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Epidemiologic monitoring of wild animals is always an important step in defining potential zoonoses that can threaten humans. Particular emphasis should be given to those zoonotic agents permanently cycling within wild animal populations and represent a permanent reservoir for other wild or domesticated animals that can be direct sources of disease for humans. In Croatia, there are two European jackal populations: the Dalmatian population (DP) that has been inhabiting the islands and coastal area along the Adriatic Sea since medieval times, and the South East European population (SEEP) that is found in continental Croatia. Research on Trichinella infections in jackal populations in Croatia was conducted from 2008 to 2022. During this 15-year period, we tested 186 jackal samples and confirmed infection in 47 individuals (25.3 %). The dominant species was T. spiralis, identified in 28 samples (60 %), T. britovi was found in 13 samples (28 %), while for six samples (12 %) the PCR test was unsuccessful. In both populations, the Trichinella species of the domestic cycle (T. spiralis) was found, though in varying ratios: in DP the ratio of identified species was 10:6 in favour of T. britovi, as opposed to 22:3 in favour of T. spiralis in SEEP. The frequency of infection with parasites from the genus Trichinella was significantly different in DP (22.9 %) than in SEEP (26.7 %) (p<0.001), while the larval count in analysed tissue did not differ by type of Trichinella species (p=0.1028). Infected jackals were found in nine of ten tested counties. The results were analysed statistically and the origin of tested and positive samples shown on a map of Croatia. Based on these findings, both jackal populations can be considered to represent an exceptionally important indicators of parasites from the genus Trichinella in Croatia, both for the sylvatic and domestic cycles. There is an evident need for epidemiological monitoring for members of both populations., Competing Interests: Declaration of Competing Interest The authors declared that there is no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In some Southeastern European (SEE) countries, like Serbia, Romania, and Bulgaria, trichinellosis is one of the most important foodborne zoonotic diseases. In those countries, EU regulation and local authorities require the staff of laboratories performing official controls on meat to be properly trained and to check their competence by participating regularly in proficiency testing (PT). PTs are organized by National Reference Laboratories for Trichinella of each country and involve all official laboratories testing meat. In Romania and Bulgaria, the organization of PT for the detection of Trichinella larvae in meat by Magnetic Stirrer Method (MSM) started in 2012. In Croatia and Serbia PT was first organized in 2015 and 2017, respectively. This study presents data on the performance obtained by official laboratories of SEE countries that organize PT at national level and compares the performance obtained by laboratories belonging to different countries. Results suggest that the constant participation in PT leads to an increase in the performance of participating laboratories by positively affecting the staff accuracy in sample testing by MSM. Since the percentage of recovered larvae was in some cases suboptimal (<80%) and occasionally very poor (<40%), there is room for improvement. The regular participation in PT by laboratories involved in official controls on meat intended for human consumption is fundamental to guarantee consumer safety., Competing Interests: Declaration of Competing Interest All authors declare no conflict of interest in this study., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In Europe, Trichinella spiralis, the most dangerous species for humans of the genus Trichinella, has a patchy distribution with important foci in Eastern countries and Spain. This zoonotic pathogen was apparently not circulating among wild and domestic animals of Italy. In 2016, muscle larvae belonging to this nematode species were detected in a red fox (Vulpes vulpes) shot in the Piacenza province (Northern Italy). This parasite may have been introduced into northern Italy from eastern Europe by hunters, by a hunting dog, or by immigrants, who illegally carried infected meat in their personal baggage. In the same year, T. spiralis infected sausages illegally introduced by personal baggage into Italy from Romania, were inadequately disposed of in the garbage of a central Italian town. Even though these two episodes may not be connected in time and space, they represent an increased risk of infection for domestic and wild swine, which are highly susceptible to this pathogen. In these animals, T. spiralis shows a higher larval burden and a longer survival time than other Trichinella species. Since most of the Italian pig production plants are in northern Italy, the circulation of T. spiralis should be strictly monitored in wildlife living in these areas., (Copyright © 2017. Published by Elsevier B.V.)

