In vitro assay to determine inactivation of Toxoplasma gondii in meat samples.

Consumption of raw and undercooked meat is considered as an important source of Toxoplasma gondii infections. However, most non-heated meat products contain salt and additives, which affect T. gondii viability. It was our aim to develop an in vitro method to substitute the mouse bioassay for determining the effect of salting on T. gondii viability. Two sheep were experimentally infected by oral inoculation with 6.5 × 104 oocysts. Grinded meat samples of 50 g were prepared from heart, diaphragm, and four meat cuts. Also, pooled meat samples were either kept untreated (positive control), frozen (negative control) or supplemented with 0.6 %, 0.9 %, 1.2 % or 2.7 % NaCl. All samples were digested in pepsin-HCl solution, and digests were inoculated in duplicate onto monolayers of RK13 (a rabbit kidney cell line). Cells were maintained for up to four weeks and parasite growth was monitored by assessing Cq-values using the T. gondii qPCR on cell culture supernatant in intervals of one week and ΔCq-values determined. Additionally, 500 µL of each digest from the individual meat cuts, heart and diaphragm were inoculated in duplicate in IFNγ KO mice. Both sheep developed an antibody response and tissue samples contained similar concentrations of T. gondii DNA. From all untreated meat samples positive ΔCq-values were obtained in the in vitro assay, indicating presence and multiplication of viable parasites. This was in line with the mouse bioassay, with the exception of a negative mouse bioassay on one heart sample. Samples supplemented with 0.6 %-1.2 % NaCl showed positive ΔCq-values over time. The frozen sample and the sample supplemented with 2.7 % NaCl remained qPCR positive but with high Cq-values, which indicated no growth. In conclusion, the in vitro method has successfully been used to detect viable T. gondii in tissues of experimentally infected sheep, and a clear difference in T. gondii viability was observed between the samples supplemented with 2.7 % NaCl and those with 1.2 % NaCl or less.

Proof of concept for multiplex detection of antibodies against Chlamydia species in chicken serum using a bead-based suspension array with peptides as antigens.

The available differentiating tests for Chlamydia are based on detection of genetic material and only give information about the actual infection status, but reveal nothing of past infections. As the use of serological methods increases the window of detection, the goal of this study was to investigate if it is possible to develop a differentiating serological test for antibodies against Chlamydia species in chicken sera. Focus was on C. psittaci, C. gallinacea, and two closely related species, i.e. C. abortus and C. avium. To enable differentiating serology, a bead-based Luminex suspension array was constructed, using peptides as antigens, derived from known immunoreactive Chlamydia proteins. For the majority of these peptides, species-specific seroreactivity in mammalian sera has been reported in literature. The suspension array correctly identified antibodies against various Chlamydia species in sera from experimentally infected mice, and was also able to differentiate between antibodies against C. psittaci and C. gallinacea in sera from experimentally infected chickens. In field sera, signals were difficult to interpret as insufficient sera from experimentally infected chickens were available for evaluating the seroreactivity of all peptides. Nevertheless, results of the suspension array with field sera are supported by published data on the occurrence of C. gallinacea in Dutch layers, thereby demonstrating the proof of concept of multiplex serology for Chlamydial species in poultry.

Methods to assess the effect of meat processing on viability of Toxoplasma gondii: towards replacement of mouse bioassay by in vitro testing.

Consumption of meat containing viable tissue cysts is considered one of the main sources of human infection with Toxoplasma gondii. In contrast to fresh meat, raw meat products usually undergo processing, including salting and mixing with other additives such as sodium acetate and sodium lactate, which affects the viability of T. gondii. However, the experiments described in the literature are not always performed in line with the current processing methods applied in industry. It was our goal to study the effect of salting and additives according to the recipes used by industrial producers. Mouse or cat bioassay is the 'gold standard' to demonstrate the presence of viable T. gondii. However, it is costly, time consuming and for ethical reasons not preferred for large-scale studies.Therefore, we first aimed to develop an alternative for mouse bioassay that can be used to determine the effect of processing on the viability of T. gondii tissue cysts. The assays studied were (i) a cell culture method to determine the parasite's ability to multiply, and (ii) a propidium monoazide (PMA) dye-based assay to selectively detect DNA from intact parasites. Processing experiments were performed with minced meat incubated for 20 h with low concentrations of NaCl, sodium lactate and sodium acetate. NaCl appeared to be the most effective ingredient with only one or two out of eight mice infected after inoculation with pepsin-digest of portions processed with 1.0, 1.2 and 1.6% NaCl. Results of preliminary experiments with the PMA-based method were inconsistent and did not sufficiently discriminate between live and dead parasites. In contrast, the cell culture method showed promising results, but further optimization is needed before it can replace or reduce the number of mouse bioassays needed. In future, standardised in vitro methods are necessary to allow more extensive testing of product-specific processing methods, thereby providing a better indication of the risk of T. gondii infection for consumers.

