The Birth of a Trichinella Britovi Focus on the Mediterranean Island of Sardinia (Italy)

Veterinary Parasitology 159 (2009) 361–363

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Veterinary Parasitology

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The birth of a Trichinella britovi focus on the Mediterranean island of Sardinia (Italy)

Edoardo Pozio a,*, Pasquale Cossu b, Gianluca Marucci a, Marco Amati a, Alessandra Ludovisi a, Maria Angeles Gomez Morales a, Giuseppe La Rosa a, Tonino Firinu b a Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanita`, viale Regina Elena 299, 00161 Rome, Italy b Istituto Zooproﬁlattico Sperimentale della Sardegna, via Duca degli Abruzzi 8, 07100 Sassari, Italy

ARTICLE INFO ABSTRACT

Keywords: For 60 years, the islands of the Mediterranean basin were considered to be Trichinella-free. Mediterranean islands In April 2005, an outbreak of human trichinellosis due to the consumption of infected pork Sardinia involved 11 persons in the villages of Orgosolo and Lanusei (Nuoro province) on the island Trichinella britovi of Sardinia (Italy). We conducted an investigation to identify free-range and backyard pigs Free-range pigs and other humans with Trichinella infection in the area of the 2005 outbreak. We also Trichinellosis tested wild animals from various parts of Sardinia. In December 2005, eight persons were Outbreaks Human infections found to have been infected, and in May 2007 there was a single case of infection. The sources of all infections were domestic pigs. Artiﬁcial digestion of muscle samples from 681 pigs (325 free-range and 356 backyard pigs) revealed Trichinella sp. larvae in four sows (1.2%). All larvae, including those from the consumed pork products, were identiﬁed as Trichinella britovi. All infected pigs originated from the Orgosolo municipality. None of the 6188 wild boars (Sus scrofa) or 13 red foxes (Vulpes vulpes) examined were positive for Trichinella sp., suggesting that this parasite is restricted to free-range pigs. The origin of infected animals on Sardinia remains to be determined, although it could be related to the presence of T. britovi-infected animals on the island of Corsica (France). ß 2008 Elsevier B.V. All rights reserved.

1. Introduction made with pork from a single free-range pig occurred on the Mediterranean island of Sardinia, Italy (Pozio et al., Until very recently the only documented Trichinella sp. 2006). In early 2006, the hospital in the area where the infections on islands in the Mediterranean, were those due outbreak had occurred reported other cases of suspected to Trichinella spiralis reported between 1933 and 1946 in Trichinella infection. Although it is illegal to raise free- Sicily in humans and backyard pigs (Pozio and La Rosa, range swine in Sardinia because of the presence of both 1998). Since then, the only other Trichinella outbreak on a classic and African swine fever, in remote areas of the Mediterranean island was the 2004 Trichinella britovi island there are thousands of free-range pigs which are outbreak in free-range domestic pigs, wild boars, and a red never subjected to veterinary controls. In light of these fox in a remote area of Corsica (Boireau and Valle´e, 2004). considerations, we conducted an investigation to identify In April 2005, an outbreak of T. britovi infection free-range and backyard pigs and other humans with involving 11 persons who had consumed raw sausage Trichinella infection in the area of the 2005 outbreak. We also tested wild animals from various parts of Sardinia. In this report, we summarize the results of this investigation * Corresponding author. Tel.: +39 06 4990 2304; fax: +39 06 4990 3561. and the present situation with regard to the circulation of E-mail address: [email protected] (E. Pozio). Trichinella sp. on Sardinia.

