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Background: To offer an alternative to conventional techniques of lateral prosthetic anastomosis on arteries which require a long training, and impose an extensive open surgery, we are proposing the clampless 2 device (C2D) implanted by a simple arterial puncture and allows a lateral implantation of a polytetrafluoroethylene (PTFE) vascular graft in an artery, without arterial clamping or suture., Methods: C2D is a "T" shaped 25-mm long and 8-mm diameter Nitinol stent with a 6-mm PTFE graft prosthesis mounted laterally on the stent, and implanted in an artery, via a 21-French sheath, and a compliant balloon inflation. In vitro testing of the C2D was first performed on a bench including a segment of a 7-mm internal diameter pig abdominal aorta. A series of 5 consecutive C2D implantation was analyzed with evaluation of the implantation time and the fluid losses at a fluid pressure of 80 and 150 mm Hg. The C2D implantation was finally controlled by angioscopy. An aorto-iliac bypass was then secondly performed on 8 living sows, with a side-to-end C2D implantation in the infrarenal abdominal aorta, followed by a conventional end-to-end prosthetic left iliac trunk anastomosis. The C2D and distal conventional anastomotic times were evaluated, as well as the total operative time and blood loss. A postoperative angiogram was systematically performed., Results: The C2D was successfully implanted in all 5 in vitro tests, with an average implantation time of 2'58 (range: 2'25-3'22). The mean value of fluid losses was 84 ml (range: 67-94 ml), with no fluid leakage occurring at 80- and 150-mm Hg pressure. All anastomoses were patent after macroscopic study by angioscopy with a perfect application of the stent in the aortic wall. In 8 living sows (mean weight: 42 kg, 37-50 kg), an aorto-left iliac bypass was successfully implanted in all cases, with a total mean procedure time of 101 min (range: 90-130 min), and an average fluid loss of 77 ml (range:20-120 ml). The mean implantation time was 4'39 (range 3'29-5'52) for C2D and 16 min (range 12-17 min) to perform the conventional distal prosthetic-iliac anastomosis. Systematic arteriographic and angioscopy control showed perfect patency of the C2D implantations., Conclusions: Preliminary in vitro and acute in vivo testing of C2D implantation show good early results, allowing further long-lasting pig experiments on the way to human homologation., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Intro: Toxoplasmosis has high disease burden in the Netherlands and in the rest of Europe. It can be acquired directly by ingestion of Toxoplasma gondii (T. gondii) oocysts shed by infected cats, or indirectly via consumption of undercooked meat from infected livestock. Cat vaccination has been proposed for reducing oocyst-acquired human infections but it remains unclear whether such an intervention can be effective. In this study we quantified the effects of using cat vaccination on reducing oocyst-originated T. gondii human infections., Method: By using a disease dynamics compartmental model for T. gondii infections in cats and mice we studied the effects of a hypothetical cat vaccine on the presence of T. gondii oocysts in the environment. A fitted dose response model was used to assess the effect of oocyst reduction on the expected human infections., Results: For rats, mice and pigs, and possibly intermediate hosts in general, ingestion of one oocyst provides 30%-60% probability of T. gondii infection. Assuming a favourable ideal scenario where vaccination completely prevents oocyst shedding and predation rate is of one mouse per week per cat, eight cats can be left susceptible in order to achieve elimination and stop oocyst-originated transmission, independent of the total cat population. Considering populations of 1000, 100, 50 and 20 cats, cat vaccination coverage of 94%, 68%, 54% and 35%, respectively, would reduce expected oocyst-originated human cases by 50%., Conclusion: For attaining elimination of oocyst-originated human infections, only few cats may remain unvaccinated, regardless of the cat-population size, and only a few more cats may remain unvaccinated for reducing infections substantially. Such vaccination coverages can in practice be achieved only when small cat-populations are considered, but in larger cat-populations the large efficacy and vaccination coverage needed are unfeasible., (Copyright © 2019. Published by Elsevier B.V.)

