Variability in results of the microscopic agglutination test in dogs with clinical leptospirosis and dogs vaccinated against leptospirosis

Background: The microscopic agglutination test (MAT) is commonly used for the diagnosis of canine leptospirosis. In dogs it is sometimes suggested that the serogroup with the highest MAT titer is the infecting serogroup; however, this is not true in humans with confirmed leptospirosis. We sought to investigate the value of MAT results in predicting the infecting serogroup by comparing results across several laboratories and within individual dogs over time. Objectives: To examine the variability in MAT results across different laboratories in dogs recently vaccinated against leptospirosis, and in dogs with clinical leptospirosis, and to investigate variability over time in MAT results in individual dogs with leptospirosis. Animals: Eighteen dogs from a research colony, 9 of which had been vaccinated against leptospirosis, and 17 dogs clinically diagnosed with leptospirosis. Methods: Serum samples were submitted to up to 5 veterinary diagnostic laboratories for MAT titers from each dog on at least 1 occasion. MAT results also were followed over time in 6 dogs diagnosed with leptospirosis. Results: MAT results were discordant across different laboratories in dogs recently vaccinated against leptospirosis and in dogs with clinical leptospirosis. MAT results varied over time in individual dogs with the disease. Conclusions and Clinical Importance: The MAT is a valuable test for the diagnosis of leptospirosis in dogs, but it is unlikely that test results can be used to predict the infecting serogroup. Laboratories offering the MAT should consider participation in a proficiency scheme.     

New enzyme-linked immunoassay for the detection of specific antibodies against multiple Leptospira serogroups in bovine sera

Leptospirosis is a zoonotic disease with worldwide endemicity in Argentina, it is a significant public health problem in low-income populations. Bovine leptospirosis is a serious economic problem for cattle production, causing abortions, reduced milk yield, mortality in calves and decreased daily weight gain. We developed an enzyme-linked immunosorbent assay (ELISA) with sonicated Leptospira interrogans serogroup Icterohaemorrhagiae serovar Copenhageni M 20. We evaluated its performance for the detection of specific antibodies against multiple Leptospira serogroups in bovine. The microscopic agglutination test (MAT) was used as the gold standard. The performance of this ELISA was evaluated with a panel of sera (118 MAT confirmed positive and 97 MAT negative). The overall sensitivity was close to 85.6 % and the specificity was 83.5 %, according to the MAT reference method. Analytical specificity of the IgG-ELISA was evaluated using 50 bovine serum samples from animals showing serum antibodies against other pathogens that cause abortion in bovine, such as Brucella sp., Neospora sp. and Bovine Viral Diarrhea (BVD), and no cross-reaction was observed. This IgG-ELISA can be an alternative to the MAT for diagnosis of leptospiral infection in bovine.     

Prevalence of serum antibodies against six Leptospira serovars in healthy dogs

OBJECTIVE: To determine the prevalence of antibodies against 6 Leptospira serovars and determine risk factors associated with positive Leptospira titers in healthy client-owned dogs in Michigan. DESIGN: Cross-sectional study. ANIMALS: 1,241 healthy dogs at least 4 months of age. PROCEDURES: Dogs were examined by veterinarians at private practices. Vaccinated and unvaccinated dogs were enrolled in the study, which occurred prior to the availability of a 4-serovar (Canicola, Grippotyphosa, Icterohaemorrhagiae, and Pomona) Leptospira vaccine. Sera were tested by use of the microscopic agglutination test to determine antibody titers against Leptospira serovars Bratislava, Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, and Pomona. A questionnaire was used to collect demographic information about each dog to identify risk factors associated with seropositive status. RESULTS: 309 of 1,241 (24.9%) dogs had antibody titers against at least 1 of the 6 Leptospira serovars, which suggested exposure to Leptospira spp. Prevalence of antibodies was highest to serovar Grippotyphosa, followed by Bratislava, Canicola, Icterohaemorrhagiae, and Pomona. Age, travel outside Michigan, exercise outside fenced yards, and exposure to livestock and wildlife were significant risk factors for positive titers. CONCLUSIONS AND CLINICAL RELEVANCE: Among healthy dogs from the lower peninsula of Michigan, > 20% have antibodies against leptospiral serovars historically considered uncommon but more recently incriminated as causing clinical canine leptospirosis. Wildlife and livestock may be of increasing importance as reservoirs for canine leptospirosis as urbanization continues to occur. Expanded vaccination strategies may partially mitigate these trends.     

