In vitro culture of the zoonotic nematode Anisakis pegreffii (Nematoda, Anisakidae).

BACKGROUND: Anisakiasis is a foodborne disease caused by the third-stage larvae (L3) of two species belonging to the genus Anisakis: Anisakis pegreffii and Anisakis simplex sensu stricto. Both species have been the subject of different -omics studies undertaken in the past decade, but a reliable in vitro culture protocol that would enable a more versatile approach to functional studies has never been devised. In nature, A. pegreffii shows a polyxenous life-cycle. It reproduces in toothed whales (final host) and disseminates embryonated eggs via cetacean faeces in the water column. In the environment, a first- (L1) and second-stage larva (L2) develops inside the egg, and subsequently hatched L2 is ingested by a planktonic crustacean or small fish (intermediate host). In the crustacean pseudocoelom, the larva moults to the third stage (L3) and grows until the host is eaten by a fish or cephalopod (paratenic host). Infective L3 migrates into the visceral cavity of its paratenic host and remains in the state of paratenesis until a final host preys on the former. Once in the final host's gastric chambers, L3 attaches to mucosa, moults in the fourth stage (L4) and closes its life-cycle by becoming reproductively mature. METHODS: Testing two commercially available media (RPMI 1640, Schneider's Drosophila) in combination with each of the six different heat-inactivated sera, namely foetal bovine, rabbit, chicken, donkey, porcine and human serum, we have obtained the first reliable, fast and simple in vitro cultivation protocol for A. pegreffii. RESULTS: Schneider's Drosophila insect media supplemented with 10% chicken serum allowed high reproducibility and survival of adult A. pegreffii. The maturity was reached already at the beginning of the third week in culture. From collected eggs, hatched L2 were maintained in culture for 2 weeks. The protocol also enabled the description of undocumented morphological and ultrastructural features of the parasite developmental stages. CONCLUSIONS: Closing of the A. pegreffii life-cycle from L3 to reproducing adults is an important step from many research perspectives (e.g., vaccine and drug-target research, transgenesis, pathogenesis), but further effort is necessary to optimise the efficient moulting of L2 to infective L3.

Microsatellite profiling of hosts from parasite-extracted DNA illustrated with raccoons (Procyon lotor) and their Baylisascaris procyonis roundworms.

BACKGROUND: Important information on movement pathways and introduction routes of invasive parasites can be obtained by comparing the genetic makeup of an invader with its spatial genetic structure in other distribution areas. Sometimes, the population genetic structure of the host might be more informative than that of the parasite itself, and it is important to collect tissue samples of both host and parasite. However, host tissue samples are frequently not available for analysis. We aimed to test whether it is possible to generate reliable microsatellite profiles of host individuals by amplifying DNA extracted from a nematode parasite, using the raccoon (Procyon lotor) and the raccoon roundworm (Baylisascaris procyonis) as a test case. METHODS: Between 2020 and 2021, we collected tissue as well as a single roundworm each from 12 raccoons from central Germany. Both the raccoon and the roundworm DNA extracts were genotyped using 17 raccoon-specific microsatellite loci. For each roundworm DNA extract, we performed at least eight amplification reactions per microsatellite locus. RESULTS: We extracted amplifiable raccoon DNA from all 12 roundworms. We obtained at least two amplification products for 186 of the 204 possible genotypes. Altogether 1077 of the 1106 genotypes (97.4%) matched the host-DNA derived reference genotypes and thus did not contain genotyping errors. Nine of the 12 roundworm-derived genetic profiles matched the reference profiles from the raccoon hosts, with one additional genetic profile containing genotyping errors at a single locus. The remaining two genetic profiles were deemed unsuitable for downstream analysis because of genotyping errors and/or a high proportion of missing data. CONCLUSIONS: We showed that reliable microsatellite-based genetic profiles of host individuals can be obtained by amplifying DNA extracted from a parasitic nematode. Specifically, the approach can be applied to reconstruct invasion pathways of roundworms when samples of the raccoon hosts are lacking. Further research should assess whether this method can be replicated in smaller species of parasitic nematodes and other phyla of parasites more generally.