2. Materials and methods sion, 2005). Trichinella sp. larvae were identified at the species level by a multiplex-PCR analysis, following a 2.1. Investigation area and sample collection previously published protocol (Pozio and La Rosa, 2003). Reference larvae of the following strains were used for the This investigation was conducted in the area of Orgosolo, comparison in the PCR analysis: T. spiralis (ISS003), T. the village in which the infected pig responsible for the first britovi (ISS002), and T. pseudospiralis (ISS013) (http:// 2005 outbreak (Pozio et al., 2006) was slaughtered and www.iss.it/site/Trichinella/). where most of the persons involved in the outbreak and all Serum samples were tested by both an ELISA and a suspected cases reported by the local hospital lived; Western blot using excretory/secretory antigens according Orgosolo borders on Barbagia, a remote mountain area to standard protocols (Pozio et al., 1993). The case covered by forest. We interviewed the individuals with definition of trichinellosis was based on the algorithm suspected infection to identify the origin of the meat and proposed by Dupouy-Camet and Bruschi (2007). other persons who had consumed the same meat. We collected serum samples at different times from the 3. Results infection from all individuals with signs and/or symptoms consistent with trichinellosis and from persons who had In December 2005, eight cases of Trichinella infection consumed the same pork or pork-derived products in the were confirmed in the village of Orgosolo, and in May 2007 course of the two human outbreaks (April and December a single case was confirmed in the same village. When also 2005) and of the single human infection (May 2007). We considering the 11 persons involved in the outbreak in collected samples of the pork or pork-derived products from April 2005, five of whom lived in the nearby village of these individuals’ refrigerators or freezers or from the Lanusei (Nuoro Province), there was a total of 20 confirmed person who had provided them with the meat. We also cases of trichinellosis between 2005 and 2007 (Table 1). collected muscle samples from the diaphragm pillars and/or For the 20 human cases, seroconversion occurred the tongue (100 g total) from free-range and backyard pigs, between 25 and 60 days after consumption of the infected all of which were home-slaughtered in the Orgosolo pork. All of these persons had been hospitalised, with an municipality from January to March 2006. Muscle samples average hospital stay of 3 days. Infection was clinically were also collected from the diaphragm pillars and/or the mild in all but two cases, for whom it was severe; both of tongue (100 g total) of wild boars (Sus scrofa) and from the the cases with severe infection were involved in the anterior leg and/or the tongue (100 g total) of red foxes outbreak of April 2005. (Vulpes vulpes), which had been killed by hunters in different For the two outbreaks in 2005 and the single case of areas of Sardinia, including Orgosolo. infection in 2007, the source of infection was a free-range sow (Tables 1 and 2). All of the samples of pork and pork- 2.2. Diagnostic tests derived products (sausages) collected from the homes of infected persons were positive for Trichinella sp. larvae. To detect Trichinella spp. larvae in the samples of Of the 681 muscle samples collected from the home- muscle, pork or pork-derived products, the pepsin– slaughtered pigs of the Orgosolo municipality between hydrochloric acid magnetic stirrer method described in January and March 2006, 325 were from free-range EU regulation 2075/2005 was used (European Commis- animals and 356 were from backyard animals (Table 3).

Table 1 Human foci of trichinellosis, Sardinia (Italy), 2005–2007.

Month, year No. of infected persons Municipality of the human outbreak Place of origin of the pig that was the source of infection

April, 2005 11 Orgosolo (6), Lanusei (5) Carvacone (Orgosolo municipality) December, 2005 8 Orgosolo Sos pireddas (Orgosolo municipality) May, 2007 1 Orgosolo Su haharu (Orgosolo municipality)

Table 2 Epidemiological data for seven Trichinella britovi-infected sows including those detected during the survey of free-ranging pigs of the Orgosolo municipality (Nuoro province, Sardinia region, Italy) and those which were the source of infection for the two human outbreaks and the single case of trichinellosis which occurred in the Sardinia island from 2005 to 2007.

Pig code Age (years) No. larvae/g Breeding type Locality of origin Source of infection for humans (muscle or pork product) of infected pig

1 4 20 (sausage) Free-ranging Carvacone Yes (April 2005) 2 8 43 (neck muscle) Free-ranging Sos pireddas Yes (December 2005) 3 5 15 (intercostals) Free-ranging Su haharu Yes (May 2007) 4 9 34 (diaphragm) Free-ranging Su haharu No 5 10 4 (diaphragm) Free-ranging Su haharu No 6a 2 0.9 (diaphragm) Backyard Locoi No 7a 2 0.1 (diaphragm) Backyard Locoi No a Pig fed with pork scraps from pig no. 1. E. Pozio et al. / Veterinary Parasitology 159 (2009) 361–363 363

Table 3 Domestic and wild animals tested for Trichinella sp. larvae.

Host species No. of positive/tested (%) Animal origin Breeding type

Domestic pig 2/356 (0.6) Orgosolo municipality Backyard Domestic pig 2/325 (0.6) Orgosolo municipality Free-ranging Wild boar 0/6188 Sardinia (different areas) Wild Red fox 0/13 Orgosolo municipality Wild

