A proteomic approach to the development of DIVA ELISA distinguishing pigs infected with Salmonella Typhimurium and pigs vaccinated with a Salmonella Typhimurium-based inactivated vaccine.

BACKGROUND: Salmonella enterica serovar Typhimurium is one of the most common enteropathogenic bacteria found in pigs in Europe. In our previous work, we demonstrated the protective effects in suckling piglets when their dams had been vaccinated with an S. Typhimurium-based inactivated vaccine. This study is focused on a procedure leading to serological discrimination between vaccinated and infected pigs. As we supposed, distinct environment during natural infection and in bacterial cultures used for vaccine preparation led to a slightly different spectrum of expressed S. Typhimurium proteins. The examination of porcine antibodies produced after the experimental infection with S. Typhimurium or after vaccination with S. Typhimurium-based inactivated vaccine by affinity chromatography and mass spectrometry revealed differences in antibody response applicable for serological differentiation of infected from vaccinated animals. RESULTS: Antibodies against Salmonella SipB, SipD and SseB proteins were detected at much higher levels in post-infection sera in comparison with control and post-vaccination sera. On the other hand, proteins BamB, OppA and a fragment of FliC interacted with antibodies from post-vaccination sera with a much higher intensity than from control and post-infection sera. In addition, we constructed ELISA assays using post-infection antigen - SipB protein and post-vaccination antigen - FliC-fragment and evaluated them on a panel of individual porcine sera. CONCLUSIONS: The analysis of antibody response of infected and vaccinated pigs by proteomic tools enabled to identify S. Typhimurium antigens useful for distinguishing infected from vaccinated animals. This approach can be utilized in other challenges where DIVA vaccine and a subsequent serological assay are required, especially when genetic modification of a vaccine strain is not desirable.

Survival of experimentally induced Toxoplasma gondii tissue cysts in vacuum packed goat meat and dry fermented goat meat sausages.

Ingestion of raw or undercooked meat is a potential source of human toxoplasmosis. The aim of this study was to determine the viability of Toxoplasma gondii cysts in vacuum packed (VP) goat meat and in dry fermented sausages (DFS), and evaluate certain physical and chemical parameters, like water activity (aw), pH value, content of salt, dry matter and fat. A portion of muscle tissue from experimentally infected animals was used for production of VP meat with or without addition of 2.5% curing salt, and stored at 4 °C or at -20 °C. Results of bioassay showed that, samples of vacuum packed Toxoplasma positive meat without salt addition were alive after six weeks at 4 °C. Incubation at -20 °C supported the viability after 3 h, but not after 4 h. After 7 days in 2.5% of curing salt, samples of T. gondii VP goat meat were still viable, but not after 14 days at 4 °C. All the DFS samples were not positive for infective cysts which mean that, they do not pose a risk of T. gondii transmission. These data suggest that vacuum packaging increases the survival of T. gondii cysts.

Effective Control of Johne's Disease in Large Czech Dairy Herds.

Introduction: Johne's disease, caused by infection with Mycobacterium avium subsp. paratuberculosis (MAP), causes economic losses in dairy herds due to reduced milk production and premature culling. A test-and-cull strategy coupled with changes in calf rearing management preventing new infections has been introduced into infected herds to control MAP prevalence. This study appraised the effectiveness of these practice changes. Material and Methods: In 19 large dairy herds (of a median 470 milk-producing cows), implementing MAP control measures for 3-7 years, a serum ELISA was used to detect infected cows in their dry-off period. The number of ELISA-positive animals per year (EPAY) was calculated and statistical analysis was used to test whether the EPAY total decreased during the control period and to analyse the EPAY in relationship to the duration of the control programme. Results: Statistical support was found for a decrease of EPAY over time (P < 0.01, odds ratio 0.756) and in 14 herds a significant fall in the percentages of EPAY during the test period (P ≤ 0.05) was noted. Conclusion: Our results demonstrated the effectiveness of the control measures in place to reduce MAP infection in herds with initial EPAY ≥3.36%. The missing decreasing trend in the remaining five herds with low average initial EPAY suggested the need for additional measures to reduce the number of infected animals in these herds.

Characterisation of immunosuppression in rabbits after infection with myxoma virus.

