Application of the Leishmania infantum 21-kDa recombinant protein for the development of an immunochromatographic test.

BACKGROUND: Visceral leishmaniasis (VL), caused by Leishmania infantum, is a systemic parasitic disease and presents a global health problem which can be fatal if not diagnosed and treated. Dogs are the main hosts and provide reservoirs for the transmission of the disease to humans. METHODS: In this study, the gene encoding a 21-kDa protein was cloned and expressed as a fusion protein in Escherichia coli strain BL21 (DE3) for developing a rapid immunochromatographic test (ICT) to identify infected dogs. The expression of the recombinant 21-kDa protein (r21) was investigated using SDS-PAGE and Western blot methods. The purified r21-kDa protein was spotted onto ICT strips and tested by sera from experimentally infected, naturally infected and uninfected dogs. RESULTS: The SDS-PAGE and Western blot methods showed the successful expression of r21-kDa protein. The ICT strip test revealed that the r21-kDa protein was detected by the sera of experimentally and naturally infected dogs. The specificity tests also confirmed no cross-reactivity with animals infected with Trypanosoma cruzi, Toxoplasma gondii and Ehrlichia canis. CONCLUSIONS: Based on these findings, the new r21-kDa protein may be a suitable target for developing a new simple, specific and rapid serological method to detect VL in infected dogs.

Evaluation of a rapid immunochromatographic test kit to the gold standard fluorescent antibody test for diagnosis of rabies in animals in Bhutan.

BACKGROUND: Rabies kills approximately 59,000 people each year worldwide. Rapid and accurate diagnosis of rabies is important for instituting rapid containment measures and for advising the exposed people for postexposure treatment. The application of a rapid diagnostic tests in the field can greatly enhance disease surveillance and diagnostic activities, especially in resource poor settings. In this study, a total of 179 brain tissue samples collected from different rabies suspect animal species (113 dogs, 50 cattle, 10 cats, 3 goats, 2 horses, and 1 bear) were selected and tested using both rapid immunochromatographic kit and the reference standard fluorescent antibody test (FAT). We evaluated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of a rapid antigen detection test kit produced by BioNote, Inc. (Hwaseong-si, Korea) relative to a FAT for its fit-for-purpose for confirmation of clinical cases of rabies for early response and enhancing rabies surveillance. RESULTS: Among 179 samples examined in this study, there was a concordance in results by the rapid test and FAT in 115 positive samples and 54 negative samples. Test results were discordant in 10 samples which were positive by FAT, but negative (false negative) by rapid kit. The rapid test kit showed a sensitivity of 92% (95% CI: 85.9-95.6) and specificity of 100% (95% CI: 93.4-100) using FAT as the reference standard. The positive and negative predictive values were found to be 100% (95% CI:96.7-100) and 84.4% (95% CI: 73.6-91.3), respectively. Overall, there was 94.4% (95% CI: 90-96.9) test agreement between rapid test and FAT (Kappa value = 0.874) with a positive percent agreement and negative percent agreement of 92 and 100%, respectively. CONCLUSIONS: Our finding demonstrated that the rapid test kit (BioNote) can be used for rabies surveillance and confirming clinical case of rabies in animals for making rapid decisions particularly controlling rabies outbreaks in resource poor settings.

Dual-mode immunoassay system based on glucose oxidase-triggered Fenton reaction for qualitative and quantitative detection of danofloxacin in milk.

