Comparative multi-omics analyses reveal differential expression of key genes relevant for parasitism between non-encapsulated and encapsulated Trichinella.

Genome assemblies provide a powerful basis of comparative multi-omics analyses that offer insight into parasite pathogenicity, host-parasite interactions, and invasion biology. As a unique intracellular nematode, Trichinella consists of two clades, encapsulated and non-encapsulated. Genomic correlation of the distinct differences between the two clades is still unclear. Here, we report an annotated draft reference genome of non-encapsulated Trichinella, T. pseudospiralis, and perform comparative multi-omics analyses with encapsulated T. spiralis. Genome and methylome analyses indicate that, during Trichinella evolution, the two clades of Trichinella exhibit differential expansion and methylation of parasitism-related multi-copy gene families, especially for the DNase II members of the phospholipase D superfamily and Glutathione S-transferases. Further, methylome and transcriptome analyses revealed divergent key excretory/secretory (E/S) genes between the two clades. Among these key E/S genes, TP12446 is significantly more expressed across three life stages in T. pseudospiralis. Overexpression of TP12446 in the mouse C2C12 skeletal muscle cell line could induce inhibition of myotube formation and differentiation, further indicating its key role in parasitism of T. pseudospiralis. This multi-omics study provides a foundation for further elucidation of the mechanism of nurse cell formation and immunoevasion, as well as the identification of pharmacological and diagnostic targets of trichinellosis.

Immunoproteomic analysis of Trichinella spiralis and Trichinella britovi excretory-secretory muscle larvae proteins recognized by sera from humans infected with Trichinella.

The present study compares the immunogenic patterns of muscle larvae excretory-secretory proteins (ML E-S) from T. spiralis and T. britovi recognized by Trichinella-infected human sera. Samples were analyzed using two-dimensional electrophoresis (2-DE) coupled with 2D-immunoblot and liquid chromatography-tandem mass spectrometry LC-MS/MS analysis, two ELISA procedures and a confirmatory 1D-immunoblot test. Sera were obtained from nine patients with a history of ingestion of raw or undercooked meat who presented typical clinical manifestations of trichinellosis and from eleven healthy people. Specific anti-Trichinella IgG antibodies were detected in all samples tested with the Home-ELISA kits, but in only four samples for the commercially-available kit. The 1D-immunoblot results indicated that all nine serum samples were positive for T. spiralis ML E-S antigens, expressed as the presence of specific bands. In contrast, eight of the serum samples with T. britovi E-S ML antigens were positive, with one serum sample taken from a patient at 33dpi (days post infection) being negative. To identify immunoreactive proteins that are specifically recognized by host antibodies, both species of ML E-S proteins were subjected to 2D-immunoblotting with human serum taken at 49 dpi. The sera recognized 22 protein spots for T. spiralis and 18 for T. britovi in 2D-immunoblot analysis. Their molecular weights (MW) ranged from 50 to 60 kDa. LC-MS/MS analysis identified both common and specifically-recognized immunoreactive proteins: transmembrane serine protease 9, serine protease, antigen targeted by protective antibodies and Actin-1 partial were shared for both Trichinella species; hypothetical protein T01_7775 and P49 antigen, partial those specific to T. spiralis; deoxyribonuclease-2-alpha and hypothetical protein T03_17187/T12_7360 were specific to T. britovi. Our results demonstrate the value of 2-DE and 2D-immunblot as versatile tools for pinpointing factors contributing to the parasite-host relationship by comparing the secretomes of different Trichinella species.

Trichinella spiralis y Trichinella patagoniensis: cinética de desialización eritrocitaria / Trichinella spiralis and Trichinella patagoniensis: kinetics of erythrocyte desialization / Trichinella spiralis e Trichinella patagoniensis: cinética de dessialização eritrocitária

El objetivo del trabajo fue estudiar la cinética de desialización eritrocitaria producida por larvas infectantes de Trichinella spiralis y Trichinella patagoniensis. Se trabajó con 7 suspensiones eritrocitarias incubadas con 1.000±200 larvas musculares/mL, durante 2 horas, tomando muestra al tiempo inicial y cada 15 minutos. Los respectivos eritrocitos controles se incubaron de la misma manera con solución salina. Se aplicaron el método de titulación por Polibrene calculando el CexpST y un análisis de varianza (ANOVA) con las comparaciones múltiples según Tukey. Los resultados mostraron que el valor promedio de CexpST disminuyó con el aumento del tiempo de incubación, para ambas especies. En el tratamiento con T. spiralis no hubo diferencias significativas entre el valor medio del coeficiente a tiempo 60 y 75 minutos, mientras que con T. patagoniensis, a 45 y 60 minutos. Todas las restantes diferencias fueron significativas. La comparación entre los tratamientos, para cada uno de los tiempos, mostró que al tiempo inicial el coeficiente promedio no difirió entre las especies, pero que a todos los otros tiempos fue significativamente menor en la incubación de los eritrocitos con T. spiralis. Se concluye que la relación hospedador-parásito que se establece en ambos casos es distinta y probablemente también la capacidad de adaptación y de daño al hombre.