Clonal expansion of a virulent Streptococcus suis serotype 9 lineage distinguishable from carriage subpopulations.

Streptococcus suis is a porcine pathogen, causing severe invasive infections. S. suis serotype 9 is increasingly causing disease in Dutch and Chinese pig herds, but it is unknown whether all serotype 9 isolates are equally virulent and markers that can identify virulent strains are not available. Therefore, discrimination between virulent isolates and carriage isolates typically not associated with disease, is currently not possible. We collected tonsillar S. suis isolates from 6 herds not previously diagnosed with S. suis infections, and clinical S. suis isolates of previously diseased pigs. We confirmed the virulence of a virulent type strain and one representative clinical isolate, and the lack of virulence of two carriage isolates, in a pig infection model. Phylogenetic analysis of whole genome sequences of 124 isolates resulted in 10 groups, of which two were almost uniquely populated by clinical isolates. The population structure of S. suis serotype 9 appears highly diverse. However, analysis of the capsule loci sequences showed variation in a single region which fully correlated with a virulent genotype. Transmission electron microscopy suggested differences in capsule thickness between carriage and clinical genotypes. In conclusion, we found that that the S. suis serotype 9 population in the Netherlands is diverse. A distinct virulence-associated lineage was identified and could be discriminated based on the capsule locus sequence. Whilst the difference in virulence cannot be directly attributed to the DNA sequence, the correlation of capsule locus sequence with virulence could be used in the development of diagnostic tests to identify potential virulent S. suis serotype 9 in pigs.

Prevalence of tick-borne viruses in Ixodes ricinus assessed by high-throughput real-time PCR.

Ticks are one of the principal arthropod vectors of human and animal infectious diseases. Whereas the prevalence of tick-borne encephalitis virus in ticks in Europe is well studied, there is less information available on the prevalence of the other tick-borne viruses (TBVs) existing worldwide. The aim of this study was to improve the epidemiological survey tools of TBVs by the development of an efficient high-throughput test to screen a wide range of viruses in ticks.In this study, we developed a new high-throughput virus-detection assay based on parallel real-time PCRs on a microfluidic system, and used it to perform a large scale epidemiological survey screening for the presence of 21 TBVs in 18 135 nymphs of Ixodes ricinus collected from five European countries. This extensive investigation has (i) evaluated the prevalence of four viruses present in the collected ticks, (ii) allowed the identification of viruses in regions where they were previously undetected.In conclusion, we have demonstrated the capabilities of this new screening method that allows the detection of numerous TBVs in a large number of ticks. This tool represents a powerful and rapid system for TBVs surveillance in Europe and could be easily customized to assess viral emergence.

Exploring target-specific primer extension in combination with a bead-based suspension array for multiplexed detection and typing using Streptococcus suis as a model pathogen.

We investigated the feasibility of an assay based on target-specific primer extension, combined with a suspension array, for the multiplexed detection and typing of a veterinary pathogen in animal samples, using Streptococcus suis as a model pathogen. A procedure was established for simultaneous detection of 6 S. suis targets in pig tonsil samples (i.e., 4 genes associated with serotype 1, 2, 7, or 9, the generic S. suis glutamate dehydrogenase gene [ gdh], and the gene encoding the extracellular protein factor [ epf]). The procedure was set up as a combination of protocols: DNA isolation from porcine tonsils, a multiplex PCR, a multiplex target-specific primer extension, and finally a suspension array as the readout. The resulting assay was compared with a panel of conventional PCR assays. The proposed multiplex assay can correctly identify the serotype of isolates and is capable of simultaneous detection of multiple targets in porcine tonsillar samples. The assay is not as sensitive as the current conventional PCR assays, but with the correct sampling strategy, the assay can be useful for screening pig herds to establish which S. suis serotypes are circulating in a pig population.

