Trichinella britovi in wild boar meat from Italy, 2015-2021: A citizen science approach to surveillance.

As a result of the increase of game meat intended for human consumption through Europe, a plethora of food-borne diseases, including trichinellosis, may occur in consumers, posing a relevant public health threat. Thus, this study aims to a citizen science approach to monitor the occurrence of Trichinella spp. in wild boar meat intended for human consumption, evaluating the risk of infection for consumers. Following the European Regulation 2015/1375 (laying down specific rules on official controls for Trichinella in meat), from 2015 to 2021, hunters (n = 478) were involved to collect diaphragm pillar samples of wild boars from mainland southern Italy, which were tested for Trichinella spp. L1 larvae via HCl-pepsin digestion and Multiplex PCR. Overall, 139,160 animals were collected (average of 19,880 per year), being 14 (i.e., 0.01%) tested positive to Trichinella britovi by the combined biochemical and molecular approach. An average larval burden of 28.4 L1 per gram of meat was found (minimum 3.2 - maximum 132.6). A statistically significant difference was found in the prevalence according to hunting seasons (p < 0.01, with higher values in 2016 and 2021) and regions of the study area (p < 0.01). No statistically significant decrease in the prevalence of T. britovi throughout the study period was found (p = 0.51), except in Apulia region (p < 0.01). These findings revealed a stable prevalence of T. britovi in wild boar meat intended for human consumption, suggesting a risk of infection for consumers, especially hunters and local markets users. Citizen science surveillance models could be promoted to improve trichinellosis control and prevention in a One Health perspective.

Detection of Trichinella murrelli and Trichinella pseudospiralis in bobcats (Lynx rufus) from Oklahoma.

Trichinella spp. infect wild carnivores throughout the world. We determined the prevalence and mean infection intensity of Trichinella spp. in bobcats (Lynx rufus) from 41 counties in Oklahoma (USA). Tongues from 306 bobcats were examined using artificial tissue digestion. The prevalence (95% confidence interval) of Trichinella spp. was 5.9% (3.7%-9.2%) in which 18 of the 301 bobcats were infected. Bobcats infected with Trichinella spp. were detected in 10 of the 41 (24.4%; 13.7%-39.5%) counties sampled. Although variable, a statistically significant difference was not detected in the prevalence of Trichinella spp. among counties where bobcats were collected. The mean (standard deviation) and median (range) infection intensity of Trichinella sp. larvae were 30.9 (39.8) and 9.6 (0.6-119.9) larvae per gram of tissue examined. Genotyping results demonstrated that 17 bobcats were infected with T. murrelliand one bobcat was infected with T. pseudospiralis. This is the first report of T. pseudospiralis in bobcats and in Oklahoma. These data suggest the bobcat, as an obligate carnivore, is likely an important host in maintaining T. murrelli sylvatic cycles in Oklahoma.

Animal welfare and zoonosis risk: anti-Trichinella antibodies in breeding pigs farmed under controlled housing conditions.

BACKGROUND: Domesticated pigs are the main source of Trichinella sp. infections for humans, particularly when reared in backyards or free-ranging. In temperate areas of southern Europe, most pigs are farmed under controlled housing conditions, but sows and sometimes fattening pigs have access to outdoors to improve animal welfare. The aim of the present study was to investigate whether outdoor access of breeding pigs farmed under controlled housing conditions can represent a risk for Trichinella sp. transmission when the farm is located in an agricultural area interspersed with wooded areas and badlands, where Trichinella spp. could be present in wildlife. METHODS: Serum samples were collected from 63 breeding sows and one boar before and after their access to an open fenced area for 2 months and from 84 pigs that never had outdoor access. Samples were screened for anti-Trichinella antibodies by ELISA, and positive sera were confirmed using Western blot (Wb) excretory/secretory antigens. To detect Trichinella sp. larvae, muscle tissues from serologically positive and negative pigs were tested by artificial digestion. RESULTS: Thirteen (20.6%) sows and one boar tested positive with both ELISA and Wb. No larvae were detected in muscle samples of serologically positive and serologically negative pigs. Positive serum samples were then tested by Wb using crude worm extract as antigens. The Wb banding pattern displayed was that characteristic of encapsulated species (Trichinella spiralis or Trichinella britovi). CONCLUSIONS: The detection of anti-Trichinella antibodies without larvae in the pig muscles, supported by epidemiological data, suggests that pigs may have been exposed to T. britovi. This study stresses the importance of instigating monitoring systems at farm level to prevent Trichinella sp. transmission and to investigate, through a landscape parasitological study, the suitability of a site before the planting of a high containment level pig farm in which the sows can have outside access to improve their welfare during pregnancy.

