Corynosoma strumosum (Acanthocephala) infection in marine foraging mink (Neogale vison) and river otter (Lontra canadensis) and associated peritonitis in a juvenile mink

Corynosoma strumosum (Acanthocephala), a widespread parasite of pinnipeds, is reported in marine foraging North American mink (Neogale vison) and river otter (Lontra canadensis) on Vancouver Island, British Columbia. This is the first confirmed case of infection by C. strumosum in river otters on the west coast of North America and may be the first confirmed case of infection in wild North American mink; C. strumosum has previously been reported in river otters in Europe (Lutra lutra) and in farmed mink fed with marine fish. We also detected a case of acanthocephalan associated peritonitis in a juvenile mink. Furthermore, though infections with Corynosoma spp. are often assumed to be accidental in mustelids, some C. strumosum individuals found in mink showed signs of reproductive activity. These findings indicate that mink may be a competent definitive host and represent a reservoir in coastal habitats although further research is needed to confirm this. Investigating whether river otters may be competent hosts and determine the prevalence of infection in coastal populations would determine the potential implications of C. strumosum for coastal otters and minks. Our report indicates that mink and possibly river otter living in coastal areas are vulnerable to this previously unreported parasitic infection with mortality risk, at least in juvenile individuals.     

Conservation genomics reveals possible illegal trade routes and admixture across pangolin lineages in Southeast Asia

The use of genome-wide genetic markers is an emerging approach for informing evidence-based management decisions for highly threatened species. Pangolins are the most heavily trafficked mammals across illegal wildlife trade globally, but critically endangered Sunda pangolins (Manis javanica) have not been widely studied in insular Southeast Asia. We used?>?12,000 single nucleotide polymorphic markers (SNPs) to assign pangolin seizures from illegal trade of unknown origin to possible geographic sources via genetic clustering with pangolins of known origin. Our SNPs reveal three previously unrecognized genetic lineages of Sunda pangolins, possibly from Borneo, Java and Singapore/Sumatra. The seizure assignments suggest the majority of pangolins were traded from Borneo to Java. Using mitochondrial markers did not provide the same resolution of pangolin lineages, and to explore if admixture might explain these differences, we applied sophisticated tests of introgression using?>?2000 SNPs to investigate secondary gene flow between each of the three Sunda pangolin lineages. It is possible the admixture which we discovered is due to human-mediated movements of pangolins. Our findings impact a range of conservation actions, including tracing patterns of trade, repatriation of rescue animals, and conservation breeding. In order to conserve genetic diversity, we suggest that, pending further research, each pangolin lineage should as a precaution be protected and managed as an evolutionarily distinct conservation unit.     

Molecular basis of cross‐species ACE2 interactions with SARS‐CoV‐2‐like viruses of pangolin origin

Pangolins have been suggested as potential reservoir of zoonotic viruses, including SARS‐CoV‐2 causing the global COVID‐19 outbreak. Here, we study the binding of two SARS‐CoV‐2‐like viruses isolated from pangolins, GX/P2V/2017 and GD/1/2019, to human angiotensin‐converting enzyme 2 (hACE2), the receptor of SARS‐CoV‐2. We find that the spike protein receptor‐binding domain (RBD) of pangolin CoVs binds to hACE2 as efficiently as the SARS‐CoV‐2 RBD in vitro. Furthermore, incorporation of pangolin CoV RBDs allows entry of pseudotyped VSV particles into hACE2‐expressing cells. A screen for binding of pangolin CoV RBDs to ACE2 orthologs from various species suggests a broader host range than that of SARS‐CoV‐2. Additionally, cryo‐EM structures of GX/P2V/2017 and GD/1/2019 RBDs in complex with hACE2 show their molecular binding in modes similar to SARS‐CoV‐2 RBD. Introducing the Q498H substitution found in pangolin CoVs into the SARS‐CoV‐2 RBD expands its binding capacity to ACE2 homologs of mouse, rat, and European hedgehog. These findings suggest that these two pangolin CoVs may infect humans, highlighting the necessity of further surveillance of pangolin CoVs.     

