Distribution and Prevalence of the Australian Non-Pathogenic Rabbit Calicivirus Is Correlated with Rainfall and Temperature

Background

Australia relies heavily on rabbit haemorrhagic disease virus (RHDV) for the biological control of introduced European wild rabbits Oryctolagus cuniculus, which are significant economic and environmental pests. An endemic non-pathogenic rabbit calicivirus termed RCV–A1 also occurs in wild rabbits in Australian and provides partial protection against lethal RHDV infection, thus interfering with effective rabbit control. Despite its obvious importance for rabbit population management, little is known about the epidemiology of this benign rabbit calicivirus.

Methods

We determined the continent-wide distribution and prevalence of RCV-A1 by analysing 1,805 serum samples from wild rabbit populations at 78 sites across Australia for the presence of antibodies to RCV-A1 using a serological test that specifically detects RCV-A1 antibodies and does not cross-react with co-occurring RHDV antibodies. We also investigated possible correlation between climate variables and prevalence of RCV-A1 by using generalised linear mixed effect models.

Results

Antibodies to RCV-A1 were predominantly detected in rabbit populations in cool, high rainfall areas of the south-east and south-west of the continent. There was strong support for modelling RCV-A1 prevalence as a function of average annual rainfall and minimum temperature. The best ranked model explained 26% of the model structural deviance. According to this model, distribution and prevalence of RCV-A1 is positively correlated with periods of above average rainfall and negatively correlated with periods of drought.

Implications

Our statistical model of RCV-A1 prevalence will greatly increase our understanding of RCV-A1 epidemiology and its interaction with RHDV in Australia. By defining the environmental conditions associated with the prevalence of RCV-A1, it also contributes towards understanding the distribution of similar viruses in New Zealand and Europe.     

Evolution and Phylogeography of the Nonpathogenic Calicivirus RCV-A1 in Wild Rabbits in Australia