In this study, a novel colorimetric and fluorescent dual-mode ELISA based on glucose oxidase (GOx)-triggered Fenton reaction was developed for the qualitative and quantitative detection of danofloxacin (DAN). In this system, streptavidin-linked biotinylated anti-DAN-monoclonal antibody (SA-Bio-mAb) and biotinylated GOx (Bio-GOx) form the immune complex mAb-Bio-SA-Bio-GOx. In the absence of DAN, the mAb-Bio-SA-Bio-GOx would be immobilized by combining with coated DAN-BSA and catalyzed glucose to generate H2O2. The Fenton reaction between H2O2 and Fe2+ generated hydroxyl radicals, which oxidized the o-phenylenediamine to 2,3-diamino-phenazine. A dual-signal immunoassay with colorimetry and fluorescence as the signal readout was established. In the presence of DAN, DAN and DAN-BSA competed with Bio-mAb, decreasing the connection between immune complexes and DAN-BSA and finally resulting in lower signal of colorimetry and fluorescence. Under optimal conditions, the limit of detection of the fluorescence immunoassay was 0.337 ng/mL and was 5.24-fold lower than that of traditional ELISA. The colorimetric immunoassay cut-off value was 30 ng/mL in milk. The average recoveries of the method for milk samples that are spiked with different concentrations of DAN were 91.1 to 128.3%, with a coefficient of variation of 0.7 to 8.2%. These results of the method exhibited good agreement with those of liquid chromatography-tandem mass spectrometry system (LC-MS/MS) method. In brief, this work provides an improved screening strategy with high sensitivity and accuracy for the qualitative or quantitative detection of DAN in milk monitoring.

Molecular characterization and immunodiagnostics of Dicrocoelium dendriticum species isolated from sheep of north-west Himalayan region.

Despite its extensive presence among grazing ruminants, dicrocoeliosis, also known as 'small liver fluke' disease, is poorly known and often underestimated by researchers and practitioners in many countries. The accurate identification and prepatent diagnosis of Dicrocoelium dendriticum infection is an essential prerequisite for its prevention and control. In the present study, the morphologically identified specimens isolated from the bile ducts of sheep (Ovis aries) were validated through molecular data. The sequence analysis of the second internal transcribed spacer (ITS-2) of our isolates showed a high degree of similarity with D. dendriticum using the BLAST function of the National Center for Biotechnology Information (NCBI). The phylogenetic analysis of our isolates showed a close relationship with previously described D. dendriticum isolates from different countries. The antigenic profiles of somatic and excretory/secretory (E/S) antigens of D. dendriticum were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from sheep naturally infected with D. dendriticum. By SDS-PAGE, 16 distinct bands were revealed from crude somatic fraction. Immunoblotting analysis of these proteins with positive sera exhibited six seroreactive bands ranging from 27 to 130 kDa. Among these, the 84 and 130 kDa bands were quite specific, with high diagnostic specificity and sensitivity. The E/S fraction comprised nine distinct bands, as revealed by SDS-PAGE analysis. Immunoblotting analysis of these proteins with positive sera exhibited five antigenic bands ranging from 27 to 130 kDa. Among these, the 130 kDa band was found to be quite specific, with high diagnostic specificity and sensitivity. The present study concludes that the protein bands of 84 and 130 kDa in somatic fraction and 130 kDa in E/S fraction can be used for the immunodiagnostic purpose for this economically important parasite, which may also encourage further studies regarding their vaccine potential.

Feline heartworm (Dirofilaria immitis) infection: first case report of serological diagnosis in Brazil, confirmed by molecular assay.

The clinical importance of heartworm infection in cats has indeed increased in recent years. Dirofilaria immitis infection has been reported worldwide in cats and continues to be regularly diagnosed in endemic areas. The diagnosis can be overlooked easily, especially in Brazil, where there is not a specific feline immunodiagnostic test, forcing the veterinarians to use a test made for the canine host. In 2015, a 10-year-old female neutered cat was diagnosed with D. immitis using an antigen serological test, based on imunocromatography and designed for dogs. The modified Knott test was negative. As the disease progressed, the cat showed clinical signals of respiratory distress, such as dyspnoea and polypnea in addition to prostration and emaciation, and died a few weeks after the diagnosis. During necropsy, one adult nematode was found in the pulmonary artery. D. immitis infection was confirmed by molecular amplification, performed in the worm fragment. This is the first report of serological diagnosis of feline dirofilariasis in Brazil. A chemoprophylaxis routine in cats should be done, as is done in dogs from endemic areas.

Immunodiagnosis of paramphistomosis using monoclonal antibody-based sandwich ELISA for detection of Paramphistomum gracile circulating 16 kDa antigen.

