Development of enzyme-linked immunosorbent assays based on recombinant MSP1a and MSP2 of Anaplasma marginale

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant MSP1a and MSP2 from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. The high sensitivities (99% for both tests) and specificities (100% for both tests) were confirmed with sera from cattle positive or negative for A. marginale antibodies, respectively, by immunofluorescent antibody test. By the analysis of 583 sera from cattle of three regions of the state of Pernambuco, Brazil, the agreement between both tests was high, with a kappa index of 0.89. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.     

Use of a pre-selected epitope of cathepsin-L1 in a highly specific peptide-based immunoassay for the diagnosis of Fasciola hepatica infections in cattle.

A peptide-based indirect ELISA to detect cattle antibodies against Fasciola hepatica was developed and evaluated for its sensitivity and specificity. An immunogenic antigen released in vitro by F. hepatica was purified. After purification the sequence of the first 20 N-terminal aa of this protein showed considerable homology with cathepsin L-like proteinase. Based on its homology with cathepsin-L1, we further focused on this protein for diagnostic purpose. Predicted B-cell epitopes of cathepsin-L1 were synthesised as single synthetic peptides and tested with respect to their diagnostic potential. An indirect ELISA based on one of these peptides was (i) evaluated further and (ii) compared to the potential of an indirect ELISA with excretion/secretion antigens from adult F. hepatica, or (iii) purified cathepsin-L1. Specificity and sensitivity of the three ELISAs were assessed using sera from calves experimentally infected with pure isolates of Dictyocaulus viviparus, Ostertagia ostertagi, Cooperia oncophora, Nematodirus helvetianus, Schistosoma mattheei, Ascaris suum, Taenia saginata or F. hepatica, respectively, and sera from parasite-naive calves. In addition, sera were analysed from calves naturally infected with F. hepatica. The sensitivities of all three ELISAs were also very high, 98.9% (i), 100% (ii) and 100% (iii). The specificity of the peptide ELISA was very high, 99.8%, whereas specificities of the ES antigens and cathepsin-L1 ELISAs were only 82.8% and 94.6%. In experimentally infected cattle, F. hepatica-specific antibodies were first detected between days 21 and 28 p.i. with all three ELISAs, and the antibody levels persisted in the peptide ELISA until day 183 p.i. All sera from naturally infected calves were positive in the peptide ELISA. These results demonstrate that the peptide-based F. hepatica ELISA is a useful method for detecting antibodies in the sera from cattle infected with F. hepatica. This type of immunodiagnostic will therefore contribute to more accurate diagnosis and to timely curative treatment of animals.     

Serological survey of Babesia bovis, Babesia bigemina, and Anaplasma marginale antibodies in cattle from the semi-arid region of the state of Bahia, Brazil, by enzyme-linked immunosorbent assays

The objectives of this work were to determine the prevalence of Babesia bovis, Babesia bigemina, and Anaplasma marginale detecting antibodies in cattle raised in the semi-arid region of the state of Bahia, Brazil, through indirect enzyme linked immunosorbent assays (ELISA) and to compare the performances of indirect enzyme-linked immunosorbent assays with crude (I-ELISA-CrAnaAg) and recombinant major surface protein-5 (I-ELISA-MSP-5Ag), as antigens to detect antibodies against A. marginale. An stable enzootic area was found in Senhor do Bonfim and Euclides da Cunha for B. bovis that showed 86 and 95.5% of prevalence, respectively, and for B. bigemina with 90.8 and 91.3%. On the other hand, Uauá and Juazeiro, were characterized as enzootically unstable areas, since prevalences were: B. bovis--63.7 and 56.4% and B. bigemina--53 and 54.8%, respectively. The prevalence of A. marginale in the four municipalities was above 97% with I-ELISA-CrAnaAg and 94.8% with I-ELISA-MSP-5Ag which is an indication of stable enzootic condition for the rickettsia. The I-ELISA-CrAnaAg and I-ELISA-MSP-5Ag showed a highly significant association (r = 0.977), which means that both ELISA tests are suitable for epidemiological studies of A. marginale.     

Evaluation of the anaplasmosis rapid card agglutination test for detecting experimentally-infected elk.