Morphological and molecular characterization of Hysterothylacium spp. parasitizing Pomatomus saltatrix and Pagrus pagrus of the State of São Paulo, Brazil.

Raphidascarid nematodes have been the focus of several studies, mainly due to the zoonotic potential of some species, even though the cases are underreported. Due to the difficulty in identifying their larvae, the use of diagnostic techniques involving morphological and molecular analyses has grown in the last 20 years. The present study had as objective the morphological and molecular characterization of the L3 larval types of Hysterothylacium collected in Pomatomus saltatrix and Pagrus pagrus from the Brazilian coast, close to the municipality of Santos, State of São Paulo. Twenty specimens of P. saltatrix were necropsied and Hysterothylacium type V (n = 257) and Hysterothylacium type X (n = 5) larvae were found. Five specimens of P. pagrus were necropsied and all were parasitized by Hysterothylacium type V larvae. The analyses showed a genetic proximity relationship between Hysterothylacium types V with other Hysterothylacium V and with H. deardorffoverstreetorum, although this is a species inquirenda. Haplotypes for Hysterothylacium type X found in the present study formed a monophyletic group with other Hysterothylacium X, H. amoyense, and H. zhoushanense. Through this study, new hosts and localities were registered for Hysterothylacium type V and Hysterothylacium type X.

First morphological and molecular report of Lagochilascaris minor (Nematoda, Ascarididae) in a domestic cat from Veracruz, Mexico.

The clinical presentation and the morphological and molecular findings of Lagochilascaris minor in a domestic cat from Veracruz, Mexico are described. In the physical examination of the cat, a fistula with purulent exudate was observed in the ventral middle and left lateral region of the neck, with the presence of nematodes inside. Thirty-two worms were recovered from the lesion, which were identified based on their morphological characteristics as Lagochilascaris spp. To determine the species of the nematode, DNA was extracted from the worms and a fragment of the cytochrome C oxidase subunit 1 was amplified. The product obtained was 100% similar with L. minor from Costa Rica. This is the first morphological and molecular report of L. minor parasitizing a domestic cat from Veracruz, Mexico, and emphasizes the circulation of this zoonotic parasite in the country.

Morphological and molecular identification of third-stage larvae of Anisakis typica (Nematoda: Anisakidae) from Red Sea coral trout, Plectropomus areolatus.

Anisakidosis is a foodborne zoonotic infection induced by members of the family Anisakidae via the consumption of raw or undercooked fish such as sushi and sashimi. Identifying anisakid larval species is critical for the epidemiology and diagnosis of diseases caused by them. This study aimed at identifying Anisakis larvae collected from marine fish in Egyptian waters based on morphological characteristics and molecular analysis. Thirty marine fish coral trout, Plectropomus areolatus, were collected from Hurghada, Red Sea, Egypt, to investigate larval nematodes of the genus Anisakis. The larvae were detected encapsulated in the peritoneal cavity and muscle of the fish host. This examination revealed that anisakid larvae naturally infected 19 fish specimens with a prevalence of 63.33% and a mean intensity of 4.1 ± 0.40. Most of them (68 larvae: 71.57%) were found in the musculature. Morphological and morphometric analyses using light and scanning electron microscopy revealed a head region with a prominent boring tooth, inconspicuous lips, and a characteristic protruded cylindrical mucron. All larvae in this study possessed the same morphology as Anisakis Larval type I. Molecular analysis based on ITS region using maximum likelihood and Bayesian phylogenetic methods confirmed them as Anisakis typica. This is the first study to identify A. typica larvae from the commercial fish coral trout P. areolatus in Egyptian waters using morphological and molecular methods.

Epidemiological study on factors influencing the occurrence of helminth eggs in horses in Germany based on sent-in diagnostic samples.