The reference method for Trichinella detection at meat inspection is the magnetic stirrer method (MSM) utilising HCl-pepsin for pooled sample digestion. Due to availability and quality issues with pepsin, alternative digestion methods are being offered, such as the Priocheck Trichinella AAD kit (T-AAD), based on serine endopeptidase digestion. In this study the T-AAD kit was compared to the reference method. Minced pork samples were spiked with T. spiralis muscle larvae (ML) with- and without capsule or T. pseudospiralis ML, and analysed with both tests. Test results of individually spiked test samples were analysed by generalised linear modelling. The T-AAD test kit was comparable to the reference method for the qualitative detection of T. spiralis in pigs, but not quantitatively. Overall, 94% of spiked T. spiralis were recovered using MSM against 75.2% when using T-AAD (p < 0.0001). Using the MSM 80.0% of spiked T. pseudospiralis were recovered against 20% with the T-AAD (p < 0.0001). Based on our experience with the T-AAD kit, we strongly recommend validating the method on site prior to introduction into routine diagnostic laboratories, but this will not alleviate the poor test sensitivity of the T-AAD for the detection of T. pseudospiralis., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The performance of a 400-μm-mesh-size sieve (sieve400) has not previously been compared with that of a 180-μm-mesh-size sieve (sieve180). Using pork samples spiked with 0 to 10 Trichinella muscle larvae and an artificial digestion method, sieve performance was evaluated for control of Trichinella in meat-producing animals. The use of a sieve400 resulted in 12% lower larval counts, 147% more debris, and 28% longer counting times compared with the use of a sieve180. Although no false-negative results were obtained, prolonged counting times with the sieve400 may have an impact on performance in a high-throughput environment such as a slaughterhouse laboratory. Based on our results, the sieve180 remains the sieve of choice for Trichinella control in meat in slaughterhouse laboratories, according to the European Union reference method (European Commission regulation 2075/2005). Furthermore, the results of the present study contribute to the discussion of harmonization of meat inspection requirements among countries.

