Mucosal Immunity of Major Gastrointestinal Nematode Infections in Small Ruminants Can Be Harnessed to Develop New Prevention Strategies.

Gastrointestinal parasitic nematode (GIN) infections are the cause of severe losses to farmers in countries where small ruminants such as sheep and goat are the mainstay of livestock holdings. There is a need to develop effective and easy-to-administer anti-parasite vaccines in areas where anthelmintic resistance is rapidly rising due to the inefficient use of drugs currently available. In this review, we describe the most prevalent and economically significant group of GIN infections that infect small ruminants and the immune responses that occur in the host during infection with an emphasis on mucosal immunity. Furthermore, we outline the different prevention strategies that exist with a focus on whole and purified native parasite antigens as vaccine candidates and their possible oral-nasal administration as a part of an integrated parasite control toolbox in areas where drug resistance is on the rise.

Next-generation diagnostic test for dengue virus detection using an ultrafast plasmonic colorimetric RT-PCR strategy.

The current global COVID-19 pandemic once again highlighted the urgent need for a simple, cost-effective, and sensitive diagnostic platform that can be rapidly developed for distribution and easy access in resource-limited areas. Here, we present a simple and low-cost plasmonic photothermal (PPT)-reverse transcription-colorimetric polymerase chain reaction (RTcPCR) for molecular diagnosis of dengue virus (DENV) infection. The assay can be completed within 54 min with an estimated detection limit of 1.6 copies/µL of viral nucleic acid. The analytical sensitivity and specificity of PPT-RTcPCR were comparable to that of the reference RT-qPCR assay. Moreover, the clinical performance of PPT-RTcPCR was evaluated and validated using 158 plasma samples collected from patients suspected of dengue infection. The results showed a diagnostic agreement of 97.5% compared to the reference RT-qPCR and demonstrated a clinical sensitivity and specificity of 97.0% and 100%, respectively. The simplicity and reliability of our PPT-RTcPCR strategy suggest it can provide a foundation for developing a field-deployable diagnostic assay for dengue and other infectious diseases.

Phylogeographic genetic variation of Indoplanorbis exustus (Deshayes, 1834) (Gastropoda: Planorbidae) in South and Southeast Asia.

The freshwater snail Indoplanorbis exustus play an important role as the sole intermediate host of several medically- and economically-important trematodes, especially zoonotic schistosomes and echinostomes, which can infect and cause diseases in livestock and people. This study aims to explore the mitochondrial cytochrome c oxidase subunit 1 sequence variation of I. exustus collected from new geographical areas; 459 specimens of I. exustus were collected from 43 localities in South and Southeast Asia. The 42 haplotypes (Ie1 - Ie42) we detected were classified into haplogroups I - V. Phylogenetic analyses revealed five major clades, A - E, in concordance with all previous studies. Clade E contained two subclades, E1 (haplogroup I) and E2 (haplogroup II). The most widespread genetic group was subclade E1. Clade A, clade B (haplogroup V), and clade C (haplogroup IV) were found only in South Asia, whereas clade D (haplogroup III) was specifically found in Southeast Asia. In Thailand, I. exustus showed high genetic divergence with 21 haplotypes. Several isolates showed significant genetic differences from others with unique haplotype(s). Hence, we confidently conclude our findings support all previous studies that I. exustus is a species complex with at least four major lineages and five haplogroups. Our additional analyses of 35 samples from Sri Lanka showed these were indeed an independent genetic group as previously found, but they can now be classified as a unique group forming subclade E2 (haplogroup II) of I. exustus sensu lato.

Divergence across mitochondrial genomes of sympatric members of the Schistosoma indicum group and clues into the evolution of Schistosoma spindale.

