Development and evaluation of loop-mediated isothermal amplification assay for rapid and sensitive detection of canine parvovirus DNA directly in faecal specimens

Aims: To develop a specific and highly sensitive loop-mediated isothermal amplification (LAMP) technique for the rapid detection of canine parvovirus (CPV) DNA directly in suspected faecal samples of dogs by employing a simple method of template preparation. Methods and Results: LAMP reaction was developed by designing two sets of outer and inner primers, which target a total of six distinct regions on VP2 gene of CPV. The template DNA was prepared by a simple boiling and chilling method. Of the 140 faecal samples screened by the developed LAMP and the conventional PCR assays, 104 samples (74·28%) were found positive by LAMP, whereas 81 samples (57·85%) were found positive by PCR. The specificity of the LAMP assay was tested by cross-examination of common pathogens of dogs and further confirmed by sequencing. The detection limit of the LAMP was 0·0001 TCID(50) ml(-1) , whereas the detection limit of the PCR was 1000 TCID(50) ml(-1) . Conclusions: The developed LAMP assay detects CPV DNA in faecal specimens directly within an hour by following a simple and rapid boiling and chilling method of template preparation. The result also shows that the developed LAMP assay is specific and highly sensitive in detecting CPV. Significance and Impact of the Study: The result indicates the potential usefulness of LAMP which is a simple, rapid, specific, highly sensitive and cost-effective field-based method for direct detection of CPV from the suspected faecal samples of dogs.     

犬、猫、貂细小病毒通用快速诊断盒的研制

本文报道,由犬细小病毒灭活抗原、特异抗体、戊二醛醛化猪红细胞及微量血凝板、稀释棒等组成的犬、猫、貂细小病毒通用快诊盒,可用于犬细小病毒性肠炎、猫泛白细胞减少症和水貂肠炎的特异诊断,适合于基层和野外应用,并可在接到病料后4小时内报告结果,有关试剂的有效期在一年以上。     

Isolation and Typing of Canine Parvovirus in CRFK Cell Line in Puducherry,South India

A total of 128 faecal samples/rectal swabs were collected from dogs showing signs of diarrhea/enteritis in and around Puducherry, South India. Eighteen clinical samples, showing high HA titre of 1:512 and above and positivity by polymerase chain reaction (PCR) with CPV-2ab primers, were subjected to virus isolation in CRFK cell line. Of the 18 samples processed, 3 samples (16.6%) were positive for CPV and were confirmed by haemagglutination, dot-ELISA, and IFAT. The three cell culture isolates were characterized as CPV-2b types by multiplex PCR as well as by monoclonal antibody typing.     

Multiplex PCR for rapid detection of minute virus of mice,bovine parvovirus,and bovine herpesvirus during the manufacture of cell culture-derived biopharmaceuticals

Validation of viral safety is essential in ensuring the safety of mammalian cell culture-derived biopharmaceuticals since numerous adventitious viruses get contaminated during the manufacturing process. In particular, Chinese hamster ovary (CHO) cells are highly susceptible to minute virus of mice (MVM), bovine parvovirus (BPV), and bovine herpesvirus (BHV). Therefore, viral detection during CHO cell culturing is necessary to ensure the safety of biopharmaceuticals against viruses. In this study, a multiplex PCR assay was developed and subsequently evaluated for its effectiveness to simultaneously detect MVM, BPV and BHV during the manufacture of cell culture-derived biopharmaceuticals. Specific primers for MVM, BPV, and BHV were selected, and a multiplex PCR was optimized. The sensitivity of the assay was 6.49 × 10¹ TCID₅₀/mL for MVM, 7.23 × 10² TCID₅₀/mL for BPV, and 5.80 × 10¹ TCID₅₀/mL for BHV. The multiplex PCR assay was very specific to MVM, BPV, and BHV and was subsequently applied to the validation of CHO cells artificially infected with each virus. It could detect each viral DNA from CHO cells as well as culture supernatants. Therefore, we concluded that the multiplex PCR assay is invaluable for detecting adventitious viruses during the manufacture of cell culture-derived biopharmaceuticals.     

