Trichinella pseudospiralis in a red kite (Milvus milvus) from Italy.

Trichinella pseudospiralis is a non-encapsulated species infecting both mammals and birds. In Italy, this species has been reported so far only in central regions (two nocturnal birds of prey, one red fox, and one wild boar) and in northeast regions (four wild boars). In November 2020, Trichinella sp. larvae were isolated by enzymatic digestion from muscle tissues of a red kite (Milvus milvus) specimen belonging to a population residing in the Basilicata region (Southern Italy). The parasite was identified as T. pseudospiralis by multiplex PCR, and the sequencing of the expansion segment V (ESV) region of the nuclear large subunit ribosomal DNA showed, in the microsatellite region, the polymorphism characteristic of the Palearctic population. This represents the first record of T. pseudospiralis in a red kite and the first report of this parasite in Southern Italy. The isolation of the parasite in a resident bird confirms that T. pseudospiralis is present, even if at low prevalence, in the Italian avifauna.

Species identification of Trichinella originated from various host and different geographical location by MALDI-TOF.

The foodborne zoonotic nematode Trichinella spp. can cause human trichinellosis when raw or undercooked contaminated meat is ingested. To date, twelve Trichinella species/genotypes have been described. According to EU regulation any Trichinella larvae detected during mandatory routine examinations need to be identified at a species level by a competent laboratory. Currently, Trichinella species identification is performed using molecular biology tools such as multiplex PCR, PCR-sequencing or PCR-RFLP. These techniques require high level of skills for good interpretation of the results. Due to its rapidness and ease of use a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) protocol was previously developed for the identification of Trichinella species. Using this method, spectra from different Trichinella species and strains were acquired allowing to generate new Main Spectra (MSP). Finally a new MSP database from Trichinella spp. Samples of different countries (France, Germany and Poland), including field samples, was generated. Comparing the different main spectra, Trichinella worms were identified at the species level and differences in the genetic diversities within the different species are discussed. In conclusion, using the previously described method on field samples is a reliable, rapid, easy-to-use and cheap tool for Trichinella species identification. The new Trichinella database could be incremented with new samples. It constitutes a tool, which could be used as an alternative method to replace the actual molecular methods for Trichinella species identification.

A two-step morphology-PCR strategy for the identification of nematode larvae recovered from muscles after artificial digestion at meat inspection.

To ensure that meat from livestock and game is safe for human consumption, European legislation lays down rules for mandatory testing. Helminth larvae are a category of zoonotic foodborne pathogens that can contaminate meat. Among helminths, the only zoonotic nematode regulated in Europe regarding meat inspection is Trichinella spp.. It is precisely during Trichinella testing that other potentially zoonotic larvae can be found. Due to current lack of tools, their identification is often very complicated. Nematode larvae other than Trichinella, recovered from artificial digestions of pig and wild boar muscles from France and Germany, were subjected to a newly developed two-step identification scheme, which includes both morphological examination and molecular assays. The first step is a general orientation towards a broad taxonomic group; the second step consists of targeted identification based on the results of first step. Different parasites were identified, some of which were not zoonotic such as Metastrongylus spp. and Angiostrongylus vasorum, but others are known to be zoonotic such as Toxocara cati, Ascaris suum, and Uncinaria stenocephala. The strategy is efficient for the identification of nematode larvae recovered from muscles but could also be applied for larvae from other sources.

Survey of Toxoplasma gondii and Trichinella spp. in hedgehogs living in proximity to urban areas in the Czech Republic.

Hedgehogs (Mammalia: Erinaceidae) are omnivorous nocturnal animals typically living in anthropogenic areas. They may be suitable as sentinels for a wide range of zoonotic infections. Only a few studies have investigated hedgehogs (and then as representative wildlife species) to establish their role in the life cycle of such tissue parasites with zoonotic potential as Toxoplasma gondii or Trichinella spp. Working with frozen hedgehog cadavers, we tested for these parasites using T. gondii DNA-specific magnetic capture isolation plus polymerase chain reaction and Trichinella spp. digestion assay. All of 50 examined hedgehogs were negative for Trichinella spp. larvae in their muscles, but brain tissue from 5 out of 26 Erinaceus europaeus (19.2%) and 4 out of 24 E. roumanicus (16.6%) tested positive for T. gondii DNA. Frequency of T. gondii for both hedgehog species was equal, as was distribution between males and females and across age categories. Although a few studies have suggested the possibility of Trichinella spp. infection in hedgehogs, the zero prevalence in the tested hedgehogs is not surprising in view of the generally low prevalence of Trichinella spp. in Central Europe. Our results show that hedgehogs are susceptible to infection by T. gondii and can be used as indicator wildlife animal species in anthropogenic ecosystems.