In this study, we have produced a monoclonal antibody (MoAb) against 16 kDa antigen of Paramphistomum gracile (16 kDaAgPg), and developed an accurate sandwich enzyme-linked immunosorbent assay (sandwich ELISA) for the detection of circulating 16 kDaAg in the serum and fecal samples from cattle naturally infected with P. gracile. MoAb 1D10 was immobilized on a microtitre plate, and the antigen in the samples was captured and detected with biotinylated rabbit anti-16 kDaAgPg antibody. The lower detection limit of sandwich ELISA was 3·5 pg mL-1, and no cross-reaction with other parasite antigens was evaluated. The reliability of the assay was examined using the serum and fecal samples from cattle naturally infected with P. gracile, Fasciola gigantica, Moniezia benedeni, Trichuris sp., Strongyloides sp., strongylids and non-infected animals. The sandwich ELISA showed the sensitivity, specificity and accuracy at 98·33, 100 and 99·55% (serum samples), and 96·67, 100 and 99·09% (fecal samples). Therefore, this detection method is a rapid and excellent potential assay for the accurate diagnosis of paramphistomosis.

Critically appraised topic on adverse food reactions of companion animals (4): can we diagnose adverse food reactions in dogs and cats with in vivo or in vitro tests?

BACKGROUND: The gold standard to diagnose adverse food reactions (AFRs) in the dog and cat is currently an elimination diet with subsequent provocation trials. However, those trials are inconvenient and client compliance can be low. Our objective was to systematically review the literature to evaluate in vivo and in vitro tests used to diagnose AFR in small animals. RESULTS: We searched three databases (CAB Abstracts, MEDLINE and Web of Science) for pertinent references on September 16, 2016. Among 71, 544 and 41 articles found in the CAB Abstract, MEDLINE and Web of Science databases, respectively, we selected 22 articles and abstracts from conference proceedings that reported data usable for evaluation of tests for AFR. Serum tests for food-specific IgE and IgG, intradermal testing with food antigens, lymphocyte proliferation tests, fecal food-specific IgE, patch, gastroscopic, and colonoscopic testing were evaluated. CONCLUSIONS: Testing for serum food-specific IgE and IgG showed low repeatability and, in dogs, a highly variable accuracy. In cats, the accuracy of testing for food-specific IgE was low. Lymphocyte proliferation tests were more frequently positive and more accurate in animals with AFR, but, as they are more difficult to perform, they remain currently a research tool. All other reported tests were only evaluated by individual studies with small numbers of animals. Negative patch test reactions have a very high negative predictability in dogs and could enable a choice of ingredients for the elimination diet in selected patients. Gastroscopic and colonoscopic testing as well as food-specific fecal IgE or food-specific serum IgG measurements appear less useful. Currently, the best diagnostic procedure to identify AFRs in small animals remains an elimination diet with subsequent provocation trials.

Hematological and Immunological plasma assays for grass carp (Ctenopharyngodon idella) infected with Aeromonas hydrophila as an immune model in carp aquaculture.

Aeromonas hydrophila is the causative agent of bacterial septicemia, a common disease observed in grass carp, Ctenopharyngodon idella. In our study, C. idella specimens were infected with A. hydrophila, and parameters of Hematological and Immunological plasma parameters were monitored. At blood cell level, levels of red blood cells (RBCs), hematocrit (HCT), and mean corpuscular volume (MCV) showed no differences between the treatment and control groups, but levels of white blood cells (WBCs) increased. The monocyte and neutrophil varied significant according to stimulation by A. hydrophila at 1 DPI, the thrombocyte and lymphocyte at 14 and 21 DPI. At serum level, total protein, lysozyme, and IgM increased at the early infection phase and then decreased at other time points; however, peroxidase levels were significantly lower in the treatment group than that in the control group during the early infection phase. ACH50 was significantly higher in the treatment group than that in the control group during the late infection phase. On the basis of the results, we suggest that innate and adaptive immune mechanisms of C. idella are able to neutralize the virulence factors secreted by A. hydrophila. Our findings would help in understanding the mechanisms underlying resistance to infection by A. hydrophila.