Development of a Cryptosporidium oocyst assay using an automated fiber optic-based biosensor

An intestinal protozoan parasite, Cryptosporidium parvum, is a major cause of waterborne gastrointestinal disease worldwide. Detection of Cryptosporidium oocysts in potable water is a high priority for the water treatment industry to reduce potential outbreaks among the consumer populace. Anti-Cryptosporidium oocyst polyclonal and monoclonal antibodies were tested as capture and detection reagents for use in a fiber optic biosensor assay for the detection of Cryptosporidium oocysts. Antibodies were validated using enzyme-linked immunosorbent assays, flow cytometry, Western blotting and fluorescent microscopy. Oocysts could be detected at a concentration of 10⁵ oocysts/ml when the polyclonal antibodies were used as the capture and detection reagents. When oocysts were boiled prior to detection, a ten-fold increase in sensitivity was achieved using the polyclonal antibody. Western blotting and immunofluorescence revealed that both the monoclonal and polyclonal antibodies recognize a large (>300 kDa) molecular weight mucin-like antigen present on the surface of the oocyst wall. The polyclonal antibody also reacted with a small (105 kDa) molecular weight antigen that was present in boiled samples of oocysts. Preliminary steps to design an in-line biosensor assay system have shown that oocysts would have to be concentrated from water samples and heat treated to allow detection by a biosensor assay.     

Effect of Porcine Epidemic Diarrhea Virus Infectious Doses on Infection Outcomes in Na?ve Conventional Neonatal and Weaned Pigs

Porcine epidemic diarrhea virus (PEDV) was identified in the United States (U.S.) swine population for the first time in April 2013 and rapidly spread nationwide. However, no information has been published regarding the minimum infectious dose (MID) of PEDV in different pig models. The main objective of this study was to determine the oral minimum infectious dose of PEDV in naïve conventional neonatal piglets and weaned pigs. A U.S. virulent PEDV prototype isolate (USA/IN19338/2013) with known infectious titer was serially ten-fold diluted in virus-negative cell culture medium. Dilutions with theoretical infectious titers from 560 to 0.0056 TCID₅₀/ml together with a medium control were orogastrically inoculated (10ml/pig) into 7 groups of 5-day-old neonatal pigs (n = 4 per group) and 7 groups of 21-day-old weaned pigs (n = 6 per group). In 5-day-old pigs, 10ml of inoculum having titers 560–0.056 TCID₅₀/ml, corresponding to polymerase chain reaction (PCR) cycle threshold (Ct) values 24.2–37.6, resulted in 100% infection in each group; 10ml of inoculum with titer 0.0056 TCID₅₀/ml (Ct>45) caused infection in 25% of the inoculated pigs. In 21-day-old pigs, 10ml of inoculum with titers 560–5.6 TCID₅₀/ml (Ct 24.2–31.4) resulted in 100% infection in each group while 10ml of inoculum with titers 0.56–0.0056 TCID₅₀/ml (Ct values 35.3 –>45) did not establish infection in any pigs under study conditions as determined by clinical signs, PCR, histopathology, immunohistochemistry, and antibody response. These data reveal that PEDV infectious dose is age-dependent with a significantly lower MID for neonatal pigs compared to weaned pigs. This information should be taken into consideration when interpreting clinical relevance of PEDV PCR results and when designing a PEDV bioassay model. The observation of such a low MID in neonates also emphasizes the importance of strict biosecurity and thorough cleaning/disinfection on sow farms.     

Severe Fever with Thrombocytopenia Syndrome Virus Antigen Detection Using Monoclonal Antibodies to the Nucleocapsid Protein

Background

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease with a high case fatality rate, and is caused by the SFTS virus (SFTSV). SFTS is endemic to China, South Korea, and Japan. The viral RNA level in sera of patients with SFTS is known to be strongly associated with outcomes. Virological SFTS diagnosis with high sensitivity and specificity are required in disease endemic areas.

Methodology/Principal Findings

We generated novel monoclonal antibodies (MAbs) against the SFTSV nucleocapsid (N) protein and developed a sandwich antigen (Ag)-capture enzyme-linked immunosorbent assay (ELISA) for the detection of N protein of SFTSV using MAb and polyclonal antibody as capture and detection antibodies, respectively. The Ag-capture system was capable of detecting at least 350–1220 TCID₅₀/100 μl/well from the culture supernatants of various SFTSV strains. The efficacy of the Ag-capture ELISA in SFTS diagnosis was evaluated using serum samples collected from patients suspected of having SFTS in Japan. All 24 serum samples (100%) containing high copy numbers of viral RNA (>10⁵ copies/ml) showed a positive reaction in the Ag-capture ELISA, whereas 12 out of 15 serum samples (80%) containing low copy numbers of viral RNA (<10⁵ copies/ml) showed a negative reaction in the Ag-capture ELISA. Among these Ag-capture ELISA-negative 12 samples, 9 (75%) were positive for IgG antibodies against SFTSV.