First report of Trichinella pseudospiralis in a wolf (Canis lupus italicus).

Within the genus Trichinella, Trichinella pseudospiralis is the only recognized non-encapsulated species known to infect mammals and birds. In October 2020, larvae recovered from muscle tissues of a wolf (Canis lupus italicus) originating from Molise Region, Central Italy, were molecularly confirmed as those of Trichinella britovi and T. pseudospiralis. This is the first detection of T. pseudospiralis from a wolf. In Italy, this zoonotic nematode was detected in a red fox (Vulpes vulpes), three birds (Strix aluco, Athene noctua, Milvus milvus) and five wild boars (Sus scrofa), and was also identified as the etiological agent of a human outbreak of trichinellosis in 2015. Since T. pseudospiralis is rarely reported from carnivore mammals in comparison to the encapsulated species frequently detected in these hosts, this finding opens the question of the role of carnivores as reservoirs for this parasite.

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.

Differences in larval survival and IgG response patterns in long-lasting infections by Trichinella spiralis, Trichinella britovi and Trichinella pseudospiralis in pigs.

BACKGROUND: Domesticated and wild swine play an important role as reservoir hosts of Trichinella spp. and a source of infection for humans. Little is known about the survival of Trichinella larvae in muscles and the duration of anti-Trichinella antibodies in pigs with long-lasting infections. METHODS: Sixty pigs were divided into three groups of 20 animals and infected with 10,000 larvae of Trichinella spiralis, Trichinella britovi or Trichinella pseudospiralis. Four pigs from each group were sacrificed at 2, 6, 12, 18 and 24 months post-infection (p.i.) and the number of larvae per gram (LPG) of muscles was calculated. Serum samples were tested by ELISA and western blot using excretory/secretory (ES) and crude antigens. RESULTS: Trichinella spiralis showed the highest infectivity and immunogenicity in pigs and larvae survived in pig muscles for up to 2 years p.i. In these pigs, the IgG level significantly increased at 30 days p.i. and reached a peak at about 60 days p.i., remaining stable until the end of the experiment. In T. britovi-infected pigs, LPG was about 70 times lower than for T. spiralis at 2 months p.i. and only very few infecting larvae were detected at 6 months p.i., whereas no larvae were detected at 12, 18 and 24 months p.i. At 6 months p.i., degenerated/calcified larvae and cysts were detected in the muscles by trichinoscopy and histology. The IgG pattern showed by T. britovi-infected pigs was similar to that of T. spiralis-infected pigs, although seroconversion occurred some days later. The larval burden of T. pseudospiralis was slightly greater than for T. britovi at 2 months p.i., but no larvae were detected at 6 and 12 months p.i. In T. pseudospiralis-infected pigs, seroconversion occurred slowly, as in T. britovi-infected pigs. The IgG level showed a significant drop at 6 months p.i. and declining to the cut-off value at 12 months p.i. CONCLUSIONS: The longer survival of T. spiralis in pigs in comparison with the other two species highlights its exceptional dissemination potential. These results provide an explanation of the controversial data collected by parasitological and serological tools in the course of epidemiological investigations.

The subnivium, a haven for Trichinella larvae in host carcasses.