Short communication: Cytokine profiles from blood mononuclear cells of dairy cows classified with divergent immune response phenotypes.

Genetic selection for enhanced immune response has been shown to decrease disease occurrence in dairy cattle. Cows can be classified as high (H), average, or low responders based on antibody-mediated immune response (AMIR), predominated by type-2 cytokine production, and cell-mediated immune response (CMIR) through estimated breeding values for these traits. The purpose of this study was to identify in vitro tests that correlate with in vivo immune response phenotyping in dairy cattle. Blood mononuclear cells (BMC) isolated from cows classified as H-AMIR and H-CMIR through estimated breeding values for immune response traits were stimulated with concanavalin A (ConA; Sigma Aldrich, St. Louis, MO) and gene expression, cytokine production, and cell proliferation was determined at multiple time points. A repeated measures model, which included the effects of immune response group, parity, and stage of lactation, was used to compare differences between immune response phenotype groups. The H-AMIR cows produced more IL-4 protein than H-CMIR cows at 48 h; however, no difference in gene expression of type-2 transcription factor GATA3 or IL4 was noted. The BMC from H-CMIR cows had increased production of IFN-γ protein at 48, 72, and 96 h compared with H-AMIR animals. Further, H-CMIR cows had increased expression of the IFNG gene at 16, 24, and 48 h post-treatment with ConA, although expression of the type-1 transcription factor gene TBX21 did not differ between immune response groups. Although proliferation of BMC increased from 24 to 72 h after ConA stimulation, no differences were found between the immune response groups. Overall, stimulation of H-AMIR and H-CMIR bovine BMC with ConA resulted in distinct cytokine production profiles according to genetically defined groups. These distinct cytokine profiles could be used to define disease resistance phenotypes in dairy cows according to stimulation in vitro; however, other immune response phenotypes should be assessed.

The effect of oyster mushroom ß-1.3/1.6-D-glucan and oxytetracycline antibiotic on biometrical, haematological, biochemical, and immunological indices, and histopathological changes in common carp (Cyprinus carpio L.).

The aim of the study was to evaluate the effect of micronized ß-1.3/1.6-D-glucan (BG) derived from the oyster mushroom Pleurotus ostreatus Hiratake and tetracycline antibiotic oxytetracycline (OTC) on biometrical, haematological, biochemical, and immunological indices, and histopathological changes in tissues of one- to two-year-old common carp (Cyprinus carpio L.). The fish tested were divided into five experimental groups and one control. Carp in the control group were fed commercial carp feed pellets. Fish in the five experimental groups were fed the same pellets supplemented with either OTC, a combination of OTC and BG, or BG as follows: 75 mg oxytetracycline kg(-1) bw (OTC group), 75 mg oxytetracycline kg(-1) bw and 0.5% ß-glucan (OTC + 0.5% BG group), 75 mg oxytetracycline kg(-1) bw and 2.0% ß-glucan (OTC + 2.0% BG group), 0.5% ß-glucan (0.5% BG group), and 2.0% ß-glucan (2.0% BG group). OTC- and BG-supplemented diets and the control diet were administered to experimental and control carp for 50 days (i.e. samplings 1-3, the exposure period); for the following 14 days, fish were fed only control feed pellets with no OTC or BG supplementation (i.e. sampling 4, the recovery period). Blood and tissue samples were collected both during, and at the end of the study. No significant changes in biometrical indices (i.e. total length, standard length, total weight, hepatosomatic and spleen somatic index, and Fulton's condition factor) were found in experimental carp compared to control in any sampling. In haematological indices, significant changes were found only in sampling 2, in which shifts in PCV (P < 0.01), Hb (P < 0.01), and WBC (P < 0.01), and in the counts of lymphocytes (P < 0.01), monocytes (P < 0.01), and neutrophil granulocytes-segments (P < 0.05) were revealed. As for biochemical profiling, plasma concentrations of glucose, albumins, cholesterol, natrium, and chlorides (all P < 0.01), and total proteins, lactate, phosphorus, and potassium (all P < 0.05) as well as the catalytic activity of ALP (P < 0.05) were altered in common carp. A significant change in induced (opsonizedzymosan particles, OZP) chemiluminescence (P < 0.05) in sampling 3 and no shifts in serum immunoglobulins concentration were found in the immunological analysis. Histopathological examination of skin, gills, liver, spleen, and cranial and caudal kidneys revealed no obvious specific changes in any tissue analysed. The use of ß-glucans in clinically healthy aquaculture remains an issue. Nevertheless, their use in breeding endangered by stress stimuli, infectious disease, or adverse environmental factors is defensible.