Captive Agamid lizards in Germany: Prevalence,pathogenicity and therapy of gastrointestinal protozoan and helminth infections

Reptiles are becoming popular pets in many parts of the world. They are also known to harbor numerous gastrointestinal parasites. We used faecal smears to examine 748 stool samples from 14 different agamid lizard species. In addition, we used coproantigen ELISA tests (11 samples) and immunofluorescence assays (IFA) (19 samples) to detect reptile Cryptosporidium infections. In 28 cases, veterinarians requested therapy to treat oxyurid- and/or Isospora amphiboluri-infections and resent fecal samples after proposed therapy and anti-parasitic treatments had been applied. We also performed complete dissections of 24 deceased agamas in order to specify protozoan and helminth parasite infections.Overall, the examined fecal samples contained 6 different taxa. Oxyurids (Pharyngodonidae) were the most prevalent nematodes (41.2%), followed by I. amphiboluri (17.0%), Entamoeba spp. (0.8%), Choleoeimeria spp. (0.5%), Trichomonas spp. (0.3%), Cryptosporidium spp. (0.3%) and Strongyloides-like nematodes (0.1%). I. amphiboluri infections were significantly more prevalent (Chi-square test: χ² = 21,5, df = 1, P < 0.001) in juvenile agamid lizards (31.9%) than in adults (14.2%). One of 11 (9.1%) coproantigen ELISA-examined samples was positive for Cryptosporidium. In 10.5% of the samples we found oocysts of Cryptosporidium. Thirteen (54.2%) of necropsied agamid lizards were infected with endoparasites and it is likely that three (12.5%) of them died due to severe parasitic infections. 74.0% of the samples that were submitted after therapy had been applied were negative. The high prevalences and pathological findings of several clinical parasitoses observed in these exotic reptiles calls for more detailed investigations on agamid gastrointestinal parasite fauna.     

Improved detection of equine antibodies against Sarcocystis neurona using polyvalent ELISAs based on the parasite SnSAG surface antigens

Equine protozoal myeloencephalitis (EPM) is a common neurologic disease of horses that is caused by the apicomplexan pathogen Sarcocystis neurona. To help improve serologic diagnosis of S. neurona infection, we have modified existing enzyme-linked immunosorbent assays (ELISAs) based on the immunogenic parasite surface antigens SnSAG2, SnSAG3, and SnSAG4 to make the assays polyvalent, thereby circumventing difficulties associated with parasite antigenic variants and diversity in equine immune responses. Two approaches were utilized to achieve polyvalence: (1) mixtures of the individual recombinant SnSAGs (rSnSAGs) were included in single ELISAs; (2) a collection of unique SnSAG chimeras that fused protein domains from different SnSAG surface antigens into a single recombinant protein were generated for use in the ELISAs. These new assays were assessed using a defined sample set of equine sera and cerebrospinal fluids (CSFs) that had been characterized by Western blot and/or were from confirmed EPM horses. While all of the polyvalent ELISAs performed relatively well, the highest sensitivity and specificity (100%/100%) were achieved with assays containing the rSnSAG4/2 chimera (Domain 1 of SnSAG4 fused to SnSAG2) or using a mixture of rSnSAG3 and rSnSAG4. The rSnSAG4 antigen alone and the rSnSAG4/3 chimera (Domain 1 of SnSAG4 fused to Domain 2 of SnSAG3) exhibited the next best accuracy at 95.2% sensitivity and 100% specificity. Binding ratios and percent positivity (PP) ratios, determined by comparing the mean values for positive versus negative samples, showed that the most advantageous signal to noise ratios were provided by rSnSAG4 and the rSnSAG4/3 chimera. Collectively, our results imply that a polyvalent ELISA based on SnSAG4 and SnSAG3, whether as a cocktail of two proteins or as a single chimeric protein, can give optimal results in serologic testing of serum or CSF for the presence of antibodies against S. neurona. The use of polyvalent SnSAG ELISAs will enhance the reliability of serologic testing for S. neurona infection, which should lead to improved diagnosis of EPM.     