Parasite nematodes of the genus Trichinella are transmitted from one host to another through the ingestion of larvae present in striated muscles. The longer the survival of muscle larvae in host carcasses, the higher the probability of being ingested by a scavenging host. Thereby, these nematodes developed an anaerobic metabolism favouring their survival in decaying tissues. In addition, muscle larvae of three taxa, namely Trichinella nativa, Trichinella britovi and Trichinella T6, can survive freezing for several months to several years depending on the taxon. The aim of the present work was to investigate the survival time of T. britovi larvae in naturally infected host carcasses preserved beneath or above the snow. Fox and raccoon dog carcasses naturally infected with T. britovi larvae were preserved beneath or above the snow in a cold mountainous area. Temperature and relative humidity were recorded. Every 14 days, muscle samples collected from each carcass, were digested and larvae were counted and given per os to laboratory mice to evaluate their reproductive capacity index (RCI). The RCI of larvae in carcasses preserved beneath the snow (the subnivium) ranged from 23 to 25 at day 0, to 12-18 after 112 days. In contrast, the RCI of larvae in carcasses preserved above the snow ranged from 22 to 27 at day 0, to 0.0 after 112 days. The difference between the RCIs of larvae beneath the snow and above the snow was statistically significant (P < 0.01). These data corroborate the hypothesis that the subnivium with its environmental stability favours the survival of Trichinella larvae in host muscles, increasing the probability of their transmission to other hosts. On the other hand, the environment above the snow, characterized by sudden temperature variations, causes strong environmental stress for larvae in host carrions causing their death.

Trichinella spiralis prevalence among wildlife of a boreal region rapidly reduced in the absence of spillover from the domestic cycle.

Carnivore and omnivore animals are the main reservoir hosts of nematodes of the genus Trichinella. Out of the twelve taxa identified so far in the world, four, namely Trichinella spiralis, Trichinella nativa, Trichinella britovi and Trichinella pseudospiralis, are circulating in Europe. All these four species were detected in Finnish wild animals and T. spiralis also in domestic pigs and synanthropic rats of Finland up to 2004, when the last T. spiralis infection was documented in a domestic pig. In the periods 1993-1997 and 1999-2005, T. spiralis was detected in 13.6% (6/44) and in 14.5% (46/317) of infected wild animals, respectively. The aims of the present study were to investigate the prevalence of Trichinella spp., especially T. spiralis in wild carnivores of Finland, 7-9 years after the eradication of Trichinella infection from Finnish domestic pigs, and to evaluate the impact of the disappearance of domestic cycle of T. spiralis on the sylvatic Trichinella cycle(s). Trichinella spp. larvae were detected in 34.7% (1081/3112) wild animals and 835 (77.2%) Trichinella spp. isolates were identified at the species level. Trichinella spiralis was detected in 9 animals (1.0% of identified infections) as single, double or triple infections; whereas, T. spiralis had been detected in 13.6% and in 14.5% of infected wild animals in the course of the two previous investigations. The reduction of T. spiralis prevalence from 1993-1997 to 2011-2013 is statistically significant (p < 0.001) both per total tested animals and per host species. The results of this study support the hypothesis of spillover from domestic pigs as a source of T. spiralis in wildlife. The eradication of T. spiralis from the domestic cycle in Finland in 2004 has probably established a virtuous cycle reducing the prevalence of this pathogen among wild carnivores. We can expect that within some years and in absence of T. spiralis reintroduction in the domestic habitat, it might even disappear from the Finnish wildlife.

Trichinella spiralis a new alien parasite in Italy and the increased risk of infection for domestic and wild swine.

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.

Two haplotype clusters of Echinococcus granulosus sensu stricto in northern Iraq (Kurdistan region) support the hypothesis of a parasite cradle in the Middle East.

Human cystic echinococcosis (CE) caused by Echinococcus granulosus s.s. is a major public health problem in Iraqi Kurdistan with a reported surgical incidence of 6.3 per 100,000 Arbil inhabitants. A total of 125 Echinococcus isolates retrieved from sheep, goats and cattle were used in this study. Our aim was to determine species/genotypes infecting livestock in Iraqi Kurdistan and examine intraspecific variation and population structure of Echinococcus granulosus s.s. in this region and relate it to that of other regions worldwide. Using nucleotide sequences of the mitochondrial cytochrome c oxidase subunit 1 (cox 1) we identified E. granulosus s.s. as the cause of hydatidosis in all examined animals. The haplotype network displayed a double-clustered topology with two main E. granulosus s.s. haplotypes, (KU05) and (KU33). The 'founder' haplotype (KU05) confirmed the presence of a common lineage of non-genetically differentiated populations as inferred by the low non-significant fixation index values. Overall diversity and neutrality indices indicated demographic expansion. We used E. granulosus s.s. nucleotide sequences from GenBank to draw haplotype networks for the Middle East (Iran, Jordan and Turkey), Europe (Albania, Greece, Italy, Romania and Spain), China, Mongolia, Russia, South America (Argentina, Brazil, Chile and Mexico) and Tunisia. Networks with two haplotype clusters like that reported here for Iraqi Kurdistan were seen for the Middle East, Europe, Mongolia, Russia and Tunisia using both 827bp and 1609bp cox1 nucleotide sequences, whereas a star-like network was observed for China and South America. We hypothesize that the double clustering seen at what is generally assumed to be the cradle of domestication may have emerged independently and dispersed from the Middle East to other regions and that haplotype (KU33) may be the main haplotype within a second cluster in the Middle East from where it has spread into Europe, Mongolia, Russia and North Africa. Further studies using metacestodes of human origin are required to investigate the biological importance of E. granulosus s.s. haplotypes/clusters and their association, if any with clinical manifestations of CE infection.