Schistosoma spindale and Schistosoma indicum are ruminant-infecting trematodes of the Schistosoma indicum group that are widespread across Southeast Asia. Though neglected, these parasites can cause major pathology and mortality to livestock leading to significant welfare and socio-economic issues, predominantly amongst poor subsistence farmers and their families. Here we used mitogenomic analysis to determine the relationships between these two sympatric species of schistosome and to characterise S. spindale diversity in order to identify possible cryptic speciation. The mitochondrial genomes of S. spindale and S. indicum were assembled and genetic analyses revealed high levels of diversity within the S. indicum group. Evidence of functional changes in mitochondrial genes indicated adaptation to environmental change associated with speciation events in S. spindale around 2.5 million years ago. We discuss our results in terms of their theoretical and applied implications.

Comparative mitogenomics of the zoonotic parasite Echinostoma revolutum resolves taxonomic relationships within the 'E. revolutum' species group and the Echinostomata (Platyhelminthes: Digenea).

The complete mitochondrial sequence of 17,030 bp was obtained from Echinostoma revolutum and characterized with those of previously reported members of the superfamily Echinostomatoidea, i.e. six echinostomatids, one echinochasmid, five fasciolids, one himasthlid, and two cyclocoelids. Relationship within suborders and between superfamilies, such as Echinostomata, Pronocephalata, Troglotremata, Opisthorchiata, and Xiphiditata, are also considered. It contained 12 protein-coding, two ribosomal RNA, 22 transfer RNA genes and a tandem repetitive consisting non-coding region (NCR). The gene order, one way-positive transcription, the absence of atp8 and the overlapped region by 40 bp between nad4L and nad4 genes were similar as in common trematodes. The NCR located between tRNAGlu (trnE) and cox3 contained 11 long (LRUs) and short repeat units (SRUs) (seven LRUs of 317 bp, four SRUs of 207 bp each), and an internal spacer sequence between LRU7 and SRU4 specifying high-level polymorphism. Special DHU-arm missing tRNAs for Serine were found for both tRNAS1(AGN) and tRNAS2(UCN). Echinostoma revolutum indicated the lowest divergence rate to E. miyagawai and the highest to Tracheophilus cymbius and Echinochasmus japonicus. The usage of ATG/GTG start and TAG/TAA stop codons, the AT composition bias, the negative AT-skewness, and the most for Phe/Leu/Val and the least for Arg/Asn/Asp codons were noted. Topology indicated the monophyletic position of E. revolutum to E. miyagawai. Monophyly of Echinostomatidae and Fasciolidae was clearly solved with respect to Echinochasmidae, Himasthlidae, and Cyclocoelidae which were rendered paraphyletic in the suborder Echinostomata.

The Venom of Spectacled Cobra (Elapidae: Naja naja): In Vitro Study from Distinct Geographical Origins in Sri Lanka.

Several countries residing envenomation due to Naja naja had revealed a disparity in the venom composition according to their geographic location and Sri Lankan cobra still lacks the evidence to support this. Therefore, the current study was focused on addressing relationship between the histopathological changes according to geographic variation of Sri Lankan N. naja venom. The histopathological changes in vital organs and muscle tissues following intramuscular administration of venom of N. naja were studied using BALB/c mice. The median lethal dose of venom of N. naja in the present study was determined to be 0.55, 0.66, 0.68, 0.62, and 0.7 mg/kg for North (NRP), Central (CRP), Western, Southern, and Sabaragamuwa Regional Population venoms, respectively. Histopathological changes were observed in different levels in vital organs and muscle tissues of mice. NRP accompanied significantly higher infiltration of inflammatory and necrotic cells into skeletal muscle and CRP venom demonstrated high level of cardiotoxic effects comparing to other regions. This study revealed a certain extent of variations in the pathological effects of N. naja venom samples according to their geographical distribution.

Toxocara pteropodis in Free-Ranging Indian Flying Foxes (Pteropus medius) in Sri Lanka.

Toxocara pteropodis, an intestinal nematode, occurs in several captive and free-ranging pteropid bat species. We report infection in free-ranging Indian flying foxes (Pteropus medius) in Sri Lanka and contribute to our understanding of parasites in free-ranging P. medius .

A Pan-Dengue Virus Reverse Transcription-Insulated Isothermal PCR Assay Intended for Point-of-Need Diagnosis of Dengue Virus Infection by Use of the POCKIT Nucleic Acid Analyzer.