Molecular delineation of the Y-borne Sry gene in the Formosan pangolin (Manis pentadactyla pentadactyla) and its phylogenetic implications for Pholidota in extant mammals

The systematic status of Pholidota has been a matter of debate, particularly regarding the apparent inconsistency between morphological and molecular studies. The Sry gene, a master regulator of male sex determination in eutherian mammals, has not yet been used for phylogenetic analyses of extant mammals. The objective of the present study was to clone and characterize the complete gene (1300 base pairs; bp) and amino acid sequences (229 residues) of Sry from the Formosan pangolin (Manis pentadactyla pentadactyla), a member of Pholidota. The Sry amino acid identity between pangolin and other reported species ranged from 42.5% (mouse, Mus musculus) to 84.1% (European hare, Lepus europaeus). Sequence conservation was primarily in the high motility group (HMG) box (234 bp), whereas homology outside the HMG box was low. The cloned Sry was mapped to the pangolin Y chromosome by fluorescence in situ hybridization (FISH); this was confirmed to be the first Y-borne molecular marker identified in Pholidota. Based on Bayesian phylogenetic analysis for Sry HMG sequences from 36 representative taxa, including the Formosan pangolin, Pholidota was more closely related to Carnivora than to Xenarthra, consistent with the emerging molecular tree inferred from markers not located on the Y chromosome. In conclusion, this study characterized the gene structure of Sry of the Formosan pangolin and provided insights into the phylogenetic position of Pholidota.     

Phylogenetic analyses of endoparasitic Acanthocephala based on mitochondrial genomes suggest secondary loss of sensory organs

The metazoan taxon Syndermata (Monogononta, Bdelloidea, Seisonidea, Acanthocephala) comprises species with vastly different lifestyles. The focus of this study is on the phylogeny within the syndermatan subtaxon Acanthocephala (thorny-headed worms, obligate endoparasites). In order to investigate the controversially discussed phylogenetic relationships of acanthocephalan subtaxa we have sequenced the mitochondrial (mt) genomes of Echinorhynchus truttae (Palaeacanthocephala), Paratenuisentis ambiguus (Eoacanthocephala), Macracanthorhynchus hirudinaceus (Archiacanthocephala), and Philodina citrina (Bdelloidea). In doing so, we present the largest molecular phylogenetic dataset so far for this question comprising all major subgroups of Acanthocephala. Alongside with publicly available mt genome data of four additional syndermatans as well as 18 other lophotrochozoan (spiralian) taxa and one outgroup representative, the derived protein-coding sequences were used for Maximum Likelihood as well as Bayesian phylogenetic analyses. We achieved entirely congruent results, whereupon monophyletic Archiacanthocephala represent the sister taxon of a clade comprising Eoacanthocephala and monophyletic Palaeacanthocephala (Echinorhynchida). This topology suggests the secondary loss of lateral sensory organs (sensory pores) within Palaeacanthocephala and is further in line with the emergence of apical sensory organs in the stem lineage of Archiacanthocephala.     

The complete mitochondrial genome of Temminck's ground pangolin (Smutsia temminckii; Smuts, 1832) and phylogenetic position of the Pholidota (Weber, 1904)

Temmincki's ground pangolin is primarily a nocturnal mammal belonging to the order Pholidota. The body is covered in hard overlapping scales and these animals find refuge in burrows, feeding only on termites and ants. In this study, the whole mtDNA of Temmincki's ground pangolin was sequenced and the phylogenetic position of Pholidota determined within Eutheria, using whole mtDNA sequences from various representative species. The results indicate that the whole mtDNA of Temmincki's ground pangolin is 16,559 bp long and shared some similarities with the whole mtDNA of the back-bellied tree pangolin and the Chinese pangolin. Phylogenetic analysis indicate that the order Pholidota is closely related and share a recent common ancestor with the order Carnivora rather than with the ant/insect eating order Xenarthra and the group Afrotheria. A time measured phylogeny of Pholidota estimated a split from Carnivora at around 87 mya, followed by a split of the African pangolins from their Asian counterparts such as the Chinese pangolin at around 47 mya. This suggests a Laurasian origin and convergent evolution of the Pholidota with respect to Xenarthra and Afrotheria.     