Despite its potential importance for the biological control of European rabbits, relatively little is known about the evolution and molecular epidemiology of rabbit calicivirus Australia 1 (RCV-A1). To address this issue we undertook an extensive evolutionary analysis of 36 RCV-A1 samples collected from wild rabbit populations in southeast Australia between 2007 and 2009. Based on phylogenetic analysis of the entire capsid sequence, six clades of RCV-A1 were defined, each exhibiting strong population subdivision. Strikingly, our estimates of the time to the most recent common ancestor of RCV-A1 coincide with the introduction of rabbits to Australia in the mid-19th century. Subsequent divergence events visible in the RCV-A1 phylogenies likely reflect key moments in the history of the European rabbit in Australia, most notably the bottlenecks in rabbit populations induced by the two viral biocontrol agents used on the Australian continent, myxoma virus and rabbit hemorrhagic disease virus (RHDV). RCV-A1 strains therefore exhibit strong phylogeographic separation and may constitute a useful tool to study recent host population dynamics and migration patterns, which in turn could be used to monitor rabbit control in Australia.Lagoviruses form a genus within the Caliciviridae family of RNA viruses (16). All representatives of this genus are highly species specific and infect only their respective hosts, i.e., rabbits and hares. The prototype species of the genus Lagovirus is rabbit hemorrhagic disease virus (RHDV), which was first described in China in 1984 when a severe infectious necrotizing hepatitis with mortality rates up to 90% was observed in angora rabbits (22). However, phylogenetic analysis indicates that the pathogenic strains of RHDV likely evolved from nonpathogenic lagoviruses several decades before they were initially described (20). Today, RHDV is found on most continents, causing ongoing damage to the rabbit meat industry (24) and threatening wild native rabbit populations in Europe. On the Iberian peninsula the rabbit is considered an endangered species and is itself a staple food of endangered predators, such as the imperial eagle and the Iberian lynx (10).In marked contrast, since 1995 Australia has been using RHDV as a successful viral biocontrol agent for rabbits (7), which cause severe environmental and economic damage in this country. The use of a viral biocontrol agent for a vertebrate species has been and remains controversial, but there is little doubt that since its release RHDV has generated close to $6 billion of savings to the Australian agricultural industry (41), as well as some much needed relief for the regeneration of many native plant species (35).Notably, RHDV-induced mortality is lower in certain areas of Australia, namely, the cooler and more humid southeast region of the continent, which is believed to be in part due to the presence of related but nonpathogenic lagoviruses circulating in the population (8). Recently, such a virus was identified in Australian wild rabbits (38). This new member of the genus Lagovirus, termed rabbit calicivirus Australia 1 (RCV-A1), is a nonpathogenic virus causing a predominantly enteric infection in rabbits. Other benign or moderately pathogenic RCV strains have also been described in the United States and Europe (1, 5, 14, 15). While the Italian nonpathogenic RCV provides complete cross-immunity to RHDV, only partial protection is conveyed by the Australian virus RCV-A1 (37) although this may be sufficient to reduce overall RHDV-induced mortality.Like all lagoviruses, RCV-A1 has a single-stranded positive-sense RNA genome of approximately 7.5 kb that is polyadenylated and has a viral protein (VpG) covalently bound to its 5′ end (25). The genome is organized into two open reading frames (ORFs) (6, 39). ORF1, which represents the majority (7 kb) of the genome, encodes a polyprotein that is auto-proteolytically cleaved during posttranslational processing into several smaller proteins, including the helicase, protease, and polymerase (26). In contrast, ORF2 is only 351 nucleotides (nt) long, and the function of its VP10 gene product is unknown although it is present in small amounts in the virion (39).Given the potential interactions between RCVs and pathogenic RHDV, which could impact rabbit survival in both Australia and Europe, it is clearly important to understand the evolutionary history, genetic diversity, and geographic distribution of RCV-A1 and other benign lagoviruses. In Australia, growing reports of rising rabbit numbers mean that there is a clear need for improved strategies to monitor and control rabbit populations. In addition, because RCV-A1 does not cause any fitness decrease in its rabbit host (37), it is possible that this virus could be used as a population genetic marker to track movements and changes in the distribution of the European rabbit across the Australian continent. Indeed, other microorganisms have proven to be useful indicators of the population dynamics of their hosts (3, 42). Microbial markers are particularly informative for the study of population processes in the very recent past since genetic changes will not have had sufficient time to be recorded in the more slowly evolving host genome (2).The host-pathogen interaction of Australian rabbits and their viruses is unique in that both were introduced only once (or possibly a few times in a limited period of time). In addition, 150 years of historical records are available documenting the introduction, spread, and control efforts of rabbits in Australia. Herein, we explore the evolutionary history and dynamics of RCV-A1 in Australia, with a particular focus on revealing the phylogeographic distribution of the virus across this continent, the evolutionary processes that have shaped its diversity, and its suitability as a marker to study host distribution and migration patterns.     

实验兔出血症病毒检测方法的建立与免疫效果调查

目的检测全省六个实验兔场的兔子所携带的兔出血症病毒（RHDV）情况,调查实验兔RHDV抗体水平,评价不同疫苗的免疫效果,比较HAI与ELISA两种方法的符合率。方法采用HAI、ELISA方法对1168份实验兔RHDV抗体进行了检测,并与RT-PCR方法的检测结果进行对比分析。结果我省实验兔免疫情况较好,不同饲养场的实验兔免疫合格率虽有不同,但未发生疫情。通过比较发现ELISA法检测的抗体合格率明显高于HAI法。结论LISA、HAI和RT-PCR方法均适合实验兔RHDV的检测。     

Horizontal transmissible protection against myxomatosis and rabbit hemorrhagic disease by using a recombinant myxoma virus

We have developed a new strategy for immunization of wild rabbit populations against myxomatosis and rabbit hemorrhagic disease (RHD) that uses recombinant viruses based on a naturally attenuated field strain of myxoma virus (MV). The recombinant viruses expressed the RHDV major capsid protein (VP60) including a linear epitope tag from the transmissible gastroenteritis virus (TGEV) nucleoprotein. Following inoculation, the recombinant viruses induced specific antibody responses against MV, RHDV, and the TGEV tag. Immunization of wild rabbits by the subcutaneous and oral routes conferred protection against virulent RHDV and MV challenges. The recombinant viruses showed a limited horizontal transmission capacity, either by direct contact or in a flea-mediated process, promoting immunization of contact uninoculated animals.     

