whether horses living in tick-infested areas of northeastern United States with clinical signs of borreliosis or granulocytic ehrlichiosis had detectable serum antibodies to both Borrelia burgdorferi and Ehrlichia equi. DESIGN: Prospective study. ANIMALS: Serum samples from 51 clinically normal horses, 14 horses with clinical signs of borreliosis, and 17 horses with clinical signs of granulocytic ehrlichiosis. PROCEDURE: Serum B burgdorferi or E equi antibodies were measured by use of an ELISA, immunoblot analysis, or indirect fluorescent antibody (IFA) staining. RESULTS: Of the 82 serum samples tested, 37 (45.1%) and 13 (15.9%) had detectable antibodies to B burgdorferi or E equi, respectively. Test results indicated that 12 horses had been exposed to both agents, 11 of these horses had granulocytic ehrlichiosis. The ELISA regularly detected antibodies to the following recombinant protein (p) antigens of B burgdorferi: p29, p37, p39, and p41-G. The use of immunoblot analysis confirmed ELISA results by indicating antibody reactivities to antigens of whole-cell B burgdorferi having molecular masses of predominantly 31, 34, 37, 39, 41, 58, and 93 kd. CONCLUSIONS AND CLINICAL RELEVANCE: Horses living in areas where ticks (Ixodes scapularis) abound are sometimes exposed to multiple pathogens. Analyses for specific recombinant borrelial antibodies using an ELISA can help separate horses with borreliosis from those with granulocytic ehrlichiosis, even when antibodies to both etiologic agents are detected in serum samples. Analysis using immunoblots is sensitive, and along with ELISA or IFA procedures, is suitable for confirming a clinical diagnosis of each disease.     

35 leptospira isolated from the vitreous body of 32 horses with recurrent uveitis (ERU)

130 vitreous samples, systematically collected in 1998 from 117 horses during vitrectomy, were cultured for the presence of leptospires. All horses suffered from equine recurrent uveitis (ERU), also known as periodic ophthalmia or moon blindness, and were treated surgically to combat painful attacks, and to preserve vision. In 35 out of 130 vitreous samples (35/130 = 26.9%), leptospires could be isolated. These isolates belong to the grippotyphosa serogroup (n = 31) and to the australis serogroup (n = 4). So, for the first time, leptospires were recovered from eyes in vivo in a large number of horses with ERU. Vitreous samples and one serum sample from each horse were also tested for leptospiral antibodies using the microscopic agglutination test (MAT). In 92 vitreous samples (92/130 = 70.7%) and 96 serum samples (96/117 = 82.0%) leptospiral antibodies were detected at a dilution of > 1:100. The presence of intact leptospires and specific antibodies in eyes affected with ERU demonstrates a local antibody production to leptospiral antigen. These results indicate an important etiological role of leptospires in equine recurrent uveitis.     

<Emphasis Type="Italic">Theileria</Emphasis> (<Emphasis Type="Italic">Babesia</Emphasis>) <Emphasis Type="Italic">equi</Emphasis> and <Emphasis Type="Italic">Babesia caballi</Emphasis> Infections in Horses in Galicia,Spain

The control of equine piroplasmosis is becoming increasingly important to maintain the international market open to the horse industry. The purpose of this study was to demonstrate the occurrence of equine piroplasmosis (Theileria equi and Babesia caballi) in Galicia, north-west Spain, and to compare haematological and serum biochemistry parameters between non-parasitaemic horses and horses parasitaemic with T. equi and B. caballi. Sixty serum samples (control group) were taken from healthy horses pastured on two farms, and examined for evidence of equine T. equi and B. caballi infection by indirect fluorescent antibody test (IFAT). Of the 60 samples, 24 (40%) and 17 (28.3%) samples were positive for T. equi and B. caballi, respectively. Twelve (20%) samples were positive for both parasites. Haematology and serum biochemistry were compared between controls and a series of 36 horses clinically affected by T. equi (25) or B. caballi (11). Compared with the healthy group, there was a 43% and 37% decrease in the haematocrit for T. equi and B. caballi infection, respectively. Parasitaemic horses presented an intense anaemia and serum biochemistry signs of liver damage. The anaemia was more severe in T. equi-infected than in B. caballi-infected horses. Our results suggest that equine piroplasmosis is widespread in the region and is a cause for concern.     