Myxoma virus (MXV) causes the systemic disease myxomatosis in the European rabbit. Despite many in vitro studies on the function of MXV immunomodulatory proteins and detailed molecular knowledge of virus, little is known about the dynamics of interaction of the virus with the integrated host-immune system during infection. In this study changes in haematological profile, changes in lymphocyte subset distribution and non-specific proliferation activity of lymphocytes from different lymphoid compartments on the 2nd, 4th, 6th, 9th and 11th day after experimental infection of rabbits with MXV strain Lausanne was characterised. The relationship between alterations of immune parameters and dynamic of virus dissemination through the body was investigated. Haematological changes included moderate leucopenia with significant lymphopenia, neutrophilia, monocytosis and eosinopenia. A decrease of T cells including CD4+ and CD8+ and increase of CD79alpha+ were observed in draining popliteal lymph node 4 days after virus inoculation. From day 6, comparable changes were seen in collateral popliteal lymph node, spleen and peripheral blood. From day 9, the mentioned lymphocyte subsets tended to reach their original state in all of these lymphocyte compartments except draining popliteal lymph node. In thymus, MXV infection affected mainly CD4+CD8+ double positive thymocytes. On the other hand, proliferation activity of lymphocytes determined by the proliferation assay with plant-derived mitogens was significantly reduced from day 4 or 6 and remained reduced until the end of experiment in all observed lymphoid organs. Presence of MXV in respective lymphoid compartments preceded changes in lymphocyte subset distribution or lymphocyte activity.

Antiviral activities of 2,6-diaminopurine-based acyclic nucleoside phosphonates against herpesviruses: In vitro study results with pseudorabies virus (PrV, SuHV-1).

Pseudorabies virus (PrV), a causative agent of Aujeszky's disease, is deadly to most mammals with the exception of higher primates and men. This disease causes serious economic loses among farm animals, especially pigs, yet many European countries are today claimed to be Aujeszky's disease free because of the discovery of an efficient vaccination for pigs. In reality, the virus is still present in wild boar. Current vaccines are neither suitable for dogs nor are there anti-PrV drugs approved for veterinary use. Therefore, the disease still represents a high threat, particularly for expensive hunting dogs that can come into close contact with infected boars. Here we report on the anti-PrV activities of a series of synthetic diaminopurine-based acyclic nucleoside phosphonate (DAP-ANP) analogues. Initially, all synthetic DAP-ANPs under investigation are shown to exhibit minimal cytotoxicity by MTT and XTT tests (1-100µM range). Thereafter in vitro infection models are established using PrV virus SuHV-1, optimized on PK-15 and RK-13 cell lines. Out of the six DAP-ANP analogues tested, analogue VI functionalized with a cyclopropyl group on the 6-amino position of the purine ring proves the most effective antiviral DAP-ANP analogue against PrV infection, aided by sufficient hydrophobic character to enhance bioavailability to its cellular target viral DNA-polymerase. Four other DAP-ANP analogues with functional groups introduced to the C2'position are shown ineffective against PrV infection, even with favourable hydrophobic properties. Cidofovir(®), a drug approved against various herpesvirus infections, is found to exert only low activity against PrV in these same in vitro models.

Evidence of passive faecal shedding of Mycobacterium avium subsp. paratuberculosis in a Limousin cattle herd.

It has been suggested that passive shedding of Mycobacterium avium subsp. paratuberculosis (MAP) in faeces may occur, but reliable data are missing. Passive shedding assumes the ingestion of MAP in contaminated feed and passive passage through the gastrointestinal tract without causing infection. In this study the presence of MAP in faeces in a closed herd of Limousin cattle was monitored for 53 months using quantitative real time PCR (qPCR) and culture. The initial prevalence of MAP in the herd was determined to be 63.4% and 4.9% using qPCR and culture, respectively. After the removal of two culture- and qPCR-positive (>10(4) MAP cells/g) cows, the prevalence of MAP using qPCR decreased to 42.1% and later to 15.6% and 6.7%. The continuous removal of suspected animals from the herd during the monitoring period minimised the presence of MAP in faeces to sporadic, which may have resulted from a decrease in the environmental infectious pressure. The findings suggest that the presence of low numbers of MAP in bovine faeces may not necessarily be caused by real infection, but rather by passive passage of MAP. This phenomenon should therefore be considered when interpreting MAP qPCR data.

Seroprevalence of Toxoplasma gondii and Encephalitozoon cuniculi in rabbits from different farming systems.