The objective of this work was to study the kinetics of erythrocyte desialization produced by infective larvae of Trichinella spiralis and Trichinella patagoniensis. It was performed on 7 erythrocyte suspensions incubated with 1,000±200 muscle larvae/ mL for 120 minutes, taking samples at the initial time and every 15 minutes. The respective control erythrocytes were incubated in the same way with saline solution. The Polybrene Titration method calculating the CexpST and variance analysis (ANOVA) with the multiple comparisons according to Tukey were applied. The results showed that the average value of CexpST decreased with the increase in incubation time, for both species. There were no significant differences between the mean value of the coefficient at 60 and 75 minutes in the treatment with T. spiralis, while neither were there any differences between 45 and 60 minutes in the incubation with T. patagoniensis. All other differences were significant. The comparison between the two treatments, for each of the times, showed that at the initial time the average coefficient did not differ between the species, but at all other times it was significantly lower in the incubation of the erythrocytes with T. spiralis. It is concluded that the parasite host relationship that is established in both cases is different and probably also is the ability to adapt and cause harm to man.

O objetivo do trabalho foi estudar a cinética de dessialização eritrocitária. produzida por larvas infectantes de Trichinella spiralis e Trichinella patagoniensis. O trabalho foi feito com 7 suspensões eritrocitárias incubadas com 1.000±200 larvas musculares/mL por 2 horas, colhendo amostras no tempo inicial e a cada 15 minutos. Os respectivos eritrócitos-controle foram incubados da mesma forma com solução salina. Foi aplicado o método de titulação por Polibreno calculando o CexpST e também uma análise da variância (ANOVA) com as comparações múltiplas de acordo com Tukey. Os resultados mostraram que o valor médio de CexpST diminuiu com o aumento do tempo de incubação para ambas as espécies. No tratamento com T. spiralis não houve diferenças significativas entre o valor médio do coeficiente no tempo 60 e 75 minutos, ao passo que com T. patagoniensis, aos 45 e 60 minutos. Todas as diferenças restantes foram significativas. A comparação entre os tratamentos, para cada um dos tempos, mostrou que no tempo inicial o coeficiente médio não diferiu entre as espécies, mas que em todos os outros tempos foi significativamente menor na incubação dos eritrócitos com T. spiralis. A conclusão é que a relação hospedeiro-parasita, estabelecida em ambos os casos, é diferente e provavelmente também a capacidade de adaptação e dano ao homem.

Trichinella britovi in Game Meat Linked to Human Trichinellosis Outbreak in Serbia.

After a human trichinellosis outbreak in Zlatibor District, Serbia, in 2016, Trichinella larvae were found in wild boar ( Sus scrofa) meat products. One hundred and fourteen people were infected during the outbreak. The larvae were determined to be Trichinella britovi using the polymerase chain reaction method. Trichinella britovi has previously been identified in Serbia, but this is the first case of the species being confirmed in food samples linked to human trichinellosis. The results of the study confirmed that the T. britovi is able to affect human health. In addition, this study suggests the role of wild boars as reservoirs of T. britovi in Serbia.

Analyses of Compact Trichinella Kinomes Reveal a MOS-Like Protein Kinase with a Unique N-Terminal Domain.

Parasitic worms of the genus Trichinella (phylum Nematoda; class Enoplea) represent a complex of at least twelve taxa that infect a range of different host animals, including humans, around the world. They are foodborne, intracellular nematodes, and their life cycles differ substantially from those of other nematodes. The recent characterization of the genomes and transcriptomes of all twelve recognized taxa of Trichinella now allows, for the first time, detailed studies of their molecular biology. In the present study, we defined, curated, and compared the protein kinase complements (kinomes) of Trichinella spiralis and T. pseudospiralis using an integrated bioinformatic workflow employing transcriptomic and genomic data sets. We examined how variation in the kinome might link to unique aspects of Trichinella morphology, biology, and evolution. Furthermore, we utilized in silico structural modeling to discover and characterize a novel, MOS-like kinase with an unusual, previously undescribed N-terminal domain. Taken together, the present findings provide a basis for comparative investigations of nematode kinomes, and might facilitate the identification of Enoplea-specific intervention and diagnostic targets. Importantly, the in silico modeling approach assessed here provides an exciting prospect of being able to identify and classify currently unknown (orphan) kinases, as a foundation for their subsequent structural and functional investigation.