Prevalence and molecular identification of Trichinella species isolated from wildlife originating from Limpopo and Mpumalanga provinces of South Africa.

Trichinella species are widely distributed on all continents with the exception of Antarctica, although the full spectrum of Trichinella species found in sub-Saharan African countries, and their hosts, has not been fully documented. This study was conducted to determine the prevalence of Trichinella in wildlife from the Greater Kruger National Park (GKNP) and adjacent areas located in the Limpopo and Mpumalanga provinces of South Africa, and to identify the species and/or genotypes of Trichinella larvae isolated from muscle tissues, using molecular techniques. A review of Trichinella spp. and their wildlife hosts reported during 1964-2011 was also conducted and the results were compared with our current study. Ninety samples representing 15 mammalian, two bird and three reptile species were screened for Trichinella infection during 2012-2016, using artificial digestion. Isolates detected were identified using a multiplex polymerase chain reaction (PCR) amplification of the internal transcriber spacers ITS1 and ITS2, and expansion segment V (ESV) regions of ribosomal DNA, followed by molecular analysis of the sequences. Twenty samples from seven wildlife species were positive for Trichinella spp. larvae, with an overall prevalence of 21.1% (20/90). The prevalence was higher in carnivores (18.9%, 18/90) than in omnivores (2.2%, 2/90). Analysis of sequences showed that eight of the isolates - two from spotted hyaena (Crocuta crocuta) (2/8), three from lion (Panthera leo) (3/13), one from leopard (Panthera pardus) (1/6), one from small spotted genet (Genetta genetta) (1/2) and one Nile monitor lizard (Varanus niloticus) (1/2) - conformed to Trichinella zimbabwensis. One isolate from a hyaena was grouped under the encapsulated species clade comprising T. nelsoni and genotype Trichinella T8 reported to be present in South Africa. This is the first report confirming natural infection by T. zimbabwensis in hyaena, leopard, genet and Nile monitor lizard, adding to the body of knowledge on the epidemiology of Trichinella infections in the Greater Kruger National Park of South Africa. Ten Trichinella-like larval isolates recovered after digestion from four wildlife species in this study (2012-2016) revealed inconclusive results due to DNA degradation resulting from poor storage or too few larvae for analysis, in comparison to 20 unidentified isolates from five wildlife species during the 1964-2011 period.

Outbreak of Trichinella T9 Infections Associated with Consumption of Bear Meat, Japan.

An outbreak of trichinellosis occurred in Japan in December 2016. All case-patients had eaten undercooked bear meat, from which Trichinella larvae were subsequently isolated. DNA sequencing analysis of the mitochondrial genes cytochrome c-oxidase subunit 1 and internal transcribed spacer 2 confirmed that Trichinella T9 had caused the outbreak.

Multiplex TaqMan qPCR assay for specific identification of encapsulated Trichinella species prevalent in North America.