Taenia solium taeniasis/cysticercosis is a neglected zoonotic disease complex occurring primarily in developing countries. Though claimed eradicated from the European Union (EU), an increasing number of human neurocysticercosis cases is being detected. Risk factors such as human migration and movement of pigs/pork, as well as the increasing trend in pig rearing with outside access are discussed in this review. The entry of a tapeworm carrier into the EU seems a lot more plausible than the import of infected pork. The establishment of local transmission in the EU is presently very unlikely. However, considering the potential changes in risk factors, such as the increasing trend in pig farming with outdoor access, the increasing human migration from endemic areas into the EU, this situation might change, warranting the establishment of an early warning system, which should include disease notification of taeniasis/cysticercosis both in human and animal hosts. As currently human-to-human transmission is the highest risk, prevention strategies should focus on the early detection and treatment of tapeworm carriers, and should be designed in a concerted way, across the EU and across the different sectors., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The emergence of the zoonotic tapeworm Echinococcus multilocularis, causative agent of alveolar echinococcosis (AE), poses a public health risk. A previously designed risk map model predicted a spread of E. multilocularis and increasing numbers of alveolar echinococcosis patients in the province of Limburg, The Netherlands. This study was designed to determine trends in the prevalence and worm burden of E. multilocularis in foxes in a popular recreational area in the southern part of Limburg to assess the risk of infection for humans and to study the prevalence of E. multilocularis in dogs in the adjacent city of Maastricht. Thirty-seven hunted red foxes were tested by the intestinal scraping technique and nested PCR on colon content. Additionally, 142 fecal samples of domestic dogs from Maastricht were analyzed by qPCR for the presence of E. multilocularis. In foxes, a significantly increased prevalence of 59% (95% confidence interval 43-74%) was found, compared to the prevalence of 11% (95% CI 7-18%) in 2005-2006. Average worm burden increased to 37 worms per fox, the highest since the first detection, but consistent with the prediction about the parasite population for this region. Updated prediction on the number of AE cases did not lead to an increase in previous estimates of human AE cases up to 2018. No dogs in the city of Maastricht tested positive, but results of questionnaires showed that deworming schemes were inadequate, especially in dogs that were considered at risk for infection., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Sporadic nontravel-related hepatitis E virus (HEV) infections have been reported in industrialized countries. These infections are caused by zoonotic HEV genotypes 3 and 4 that circulate in swine, wild boar, and deer. In The Netherlands, HEV RNA has been detected in >50% of the pig farms, and HEV-specific antibodies were detected in ∼70% of the slaughter pigs. In the current study, HEV seroprevalences were investigated in pigs raised on conventional, free-range, and organic farms in The Netherlands. Differences in seroprevalence may indicate different exposure routes or transmission dynamics within pig herds for HEV. In 2004, serum samples of 846 fattening pigs were obtained from farms that applied conventional (265 pigs at 24 farms), organic (417 pigs at 42 farms), and free-range (164 pigs at 12 farms) farming. HEV-specific antibodies were detected in samples from all conventional and free-range pig farms and in 41 of 42 organic pig farms, indicating that the probability of introducing HEV on a farm appeared to be equal for the different farming types. The estimated average within-herd seroprevalence was significantly higher for pigs raised on organic farms (89%) than for pigs raised on conventional farms (72%, P = 0.04) and nearly significant for pigs raised on free-range farms (76%, P = 0.06). Six of ten organic farms were estimated to have a withinherd seroprevalence of >95%, compared with 1 of 10 and 4 of 10 of the free-range and conventional pig farms, respectively. This suggests a higher force of infection with HEV for pigs reared on organic farms compared with pigs reared on conventional or free-range farms. This may be due to repetitive exposure to HEV caused by farming system-specific housing conditions, such as a greater contact frequency between pigs and more exposure to pig manure, increasing the transmission rate.

The occurrence of trichinellosis in a resident of the Netherlands prompted us to examine the likelihood of this originating from infected rats in spite of prevailing biosecurity and testing procedures. In so doing, we sought to calculate the possible risks for trichinellosis in countries deemed non-endemic. The infection risk was determined by simulating a scenario from a reservoir of minimally contaminated wildlife to pigs to humans. Results indicate that humans might become infected even in the event that artificial digestion had been performed on individually tested pig carcasses. Our conclusions justify reconsidering Trichinella control strategies based on the current testing protocol, and emphasize the importance of proper cooking as further insurance against human infection., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Therapy of relapsed pediatric acute lymphoblastic leukemia (ALL) is hampered by low remission rates and high toxicity, especially in second and subsequent relapses. Our phase 1 study, T2005-003, showed that the combination of bortezomib with vincristine, dexamethasone, pegylated asparaginase, and doxorubicin had acceptable toxicity. We report the phase 2 expansion of this combination in patients with relapsed ALL who failed 2-3 previous regimens. Twenty-two patients with relapsed ALL were treated with bortezomib combined with this regimen; their ages ranged from 1 to 22 years, and they had either B-precursor ALL (n = 20) or T-cell ALL (n = 2). Grade 3 peripheral neuropathy developed in 2 (9%) patients. After 3 patients died from bacterial infections, treatment with vancomycin, levofloxacin, and voriconazole prophylaxis resulted in no further infectious mortality in the last 6 patients. Fourteen patients achieved complete remission (CR), and 2 achieved CR without platelet recovery, for an overall 73% response rate, meeting predefined criteria allowing for early closure. B-precursor patients faired best, with 16 of 20 (80%) CR + CR without platelet recovery, whereas the 2 patients with T-cell ALL did not respond. Thus, this combination of bortezomib with chemotherapy is active in B-precursor ALL, and prophylactic antibiotics may be useful in reducing mortality. Bortezomib merits further evaluation in combination therapy in pediatric B-precursor ALL. This study is registered at http://www.clinicaltrials.gov as NCT00440726.