Conclusions

The newly developed Ag-capture ELISA is useful for SFTS diagnosis in acute phase patients with high levels of viremia.     

Comparison of next-generation droplet digital PCR (ddPCR) with quantitative PCR (qPCR) for enumeration of Cryptosporidium oocysts in faecal samples

Clinical microbiology laboratories rely on quantitative PCR for its speed, sensitivity, specificity and ease-of-use. However, quantitative PCR quantitation requires the use of a standard curve or normalisation to reference genes. Droplet digital PCR provides absolute quantitation without the need for calibration curves. A comparison between droplet digital PCR and quantitative PCR-based analyses was conducted for the enteric parasite Cryptosporidium, which is an important cause of gastritis in both humans and animals. Two loci were analysed (18S rRNA and actin) using a range of Cryptosporidium DNA templates, including recombinant plasmids, purified haemocytometer-counted oocysts, commercial flow cytometry-counted oocysts and faecal DNA samples from sheep, cattle and humans. Each method was evaluated for linearity, precision, limit of detection and cost. Across the same range of detection, both methods showed a high degree of linearity and positive correlation for standards (R² ⩾ 0.999) and faecal samples (R² ⩾ 0.9750). The precision of droplet digital PCR, as measured by mean Relative Standard Deviation (RSD;%), was consistently better compared with quantitative PCR, particularly for the 18S rRNA locus, but was poorer as DNA concentration decreased. The quantitative detection of quantitative PCR was unaffected by DNA concentration, but droplet digital PCR quantitative PCR was less affected by the presence of inhibitors, compared with quantitative PCR. For most templates analysed including Cryptosporidium-positive faecal DNA, the template copy numbers, as determined by droplet digital PCR, were consistently lower than by quantitative PCR. However, the quantitations obtained by quantitative PCR are dependent on the accuracy of the standard curve and when the quantitative PCR data were corrected for pipetting and DNA losses (as determined by droplet digital PCR), then the sensitivity of both methods was comparable. A cost analysis based on 96 samples revealed that the overall cost (consumables and labour) of droplet digital PCR was two times higher than quantitative PCR. Using droplet digital PCR to precisely quantify standard dilutions used for high-throughput and cost-effective amplifications by quantitative PCR would be one way to combine the advantages of the two technologies.     

Patterns of Exposure of Iberian Wolves (<Emphasis Type="Italic">Canis lupus</Emphasis>) to Canine Viruses in Human-Dominated Landscapes

Wildlife inhabiting human-dominated landscapes is at risk of pathogen spill-over from domestic species. With the aim of gaining knowledge in the dynamics of viral infections in Iberian wolves (Canis lupus) living in anthropized landscapes of northern Spain, we analysed between 2010 and 2013 the samples of 54 wolves by serology and polymerase chain reaction (PCR) for exposure to four pathogenic canine viruses: canine distemper virus (CDV), canine parvovirus-2 (CPV), canine adenovirus 1 and 2 (CAV-1 and CAV-2) and canine herpesvirus. Overall, 76% of the studied wolves presented evidence of exposure to CPV (96% by HI, 66% by PCR) and 75% to CAV (75% by virus neutralization (VN), 76% by PCR, of which 70% CAV-1 and 6% CAV-2). This represents the first detection of CAV-2 infection in a wild carnivore. CPV/CAV-1 co-infection occurred in 51% of the wolves. The probability of wolf exposure to CPV was positively and significantly correlated with farm density in a buffer zone around the place where the wolf was found, indicating that rural dogs might be the origin of CPV infecting wolves. CPV and CAV-1 appear to be enzootic in the Iberian wolf population, which is supported by the absence of seasonal and inter-annual variations in the proportion of positive samples detected. However, while CPV may depend on periodical introductions by dogs, CAV-1 may be maintained within the wolf population. All wolves were negative for exposure to CDV (by VN and PCR) and CHV (by PCR). The absence of acquired immunity against CDV in this population may predispose it to an elevated rate of mortality in the event of a distemper spill-over via dogs.     