Epidemiological study of equine piroplasmosis in Mongolia

The purpose of this study was to demonstrate the occurrence of equine piroplasmosis in Mongolia, a country in which the disease occurs epidemically in different climatic conditions. Antibodies to Babesia equi and B. caballi were determined in serum samples of 254 pastured horses in different locations of Mongolia using an enzyme-linked immunosorbent assay with recombinant antigens. One hundred and eighty-five (72.8%) and 102 (40.1%) of all serum samples were positive for B. equi and B. caballi infections, respectively. In addition, 78 (30.7%) samples were positive for both B. equi and B. caballi infections. These results indicate that equine piroplasmosis is widespread in Mongolia. To our knowledge, this is the first report describing an epidemiological study on equine piroplasmosis in different geographic regions in Mongolia.     

Evaluation of a polyvalent enzyme-linked immunosorbent assay incorporating a recombinant p44 antigen for diagnosis of granulocytic ehrlichiosis in dogs and horses

OBJECTIVE: To develop and evaluate a polyvalent ELISA incorporating a highly specific recombinant antigen (p44) for diagnosis of granulocytic ehrlichiosis in dogs and horses. ANIMALS: 32 dogs and 43 horses. PROCEDURE: Results of the ELISA were compared with results of indirect fluorescent antibody (IFA) staining and western immunoblotting incorporating whole-cell antigen. RESULTS: For the canine and equine samples, percentages of samples with positive IFA staining, western immunoblotting, and ELISA results were similar. For 29 (91 %) canine samples and 30 (70%) equine samples, results of IFA staining, western immunoblotting, and the ELISA were in complete agreement. Results of the ELISA for 3 canine serum samples known to contain antibodies to Ehrlichia canis and 12 equine serum samples known to contain antibodies to E risticii were negative. CONCLUSIONS AND CLINICAL RELEVANCE: Results of the present study suggest that a polyvalent ELISA incorporating a recombinant p44 antigen is suitable for detecting antibodies to E equi in dogs and horses.     

Accuracy of an indirect fluorescent-antibody test and of a complement-fixation test for the diagnosis of Babesia caballi in field samples from horses

We evaluated the indirect fluorescent-antibody (IFA) test and complement-fixation (CF) test for diagnosis of equine piroplasmosis in the absence of a gold standard. Using Evan's blue, we estimated the specificity of the IFA test on a parasite-free, field horse population to be 98% (95% confidence interval=97, 99). We observed an excellent test agreement (kappa=0.83) between two collaborating laboratories when the IFA test was performed on identical samples from an endemic area. Using Bayesian analysis with informative prior probability distributions, we estimated the sensitivity of the IFA test to be 92% (95% probability interval, PI=81, 98), and specificity to be 95% (95% PI=88, 99). The CF test sensitivity and specificity estimates were 28% (95% PI=15, 47) and 99% (95% PI=96, 100), respectively. We found the IFA to be superior to the CF test, and the inclusion of Evan's blue in test protocol improved the performance of the IFA test. We conclude that the IFA test for Babesia caballi is a sensitive and specific test for the diagnosis of equine piroplasmosis.     

Immunoglobulin M-capture enzyme-linked immunosorbent assay testing of cerebrospinal fluid and serum from horses exposed to west nile virus by vaccination or natural infection

