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.

Zoonotic Pathogens in Eurasian Beavers (Castor fiber) in the Netherlands.

Successful repopulation programs of Eurasian beavers (Castor fiber) have resulted in an increase in beaver populations throughout Europe. This may be of public health relevance because beavers can host multiple zoonotic pathogens. From March 2018 to March 2020, opportunistic testing of dead beavers was performed for hepatitis E virus, orthohantavirus, Anaplasma phagocytophilum, Bartonella spp., extended-spectrum-betalactamase or AmpC (ESBL/AmpC-)-producing Enterobacteriaceae, Francisella tularensis, Leptospira spp., Neoehrlichia mikurensis, Babesia spp., Echinococcus multilocularis, Toxoplasma gondii, and Trichinella spp. From the 24 beavers collected, three zoonotic pathogens were detected. One beaver was positive for T. gondii, one was positive for ESBL/AmpC-producing Enterobacteriaceae, and one was positive for N. mikurensis. The latter finding indicates that beavers can be bitten by Ixodes ricinus and be exposed to tick-borne pathogens. The detected ESBL/AmpC-gene was blaCMY-2 in an Escherichia coli ST6599. The findings suggest that the role of beavers in the spread of zoonotic diseases in the Netherlands is currently limited.

Population genetics, invasion pathways and public health risks of the raccoon and its roundworm Baylisascaris procyonis in northwestern Europe.

The geographic range of the zoonotic raccoon roundworm (Baylisascaris procyonis) is expanding together with the range of its host, the raccoon (Procyon lotor). This creates a new public health risk in parts of Europe where this parasite was previously absent. In the Netherlands, a raccoon population is becoming established and incidental findings of B. procyonis have been reported. To assess the risk to public health, the prevalence of B. procyonis was determined in the province of Limburg, where currently the largest Dutch raccoon population is present, as well as in the adjoining region of southern Belgium. Furthermore, genetic methods were employed to assess invasion pathways of both the raccoon and B. procyonis to aid in the development of control measures. Macroscopic analysis of intestinal content and testing of faecal samples were performed to detect B. procyonis adults and eggs. The population genetics of both B. procyonis and its raccoon host were analysed using samples from central and northwestern Europe. B. procyonis was found in 14/23 (61%, 95% CI: 41%-78%) raccoons from Limburg, but was not detected in 50 Belgian raccoons. Genetic analyses showed that the majority of the Dutch raccoons and their roundworms were introduced through ex-captive individuals. As long as free-living raccoon populations originate from captivity, population control methods may be pursued. However, natural dispersal from the border regions will complicate prolonged population control. To reduce the public health risk posed by B. procyonis, public education to increase awareness and adapt behaviour towards raccoons is key.

Prevalence, risk factors and genetic traits of Salmonella Infantis in Dutch broiler flocks.

Salmonella Infantis is a poultry-adapted Salmonella enterica serovar that is increasingly reported in broilers and is also regularly identified among human salmonellosis cases. An emerging S. Infantis mega-plasmid (pESI), carrying fitness, virulence and antimicrobial resistance genes, is also increasingly found. We investigated the prevalence, genetic characteristics and risk factors for (pESI-carrying) S. Infantis in broilers. Faecal samples from 379 broiler flocks (in 198 farms with ≥3000 birds) in the Netherlands were tested. A questionnaire about farm characteristics was also administered. Sampling was performed in July 2018-May 2019, three weeks before slaughter. Fourteen flocks (in 10 farms) were S. Infantis-positive, resulting in a 3.7 % flock-level and 5.1 % farm-level prevalence. Based on multi-locus sequence typing (MLST), all isolates belonged to sequence type 32. All but one isolate carried a pESI-like mega-plasmid. Core-genome MLST showed considerable heterogeneity among the isolates, even within the same farm, with a few small clusters detected. The typical pESI-borne multi-resistance pattern to aminoglycosides, sulphonamide and tetracycline (93 %), as well as trimethoprim (71 %), was found. Additionally, resistance to (fluoro)quinolones based on gyrA gene mutations was detected. S. Infantis was found more often in flocks using salinomycin as coccidiostat, where flock thinning was applied or litter quality was poor, whereas employing external cleaning companies, wheat in feed, and vaccination against infectious bronchitis, were protective. Suggestive evidence for vertical transmission from hatcheries was found. A heterogeneous (pESI-carrying) S. Infantis population has established itself in Dutch broiler flocks, calling for further monitoring of its spread and a comprehensive appraisal of control options.

Digging into Toxoplasma gondii infections via soil: A quantitative microbial risk assessment approach.