海南尖峰岭中华穿山甲的分布与保护现状

中华穿山甲(Manis pentadactyla)是国家I级重点保护野生动物, 被IUCN红色名录列为极危(CR)物种, 也被列入CITES附录I。分布现状信息的匮乏是制约该物种保护规划制定与保护行动开展的关键因素。本文利用红外相机陷阱法和样线调查法, 于2020年8月至2021年11月在海南尖峰岭林区进行中华穿山甲海南亚种(M. p. pusilla)的监测调查, 分析评估了其分布与保护现状。调查发现: (1)尖峰岭林区7个公里网格内的8台红外相机拍摄到10次中华穿山甲出没的影像, 且在11个网格内见到35个觅食洞穴, 其中在红外相机损失率较低的南中区域记录到的中华穿山甲实体数量和洞穴数最多; (2)中华穿山甲主要分布于尖峰岭林区400-1,000 m海拔区域, 林区的国家公园一般控制区内仍然有3台红外相机拍摄到中华穿山甲及调查记录到18个觅食洞穴。结果表明: 海南尖峰岭林区仍然存在中华穿山甲种群, 目前可能被海拔等因素隔离为3个小种群; 人为干扰是影响该物种种群恢复的重要因素之一。因此, 本文提出如下建议: (1)管理部门要落实各项管护制度并加强巡护管理力度以把人为干扰程度降到最低; (2)管理部门在今后国家公园总体规划调整时可将有中华穿山甲等极危物种分布的一般控制区调整为核心保护区, 而在未调整规划前则要重点加强该区域的管理, 对建设项目布局等要尽量避开该区域; (3)尽快开展尖峰岭林区中华穿山甲的生境适宜性分析及廊道研究, 以使该物种得到更有效的保护和管理; (4)今后要定期开展其监测与保护研究, 明确尖峰岭林区中华穿山甲种群数量的动态变化; (5)在海南岛范围内开展中华穿山甲资源调查, 明确该物种在海南的种群数量及分布等情况。     

Parasite-host relations and new reports of helminths for Rhinella dorbignyi (Duméril & Bibron, 1841) (Anura: Bufonidae) from Neotropical region

This study aimed to analyze helminth assemblage in Rhinella dorbignyi regarding host gender, size and mass in two sampling sites in southern Brazil, and to report new parasite associations. Anurans (n = 100) were collected in two localities from Rio Grande do Sul (RS) state, Brazil, from 2017 to 2020. Nineteen taxa (adults and larval forms) belonging to Nematoda, Acanthocephala, Digenea, and Cestoda were found in different infection sites. Cosmocercidae gen. spp., Physaloptera liophis, Catadiscus sp., and Cylindrotaenia americana were the dominant taxa in helminth assemblage. Female anurans showed higher helminth species richness than males considering the total sample (two localities) or one of the two localities. However, prevalence and mean intensity of infection did not show any significant difference between genders. Mean intensity of infection was significantly higher (19.52) in Laranjal locality. Host body size does not influence helminth abundance, as infections did not show significant correlation snout-vent length (SVL) nor body mass (BM) of anurans. The findings reveal anurans of R. dorbignyi may be intermediate, paratenic and definitive host for these parasites. Plagiorchioidea helminths (Digenea), Physaloptera liophis, larvae of Acuariidae and Spiroxys sp. (Nematoda), and cystacanth of Lueheia sp. (Acanthocephala) constitute new records for R. dorbignyi. Additionally, this is the first record of Cylindrotaenia americana larvae in this host species. Resulting information increases the knowledge on biodiversity and parasite-host relations and may help future conservation programs developed in ecosystems in the extreme south of Brazil.     

