Serological and faecal detection of Angiostrongylus vasorum in dogs from Austria.

Canine angiostrongylosis is a potentially lethal parasitic disease that can manifest itself with a broad spectrum of clinical signs, including respiratory distress, neurological and bleeding disorders, or non-specific signs. The occurrence of Angiostrongylus vasorum is widely reported in Europe, but very little is known about its presence in Austria. In this first large-scale survey, 1279 sera were collected from Austrian dogs and tested by an ELISA for the detection of circulating antigen of A. vasorum (sensitivity: 95.7%, specificity 94.0%) and by a separate ELISA detecting specific antibodies (sensitivity 81.0%, specificity 98.8%). Furthermore, 1040 faecal samples were tested for the presence of lungworm first stage larvae (L1). One dog (0.1%, 95% confidence intervals [CI]: 0.0-0.4%) was positive in both ELISAs, while 1.2% (n = 15, CI: 0.7-1.9%) of the tested dogs were antigen-positive and 1.5% (n = 19, CI: 0.9-2.3%) were positive for specific antibodies. Overall, 13 dogs (1.3%; CI: 0.7-2.1%) were positive for A. vasorum L1 while 31 dogs were positive for Crenosoma vulpis L1 (3.0%; CI: 2.0-4.2%). One dog shed L1 from both A. vasorum and C. vulpis (0.1%, CI: 0.0-0.5%). Dogs positive for A. vasorum originated from northeast, southeast and south Austria (antigen and/or antibody detection), but also from north, west and southwest Austria (antibody detection) and from northeast and west Austria (L1 detection). One of 88 blood samples (1.1%, CI: 0.0-6.2%) submitted from the eastern part of Austria was positive by a rapid assay for A. vasorum antigen detection (Angio Detect™). Crenosoma vulpis positive samples originated from northwest, north, northeast, south and west Austria. These results confirm the very sporadic occurrence of A. vasorum in the investigated areas of the country. However, due to the substantial infectious pressure from the surrounding countries and the free circulation of dogs and foxes acting as wildlife reservoirs and due to clinical relevance for infected dogs, it is crucial to maintain disease awareness also in areas where the parasite has not yet been detected.

Vector-borne pathogens in clinically healthy military working dogs in eastern Austria.

Military working dogs have an increased risk of acquiring an infection with vector-borne pathogens due to kennel housing and regular exposure to wildlife and vectors. To evaluate the level of infections in clinically healthy dogs of the Austrian Armed Forces, 94 individuals of the Military Working Dog Training Centre (MWDTC) Kaisersteinbruch/eastern Austria were examined in August 2016, February 2019 and August 2019. A modified Knott test was used to determine the presence of microfilariae, PCR for DNA detection of filarioid nematodes (incl. Dirofilaria), Leishmania spp., piroplasms, Borrelia spp., Bartonella spp. and Anaplasmataceae, and serological examination for antibodies against Borrelia burgdoferi s. l. and Leishmania infantum in all dogs. Two dogs were positive for Dirofilaria repens in the Knott test, and one of them also by PCR. Six clinically healthy dogs (4.2%) were positive for Babesia canis (PCR). In serology, 10 (10.6%) of the dogs were positive for specific antibodies against Borrelia burgdoferi s. l. The results suggest that the current measures against arthropod vector exposure and the pathogens they can transmit are not fully sufficient for these dogs. Further investigations of the tick and mosquito fauna in this area will shed more light on the risk of exposure for both the dogs and the staff of the MWDTC.

Reduction of Allergic Lung Disease by Mucosal Application of Toxoplasma gondii-Derived Molecules: Possible Role of Carbohydrates.

Background: The hygiene hypothesis suggests a link between parasitic infections and immune disorders, such as allergic diseases. We previously showed that infection with Toxoplasma gondii or systemic application of T. gondii tachyzoites lysate antigen (TLA) in a prophylactic, but not therapeutic protocol, prevented allergic airway inflammation in mice. Here we tested the effect of prophylactic and therapeutic application of TLA via the mucosal route. Methods: Mice were intranasally treated with TLA either i) prior to sensitization, ii) during sensitization and challenge, or iii) after sensitization with ovalbumin (OVA). Recruitment of inflammatory cells to the lung, cytokine levels in restimulated lung and spleen cell cultures as well as levels of OVA-specific antibodies in serum were measured. In parallel, the effect of native TLA, heat-inactivated (hiTLA) or deglycosylated TLA (dgTLA) on sensitized splenocytes was evaluated ex vivo. Results: When applied together with OVA i) during systemic sensitization and local challenge or ii) exclusively during local challenge, TLA reduced infiltration of eosinophils into the lung, OVA-specific type 2 cytokines in restimulated lung cell cultures, and partially, type 2 cytokines in restimulated spleen cell cultures in comparison to allergic controls. No beneficial effect was observed when TLA was applied prior to the start of sensitization. Analysis of epitope sugars on TLA indicated a high abundance of mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. Deglycosylation of TLA, but not heat-inactivation, abolished the potential of TLA to reduce type 2 responses ex vivo, suggesting a significant role of carbohydrates in immunomodulation. Conclusion: We showed that mucosal application of TLA reduced the development of experimental allergy in mice. The beneficial effects depended on the timing of the application in relation to the time point of sensitization. Not only co-application, but also therapy in sensitized/allergic animals with native TLA reduced local allergic responses. Furthermore, we show that TLA is highly glycosylated and glycoconjugates seem to play a role in anti-allergic effects. In summary, given the powerful modulatory effect that TLA exhibits, understanding its exact mechanisms of action may lead to the development of novel immunomodulators in clinical application.