[Assessment of transmammary transmission of Trichinella patagoniensis in BALB/c mice]. / Evaluación de la transmisión galactógena de Trichinella patagoniensis en ratones BALB/c.

Since 1916 to date, it has been suspected that vertical transmission of parasites from the genus Trichinella could occur in pregnant or lactating women during the parenteral phase of infection. The aim of the present study was to evaluate the transmammary transmission of T. patagoniensis in BALB/c mice. Twenty 7-week-old BALB/c mice were distributed into two groups of 10 individuals each, depending on the time of gestation when they were infected, 15 or 18 days after detection of the vaginal plug. Each group was subdivided into two subgroups of 5 mice each, which were given an oral dose of 100 or 500 infective larvae respectively. Euthanasia and subsequent artificial digestion was performed in the pups and the dams. No T. patagoniensis L1 larvae were found in any of the offsprings analyzed. The observed results suggest that vertical transmission of T. patagoniensis would not be possible in BALB/c mice.

[ROLE OF ANIMALS AND HUMAN BEINGS IN THE SPREAD OF TRICHINOSIS IN THE KURSK REGION].

Trichinosis is natural focal invasion in the Kursk Region. Porkworms (Trichinella) circulate in natural biocenoses among wild carnivorous mammals, wild boars, and rodents. Trichinosis cases are recorded in synanthropic animals. Carnivorous mammals form the basis for the parasitic system of trichinosis. The animals are infected with Trichinella through carnivorism, necrophagy, and cannibalism. The transport Trichinella vectors, necrophagous insects, naturally play an insignificant role-in the spread of trichinosis. Trichinella infection in animals occurs more commonly through necrophagia and cannibaism during winter months. Not only animals, but also man, play a great role in the spread of trichinosis. Infested wild and synanthropic aninals inhabiting the Kursk Region may carry the risk for Trichinella infection in the population.

Clinical & biochemical profile of trichinellosis outbreak in north India.

BACKGROUND & OBJECTIVES: Trichinellosis is a parasitic infection caused by Trichinella nematodes, acquired from consumption of raw meat. However, data from Indian subcontinent are limited. The aim of this study was to investigate the clinical and biochemical profile of a suspected trichinellosis outbreak in a village in Tehri Garhwal district of Uttarakhand state in north India. METHODS: Three index cases presenting as acute febrile myalgia syndrome with eosinophilia, after consumption of uncooked pork in a common feast, were confirmed as trichinellosis on muscle biopsy. A detailed epidemiological survey was carried out in the affected community and all the people who participated in the feast were investigated for clinical and biochemical profile. RESULTS: A total of 54 patients were evaluated in the study. The type of pork consumed included uncooked in 24 per cent (n=13), open fire roasted in 39 per cent (n=21) and fried in 37 per cent (n=20). Clinical symptoms were found in those who consumed pork in uncooked or open fire roasted form (n=34). These included fever with chills and myalgia (100%), periorbital oedema (67%), dyspnoea (9%), and dysphagia (3%). Laboratory parameters studied in both symptomatic and asymptomatic patients showed eosinophilia in 90 per cent (n=41), raised ESR in 98 per cent (n=45), and an elevated creatinine phosphokinase (CPK) level in 85 per cent (n=39). All symptomatic patients were treated with a short course of oral steroids and albendazole therapy. CONCLUSIONS: Trichinella infection is not uncommon in India, and should be suspected in case of acute febrile myalgia especially in areas, where habits of consumption of raw meat is more prevalent.

[On the possibility of vertical transmission of Trichinella and the impact of its invasion].

Whether experimentally infected female rats can transmit Trichinella in utero and its invasion may affect the immunity of their offspring has been investigated. There is evidence that maternal antibodies have a significant impact on the humoral immunity of the offspring and as a result on the number of Trichinella larvae accustomed in the muscles. Immune interlayer formation in the populations of carnivorous mammals in the endemic areas occurs due to reinvasions and passive transmission of maternal antibodies to the offspring. High-tension immunity in the population and animals contributes to a reduction in the onset of invasion and to a predominance of the subclinical course of the disease and asymptomatic carriage in a specific focus. Passive immunity transmitted from the Trichinella-infected mother to its offspring is of importance in the development of epidemic and epizootic processes in the natural and synanthropic foci of trichinosis.

Neurotrichinellosis.

