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Botulinum neurotoxins (BoNTs) are highly toxic proteins that require high-affinity immunocapture reagents for use in endopeptidase-based assays. Here, 30 novel and 2 earlier published llama single-domain antibodies (VHHs) against the veterinary-relevant BoNT serotypes C and D were yeast-produced. These VHHs recognized 10 independent antigenic sites, and many cross-reacted with the BoNT/DC and CD mosaic variants. As VHHs are highly suitable for genetically linking to increase antigen-binding affinity, 52 VHH multimers were produced and their affinity for BoNT/C, D, DC, and CD was determined. A selection of 15 multimers with high affinity (KD < 0.1 nM) was further shown to be resilient to a high salt wash that is used for samples from complex matrices and bound native BoNTs from culture supernatants as shown by Endopep-MS. High-affinity multimers suitable for further development of a highly sensitive Endopep-MS assay include four multimers that bind both BoNT/D and CD with KD of 14–99 pM, one multimer for BoNT/DC (65 pM) that also binds BoNT/C (75 pM), and seven multimers for BoNT/C (<1–19 pM), six of which also bind BoNT/DC with lower affinity (93–508 pM). In addition to application in diagnostic tests, these VHHs could be used for the development of novel therapeutics for animals or humans.

In assessing species susceptibility for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and in the search for an appropriate animal model, multiple research groups around the world inoculated a broad range of animal species using various SARS-CoV-2 strains, doses and administration routes. Although in silico analyses based on receptor binding and diverse in vitro cell cultures were valuable, exact prediction of species susceptibility based on these tools proved challenging. Here, we assessed whether precision-cut lung slices (PCLS) could facilitate the selection of animal models, thereby reducing animal experimentation. Pig, hamster and cat PCLS were incubated with SARS-CoV-2 and virus replication was followed over time. Virus replicated efficiently in PCLS from hamsters and cats, while no evidence of replication was obtained for pig PCLS. These data corroborate the findings of many research groups that have investigated the susceptibility of hamsters, pigs and cats towards infection with SARS-CoV-2. Our findings suggest that PCLS can be used as convenient tool for the screening of different animal species for sensitivity to newly emerged viruses. To validate our results obtained in PCLS, we employed the hamster model. Hamsters were inoculated with SARS-CoV-2 via the intranasal route. Susceptibility to infection was evaluated by body weight loss, viral loads in oropharyngeal swabs and respiratory tissues and lung pathology. The broadly used hamster model was further refined by including activity tracking of the hamsters by an activity wheel as a very robust and sensitive parameter for clinical health. In addition, to facilitate the quantification of pathology in the lungs, we devised a semi-quantitative scoring system for evaluating the degree of histological changes in the lungs. The inclusion of these additional parameters refined and enriched the hamster model, allowing for the generation of more data from a single experiment. View Full-Text

BackgroundPasteurella multocida, Mannheimia haemolytica, Histophilus somni and Trueperella pyogenes are four bacterial agents commonly associated with bovine respiratory disease (BRD). In this study a bacterial multiplex real-time PCR (the RespoCheck PCR) was evaluated for the detection in bronchoalveolar lavage fluid (BALF) of these four bacterial agents.ResultsThe analytical sensitivity of the multiplex real-time PCR assay determined on purified DNA and on bacterial cells of the four target pathogens was one to ten fg DNA/assay and 4 × 10−1 to 2 × 100 CFU/assay. The analytical specificity of the test was, as evaluated on a collection of 118 bacterial isolates, 98.3% for M. haemolytica and 100% for the other three target bacteria. A set of 160 BALF samples of calves originating from ten different herds with health problems related to BRD was examined with bacteriological methods and with the RespoCheck PCR. Using bacteriological examination as the gold standard, the diagnostic sensitivities and specificities of the four bacterial agents were respectively between 0.72 and 1.00 and between 0.70 and 0.99. Kappa values for agreement between results of bacteriological examination and PCRs were low for H. somni (0.17), moderate for P. multocida (0.52) and M. haemolytica (0.57), and good for T. pyogenes (0.79). The low and moderate kappa values seemed to be related to limitations of the bacteriological examination, this was especially the case for H. somni.ConclusionIt was concluded that the RespoCheck PCR assay is a valuable diagnostic tool for the simultaneous detection of the four bacterial agents in BALF of calves.