Associations between Campylobacter fetus, Haemophilus somnus, Leptospira hardjo and bovine viral diarrhea virus antibody titers in dairy cattle from a California herd

In a cross-sectional study, the associations of age, serological status against Campylobacter fetus (CF), Haemophilus somnus (HS) and Leptospira hardjo (LH) with the seroprevalence of bovine viral diarrhea virus (BVDV) in dairy cattle were evaluated. Six hundred and thirty cattle from a herd in Tulare county of California were included in the study. Blood samples from the cattle were collected in December 1986. Enzyme-linked immunosorbant assay (ELISA) was used to determine the positive or negative status of cows against CF, HS, LH and BVDV antigens. Loglinear analysis was used to describe the relationships of four categorical variables with the seroprevalence of BVDV. The final loglinear model was interpreted as a logit model and obtained by conditioning on explanatory variables in the model found to relate significantly with BVDV seropositivity. The hierarchical logit model included two two-way interactions (BVDV × HS and BVDV × LH) and a three-way interaction (BVDV × CF × age). HS-seronegative cattle had 4.7 times greater odds of being BVDV-seropositive compared with HS-seropositive cattle. In contrast, LH-seropositive cattle had 2.7 times increased odds of BVDV seropositivity compared with LH-seronegative cattle. The presence of the three-way interaction term in the model involving age, CF and BVDV seroprevalence implies that among heifers, CF-seronegative and CF-seropositive animals had about the same odds of BVDV seropositivity (e.g. adjusted odds ratios = 20.5 vs. 24.5), whereas, among cows, CF-seronegative animals had at least twice the odds of being BVDV-seropositive than those of CF-seropositive animals (e.g. adjusted odds ratios = 23.0 vs. 11.0).     

Use of the Leptospira sp. ligB C-terminus coding region as a diagnostic tool of animal leptospirosis

Leptospirosis is the most widespread zoonosis worldwide, and it can cause reproductive failures in livestock, while in humans may vary from a mild fever to multi-organ failure and death. Due to this, in this study, we evaluated the usefulness of the segment encoding LigB C-terminus region, only present in pathogenic as target for a diagnostic PCR.This new PCR yielded a 100 % positivity for pathogenic Leptospira species and no cross-reactivity was found with intermediate or non-pathogenic species, or with other microorganisms, demostrating its high analytical specificity.The estimated analytical sensitivity was higher in serum samples than in blood or urine samples (6−9 × 10² lept/mL and 6−9 × 10⁵ and 6−9 × 10⁶ lept/mL, respectively). Multiple sequence alignment of the target region from different pathogenic Leptospira species confirmed that this gene region is highly conserved among these species, with few single nucleotide polymorphisms.The ligb-ct PCR here developed appears as a useful tool for the molecular diagnosis of leptospirosis.     

Prevalence of antibodies of Leptospira serovars in beef cattle in central Queensland.

OBJECTIVE: To obtain up-to-date data on the prevalence of antibodies to Leptospira serovars in central Queensland beef herds preliminary to assessing their role in bovine subfertility and the role of cattle as a zoonotic reservoir. DESIGN: Sera from 2857 female cattle in 68 central Queensland beef herds were tested for antibodies to 14 Leptospira serovars using the microscopic agglutination test. Vaccination use and age of cattle were collected to enable the calculation of crude and age-stratified seroprevalences. RESULTS: The most commonly detected antibodies were to serovars hardjo (15.8% crude seroprevalence), tarassovi (13.9%), pomona (4.0%) and szwajizak (2.2%). Vaccinates were omitted from the hardjo and pomona seroprevalence data. The seroprevalence for hardjo and pomona tended to increase with age of the animals. CONCLUSION: These results are broadly similar to those of previous serological surveys. The data suggest that serovars other than hardjo, pomona and tarassovi, are unlikely to have a significant role in bovine subfertility and that cattle are unlikely to be a source of human infection with them in central Queensland.     

Detection and characterization of Leptospira spp. in dogs diagnosed with kidney and/or liver disease in Selangor,Malaysia

Leptospirosis is a serious bacterial disease that affects both humans and animals. A wide range of symptoms have been described in humans; the disease in dogs is commonly associated with kidney and/or liver disease. In Malaysia, information about the common serovars infecting dogs is limited. Therefore, we investigated the occurrences of leptospirosis in 124 pet dogs diagnosed with kidney and/or liver disease. Blood, urine, abdominal effusion, and/or kidney and liver were collected from the dogs. Based on microscopic agglutination testing, 53 of 124 (42.7%) dogs were seropositive for leptospiral exposure. Sera were frequently positive to serovars Bataviae (n = 12), Javanica (n = 10), and Icterohaemorrhagiae (n = 10). Direct detection using PCR showed that 42 of 124 (33.9%) of the whole blood and 36 of 113 (31.9%) urine samples were positive for pathogenic Leptospira spp. By PCR, 2 of 23 (9.1%) kidney and 2 of 23 (9.1%) liver were positive for pathogenic Leptospira spp. Abdominal effusion from 4 dogs were PCR-positive for pathogenic Leptospira spp. The species detected were L. interrogans, L. borgpetersenii, L. kirschneri, and L. kmetyi by partial 16S rRNA sequencing. We further identified and characterized 11 Leptospira spp. isolates from 8 dogs as serovars Bataviae, Javanica, and Australis. The mortality rate of the Leptospira-infected dogs was high (18 of 53; 34%).     