Anaplasma marginale was experimentally transmitted from cattle to elk to cattle. Six intact adult elk (Cervus canadensis canadensis) inoculated with freshly collected heparinized blood from cattle chronically infected with A. marginale became asymptomatic carriers. Although the elk did not develop clinical or hematologic evidence of infection, they became seropositive by the serum(SRCA) and plasma rapid card agglutination (PRCA) tests. Blood from the experimentally-infected elk produced disease in splenectomized bovine calves and the carrier state persisted for at least one year. Infection did not occur when two elk were inoculated with 0.5 ml of frozen blood from known bovine carriers. The blood had been frozen for four weeks in liquid nitrogen with 6% dimethyl-sulfoxide. The bovine SRCA and PRCA tests were adapted for use with elk serum. To obtain accurate test results, serum collected from clotted elk blood had to be held for at least 72 h at 21-27 C before performance of the SRCA test. Comparative serologic and infectivity studies indicated that the carrier (reactor) status of elk was accurately identified with the serologic tests in 61 of 68 samples evaluated. Incorrect serologic results with the SRCA and PRCA tests were false-negative readings. In no case were uninfected elk identified as seropositive.     

Cloning, sequencing, expression, and antigenic characterization of rMSP4 from Anaplasma marginale isolated from Paraná State, Brazil

Anaplasmosis is a bovine intraerythrocytic disease caused by the bacterium Anaplasma marginale; it causes significant economic losses in tropical and subtropical regions, worldwide. The msp4 gene of an A. marginale strain isolated in Paran , Brazil, was amplified by PCR and sequenced; its cloning into the pET102/D-TOPO vector produced an msp4-6xHis-V5-HP thioredoxin fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21(DE-3); the expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in the cell lysate. The inclusion bodies were solubilized with urea and the recombinant protein was purified by Ni-NTA column and dialyzed. This method produced a relatively high yield of rMSP4, which was used to immunize rabbits. The deduced amino acid sequence encoded by MSP4 showed 99% homology to A. marginale isolates from Florida, USA, and from Minas Gerais, Brazil. Both rMSP4 and native MSP4 were recognized by post-immunization rabbit serum, showing that rMSP4 has conserved epitopes. As antigenicity was preserved, rMSP4 might be useful for the development of vaccine against anaplasmosis.     

Seroprevalence of Neospora caninum antibodies in cattle and water buffaloes in India

Neospora caninum is now recognized as a major cause of abortion in cattle worldwide, but there is no report of N. caninum infection in cattle in India. Serum samples from 427 dairy cattle and 32 dairy water buffaloes from 7 organized dairy farms located in Punjab, India, were tested for N. caninum antibodies using a commercial monoclonal antibody-based competitive enzyme-linked immunosorbent assay (ELISA). Antibodies to N. caninum were found in 35 of 427 cattle from 6 of the 7 farms; 9.6% of cows, 5.1% of heifers, and 5.0% of calves were seropositive, suggesting postnatal transmission of N. caninum on the farm. Antibodies to N. caninum were found in 16 of 32 buffaloes tested from 2 dairy farms. In total, 64 cattle and 16 buffalo sera already tested by ELISA were also evaluated by an indirect fluorescent antibody test (IFAT) to verify ELISA results. Of the 64 cattle samples, 29 sera were negative by both tests and of the 35 ELISA-positive sera, 12 had IFAT titers of 1:100 or higher (1 had IFAT titer of 100, 2 had IFAT titer of 200, and 9 had IFAT titers of 400 or higher). Of the 16 buffalo sera positive by ELISA, 1 had an IFAT titer of 1:400. Thus, antibodies to N. caninum were demonstrated in cattle sera by 2 serologic methods. To our knowledge this is the first report of N. caninum infection in cattle and buffaloes in India.     

Seroepidemiology of Anaplasma marginale in white-tailed deer (Odocoileus virginianus) from Louisiana

Antibodies to Anaplasma marginale were detected by the indirect fluorescent antibody test (IFA) in six of 331 (2%) serum samples of white-tailed deer (Odocoileus virginianus) from Louisiana. None of the serum samples were positive using the A. marginale modified rapid card agglutination test. Of the six IFA positive sera retested by the complement fixation test four sera gave anticomplementary and two gave seropositive reactions. The low A. marginale reactor rate in this white-tailed deer population was probably a reflection of the lack of cohabitation between cattle and deer and the fact that the primary arthropod vectors in Louisiana are tabanids. The validity of the indirect fluorescent antibody test for A. marginale antibodies in white-tailed deer should be evaluated.     