The breeding of domestic rabbit (Oryctolagus cuniculus) for human consumption has a long tradition mainly in European and Asian countries. Infections that can affect the production of meat or even be transmitted from animals to humans are important to monitor, especially for public health reasons as well as for their impact on animals health. This study aimed to collect sera from rabbits bred in different conditions and test the presence of Toxoplasma gondii and Encephalitozoon cuniculi antibodies. Whether infections were active or latent was assessed by determining the occurrence of IgM or IgM together with IgG antibodies which indicated active infection whereas latent infection was characterized by finding IgG antibodies only. An ELISA test was performed with 1883 sera samples collected throughout the Czech and Slovak Republics. The seroprevalence of T. gondii in 902 samples from 6 commercial farms (CF) was very low with only 4 rabbits (0.4%) being positive. In total 99 (10.1%) individuals out of 981 samples from 29 household farms (HF) were positive for T. gondii antibodies. Only 2 (50%) of the T. gondii positive CF rabbits had active infections while the rest were latently infected. The serological results showed that 35 (35.4%) rabbits from the T. gondii positive HF group suffered from active infection. Out of CF samples 185 (20.5%) were positive for E. cuniculi. Antibodies of E. cuniculi were detected in 497 (50.7%) HF rabbits. Active E. cuniculi infections were determined in 85.9% of CF and 56.3% of HF rabbits; respectively. Interestingly, the E. cuniculi positive rabbits were significantly more often positive for anti-T. gondii antibodies in comparison to E. cuniculi negative individuals. Prevalence of T. gondii in CF rabbits was negligible. According to our results meat of HF rabbits still poses a risk of T. gondii infection. Nevertheless, the risk is on its lowest level in 20 years which is apparently caused due to changes in feeding practices. The occurrence of E. cuniculi antibodies was significantly lower in rabbits from commercial farms, apparently because of better hygiene conditions.

Quantification of Toxoplasma gondii in tissue samples of experimentally infected goats by magnetic capture and real-time PCR.

Undercooked meat containing tissue cysts is one of the most common sources of Toxoplasma gondii infection in humans. Goats are very susceptible to clinical toxoplasmosis, and especially kids are common food animals, thereby representing a risk for human infection. A sequence-specific magnetic capture method was used for isolation of T. gondii DNA from tissue samples from experimentally infected goat-kids and real-time PCR for the 529 bp repeat element allowed quantification of T. gondii DNA. The contamination level in different types of tissue and in two groups of goats euthanized 30 and 90 dpi was compared. The highest concentration of T. gondii DNA in both groups of goats was found in lung tissue, but only the higher parasite count in lung tissue compared to other organs in group A (euthanized 30 dpi) was statistically significant. T. gondii concentrations were higher in liver and dorsal muscle samples from goats euthanized 90 dpi than in goats euthanized at 30 dpi, while the T. gondii concentration in hearts decreased. This study describes for the first time distribution of T. gondii parasites in post-weaned goat kids. New information about T. gondii predilection sites in goats and about the progression of infection between 30 and 90 dpi was achieved.

Antiviral effect of HPMPC (Cidofovir®), entrapped in cationic liposomes: in vitro study on MDBK cell and BHV-1 virus.

We designed and synthesised a series of new cationic lipids based on spermine linked to various hydrophobic anchors. These lipids could be potentially useful for the preparation of stable cationic liposomes intended for the construction of drug targeting systems applicable in the field of anticancer/antiviral therapy, vaccine carriers, and vectors for the gene therapy. Low in vitro toxicity was found for these compounds, especially for LD1, in several cell lines. The delivery of both a fluorescence marker (calcein) and antiviral drugs into cells has been achieved owing to a large extent of internalization of cationic liposomes (labelled by Lyssamine-Rhodamine PE or fluorescein-PE) as demonstrated by fluorescent microscopy and quantified by flow cytometry. The bovine herpes virus type 1 (BHV-1) virus infection in vitro model using MDBK cells was employed to study the effect of the established antiviral drug HPMPC (Cidofovir®) developed by Prof. A. Holý. Inhibition of BHV-1 virus replication was studied by quantitative RT-PCR and confirmed by both Hoffman modulation contrast microscopy and transmission electron microscopy. We found that in vitro antiviral activity of HPMPC was significantly improved by formulation in cationic liposomes, which decreased the viral replication by about 2 orders of magnitude.

Usefulness of detection of specific IgM and IgG antibodies for diagnosis of clinical encephalitozoonosis in pet rabbits.

Encephalitozoon cuniculi is an obligate intracellular pathogen that has wide host distribution, but primary affects rabbits. This study presents a seroepidemiological study of E. cuniculi infection in 500 pet rabbits from the Czech Republic using ELISA capable of measuring IgM and IgG antibodies. Specific IgM antibodies, reflecting acute, reactivated infection or reinfection, were detected in 32.4% of all rabbits. IgG antibodies indicating chronic infection, were presented in 68.0% of all rabbits. The highest detection rate of IgM (54.4%) and IgG (86.1%) antibodies was ascertained in rabbits with neurological symptoms (n=79, group I). In rabbits with renal disorders (n=47, group II) 36.2% animals were specific IgM and 80.9% IgG positive. Out of 9 rabbits with ocular disorders (group III), 44.4% were positive for anti-E. cuniculi IgM and 77.8% for IgG antibodies. In rabbits with multiple signs (neurological and renal or ocular, n=16, group IV), 43.8% animals were specific IgM and 68.8% IgG positive. Out of 287 rabbits with other disease (group V), 26.5% were positive for anti-E. cuniculi IgM and 64.1% for IgG antibodies. However, the high presence of IgM (24.2%) and IgG (51.6%) antibodies was detected in clinically healthy rabbits (n=62, group VI). Toxoplasma gondii infection should be considered as a differential diagnosis for neurological and ocular disorders in rabbits. Using ELISA, 19.2% from all rabbits were positive for specific anti-T. gondii IgG. The highest seropositivity was detected in group III (44.4%). Simultaneous testing of IgM and IgG specific antibodies give an indication of the infection status. Presence of IgM antibodies is indicative for active infection with requirement to institute proper antimicrosporidial therapy. As active infection was detected in considerably high numbers of rabbits with clinical signs that are not usually associated with E. cuniculi, and even in asymptomatic rabbits, detection of both isotypes of specific antibodies should be a routine part of a health check in rabbits.