Differentiation of feline immunodeficiency virus vaccination, infection, or vaccination and infection in cats

BACKGROUND: Serodiagnosis of feline immunodeficiency virus (FIV) is complicated by the use of a formalin-inactivated whole-virus FIV vaccine. Cats respond to immunization with antibodies indistinguishable from those produced during natural infection by currently available diagnostic tests, which are unable to distinguish cats that are vaccinated against FIV, infected with FIV, or both. HYPOTHESIS: An enzyme-linked immunosorbent assay (ELISA) detecting antibodies against formalin-treated FIV whole virus and untreated transmembrane peptide will distinguish uninfected from infected cats, regardless of vaccination status. ANIMALS: Blood samples were evaluated from uninfected unvaccinated cats (n = 73 samples), uninfected FIV-vaccinated cats (n = 89), and FIV-infected cats (n = 102, including 3 from cats that were also vaccinated). METHODS: The true status of each sample was determined by virus isolation. Plasma samples were tested for FIV antibodies by a commercial FIV diagnostic assay and an experimental discriminant ELISA. RESULTS: All samples from uninfected cats were correctly identified by the discriminant ELISA (specificity 100%). Of the samples collected from FIV-infected cats, 99 were correctly identified as FIV-infected (sensitivity 97.1%). CONCLUSIONS AND CLINICAL IMPORTANCE: With the exception of viral isolation, the discriminant ELISA is the most reliable assay for diagnosis of FIV. A practical strategy for the diagnosis of FIV infection would be to use existing commercial FIV antibody assays as screening tests. Negative results with commercial assays are highly reliable predictors for lack of infection. Positive results can be confirmed with the discriminant ELISA. If the discriminant ELISA is negative, the cat is probably vaccinated against FIV but not infected. Positive results are likely to represent infection.     

Prevalence of antibodies to Leptospira serovars in sheep and goats in Alto Adige-South Tyrol

Serum samples from 313 sheep and 95 goats were collected during November 1993 in 26 localities in Alto Adige-South Tyrol and tested by microscopic agglutination test for antibodies to 28 serovars of the genus Leptospira. At the time of blood collection all the animals appeared healthy with no clinical sign suggestive of leptospirosis. The observed seroprevalence in sheep was 6.1%, whereas the seropositivity rate for goat serum samples was 2.1%. The highest serological prevalence in sheep was recorded for serovar castellonis, followed by poi, sejroe, hardjo subtype hardjobovis, copenhageni, and cynopteri. Titres to poi were the only ones found in goats. These findings, which are proof of Leptospira infection in Alto Adige-South Tyrol, indicate that foci of several serovars exist in this region.     

Prevalence of serum antibodies to bovine herpesvirus-1 and bovine viral diarrhea virus in beef cattle in Uruguay

Our objective was to determine the prevalence of serum antibodies to bovine herpesvirus-1 (BHV-1) and bovine viral diarrhea (BVD) virus in beef cattle in Uruguay. A random sample of 230 herds selected with probability proportional to population size based on the number of cattle was chosen from a list frame of all registered livestock farms as of June 1999. Sera from up to 10 heifers, cows and bulls (up to 30 sera total per herd) were collected on selected farms between March 2000 and March 2001 and evaluated by means of enzyme-linked immunosorbent assays (ELISAs). Overall, 6358 serum samples were evaluated. We also collected data on previous diagnosis of BHV-1 or BVD infections and on the use of vaccines against these agents.

The estimated prevalence of exposure to BHV-1 and BVD at the herd level for the Uruguayan beef population was 99% and 100%, respectively. Approximately 37% of beef cattle in Uruguay have been exposed to BHV-1 and 69% to BVD virus. Only 3% of beef herds in Uruguay regularly (typically, annually) use vaccines against either of these agents.     