Dengue virus (DENV) infection is considered a major public health problem in developing tropical countries where the virus is endemic and continues to cause major disease outbreaks every year. Here, we describe the development of a novel, inexpensive, and user-friendly diagnostic assay based on a reverse transcription-insulated isothermal PCR (RT-iiPCR) method for the detection of all four serotypes of DENV in clinical samples. The diagnostic performance of the newly established pan-DENV RT-iiPCR assay targeting a conserved 3' untranslated region of the viral genome was evaluated. The limit of detection with a 95% confidence was estimated to be 10 copies of in vitro-transcribed (IVT) RNA. Sensitivity analysis using RNA prepared from 10-fold serial dilutions of tissue culture fluid containing DENVs suggested that the RT-iiPCR assay was comparable to the multiplex real-time quantitative RT-PCR (qRT-PCR) assay for DENV-1, -3, and -4 detection but 10-fold less sensitive for DENV-2 detection. Subsequently, plasma collected from patients suspected of dengue virus infection (n = 220) and individuals not suspected of dengue virus infection (n = 45) were tested by the RT-iiPCR and compared to original test results using a DENV NS1 antigen rapid test and the qRT-PCR. The diagnostic agreement of the pan-DENV RT-iiPCR, NS1 antigen rapid test, and qRT-PCR tests was 93.9%, 84.5%, and 97.4%, respectively, compared to the composite reference results. This new RT-iiPCR assay along with the portable POCKIT nucleic acid analyzer could provide a highly reliable, sensitive, and specific point-of-need diagnostic assay for the diagnosis of DENV in clinics and hospitals in developing countries.

Isolation and Molecular Characterization of Entamoeba nuttalli Strains Showing Novel Isoenzyme Patterns from Wild Toque Macaques in Sri Lanka.

We have proposed the revival of the name Entamoeba nuttalli for a virulent ameba strain, P19-061405, from a rhesus macaque and located it phylogenetically between E. histolytica and E. dispar. As E. nuttalli was originally described for an ameba found in a toque macaque in Sri Lanka, the prevalence and characteristics of Entamoeba species in wild toque macaques were examined. PCR analysis of 227 stool samples from six locations showed positive rates for E. nuttalli, E. dispar, and E. histolytica of 18.5%, 0.4%, and 0%, respectively. Fifteen E. nuttalli strains were cultured successfully from five locations. The 18S ribosomal RNA gene showed only three nucleotide differences in comparison with P19-061405 strain. In isoenzyme analysis, the pattern of hexokinase in Sri Lankan strains was different from that of P19-061405 strains and the difference was confirmed by analysis of the genes. Hepatic inoculation of one of the Sri Lankan E. nuttalli strains in hamsters resulted in amebic abscess formation and body weight loss. These results demonstrate that E. nuttalli is prevalent in wild toque macaques and that several characteristics of the strains are unique. We conclude that use of the name E. nuttalli is appropriate for the new Entamoeba species found in nonhuman primates.

First molecular characterization of Cryptosporidium and Giardia from bovines (Bos taurus and Bubalus bubalis) in Sri Lanka: unexpected absence of C. parvum from pre-weaned calves.

