Enhanced Genomic and Transcriptomic Resources for Trichinella pseudospiralis and T. spiralis to Underpin the Discovery of Molecular Differences between Stages and Species.

Nematodes of the genus Trichinella are important pathogens of humans and animals. This study aimed to enhance the genomic and transcriptomic resources for T. pseudospiralis (non-encapsulated phenotype) and T. spiralis (encapsulated phenotype) and to explore transcriptional profiles. First, we improved the assemblies of the genomes of T. pseudospiralis (code ISS13) and T. spiralis (code ISS534), achieving genome sizes of 56.6 Mb (320 scaffolds, and an N50 of 1.02 Mb) and 63.5 Mb (568 scaffolds, and an N50 value of 0.44 Mb), respectively. Then, for each species, we produced RNA sequence data for three key developmental stages (first-stage muscle larvae [L1s], adults, and newborn larvae [NBLs]; three replicates for each stage), analysed differential transcription between stages, and explored enriched pathways and processes between species. Stage-specific upregulation was linked to cellular processes, metabolism, and host-parasite interactions, and pathway enrichment analysis showed distinctive biological processes and cellular localisations between species. Indeed, the secreted molecules calmodulin, calreticulin, and calsyntenin-with possible roles in modulating host immune responses and facilitating parasite survival-were unique to T. pseudospiralis and not detected in T. spiralis. These insights into the molecular mechanisms of Trichinella-host interactions might offer possible avenues for developing new interventions against trichinellosis.

The illegal rearing and slaughtering of pigs in the wild on the Mediterranean island of Sardinia favor an increase in the biomass of Trichinella britovi in wild boars (Sus scrofa) but do not affect the serological prevalence of infection.

BACKGROUND: Worms of the nematode genus Trichinella are zoonotic pathogens with a worldwide distribution. The first report of Trichinella on the Mediterranean island of Sardinia was for Trichinella britovi, one of the four species of this genus circulating in Europe, which was identified in 2005 following an outbreak of trichinellosis in humans due to the consumption of pork from pigs reared in the wild. Since then, T. britovi larvae have been repeatedly isolated from free-ranging pigs, foxes (Vulpes vulpes) and wild boars (Sus scrofa) sampled in the central-eastern region of the island (Orgosolo municipality), but have never been isolated from samples from other areas of the island. The aim of this study was to investigate the parasitological and serological prevalence of T. britovi infection in wild boars in Sardinia over space [eight wild boar hunting management units (HMUs)] and time (seven wild boar hunting seasons). METHODS: Muscle and serum samples of boars killed in the 2014-2015 to 2020-2121 hunting seasons were collected from eight HMUs of central and south-western Sardinia. Trichinella sp. larvae were detected by artificial digestion of predilection muscles. A total of 4111 serum samples of wild boar were collected from the investigated HMUs and tested by enzyme-linked immunosorbent assay as a screening test and by western blot as a confirmatory test using excretory/secretory antigens. RESULTS: Trichinella britovi muscle larvae were detected in six (0.03%) of the 17,786 wild boars tested. All of the Trichinella sp.-positive wild boars had been hunted in Orgosolo municipality (central-eastern area of the island), except for one, hunted in a neighboring municipality. An overall serological prevalence of 3.8% (95% confidence interval, 3.3-4.5) was detected by western blot. No statistical differences were detected between the HMUs where T. britovi larvae were detected in wild boars, foxes, and free-ranging pigs and those where wild boars, foxes and free-ranging pigs tested negative. CONCLUSIONS: The serological prevalence did not vary between the wild boar populations in which the larval load was detectable by artificial digestion (Orgosolo municipality) and those in which the larval load was below the detection limit. Furthermore, the serological prevalence of anti-Trichinella immunoglobulin G in the wild boar populations remained constant during the study period, which covered seven wild boar hunting seasons. As the transmission events (i.e., the serological prevalence) are stable, the high biomass of the parasite in Orgosolo municipality can only have arisen as a consequence of factors independent of its natural cycle, i.e., the presence of a high number of free-ranging pigs, and the concomitant presence of African swine fever, due to illegal pig slaughtering in the field. This epidemiological situation suggests that the natural cycle of T. britovi may be influenced by inappropriate pig husbandry and slaughtering practices.

The impact of globalization and climate change on Trichinella spp. epidemiology.