The West Nile (WN) virus, present in the United States since 1999, is a cause of encephalomyelitis in birds, alligators, humans, and horses. No data exist regarding detection of anti-WN virus immunoglobins in equine cerebrospinal fluid (CSF). The aims of this study were to evaluate the blood-brain barrier (BBB) in WN virus-infected (WNE) horses, to compare diagnostic testing in serum and CSF, and to describe the immunoglobulin M (IgM) response in serum and CSF of vaccinated horses. CSF was collected from the lumbosacral (LS) space (n = 13) or the allanto-occipital (AO) space (n = 14) of WNE horses. The albumin quotient (AQ) and IgG index were calculated, and the IgM-capture-enzyme-linked immunosorbent assay (MAC-ELISA) was used to detect anti-WN virus IgM in serum and CSF. CSF collected from the LS site had a higher (P < .02) IgG index compared to the AO site (0.34 +/- 0.04 versus 0.22 +/- 0.04 [mean +/- SE], respectively). The mean AQ, irrespective of collection site, did not exceed reference values. There was 100% agreement between CSF and serum testing for IgM by MAC-ELISA testing. However, the positive to negative antigen ratios were higher (P < .001) in CSF (34.5) versus serum (8.5), indicating lower nonspecific reactivity in CSF samples. Horses vaccinated against WN virus did not develop an IgM response at 1:400 mg/dL in serum; however, a few horses developed a weak IgM response in serum but not in CSF. In conclusion, MAC-ELISA testing of serum and CSF were equivocal. Also, examination of CSF data from WNE horses suggests a normal BBB integrity and increased intrathecal production of antibodies.     

Serological prevalence of Babesia caballi and Theileria equi in horses of Lara State, Venezuela

The main objective of this study was to demonstrate the occurrence of equine piroplasmosis (EP) in horses of Lara State, Venezuela, and to correlate it with the factors host's sex and age in order to know the epidemiology of this disease at the Venezuelan Centroccidental Region. Antibody levels to Babesia caballi and Theileria equi were assessed in 360 equine serum samples, collected from 9 municipalities of Lara State, using an ELISA technique with recombinant antigens and monoclonal antibodies (Mabs). Antibodies to B. caballi were found in 254 horses (70.6%), whereas 181 animals (50.3%) were detected as seropositives to T. equi. In addition, 128 samples (35.56%) were seropositives to both hemoparasites. There were no significant differences between the seropositivity to B. caballi and T. equi with the factors sex and age of the horses. These results show that Lara State is an enzootic area for equine piroplasmosis, and are a contribution to a partial knowledge of the dynamic of this disease in Venezuela.     

Oral vesicular lesions in horses without evidence of vesicular stomatitis virus infection

OBJECTIVE: To report clinical and serologic findings in horses with oral vesicular lesions that were consistent with vesicular stomatitis (VS) but apparently were not associated with VS virus (VSV) infection. DESIGN: Serial case study. ANIMALS: 8 horses. PROCEDURE: Horses were quarantined after appearance of oral lesions typical of VS. Severity of clinical signs was scored every 2 to 5 days for 3 months. Serum samples were tested for antibodies by use of competitive ELISA (cELISA), capture ELISA for IgM, serum neutralization, and complement fixation (CF). Virus isolation was attempted from swab specimens of active lesions. RESULTS: 2 horses with oral vesicular lesions on day 1 had antibodies (cELISA and CF) against VSV; however, results of CF were negative by day 19. Five of the 6 remaining horses were seronegative but developed oral lesions by day 23. Virus isolation was unsuccessful for all horses. CONCLUSIONS AND CLINICAL RELEVANCE: Horses were quarantined for 75 days in compliance with state and federal regulations. However, evidence suggests that oral lesions were apparently not associated with VSV infection. The occurrence in livestock of a vesicular disease that is not caused by VSV could confound efforts to improve control of VS in the United States and could impact foreign trade. Vesicular stomatitis is of substantial economic and regulatory concern.     

Role of bacteria in the pathogenesis of recurrent uveitis in horses from the southeastern United States