Discovery of novel papillomaviruses in the critically endangered Malayan and Chinese pangolins

Pangolins are scaly and toothless mammals which are distributed across Africa and Asia. Currently, the Malayan, Chinese and Philippine pangolins are designated as critically endangered species. Although few pangolin viruses have been described, their viromes have received more attention following the discovery that they harbour sarbecoviruses related to SARS-CoV-2. Using large-scale genome mining, we discovered novel lineages of papillomaviruses infecting the Malayan and Chinese pangolins. We were able to assemble three complete circular papillomavirus genomes with an intact coding capacity and five additional L1 genes encoding the major capsid protein. Phylogenetic analysis revealed that seven out of eight L1 sequences formed a monophyletic group which is the sister lineage to the Tupaia belangeri papillomavirus 1, isolated from Yunnan province in China. Additionally, a single L1 sequence assembled from a Chinese pangolin was placed in a clade closer to Alphapapillomavirus and Omegapapillomavirus. Examination of the SRA data from 95 re-sequenced genomes revealed that 49.3% of Malayan pangolins and 50% of Chinese pangolins were positive for papillomavirus reads. Our results indicate that pangolins in South-East Asia are the hosts of diverse and highly prevalent papillomaviruses, and highlight the value of in silico mining of host sequencing data for the discovery of novel viruses.     

A case of congenital toxoplasmosis-associated miscarriage with maternal infection four months prior to conception

BackgroundWe report a case of fatal congenital toxoplasmosis with maternal infection dated four months before pregnancy in the absence of any specific immunosuppressive condition.CaseMs. D. experienced submaxillary lymphadenitis in February 2018. The medical workup performed revealed an acute T. gondii infection. She became pregnant in June 2018 while she still had adenopathy. The second obstetrical ultrasound, performed at 16 weeks of pregnancy, revealed a fetal death. The research for T. gondii by PCR was positive in the products of conception.ConclusionDiagnosis of toxoplasmosis should be discussed in case of miscarriage with lymphadenitis. As lymph nodes in T. gondii infection could be responsible for iterative release of parasites and fetal death, symptomatic toxoplasmosis should be treated in women of childbearing age.     

The first authenticated record of the pangolin tick <Emphasis Type="Italic">Amblyomma javanense</Emphasis> (Acari: Ixodidae) in Singapore,with notes on its biology and conservation

The first authenticated record of the co-endangered pangolin tick (Amblyomma javanense) is reported from the Republic of Singapore, based on specimens of males and females collected from the Sunda pangolin (Manis javanica). The biology of the species is reviewed and steps for its conservation are outlined to ensure its continued survival across its range.     

Immunohistochemical Demonstration of Keratins in the Epidermal Layers of the Malayan Pangolin (Manis Javanica), with Remarks on the Evolution of the Integumental Scale Armour

Using immunohistochemistry, the study demonstrates the distribution of keratins (pankeratin with CK1-8, 10, 14-16, 19; keratins CK1, 5, 6, 9, 10; hair keratins AE13, AE14) in the epidermis of the Malayan pangolin (Manis javanica). A varying reaction spectrum was observed for pan-keratin, with body region-dependent negative to very strong reaction intensities. The dorsolateral epidermis exhibited positive reactions only in its vital layers, whereas the abdominal epidermis showed strong positive reactions in the soft two outer strata. The single acidic and basic-to-neutral (cyto)keratins produced clear variations compared to the pan-keratin tinging. For example, CK1 appeared in all epidermal layers of both body regions, except for the ventral stratum corneum, whereas CK5, 6, 9, 10 were restricted to the soft ventral epidermis. Here, distinctly positive reactions were confined to the stratum granulosum, except for CK6 that appeared in the soft stratum corneum. A different staining pattern was obvious for the hair keratins, i.e., positive reactions of AE13 concentrated only in the granular layer of the dorsal epidermis. In the abdominal epidermis, remarkable tinging for AE14 was visible in the stratum basale, decreasing toward the corneal layer, but was also found in the outer root sheath cells of the hair follicles in the ventral body part. Our findings are discussed related to the evolution of the horny dorsal scales of the pangolin, which may have started from the tail root, projecting forward to the head.Key words: keratinisation, epidermis, scale evolution, Malayan pangolin, immunohistochemistry     