BACKGROUND Human trichinellosis is a foodborne parasitic zoonotic disease caused by ingestion of raw or undercooked meat infected with nematode larvae of the genus Trichinella. In the USA, sporadic cases and outbreaks caused by the consumption of wild game meat infected with Trichinella have been reported. The current methods for diagnosis such as serology and microscopy are not specific, may result in false negative results, and cannot differentiate encapsulated Trichinella larvae to species level. The molecular protocols currently available for the differentiation of all encapsulate Trichinella species prevalent in North America have some limitations such as the inability to identify and resolve the presence of several Trichinella species in a single test. OBJECTIVES/METHODS In this study we developed and evaluated a multiplex TaqMan quantitative real-time polymerase chain reaction (qPCR) assay, which can simultaneously detect, identify and differentiate all species of encapsulated Trichinella occurring in North America i.e., T. nativa, T. spiralis, T. murrelli and Trichinella T6, even in cases of multiple infection in a single sample. We investigated two human biopsies and 35 wild animal meat samples considered as having a high likelihood of harboring Trichinella larvae obtained from the United States during 2009-2017. FINDINGS Using the multiplex assay describe here, 22 (59%) samples that tested positive contained Trichinella spp., were identified as: T. nativa (n = 7, including a human biopsy), T. spiralis (n = 9, including a human biopsy), T. murrelli (n = 3), Trichinella T6 (n = 1). Results also included two rare mixed infection cases in bears, a T. nativa/T. spiralis from Alaska and a T. spiralis/Trichinella T6 from California. The species identifications were confirmed using a conventional PCR targeting the rRNA ITS1-ITS2 region, followed by DNA sequencing analysis. The estimated limit of detection (LOD) was approximately seven larvae per gram of meat. MAIN CONCLUSIONS Differentiation of Trichinella spp. is needed to improve efforts on identification of case, optimize food safety control and better understand the geographic distribution of Trichinella species. The Trichinella qPCR multiplex proved to be a robust, easy to perform assay and is presented as an improved technique for identification of all known encapsulated species occurring in North America continent.

Molecular identification of Sarcocystis lutrae in the European otter (Lutra lutra) and the European badger (Meles meles) from the Czech Republic.

Muscular sarcosporidial infections by Sarcocystis lutrae (Apicomplexa: Sarcocystidae) from the otter (Lutra lutra) and badger (Meles meles) (Carnivora: Mustelidae) were found in the Czech Republic. As part of a diversity evaluation of Sarcocystis in wild carnivores during 2016-2017, samples of diaphragm, tongue and hind-limb muscles were collected from nine districts, examined by compression and characterized molecularly. Cyst walls were thin, with no visible protrusions, and histological sections of infected muscle tissue showed no host responses. Fourteen of 17 badgers (82% prevalence) and one otter (100% prevalence) were positive for sarcocysts. Sequence analyses at four loci (18S rRNA, 28S rRNA, ITS1 and cox1) confirmed the identity as S. lutrae. This is also the first report of a co-infection with muscular sarcocystosis and Trichinella in badger. The finding of Trichinella is important from the zoonotic point of view, since badgers are used for meat consumption. Similar and future monitoring of both parasitic taxa are needed.

Occurrence of selected zoonotic food-borne parasites and first molecular identification of Alaria alata in wild boars (Sus scrofa) in Italy.

Wild boar is a source of human infections with zoonotic pathogens, including food-borne parasites. With the aim of a characterization of the human exposure risk, a survey on wild boars intended for human consumption was planned, selecting three pathogens, Toxoplasma gondii, Alaria alata, and Trichinella spp., as markers of meat infection. Diaphragm muscle samples from 100 wild boars hunted in Piedmont region (Northern Italy) in two hunting seasons (2015-2016) were collected. Concerning T. gondii, a combined approach of antibody detection and molecular techniques with genotyping was performed. For the detection of A. alata and Trichinella spp., the larva migration technique and the magnetic stirrer method were employed, respectively; in addition, molecular confirmation of the morphological identification of the recovered specimen was performed. Anti-T. gondii antibodies were found in meat juice samples (43.3%) and T. gondii DNA (type II) was detected in three animals (7.1%) out of 42 seropositive examined. In none of the sampled wild boars (0%), Trichinella spp. larvae were found, whereas one animal (1%) scored positive to A. alata mesocercariae. The molecular diagnosis proved the morphological identification of the trematode. This is the first finding of A. alata in Italian wild boar population. The present study confirmed the role of wild boars as a source of parasitic zoonotic diseases and thus the risk derived for humans posed by the consumption of game meat. Considering the zoonotic implications, the results underline the importance of monitoring and surveillance of zoonotic parasites in Italian wild boar populations.

Evaluation of the infectivity and the persistence of Trichinella patagoniensis in muscle tissue of decomposing guinea pig (Cavia porcellus).