Serological evidence for the presence of non-pathogenic rabbit haemorrhagic disease virus-like strains in rabbits (Oryctolagus cuniculus) of the Kerguelen archipelago

Antibodies raised against a Lagovirus were found in healthy rabbits Oryctolagus cuniculus sampled in 2003 and 2004 in the Kerguelen archipelago. The serological test we used enabled the detection of antibodies due to both pathogenic and non-pathogenic viruses related to the rabbit haemorrhagic disease virus (RHDV). The overall proportion of seropositive rabbits was 35% and differed between sites. Since previous studies have failed to detect antibodies raised against pathogenic RHDV strains, the antibodies detected in the present study were likely due to non-pathogenic strains. The interest of these strains circulating in the Kerguelen archipelago is that they have evolved independently of those detected in the other parts of the world and should derive from an ancestral RHDV precursor. Their characterization may help understanding the evolution of the virus and the emergence of the disease.     

High adaptive variability and virus-driven selection on major histocompatibility complex (MHC) genes in invasive wild rabbits in Australia

The rabbit haemorrhagic disease virus (RHDV) was imported into Australia in 1995 as a biocontrol agent to manage one of the most successful and devastating invasive species, the European rabbit (Oryctolagus cuniculus cuniculus). During the first disease outbreaks, RHDV caused mortality rates of up to 97% and reduced Australian rabbit numbers to very low levels. However, recently increased genetic resistance to RHDV and strong population growth has been reported. Major histocompatibility complex (MHC) class I immune genes are important for immune responses against viruses, and a high MHC variability is thought to be crucial in adaptive processes under pathogen-driven selection. We asked whether strong population bottlenecks and presumed genetic drift would have led to low MHC variability in wild Australian rabbits, and if the retained MHC variability was enough to explain the increased resistance against RHD. Despite the past bottlenecks we found a relatively high number of MHC class I sequences distributed over 2–4 loci. We identified positive selection on putative antigen-binding sites of the MHC. We detected evidence for RHDV-driven selection as one MHC supertype was negatively associated with RHD survival, fitting expectations of frequency-dependent selection. Gene duplication and pathogen-driven selection are possible (and likely) mechanisms that maintained the adaptive potential of MHC genes in Australian rabbits. Our findings not only contribute to a better understanding of the evolution of invasive species, they are also important in the light of planned future rabbit biocontrol in Australia.     

Resistance to RHD virus in wild Australian rabbits: Comparison of susceptible and resistant individuals using a genomewide approach

Deciphering the genes involved in disease resistance is essential if we are to understand host–pathogen coevolutionary processes. The rabbit haemorrhagic disease virus (RHDV) was imported into Australia in 1995 as a biocontrol agent to manage one of the most successful and devastating invasive species, the European rabbit (Oryctolagus cuniculus). During the first outbreaks of the disease, RHDV caused mortality rates of up to 97%. Recently, however, increased genetic resistance to RHDV has been reported. Here, we have aimed to identify genomic differences between rabbits that survived a natural infection with RHDV and those that died in the field using a genomewide next‐generation sequencing (NGS) approach. We detected 72 SNPs corresponding to 133 genes associated with survival of a RHD infection. Most of the identified genes have known functions in virus infections and replication, immune responses or apoptosis, or have previously been found to be regulated during RHD. Some of the genes identified in experimental studies, however, did not seem to play a role under natural selection regimes, highlighting the importance of field studies to complement the genomic background of wildlife diseases. Our study provides a set of candidate markers as a tool for the future scanning of wild rabbits for their resistance to RHDV. This is important both for wild rabbit populations in southern Europe where RHD is regarded as a serious problem decimating the prey of endangered predator species and for assessing the success of currently planned RHDV variant biocontrol releases in Australia.     