Developing a framework for risk-based surveillance of tuberculosis in cattle: a case study of its application in Scotland.

Due to its substantially lower prevalence of bovine tuberculosis (bTB) relative to other areas of Great Britain, Scotland was designated as an officially (bovine) TB-free region in 2009. This paper investigates resultant possibilities for reducing surveillance by developing risk-based alternatives to current 4-year testing of eligible herds. A model of freedom of infection was used to develop strategies that specifically tested herds that are at risk of infection but would probably not be identified by slaughterhouse meat inspection. The performance of current testing is mimicked by testing all herds that slaughter fewer than 25% of their total stock per year and regularly import animals from high-incidence areas of England and Wales or from Ireland. This system offers a cost reduction by requiring 25% fewer herd and animal tests and 25% fewer false positives.

New developments in the immunodiagnosis of brucellosis in livestock and wildlife.

Although relatively effective diagnostic tests for brucellosis have been in existence for more than 100 years, it remains a serious, embedded and also a re-emerging disease in many parts of the globe. There are many factors besides suboptimal diagnosis that impede the complete and sustained eradication of animal brucellosis. In this review a case for the continued improvement of diagnostic methods is made through identifying existing shortcomings and considering what impact these have upon control and eradication. The focus is on developments in immunodiagnostics as these seem more likely to yield the pragmatic solutions needed. Moreover, developments in DNA detection methods have been neatly and recently reviewed elsewhere. This article reviews issues such as test cost, mobility, sensitivity and specificity. Advances in low-cost materials, high-throughput testing, assay multiplexing and the quantification of pen-side tests are described and their relevance to disease control considered. Poor test specificity when resolving positive serology, due to infection with cross-reactive bacteria and vaccination with smooth Brucella strains, is also an impediment to efficient disease eradication. A case for the development of novel discrete epitope antigens to address this is presented alongside in silico methods of selection and tools that enable increased analytical sensitivity that may be required to detect relatively low, but potentially significant, analytes. References have been drawn from the study of brucellosis wherever possible. However, in some cases new technological developments worthy of discussion have been included via the use of pertinent alternative examples. In conclusion, despite developments and innovations the classical serological tests seem under no imminent danger of mass extinction but there is potential for significant improvement and supplementation.

Development and validation of a method for purification of mallein for the diagnosis of glanders in equines.

BACKGROUND: The allergic test of mallein is one of the most frequently used tests, together with the Complement Fixation Test (CFT), for the diagnosis of glanders in endemic areas. Mallein, a purified protein derivative (PPD), is produced similarly to PPD tuberculin and the end product is a primarily proteic antigen, which is only poorly purified. The immuno-allergic activity of mallein is believed to be due to a high molecular weight group of proteins present in the antigen. To improve the quality of the antigen, in terms of sensitivity and specificity, a new method of mallein production was developed, in which purification was accomplished by ultrafiltration in a Tangential Flow Filtration system (TFF). RESULTS: The TFF methodology efficiently separated the high and low molecular weight protein groups of mallein. The five TFF-purified malleins, produced from Burkholderia mallei strains isolated from clinical cases of glanders in Brazil, proved to be more potent than standard mallein in the induction of an allergic reaction in sensitized animals. Regarding specificity, two of the purified malleins were equivalent to the standard and three were less specific. CONCLUSION: Some of the TFF-purified malleins showed considerable potential to be used as an auxiliary test in the diagnosis of glanders.