Seroprevalence Survey for Microsporidia in Common Bottlenose Dolphin ( Tursiops truncatus): Example of a Quantitative Approach Based on Immunoblotting.

Little is known about microsporidiosis pathogenicity in cetaceans. Here we report seroprevalence of 76% for microsporidia in blood samples from common bottlenose dolphins ( Tursiops truncatus), from animals managed under human care ( n=108) or captured for health assessments ( n=13) and released.

Toxoplasma gondii tachyzoite-extract acts as a potent immunomodulator against allergic sensitization and airway inflammation.

Epidemiological and experimental studies have shown an inverse relationship between infections with certain parasites and a reduced incidence of allergic diseases. We and others have shown that infection with Toxoplasma gondii prevents the development of allergy in mice. To establish whether this beneficial effect could be recapitulated by soluble products of this parasite, we tested an extract derived from T. gondii tachyzoites. Immunization of BALB/c mice with tachyzoites lysate antigen (TLA) elicited mixed Th1/Th2 responses. When TLA was applied together with the sensitizing ovalbumin (OVA), the development of allergic airway inflammation was reduced, with decreased airway hyperresponsiveness associated with reduced peribronchial and perivascular cellular infiltration, reduced production of OVA-specific Th2 cytokines in lungs and spleens and reduced levels of serum OVA-specific IgG1 as well as IgE-dependent basophil degranulation. Of note, TLA retained its immunomodulatory properties, inducing high levels of IL-6, TNFα, IL-10 and IL-12p70 in bone marrow-derived dendritic cells after heat-inactivation or proteinase K-treatment for disruption of proteins, but not after sodium metaperiodate-treatment that degrades carbohydrate structures, suggesting that carbohydrates may play a role in immunomodulatory properties of TLA. Here we show that extracts derived from parasites may replicate the benefits of parasitic infection, offering new therapies for immune-mediated disorders.

Application of mass spectrometry to elucidate the pathophysiology of Encephalitozoon cuniculi infection in rabbits.

Encephalitozoon cuniculi is a microsporidian species which can induce subclinical to serious disease in mammals including rabbits, a definitive natural host. The pathophysiology of infection has not been comprehensively elucidated. In this exploratory study, we utilized two mass spectrometry approaches: first, the analysis of the humoral response by profiling the microsporidian antigens as revealed by Western blot screening, and second, implementing the iTRAQ®-labeling protocol to focus on the changes within the host proteome during infection. Seven E. cuniculi proteins were identified at one-dimensional gel regions where specific seropositive reaction was observed by Western blot, including polar tube protein 3, polar tube protein 2, and for the first time reported: heat shock related 70kDa protein, polysaccharide deacetylase domain-containing protein, zinc finger protein, spore wall and anchoring disk complex protein EnP1, and translation elongation factor 1 alpha. In addition, there was a significant increase of nine host proteins in blood samples from E. cuniculi-diseased rabbits in comparison with non-diseased control subjects undergoing various inflammatory processes. This included serum paraoxonase, alpha-1-antiproteinase F precursor and alpha-1-antiproteinase S-1 which have presumptive catalytic activity likely related to infection control, and cystatin fetuin-B-type, an enzyme regulator that has been poorly studied to date. Notably, 11 proteins were found to be statistically increased in rabbits with neurological versus renal clinical presentation of E. cuniculi infection. Overall, this novel analysis based on mass spectrometry has provided new insights on the inflammatory and humoral responses during E. cuniculi infection in rabbits.

Application of Western blot analysis for the diagnosis of Encephalitozoon cuniculi infection in rabbits: example of a quantitative approach.