Identification of antigenic differences that discriminate between cattle vaccinated with Anaplasma centrale and cattle naturally infected with Anaplasma marginale

Monoclonal antibodies were raised against the vaccine strain of Anaplasma centrale used in Australia. A monoclonal antibody that reacted with an 80 kDa antigen was used to develop an A. centrale-specific fluorescent antibody test that will be useful for confirming species identity in patent infections. Another monoclonal antibody that reacted with a 116 kDa antigen was used to develop an A. centrale-specific competitive inhibition enzyme-linked immunosorbent assay (ELISA) for the serological identification of vaccinated cattle. The sensitivity of the ELISA was 100% in cattle experimentally infected with A. centrale, 97.1% in a vaccinated beef herd and 98.3% in a vaccinated dairy herd. The specificity of the ELISA was 98.6% in non-vaccinated cattle outside the Anaplasma marginale-endemic area, 97.9% in non-vaccinated cattle within the A. marginale-endemic area and 100% in cattle experimentally infected with A. marginale. The ELISA detected antibodies to A. centrale in cattle up to 9 years after vaccination with no apparent decrease in sensitivity. The assay has proved extremely valuable in Australia for investigating reported failures of multivalent live vaccines used to protect cattle against anaplasmosis and babesiosis, and should be similarly useful elsewhere in the world where these types of vaccines are used, e.g. Israel and South America.     

Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease

A diagnostic ELISA with recombinant Fasciola hepatica cathepsin L-like protease as antigen was developed to detect antibodies against F. hepatica in sheep and cattle. The recombinant cathepsin L-like protease was generated by functional expression of the cDNA from adult stage F. hepatica flukes in Saccharomyces cerevisiae. Specificity and sensitivity of the cathepsin L enzyme-linked immunosorbent assay (ELISA) was assessed using sera from sheep and calves experimentally or naturally mono-infected with F. hepatica and six-seven other parasites. The sensitivity of the cathepsin L ELISA for sheep and cattle sera was 99.1 and 100%, respectively. In the experimental setting with established mono-infections, the specificity of the cathepsin L ELISA was 98.5% for cattle sera and 96.5% for sheep sera. In experimentally infected cattle and sheep, the first detection of F. hepatica-specific antibodies appeared first between 5 and 7 weeks post-infection, but depended on the infectious dose of F. hepatica. In ELISA the detection preceded first detection of the infection based on egg counts and remained detectable till at least 23 weeks after a primary F. hepatica infection. Detection of Fasciola gigantica infections was similar to detection of F. hepatica. The first detection occurred at week 5 and signals persisted for at least 20 weeks. All sera from naturally F. hepatica infected sheep were seropositive in the cathepsin L-like ELISA. The relevance of this ELISA format was also evaluated using sera from naturally infected cattle in the Netherlands, Ecuador and Vietnam and compared with results from egg-counts. For the latter two endemic areas with mixed parasitic infections the 'apparent' sensitivity of the cathepsin L ELISA was calculated for all serum samples together to be 90.2%. The 'apparent' specificity under these conditions was calculated to be 75.3%. In cattle, the cathepsin L ELISA was superior to the concurrently evaluated peptide ELISA format using a single epitope as the antigen both in controlled natural infections as well as in infections in endemic areas. The present ELISA-format contributes a relatively sensitive and reliable tool for the early serodiagnosis of bovine and ovine fasciolosis.     

The modified card agglutination test: an accurate tool for detecting anaplasmosis in Columbian black-tailed deer

Inoculation of susceptible calves confirmed that the modified card agglutination test accurately detected the anaplasmosis infection status of each of 35 Columbian black-tailed deer (Odocoileus hemionus columbianus). Anaplasma marginale, and specific antibodies, were demonstrated only in calves which received blood from deer that were positive by the card test. The modified card agglutination testing of deer serum was performed in the manner recommended for testing cattle serum with bovine-origin antigen and bovine serum factor.     

Association between Coxiella burnetii seropositivity and abortion in dairy cattle of Northern Italy

Coxiella burnetii, the agent of Q fever in humans, has been associated with abortion in cattle. In this study 650 sera from cattle with abortion and 600 randomly-selected control sera were examined for antibodies to C. burnetii by ELISA. Two hundred and ninety-two (44.9%) out of 650 animals which experienced abortion were seropositive versus 132 (22%) out of 600 of the control group. A statistically significant difference resulted from the comparison of the seroprevalence of aborted cattle with that of controls (p < 0.001). Moreover, a significant higher prevalence was disclosed in cattle which aborted during late gestation (p < 0.002) and in the autumn (p < 0.001).     