Echinococcus multilocularis in foxes and raccoon dogs: an increasing concern for Baltic countries.

BACKGROUND: In Europe, the life-cycle of Echinococcus multilocularis is predominantly sylvatic, involving red foxes (Vulpes vulpes) as the main definitive hosts and rodents such as muskrats and arvicolids as intermediate hosts. The parasite is the etiological agent of human alveolar echinococcosis, a malignant zoonotic disease caused by the accidental ingestion of eggs shed by definitive hosts in their faeces. The aims of this study were to investigate the prevalence of E. multilocularis in red foxes and raccoon dogs (Nyctereutes procyonoides) and to study the environmental factors favouring the perpetuation of the parasite in Latvia. METHODS: A total of 538 red foxes and 407 raccoon dogs were collected across Latvia from 2010 to 2015. The sedimentation and counting technique was used for collecting E. multilocularis adult worms from fox and raccoon dog intestines. The morphological identification of the parasite was confirmed by molecular analysis. RESULTS: The prevalence of E. multilocularis was significantly higher in foxes (17.1%; intensity of infection 1-7,050 worms) (P < 0.001) than in raccoon dogs (8.1%; intensity of infection 5-815 worms). In foxes, a significant positive correlation (r (10) = 0.7952, P = 0.001) was found between parasite prevalence and the intensity of infection. A positive relationship (R s = 0.900, n = 5, P = 0.037) between parasite prevalence and precipitation was also observed. In raccoon dogs, a significant negative relationship (F (1,8) = 9.412, P = 0.015) between animal density and parasite prevalence, and a significant positive relationship (F (1,8) = 7.869, P = 0.023) between parasite prevalence and agricultural land cover, were detected. CONCLUSIONS: The results of this study confirm the red fox as the most important definitive host of E. multilocularis and, consequently, as the main target for control programmes in the Baltic countries. Raccoon dogs seem to play a secondary role in the life-cycle of E. multilocularis within the investigated European region.

First insights into the genetic diversity of Echinococcus granulosus sensu stricto (s.s.) in Serbia.

Cystic echinococcosis (CE) caused by Echinococcus granulosus sensu stricto (s.s.) is a cosmopolitan zoonotic infection which is endemic in Serbia where it is subject to mandatory reporting. However, information on the incidence of the disease in humans and prevalence of hydatid infection in livestock remains limited. We used sequenced data of the cytochrome c oxidase subunit 1 (cox 1) mitochondrial gene to examine the genetic diversity and population structure of E. granulosus (s.s.) from intermediate hosts from Serbia. We also compared our generated nucleotide sequences with those reported for neighbouring European countries. Echinococcus canadensis was molecularly confirmed from pig and human hydatid isolates. E. granulosus (G1) was confirmed from sheep and cattle hydatid isolates as well as the first molecular confirmation in Serbia of E. granulosus G2 in sheep and E. granulosus G3 in sheep and cattle hydatid isolates. The Serbian E. granulosus (s.s.) parsimony network displayed 2 main haplotypes (SB02 and SB05) which together with the neutrality indices were suggestive of bottleneck and/or balancing selection. Haplotype analysis showed the presence of the common E. granulosus haplotype described from other worldwide regions. Investigation of the pairwise fixation (Fst) index suggested that Serbian populations of E. granulosus (s.s.) from sheep and cattle hosts showed moderate genetic differentiation. Six of the Serbian haplotypes (SB02-SB07) were shared with haplotypes from Bulgaria, Hungary and/or Romania. Further studies using a larger number of hydatid isolates from various locations across Serbia will provide more information on the genetic structure of E. granulosus (s.s.) within this region.