Diagnosis of Encephalitozoon cuniculi infection in rabbits remains a major veterinary issue. ELISA or immunofluorescence assays are the current reference standards of serological tests. However, these conventional techniques suffer from a lack of accuracy for distinguishing active from past infections, as a positive serostatus is common in clinically normal rabbits. In this study, we assessed the diagnostic performance of Western blot (WB) to detect both anti-E. cuniculi immunoglobulin G (IgG) and immunoglobulin M (IgM) in comparison with ELISA and to address the intensity of the immune response through a quantitative approach. Positive WB results were highly correlated with the E. cuniculi-related diseased status (P < 0.0001). Although it was more labor intensive and less standardized, quantitative WB provided detailed comparable analysis regarding the humoral response and diagnostic performance similar to ELISA testing with statistically higher sensitivity (88.4 vs. 76.1% for IgG detection and 84.3 vs. 70.4% for IgM, P < 0.01). Several specific WB bands were shown to be significantly associated with concomitant clinical signs, like the one located at 50 kDa (OR = 8.2, [2.4-27.7], P = 0.0008) for IgG and (OR = 27.9, [4.2-187.9], P = 0.0006) for IgM. Therefore, the quantitative WB may have application in veterinary diagnostic laboratories to increase the accuracy of the clinical diagnosis of E. cuniculi infection. In addition, this tool may help to further understand the development and function of the humoral immune response to this infectious agent.

Prime-boost vaccination with toxoplasma lysate antigen, but not with a mixture of recombinant protein antigens, leads to reduction of brain cyst formation in BALB/c mice.

INTRODUCTION: Infection with the ubiquitous parasite Toxoplasma gondii is a threat for immunocompromised patients and pregnant women and effective immune-prophylaxis is still lacking. METHODS: Here we tested a mixture of recombinant T. gondii antigens expressed in different developmental stages, i.e., SAG1, MAG1 and GRA7 (SMG), and a lysate derived from T. gondii tachyzoites (TLA) for prophylactic vaccination against cyst formation. Both vaccine formulations were applied systemically followed by an oral TLA-booster in BALB/c mice. RESULTS: Systemic priming with SMG and oral TLA-booster did not show significant induction of protective immune responses. In contrast, systemic priming and oral booster with TLA induced higher levels of Toxoplasma-specific IgG, IgG1 and IgG2a in sera as well as high levels of Toxoplasma-specific IgG1 in small intestines. Furthermore, high levels of Toxoplasma-specific Th1-, Th17- and Th2-associated cytokines were only detected in restimulated splenocytes of TLA-vaccinated mice. Importantly, in mice orally infected with T. gondii oocysts, only TLA-vaccination and booster reduced brain cysts. Furthermore, sera from these mice reduced tachyzoites invasion of Vero cells in vitro, indicating that antibodies may play a critical role for protection against Toxoplasma infection. Additionally, supernatants from splenocyte cultures of TLA-vaccinated mice containing high levels of IFN-γ lead to substantial production of nitric oxide (NO) after incubation with macrophages in vitro. Since NO is involved in the control of parasite growth, the high levels of IFN-γ induced by vaccination with TLA may contribute to the protection against T. gondii. CONCLUSION: In conclusion, our data indicate that prime-boost approach with TLA, but not with the mixture of recombinant antigens SMG, induces effective humoral and cellular Toxoplasma-specific responses and leads to significant reduction of cerebral cysts, thereby presenting a viable strategy for further vaccine development against T. gondii infection.

Comparison of an indirect fluorescent antibody test with Western blot for the detection of serum antibodies against Encephalitozoon cuniculi in cats.

Current clinical research indicates that Encephalitozoon (E.) cuniculi infections in cats may be underdiagnosed, especially in animals with typical ocular signs (cataract/anterior uveitis). Although molecular detection of the pathogen in tissue appears promising, serology remains the major diagnostic tool in the living animal. While serological tests are established for the main host of E. cuniculi, the rabbit, the routine serological diagnosis for cats still needs validation. The aim of the study was to evaluate the consistency of indirect fluorescence antibody test (IFAT) and Western blot (WB) for the detection of IgG antibodies against E. cuniculi in the serum of 84 cats. In addition, PCR of liquefied lens material or intraocular fluid was performed in those of the cats with a suspected ocular E. cuniculi infection. Twenty-one cats with positive PCR results were considered as a positive reference group. Results obtained by IFAT and WB corresponded in 83/84 serum samples, indicating a very good correlation between both serological methods. Using WB as the standard reference, sensitivity and specificity for the detection of antibodies against E. cuniculi by the IFAT were 97.6 and 100%, respectively. The positive and negative predictive values for the IFAT were 100 and 97.7%, respectively. The accuracy (correct classified proportion) for the detection of IgG antibodies against E. cuniculi in cats was 98.8%. The comparison of both serological methods with the PCR results also revealed a good agreement as 20 out of 21 PCR-positive samples were seropositive both in IFAT and WB. Both tests can be considered as equally reliable assays to detect IgG antibodies against E. cuniculi in cats. As the IFAT is quicker and easier to perform, it is recommended for routine use in the diagnosis of feline encephalitozoonosis.