Prevalence of Mycobacterium avium in slaughter pigs based on serological monitoring results and bacteriological validation.

Mycobacterium avium (MA) is a potential food safety hazard in pigs. Blood samples of slaughtered pigs in the Netherlands and Germany were tested for the presence of MA antibodies to estimate the serological prevalence in the tested population. In the Dutch and German population 1.0% and 1.7% samples were positive, and 0.5% and 17.4% of the herds were at risk for having a MA infection respectively. The validity of the applied MA-ELISA was evaluated under field conditions. The specificity of the MA-ELISA was high (>98.4%). The average herd sensitivity was 18%. In the affected herds on average 50% of the animals were tested bacteriological positive for MA. It can be concluded that serological screening for the presence of MA antibodies is capable of identifying pig populations that are at risk for a MA infection.

Isolation of mycobacteria other than Mycobacterium avium from porcine lymph nodes.

Mycobacterium avium causes lymphadenitis in pigs. This presents an economical burden, as these pigs meat is considered inappropriate for consumption. In humans, lymphadenitis due to nontuberculous mycobacteria (NTM) primarily affects children and is caused by a variety of NTM, though M. avium predominates. Mycobacterial culture was undertaken on lymph nodes of 107 slaughter pigs from a single pig farm. A high number of pigs with mycobacterial lymphadenitis were identified by culture. A commercial line probe assay and 16S rDNA gene sequencing were used to assess the frequency of disease due to mycobacteria other than M. avium. Forty-five pigs had mandibular lymph node samples yielding mycobacteria in culture. The majority yielded M. avium (39; 87%) only. One yielded M. avium and Mycobacterium palustre, five yielded only NTM other than M. avium (2yielded Mycobacterium malmoense, 1Mycobacterium bohemicum, 1Mycobacterium heckeshornense and a possibly novel species related to Mycobacterium scrofulaceum, and 1 grew a possibly novel species related to M. palustre). Several NTM species other than M. avium were cultured from porcine lymph nodes. The species distribution shows interesting parallels with human NTM lymphadenitis. Molecular typing and environmental sampling studies are required to identify the sources of these infections.

Serodiagnosis of Mycobacterium avium infections in pigs.

The aim of this study is the development and evaluation of a serodiagnostic assay for Mycobacterium avium (MA). After screening MA lipid fractions in an ELISA format, a polar lipid fraction was selected as antigen because of its superior recognition by serum antibodies in experimentally infected pigs. The resulting MA-ELISA was evaluated as an alternative for detection of MA infection by traditional pathological examination of pig lymph nodes for granulomatous lesions by meat inspectors. By comparing with bacteriological examination, the MA-ELISA showed significantly better sensitivity (69%) as compared to pathological examination (31%) in experimentally infected pigs. The MA-ELISA also appeared significantly more specific in a set of serum samples from MA negative pigs: only 1 out of these 153 serum samples reacted positive, whereas 99 (65%) of these had displayed false positive results by detection of lymph nodes lesions that appeared not to be associated with MA (Komijn et al., 2007). The MA-ELISA was subsequently evaluated using serum samples from two farms with pigs known to be infected with MA. Bacteriological examination of the sub-maxillary and mesenteric lymph nodes showed that 56% (103/184) and 35% (41/117) of the pigs, respectively were positive for MA in these farms. In the first farm, 16% (29/184) of the pigs tested positive in MA-ELISA and 31% (57/184) by pathological examination. On the contrary, in the second farm, more pigs tested positive 17% (15/117) in MA-ELISA with 8% (9/117) positivity by pathological examination. Taking the results on both farms together, the sensitivity of the MA-ELISA was 14% and the specificity 83%, whereas the sensitivity of the pathological examination was 31% and the specificity 86%. For practical reasons use of a serological test as the MA-ELISA may be preferred over pathological or bacteriological examinations. Our studies in experimentally infected and negative "field" sera indicate that the MA-ELISA is significantly more specific and more sensitive than detection by classical pathological examination. However, the studies in two MA infected farms show a variable picture with pathological examination overall performing better. Study in a wider range of "positive" farms will be needed to provide a more comprehensive view of the quality of both tests for detection of MA in infected farms. At the same time further optimization of MA-ELISA with use of lipid antigens from a broader range of serotypes may improve its performance in the face of infections with different MA serotypes.