The main reservoir hosts of nematodes of the genus Trichinella are wild carnivores, although most human infections are caused by the consumption of pork. This group of zoonotic parasites completes the entire natural life cycle within the host organism. However, there is an important phase of the cycle that has only been highlighted in recent years and which concerns the permanence of the infecting larvae in the striated muscles of the host carcasses waiting to be ingested by a new host. To survive in this unique biological niche, Trichinella spp. larvae have developed an anaerobic metabolism for their survival in rotting carcasses and, for some species, a resistance to freezing for months or years in cold regions. Climate changes with increasingly temperatures and reduction of environmental humidity lower the survival time of larvae in host carcasses. In addition, environmental changes affect the biology and ecology of the main host species, reducing their number and age composition due to natural habitat fragmentation caused by increasing human settlements, extensive monocultures, increasing number of food animals, and reduction of trophic chains and biodiversity. All of these factors lead to a reduction in biological and environmental complexity that is the key to the natural host-parasite balance. In conclusion, Trichinella nematodes can be considered as an indicator of a health natural ecosystem.

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.

Genetic evidence substantiates transmission of Trichinella spiralis from one swine farm to another.

BACKGROUND: Trichinella spiralis ranks seventh in the risk posed by foodborne parasites. It causes most human cases of trichinellosis and is the most frequent cause of Trichinella outbreaks on pig farms and in wild boar, worldwide. Veterinary inspectors seek the source of outbreaks in hopes of limiting the spread. Established molecular tools are inadequate for distinguishing among potential T. spiralis infection sources because genetic variability in these zoonotic pathogens is limited in Europe. Microsatellite markers proved successful in tracing an outbreak of T. britovi, a related parasite harboring much more genetic variation. Here, we successfully employed microsatellite markers to determine the genetic structure of T. spiralis isolates from two pig outbreaks, discovering notable uniformity among parasites within each farm and discovering an epidemiological link between these two outbreaks. METHODS: The individual larvae from five isolates of T. spiralis from two pig farms and from ten wild boars were genotyped using nine microsatellite markers to examine their genetic structure. RESULTS: Notably uniform parasite populations constituted each farm outbreak, and the parasites from the first and second outbreaks resembled each other to a notable degree, indicating an epidemiological link between them. Wild boar harbored more genetically variable larval cohorts, distinguishing them from parasites isolated from domestic pigs. CONCLUSIONS: Microsatellite markers succeeded in distinguishing isolates of the highly homogeneous T. spiralis, aiding efforts to track transmission. Each outbreak was composed of a homogenous group of parasites, suggesting a point source of contamination.

Collaborative Studies for the Detection of Taenia spp. Infections in Humans within CYSTINET, the European Network on Taeniosis/Cysticercosis.

Laboratory tools for diagnosing taeniosis/cysticercosis in non-endemic countries are available; however, there is little data on their performance. To provide information on the sensitivity, specificity, and reproducibility of these tools, inter-laboratory studies were organized within the EU COST-Action CYSTINET (TD1302). Two serological and one coprological Ring Trials (RTs) were organized to test a panel of human-derived sera and stool samples using assays routinely conducted by the participating laboratories to detect Taenia spp. infections. Four Western blots (WBs) and five ELISAs were used by nine laboratories for cysticercosis diagnosis. In the first serological RT, the overall sensitivity was 67.6% (95% CI, 59.1-75.4), whereas specificity was 97% (95% CI, 89.8-99.6). WBs recorded the best accuracy. A second serological RT was organized, to assess the three tests most frequently used during the first RT. Two out of six laboratories performed all the three tests. The overall sensitivity and specificity were 52.8% (95% CI, 42.8-62.7) and 98.1% (95% CI, 93.2-99.7), respectively. Laboratory performance strongly affected test results. Twelve laboratories participated in the coprological RT using conventional microscopy and six laboratories used molecular assays. Traditional diagnosis by microscopy yielded better results than molecular diagnosis. This may have been influenced by the lack of standardization of molecular tests across participating laboratories.

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.

A Bead-Based Assay for the Detection of Antibodies against Trichinella spp. Infection in Humans.

Human trichinellosis can be diagnosed by a combination of medical history, clinical presentation, and laboratory findings, and through detection of anti-Trichinella IgG in the patient's sera. ELISA using excretory-secretory (E/S) antigens of Trichinella spiralis larvae is currently the most used assay to detect Trichinella spp. antibodies. Bead-based assay can detect antibodies to multiple antigens concurrently; the ability to detect antibody to T. spiralis using a bead assay could be useful for diagnosis and surveillance. We developed and evaluated a bead assay to detect and quantify total IgG or IgG4 Trichinella spp. antibodies in human serum using T. spiralis E/S antigens. The sensitivity and specificity of the assay were determined using serum from 110 subjects with a confirmed diagnosis of trichinellosis, 140 subjects with confirmed infections with other tissue-dwelling parasites, 98 human serum samples from residents of the United States with no known history of parasitic infection, and nine human serum samples from residents of Egypt with negative microscopy for intestinal parasites. Sensitivity and specificity were 93.6% and 94.3% for total IgG and 89.2% and 99.2% for IgG4, respectively. Twelve percent of sera from patients with confirmed schistosomiasis reacted with the IgG Trichinella bead assay, as did 11% of sera from patients with neurocysticercosis. The Trichinella spp. bead assay to detect IgG total antibody responses has a similar performance as the Trichinella E/S ELISA. The Trichinella spp. bead assay shows promise as a method to detect trichinellosis with a possibility to be used in multiplex applications.