High prevalence of Trichinella pseudospiralis in Florida panthers (Puma concolor coryi).

BACKGROUND: Parasites of the genus Trichinella are zoonotic nematodes common in carnivores throughout the world. We determined the prevalence and species of Trichinella infections in Florida panthers (Puma concolor coryi). METHODS: Tongues from Florida panthers were collected at necropsy and examined by pepsin-HCl artificial digestion for infection with Trichinella spp. DNA was extracted from larvae and multiplex PCR using Trichinella species-specific primers was used to genotype the worms. RESULTS: Trichinella spp. larvae were detected in 24 of 112 (21.4%; 14.6%-30.3%) panthers. Sixteen of the panthers (14.3%) were infected with T. pseudospiralis, 1 (0.9%) was infected with T. spiralis, and 2 (1.8%) had mixed infections of T. pseudospiralis and T. spiralis. Trichinella spp. larvae from 5 panthers were not identified at the species level due to degraded DNA. CONCLUSIONS: This is the highest prevalence of T. pseudospiralis detected in North America up to now and suggests the Florida panther is a key mammalian reservoir of this parasite in southern Florida. Trichinella pseudospiralis can infect both mammals and birds indicating the source of infection for Florida panthers could be broader than believed; however, birds represent a small percentage (0.01%) of the cat's diet. Since wild pigs (Sus scrofa) can be parasitized by both T. pseudospiralis and T. spiralis and these swine can comprise a large portion (~40%) of a panther's diet in Florida, we believe that Florida panthers acquired these zoonotic parasites from feeding on wild pigs.

Validation of a latex agglutination test for the detection of Trichinella infections in pigs.

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.

Molecular identification of nematode larvae different from those of the Trichinella genus detected by muscle digestion.

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).

Genetic variability of Echinococcus granulosus sensu stricto in Europe inferred by mitochondrial DNA sequences.

The genetic diversity of Echinococcus granulosus sensu stricto (s.s.) metacestodes from four European countries was evaluated by the DNA sequence analysis of the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene. Of the 312 organisms investigated, 132 were from Bulgaria, 35 from Hungary, 89 from Italy and 56 from Romania. Considerable intraspecific variation was observed in the mitochondrial cox1 sequences: 24 haplotypes were detected in the Eastern European population and seven in the Italian population. The Eastern European population parsimony network displayed a star-like features consisting of the most common haplotype EG1 (G1 genotype) and the three major haplotypes: EG2, EG3 and EG4. The EG1 was also the major haplotype in the Italian population network, though with a higher prevalence (73%) compared to the Eastern European network. The percentage of the population constituted by the G1 genotype was used as an indirect index to evaluate the genetic diversity within E. granulosus s.s. populations of Eurasia. A clinal correlation between the percentage of the G1 genotype and the geographical regions of Eurasia was observed: the G1 genotype is highly represented in the Mediterranean Basin; it decreases in Eastern Europe and South-West Asia and increases in China. This clinal correlation could reflect the spreading of livestock domestication from Southern-Western Asia during the Neolithic period, beginning around 12,000 BC.

Detection of Trichinella murrelli in coyotes (Canis latrans) from Oklahoma and North Texas.

We determined the prevalence and mean intensity of Trichinella sp. infection in coyotes from six counties in Oklahoma and one in northern Texas. Tongues from 77 coyotes were examined using histology and artificial tissue digestion. Histological examination showed a prevalence of 3.9% (3 of 77) whereas the prevalence was 6.5% (5 of 77) based on artificial digestion of 5.0 g of muscle from coyote tongues. One sample was positive for Trichinella sp. on histology but negative by artificial digestion. Combining data from both diagnostic techniques showed that six of 77 (7.8%) coyotes were infected with Trichinella spp. The mean intensity of Trichinella sp. larvae ranged from 0.2 to 66.2 with an average of 16.0 larvae per gram (LPG) of tongue. Genotyping results demonstrated that the coyotes were infected with Trichinella murrelli. This is the first report of T. murrelli infection in coyotes in Oklahoma. T. murrelli had previously been isolated from coyotes in Texas.