OBJECTIVE: To determine the role of intraocular bacteria in the pathogenesis of equine recurrent uveitis (ERU) in horses from the southeastern United States by evaluating affected eyes of horses with ERU for bacterial DNA and intraocular production of antibodies against Leptospira spp. SAMPLE POPULATION: Aqueous humor, vitreous humor, and serum samples of 24 clinically normal horses, 52 horses with ERU, and 17 horses with ocular inflammation not associated with ERU (ie, non-ERU inflammation). PROCEDURES: Ribosomal RNA quantitative PCR (real-time PCR) assay was used to detect bacterial DNA in aqueous humor and vitreous humor from clinically normal horses (n = 12) and horses with chronic (> 3-month) ERU (28). Aqueous humor and serum were also evaluated for anti-Leptospira antibody titers from clinically normal horses (n = 12), horses with non-ERU inflammation (17), and horses with confirmed chronic ERU (24). RESULTS: Bacterial DNA was not detected in aqueous humor or vitreous humor of horses with ERU or clinically normal horses. No significant difference was found in titers of anti-Leptospira antibodies in serum or aqueous humor among these 3 groups. Only 2 horses, 1 horse with ERU and 1 horse with non-ERU inflammation, had definitive intraocular production of antibodies against Leptospira organisms. CONCLUSIONS AND CLINICAL RELEVANCE: In horses from the southeastern United States, Leptospira organisms may have helped initiate ERU in some, but the continued presence of the organisms did not play a direct role in the pathogenesis of this recurrent disease.     

Evaluation of plasma carboxy-terminal cross-linking telopeptide of type I collagen concentration in horses

OBJECTIVE: To evaluate a human assay for quantification of carboxy-terminal cross-linking telopeptide of type I collagen (CTX-I), assess the influence of age on plasma CTX-I concentration, investigate the relationship between plasma CTX-I and serum osteocalcin concentrations, and determine whether concentrations of plasma CTX-I or serum osteocalcin fluctuate in circadian manner in horses. HORSES: 75 clinically normal horses. PROCEDURE: Cross-reactivity between equine serum CTX-I and CTX-I antibodies in an automated electrochemiluminescent sandwich antibody assay (ECLIA) was evaluated via a specificity test (ie, dilution test) and recovery calculation. Serum osteocalcin concentration was measured with an equine-specific osteocalcin radioimmunoassay. To analyze diurnal variations in plasma CTX-I and serum osteocalcin concentrations, blood samples were obtained hourly during a 24-hour period. RESULTS: Results of the dilution test indicated good correlation (r > 0.99) between expected serum CTX-I concentrations and measured serum CTX-I concentrations. The calculated CTX-I recovery was 97.6% to 109.9%. Plasma CTX-I and serum osteocalcin concentrations were correlated. Plasma CTX-I concentration was inversely correlated with age of the horse. No significant circadian variations in plasma CTX-I and serum osteocalcin concentrations were detected. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the fully automated CTX-I ECLIA can be used for evaluation of plasma and serum samples from horses and may be a useful tool to monitor bone metabolism changes. Horses in this study did not have notable diurnal fluctuations in serum osteocalcin and plasma CTX-I concentrations.     

Seroepidemiologic studies on Babesia equi and Babesia caballi infections in horses in Jilin province of China

The prevalence of equine piroplasmosis caused by Babesia equi and Babesia caballi in northeast China has remained unknown, although the People's Republic of China is recognized as an endemic country for the diseases. In the present study, we investigated the prevalence of equine piroplasmosis in Jilin province, a part of northeast China. A total of 111 serum samples were taken from horses in eastern Jilin, and examined for diagnosis of B. equi and B. caballi infections by the enzyme-linked immunosorbent assays with recombinant antigens, equi merozoite antigen-1 and P48, respectively. Of the 111 samples, 38 (34%) and 36 (32%) samples were sero-positive for B. equi infection and B. caballi infection, respectively. In addition, 14 (12%) samples were sero-positive for both B. equi and B. caballi infections. These results indicate that equine piroplasmosis is widespread and therefore a cause for serious concern in northeast China.     