Genetic characterization of Anaplasma ovis strains from bighorn sheep in Montana

Wildlife reservoir species and genetic diversity of Anaplasma ovis (Rickettsiales: Anaplasmataceae) have been poorly characterized. Bighorn sheep (Ovis canadensis), captured in Montana from December 2004 to January 2005, were tested for antibodies to Anaplasma spp.; the presence of A. ovis was determined by the characterization of major surface protein msp4 sequences. Anaplasma antibodies were detected in 25/180 (14%) sampled bighorn sheep and A. ovis msp4 sequences were amplified by polymerase chain reaction (PCR) and sequenced from 9/23 (39%) of seropositive animals. All animals were negative by PCR for the related pathogens, Anaplasma phagocytophilum and Anaplasma marginale. All msp4 sequences identified in the bighorn sheep were identical and corresponded to a single A. ovis genotype that was identical to a sheep isolate reported previously from Idaho. The finding of a single genotype of A. ovis in this wild herd of bighorn sheep was in contrast to the genetic diversity reported for A. marginale in cattle herds in the western United States and worldwide. These results demonstrated that bighorn sheep may be a wildlife reservoir of A. ovis in Montana.     

Bluetongue virus serotype 8 (BTV-8) infection reduces fertility of Dutch dairy cattle and is vertically transmitted to offspring

In 2007, BTV-8 re-emerged for the second year in the Netherlands and caused morbidity and increased mortality in cattle herds. In addition, cattle farmers reported reduced fertility in their cows. For this study, fifteen herds that were not vaccinated were selected. These were matched to 10 vaccinated herds by geographic region. At the start of the study, in July 2008, all cattle in the non-vaccinated herds >1 year old were sampled. All seronegative cows entered the study program and blood samples from these cows were tested for antibodies against BTV-8 in an ELISA. Cows were sampled at intervals of three weeks and sampling was stopped once a cow tested seropositive. Sampling ceased in all remaining cows in December 2008.Newborn calves originating from infected dams or from vaccinated dams were tested by PCR for BTV-8. Fertility data were obtained from the Royal Dutch Cattle Syndicate (CRV). Multi-level generalized latent and linear models were used for analyses.In 2008, 185 (17.2%) out of 1,074 initially seronegative non-vaccinated cattle seroconverted and were assumed to be infected with BTV-8. Infected cows were 5 (95% CI: 1.9-14.3) times more likely to return for insemination within 56 days after first insemination. In addition, these cows needed 1.7 (95% CI: 1.4-2.0) times more inseminations for an assumed pregnancy, and needed 2.5 (95% CI: 2.4-2.6) times more days between first and last insemination compared to the period prior to seroconversion and compared to cows not infected by BTV-8 in 2008. No association between BTV-8 infection and the chance to abort between 100 and 260 days after last insemination was found.In total, 48 calves originating from infected cows were tested by PCR for the presence of BTV-8. Ten (20.8%) out of these 48 calves were born PCR-positive. None of 256 calves from vaccinated dams tested PCR-positive. Further, cows infected during the second half of gestation had a 15.5 times (95% CI: 1.3-190.4) higher chance of a PCR-positive newborn calf compared to cows infected in the first half of gestation. This study showed that BTV-8 has a negative effect on fertility of dairy cattle.     

Serological diagnosis of neosporosis in a herd of dairy cattle in southern Brazil.

Neospora caninum-specific antibodies were detected in 60 of 172 (34.8%) dairy cattle by enzyme-linked immunosorbent assay (ELISA) in a herd from Parana State, Brazil. The seropositive animals included 47 of 126 (37.3%) adult cows, 7 of 29 (24%) heifers (1-2 yr), 4 of 15 (27%) heifers (5 mo-1 yr), and 2 precolostral samples. Data collected over a 9-yr follow-up period revealed that the proportion of pregnancies ending in abortion was 20% (31/154) among ELISA seropositive cows and 8% (15/193) among seronegative cows. The farm recorded 46 abortions, of which 31 (67.3%) were from seropositive cows. All sera positive by ELISA (n = 60) and sera from cows (n = 11) that were ELISA-negative but that had aborted were tested by the indirect fluorescent antibody test (IFAT) at dilutions from 1:25 to 1:200. All sera from ELISA-positive cows (n = 47) had an IFAT titer of 1:25:35 (74%) of these sera were also seropositive at a dilution of 1:200 (IFAT). Cows seropositive by ELISA had a 4-fold increased risk of having aborted at least once, compared to ELISA-seronegative cows. This association was statistically significant (P = 0.0016). The attributable fraction for this association indicated that approximately 76% of the risk for a cow having a history of abortion was attributable to seroconversion to N. caninum.     