猪瘟单克隆抗体的制备及ACI-ELISA检测猪瘟病毒的研究

本研究用猪瘟石门毒(CSFV-Shimen)免疫BALB/C小鼠,按常规单克隆抗体(McAb)技术方法制作,最终获得4株McAb,分别命名为AC9、CF8、DG5和EC9,4株McAb与基因工程CSFV E2蛋白反应结果表明:AC9、CF8和EC9是抗CS-FV E2蛋白的McAb.用AC9和CF8McAb对CSFV进行抗原捕获间接ELJSA试验(ACI-ELISA),通过一元McAb和二元McAb CAI-ELISA试验的比较,结果表明AC9与CF8两种McAb有协同作用,其捕获CSFV的能力比一元McAb显著提高.方阵试验结果表明:McAb和血清多抗(PcAb)的最佳工作稀释度分别为1:400和1:200.特异性试验和敏感性试验结果显示本法特异性强,敏感性高.最后用ACI-ELISA与PCR对30份病料的检测结果比较,表明ACI-ELISA与PCR检测结果相符.上述结果说明本研究所获得AC9和CF8可用作猪瘟诊断试剂盒的研制,是检测CSFV的有效方法.     

Prevalence of Leptospira interrogans antibodies in free-ranging Tayassu pecari of the Southern Pantanal, Brazil, an ecosystem where wildlife and cattle interact

We surveyed a wild population of white-lipped peccaries (Tayassu pecari) in the Brazilian Pantanal for evidence of Leptospira interrogans. Serum samples from 71 free-ranging T. pecari were obtained between 2003 and 2005 in the southern Pantanal of Mato Grosso do Sul state. We used microscopic microagglutination to test for antibodies against 14 L. interrogans serovars (antibody titers ≥1:100 were considered seropositive). Seventy percent of captured animals tested positive for leptospirosis antibodies. Antibodies against icterohaemorrhagiae and autumnalis serovars were the most prevalent. We used log-linear analyses to test for associations among seropositivity, age class, and sex of captured animals. Seropositivity was strongly associated with animal age class, but independent of sex. Forty-six percent of animals less than 2 years old, 63% of adults during peak reproductive years, and 100% of the oldest age class were seropositive. A nonparametric multivariate procedure (MRPP) showed that the composition of serovar antibody types changed with age, and ANOVA models demonstrated that antibody titers increased with age, suggesting long-term exposure to a greater number and variety (i.e., serovar types) of L. interrogans infections. This study presents the first quantitative survey of antibodies against L. interrogans serovars in a T. pecari population of the Pantanal. The high prevalence of leptospirosis antibodies in free-ranging white-lipped peccaries and the potential impacts on reproduction and population dynamics emphasize the need for further studies investigating the roles of Pantanal wildlife and livestock in the transmission and maintenance of L. interrogans in the environment.     

Indirect ELISA for the detection of antibodies against Opisthorchis felineus (Rivolta, 1884) and Metorchis bilis (Braun, 1790) in foxes

Serological investigations focused on the detection of specific opisthorchiid liver fluke antibodies in silver foxes (Vulpes vulpes fulva). Animals were experimentally infected with Opisthorchis felineus (nos. 1 and 2) and Metorchis bilis (nos. 3–8) by feeding fish with a counted number of metacercariae. Four foxes remained as non-infected negative controls (nos. 9–12). For the indirect ELISA, an excretory–secretory antigen was produced by in vitro cultivation of O. felineus and M. bilis adults isolated from livers of experimentally infected hamsters.

Immunoglobulin G (IgG) seroconversion against homologous antigen took place between weeks 2 and 6 postinfection (p.i.) and foxes remained seropositive up to the end of the trial at week 41 p.i. In contrast, IgG titres against heterologous antigen remained significantly lower and stayed near the cut-off. All infected animals excreted opisthorchiid eggs, starting between weeks 2 and 4 p.i. The number of liver flukes found at necropsy was relatively low, except in one fox that was sacrificed at the week 11 p.i. These results suggest that the ELISA is a suitable tool for the detection of specific O. felineus and M. bilis antibodies in the fox.     

Improvement of Leptospira spp. diagnosis in aborted bovine fetuses by qPCR

Leptospirosis is a disease with major economic impact on livestock industry. The objective of this work was to determine the presence of Leptospira spp. DNA by qPCR in bovine fetuses with presumptive diagnosis of leptospirosis as the cause of abortion. Leptospira spp. DNA was detected by qPCR in 11 out of 34 fetuses. These specimens (10/11) had histopathological findings in hepatic and/or renal tissues compatible with leptospirosis. qPCR detection rate (32.4 %) was higher compared with direct immuno-fluorescence antibody test (DFAT) (11.8 %). The concordance coefficient between both techniques was 0.44. qPCR is a rapid and sensitive technique for the diagnosis of leptospirosis and improved the detection rate in fetal tissues compared with DFAT. Implementation of molecular techniques may increase the accurate detection of leptospirosis as a cause of bovine abortion allowing the application of rapid therapeutic and prophylactics measures in order to reduce the impact of this zoonotic disease.