Gastrointestinal nematodes are ubiquitous parasites of grazing equines with Parascaris spp., and strongyles being the most relevant ones regarding the prevalence and potential disease severity. Despite their importance, epidemiological data regarding the presence and egg-shedding intensities of these parasites are scarce. Data from 1067 horse samples collected on German horse farms initially to compare diagnostic methods were used for epidemiological analyses. Due to its higher sensitivity, presence/absence data were based on a combined sedimentation/flotation technique while faecal egg counts were based on Mini-FLOTAC. For strongyles, 46.5% of the samples were positive and the median egg-shedding intensity was 40 (range 5-2590). In multivariate analyses, prevalence and egg-shedding intensity were significantly influenced by season, age group and sample type. The drug used for the last treatment and the number of foals on the yard only affected prevalence while the number of horses on the yard and sex were only significant for egg-shedding intensity. For Parascaris spp., a prevalence of 4.6% and a median egg-shedding intensity of 0 (range 5-905) were observed. In multivariate analyses, the age group, the time since the last anthelmintic treatment, presence and number of foals had significant effects on ascarid prevalence whereas egg-shedding intensity was significantly influenced by age group and season only. Parascaris occurred only on yards with foals, but with an increasing number of foals, Parascaris egg-shedding intensity decreased. Prevalence and egg-shedding intensity were influenced by different but partially overlapping variables for Parascaris and strongyles.

Cucullanus pinnai pinnai and C. pinnai pterodorasi (Nematoda Cucullanidae): what does the integrative taxonomy tell us about these species and subspecies classification?

Cucullanus pinnai has been divided in two subspecies (C. pinnai pinnai and C. pinnai pterodorasi) based on the morphology of oesophastome. While C. pinnai pinnai apparently shows low host specificity and broad geographic occurrence, with certain morphological variations, C. pinnai pterodorasi was reported once, parasitizing Pterodoras granulosus. We used an integrative taxonomic approach to evaluate whether or not populations of C. pinnai pinnai from Trychomycterus spegazzinii (Escoipe River, Argentina) and Pimelodus fur (Miranda River, Brazil), and of C. pinnai pterodorasi from Pterodoras granulosus (Miranda River, Brazil) are conspecific. Parasites were observed using light microscopy and genetically characterized based on partial sequences of the 18S and 28S rDNA, ITS1-5.8S-ITS2, and COI mtDNA. Phylogenies were reconstructed and the Generalized Mixed Yule Coalescent (GMYC), Poisson Tree Process (bPTP), and Automatic Barcode Gap Discovery (ABGD) were used for species delimitation purposes. The present samples formed well-supported monophyletic assemblages, corroborating in part the results of morphological analyses; however, they grouped according to geographic origin. Species delimitation suggested conspecificity of C. pinnai pinnai with C. pinnai pterodorasi from Brazil; consequently, the morphology of oesophastome may be an intraspecific variation. Results also indicated that C. pinnai may represent a species complex as samples from Argentina were suggestive of an independent specific entity. However, definitive affirmations are premature, since there is no autapomorphy for separating C. pinnai from Brazil and Argentina and sampling was limited to three host species from two river basins. The phylogenetic reconstructions also confirmed the artificiality of some genera within Cucullanidae.

Metazoan endoparasites of Hoplias malabaricus (Bloch, 1794) (Actinopterygii: Erythrinidae) from upper and middle São Francisco river basin, Minas Gerais State, Brazil.