Characterization of Foot-And-Mouth Disease Viruses (FMDVs) from Ugandan Cattle Outbreaks during 2012-2013: Evidence for Circulation of Multiple Serotypes

To investigate the foot-and-mouth disease virus (FMDV) serotypes circulating in Uganda’s cattle population, both serological and virological analyses of samples from outbreaks that occurred during 2012–2013 were performed. Altogether, 79 sera and 60 oropharyngeal fluid (OP)/tissue/oral swab samples were collected from herds with reported FMD outbreaks in seven different Ugandan districts. Overall, 61/79 (77%) of the cattle sera were positive for antibodies against FMDV by PrioCHECK FMDV NS ELISA and solid phase blocking ELISA detected titres ≥ 80 for serotypes O, SAT 1, SAT 2 and SAT 3 in 41, 45, 30 and 45 of these 61 seropositive samples, respectively. Virus neutralisation tests detected the highest levels of neutralising antibodies (titres ≥ 45) against serotype O in the herds from Kween and Rakai districts, against SAT 1 in the herd from Nwoya district and against SAT 2 in the herds from Kiruhura, Isingiro and Ntungamo districts. The isolation of a SAT 2 FMDV from Isingiro was consistent with the detection of high levels of neutralising antibodies against SAT 2; sequencing (for the VP1 coding region) indicated that this virus belonged to lineage I within this serotype, like the currently used vaccine strain. From the Wakiso district 11 tissue/swab samples were collected; serotype A FMDV, genotype Africa (G-I), was isolated from the epithelial samples. This study shows that within a period of less than one year, FMD outbreaks in Uganda were caused by four different serotypes namely O, A, SAT 1 and SAT 2. Therefore, to enhance the control of FMD in Uganda, there is need for efficient and timely determination of outbreak virus strains/serotypes and vaccine matching. The value of incorporating serotype A antigen into the imported vaccines along with the current serotype O, SAT 1 and SAT 2 strains should be considered.     

Development of an ELISA system for tick-borne encephalitis virus infection in rodents

Tick-borne encephalitis (TBE) virus causes severe encephalitis with serious sequelae in humans. An epizootiological survey of wild rodents is effective to detect TBE virus-endemic areas; however, limited serological diagnostic methods are available to detect anti-TBE virus antibodies in wild rodents. In this study, ELISAs for the detection of rodent antibodies against the TBE virus were developed using two recombinant proteins, domain III of the E protein (EdIII) and subviral particles (SPs), as the antigens. As compared with the neutralization test, the ELISA using EdIII had 77.1% sensitivity and 80.0% specificity, and the ELISA using SPs had 91.4% sensitivity and 100% specificity. Furthermore, when the ELISAs were applied to the epizootiological survey in the TBE virus-endemic area, both of the ELISAs was able to detect wild rodents with TBE virus-specific antibodies. This is the first study to show that ELISAs using recombinant antigens can be safe and useful in the detection of TBE virus-infected wild rodents in epizootiological research.     

Do commercial serologic tests for Trypanosoma cruzi infection detect Mexican strains in women and newborns?

We sought to determine the serological test that could be used for Trypanosoma cruzi seroprevalence studies in Mexico, where lineage I predominates. In a previous study among pregnant women and their newborns in the states of Yucatan and Guanajuato, we reported a 0.8-0.9% of prevalence for T. cruzi -specific antibodies by Stat-Pak and Wiener ELISA. We have expanded this study here by performing an additional non-commercial ELISA and confirming the seropositives with Western blot, using whole antigens of a local parasite strain. We found a seroprevalence of 0.6% (3/500) in Merida and 0.4% in Guanajuato (2/488). The 5 seropositive umbilical cord samples reacted to both non-commercial ELISA and Western blot tests, and only 1 of the maternal samples was not reactive to non-commercial ELISA. A follow-up of the newborns at 10 mo was performed in Yucatan to determine the presence of T. cruzi antibodies in children as evidence of congenital infection. None of the children was seropositive. One newborn from an infected mother died at 2 wk of age of cardiac arrest, but T. cruzi infection was not confirmed. The T. cruzi seroprevalence data obtained with both commercial tests (Stat-Pak and ELISA Wiener) are similar to those from non-commercial tests using a local Mexican strain of T. cruzi.     