The biology of the thorny-headed worms, parasites of trout acclimatized in the bodies of water of the Tien Shan]

A new species of Acanthocephala, a parasite of local Naemacheilus stoliczkai, was recorded from Salmo gairdneri after 9 years of its introduction into mountain water bodies of Tien Shan. Gammarus lacustris serves as an intermediate host. Dynamics of infection of trout for 9 years is shown.     

A case of Moniliformis moniliformis (Acanthocephala) infection in Iran

Only a few cases of Acanthocephala infections have been reported in humans, and Moniliformis moniliformis is the most common species around the world. We report here a case of infection with M. moniliformis, which passed in the stool of a 2-year-old girl in Iran. The patient had abdominal pain, diarrhea, vomiting, and facial edema. According to her mother, the patient had habit of eating dirt and once a cockroach was discovered in her mouth. In stool examination, eggs of M. moniliformis were not found. She was treated with levamisole and the clinical symptoms reduced within 2 weeks. The specimen contained 2 pieces of a female worm with a total length of 148 mm lacking the posterior end. The spiral musculature of the proboscis receptacle and the shape of the trunk allowed its generic determination. Previously 2 cases of M. moniliformis infection were reported in Iran. This is the 3rd case of M. moniliformis infection in Iran.     

Fatal infection with Taenia martis metacestodes in a ring-tailed lemur (Lemur catta) living in an Italian zoological garden

A case of fatal infection caused by larval forms of Taenia martis in a ring-tailed lemur (Lemur catta) living in the Rome zoological garden is described. The animal, living in a semi-natural pen with other 15 conspecific individuals and being fed with fresh fruit and vegetables, yoghurt and eggs, was transported to the Istituto Zooprofilattico of Rome for post-mortem examination. The anamnesis included, ten days before the death, apathy, lack of appetite, abdominal distension and diarrhoea. A severe exudative fibrinous-purulent peritonitis with numerous adhesions between the abdominal wall and the bowel loops was detected. After intestine removal, two free and viable, 4 cm long, whitish, leaf-like parasitic forms were pinpointed. Macroscopic examination of the two parasites allowed their identification as larval stages of cestodes, identified via molecular analysis as T. martis metacestodes. This report represents the first record of T. martis infection in the host species and in a zoological garden and for the pathological relevance of the infection.     

Mutations derived from horseshoe bat ACE2 orthologs enhance ACE2-Fc neutralization of SARS-CoV-2

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002–2003. Horseshoe bats (genus Rhinolophus) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related to SARS-CoV-2, has been identified in one horseshoe-bat species. Here we characterize the ability of the S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, pangolin coronavirus (PgCoV), RaTG13, and LyRa11, a bat virus similar to SARS-CoV-1, to bind a range of ACE2 orthologs. We observed that the PgCoV RBD bound human ACE2 at least as efficiently as the SARS-CoV-2 RBD, and that both RBDs bound pangolin ACE2 efficiently. We also observed a high level of variability in binding to closely related horseshoe-bat ACE2 orthologs consistent with the heterogeneity of their RBD-binding regions. However five consensus horseshoe-bat ACE2 residues enhanced ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 pseudoviruses by an enzymatically inactive immunoadhesin form of human ACE2 (hACE2-NN-Fc). Two of these mutations impaired neutralization of SARS-CoV-1 pseudoviruses. An hACE2-NN-Fc variant bearing all five mutations neutralized both SARS-CoV-2 pseudovirus and infectious virus more efficiently than wild-type hACE2-NN-Fc. These data suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of soluble ACE2.     