Evaluation of a rapid and inexpensive liquid culture system for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faeces

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis, a chronic granulomatous enteric disease of ruminants. MAP detection by faecal culture provides the definitive diagnosis of the infection. Automated liquid systems for MAP culture are more sensitive and rapid than culture on solid media, but they are expensive and require specialised equipment. In this study, a non-automated culture method using a modified Middlebrook 7H9 liquid medium (7H9+) was compared with Herrold's solid medium (HEYM) and direct real-time PCR on dairy cattle faeces. MAP growth in 7H9+ was monitored weekly by real-time PCR until the 12th week post-inoculation. The analytical sensitivity of the three methods was evaluated using faecal samples from a healthy cow spiked with ten-fold dilutions of MAP organisms (10⁴-10^− 1) and naturally MAP-infected faeces serially diluted 1 to 10 in negative faecal samples. The limits of detection of the solid culture and direct real-time PCR were 10² and 10³ MAP/g, respectively. In comparison, the 7H9+ culture revealed as few as 1 MAP/g. A marked reduction in time to detection of the pathogen, compared with HEYM culture, was obtained. In addition, the three methods were applied to environmental faecal samples collected from a high- and a low-prevalence herd. The culture in 7H9+ showed to be the most sensitive test in the low-prevalence herd and provided faster results than HEYM. In the high-prevalence herd the three methods showed the same sensitivity and the real-time PCR had the shortest turnaround time. In conclusion, the use of 7H9+ for MAP-detection from cattle faeces maximizes diagnostic sensitivity and reduces turnaround time and, therefore, could replace culture in solid medium. Hence, we propose a two-step protocol for the assessment of MAP faecal excretion based on: 1) direct real-time PCR on all samples; and 2) inoculation of negative samples into 7H9+ and analysis after 3 and, if necessary, 6 weeks by real-time PCR.     

Evaluation of the analytical sensitivity of a polymerase chain reaction assay for the detection of chicken infectious anemia virus in avian vaccines

Chicken infectious anemia virus (CAV) is a ubiquitous pathogen of chickens causing significant disease in commercial flocks worldwide. During CAV outbreaks, the Center for Veterinary Biologics requires manufacturers of veterinary biologicals to test materials derived from infected flocks for extraneous CAV by polymerase chain reaction (PCR). The analytical sensitivity of a PCR assay for detection of CAV was determined and the applicability of a CAV DNA standard as a positive control for assay validity was evaluated. The analytical sensitivity of the CAV PCR assay was assessed to be 100 copies per reaction for the DNA standard and 1 × 10^1.9 TCID₅₀/reaction for infectious virus. Establishing the analytical sensitivity of this CAV PCR assay and the inclusion of internal and external positive controls for validity provide a basis for determining whether suspect materials are safe for use in the production of veterinary biologics.     

The replication of a cytoplasmic polyhedrosis virus from Chrysodeixis eriosoma (Lepidoptera: Noctuidae) in Spodoptera frugiperda cells

A cytoplasmic polyhedrosis virus (CPV) from Chrysodeixis eriosoma (Lepidoptera: Noctuidae) replicated in Spodoptera frugiperda cells. Low rates of infection were achieved, even at high multiplicities of infection and TCID₅₀ assays showed that there was negligible release of virus particles from infected cells. In an infected focus assay, based on formation of PIB, the dose-response data demonstrated that a single particle could initiate infection. No loss of infectivity occurred in virus preparations stored at 4°, ?20°, or ?90°C, but infectivity of virus stored at 20°C declined sharply. A small isometric virus contaminant was present in some CPV preparations and its interaction with the CPV is discussed. Limited CPV infection was achieved in Trichoplusia ni cells, but attempts to infect Aedes aegypti cells were unsuccessful.     

Generation and Immunity Testing of a Recombinant Adenovirus Expressing NcSRS2-NcGRA7 Fusion Protein of Bovine Neospora caninum

Neospora caninum is the etiologic agent of bovine neosporosis, which affects the reproductive performance of cattle worldwide. The transmembrane protein, NcSRS2, and dense-granule protein, NcGRA7, were identified as protective antigens based on their ability to induce significant protective immune responses in murine neosporosis models. In the current study, NcSRS2 and NcGRA7 genes were spliced by overlap-extension PCR in a recombinant adenovirus termed Ad5-NcSRS2-NcGRA 7, expressing the NcSRS2-NcGRA7 gene, and the efficacy was evaluated in mice. The results showed that the titer of the recombinant adenovirus was 10⁹TCID₅₀/ml. Three weeks post-boost immunization (w.p.b.i.), the IgG antibody titer in sera was as high as 1:4,096. IFN-γ and IL-4 levels were significantly different from the control group (P<0.01). This research established a solid foundation for the development of a recombinant adenovirus vaccine against bovine N. caninum.     