The endoparasitic fauna of Hoplias malabaricus (which is a species of paramount importance in the fishing and human food sectors) from Três Marias reservoir, São Francisco river, and from eleven marginal lagoons in the upper and middle São Francisco river basin, Brazil, was herein recorded for the first time. In total, 13 endoparasite species belonging to ten different families were found in 147 analyzed H. malabaricus specimens. The identified taxa comprised individuals belonging to phyla Apicomplexa-Calyptosporidae-Caplyptospora sp. (oocysts); Platyhelminthes-Trematoda-Diplostomidae (metacercariae)-Austrodiplostomum sp. and Sphincterodiplostomum musculosum, Clinostomidae (metacercariae)-Clinostomum sp., Gorgoderidae (adults)-Phyllodistomum spatula, and Eucestoda-Proteocephalidae gen. sp. (plerocercoids larvae); and Nematoda-Anisakidae (larvae)-Contracaecum sp. Types 1 and 2 and Hysterothylacium sp., Gnathostomatidae (larvae)-Spiroxys sp., Camallanidae (juveniles/adults)-Procamallanus (Spirocamallanus) inopinatus, Guyanemidae (juveniles/adults)-Guyanema baudi, and Cystidicolidae (juveniles/adults)-Cystidicoloides fischeri. Proteocephalidae gen. sp. and Contracaecum sp. Type 1 were the species presenting expressive parasitic indexes in the reservoir, in the river, and in nine of the eleven lagoons. Cystidicoloides fischeri was recorded for the first time in H. malabaricus. Guyanema baudi and S. musculosum had their geographic distribution expanded to São Francisco river basin.

Parasitism of Arapaima gigas (Schinz, 1822) in fish farms of the state of Amazonas, Brazil.

The objective of this study was to investigate the parasites occurrences and to determine the rates of infestation/parasitic infection in juvenile Arapaima gigas from seven fish farms in the state of Amazonas, relating to the characteristics of these fish farms. Of the 70 A. gigas evaluated, 43 were infested/infected, with a total of 133 parasites collected. Three fish farms (2, 4, 6) showed the highest levels of prevalence of parasites (100%, 70%, 70%), mean intensity (4.1±2.6, 8.1±9.2, and 2.1±1.3), and mean abundance (4.1±2.6, 5.7±8.1, and 1.5±1.5), respectively. Prevalence ranged from 30% to 100%, mean intensity from 1.0 to 8.1 and mean abundance from 0.3 to 5.7. Monogenea, Digenea, Nematoda (Hysterothylacium sp. and Spirocamallanus sp.) and Acanthocephala (Neoechinorhynchus sp. and Polyacanthorhynchus sp.) were identified. The parasites Hysterothylacium sp. and Neoechinorhynchus sp. were the most prevalent parasites with 31.43% and 15.71%, respectively. The fish presented negative allometry in growth and constant condition factor. Measures to prevent and control the parasitic diseases diagnosed are discussed as well prophylactic practices that contribute to the biosecurity of the farms.

The microbial community associated with Parascaris spp. infecting juvenile horses.

BACKGROUND: Parasitic nematodes, including large roundworms colloquially known as ascarids, affect the health and well-being of livestock animals worldwide. The equine ascarids, Parascaris spp., are important parasites of juvenile horses and the first ascarids to develop widespread anthelmintic resistance. The microbiota has been shown to be an important factor in the fitness of many organisms, including parasitic nematodes, where endosymbiotic Wolbachia have been exploited for treatment of filariasis in humans. METHODS: This study used short-read 16S rRNA sequences and Illumina sequencing to characterize and compare microbiota of whole worm small intestinal stages and microbiota of male and female intestines and gonads. Diversity metrics including alpha and beta diversity, and the differential abundance analyses DESeq2, ANCOM-BC, corncob, and metagenomeSeq were used for comparisons. RESULTS: Alpha and beta diversity of whole worm microbiota did not differ significantly between groups, but Simpson alpha diversity was significantly different between female intestine (FI) and male gonad (MG) (P= 0.0018), and Shannon alpha diversity was significantly different between female and male gonads (P = 0.0130), FI and horse jejunum (HJ) (P = 0.0383), and FI and MG (P= 0.0001). Beta diversity (Fig. 2B) was significantly different between female and male gonads (P = 0.0006), male intestine (MI) and FG (P = 0.0093), and MG and FI (P = 0.0041). When comparing organs, Veillonella was differentially abundant for DESeq2 and ANCOM-BC (p < 0.0001), corncob (P = 0.0008), and metagenomeSeq (P = 0.0118), and Sarcina was differentially abundant across four methods (P < 0.0001). Finally, the microbiota of all individual Parascaris spp. specimens were compared to establish shared microbiota between groups. CONCLUSIONS: Overall, this study provided important information regarding the Parascaris spp. microbiota and provides a first step towards determining whether the microbiota may be a viable target for future parasite control options.