Dioctophyme renale: in vitro culture, production of excretory and secretory antigens, and their use in immunodiagnosis.

The nematode Dioctophyme renale affects mainly the right kidney of domestic and wild mammals, in addition to having zoonotic potential. Therefore, this study aimed to produce excretion and secretion antigens of adult D. renale (DES) to diagnose canine dioctophimosis. To obtain the excretion and secretion antigens (DES), five D. renale adults were maintained for three weeks in supplemented RPMI medium with weekly supernatant collections. After the DES concentration, 2200 mL of the collected supernatant was used, which was centrifuged, followed by two filtrations and dialyzed, resulting in 12.5 mL of DES with a protein concentration of 0.59 mg/mL. The polyacrylamide gel electrophoresis (SDS-PAGE) of the DES antigen showed fractions with molecular weights ranging from 30 to 250 Kda. In the indirect ELISA with the DES antigen, the mean absorbance of the positive sera (38) was 0.839 ± 0.267, and the mean of the negative control sera (7) was 0.208 ± 0.083; the specificity and sensitivity of the ELISA were 100 and 97.4 %, respectively, being as effective as the surgical method in the presence of viable parasites. Thus, for the first time, this study describes the production of excretion and secretion antigens of adult D. renale and standardizes an indirect ELISA to diagnose dioctophimosis.

Proteomics and immunoblotting analyses reveal antigens that optimize the immunodiagnosis of the infection by Toxocara spp.

Toxocariasis is an infection caused by the round worms Toxocara canis and Toxocara cati. It occurs worldwide though it is more prevalent in developing countries. For the diagnosis of toxocariasis, the most used method is the indirect enzyme-linked immunosorbent assay (indirect ELISA), based on the detection of specific antibodies using the excreted/secreted products from T. canis larvae (TES) as antigens, but it cross-reacts with several helminth infections. For this reason, there is a need to investigate species-specific immunoreactive proteins, which can be used for the development of a more sensitive and specific diagnosis. This study aims to investigate immunoreactive protein candidates to be used for the development of a more sensitive and specific diagnosis of Toxocara spp. infection in humans. We have used immunoblotting and mass spectrometry to select four Toxocara canis immunoreactive proteins that were recombinantly expressed in bacteria and evaluated as potential new diagnostic antigens (rMUC3, rTES 26, rTES32 and rCTL4). The recognition of these recombinant proteins by total serum IgG and IgG4 was assayed using the purified proteins in an isolated manner or in combination. The IgG ELISAs performed with individual recombinant antigens reached values of sensitivity and specificity that ranged from 91.7% to 97.3% and 94.0% to 97.9%, respectively. Among the analyses, the IgG4 immunoassay was proven to be more effective, revealing a sensitivity that ranged from 88.8% to 98.3% and a specificity of 97.8%-97.9%. The IgG4 ELISA was shown to be more effective and presented no cross-reactivity when using combinations of the rTES 26 and rCTL4 recombinant proteins. The combination of these two molecules achieved 100% sensitivity and specificity. The use of only two recombinant proteins can contribute to improve the current panorama of toxocariasis immunodiagnosis for, with a better optimization and reduced cost.

Use of blood matrices and alternative biological fluids for antibody detection in animal tuberculosis.