Unusual outcome of in utero infection and subsequent postnatal super-infection with different PCV2b strains

VC2002, isolated from postweaning multisystemic wasting syndrome (PMWS)-affected pig, is a mixture of two porcine circovirus genotype 2b (PCV2b) viruses, K2 and K39. Preliminary experiments disclosed short-term adverse effects of K39, but not K2, on porcine foetuses. These findings led to the hypothesis that infection of immuno-incompetent foetuses with K2 confers a status of immunotolerance, and postnatal super-infection with K39 triggers PMWS. To explore this hypothesis, nine 55-day-old foetuses were inoculated in utero (three with K2-10^4.3TCID₅₀, three with K39-10^4.3TCID₅₀ and three with medium), and foeto-pathogenicity examined. At 21 days post-inoculation (dpi), K2 did not induce pathology, whereas pathological effects of K39 were evident. Twenty-four 45-day-old foetuses were subsequently inoculated to examine the long-term effect of K2, including six with K2-high dose-10^4.3TCID₅₀, six with K2-low dose-10^2.3TCID₅₀ and 12 mock-inoculated controls. Both doses resulted in five mummified foetuses and one live-born piglet each (69dpi). K2 was recovered from all mummies. K2 and K2-specific antibodies were not detected in serum of the two live-born piglets at birth, indicating full control of K2 infection. The K2-low dose-infected piglet was immunostimulated at day 2, but not the K2-high dose-infected piglet. Both non-stimulated and stimulated K2-infected piglets were super-inoculated with K39 at day 6 or 8 (taken as 0 days post super-inoculation). Low viral replication was observed in the non-stimulated K2-K39 piglet (up to 10^3.3TCID₅₀/g; identified as K39). In contrast, viral replication was extremely high in the stimulated K2-K39 piglet (up to 10^5.6TCID₅₀/g) and identified as K2, indicating that K2 infection is controlled during foetal life, but emerges after birth upon immunostimulation. However, none of the piglets showed any signs of PMWS.     

Self-assembly of virus-like particles of canine parvovirus capsid protein expressed from Escherichia coli and application as virus-like particle vaccine

Canine parvovirus disease is an acute infectious disease caused by canine parvovirus (CPV). Current commercial vaccines are mainly attenuated and inactivated; as such, problems concerning safety may occur. To resolve this problem, researchers developed virus-like particles (VLPs) as biological nanoparticles resembling natural virions and showing high bio-safety. This property allows the use of VLPs for vaccine development and mechanism studies of viral infections. Tissue-specific drug delivery also employs VLPs as biological nanomaterials. Therefore, VLPs derived from CPV have a great potential in medicine and diagnostics. In this study, small ubiquitin-like modifier (SUMO) fusion motif was utilized to express a whole, naturalVP2 protein of CPV in Escherichia coli. After the cleavage of the fusion motif, the CPV VP2 protein has self-assembled into VLPs. The VLPs had a size and shape that resembled the authentic virus capsid. However, the self-assembly efficiency of VLPs can be affected by different pH levels and ionic strengths. The mice vaccinated subcutaneously with CPV VLPs and CPV-specific immune responses were compared with those immunized with the natural virus. This result showed that VLPs can effectively induce anti-CPV specific antibody and lymphocyte proliferation as a whole virus. This result further suggested that the antigen epitope of CPV was correctly present on VLPs, thereby showing the potential application of a VLP-based CPV vaccine.     