Surveillance for gastrointestinal, subcutaneous, and ectoparasites of invasive North American raccoons (Procyon lotor) in central Spain.

The American raccoon (Procyon lotor) is an invasive meso-carnivore which has been introduced and established in many European countries. Although the presence of the raccoon in the Iberian Peninsula was confirmed around 20 years ago, there are few data on pathogens of these animals in this region. For this work, 72 American raccoons from two subpopulations in the central region of the Iberian Peninsula were examined for selected parasites. Ectoparasite species richness (both fleas and ticks) increased during the sampling season and was highest in the Henares subpopulation and on males. Similarly, ectoparasite abundance increased during the sampling season and was highest in Henares and on adult raccoons. Four species of ticks were detected including Rhipicephalus pusillus (71%), followed by R. sanguineus sensu lato (24%), Ixodes ventalloi (3%), and Dermacentor marginatus (1.4%). Four species of fleas were detected including Pulex irritans (44%), Ctenocephalides felis (3%), C. canis (1.4%), and Paraceras melis (1.4%) infestations. A subset of raccoons (n = 56) was examined for intestinal parasites; low prevalence and diversity were found including Strongyloides procyonis (4%), Dilepis sp. (5%), Plagiorchis sp. (2%), and Moniliformis moniliformis (2%). Importantly, Baylisascaris procyonis was not found. Finally, no subcutaneous nematodes (i.e., Dracunculus and Dirofilaria spp.) were found in the 56 raccoons examined. The results of this work show that the invasive North American raccoons currently are infected with few endoparasites but are commonly infested with native ectoparasites, several of which can transmit pathogens relevant for public and veterinary health. However, the geographically distinct populations of raccoons in Spain have different introduction histories, thus additional surveillance for parasites is warranted.

Emerging Lagochilascaris minor infections in domestic cats from Costa Rica: A zoonotic threat for the region.

Two cases of lagochilascariasis minor in domestic cats from Costa Rica within a period of two months are recorded for the first time in Central America. Clinical findings included purulent discharge and a tumor in the left ear in one of the cats, whereas the other cat had an ulcerated cervical lesion. Both patients underwent surgical procedures during which nematode worms were collected and analyzed. The collected nematodes were identified using a combination of morphological and molecular assays, which revealed a 99.1% similarity in the cytochrome oxidase subunit 1 with L. minor from Mexico. The lack of information on this parasitosis, as well as the enormous harm it does to animal and human hosts, highlights the need for more research and awareness in Costa Rica and Central America. Furthermore, the unexpected occurrence of these instances in the same location emphasizes the imminent zoonotic risk to humans and the active circulation of the parasite.

Characterization of Pseudoterranova ceticola (Nematoda: Anisakidae) larvae from meso/bathypelagic fishes off Macaronesia (NW Africa waters).