Characterisation of Sarcoptes scabiei antigens.

In pig herds, the status of Sarcoptes scabiei infections is routinely monitored by serodiagnosis. Crude antigen for ELISA is usually prepared from S. scabiei var. canis or other variations and may lead to variations in the outcome of different tests, making assay standardisation difficult. This study was performed to investigate the antigen profiles of S. scabiei, including differences between hydrophilic and more hydrophobic protein fractions, by Western blotting with sera from pigs with defined infection status. Potential cross-reactivity among S. scabiei (var. canis, suis and bovis), Dermatophagoides farinae and Tyrophagus putrescentiae was also analysed. Hydrophobic S. scabiei antigens were detectable in the range of 40-50 kDa, whilst the hydrophilic fraction showed no specific antigenicity. In the hydrophobic fractions of D. farinae and T. putrescentiae, two major protein fractions in a similar size range could be identified, but no cross-reactivity with Sarcoptes-positive sera was detectable. However, examination of the hydrophilic fractions revealed cross-reactivity between Sarcoptes-positive sera and both the house dust mite and the storage mite in the range of 115 and 28/38 kDa. Specific bands in the same range (42 and 48 kDa) could be detected in blots from hydrophobic fractions of all three tested variations of S. scabiei (var. canis, bovis and suis). These results show that there are considerable differences in mange antibody reactivity, including reactions with proteins from free-living mites, which may interfere with tests based on hydrophilic antigens. Further refinement of antigen and the use of specific hydrophobic proteins could improve ELISA performance and standardisation.

Parasites on paper--The use of FTA Elute((R)) for the detection of Dirofilaria repens microfilariae in canine blood.

Storage of blood samples for subsequent DNA extraction without loss of integrity can be difficult under field conditions. Filter-based technologies are known to deliver good results for viral, bacterial and protozoan material but have not been tested for blood-dwelling stages of nematodes. In this study Whatman FTA Elute (Whatman plc, Middlesex, UK) filter technology was tested for its ability to stabilise DNA from blood samples of dogs naturally infected with Dirofilaria repens for storage prior to PCR. The concentration of microfilariae per 100microl of blood was evaluated and the blood was diluted to determine the lowest detectable number of parasites by real-time PCR (qPCR). In parallel, negative dog blood was prepared in the same way. A parasitaemia of 6+/-0.43 larvae per 100microl of blood containing EDTA could be detected using the FTA Elute filter cards, although quantification of the larvae by using the qPCR was not possible. Inasmuch as the average microfilaremia in a pool of positive dogs was 311+/-21.72 larvae per 100microl, these cards provide an effective tool for parasitological field studies because blood samples are stable for extended periods of time at room temperature without loss of DNA integrity.

[Sarcoptes scabiei var. suis in a closed pig breeding and fattening herd and control possibilities after treatment]. / Sarcoptes scabiei var. suis in einem geschlossenen Schweinezucht- und Mast- betrieb und Kontrollmöglichkeiten nach der Behandlung.

On an Austrian pig breeding and finishing farm containing 13,000 pigs a mange prevalence of 38.7% according to the results of the skin scraping and 28.2% based on serology was determined. Due to the insufficient treatment (single treatment of the sows using Phoxim [Sebacil pour on]), sustainable control was impossible. That could be confirmed by the high number of mange positive gilts and finishing pigs. Before eradication started the following prevalences of mange could be found: sows 6.74% (skin scrapings), respectively 6.18% (serologically), gilts 18.18% resp 28.67%, finishing pigs 54.35% and 38.58%. The breeding stock for eradication was treated with doramectin (Dectomax) injectable solution and the finishing pigs with Ivomec-praemix, both applied twice. The success of treatment of the different farm units and of different age groups was controlled for the following ten months by combined diagnostic methods. In addition to skin scrapings, serum and colostral samples were carried out using a commercially available ELISA licensed for investigation of blood serum and colostrum. After treatment antibodies in the serum of the sows and gilts and Sarcoptes mites in their skin scrapings were detectable for up to four months after treatment. In serum samples of piglets and colostrum samples antibodies against Sarcoptes mites were detectable up to five months after final treatment. Due to the higher level and longer verifiability of antibodies in blood samples of piglets for five months after treatment and high prevalences their use as a diagnostic tool can be recommended. In contrast the use of colostral samples for routine diagnosis should be investigated more thoroughly. The comparison of the results of different diagnostic methods showed that for reliable mange diagnosis combined methods are recommended.