The aim of this study was to examine the dynamics of parasite specific antibody development in Trichinella spiralis and Toxoplasma gondii co-infections in pigs and to compare these with antibody dynamics in T. spiralis and T. gondii single infections. In this experiment, fifty-four pigs were divided into five inoculated groups of ten animals, and one control group of four animals. Two groups were inoculated with a single dose of either T. gondii tissue cysts or T. spiralis muscle larvae, one group was inoculated simultaneously with both parasites and two groups were successively inoculated at an interval of four weeks. Specific IgG responses to the parasites were measured by ELISA. T. gondii burden was determined by MC-PCR carried out on heart muscle and T. spiralis burden by artificial digestion of diaphragm samples. Specific IgG responses to T. gondii and T. spiralis in single and simultaneously inoculated animals showed a respective T. gondii and T. spiralis inoculation effect but no significant interaction of these parasites to the development of specific antibodies with the serum dilutions used. Moreover, our data showed that the specific IgG response levels in groups of animals successively or simultaneously co-infected were independent of a respective previous or simultaneous infection with the other parasite. Additionally, no differences in parasite burden were found within groups inoculated with T. gondii and within groups inoculated with T. spiralis. Conclusively, for the infection doses tested in this experiment, the dynamics of specific antibody development does not differ between single and simultaneous or successive infection with T. gondii and T. spiralis. However, lower parasitic doses and other ratios of doses, like low-low, low-high and high-low of T. gondii and T. spiralis in co-infection, in combination with other time intervals between successive infections may have different outcomes and should therefore be studied in further detail., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The Mediterranean island of Corsica was considered Trichinella-free until 2004, when T. britovi larvae were discovered in domestic pigs at meat inspection. One red fox was also found infected the same year and in the same area than the infected pigs. This last finding highlighted the presence of trichinellosis in Corsican wildlife. A Trichinella survey was thus performed in wild boar (Sus scrofa) and fox (Vulpes vulpes), the two large wild species present on the island, to determine prevalence of muscle larvae and antibodies. Diaphragm muscles of 1881 wild boars and 74 forelegs of foxes were tested by artificial digestion. No Trichinella larva was identified. The highly sensitive ELISA was used to test muscle fluid samples of 1492 wild boars. The apparent serological prevalence of Trichinella infections in wild boar was 2.01% (95% CI: 1.36-2.86). The present results suggest that wildlife is currently exposed to Trichinella in Corsica. In this context, adequate cooking and veterinary controls of meat offer the only complete sanitary warranties to consumers., ((c) 2010 Elsevier B.V. All rights reserved.)

In this paper we evaluate serology as a tool to monitor Trichinella-free pig herds. Indoor, industrial-raised fattening pigs in the Netherlands are practically Trichinella-free, and were used as a negative reference cohort. A positive cohort was not available but we used sera from an endemic region in Argentina to model a plausible distribution of serological responses (as OD levels) in positive sera, employing the difference between the endemic sera and the negative Dutch sera. We describe a method for correcting for variation among ELISA plates using on-plate reference sera, and demonstrate how to apply these corrections to a collection of test sera from pig farms. The positive and negative reference distributions can be used to estimate fractions true and false positives, necessary for defining appropriate cutoffs to be used for classifying positive and negative animals. Based on this analysis, the serological test was shown to lack the predictive power required for its large scale deployment. The properties of the serological test were also compared to the conventional digestion assay, which is highly specific but considerably less sensitive.