The genus Pseudoterranova includes parasite species of cetaceans and pinnipeds. The third stage larva (L3) of seal-infecting species occur in second intermediate or paratenic fish hosts mainly in neritic waters. This study firstly describes a Pseudoterranova L3 from meso/bathypelagic fishes off Macaronesia. L3s were morphologically and genetically studied by light microscopy and sequencing of the mtDNA cox2 and entire ITS rDNA genes. Bayesian inferences were performed with sequences from the larvae and selected sequences from GenBank. The nematode L3s were molecularly identified as Pseudoterranova ceticola, a parasite of kogiid whales. Such larvae were collected from Bolinichthys indicus, Chauliodus danae, Eupharynx pelecanoides, Diaphus rafinesquii, D. mollis, Diretmus argenteus and Maulisia argipalla. They mainly occurred in the viscera of these fishes. Pseudoterranova ceticola L3 were small (< 12 mm) and whitish, and a prominent characteristic is a circumoral ridge extending from the ventral boring tooth which differentiate them from Pseudoterranova spp. L3 maturing in pinnipeds and Terranova sensu lato larvae that mature in poikilotherms. The shape of the tail: conical, long, pointed, ventrally curved and lacking mucron also distinguish these larvae from those of the pinniped-infecting Pseudoterranova spp. Phylogenetic analyses based on mtDNA cox2 and ITS rDNA sequences suggest that P. ceticola is closely related to Skrjabinisakis spp., and not with Pseudoterranova spp. parasitizing pinnipeds. The related species Skrjabinisakis paggiae, S. brevispiculata and S. physeteris (until recently belonging to genus Anisakis), are as P. ceticola also parasites of physeteroid cetaceans. The morphology and morphological variation of the larvae of the cetacean parasite P. ceticola is thoroughly described for the first time. These L3 can readily be morphologically distinguished from those of the pinniped-infecting Pseudoterranova spp. The parasite likely completes its life cycle in the mesopelagic and bathypelagic realm, with meso/bathypelagic fish as 2nd intermediate or paratenic hosts and kogiids as final host. Thus, Pseudoterranova from cetaceans appear to be morphologically, genetically, and ecologically differentiated to those from pinnipeds, suggesting that they are not congeneric.

Infection process, viability and establishment of Anisakis simplex s.l. L3 in farmed fish; A histopathological study in gilthead seabream.

Anisakis spp. (Nematoda, Anisakidae) are parasites known by their economic and health impacts, as their L3 larval stages infect a variety of fish species, many of them commercial species, sometimes causing zoonotic episodes due to consumption of raw or undercooked fish. The aim of this study is to determine the infection process and the potential impact of A. simplex s.l. L3 on gilthead seabream (Sparus aurata L.), one of the most important fish species in Mediterranean aquaculture, by periodic histological monitoring of the infection process. For this, fish were experimentally infected with A. simplex s.l. L3 and periodically analysed for L3 larvae, collecting samples at different time points (hours post ingestion, hpi): 3, 6, 12, 18, 24, 36, 48, 72, 96, 120, 144, 168 and 192, up to 6 months post infection (mpi). All samples were observed under a stereomicroscope and later fixed for histological examination. A. simplex s.l. L3 were only found on the visceral surface and mesenteric tissue, but never free or encapsulated in muscle. Chronological events were found to occur faster than those reported in previous studies. They were first observed 6 hpi in the coelomic cavity, being present up to 48 hpi. While the earliest evidence of fibrocytes surrounding A. simplex s.l. L3 larvae were observed at 18 hpi, complete spiral encapsulation occurred by 72 hpi. Alive parasites were observed up to 6 mpi. Although the infection of gilthead seabream by Anisakis spp. larvae is feasible, it seems unlikely, especially in aquaculture given the hygienically controlled feeding systems. In the event of infection, the transmission would be unlikely due to the poor condition in which specimens of Anisakis spp. are found. Furthermore, since no larvae were detected in the fish's muscle, human infection seems improbable.

A New Species of Sprentascaris (Nematoda: Raphidascarididae) in Hypostomus commersoni (Pisces: Loricariidae) from Argentina.

PURPOSE: To report a new species: Sprentascaris pampayensis, a Raphidascarididae parasite from the Neotropical region. METHODS: During a parasitological survey, specimens were collected of a new species of Sprentascaris from Hypostomus commersoni Valenciennes from urban lakes in Santa Fe City, Santa Fe Province, in the Pampean region of Argentina. Morphological and morphometric descriptions, and molecular analyses were performed. RESULTS: The new species, S. pampayensis n. sp., is characterized by having slightly extended lateral wings in both sexes, oral structure with three well-developed lips with double papillae, ventrolateral lips with an amphidial pore. Triangular buccal opening with three well-developed lips with flattened extensions at their bases, which end in a pointed manner towards the back. Males have 17 pairs of preanal papillae, 1 pair of adanal papillae, 3 pairs of postanal papillae and 1odd median postanal papilla located between the first and second pairs, unusual shape of caudal papillae with the central tubular part conspicuously elevated. Females have pre-equatorial vulva, smooth-shelled eggs, and mature embryonated eggs. DISCUSSION: Sprentascaris pampayensis n. sp. differs morphologically and genetically from S. manherti Petter and Cassone, 1984, S. lanfrediae Melo, Santos, Giese, Santos and Santos, 2011 and S. andersoni Malta, Paiva, Elisei, Tavares and Pereira, 2018. Morphological and morphometric comparison also distinguishes it from S. pimelodi Petter and Cassone, 1984, S. hypostomi Petter and Cassone, 1984, S. marano Ramallo, 2009, and S. saltaensis Ailán-Choke, Ramallo and Davies, 2017. Sprentascaris pampayensis n. sp. is a new parasitic nematode of Hypostomus commersoni.