Trichinella spp. are zoonotic parasites transmitted to humans by the consumption of raw or insufficiently cooked meat of different animal species. Carnivore mammals are important reservoir hosts of these nematodes. The aims of this work were to establish the prevalence of Trichinella spp. and infection intensity in sylvatic carnivore mammals of Latvia, to identify the etiological agents at the species level and their circulation in the Latvian regions. From 2010 to 2014, muscle samples were collected from 1286 hunted animals (2 European badgers, 137 pine martens, 24 stone martens, 4 golden jackals, 394 raccoon dogs, 668 red foxes, 23 grey wolves, and 34 Eurasian lynxes). Trichinella spp. larvae were isolated by muscle digestion. Overall, 633 animals (49.2%; 95% CI 46.5%-52.0%) belonging to all the eight investigated species, tested positive for Trichinella spp. larvae. Trichinella britovi was the most common species (94.0%; 95% CI 91.7%-95.7%). Trichinella nativa was detected in 30 animals as single (6, 1.1%; 95% CI 0.4%-2.3%) or mixed infection (24, 4.4%; 95% CI 2.9%-6.4%) with T. britovi. Trichinella spiralis was detected in only three animals as mixed infection with T. britovi. The high prevalence of Trichinella spp. infection in sylvatic carnivore mammals suggests that they are good indicators for the risk assessment of Trichinella spp. in Latvia., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Active surveillance of red foxes for Trichinella has been undertaken in mainland Britain since 1999. Post-mortems are carried out, followed by a magnetic stirrer method for sample digestion based on European Commission (EC) Regulation 216/2014 (which amends 2075/2005). Initially samples are tested in batches of 20 foxes and in December 2013, for the first time under the surveillance programme, a batch tested positive for Trichinella at the Animal and Plant Health Agency, York. Further individual tests identified one infected fox, from the Bristol area. The larvae were identified as Trichinella pseudospiralis. This is the first report of T. pseudospiralis in Great Britain and suggests the possibility of a cycle of infection existing in wildlife., (Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.)

An antigen detection kit (Trichin-L), based on latex agglutination and developed by the Bio-Rad company was validated at five European laboratories. The validation parameters included specificity, sensitivity, robustness and reproducibility. Specificity was evaluated by testing parasite antigens from five non-Trichinella parasites in addition to the Trichinella genus. To evaluate sensitivity, 10 pork samples spiked with 1, 3, 6 or 15 Trichinella larvae were tested in each laboratory. To evaluate the robustness of the test, the solubilized antigens were maintained at room temperature and tested at different times. Reproducibility was assessed in each laboratory using 40, 100g minced pork samples, each spiked with Trichinella spiralis. The use of larval homogenates obtained from the Trichin-L kit as a template for parasite identification at the species level by a multiplex PCR, was also evaluated. The results showed a high specificity and sensitivity where solubilized antigens maintained their stability and reactivity for up to three days. Reproducibility was high, as similar results were obtained in the five laboratories. The larval homogenates obtained using the Trichin-L kit were successfully used in multiplex PCRs to identify Trichinella species., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Although larvae of the genus Trichinella are the most common parasite species detected in vertebrate muscles using artificial digestion, nematode larvae belonging to other genera are sometimes detected and incorrectly identified as Trichinella. However, it is often very difficult to identify these larvae at the species, genus or family level using microscopy because of the absence of specific morphological characters or cuticle damage, and the only means of identification is PCR and sequencing of specific molecular markers (12S mtDNA; COI; 18S rDNA; and ITS1). From 2008 to 2011, 18 nematode isolates not belonging to the genus Trichinella were collected from different host species. Eleven of these isolates were successfully identified at the species, genus or superfamily level: larvae from two common kestrels, three hooded crows, a hen harrier and a domestic pig were identified as Toxocara cati; larvae from a badger were identified as Toxocara canis; larvae from a domestic pig were identified as a free-living nematode of the genus Panagrolaimus; larvae from a wild boar were identified as belonging to the Metastrongylus genus; and larvae from a rough-legged buzzard were identified as belonging to the superfamily Filarioidea. The recovery of nematodes belonging to genera other than Trichinella during routine meat inspection suggests that the persons performing the analyses need to be informed of the possibility of false positives and that a molecular-based identification system that allows for a rapid and reliable response must be adopted (i.e., a DNA barcoding-like system)., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Trichinella pseudospiralis is a non-encapsulated species infecting both mammals and birds. In Italy, this parasite was reported only in two night-birds of prey of Central Italy. In January 2010, Trichinella larvae were detected in three wild boars (Sus scrofa) of two regions of Northern Italy by enzymatic digestion. The parasites were identified as T. pseudospiralis by multiplex-PCR. The first infected wild boar was hunted in the Emilia Romagna region and the other two infected wild boars were bred outdoors in a small family farm of the Friuli Venezia Giulia region. These new epidemiological data reinforce the role of the wild boar as the main reservoir of T. pseudospiralis in Europe., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Nematodes of the genus Trichinella are zoonotic parasites with a cosmopolitan distribution. In Iran, these parasites have mainly been detected in carnivorous mammals, yet information on the Trichinella taxa circulating in this country date back to a time when biochemical and molecular tests were not available. We describe the first detection of Trichinella larvae in a leopard (Panthera pardus saxicolor) in Asia and its identification at the species level. The larvae recovered from the leopard muscles were identified as Trichinella britovi using multiplex PCR. The detection of Trichinella infection in a leopard confirms literature data on the high prevalence of infection in carnivorous mammals in Iran.