猪伪狂犬病病毒流行株HLJ-01的分离鉴定及致病性分析

【背景】自2011年以来,猪伪狂犬病毒(pseudorabies virus,PRV)发生变异,经典的疫苗株已不能完全抵抗PRV变异株的感染,国内多个猪场出现伪狂犬病的暴发,PRV变异毒株开始在我国大规模流行。【目的】通过分离PRV流行变异毒株,并对其进行遗传进化和致病性分析,为PRV流行病学调查及疫苗研制提供实验数据。【方法】采集黑龙江某猪场感染PRV的脑组织病料,根据GenBank PRV gE和gB保守序列设计引物,进行PCR鉴定。通过对gE和gC基因进行序列测定和遗传进化分析。利用BHK-21细胞分离病毒,采用噬斑纯化方法对病毒进行纯化。通过电镜、间接免疫荧光对病毒进行鉴定,测定病毒生长曲线并进行致病性研究。【结果】经PCR和测序鉴定分离株为PRV流行株,将其命名为HLJ-01。遗传进化分析结果显示,该分离毒株与我国近几年分离的流行变异株位于同一分支;氨基酸序列分析结果显示,gE和gC存在国内流行变异株的特征序列,表明该分离毒株为流行变异株。生长曲线显示,分离株HLJ-01在感染48h时滴度最高(10^8.5TCID₅₀/mL)。电镜观察结果显示,病毒颗粒直径约150nm,呈球形,有囊膜,囊膜外有放射状纤突,呈现典型PRV病毒特征。动物感染实验结果显示,10^7.0TCID₅₀剂量感染组死亡率为100%;10^6.0TCID₅₀剂量感染组死亡率为80%;10^5.0TCID₅₀剂量感染组死亡率为60%。仔猪在接种病毒后均出现PRV感染的典型症状和病理变化,证实分离毒株对仔猪有较强致病力。【结论】分离获得一株猪伪狂犬病毒,经鉴定该分离株为流行变异株,而且具有较强的致病力,这为PRV流行病学分析及疫苗候选株的筛选奠定了基础。     

Viral Dose and Immunosuppression Modulate the Progression of Acute BVDV-1 Infection in Calves: Evidence of Long Term Persistence after Intra-Nasal Infection

Bovine viral diarrhoea virus (BVDV) infection of cattle causes a diverse range of clinical outcomes from being asymptomatic, or a transient mild disease, to producing severe cases of acute disease leading to death. Four groups of calves were challenged with a type 1 BVDV strain, originating from a severe outbreak of BVDV in England, to study the effect of viral dose and immunosuppression on the viral replication and transmission of BVDV. Three groups received increasing amounts of virus: Group A received 10^2.55TCID₅₀/ml, group B 10^5.25TCID₅₀/ml and group C 10^6.7TCID ₅₀/ml. A fourth group (D) was inoculated with a medium dose (10^5.25TCID₅₀/ml) and concomitantly treated with dexamethasone (DMS) to assess the effects of chemically induced immunosuppression. Naïve calves were added as sentinel animals to assess virus transmission. The outcome of infection was dose dependent with animals given a higher dose developing severe disease and more pronounced viral replication. Despite virus being shed by the low-dose infection group, BVD was not transmitted to sentinel calves. Administration of dexamethasone (DMS) resulted in more severe clinical signs, prolonged viraemia and virus shedding. Using PCR techniques, viral RNA was detected in blood, several weeks after the limit of infectious virus recovery. Finally, a recently developed strand-specific RT-PCR detected negative strand viral RNA, indicative of actively replicating virus, in blood samples from convalescent animals, as late as 85 days post inoculation. This detection of long term replicating virus may indicate the way in which the virus persists and/or is reintroduced within herds.     

A new faecal antigen detection system for Strongyloides venezuelensis diagnosis in immunosuppressed rats

This study was performed with the objective of developing innovative procedures for the diagnosis of strongyloidiasis. Enzyme-linked immunosorbent assay (ELISA) was conducted to detect coproantigen in the faecal samples of normal and of immunosuppressed rats using an anti-L3 polyclonal antibody produced in rabbits. Analysis revealed the kinetics of egg shedding in the non-immunosuppressed and immunosuppressed rats infected with S. venezuelensis. Further analysis verified the ability of the immune serum to detect L3 antigens in faecal samples from infected animals. The number of eggs recovered in the faeces at 8 days p.i was significantly higher for both groups. Immunosuppressed animals eliminated increased quantities of eggs. The immune serum was able to detect 0.39 μg/ml of L3 antigens. The antigen recognition in the immunosuppressed group was anticipated on the 8th day p.i. In conclusion, these results may represent a first step in the development of a rapid coproantigen detection kit for strongyloidiasis.