Scientific achievements of the last 60 years: From a single to a multispecies concept of the genus Trichinella.

The scientific basis that led to the development of a multispecies concept within the Trichinella genus originated in the 1950s, when scientists began reporting an increasing number of host-specific peculiarities among different geographic isolates. This led to speculation that important geographic variability existed within Trichinella spiralis, the only species in the genus at that time. Comparative infection results sparked great interest among investigators and led to similar studies using various geographic isolates of the parasite. In 1972, the Russian scientists V.A. Britov, S.N. Boev and B.L. Garkavi, described three new species: Trichinella nativa, Trichinella nelsoni and Trichinella pseudospiralis. This shattered the concept that the genus Trichinella was monospecific and widened the host range to include birds. The description of these new species generated an intense debate over their taxonomic validity because there were no clear morphological differences among them and because the concept of sibling species had not yet been accepted by parasitologists. The resolution of the taxonomic issues was facilitated by the adoption of new biochemical and molecular techniques for systematics research. In 1992, the first study comparing 152 isolates from various host species and geographical regions identified eight distinct taxa, coded T1 through T8; four of these represented the previously proposed species and included one new species, Trichinella britovi (T3). During the past 27 years, an increasing number of investigations in different geographical regions and hosts coupled with the availability of new and highly sensitive molecular techniques have allowed the description of four new species; Trichinella murrelli (T5), Trichinella papuae (T10), Trichinella zimbabwensis (T11) and Trichinella patagoniensis (T12), and two new genotypes Trichinella T9 and T13. Thus, the taxonomic status of Trichinella T6, T8, T9 and T13 remain unresolved. These new technologies have also advanced a more complete phylogenetic, zoogeographical and epidemiological knowledge base for future work.

Second outbreak of Trichinella pseudospiralis in Europe: clinical patterns, epidemiological investigation and identification of the etiological agent based on the western blot patterns of the patients' serum.

Trichinellosis is a zoonotic disease due to the ingestion of raw or undercooked meat from animals infected with the larvae of nematodes belonging to the genus Trichinella. In January-February 2015, an outbreak of trichinellosis occurred in Genoa, Northern Italy. The epidemiological link was traced back to a dinner served at an agritourism farm on 31 December 2014, where a majority of the 52 guests had consumed the 'beef' steak tartare. The source of infection was not traced; however, it was noted that the amount of beef purchased officially for providing at the dinner did not correspond with that served, suggesting that meat of a different origin had been added to the beef to prepare the steak tartare. Clinical and laboratory data of 30 individuals out of the 52 (57.7%), of which four were hospitalized, were consistent with that of the case definition of trichinellosis. Western blot patterns of the sera from patients with confirmed trichinellosis were similar to the diagnostic pattern identified for the reference sera of Trichinella pseudospiralis but different from those of the control sera tested for patients infected with Trichinella spiralis and Trichinella britovi. Identification of T. pseudospiralis as the aetiological agent responsible for the outbreak of trichinellosis using an indirect tool represents an advancement in the epidemiological investigation of this zoonotic disease.

Trichinella species and genotypes.

Trichinella spiralis has historically been deemed "the pig parasite" owing to its initial classification within a monospecific genus. However, in recent years, the genus has expanded to include 10 distinct species and at least 3 different genotypes whose taxonomic status remains unstipulated. In contrast to T. spiralis, however, most of these sylvatic species and genotypes do not infect pigs well. Inasmuch as morphological characters cannot be used to define species within this genus, earlier classifications were based upon host and geographical ranges, biological characters, and the presence or absence of a collagen capsule that surrounds the muscle stage larvae. Later, isoenzymes, DNA gel fragmentation patterns and DNA probes were used to help in identification and classification. Today, amidst the "-omics" revolution, new molecular and biochemical-based methodologies have improved detection, differentiation and characterization at all levels including worm populations. These efforts have discernably expanded immunological, epidemiological, and genetic studies resulting in better hypotheses on the evolution of the genus, and on global events, transmission cycles, host associations, and biogeographical histories that contributed to its cosmopolitan distribution. Reviews of this sort are best begun with a background on the genus; however, efforts will divert to the most recent knowledge available on the taxonomy, phylogeny, epidemiology and biochemistry that define this genus in the 21st century.