In addition to the pigs that were the source of infection for which were fed to domestic pigs. With regard to the origin humans, Trichinella sp. larvae were detected in two free- of the parasite circulating in Corsica, several hypotheses range and two backyard sows; the backyard sows had been can be formulated: (1) infected wild boars from France fed with pork scraps from the free-range sow that was the may have been introduced on Corsica; (2) infected pork source of infection in the first outbreak (Table 2). No larvae may have been brought by persons from Eastern Europe, were detected in 6188 wild boars from different areas of where the prevalence of infection in domestic pigs is quite Sardinia (of which 129 were from the Orgosolo munici- high; and (3) an infected hunting dog may have been pality) or in 13 foxes from the Orgosolo municipality brought from France to Corsica, where it died and was (Table 3). All Trichinella sp. larvae were identified as T. eaten by wild animals. The study of genetic polymorph- britovi (data not shown). isms of T. britovi isolates from Corsica and Sardinia will hopefully allow the origin of the parasite to be traced. 4. Discussion Acknowledgement Before the cases reported here, Trichinella sp. infection had never been found in domestic or wild animals or in This work was supported in part by the European humans in Sardinia (Arru, 1962; Pintore et al., 1996). In Project MED-VET-NET, contract Food-CT-2004-506122, light of this consideration and of the fact that there have Workpackage 27, Trichimed. been no changes in pig husbandry practices or eating habits for pork products in Sardinia, the island was Conflict of interest probably truly Trichinella-free. Since 2005, seven domestic pigs have been found to be None declared. infected with T. britovi and three of these were a source of infection for humans. The seven infected pigs were 2–10- References year-old sows (average age of 5.7 years). Given that sows have a longer life span than fattening pigs and that older Arru, E., 1962. Indagini sulla trichinosi in Sardegna. Arch. Vet. It. 13, 345– pigs, because of the hierarchy in the pig population 348. (Copado et al., 2004), have greater access to food, sows may Boireau P., Valleé I., 2004. Epidemiologie des trichinelloses animales en play an important role as a reservoir of Trichinella spp. In France. Surveillance officielle et surveillance se´rologique. Report for the French Ministry of Agriculture, 5 April 2004, pp. 1–39. this light, they could be considered as a good sentinel Boireau, P., Valleé, I., Rossi, S., Hars, J., Dupouy-Camet, J., 2007. Trichinella animal for detecting Trichinella spp. infection in the pig surveillance system in wild and domestic animals in France. Outcome population of both traditional and organic farms. of the last 10 years.In: XII International Conference on Trichinellosis. National Park Plitvice Lakes, 25–30 September, (Abstract Book), p. 66. The finding that none of the wild boars or foxes Copado, F., de Aluja, A., Mayagoitia, L., Galindo, F., 2004. The behaviour of harbored the parasite suggests that T. britovi infection may free ranging pigs in the Mexican tropics and its relationships with be confined to free-range pigs. However, now that the human faeces consumption. Appl. Anim. Behav. Sci. 88, 243–252. Dupouy-Camet, J., Bruschi, F., 2007. Management and diagnosis of human parasite has been introduced on the island, the future trichinellosis. In: Dupouy-Camet, J., Murrell, K.D. (Eds.), FAO/WHO/ spread to wildlife cannot be excluded, especially because OIE Guidelines for the Surveillance, Management, Prevention and many free-range pigs die from classic and African swine Control of Trichinellosis. pp. 37–68. European Commission, 2005. Regulation (EC) No. 2075/2005 of the fever and the carcasses of these animals are often eaten by European Parliament and of the Council of 5 December 2005 laying wild animals. The spread to wild animals would impede down specific rules on official controls for Trichinella in meat. Off. J. EC, any attempts to eradicate infection on the island. L 338, 60–82. Pintore, A., Liciardi, M., Porcu, R., Cossu, P., Cabras, P., Bozzi, F., Fois, M., With regard to human infection, these are the first cases Soddu, M., Firinu, A., 1996. Epidemiological survey on trichinellosis in of trichinellosis to be reported on a Mediterranean island in Sardinia. ISTISAN Congr. 46, 84. nearly 60 years. In 2004, 1 year before the first outbreak in Pozio, E., La Rosa, G., 1998. Identification of the likely etiological agent of Sardinia, T. britovi was documented in free-range domestic human trichinellosis in Sicily (Italy) between 1933 and 1946. Am. J. Trop. Med. Hyg. 59, 906–907. pigs, wild boars, and in a red fox in a remote area of Corsica, Pozio, E., La Rosa, G., 2003. PCR-derived methods for the identification of which until then was considered to be ‘‘Trichinella-free’’ Trichinella parasites from animal and human samples. Methods Mol. (Boireau and Valleé, 2004). Since then, no other infections Biol. 216, 299–309. Pozio, E., Mesina, P., Sechi, F., Pira, M., Liciardi, M., Cossu, P., Marucci, G., have been reported in Corsica (Boireau et al., 2007). Garippa, G., Firinu, A., 2006. Human outbreak of trichinellosis in the Although it is difficult to identify the origin of infection Mediterranean island of Sardinia, Italy. Vet. Parasitol. 140, 177–180. in pigs in Sardinia, one plausible hypothesis is that Pozio, E., Varese, P., Gomez Morales, M.A., Croppo, G.P., Pelliccia, D., Bruschi, F., 1993. Comparison of human trichinellosis caused by Sardinian hunters, who often go on hunting expeditions Trichinella spiralis and by Trichinella britovi. Am. J. Trop. Med. Hyg. to Corsica, brought back infected game meat, scraps of 48, 568–575.