The success of a Toxoplasma gondii surveillance program in European pig production systems depends partly on the quality of the test to detect infection in the population. The test accuracy of a recently developed serological bead-based assay (BBA) was investigated earlier using sera from experimentally infected animals. In this study, the accuracy of the BBA was determined by the use of sera from animals from two field subpopulations. As no T. gondii infection information of these animals was available, test accuracy was determined through a Bayesian approach allowing for conditional dependency between BBA and an ELISA test. The priors for prevalence were based on available information from literature, whereas for specificity vague non-informative priors were used. Priors for sensitivity were based either on available information or specified as non-informative. Posterior estimates for BBA sensitivity and specificity were (mode) 0.855 (Bayesian 95% credibility interval (bCI) 0.702–0.960) and 0.913 (bCI 0.893–0.931), respectively. Comparing the results of BBA and ELISA, sensitivity was higher for the BBA while specificity was higher for ELISA. Alternative priors for the sensitivity affected posterior estimates for sensitivity of both BBA and ELISA, but not for specificity. Because the difference in prevalence between the two subpopulations is small, and the number of infected animals is small as well, the precision of the posterior estimates for sensitivity may be less accurate in comparison to the estimates for specificity. The estimated value for specificity of BBA is at least optimally defined for testing pigs from conventional and organic Dutch farms.

Background: The prevalence of Toxoplasma gondii in common moles, Talpa europaea, was investigated in order to determine whether moles can serve as an indicator species for T. gondii infections in livestock. Findings: In total, 86 moles were caught from 25 different sites in the Netherlands. Five different trapping habitats were distinguished: pasture, garden, forest, roadside, and recreation area. No positive samples (brain cysts) were found during microscopic detection (n¿=¿70). Using the Latex Agglutination Test (LAT), sera of 70 moles were examined, whereby no sample reacted with T. gondii antigen. Real Time-PCR tests on brain tissue showed 2 positive samples (2.3%). Conclusions: Because of the low number of positives in our study, the use of the common mole as an indicator species for livestock infections is currently not recommended.

High numbers of Toxoplasma gondii oocysts in the environment are a risk factor to humans. The environmental contamination might be reduced by vaccinating the definitive host, cats. An experimental challenge model is necessary to quantitatively assess the efficacy of a vaccine or drug treatment. Previous studies have indicated that bradyzoites are highly infectious for cats. To infect cats, tissue cysts were isolated from the brains of mice infected with oocysts of T. gondii M4 strain, and bradyzoites were released by pepsin digestion. Free bradyzoites were counted and graded doses (1000, 100, 50, 10), and 250 intact tissue cysts were inoculated orally into three cats each. Oocysts shed by these five groups of cats were collected from faeces by flotation techniques, counted microscopically and estimated by real time PCR. Additionally, the number of T. gondii in heart, tongue and brains were estimated, and serology for anti T. gondii antibodies was performed. A Beta-Poisson dose-response model was used to estimate the infectivity of single bradyzoites and linear regression was used to determine the relation between inoculated dose and numbers of oocyst shed. We found that real time PCR was more sensitive than microscopic detection of oocysts, and oocysts were detected by PCR in faeces of cats fed 10 bradyzoites but by microscopic examination. Real time PCR may only detect fragments of T. gondii DNA without the presence of oocysts in low doses. Prevalence of tissue cysts of T. gondii in tongue, heart and brains, and anti T. gondii antibody concentrations were all found to depend on the inoculated bradyzoite dose. The combination of the experimental challenge model and the dose response analysis provides a suitable reference for quantifying the potential reduction in human health risk due to a treatment of domestic cats by vaccination or by therapeutic drug application.

Background: Avian influenza virus (AIV) is classified into two pathotypes, low pathogenic (LP) and high pathogenic ( HP), based on virulence in chickens. Differences in pathogenicity between HPAIV and LPAIV might eventually be related to specific characteristics of strains, tissue tropism and host responses. Methods: To study differences in disease development between HPAIV and LPAIV, we examined the first appearance and eventual load of viral RNA in multiple organs as well as host responses in brain and intestine of chickens infected with two closely related H7N1 HPAIV or LPAIV strains. Results: Both H7N1 HPAIV and LPAIV spread systemically in chickens after a combined intranasal/intratracheal inoculation. In brain, large differences in viral RNA load and host gene expression were found between H7N1 HPAIV and LPAIV infected chickens. Chicken embryo brain cell culture studies revealed that both HPAIV and LPAIV could infect cultivated embryonic brain cells, but in accordance with the absence of the necessary proteases, replication of LPAIV was limited. Furthermore, TUNEL assay indicated apoptosis in brain of HPAIV infected chickens only. In intestine, where endoproteases that cleave HA of LPAIV are available, we found minimal differences in the amount of viral RNA and a large overlap in the transcriptional responses between HPAIV and LPAIV infected chickens. Interestingly, brain and ileum differed clearly in the cellular pathways that were regulated upon an AI infection. Conclusions: Although both H7N1 HPAIV and LPAIV RNA was detected in a broad range of tissues beyond the respiratory and gastrointestinal tract, our observations indicate that differences in pathogenicity and mortality between HPAIV and LPAIV could originate from differences in virus replication and the resulting host responses in vital organs like the brain.

