Trichinella in wildlife and pork production: evaluation of risk-based monitoring Frits Franssen Trichinella in wildlife and pork production: evaluation of risk‐based monitoring ISBN: 978‐94‐6328‐044‐0 Dissertation Utrecht University, Utrecht, the Netherlands. The research presented was performed at the National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands. Cover image: Trichinella spiralis muscle larvae (photography by Frits Franssen) Cover design: Frits Franssen Lay‐out: Frits Franssen Printing: CPI Koninklijke Wöhrmann ©2016 Frits F.J. Franssen All rights reserved. No part of this publication may be reproduced in any form, by print, photocopying, microfilm, electronic data transmission, or any other means, without prior written permission of the author. Trichinella in wildlife and pork production: evaluation of risk-based monitoring Trichinella in wild en varkensvleesproductie: Evaluatie van risico-gestuurde monitoring (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. G.J. van der Zwaan, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op dinsdag 14 juni des middags te 12.45 uur door Friedrich Franz Joseph Franssen geboren op 4 november 1957 te Schaesberg Promotoren: Prof. dr. F. van Knapen Prof. dr. A.H. Havelaar Copromotor: Dr. J.W.B. van der Giessen Content Chapter 1. Introduction 7 Chapter 2. Analysis of long-term serological Trichinella surveillance data of wild boar from a non-endemic 23 area Chapter 3. Trend analysis of Trichinella in a red fox population from a low endemic area using a validated artificial digestion and sequential sieving technique 43 Chapter 4. Quality control of Trichinella testing at the slaughterhouse laboratory: evaluation of the use of a 400-um mesh size sieve in the magnetic stirrer 61 method Chapter 5. Genetic evidence of interspecies introgression of mitochondrial genomes between Trichinella spiralis and Trichinella britovi under natural conditions 71 Chapter 6. Increase in number of helminth species from Dutch red foxes over a 35-year period 93 Chapter 7. Antibody response against Trichinella spiralis in experimentally infected rats is dose dependent 109 Chapter 8. Zoonotic helminths in black rats (Rattus rattus) and brown rats (Rattus norvegicus) from different environments in the Netherlands 127 Chapter 9. Parasite to Patient: a Quantitative Risk Model for Trichinella spp. in pork and wild boar meat 147 Chapter 10. General Discussion 179 References 193 Summary 213 Samenvatting 221 Curriculum vitae 231 Dankwoord 235 List of Publications 241 Nowhere is it more true that ‘Prevention is better than Cure’ than in the case of Parasitic Diseases – Rudolf Leuckart – 1886 7 Chapter 1 ematodes of the genus Trichinella are parasites of many different species of mammals, birds and reptiles. Six encapsulated Trichinella species (T. spiralis, T. Nbritovi, T. nativa, T. murelli, T. patagoniensis and T. nelsoni) and three encapsulated genotypes (Trichinella T6, T8 and T9) have been recognized, which infect carnivorous and omnivorous mammals (Pozio, 2005; Pozio et al., 2009b). Three non‐encapsulated Trichinella species infect birds (T. pseudospiralis) and crocodiles (T. zimbabwensis and T. papuae). No Trichinella infections have been demonstrated in amphibians or fish (Pozio, 2005). T. spiralis and T. pseudospiralis have a cosmopolitan distribution, due to dispersion by humans and birds respectively, whereas all other species have a more restricted geographical distribution (Pozio et al., 2009a) (Table 1). 8 Introduction The Trichinella lifecycle The infective stage of encapsulated Trichinella species is a first stage larva that resides inside striated muscle tissue of an infected host within a so‐called nurse cell (Figure 1 C), consisting of a collagen capsule (mainly collagen type IV and VI) inside an infected myocyte (Despommier, 1998). Trichinella larvae (Figure 1D) are liberated in the acidic pepsin environment of the stomach after ingestion of infected meat. In the mucosa of the small intestine, the liberated larvae moult four times within 30 ‐ 36 hours and develop into adult male and female worms (Figure 1A and 1B). After mating, fertilized females produce eggs which embryonate in utero, resulting in shedding of variable numbers of newborne larvae per female from 6‐7 days (pi) to 6‐7 weeks post infection (pi), depending on the Trichinella species and age, sex and species of the host. Ultimately, the intestinal adult worms are expelled by the host (Despommiers, 2005; Pozio et al., 2003). In vitro, the newborne larval production of T. spiralis females is about 110 per 72 hours, whereas all other species produce far less, at <50 per 72 hours (Murrell et al., 2000). Newborne larvae penetrate