Low predictive value of seroprevalence of Toxoplasma gondii in cattle for detection of parasite DNA

The role of beef in human infections with Toxoplasma gondii is not clear. To get a better understanding of the value of seroprevalence as an indication of the role of beef in human infections with T. gondii we studied the seroprevalence of T. gondii in Dutch cattle and analysed the correlation between detection of antibodies and parasitic DNA. An indirect ELISA was developed and used to test a sample of the Dutch cattle population. Since validation of the ELISA was hampered by a lack of sufficient bovine reference sera, the results were analysed in two different ways: using a cut-off value that was based on the course of the OD in 27 calves followed from birth until 16 months of age, and by fitting a mixture of two normal distributions (binormal mixture model) to the log-transformed ODs observed for the different groups of cattle in the study population. Using the cut-off value, the seroprevalence was estimated at 0.5% for white veal, 6.4% for rosé veal and 25.0% for cattle. However, using the frequency distributions the prevalences were higher: 1.9% for white veal, 15.6% for rosé veal and 54.5% for cattle. Next, for 100 cattle the results with two different serological assays (ELISA and Toxo-Screen DA) were compared with detection of parasites by our recently developed sensitive magnetic capture PCR. Toxoplasma gondii DNA was detected in only two seronegative cattle. This discordance demonstrates that seroprevalence cannot be used as an indicator of the number of cattle carrying infectious parasites. Demonstrating parasitic DNA in seronegative cattle and not in seropositive cattle suggests that only recent infections are detectable. Whether beef from these PCR-positive cattle is infectious to humans remains to be studied.     

Estimation of vertical and horizontal transmission parameters of Neospora caninum infections in dairy cattle

Transmission parameters of Neospora caninum infections in dairy cattle were determined in six herds with a history of Neospora-associated abortions, using an antibody-detection ELISA to detect evidence of infection. A total of 124 seropositive dams and their calves were tested at calving to estimate vertical transmission, and 154 seronegative heifers were monitored prospectively from birth for evidence of post-natal infection. The probability of vertical transmission was very high; 95.2% (95% confidence interval: 89.8%, 98.2%) of the seropositive dams produced calves that were seropositive prior to consumption of colostrum. In the prospective study, three heifers seroconverted, by 1, 8 and 16 months of age, and the overall incidence rate of seroconversion was 1.9 per 100 heifer-years at risk, ranging from 0 to 4.1 per 100 heifer-years at risk within herds. If heifers with inconclusive ELISA results were included in the analysis, the incidence rate of seroconversion was 3.8 per 100 heifer-years at risk, ranging from 0 to 8.3 per 100 heifer-years at risk within herds. This study showed that vertical transmission was the major route involved in the spread of N. caninum in these herds, and that there was only a low detectable level of horizontal transmission. However, further studies are needed to provide parasitological evidence of infection in those heifers that seroconverted.     

Development of ELISA using recombinant antigens for specific detection of mouse parvovirus infection.

Nucleotide sequences of mouse parvovirus (MPV) isolate, named MPV/UT, and mouse minute virus (MMV) were analyzed and used for expressing recombinant proteins in E. coli. ELISA tests using recombinant major capsid protein (rVP2) and recombinant major non-structural protein (rNS1) as antigens were developed and their performance in serologic detection of rodent parvovirus infection was assessed. MPV-rVP2 and MMV-rVP2 ELISAs reacted specifically with anti-MPV and anti-MMV mouse sera, respectively. MMV-rNS1 antigen had a wide reaction range with antisera to rodent parvoviruses including MPV, MMV, Kilham rat virus (KRV) and H-1 virus. All mice oronasally infected with MPV were seropositive at 4 weeks post-infection in screening by ELISAs using MPV-rVP2 and MMV-rNS1 antigens, but were negative by conventional ELISA using whole MMV antigen. A contact transmission experiment revealed that transmission of MPV occurred up to 4 weeks post-infection, and all cage mates were seropositive in screening with MPV-rVP2 and MMV-rNS1 ELISAs. These results indicate that MPV-rVP2 and MMV-rVP2 are specific ELISA antigens which distinguish between MPV and MVM infection, while MMV-rNS1 antigen can be used in generic ELISA for a variety of rodent parvoviruses. The higher sensitivity of MPV-rVP2 ELISA than conventional ELISA for detecting seroconversion to MPV in oronasally infected mice as well as in cage mates suggests the usefulness of MPV-rVP2 ELISA in quarantine and microbiological monitoring of MPV infection in laboratory mice.