Trichinellosis is a parasitic zoonosis caused by the nematode Trichinella spp. Neurotrichinellosis represents one of the most important complications of severe trichinellosis in humans and is sometimes fatal, especially when Trichinella spiralis is involved. There are numerous mechanisms responsible for the involvement of the nervous system through direct or indirect involvement of the parasite. In the latter, inflammatory cells, especially eosinophils, appear to play a crucial role. Encephalopathy, neuromuscular disturbances, and ocular involvement represent the most frequent presentations of neurotrichinellosis, with the first being the most responsible for fatalities. The diagnosis is based on imaging (CT or MRI), which shows nodular multifocal hypodensities in serologically positive individuals with relevant epidemiological factors (e.g., consumption of raw pork). However, only direct diagnosis by muscle biopsy can give the absolute certainty of infection. Albendazole and mebendazole are the anthelminthic of choice and should be used with corticosteroids to prevent allergic manifestations.

Identifying risk factors for symptoms of severe trichinellosis--a case study of 143 infected persons in Brasov, Romania 2001-2008.

The presence of the parasite Trichinella spiralis in humans does not always manifest itself with obvious clinical symptoms; the clinical manifestations of trichinellosis are polymorphic and can cause diagnostic difficulties. Our aim was to identify risk factors that can be linked to the severity of the disease. We conducted a retrospective analysis of 143 cases of trichinellosis admitted to the Infectious Disease Hospital in Brasov, Romania during 2001-2008. We found that children 10 and younger were less prone to exhibit medium or severe symptoms. Patients with leukocytosis had a 1.75 times higher risk of developing medium to severe symptoms relative to those with normal white blood cell counts. Patients with high eosinophil counts had a 2.05 times higher risk of exhibiting average or serious symptoms relative to those with low or normal eosinophil counts. Repeated consumption of contaminated meat increased the chances of developing discernible forms of the disease by 5.25 fold when compared to those who only occasionally ate meat contaminated with Trichinella. Regular consumption of raw or undercooked meat increased the chances of developing medium or severe forms of this disease by 1.67 times when compared to those who consume meat that had been thoroughly cooked.

Trichinella infections in animals and humans in sub-Saharan Africa: a review.

The aim of this review is to provide information on Trichinella infection in humans, livestock and wildlife in sub-Saharan Africa mainly focusing on geographical distribution of species/genotypes, biology, host range, life cycles and to identify research gaps. Trichinella britovi, Trichinella nelsoni and Trichinella zimbabwensis and one genotype (Trichinella T8) are known to occur in sub-Saharan Africa. Distinct geographic ranges with overlapping of some taxa in some areas have been observed. Genetic variants of T. nelsoni has been reported to occur among parasites originating from Eastern and Southern Africa and sequence heterogeneity also occurs among T. zimbabwensis isolates originating from different regions of Zimbabwe and South Africa. Field observations so far indicate that sylvatic Trichinella infections in the region are common in carnivores (mammals and reptiles) and to a lesser extent in omnivores. Cannibalism, scavenging and predation appear to be the most important routes of transmission and maintenance of the sylvatic cycles of the Trichinella taxa. To date, human trichinellosis has been documented in only four sub-Saharan countries (8.7%, 4/46). Bushpigs and warthogs have been the source of human infection with T. britovi and T. nelsoni being the aetiological agents. An increase in bushmeat trade and the creation of Transfrontier Conservation Areas (TFCAs) may have increased the risk of human trichinellosis in the region. With the creation of TFCAs in the region, sampling of wildlife hosts from protected areas of most sub-Sahara African countries is required to fully map the distribution of Trichinella species/genotypes in this region. More structured field surveys are still needed to determine the sylvatic host distribution of the different Trichinella taxa. Biological data of the Trichinella taxa in both wild and domestic animals of sub-Saharan Africa is very limited and further research is required.

The different infectivity of Trichinella spiralis and Trichinella nativa in rat does not solely localize to enteral or parenteral phase.

Rats are selective hosts for Trichinella nativa; infection levels in their muscles remain low when compared to Trichinella spiralis. To identify in which phase of the life cycle the selective responses take place, rats were infected perorally (p.o.) and intravenously (i.v.) with T. spiralis and T. nativa. Six rats were placed in each group; three rats served as uninfected controls. The infection intensities were examined 5 or 6 weeks after the infection. T. spiralis muscle larva burden was 57 times higher compared to that of T. nativa after p.o. inoculation (mean larvae per gram ± SD, 1,243 ± 378.9 vs. 22 ± 21.5). In i.v. inoculation, 12 times more T. spiralis larvae reached the muscle than with T. nativa. Also, in vitro newborn larva (NBL) production at day 6 postinfection was analyzed for parasites originating from both rat and mouse. The mean number of in vitro-produced NBL of rat-origin T. spiralis during 24 h was 70 (95 % CI, 65-75) and of T. nativa, 23 (95 % CI, 21-24). Intriguingly, this difference was less obvious with mouse-origin female Trichinella. NBL production of mouse origin T. spiralis was 57 (95 % CI, 51-62) and T. nativa, 29 (95 % CI, 26-31). Intestine histology at day 40 post p.o. infection, serological response, and weight dynamics were indistinguishable between the Trichinella species but differed from controls. Our results show that the defense against T. nativa is not solely enteral in rats but may partly be explained by different reproduction processes for the two Trichinella species in rat.