Soil has been identified as an important source of exposure to a variety of chemical and biological contaminants. Toxoplasma gondii is one of those potential biological contaminants associated with serious health effects in pregnant women and immunocompromised patients. Gardening or consumption of homegrown vegetables may present an important route of T. gondii infection via accidental ingestion of soil. In the Netherlands, there is quantitative information on the risk of T. gondii infection via meat products, but not on the risk of infection through soil. The objective of this study was to develop a quantitative microbial risk assessment (QMRA) model for estimating the risk associated with T. gondii exposure via accidental soil ingestion in the Netherlands. In order to obtain the needed information, a magnetic capture method for detection of T. gondii oocysts in soil samples was developed, and T. gondii DNA was detected using qPCR targeting the 529 bp repeat element. The method was shown to provide 95% probability of detection (95% CI: 88-100%) when at least 34 oocysts are present in 25 g of soil. T. gondii DNA was detected in 5 of 148 soil samples with interpretable results (3%, 95% CI: 1.5-7.7%). Results for 18 samples were not interpretable due to PCR inhibition. The estimated amount of oocysts presented in qPCR positive samples was quantified by a linear model, and the amount varied from 8 to 478 in 25 g of soil. The estimated incidence rate of T. gondii infection from the QMRA model via soil varied from 0.3 to 1.8 per 1000 individuals per day. Several data gaps (e.g., soil contamination/ingestion and oocysts viability) have been identified in this study, the structure of the model can be applied to obtain more accurate estimates of the risk of T. gondii infection via soil when data become available.

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.

Detection and dissemination of Toxoplasma gondii in experimentally infected calves, a single test does not tell the whole story.

BACKGROUND: Although the detection of Toxoplasma gondii in bovine tissues is rare, beef might be an important source of human infection. The use of molecular techniques, such as magnetic capture qPCR (MC-qPCR), in combination with the gold standard method for isolating the parasite (mouse bioassay), may increase the sensitivity of T. gondii detection in infected cattle. The risk of transmission of the parasite to humans from undercooked/raw beef is not fully known and further knowledge about the predilection sites of T. gondii within cattle is needed. In the current study, six Holstein Friesian calves (Bos taurus) were experimentally infected with 106 T. gondii oocysts of the M4 strain and, following euthanasia (42 dpi), pooled tissues were tested for presence of the parasite by mouse bioassay and MC-qPCR. RESULTS: Toxoplasma gondii was detected by both MC-qPCR and mouse bioassay from distinct pools (100 g) of tissues comprising: liver, tongue, heart, diaphragm, semitendinosus (hindlimb), longissimus dorsi muscle (sirloin) and psoas major muscle (fillet). When a selection of individual tissues which had been used for mouse bioassay were examined by MC-qPCR, parasite DNA could only be detected from two animals, despite all calves showing seroconversion after infection. CONCLUSIONS: It is apparent that one individual test will not provide an answer as to whether a calf harbours T. gondii tissue cysts. Although the calves received a known number of infectious oocysts and highly sensitive methods for the detection of the parasite within bovine tissues were applied (mouse bioassay and MC-qPCR), the results confirm previous studies which report low presence of viable T. gondii in cattle and no clear predilection site within bovine tissues.

Evaluation by latent class analysis of a magnetic capture based DNA extraction followed by real-time qPCR as a new diagnostic method for detection of Echinococcus multilocularis in definitive hosts.

A new method, based on a magnetic capture based DNA extraction followed by qPCR, was developed for the detection of the zoonotic parasite Echinococcus multilocularis in definitive hosts. Latent class analysis was used to compare this new method with the currently used phenol-chloroform DNA extraction followed by single tube nested PCR. In total, 60 red foxes and coyotes from three different locations were tested with both molecular methods and the sedimentation and counting technique (SCT) or intestinal scraping technique (IST). Though based on a limited number of samples, it could be established that the magnetic capture based DNA extraction followed by qPCR showed similar sensitivity and specificity as the currently used phenol-chloroform DNA extraction followed by single tube nested PCR. All methods have a high specificity as shown by Bayesian latent class analysis. Both molecular assays have higher sensitivities than the combined SCT and IST, though the uncertainties in sensitivity estimates were wide for all assays tested. The magnetic capture based DNA extraction followed by qPCR has the advantage of not requiring hazardous chemicals like the phenol-chloroform DNA extraction followed by single tube nested PCR. This supports the replacement of the phenol-chloroform DNA extraction followed by single tube nested PCR by the magnetic capture based DNA extraction followed by qPCR for molecular detection of E. multilocularis in definitive hosts.

First findings of Trichinella spiralis and DNA of Echinococcus multilocularis in wild raccoon dogs in the Netherlands.