Bovine tuberculosis (bTB) control programs can be improved by implementation of advanced ante-mortem testing algorithms. Serodiagnostic methods using traditional blood or blood-derived specimens may benefit from the use of less invasive alternative biological fluids, provided those mirror systemic antibody responses. In the present study, we used Dual Path Platform (DPP) and Multiantigen Print Immunoassay (MAPIA) to compare antibody levels in ten sample types including whole blood (fresh and hemolyzed), plasma (fresh and leftover from Bovigam testing), serum, saliva, broncho-alveolar lavage, urine, diaphragm extract, and bile collected from cattle aerosol-infected with Mycobacterium bovis. High correlation (r = 0.97-0.99) in measurements of IgG antibodies to MPB70/MPB83 fusion antigen by DPP assay was found between all blood-derived specimens, supporting matrix equivalency. Broncho-alveolar lavage and diaphragm extract yielded positive results in all the infected animals tested, showing high correlation with matching serum data (r = 0.94 and r = 0.95, respectively) and suggesting their potential use in antibody assays. Characterized by MAPIA, the antigen reactivity patterns obtained with paired sera and alternative specimens were nearly identical, with slight differences in intensity. Antibodies were also found by DPP assay in saliva, urine, and bile from some of the infected animals, but the titers were relatively low, thus reducing the diagnostic value of such specimens. The proposed approach was evaluated in a pilot field study on warthogs diagnosed with M. bovis infection. Relative levels of antibody in tissue fluid obtained from lymph nodes or lungs were consistent with those detected in sera and detectable in all infected warthogs. The findings support the diagnostic utility of non-traditional biological fluids and tissue samples when used as alternative test specimens in serologic assays for bTB.

Comparison of three immunodiagnostic assays for antemortem detection of Mycobacterium bovis stimulation in domestic cats.

Mycobacterium bovis causes disease in numerous mammalian species including humans, thus making research, surveillance, and control important in the eradication of tuberculosis. Domestic cats are susceptible to multiple mycobacterial species including Mycobacterium bovis; however, their role in the epidemiology of bovine tuberculosis is not fully documented. The current study was an evaluation of the immune response in specific pathogen-free (SPF) cats stimulated with sensitinogen, a heat-killed M. bovis product, using the rapid test, multiple antigen print immunoassay (MAPIA), and bovine-purified protein derivative (bPPD) single skin test. Six cats were inoculated with sensitinogen subcutaneously on days 0 and 24; 2 noninoculated cats and 49 non-SPF cats were controls. Serial serum samples were collected during 135 days and assayed for M. bovis antibodies by rapid test and MAPIA. On day 123, bPPD skin test was performed and read at 48 and 72 hr. The bPPD test at 72 hr had a mean skin thickness of 0.3 mm for stimulated cats and 0.1 mm for controls. Rapid test identified 4 of 6 stimulated cats after bPPD injection. The MAPIA detected antibody against MPB83, 16/83, 16 kDa, and M. bovis culture filtrate (MBCF) antigens. All assays differentiated between stimulated and control cats; however, 7 of 49 non-SPF control cats had a reaction for either antigen MBCF or 16/83. These preliminary studies show potential for antemortem detection of M. bovis among domestic cats. Additional studies to better characterize virulent M. bovis infection in cats would be of value.

Detection of antibodies to Toxoplasma gondii in oral fluid from pigs.

Toxoplasma gondii-infected pigs play a major role as a source of infection for humans and detection of high-risk herds is essential to implement control measures at the farm level. The aim of this study was to determine whether oral fluid (OF) could be used as a matrix to detect antibodies against T. gondii in infected pigs by immunoblot (IB). For this, OF from experimentally inoculated sows (n = 8) (serial samples) and naturally exposed group-housed fatteners (n = 42 groups, one sample/group) were analysed for IgG and IgA against T. gondii-SAG1 antigen by IB. Simultaneously, each animal was serologically tested for anti-T. gondii IgG by ELISA. Specific IgG was detected in the sera of all inoculated sows from 2 to 3 weeks post inoculation (pi) and in 3.4 to 92% of the pigs in 13 out of 42 groups. Experimentally inoculated sows showed positive OF-IB results for IgA (100%) and IgG (87.5%) at 1.5 weeks pi and continued yielding positive results for IgA (87.5-75%) and IgG (50%) until 4 weeks pi; however, from 8 weeks pi the frequency of detection of both isotypes was lower, despite constantly positive IgG values in serum-ELISA. Interestingly, consecutive daily samplings for 4 days at 13 and 30 weeks pi showed inconsistent results for some sows, showing that the antibody concentration in OF is prone to timely variations. Pooled OF from groups with 91 and 92% of seropositive pigs yielded positive IB results for IgG and IgA. Fattener groups with ≤13% of seropositive pigs gave negative IB results to both isotypes. Our results showed that antibodies to T. gondii can be detected in OF from infected pigs, and that IgA seems to be a more adequate target than IgG. Although OF does not seem to be a robust matrix to assess the serological status for T. gondii in individual animals, this diagnostic approach represents an interesting non-invasive, low-cost and animal welfare friendly option as a screening method at the farm level to determine high exposure to T. gondii in the herd.