Parasite communities of the spotted rose snapper Lutjanus guttatus (Perciformes: Lutjanidae) off the Mexican Pacific coasts: Spatial and long-term inter-annual variations

A total of 802 individuals of Lutjanus guttatus (Steindachner, 1869) specimens were collected over a 10-year period (August 2012 to February 2021) from four locations on the south-central Pacific coast of Mexico. Their parasite communities were quantified and analyzed to determine if they experience significant spatial and inter-annual variations.Thirty-two taxa of metazoan parasite were recovered and identified: four species of Digenea, four Monogenea, one Cestoda, two Acanthocephala, seven Nematoda, one Hirudinea, and nine of Crustacea (six Copepoda, and three Isopoda). Species richness was greatest among the digenean group, which represented 25% of the total species recovered, followed by the nematodes (22% of total species). Species richness at the component community level (10 to 20 species) was similar to reported richness in other Lutjanus spp. The component communities and infracommunities exhibited a similar pattern: low species richness and diversity, and dominance by a single species, mainly the monogenean Haliotrematoides guttati. Parasite community structure and species composition varied through the years, as well as between sampling locations. Variations were possibly caused by a combination of biotic and abiotic factors which generated notable changes in the infection levels of several component species. However, the similarity in the parasite species composition was high locally for short-term periods (one or two years). This result, therefore, suggests that parasite communities of L. guttatus may be more predictable locally, but only for short-term periods.     

Molecular evidence for Acanthocephala as a subtaxon of Rotifera

Rotifers are free-living animals usually smaller than 1 mm that possess a characteristic wheel organ. Acanthocephalans (thorny-headed worms) are larger endoparasitic animals that use vertebrates and arthropods to complete their life cycle. The taxa Acanthocephala and Rotifera are considered separate phyla, often within the taxon Aschelminthes. We have reexamined the relationship between Rotifera and Acanthocephala using 18S rRNA gene sequences. Our results conclusively show that Acanthocephala is the sister group of the rotifer class Bdelloidea. Rotifera was nonmonophyletic in all molecular analyses, which supports the hypothesis that the Acanthocephala represent a taxon within the phylum Rotifera and not a separate phylum. These results agree with a previous cladistic study of morphological characters. Correspondence to: J.R. Garey     

Inducible suicide vector systems for Trypanosoma cruzi

Chagas disease caused by Trypanosoma cruzi is a major neglected tropical parasitic disease. The pathogenesis of this infection remains disputable. There is no suitable vaccine for the prevention. Attenuated live vaccines can provide strong protection against infection; however, there are the concerns about latent infection or reversion to virulence in such attenuated strains. A method to induce T. cruzi death would provide a critical tool for research into the pathophysiological mechanisms and provide a novel design of safe live attenuated vaccines. We established effective inducible systems for T. cruzi employing the degradation domain based on the Escherichia coli dihydrofolate reductase (ecDHFR). The DHFR degradation domain (DDD) can be stabilized by trimethoprim-lactate and can be used to express detrimental or toxic proteins. T. cruzi lines with Alpha-toxin, Cecropin A and GFP under the control of DDD with a hemagglutinin tag (HA) were developed. Interestingly, amastigotes bearing GFP-DDDHA, Alpha-toxin-DDDHA, Cecropin A-DDDHA and DDDHA all resulted in inducible cell death with these fusions, indicating that DDDHA protein is also detrimental to amastigotes. Furthermore, these strains were attenuated in mouse experiments producing no pathological changes and inoculation with these DDDHA strains in mice provided strong protection against lethal wild type infection.