兔出血症病毒VP60基因在昆虫细胞中形成病毒样颗粒及其特性

目的将兔出血症病毒（RHDV）VP60全长基因在昆虫细胞-杆状病毒系统中表达,验证重组蛋白形成病毒样颗粒（VLPs）的能力及其生物学特性,探讨VLPs作为检测抗原及亚单位疫苗的潜力。方法用Bac-to-Bac系统体外表达RHDVVP60全长基因。以免疫荧光及Western blotting检测蛋白表达情况及确定蛋白最佳表达条件;免疫电镜观察VLPs形态,并对VLPs的血凝性、免疫原性进行检测。结果SDS-PAGE电泳分析表明,表达的重组蛋白分子量大小约为68KDa,在免疫荧光、琼脂扩散、ELISA试验中均与RHD多克隆抗血清特异性反应;接种重组病毒的Sf9细胞裂解液在电镜下可观察到与RHDV形态相似的VLPs;该VLPs可凝集人“O”、“B”型红细胞,凝集可被RHD多克隆抗血清所抑制;含VLPs的Sf9细胞裂解液可不经纯化用作间接ELISA抗原,所建立的ELISA方法与进口商品化试剂盒相比,特异性良好,敏感性、检出率稍低;将含VLPs的细胞裂解液加氟氏佐剂免疫兔,HI效价可达1∶40,可经受致死量病毒攻击。结论RHDV-VLPs的获得及其良好的免疫原性,为RHD血清学检测试剂的标准化、亚单位疫苗研制应用奠定基础,同时在转移载体及RHDV受体方面研究亦有潜在应用价值。     

Serological response against myxoma virus and rabbit hemorrhagic disease virus in European wild rabbits using commercial vaccines

We carried out an experimental study to determine the serological response against myxoma virus (MV) and rabbit hemorrhagic disease virus (RHDV) in wild rabbits using commercial vaccines. Seroconversion against MV ranged between 72.7% and 97.2% in animals vaccinated by subcutaneous and intradermal route, respectively, whereas between 75.0% and 77.8% of the animals presented antibodies against RHDV after inoculation with subcutaneous and intradermal vaccines, respectively. Regardless of the inoculation route, vaccination against MV resulted in a significant increase of seropositivity 5 days post-vaccination (dpv), which did not occur in animals vaccinated against RHDV. Furthermore, seroconversion against MV was significantly higher and faster in intradermally vaccinated rabbits as compared to those inoculated subcutaneously due to either the route of application and/or the type of vaccine used. The results indicated that vaccination significantly increased the prevalence of antibodies against MV and RHDV and suggested that the vaccines currently available induce a safe and effective immune response against both diseases in wild rabbits. Vaccination may be a useful management tool to control both viral diseases in field conditions, particularly in wild rabbits captured for translocations and restocking purposes in which a large number of animals are handled. © 2011 The Wildlife Society.     

Oxidative stress in wild European rabbits naturally infected with myxoma virus and rabbit haemorrhagic disease virus

The European rabbit (Oryctolagus cuniculus) is one of the most important vertebrate species in the Mediterranean Basin ecosystem. Over the last 60 years, the arrival of two viral diseases, myxomatosis and rabbit haemorrhagic disease, have led to dramatic declines in wild rabbit populations across the Iberian Peninsula. These diseases are currently endemic. Periodic outbreaks occur and have significant impacts on wild populations. Both infection types have diverse physiological effects on their hosts that are rooted in aerobic metabolic processes. To fight off these viruses, rabbits activate their immune systems. However, the production of immune defences generates reactive oxygen species that may consequently damage host tissues. Hypothesising that immune responses increase oxidative stress, we examined whether wild rabbits naturally infected with myxoma virus (MV) and rabbit haemorrhagic disease virus (RHDV) had high oxidative stress. Using blood samples, we measured anti-MV and anti-RHDV antibody concentrations and different oxidative stress markers (i.e., glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase, and malondialdehyde). Our results show that rabbits that were seropositive for both MV and RHDV had high concentrations of malondialdehyde. Age and body condition were also positively related to dual seropositivity. No significant relationships were observed between serostatus and the concentrations of the other oxidative stress markers. Although we expected infection with MV and RHDV to be correlated with oxidative stress, the influence of external sources of oxidative stress (e.g., climatic conditions) likely made it more difficult to detect such relationships in wild rabbits.     