Evaluation of the recombinant Heat shock protein B (HspB) of Coxiella burnetii as a potential antigen for immunodiagnostic of Q fever in goats.

Coxiella burnetii is an intracellular bacterium that causes a worldwide zoonosis, the Q fever. Currently, to diagnose the infection in ruminants, whole cell antigens-based ELISAs are used. In this study a heat shock protein, the HspB, was evaluated for its ability to be recognized by the goat immune system and its capacity to sign a stage of infection. The htpB gene of C. burnetii was cloned and sequenced. A high identity (>90%) was observed among the htpB genes of four ruminant strains tested. A recombinant protein was expressed in a prokaryotic expression system. The rHspB protein was used to determine the IgG reactivity by ELISA. Sera from experimentally and naturally infected goats were tested. The rHspB is recognized early during the infection course, at 18 days post-infection. Moreover, 80-90% of the animals tested were positive at 39-60dpi. In addition, animals presenting a reactivation of the infection displayed a higher reactivity, statistically significant (p<0.05), than that of the animals in latent infection. These findings suggest that the rHspB could be a good candidate for the development of an ELISA test making possible the detection of recent C. burnetii infection in goats as well as reactivation in those with latent infection.

In vitro allergy tests compared to intradermal testing in horses with recurrent airway obstruction.

Recurrent airway obstruction (RAO) is a common condition in stabled horses characterised by small airway inflammation, airway neutrophilia and obstruction following exposure of susceptible horses to mouldy hay and straw and is thus regarded as a hypersensitivity reaction to mould spores. However, the role of IgE-mediated reactions in RAO remains unclear. The aim of the study was to investigate with a serological IgE ELISA test (Allercept), an in vitro sulfidoleukotriene (sLT) release assay (CAST) and with intradermal testing (IDT) whether serum IgE and IgE-mediated reactions against various mould, mite and pollen extracts are associated with RAO. IDT reactions were evaluated at different times in order to detect IgE-mediated immediate type reactions (type I hypersensitivity reactions, 0.5-1 h), immune complex-mediated late type reactions (type III reactions, 4-10 h) and cell-mediated delayed type reactions (type IV hypersensitivity reactions 24-48 h). In the serological test, overall the control horses displayed more positive reactions than the RAO-affected horses but the difference was not significant. Comparison of the measured IgE levels showed that the RAO-affected horses had slightly higher IgE levels against Aspergillus fumigatus than controls (35 and 16 AU, respectively, p<0.05), but all values were below the cut off (150 AU) of the test. In the sLT release assay, seven positive reactions were observed in the RAO-affected horses and four in the controls but this difference was not significant. A significantly higher proportion of late type IDT reactions was observed in RAO-affected horses compared to controls (25 of 238 possible reactions versus 12 of 238 possible reactions, respectively, p<0.05). Interestingly, four RAO-affected but none of the control horses reacted with the recombinant mould allergen A. fumigatus 8 (rAsp f 8, p<0.05), but only late phase and delayed type reactions were observed. In all three tests the majority of the positive reactions was observed with the mite extracts (64%, 74% and 88% of all positive reactions, respectively) but none of the tests showed a significant difference between RAO-affected and control animals. Our findings do not support that IgE-mediated reactions are important in the pathogenesis of RAO. Further studies are needed to investigate whether sensitisation to mite allergens is of clinical relevance in the horse and to understand the role of immune reactions against rAsp f 8.