Trichinella patagoniensis, a new species of Trichinella, is widespread in Argentina. The success of parasite transmission depends, among other factors, on the resistance of L1 larvae present in the muscle tissue (ML) of dead hosts undergoing the decomposition process in different environmental conditions. The aim of the present work was to study the infectivity of T. patagoniensis muscle larvae in Cavia porcellus and the capability of the parasite to survive in decomposed muscle tissue of guinea pigs subjected to different environmental conditions. Thirty-two female Ssi:AL guinea pigs were orally inoculated with 2000 ML of T. patagoniensis (ISS2311). All the animals were sacrificed 42 days post-infection. Twenty-six animals were eviscerated, and carcasses were placed on the surface of soil inside plastic boxes that were exposed to environmental conditions in the summer 2014-2015 and autumn of 2015 in Buenos Aires, Argentina. Carcasses from six animals were placed into a plastic box inside the refrigerator at a temperature of 4 °C. The muscle tissue samples from the carcasses were examined weekly for the presence of larvae, and the infectivity of recovered ML was tested in BALB/c mice. Our results showed for the first time the ability of T. patagoniensis to complete its life cycle in guinea pigs, thus serving as a potential natural host. Also, larvae of T. patagoniensis remained infective in muscle tissue for several weeks while undergoing decomposition under different environmental conditions.

The occurrence of Trichinella species in the cougar Puma concolor couguar from the state of Colorado and other regions of North and South America.

Trichinella species are zoonotic nematodes that infect wild carnivores and omnivores throughout the world. We examined the prevalence and species of Trichinella infections in cougars (Puma concolor couguar) from Colorado, United States. Tongues from cougars were examined by pepsin-HCl artificial digestion to detect Trichinella spp. larvae. The species or genotype of individual worms was identified by multiplex polymerase chain reaction (PCR). Trichinella spp. larvae were detected in 17 of 39 cougars (43.6% (28.7-59.5%)). Five of the cougars (12.8%) were infected with T. murrelli, 3 (7.7%) were infected with T. pseudospiralis, and 1 (2.6%) had Trichinella genotype T6. Trichinella spp. larvae from eight cougars were not identified at the species level, due to degraded DNA. The high prevalence of Trichinella spp. in cougars from Colorado and reports of the parasite in other populations of Puma spp. suggest that this large predator is a key mammalian reservoir.

Nonencapsulated Trichinella pseudospiralis Infection Impairs Follicular Helper T Cell Differentiation with Subclass-Selective Decreases in Antibody Responses.

Infectious microorganisms often modify host immunity to escape from immune elimination. Trichinella is a unique nematode of the helminth family, whose members parasitize the muscle cells inside the host without robust eliminative reactions. There are several species of Trichinella; some develop in muscle cells that become encapsulated (e.g., Trichinella spiralis) and others in cells that do not encapsulate (e.g., Trichinella pseudospiralis). It has already been established that Trichinella infection affects host immune responses in several experimental immune diseases in animal models; however, most of those studies were done using T. spiralis infection. As host immune responses to T. spiralis and T. pseudospiralis infections have been reported to be different, it is necessary to clarify how T. pseudospiralis infection influences the host immune responses. In this study, we investigated the influence on host humoral immunity in T. pseudospiralis-infected mice. We demonstrated that T. pseudospiralis infection decreased antigen-specific IgG2a and IgG2b antibody (Ab) production in mice immunized with a model antigen. This selective decrease in gamma interferon (IFN-γ)-dependent Ab production was not due to a decrease in IFN-γ production, and we instead found impaired follicular helper T (Tfh) cell differentiation. The affinity maturation of antigen-specific Ab tended to be delayed but was not significant in T. pseudospiralis-infected mice. We also observed that CD11b+ spleen cells in T. pseudospiralis-infected mice expressed CD206 and PD-L2, the phenotype of which was M2 macrophages with weak production of interleukin-6 (IL-6), possibly resulting in impaired Tfh differentiation. Taken together, our results indicate that nonencapsulated Trichinella infection induces selective dampening in humoral immunity with the suppression of Tfh differentiation.