Trichinella is a food-borne parasitic zoonoses and human cases are still reported in Europe mainly due to the consumption of pig meat originating from small backyard farms. Infections originating from industrialized pig farming have not been reported for decades in Europe, due to control measures to prevent the transmission of Trichinella from wildlife by indoor housing and good management practices. Therefore, risk-based monitoring programs might replace individual carcass control in industrialized pig farming as described in EU legislation SANCO 2075/2005. Transmission of Trichinella species between wildlife and the risk that may pose to humans via consumption of contaminated pork meat has not been studied quantitatively. One pathway by which human trichinellosis can occur is the rat-pig-human route. To evaluate the transmission risk though this pathway the dose responses of rat, pig, and human were studied. Experimental T. spiralis infection was performed in rats with doses of as few as 10 parasites and the data set was analysed using a newly developed dose response model that describes larvae per gram (LPG). Experimental T. spiralis infection in pig was analysed in a similar way. Furthermore nine published outbreaks of human trichinellosis were analysed to determine the dose response in humans. The risk of human trichinellosis via the rat-pig-human transmission was simulated by the Monte Carlo method. A pair of female and male parasites representing the lowest infection pressure from the environment, led to the probability of human trichinellosis by consumption of 100g of raw pork meat equal to 5% via the studied rat-pig-human pathway. In the absence of rodent control near the farm, a low infection pressure from wildlife presents a relatively high risk of human trichinellosis via consumption of uncooked pork meat.

In a ring trial involving five laboratories (A, B, C, D, and E), three different methods of artificial digestion were compared for the detection of non-encapsulated Trichinella pseudospiralis larvae in minced meat. Each sample panel consisted of ten 1g minced pork samples. All samples in each panel were derived from a bulk meat preparation with a nominal value of either 7 or 17 larvae per g (lpg). Samples were tested for the number of muscle larvae using the magnetic stirrer method (labs A, B, and E), stomacher method (lab B), and Trichomatic 35 (labs C and D). T. pseudospiralis larvae were found in all 120 samples tested. For samples with 7 lpg, larval recoveries were significantly higher using the stomacher method versus the magnetic stirrer method, but there were no significant differences for samples with 17 lpg. In comparing laboratory results irrespective of the method used, lab B detected a significantly higher number of larvae than lab E for samples with 7 lpg, and lab E detected significantly less larvae than labs A, B, and D in samples with 17 lpg. The lowest overall variation for quantitative results (i.e. larval recoveries which were outside the tolerance range) was achieved by using the magnetic stirrer method (22%), followed by the stomacher method (25%), and Trichomatic 35 (30%). Results revealed that T. pseudospiralis larvae in samples with a nominal value of 7 and 17 lpg can be detected by all three methods of artificial digestion.

Prevalences of parasitic infections in pigs from different housing systems may vary, due to their contact with the environment, and this might have consequences for food safety. In this study, 40 organic, 9 free-range and 24 intensive farms were selected and a total of 845 serum samples were tested for antibodies specific for Toxoplasma and Trichinella using ELISA assays. The overall seroprevalence of Toxoplasma in the total number of 845 serum samples tested is 2.6%, ranging from 0.38% in intensively raised pigs to 5.62% in free-range pigs. Of the housing systems tested, 4% (intensive farms) to 33% (free-range farms) is infected with Toxoplasma gondii. The risk of detecting Toxoplasma antibodies in a free-range farm are statistically higher (almost 16 times higher) than in an intensive farm. We observed that the risk of detecting specific antibodies is twice as high as in free-range compared with organic farms. Seropositivity of Trichinella spiralis antibodies was 0.12-0.35% (depending on the cut-off value at the 99.5% or 97.5% level). There was a tendency that Trichinella seropositivity was higher in organic pig farming (0.24%), but this was not significant. This serological study in pigs from different farming systems shows that the seroprevalence of antibodies specific for T. gondii is higher and for Trichinella equivalent in pigs raised in systems where there is contact with the environment than in pigs raised in intensive, indoor farming systems. This indicates that the prevalence of parasitic infections is higher in outdoor farming systems than in indoor farming systems. The possible consequences for food safety are discussed.