Avian influenza virus can be divided into two groups, highly pathogenic avian influenza virus (HPAI) and low pathogenic avian influenza virus (LPAI) based on their difference in virulence. To investigate if the difference in clinical outcome between LPAI and HPAI in chickens is due to immunological host responses in the lung within the first 24 hours post infection (hpi), chickens were infected with LPAI or HPAI of subtype H7N1. Virus was found in the caudal and cranial part of the lung. With LPAI, virus was localised around the intrapulmonary bronchus and secondary bronchi. In sharp contrast, HPAI was detected throughout the whole lung. However, based on viral RNA levels, no quantitative difference was observed between LPAI and HPAI infected birds. In infected areas of the lungs, an influx of CD8a+ cells as well as KUL01+ macrophages and dendritic cells (DC) occurred as fast as 8 hpi in both infected groups. No major difference between LPAI and HPAI infected birds in the induction of cytokines and interferons at mRNA level in lung tissue was found. In conclusion, the differences in lethality for chickens infected with LPAI or HPAI could be ascribed to difference in location of the virus. However similar amounts of viral RNA, similar cytokine mRNA levels, and similar influxes of CD8a+ and KUL01+ macrophages and DC were found between HPAI and LPAI in the lungs. A cytokine storm at mRNA level as described for mammals was not observed in the lungs of HPAI infected birds within 24 hpi.

A short written questionnaire was used to study certain characteristics of the organic pig production chain in The Netherlands and the circumstances on the farm that might play a role in the transmission of Toxoplasma infection to the pigs. Of the 81 certified organic slaughter pig farmers present in the Netherlands in 2006, 52 responded to the questionnaire (64 % response). The farms could be divided into two populations. One population was represented by small organic pig farms with a mean number of 55 slaughtered pigs per year. These farms covered 40% of the total number of investigated farms, but only represented 2.5 % of the total number of slaughtered pigs. The second population had a mean annual production of 1460 animals. Almost 95% of these animals are currently slaughtered and further distributed by the Vion Food Group (de Groene Weg). A small part of the pigs (4%) is directly delivered to a slaughter company in Germany (Thönes) and 1% is sold via farm home sales. For each farm an arbitrary Toxoplasma risk factor analysis was performed. Factors included the type of outdoor run (concrete or soil), feeding goat or sheep whey, number of cats, access of cats to outdoor run, stables and feed, rodent control and covering roughage fed to the animals. Calculation of the total risk score (summation of chance times severity scores for several factors) showed that many farmers already used management factors that decreased the risk for Toxoplasma infection. Analysis of a possible relation between risk score and farm size showed that a poor score was often seen on small farms. Because these farms mainly sell their meat in a frozen condition via home sales, this is not considered to represent a problem for food safety. Further research is needed to investigate whether the risk for Toxoplasma can be maintained via on farm prevention or whether a Toxoplasma monitoring program should be implemented at slaughter, possibly with post slaughter decontamination. The fact that

In organic livestock production systems, farm-management factors are thought to play an important role in the on-farm prevalence of Toxoplasma gondii. Serological results and the results of an HACCP analysis were combined to determine important risk factors for the prevalence of this protozoan parasite. Mathematical analysis demonstrated that feeding goat whey to pigs and the presence of a high number of cats were positively correlated to T. gondii seroprevalence in pigs. Not covering roughage and the farmers' assumption that pigs can come into contact with cat feces also showed a positive relationship. In order to decrease the risk of T. gondii infecting their pigs, farmers should limit the access and number of cats on their farms and refrain from feeding goat whey to their pigs.

PURPOSE. Consumption of undercooked pork meat products has been considered a major risk factor for contracting toxoplasmosis in humans. Indoor farming and improved hygiene have drastically reduced Toxoplasma infections in pigs over the past decades. Whether introduction of animal-friendly production systems will lead to a reemergence of Toxoplasma infections in pigs is not yet known. Investigating this possibility was the purpose of this study. METHODS. Blood was obtained from pigs raised for slaughter and tested for Toxoplasma antibodies by using latex agglutination and indirect immunofluorescence testing, with confirmation by immunoblotting. RESULTS. None of the slaughter pigs (n = 621) from conventional farms (n = 30) were positive, whereas 38 (2.9%) of 1295 animals from animal-friendly systems tested positive (n = 33 farms; 13 [39%] farms positive). CONCLUSIONS. The following conclusions may be derived from this study: Conventionally (indoors) raised pigs are free from Toxoplasma infection, and (2) animal-friendly production systems may lead to a reemergence of Toxoplasma infections, although many of these farms remain Toxoplasma free. Slaughterhouse monitoring of pigs from animal-friendly production systems combined with on-farm prevention strategies should be applied to ensure safety for consumers of the meat products obtained from these animals.