New finding of Trichinella britovi in a European beaver (Castor fiber) in Latvia.

We report the first finding of Trichinella britovi in a European beaver. In Latvia, beaver is a common game animal and frequently used in human diet. A high prevalence of Trichinella infections in Latvia is present in the most common hosts-carnivores and omnivores. In total, 182 European beaver muscle samples were tested for Trichinella larvae accordingly to the reference method of European Communities Commission Regulation (EC) No. 2075/2005 (2005). Trichinella britovi larvae were detected in one animal (prevalence 0.5%; intensity 5.92 larvae per gram of muscle). This finding suggests that the consumption of European beaver meat can be a risk to human health. Further studies are needed in order to determine if the present observation represents an isolated individual case or low prevalence of Trichinella infection in beavers.

Seroprevalence of Trichinella sp. in Wild Boars (Sus scrofa) from Yanggu-gun, Gangwon-do, Korea.

A total 7 outbreaks of trichinellosis have occurred in Korea, mostly as a result of consumption of raw wild boar (Sus scrofa) meat. Since only 1 serological survey on wild boars had yet been performed in Korea, the present study aimed to estimate the prevalence of trichinellosis in wild boars and some species of rodents by artificial digestion and serological examinations in Yanggu-gun, Gangwon-do, the endemic area of trichinellosis. Both the wild boar and rodent muscle samples revealed no Trichinella larvae by direct examination and artificial digestion method. However, serological examinations revealed that 4 wild boar sera samples out of 118 (3.4%) were positive to Trichinella antigen. Although the recovery of Trichinella larvae ended in a failure, it is proved for the first time that the sylvatic cycle of Trichinella has been maintained in wild boars of Gangwon-do, Korea.

Molecular differentiation of Trichinella spiralis, T. pseudospiralis, T. papuae and T. zimbabwensis by pyrosequencing.

Nematodes of the genus Trichinella which infect wildlife and domestic animals show a cosmopolitan distribution. These zoonotic parasites are the aetiological agents of a severe human disease, trichinellosis. Twelve taxa are recognized in the Trichinella genus, but they cannot be identified by morphology since they are sibling species/genotypes. For epidemiological studies, it is extremely important to identify each taxon since they have different distribution areas and host ranges. In the present study, polymerase chain reaction (PCR) amplification of the mitochondrial large subunit ribosomal RNA (lsu-RNA) gene coupled with a pyrosequencing technique was developed to distinguish among four Trichinella species: Trichinella spiralis, T. pseudospiralis, T. papuae and T. zimbabwensis. A PCR method was used to amplify the lsu-RNA of Trichinella sp. larvae in mouse muscles and single larvae collected from infected muscles by digestion. The results show that the four Trichinella species can be distinguished by using 26 nucleotides in the target region and the method is sensitive enough to identify individual larvae. The pyrosequencing provides a simple, rapid and high-throughput tool for the differentiation of Trichinella species.

[RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER 2 SEQUENCE AS A PHYLOGENETIC MARKER FOR THE IDENTIFICATION OF TRICHINELLA NEMATODES].

The results of testing several primer combinations were used to choose an optimal pair for the amplification of the internal transcribed spacer 2 (ITS2) region of ribosomal DNA (direct: Tri58s F 5 CGG TGG ATC RCT TGG CTC GTA CG and reverse: AB28 Rr (CGA CCG CTT ATT GAT ATG C). This pair of primers yields a 900 bp PCR product. Comparative analysis of obtained ITS2 sequences, for 8 Trichinella isolates from different regions of the Russian Federation permits different species and individual genotypes of these parasitic nematodes to be validly distinguished.

Matrix metalloproteinase (MMP)-2 and MMP-9 as inflammation markers of Trichinella spiralis and Trichinella pseudospiralis infections in mice.