Development and application of ELISA assays for the detection of two members of the family Luteoviridae infecting legumes: Pea enation mosaic virus (genus Enamovirus) and Bean leafroll virus (genus Luteovirus)

An antigen‐coated plate enzyme‐linked immunosorbent assay (ACP‐ELISA) method was developed and validated for the detection of Bean leafroll virus (BLRV) and Pea enation mosaic virus (PEMV), two of the important viral pathogens of several legume crops. The coat protein (CP) gene of each of the viruses was bacterially expressed as a fusion protein containing an N‐terminal hexa‐histidine tag and used as an antigen to produce antisera in rabbits. The antiserum to BLRV could detect the virus in leaf samples in up to 1:1000 dilution, and the PEMV antiserum detected the homologous virus in leaf samples of dilutions up to 1:6400. No serological cross‐reactivity was observed between anti‐BLRV and anti‐PEMV sera. The ACP‐ELISA assays were then used for estimating the prevalence of these two viruses in alfalfa, pea and vetch over a three‐state area in the US Pacific Northwest over a 2‐year period and virus incidence was mapped. Availability of rapid and sensitive ELISA assays facilitate virus disease mapping efforts and screening germplasm for virus resistance.     

Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein.

Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges.     

兔出血症细小病毒血清学的研究

应用免疫双扩散和酶联免疫吸附分析(ELISA)对七个不同地区兔出血症病原分离物进行了血清学比较研究。本试验用免疫双扩散和ELISA,证明七个兔出血症病毒分离物之间存在着基本相同的血清学关系,其中A_2R—3和H.E.二株抗原之间存在较大差异.证实我国流行的兔出血症病毒属于一个血清型,但不同地区的分离株可能出现亚型。     

Evolutionary history and molecular epidemiology of rabbit haemorrhagic disease virus in the Iberian Peninsula and Western Europe

Background  

Rabbit haemorrhagic disease virus (RHDV) is a highly virulent calicivirus, first described in domestic rabbits in China in 1984. RHDV appears to be a mutant form of a benign virus that existed in Europe long before the first outbreak. In the Iberian Peninsula, the first epidemic in 1988 severely reduced the populations of autochthonous European wild rabbit. To examine the evolutionary history of RHDV in the Iberian Peninsula, we collected virus samples from wild rabbits and sequenced a fragment of the capsid protein gene VP60. These data together with available sequences from other Western European countries, were analyzed following Bayesian Markov chain Monte Carlo methods to infer their phylogenetic relationships, evolutionary rates and demographic history.     

Adaptive changes in the genomes of wild rabbits after 16 years of viral epidemics

Since its introduction to control overabundant invasive European rabbits (Oryctolagus cuniculus), the highly virulent rabbit haemorrhagic disease virus (RHDV) has caused regular annual disease outbreaks in Australian rabbit populations. Although initially reducing rabbit abundance by 60%, continent‐wide, experimental evidence has since indicated increased genetic resistance in wild rabbits that have experienced RHDV‐driven selection. To identify genetic adaptations, which explain the increased resistance to this biocontrol virus, we investigated genome‐wide SNP (single nucleotide polymorphism) allele frequency changes in a South Australian rabbit population that was sampled in 1996 (pre‐RHD genomes) and after 16 years of RHDV outbreaks. We identified several SNPs with changed allele frequencies within or close to genes potentially important for increased RHD resistance. The identified genes are known to be involved in virus infections and immune reactions or had previously been identified as being differentially expressed in healthy versus acutely RHDV‐infected rabbits. Furthermore, we show in a simulation study that the allele/genotype frequency changes cannot be explained by drift alone and that several candidate genes had also been identified as being associated with surviving RHD in a different Australian rabbit population. Our unique data set allowed us to identify candidate genes for RHDV resistance that have evolved under natural conditions, and over a time span that would not have been feasible in an experimental setting. Moreover, it provides a rare example of host genetic adaptations to virus‐driven selection in response to a suddenly emerging infectious disease.     

An alternative, less invasive blood sample collection technique for serologic studies utilizing triatomine bugs (Heteroptera; Insecta)

The collection of blood samples for serological studies is often stressful for the focus animal. Recently, the use of bloodsucking bugs, such as Dipetalogaster maximus or Triatoma infestans (Reduviidae; Triatominae; Heteroptera), has been suggested as a new and less invasive method for blood collection. To evaluate this technique, we collected paired blood samples from 20 domestic rabbits (Oryctolagus cuniculus) during a study of rabbit hemorrhagic disease virus (RHDV). For each rabbit, blood samples were collected by the conventional method (needle and syringe from the vena auricularis) and through feeding by D. maximus. Samples were tested for RHDV antibodies using standard test kits at three different dilutions. Antibody titers were identical for 56 paired samples and differed in only four cases. The simple matching indices were 1 for the 1:10 dilution and 0.9 for the 1:100 and 1:1000 dilutions. The major advantages of the new technique are 1) the possibility to obtain blood from animals where veins are inaccessible and 2) the fact that anesthesia of focus animals may not be necessary.     