the intestinal wall and migrate via the lymph and blood vessels to striated muscle cells, which they invade. Once inside a muscle cell, the Trichinella newborne larva takes control of its new environment by down‐regulation of muscle contractile proteins, such as actin and myosin, followed by enlargement of the invaded myocyte and host cell nucleus multiplication to 40 – 60 nuclei per cell. The Trichinella larva further modifies its niche through excretion of tyvelosylated peptides that localize to the nucleoplasm of the enlarged myocyte and upregulate collagen synthesis, to build the collagen capsule of the nurse cell within approximately 26 days. A constant supply of nutrients to nourish the larva inside the nurse cells is provided by a network of small blood vessels that are attached to the outer layer of the collagen capsule, initiated by the Trichinella larva, possibly after an initial hypoxic event within the nurse cell. Default reaction of the vertebrate host to a hypoxic event is upregulation of vascular endothelial growth factor (VEGF) which in turn triggers angiogenesis (Despommier, 1998). Thus, inside its nurse cell, the mobile and metabolically active Trichinella muscle larva awaits the death of its present host and ingestion by the next, producing tyvelosylated proteins to control its environment throughout the whole infection period (Despommier, 1998). Once a host has died, the Trichinella muscle larvae (ML) are perfectly able to survive in decaying meat for long periods of time. T. spiralis ML in artificially infected pork meat that had been buried in the ground at a depth of 30‐100 cm, remained infective for rats for more than 91 days (Jovic et al., 2001; Oivanen et al., 2002). T. britovi and T. nelsoni in decaying mouse carcasses remained infective to laboratory mice after storage at room temperature for up to 45 days (Gottstein et al., 2009). Trichinella ML are susceptible to inactivation by freezing, the extent of which 9 Chapter 1 varies with Trichinella species and the host species in which the larvae developed. After freezing T. spiralis in infected wild boar meat for one week at ‐20 °C, all muscle larvae were inactivated, whereas it took eight weeks at the same temperature and in the same host to inactivate all T. britovi (Lacour et al., 2013). In a study conducted with pork infected with T. spiralis, Trichinella murrelli, Trichinella pseudospiralis and Trichinella nativa, no live larvae were obtained after 82 hours at ‐20 °C (Hill et al., 2009), whereas in another study, T. nativa larvae in rat muscle survived one week of freezing at ‐18 °C, but not four weeks (Malakauskas and Kapel, 2003a). T. spiralis in infected horsemeat is not inactivated within one week at ‐18 °C , and even after four weeks at ‐18 °C, few T. spiralis larvae were demonstrated (Hill et al., 2007). T. nativa and Table 1. Geographical distribution of Trichinella species and genotypes. Adapted from (Pozio et al., 2009a). Encapsulated species and genotypes Trichinella spp. Distribution Cycle Host range T. spiralis Cosmopolitan, except arctic Domestic and Swine, rats, seldom carnivores regions sylvatic T. nativa Arctic and subarctic areas of Sylvatic Terrestrial and marine carnivores Holarctic region T. britovi Temperate areas of Sylvatic, seldom Carnivores, seldom swine Palearctic region, northern domestic and western Africa T. murrelli Temperate areas of Nearctic Sylvatic Carnivores region T. nelsoni Ethiopic region Sylvatic Carnivores, seldom swine T. patagoniensis Argentina Sylvatic Carnivores Trichinella T6 Canada and United States Sylvatic Carnivores Trichinella T8 South Africa and Namibia Sylvatic Carnivores Trichinella T9 Japan Sylvatic Carnivores Non-encapsulated species Trichinella spp. Distribution Cycle Host range T. pseudospiralis Cosmopolitan Sylvatic, seldom Mammals and birds domestic T. papuae Papua New Guinea, Sylvatic, seldom Swine, saltwater crocodiles Thailand domestic T. zimbabwensis Ethiopia, Mozambique, Sylvatic and Nile crocodiles, Nile monitor South Africa, Zimbabwe domestic lizards, lion 10 Introduction Trichinella T6 tolerate temperatures as low as ‐15 °C for one or more years in wolf, polar bear and arctic fox muscle. T. britovi remains infective in frozen red fox muscle for up to three months at ‐15 °C (Pozio et al., 1994b). Infection with non‐encapsulated Trichinella species leads to developments comparable to those described for the encapsulated species, up to the moment where encapsulated species orchestrate the formation of a nurse cell capsule, which increases over time to 18.7 – 33.5 µm (Sacchi et al., 2001). In contrast, a nurse cell capsule is absent in muscle cells that had been invaded by T. pseudospiralis