Trichinella spiralis and Trichinella pseudospiralis exhibit differences in the host-parasite relationship such as the inflammatory response in parasitized muscles. Several studies indicate that matrix metalloproteinases (MMPs) represent a marker of inflammation since they regulate inflammation and immunity. The aim of this study was to evaluate the serum levels of gelatinases (MMP-9 and MMP-2) in mice experimentally infected with T. spiralis or T. pseudospiralis, to elucidate the involvement of these molecules during the inflammatory response to these parasites. Gelatin zymography on SDS polyacrilamide gels was used to assess the serum levels and in situ zymography on muscle histological sections to show the gelatinase-positive cells. In T. spiralis infected mice, the total MMP-9 serum level increased 6 days post-infection whereas, the total MMP-2 serum level increased onward. A similar trend was observed in T. pseudospiralis infected mice but the MMP-9 level was lower than that detected in T. spiralis infected mice. Significant differences were also observed in MMP-2 levels between the two experimental groups. The number of gelatinase positive cells was higher in T. spiralis than in T. pseudospiralis infected muscles. We conclude that MMP-9 and MMP-2 are markers of the inflammatory response for both T. spiralis and T. pseudospiralis infections.

Trichinella britovi in the jackal Canis aureus from south-west Iran.

Trichinellosis is an important helminthic food-borne zoonosis, which is caused by nematodes of the genus Trichinella. Although, Trichinella spp. has been detected frequently in Iranian wildlife, this parasitic infection is not considered a major public health problem. This is largely because Islamic codes forbid consumption of pork meat in this country. However, knowledge about this zoonotic pathogen is important because human trichinellosis has been documented in countries where most of the population is Muslim. The aims of the present work were to investigate whether Trichinella spp. was still circulating in wildlife of the Khuzestan Province (south-west Iran) about 30 years after the first investigation, to identify the aetiological agent at the species level by molecular analyses, and to review the literature on Trichinella spp. in animals of Iran. During the winter 2009-2010, muscle samples from 32 road-killed animals (14 dogs and 18 jackals, Canis aureus) were collected. Muscle samples were digested and Trichinella sp. larvae were isolated from two jackals. The Trichinella sp. larvae have been identified as Trichinella britovi by molecular analyses. These results confirm that T. britovi is the prevalent species circulating in wild animals of Iran.

Trichinella murrelli identified in red foxes (Vulpes vulpes) in Pennsylvania, USA.

Trichinella infections have been eliminated from pork where pigs are raised in biosecure facilities, but wildlife infections persist. Trichinella murrelli is the primary zoonotic species in wild carnivores in the United States, having been identified in several species of omnivores and carnivores. Here, we document its occurrence in seven of 21 (33.3%) red foxes (Vulpes vulpes) from six counties in Pennsylvania. Encysted Trichinella larvae were detected in muscle squashes (<5 g samples) of all seven foxes, and in histological sections of the tongue and limb muscle of three. Larvae from muscle squashes were pooled and tested in a multiplex PCR capable of differentiating all Trichinella species native to the USA; all samples contained only T. murrelli. This is the first identification of T. murrelli in red foxes from Pennsylvania, and the first such survey performed in the last three decades. Results indicate that Trichinella remains endemic in Pennsylvania wildlife and a threat to the health of those who consume wild game.

Invasive wild boar (Sus scrofa) as a functional reservoir for the dynamics of Trichinella in the Patagonia region.

Trichinellosis is a zoonotic disease that has been studied mainly in domestic pigs (Sus scrofa domesticus). The cycle involves infection in domestic and wild fauna, which fulfill complex ecological roles, where Trichinella spiralis is reported in wild boar (Sus scrofa). The objective of this study was to determine the prevalence of trichinellosis in wild boar and evaluate the distance of positive animals to the nearest urbanization areas in Argentina Patagonia. Necropsies were carried out on wild boar hunted in the Nahuel Huapi and Lanín National Parks and surrounding areas. Skeletal muscle samples were collected from 1,694 wild boar and artificial digestion was performed on all samples. Trichinella spp. were found in 96 (5.8%) wild boar (0.2 to 424 Larvae/g). Parasitism in wild boar depends on the distribution of the population in natural and urban areas. Infected wild boar were found near peri-urban areas, demonstrating the importance of routine epidemiological surveillance and sanitary measures in and around cities. More research is needed to identify the Trichinella species that infect wild animals. We recommend the application of active and passive epidemiological surveillance in South America on exotic and native fauna that are hunted and consumed by humans.