基因重组戊肝病毒多价复合抗原的表达及初步应用

目的：利用基因工程技术体外表达高效HEV多价复合抗原,建立一套敏感和特异的抗HEV检测体系,方法：将前期构建的基因重组戊肝病毒多价复合抗原表达质粒转化大肠埃希菌JM109。筛选鉴定阳性克隆并进行诱导表达,表达产物经分子筛层析纯化后,利用western blot作抗原特异性鉴定,以此抗原建立ELISA检测卫生部生物药品检定所HEV血清参比品及临床血清标本,同时以Genelab公司抗HEVELISA试剂盒作为对照进行对比研究。结果：重组质粒转化大肠埃希菌JM109株后的阳性克隆,经诱导在SDS-PAGE中出现一条分子量大小约为31.5kD的蛋白条带,该蛋白纯化后经Western Blotting检测证实为HEV特异性抗原,以此抗原包被酶联板建立ELISA检测53份国家卫生部标准HEV参比血清,灵敏度达100%（38/38）,特异度达93.3%(14/15)。用此方法检测68份临床标本,并与Gene4labELISA试剂盒结果比较,两者阴阳性相符的有64份,总符合率为94.1%。结论：本实验克隆表达的基因重组戊肝病毒多价复合抗原具有良好抗原性,以此抗原建立的抗HEVELISA具有灵敏度高,特异性强的特点,可望为戊型肝炎的血清学诊断和流行病学调查提供一种更为有效的检测手段。     

Development of Epitope-Blocking ELISA for Universal Detection of Antibodies to Human H5N1 Influenza Viruses

Background

Human infections with highly pathogenic H5N1 avian influenza viruses have generally been confirmed by molecular amplification or culture-based methods. Serologic surveillance has potential advantages which have not been realized because rapid and specific serologic tests to detect H5N1 infection are not widely available.

Methodology/Principal Findings

Here we describe an epitope-blocking ELISA to detect specific antibodies to H5N1 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (5F8) that binds to an epitope comprising amino acid residues 274–281 (CNTKCQTP) in the HA1 region of H5 hemagglutinin. Database search analysis of publicly available sequences revealed that this epitope is conserved in 100% of the 163 H5N1 viruses isolated from humans. The sensitivity and specificity of the epitope-blocking ELISA for H5N1 were evaluated using chicken antisera to multiple virus clades and other influenza subtypes as well as serum samples from individuals naturally infected with H5N1 or seasonal influenza viruses. The epitope-blocking ELISA results were compared to those of hemagglutinin inhibition (HI) and microneutralization assays. Antibodies to H5N1 were readily detected in immunized animals or convalescent human sera by the epitope-blocking ELISA whereas specimens with antibodies to other influenza subtypes yielded negative results. The assay showed higher sensitivity and specificity as compared to HI and microneutralization.

Conclusions/Significance

The epitope-blocking ELISA based on a unique 5F8 mAb provided highly sensitive and 100% specific detection of antibodies to H5N1 influenza viruses in human sera.     

<Emphasis Type="Italic">Blastomyces Dermatitidis</Emphasis> Antigen Detection in Urine Specimens from Dogs with Blastomycosis Using a Competitive Binding Inhibition ELISA

A competitive binding inhibition enzyme linked immunosorbent assay (ELISA) was used to detect Blastomyces dermatitidis antigens in urine specimens from dogs with blastomycosis. Sera from rabbits immunized with B. dermatitidis killed whole yeast cells were used as the primary antibody in the competitive ELISA. This initial study was performed to determine if B. dermatitidis antigen detection was possible and to test the efficacy of the rabbit sera as a primary antibody. An indirect ELISA was also performed to compare antigen detection in urine to antibody detection in the sera of the infected dogs. The results indicate 100% (36/36 specimens) detection of both antigen and antibody. Cross reactivity with Histoplasma capsulatum, as well as non-specific binding with the normal urine specimens, was observed with the competitive binding inhibition ELISA.