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.

Clinical and epidemiological descriptions from trichinellosis outbreaks in Bulgaria.

Bulgaria is one of European countries where trichinellosis continues to be regularly diagnosed and registered. The clinical and epidemiological features of 72 cases of trichinellosis associated with five outbreaks caused by Trichinella spiralis and Trichinella britovi between 2009 and 2011, are described. At hospital admission, patients were often initially treated with antibiotics, without any improvement. A range of signs and symptoms were recorded, including: myalgia, elevated temperature, arthralgia, difficulty with movement, facial oedema, conjunctival hyperaemia, ocular haemorrhages, diarrhoea, skin rash, headache, and fatigue. Due to the variable clinical course of the disease, the diagnostic process for trichinellosis is often complex and difficult. This means the diagnosis may be established late for an appropriate treatment, potentially leading to a severe course of the disease with complications. Laboratory abnormalities were expressed by marked eosinophilia (97.2%), leucocytosis (70.8%), elevated serum creatine phosphokinase levels (82%), and antibody-positive results by ELISA and indirect hemagglutination. Patients were treated with albendazole (Zentel) 10 mg/kg for 7-10 days. In two outbreaks, the aetiological agent was T. spiralis, in one outbreak T. britovi, and an unknown Trichinella species in the fourth outbreak. The sources of infection were domestic pigs, probably fed with scraps and offal of wild game. In one outbreak, T. spiralis was also detected in brown rats trapped close to where the pig had been raised in the backyard. These epidemiological factors are relevant in considering implementation of targeted control programmes.

How globalization and climate change could affect foodborne parasites.

Foodborne parasites, most of which are zoonotic, represent an important human health hazard. These pathogens which include both protozoa (e.g., Cryptosporidium spp., Cyclospora cayetanensis, Toxoplasma gondii) and helminths (e.g., liver and intestinal flukes, Fasciola spp., Paragonimus spp., Echinococcus spp., Taenia spp., Angiostrongylus spp., Anisakis spp., Ascaris spp., Capillaria spp., Toxocara spp., Trichinella spp., Trichostrongylus spp.), have accompanied the human species since its origin and their spread has often increased due to their behavior. Since both domesticated and wild animals play an important role as reservoirs of these pathogens the increase/decrease of their biomasses, migration, and passive introduction by humans can change their epidemiological patterns. It follows that globalization and climate change will have a tremendous impact on these pathogens modifying their epidemiological patterns and ecosystems due to the changes of biotic and abiotic parameters. The consequences of these changes on foodborne parasites cannot be foreseen as a whole due to their complexity, but it is important that biologists, epidemiologists, physicians and veterinarians evaluate/address the problem within a one health approach. This opinion, based on the author's experience of over 40 years in the parasitology field, takes into consideration the direct and indirect effects on the transmission of foodborne parasites to humans.

Retrospective analysis of hospital discharge records for cases of trichinellosis does not allow evaluation of disease burden in Italy.

Human trichinellosis is a disease caused by nematode worms of the genus Trichinella. In Italy, as well as in most other European countries, notification of Trichinella infections in humans is mandatory; however, no information is available on the number of cases occurring annually. The aim of the present study was to retrospectively evaluate the burden of trichinellosis in Italy from 2005 to 2016. Hospital discharge records (HDRs) showing the code for trichinellosis (124) were registered and screened. Results were then compared with yearly reports issued by the Italian National Reference Laboratory for Trichinella (NRLT), with reports from the European Centre for Disease Prevention and Control (ECDC), and with literature data. A total of 102 HDRs revealed that the 124 code was erroneously reported in 72 (70.6%) records. Out of the 30 (29.4%) records with a correct diagnosis of trichinellosis, nine cases were reported by HDRs only, 21 cases were documented by both HDRs and the NRLT, whereas the NRLT documented 100 additional cases. In the studied period, the average yearly incidence was 0.01 cases per 100,000 inhabitants. This study highlights the limitations of using HDRs to obtain a clear picture of the prevalence and incidence of trichinellosis in Italy. These findings demonstrate the need to intensify the surveillance system for trichinellosis through the development of an Italian registry. This would allow the identification of patients with severe infections and pauci-symptomatic patients, and would avoid the need for clinical analyses and unnecessary treatments, reducing the resulting economic burden on the Italian National Health Service.