Recent discovery of Trichinella zimbabwensis in crocodiles from Zimbabwe, Lake Cahora Basa, Mozambique, and from lake Abaja, Ethiopia, prompted strict control measures to curb the possible spread of the infection to humans and also to prevent its introduction to other countries, which were considered free of this pathogen. In 2006, the Chief Directorate Veterinary Services of Mpumalanga Province of South Africa launched a survey to investigate the status of wild and commercial breeding crocodiles in the province. To evaluate if T. zimbabwensis was circulating in the environments where crocodiles are living in South Africa, 9 fish, 36 reptiles (including 27 Nile crocodiles) and 4 mammals have been investigated to detect Trichinella sp. larvae in their muscles. In January 2008, a Nile crocodile from Komatipoort, sampled by means of a tail biopsy, tested positive for Trichinella larvae. In June-July 2008, Trichinella sp. larvae were also detected in four other Nile crocodiles from the Olifants River Gorge. The prevalence of Trichinella infection in the investigated wild Nile crocodiles from South Africa is 38.5%. The larvae were identified as belonging to T. zimbabwensis by multiplex-PCR. These are the first reports of T. zimbabwensis in South Africa and suggest that the distribution area of this parasite species is wider than that believed in the past.

For 60 years, the islands of the Mediterranean basin were considered to be Trichinella-free. In April 2005, an outbreak of human trichinellosis due to the consumption of infected pork involved 11 persons in the villages of Orgosolo and Lanusei (Nuoro province) on the island of Sardinia (Italy). We conducted an investigation to identify free-range and backyard pigs and other humans with Trichinella infection in the area of the 2005 outbreak. We also tested wild animals from various parts of Sardinia. In December 2005, eight persons were 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).

To control Trichinella spp. infection in the European Union, all slaughtered pigs should be tested by one of the approved digestion methods described in EU directive 2075/2005. The aim of the present work was to evaluate, by a ring trial, the sensitivity of the digestion method used at the National Reference Laboratories for Parasites (NRLP). These Laboratories are responsible for the quality of the detection method in their own country. Of the 27 EU countries, only three (Hungary, Luxembourg and Malta) did not participate in the ring trial. Each participating laboratory received 10 samples of 100g of minced pork containing 3-5 larvae (3 samples), 10-20 larvae (3 samples), 30-50 larvae (3 samples), and one negative control. In each positive sample, there were living Trichinella spiralis larvae without the collagen capsule, obtained by partial artificial digestion of muscle tissue from infected mice. No false positive sample was found in any laboratories, whereas nine laboratories (37.5%) failed to detect some positive samples with the percentage of false negatives ranging from 11 to 100%. The variation between expected and reported larval counts observed among the participating laboratories was statistically significant. There was a direct correlation between the consistency of the results and the use of a validated/accredited digestion method. Conversely, there was no correlation between the consistency of the results and the number of digestions performed yearly by the NRLP. These results support the importance of validating the test.