A new species of nematode parasite, Cucullanus tunisiensis sp. nov. (Nematoda: Cucullanidae) from Epinephelus aeneus (Perciformes: Serranidae) off Tunisia.

Cucullanus tunisiensis sp. nov., (Nematoda: Cucullanidae), collected from the intestine of the white grouper Epinephelus aeneus from waters off the coast of Tunisia is described based on light and scanning electron microscopic observations. The new species is characterized by the presence of lateral alae, ventral sucker, long unequal spicules (left spicule 2474-2789 µm long, right spicule 2357-2518 µm long). This is the sixth nominal species of the genus Cucullanus Müller, 1777 and the first representative of this genus infecting fishes of Serranidae family reported from Tunisian waters.

Contracaecum nematode parasites in hillstream loaches of the Western Ghats, India.

Nematode parasites of the family Anisakidae infect definitive hosts, such as fish-eating birds and mammals, through primary intermediate hosts like copepods and secondary intermediate hosts like fishes. However, consumption of raw or undercooked fish can lead to nematode infection called anisakidosis in humans. We observed the presence of nematode infection in hillstream loaches of families Cobitidae and Nemacheilidae available for human consumption in the local markets in the northern parts of Western Ghats, India. Scanning electron micrograph and genetic identification employing mitochondrial cytochrome oxidase subunit II, identified the nematode to the genus Contracaecum. Histology of infected host revealed the presence of the parasite in muscles. Antioxidant enzyme analysis of host liver suggested that infection leads to oxidative stress in the fish. We suspect that a gradual increase in parasite infection of the loaches in the last decade could be attributed to various anthropogenic stressors that are altering riverine habitats. Since loaches are consumed by tribal people who often prepare the fish without degutting and possibly undercooked, there is a potential threat of human infection.

Gene rearrangements in the mitochondrial genome of ten ascaris species and phylogenetic implications for Ascaridoidea and Heterakoidea families.

The Ascaridoidea family and Heterakoidea family are the most common and typical representative of large parasites. Although our understanding of these parasites' diversity has expanded by analyses of some mitochondrial genes, there is limited information on these species' evolutionary rates. Here we determined ten complete mitogenome sequences of five subfamilies of Ascaridoidea and one subfamily of Heterakoidea. The phylogenetic tree divided the Ascaridoidea into six monophyletic major clades, and the divergence time of Heterakoidea family and Ascaridoidea family can be placed during the early Carboniferous Period (300-360 Mya). The reconstruction of the ancestral state showed that the gene orders of all species in Ascaridoidea were conserved, and the Heterakoidea had obvious genome rearrangement. The conserved blocks between them were divided into five and the main types are tandem-duplication/random loss (TDRL). These results will help to better understand the gene rearrangements and evolutionary position of ascaris species.

Characterization of a novel cysteine protease inhibitor in Baylisascaris schroederi migratory larvae and its role in regulating mice immune cell response.