The recent invasion of the raccoon dog in the Netherlands may be associated with the risk of introduction and spread of zoonotic pathogens. The aim of this study was to assess whether Echinococcus multilocularis and Trichinella spp. infections are present in Dutch raccoon dogs. Between 2013 and 2014, nine raccoon dogs, mainly road kills, were collected for necropsies. One raccoon dog tested repeatedly positive in the qPCR for E. multilocularis. The positive raccoon dog was collected in the province of Flevoland, which is not a known endemic region for E. multilocularis. Another raccoon dog tested positive for Trichinella spiralis by the digestion of the forelimb musculature and the tongue. Trichinella spiralis has not been reported in wildlife since 1998 and thus far was not found in wild carnivores in the Netherlands. It shows that despite the small raccoon dog population that is present in the Netherlands and the limited number of raccoon dogs that were tested, the raccoon dog may play a role in the epidemiology of E. multilocularis and Trichinella spp. in the Netherlands.

Risk factors related to Toxoplasma gondii seroprevalence in indoor-housed Dutch dairy goats.

Toxoplasma gondii can cause disease in goats, but also has impact on human health through food-borne transmission. Our aims were to determine the seroprevalence of T. gondii infection in indoor-housed Dutch dairy goats and to identify the risk factors related to T. gondii seroprevalence. Fifty-two out of ninety approached farmers with indoor-kept goats (58%) participated by answering a standardized questionnaire and contributing 32 goat blood samples each. Serum samples were tested for T. gondii SAG1 antibodies by ELISA and results showed that the frequency distribution of the log10-transformed OD-values fitted well with a binary mixture of a shifted gamma and a shifted reflected gamma distribution. The overall animal seroprevalence was 13.3% (95% CI: 11.7­14.9%), and at least one seropositive animal was found on 61.5% (95% CI: 48.3­74.7%) of the farms. To evaluate potential risk factors on herd level, three modeling strategies (Poisson, negative binomial and zero-inflated) were compared. The negative binomial model fitted the data best with the number of cats (1­4 cats: IR: 2.6, 95% CI: 1.1­6.5; > = 5 cats:IR: 14.2, 95% CI: 3.9­51.1) and mean animal age (IR: 1.5, 95% CI: 1.1­2.1) related to herd positivity. In conclusion, the ELISA test was 100% sensitive and specific based on binary mixture analysis. T. gondii infection is prevalent in indoor housed Dutch dairy goats but at a lower overall animal level seroprevalence than outdoor farmed goats in other European countries, and cat exposure is an important risk factor.

Experimental inoculation of male rats with Coxiella burnetii: successful infection but no transmission to cage mates.

Beginning in 2007, the largest human Q fever outbreak ever described occurred in the Netherlands. Dairy goats from intensive farms were identified as the source, amplifying Coxiella burnetii during gestation and shedding large quantities during abortions. It has been postulated that wild rodents are reservoir hosts from which C. burnetii can be transmitted to domestic animals and humans. However, little is known about the infection dynamics of C. burnetii in wild rodents. The aim of this study was to investigate whether brown rats (Rattus norvegicus) can be experimentally infected with C. burnetii and whether transmission to a cage mates occurs. Fourteen male brown rats (wild type) were intratracheally or intranasally inoculated with a Dutch C. burnetii isolate obtained from a goat. At 3 days postinoculation, a contact rat was placed with each inoculated rat. The pairs were monitored using blood samples and rectal and throat swabs for 8 weeks, and after euthanasia the spleens were collected. Rats became infected by both inoculation routes, and detection of C. burnetii DNA in swabs suggests that excretion occurred. However, based on the negative spleens in PCR and the lack of seroconversion, none of the contact animals was considered infected; thus, no transmission was observed. The reproduction ratio R(0) was estimated to be 0 (95% confidence interval = 0 to 0.6), indicating that it is unlikely that rats act as reservoir host of C. burnetii through sustained transmission between male rats. Future research should focus on other transmission routes, such as vertical transmission or bacterial shedding during parturition.

Circulation of group 2 coronaviruses in a bat species common to urban areas in Western Europe.

Fecal samples of 211 bats representing 13 different bat species from 31 locations in the Netherlands were analyzed for the presence of coronaviruses (CoV) using a genus-wide reverse transcription (RT)-polymerase chain reaction. CoVs are known for their high potential for interspecies transmission, including zoonotic transmission with bats as reservoir hosts. For the first time, a group 2 CoV was found in a bat, Pipistrellus pipistrellus, in Europe. This is of particular interest for public health as the reservoir host is a species that is common to urban areas in most of Europe and notorious for its close interactions with humans. Four verspertilionid bat species were found to excrete group 1 CoVs, viz. Myotis daubentonii, M. dasycneme, P. pipistrellus, and Nyctalus noctula. The last species is a newly identified reservoir. The overall prevalence was 16.9% and positive bats were found at multiple widespread locations. The circulating group 1 CoV lineages were rather species associated than location associated.