Evaluation of ELISA and Western Blot Analysis using three antigens to detect anti-Trichinella IgG in horses

We assessed a serological method for detecting Trichinella infection in horses, specifically, an ELISA using three antigens to detect anti-Trichinella IgG (i.e. a synthetic tyvelose glycan-BSA (stg-BSA) antigen, an excretory/secretory (ES) antigen, and a crude worm extract (CWE) antigen). Serum samples were collected from 2502 horses (433 live horses from Romania and 2069 horses slaughtered in Italy and originating from Italy, Poland, Romania, and Serbia). Serum samples were also taken from horses experimentally infected with different doses of T. spiralis and T. murrelli larvae, as controls. The cut-off value of ELISA was determined on serum samples from 330 horses from Trichinella-free regions of Italy, which were also examined by artificial digestion of preferential-muscle samples. In the experimentally infected horses, the stg-BSA and ES antigens were less sensitive than the CWE antigen. Trichinella spiralis showed a higher immunogenicity than T. murrelli, and the IgG immunoresponse was dose-dependent. The kinetics of anti-Trichinella IgG were similar among all experimentally infected horses. No circulating antibodies were detected 4-5 months after experimental infection, although these horses still harbored infective larvae. Depending on the antigen used, for 4-7 of the 330 horses from Trichinella-free areas, the optical density (OD) of the serum sample was higher than the cut-off value, yet these samples were negative when subjected to Western Blot. Similar results were obtained for the 1739 horses slaughtered in Italy (originating from Italy, Poland, Romania, and Serbia) and the 433 live Romanian horses. Of the 4 horses with muscle larvae, only one was positive by ELISA and Western Blot. Because the anti-Trichinella IgG remain circulating for only a short period of time, whereas the larvae remain infective for longer periods, serology cannot be used for either diagnosing Trichinella infection in horses or estimating the prevalence of infection. Artificial digestion of at least 5 g of preferential-muscle tissue continues to be the method of choice at the slaughterhouse for preventing equine-borne trichinellosis in humans.     

新疆昭苏地区马梨形虫病病原及抗体检测

马梨形虫病是由马驽巴贝斯虫和马泰勒虫寄生于马属动物的红细胞内所引起的一类血液原虫病,呈全球性分布,尤其在新疆发病率更高,处于逐年上升趋势,对区域性马产业的发展影响极大。为了解2018年新疆昭苏养马区域马梨形虫的感染情况,随机采集昭苏县18个乡镇的马全血及血清各858份,采用PCR和间接ELISA分别进行检测,对两种方法检测的18个地区、不同年龄阶段的马驽巴贝斯虫、马泰勒虫及混合感染情况进行统计学分析。结果显示,PCR检测马驽巴贝斯虫、马泰勒虫及混合感染的阳性率分别为12.12%、13.87%和2.80%;间接ELISA检测马驽巴贝斯虫、马泰勒虫及混合感染的抗体阳性率分别为15.50%、10.14%和2.56%;不同年龄阶段筛查结果显示,在6岁以下的马匹感染马驽巴贝斯虫、马泰勒虫及混合感染的阳性率较高,并且不同地区的不同年龄阶段马匹的马梨形虫感染率存在不同程度的差异。此次获得的昭苏县马梨形虫感染情况的一线数据,可为当地养马区域马梨形虫病的综合防控提供技术支撑。     

Procedurally similar competitive immunoassay systems for the serodiagnosis of Babesia equi, Babesia caballi, Trypanosoma equiperdum, and Burkholderia mallei infection in horses.

Procedurally similar competitive enzyme-linked immunoassay (cELISA) methods were developed for the serodiagnosis of Babesia equi and Babesia caballi (piroplasmosis), Trypanosoma equiperdum (dourine), and Burkholderia mallei (glanders) infections in horses. Apparent test specificities for the B. equi, B. caballi, T. equiperdum, and B. mallei cELISAs were 99.2%, 99.5%, 98.9%, and 98.9%, respectively. Concordances and kappa values between the complement fixation (CF) and the cELISA procedures for the serodiagnosis of B. equi, B. caballi, T. equiperdum, and B. mallei infections in experimentally exposed horses were 76% and 0.55, 89% and 0.78, 97% and 0.95, and 70% and 0.44, respectively. The cELISA method may be a technically more reproducible, objective, and convenient approach for piroplasmosis, dourine, and glanders serodiagnosis in qualifying animals for international movement and disease eradication programs than the CF systems currently in use. Use of the cELISA method also obviated the problems associated with testing hemolyzed or anticomplementary sera.