Detection and zoonotic potential of Trichinella spp. from free-range pig farming in Greece.

Trichinellosis is a serious parasitic zoonosis, which is widely distributed around the world. Pork meat is still the predominant source of outbreaks of human trichinellosis in many countries. The aim of this study is to examine the impact of Trichinella spp. as an important risk factor on the free-range pig farming sector in Greece. In 2009, during routine testing for the detection of Trichinella larvae at slaughterhouses and the National Reference Laboratory for Parasites (NRL), a total of 826,426 pigs were tested with the magnetic stirrer method for Trichinella spp. at slaughterhouses, including 2,892 samples from free-range pigs. Two positive samples were detected: one positive for Trichinella britovi and one positive for Trichinella spp. (unspecified) in the samples from wild farmed free-range pigs. It is alarming that one of these cases was connected with clinical signs of trichinellosis in five persons of the same family in northeastern Greece, who consumed undercooked pork meat from a free-range pig farm. During 2010, a total number of 1,295,034 pigs were tested with same method, including 4,159 samples from free-range pig farms. Five positive samples for Trichinella spp. (unspecified) were detected from 4,159 free-range pigs tested by the Greek NRL. Moreover, 363 serum samples from free-range pigs were serologically tested with enzyme-linked immunosorbent assay (ELISA). Moreover, 363 serum samples from farmed free-range pigs were serologically tested with ELISA, and 15 samples were found positive. Finally, the present study is the first report of detection of T. britovi in Greece. In conclusion, based on the results of the present study, Trichinella spp. is a high-risk factor for the free-range pig farming in Greece.

The roots of evil: the amazing history of trichinellosis and Trichinella parasites.

This review aims to investigate, from a historical point of view, the roots of the Trichinella pathogens and the disease they produce, beginning from the times first recorded in the Bible and continuing until the scientific mysteries of the pathogen were finally unveiled. Our journey sheds light on "strange" epidemics developing before the discovery of the parasite and bearing a clinical similitude to trichinellosis; it follows the controversies that erupted after the nineteenth century discovery of the parasite and the subsequent official outbreaks, and then examines the pioneering methods used for its visualization. Finally, it tries to reveal the possible relationship of trichinellosis with the arts. Furthermore, it seems that the saga of the helminth, "destined to remain with us, both in nature and in the laboratory," will continue to haunt and fascinate scientists from both developing and developed countries, as they try to answer new questions about the parasite's "evil" nature.

Trichinella inflammatory myopathy: host or parasite strategy?

The parasitic nematode Trichinella has a special relation with muscle, because of its unique intracellular localization in the skeletal muscle cell, completely devoted in morphology and biochemistry to become the parasite protective niche, otherwise called the nurse cell. The long-lasting muscle infection of Trichinella exhibits a strong interplay with the host immune response, mainly characterized by a Th2 phenotype. The aim of this review is to illustrate the role of the Th2 host immune response at the muscle level during trichinellosis in different experimental models, such as knock-out or immuno-modulated mice. In particular, in knock-out mice a crucial role of IL-10 is evident for the regulation of inflammation intensity. The muscular host immune response to Trichinella is partially regulated by the intestinal phase of the parasite which emphasizes the intensity of the following muscle inflammation compared with animals infected by synchronized injections of newborn larvae. In eosinophil-ablated mice such as PHIL and GATA-- animals it was observed that there was an increased NOS2 expression in macrophages, driven by higher IFN-γ release, thus responsible for muscle larva damage. Besides modulation of the intestinal stage of the infection, using recombinant IL-12, increases the muscular parasite burden delaying adult worm expulsion from the intestine. Furthermore, a Th1 adjuvant of bacterial origin called Helicobacter pylori neutrophil activating protein (HP-NAP), administered during the intestinal phase of trichinellosis, alters the Th2 dependent response at muscle level. All these data from the literature delineate then a mutual adaptation between parasite and host immune response in order to achieve a strategic compromise between two evolutionary forces pointed towards the survival of both species.