In South Africa, Trichinella sp. was first discovered in 1966 in the wildlife of the Kruger National Park (KNP). Since then, both Trichinella T8 and Trichinella nelsoni have been detected in the KNP, leading to a debate on the existence of a gene flow between the two taxa. In 2006-2008, four lions were killed in the Manyeleti Game Reserve, the Mthethomusha Nature Reserve, Numbi Gate, and Skukuza, which border the KNP. Larvae were isolated from muscles by artificial digestion. The molecular identification of single larva by multiplex PCR, followed by a specific PCR to distinguish between Trichinella T8 and Trichinella britovi, revealed Trichinella T8 in the lions from Manyeleti and Skukuza, a mixed infection with T. nelsoni and Trichinella T8 in the lion from Mthethomusha, and T. nelsoni in the lion from Numbi. No larva with a hybrid pattern between the two taxa was observed. No hybrid offspring resulted when crossing single males and females of T. nelsoni and Trichinella T8 in both directions, whereas hybrid offspring were obtained when crossing T. britovi and Trichinella T8 in both directions. This is the first report of a mixed infection with two Trichinella taxa in a host from the KNP, where both Trichinella T8 and T. nelsoni circulate among wildlife. Despite the sympatry status of these two taxa, field and laboratory data seem to exclude the possibility of gene flow, confirming their evolutive separation.

In 1995, a new species of Trichinella (Trichinella zimbabwensis) was discovered in farmed Nile crocodiles (Crocodylus niloticus) in Zimbabwe, where the mode of transmission was the consumption of the meat of slaughtered crocodiles, used as feed. To determine whether T. zimbabwensis affects poikilotherm vertebrates in the wild, monitor lizards (Varanus niloticus) and Nile crocodiles were collected in Zimbabwe and Mozambique. In 5 (17.6%) of the 28 monitor lizards from Zimbabwe, T. zimbabwensis larvae were identified. For the wild Nile crocodiles from Mozambique, species-level identification was not possible, yet immunohistochemical analysis revealed that 8 (20%) of the 40 animals harboured non-encapsulated Trichinella sp. larvae, which probably belonged to T. zimbabwensis. This is the first report of T. zimbabwensis in wild reptiles, and the findings are consistent with reports that vertebrates with scavenger and cannibalistic behaviour are the most important hosts of Trichinella spp. The wide distribution of monitor lizards and crocodiles in Africa and the development of national crocodile breeding programs in many African countries should be taken into consideration when evaluating the risk of transmission of this parasite to mammals, including humans.

Trichinella sp. infection has been documented in both humans and animals in most Mediterranean countries, yet in the past 60 years no infections have been reported on the Mediterranean islands. We describe the first outbreak of Trichinella sp. infection to have been reported on the island of Sardinia. The outbreak occurred in two villages in 2005 and involved 11 persons who had eaten raw sausages made from the same free-ranging sow. All 11 persons developed signs and symptoms of trichinellosis and seroconverted within 48 days of consuming the infected meat. The etiological agent was Trichinella britovi. Sardinia, like all Mediterranean islands, had been considered to be Trichinella-free. The present report, together with a recent report of T. britovi infection in animals on the nearby island of Corsica (France), raises questions as to the validity of the concept of Trichinella-free areas or regions.

The recent discovery of Trichinella zimbabwensis in farmed crocodiles (Crocodilus niloticus) of Zimbabwe and its ability to infect mammals, and the development of both T. zimbabwensis and Trichinella papuae in experimentally infected reptiles led to an investigation of Trichinella infection in saltwater crocodiles (Crocodylus porosus) and in wild pigs (Sus scrofa) of Papua New Guinea, to see if T. papuae also, is present in both cold- and warm-blooded animals. Of 222 crocodiles examined, 47 animals (21.2%), all from Kikori, Gulf Province, were positive for non-encapsulated larvae in the muscles. The greatest number of larvae was found usually in the biceps, with an average of 7 larvae/g. One isolate from a crocodile infected successfully both laboratory rats and mice. Of 81 wild pigs examined, 9 from Bensbach river area (Western Province) and 1 from Kikori area (Gulf Province) were positive for non-encapsulated larvae in the muscles. Trichinella larvae from both saltwater crocodiles and wild pigs have been identified by multiplex-PCR analysis as T. papuae. The sequence analysis of the region within the large subunit ribosomal DNA, known as the expansion segment V, has shown the presence of a molecular marker distinguishing T. papuae isolates of Bensbach river area from those of Kikori area. This marker could be useful to trace back the geographical origin of the infected animal. The epidemiological investigation carried out in the Kikori area has shown that local people catch young crocodiles in the wild and keep them in holding pens for several months, before sending them to the crocodile farm in Lae (Morobe Province). They feed the crocodiles primarily with wild pig meat bought at the local market and also with fish. These results stress the importance of using artificial digestion for routinely screening of swine and crocodiles, and of adopting measures for preventing the spread of infection, such as the proper disposal of carcasses and the adequate freezing of meat.