Baylisascaris schroederi (B. schroederi) is a severe threat to the survival of giant pandas. Currently, the immune regulation mechanism of B. schroederi is poorly understood. Cysteine protease inhibitors (CPI) play important roles in the regulation of host immune responses against certain nematodes. In this study, a recombinant CPI of B. schroederi migratory larvae (rBsCPI-1) was cloned and expressed, and the effects of rBsCPI-1 on the physiological activities and antigen presentation of monocyte-derived macrophages (MDMs) were analyzed. We also analyzed the regulatory effects of rBsCPI-1 on the proliferation and differentiation of CD4+ T cells. And further identified the signaling pathways which play important roles in this process. The results showed that rBsCPI-1 activated the TLR2/4-small Rho GTPases-PAK1 pathway. On the one hand, it increased the phagocytosis and migration of MDMs. On the other hand, it activated downstream MAPK and NF-κB signaling pathways to induce apoptosis of MDMs. rBsCPI-1 also induced MDMs to polarize to the M2 subtype, thereby exerting an immunosuppressive effect. Meanwhile, rBsCPI-1 inhibited the antigen presentation process by decreasing the expression of MHC-II molecules, further inhibiting the proliferation of CD4+ T cells and inducing a Th1/Th2 mixed immune response. Treg cells with immunosuppressive effects were increased. The PD-L2/PD-1 and CD80/CTLA-4 signaling pathways between MDMs and CD4+ T cells were also activated by rBsCPI-1. In conclusion, this study preliminarily confirmed that rBsCPI-1 affects the physiological activities and polarization of MDMs through the TLR2/4 signaling pathway, and further interferes with antigen presentation response, inducing CD4+ T cells to play an immunosuppressive cellular response during the migratory process of B. schroederi. Thus, this study will provide a reference for elucidating the immune evasion mechanism of B. schroederi and developing new drugs and protective vaccines against B. schroederi.

A novel cysteine protease inhibitor in Baylisascaris schroederi migratory larvae regulates inflammasome activation through the TLR4-ROS-NLRP3 pathway.

BACKGROUND: Giant pandas (Ailuropoda melanoleuca) are the obligate host of the parasitic roundworm Baylisascaris schroederi. The infection of giant pandas with B. schroederi is very common. At present, little is known about the mechanism of immune interaction between B. schroederi and the host. As an important component of innate immunity, the NOD-like receptor 3 (NLRP3) inflammasome plays an important role in host immune response and the occurrence and development of infectious diseases. METHODS: We analyzed the regulation of NLRP3 inflammasome activation in monocyte-derived macrophages (MDMs) by the recombinant B. schroederi migratory larvae cysteine protease inhibitor rBsCPI-1, knowing from a previous study that the CPI-1 is highly expressed in B. schroederi migratory larvae. We first determined the effects of rBsCPI-1 and excretory-secretory products of B. schroederi migratory larvae on cell proliferation using the CCK-8 and LDH release assays. We then analyzed NLRP3 inflammasome activation, pyroptosis and pro-inflammatory cytokine release by quantitative-PCR, western blotting and enzyme-linked immunosorbent assay. The signaling pathway of rBsCPI-1 to activate NLRP3 inflammasomes was analyzed in activation and inhibition experiments. Finally, the effects of rBsCPI-1 on inflammasome activation in mice immunized with rBsCPI-1 were analyzed. RESULTS: The activation and inhibition experiments revealed that rBsCPI-1 induced inflammasome activation through the TLR4-ROS-NLRP3 signaling pathway, with reactive oxygen species (ROS) not only functioning as an activator of the NLRP3 inflammasome, but also an activation product of the NLRP3 inflammasome. rBsCPI-1 promoted the activation and assembly of the NLRP3 inflammasome, which further converted the pro-inflammatory cytokines interleukin (IL)-1ß and IL-18 into mature active forms. At the same time, caspase-1 cleaved gasdermin D to trigger cell pyroptosis. The results of animal immunization experiments further confirmed that rBsCPI-1 could induce the activation of the NLRP3 inflammasome. CONCLUSIONS: rBsCPI-1 activates the inflammasome through the TLR4-ROS-NLRP3 signaling pathway and further induces the pyroptosis of MDMs and release of pro-inflammatory factors IL-1ß and IL-18, thus promoting the occurrence and development of the inflammatory response in the host.