Serological detection of Trichinella spiralis in swine by ELISA (enzyme-linked immunosorbent assay) using an excretory-secretory (E/S) antigen.

The enzyme-linked immunosorbent assay (ELISA) method is recommended for farm surveillance programs and may be useful for epidemiological studies in wildlife or for establishing Trichinella-free areas. In this study, our interest was to compare the specificity and the time of seroconversion of excretory-secretory (E/S) antigens prepared from Trichinella spiralis. A group of eight pigs was inoculated with 500 T. spiralis larvae per animal, and blood sampling was performed at 3 and 4-day intervals during all experiments. The numbers of muscle larvae were determined in four different muscles groups. The larvae per gram burden shows that the most heavily parasitized muscles were the diaphragm [mean = 43.7 larvae per gram (lpg)] and the tongue (mean = 16.9 lpg). Antibody responses were detected by any of eight infected pigs of T. spiralis. Using the ELISA method with E/S antigen, antibodies to T. spiralis were first found on the day 21st p.i. The initial detection of antibodies varied from 21st to 31st day p.i., and the peak was reported 42nd day p.i. Dynamic of antibodies was stable or increased slightly throughout the experimental period (60 days post-inoculation). Our results represent important data for validation of a serological test, especially if blood samples are taken during early stages of infection.

Humoral immune responses during experimental infection with Fascioloides magna and Fasciola hepatica in goats and comparison of their excretory/secretory products.

This study investigated the humoral immune responses of goats experimentally infected with Fascioloides magna and Fasciola hepatica to F. magna excretory/secretory products (FmESP) or F. hepatica excretory/secretory products (FhESP), respectively. An enzyme-linked immunosorbent assay (ELISA) was used to determine serum antibody responses and for possible discrimination of F. magna and F. hepatica infections in goats. Comparison of ESPs of both flukes and evaluation of ESP antigenicity was also studied applying immunoblotting techniques. In all infected goats, antibody level was significantly increased (against negative control) since 2 weeks post infection (WPI). However, the dynamics of antibodies varied between F. magna and F. hepatica groups during the course of the infection. The cross-reaction of antibodies developed against F. magna and F. hepatica with ESP proteins was recorded by ELISA. The species-specific proteins 40, 120 kDa from FmESP and 80, 160 kDa from FhESP (with no antibody cross-reaction) were detected by two dimensional electrophoresis and immunoblot as the potential immunodiagnostic markers. Our results suggest that F. magna and F. hepatica infection could be distinguished by common immunological techniques based on species-specific antigen-antibodies interaction.

Distribution of muscle larvae and antibody dynamics in goats experimentally infected with Trichinella spiralis.

Herbivorous animals can play a very important role in spreading trichinellosis. In the study presented here, the susceptibility and distribution of Trichinella spiralis infection was examined in 16 goat kids. The goats were inoculated with 10,000 T. spiralis larvae isolated by artificial digestion methods. The animals were necropsied per two animals in weekly intervals, and the larval burdens in different muscle tissue and anti-Trichinella antibodies measured with the indirect enzyme-linked immunosorbent assay (ELISA) serological method using excretory-secretory (E/S) antigen for detecting anti-Trichinella antibodies were assessed during the experiment. T. spiralis larval burden was maximal at 6 weeks postinoculation (480-5,057 larvae/g according to locality), and the larvae were also found in the myocardium (0.77 larvae/g). In this paper, our next step was to compare the specificity and the time of seroconversion by means of ELISA based on E/S antigen prepared from T. spiralis. Antibody response was detected in all 16 goats. The ELISA test carried out showed the first increments in optical density 2 weeks postinfection (p.i.), reached their peak 4 weeks p.i., and remained elevated from that day until the end of the experiment (10 weeks p.i.). These results indicated that specific anti-Trichinella antibodies in goats persist for a relatively long time.