Since 1992, when the European Union Council Directive requires that wild boars (Sus scrofa) hunted in EU for commercial purpose should be examined for Trichinella, the infection has not been detected in wild boars from Belgium, despite serological evidence of the presence of anti-Trichinella antibodies in wildlife and previous reports of Trichinella larvae in this host species. In November 2004, Trichinella larvae were detected in a wild boar hunted near Mettet, Namur province (Southern Belgium). Larvae were identified as Trichinella britovi by polymerase chain reaction methods. This is the first report of the identification of Trichinella larvae from Belgium at the species level. The detection of T. britovi in wildlife in Belgium is consistent with findings of this parasite in other European countries and confirms the need to test game meat for Trichinella to prevent its transmission to humans.

The identification of sequence regions in the genomes of pathogens which can be useful to distinguish among species and genotypes, is of great importance for epidemiological, molecular, and phylogenetic studies. The 5S ribosomal DNA intergenic spacer region has been identified as a good target to distinguish among eight Trichinella species and genotypes. The recent discovery of two non-encapsulated species in this genus, Trichinella papuae and Trichinella zimbabwensis, which can infect both mammals and reptiles, has suggested analyzing their 5S rDNA. Amplification of the tandem repeats of the 5S rDNA intergenic region of encapsulated species of Trichinella shows a 751bp fragment, whereas the three non-encapsulated species show a fragment of 800bp with T. pseudospiralis showing an additional fragment of 522bp. Although the size of the 800bp PCR fragments of T. papuae and T. zimbabwensis are similar to that of T. pseudospiralis, there are differences in the 5S rDNA intergenic regions among the three non-encapsulated species. Phylogenetic analysis of the 5S rDNA intergenic regions shows a clustering together of the three non-encapsulated Trichinella species that is well separated from the encapsulated ones. In addition, a single PCR-based method allows distinguishing non-encapsulated and encapsulated species.

Prevalences of Cryptosporidium spp. and Giardia duodenalis in relation to age and season were investigated on a dairy farm in The Netherlands over the course of 1year. The whole herd was sampled five times, whereas calves younger than about 2 months were sampled every 2-3 weeks. Associations between diarrhoea and presence of one or more pathogens (Cryptosporidium spp., G. duodenalis, rotavirus) were investigated. Potential transmission routes of Cryptosporidium spp. were evaluated and positive samples of Cryptosporidium spp. and G. duodenalis were identified to genotype level by PCR microsatellite identification and fingerprinting. Shedding of Cryptosporidium spp. was found in all age categories but peaked in calves 1-3 weeks old (39.1%). Herd prevalence of shedding for Cryptosporidium spp. varied from 2.4% in June to 22.2% in December. Shedding of G. duodenalis was found in all age categories but peaked in animals 4-5 months old (54.5%). Herd prevalence of shedding for G. duodenalis varied from 0.8% in June to 15.5% in February. Cryptosporidium spp. and rotavirus appeared to be significantly associated with diarrhoea in calves. Microsatellite analysis showed two different subtypes (C3 and C1) of Cryptosporidium parvum calf strains. Two genotypes of G. duodenalis were found, one positive by A lineage specific PCR and thus closely related to human genotypes and one genotype, which was negative by A and B lineage specific PCR. The results indicate that cow-to-calf and indirect calf-to-calf transmission both are important routes for acquiring infection with Cryptosporidium spp.

Echinococcus multilocularis was demonstrated in 5 out of 272 foxes in The Netherlands close to the border with Germany and Belgium. Besides microscopic examination of mucosal scrapings, two different PCR assays were used based on the detection of E. multilocularis DNA in colon content. Two distinct areas in The Netherlands were positive for E. multilocularis. Two positive foxes were found in the northern province of Groningen and three positive foxes were found in the southern province of Limburg. Both PCR assays detected more positive foxes compared to microscopic examination of the intestinal content. This is the first report of E. multilocularis in foxes occurring in The Netherlands.