BACKGROUND: The genetic characterization of Cryptosporidium and Giardia has important implications for investigating their epidemiology and underpins their control. We undertook the first molecular epidemiological survey of domestic bovids in selected regions of Sri Lanka to establish whether they excreted Cryptosporidium and/or Giardia with zoonotic potential. METHODS: Faecal samples were collected from dairy calves (n = 340; Bos taurus; < 3 months of age; weekly sampling for six weeks) and water buffaloes (n = 297; Bubalus bubalis; <6 months and ≥6 months of age; one sampling) from seven different farms in Sri Lanka. Genomic DNAs were extracted from individual faecal samples and then tested for the presence of parasite DNA using a PCR-based mutation scanning-targeted sequencing-phylogenetic approach, employing genetic markers within the small subunit of nuclear ribosomal RNA and 60 kDa glycoprotein genes (designated pSSU and pgp60, respectively) for Cryptosporidium, and within the triose phosphate isomerise (ptpi) gene for Giardia. RESULTS: Based on pSSU sequence data, C. bovis, C. ryanae and six new genotypes that were genetically similar but not identical to C. andersoni (n = 1), C. bovis (n = 1), C. ryanae (n = 3) and C. suis (n = 1) were recorded in cattle. For pSSU, two other, new genotypes were defined in water buffalo, which were genetically most similar to Cryptosporidium genotypes recorded previously in this host species in other countries including Australia. Consistent with the findings for pSSU, no species or genotypes of Cryptosporidium with zoonotic potential were detected using pgp60. Based on ptpi sequence data, G. duodenalis assemblages A and E were detected in four and 137 samples from cattle, respectively, and assemblage E in two samples from water buffaloes. CONCLUSIONS: The present study showed that C. parvum, the most commonly reported zoonotic species of Cryptosporidium recognised in bovine calves globally, was not detected in any of the samples from pre-weaned calves tested in the present study. However, eight new genotypes were recorded. Future studies of different host species in various regions are required to investigate the molecular epidemiology of cryptosporidiosis and giardiasis in Sri Lanka and neighbouring countries in South Asia.

The first intermediate host of Paragonimus westermani in Sri Lanka.

Freshwater snails (family Paludomidae, genus Paludomus) were collected from streams in Hedeniya and Peradeniya (the campus of Peradeniya University), Kandy district, Central Province, Sri Lanka, and found to harbor rediae and cercariae of a Paragonimus sp. These larvae were identified as Paragonimus westermani by using ITS2 DNA sequences. The infection rates of P. westermani in Paludomus sp. in Hedeniya and Peradeniya were 0.1% (one of 1014) and 0.2% (two of 1006), respectively. The snail has not been identified to species in the present study. This is the first report of the snail host of Paragonimus in Sri Lanka.

Evolutionary origins, diversification, and biogeography of liver flukes (Digenea, Fasciolidae).

Fasciolid flukes are among the largest and best known digenetic trematodes and have considerable historical and veterinary significance. Fasciola hepatica is commonly implicated in causing disease in humans. The origins, patterns of diversification, and biogeography of fasciolids are all poorly known. We have undertaken a molecular phylogenetic study using 28S, internal transcribed spacer 1 and 2 (ITS-1 and ITS-2) of nuclear ribosomal DNA, and mitochondrial nicotinamide dehydrogenase subunit 1 (nad1) that included seven of the nine recognized species in the family. The fasciolids examined comprise a monophyletic group with the most basal species recovered from African elephants. We hypothesize fasciolids migrated from Africa to Eurasia, with secondary colonization of Africa. Fasciolids have been conservative in maintaining relatively large adult body size, but anatomical features of their digestive and reproductive systems are available. These flukes have been opportunistic, with respect to switching to new snail (planorbid to lymnaeid) and mammalian hosts and from intestinal to hepatic habitats within mammals.

Mitochondrial DNA sequence and gene order of the Sri Lankan Schistosoma nasale is affiliated to the African/Indian group.

A 1.9 kb nucleotide sequence of part of the mitochondrial (mt) genome covering the cox1-trnT-rrnL-trnC-rrnS region, and the order of the remaining mitochondrial protein-coding genes for S. nasale of Sri Lankan origin, has been determined for analysis of the possible placement of this species in the genus Schistosoma. The gene order of this species is similar to that of the African and Indian Schistosoma species, but strikingly different from the East Asian species. Analysis of an alignment of the 1.9 kb sequence with available sequences from other schistosomes indicated affinities with S. spindale (found in Sri Lanka) and African species (in particular S. intercalatum and S. haematobium). Phylogenetic trees inferred from the alignment including 1 kb of RNA (transfer RNA and ribosomal RNA) sequence for 8 other Schistosoma spp. and Fasciola hepatica as an out-group revealed that S. nasale is placed proximally to S. spindale, S. intercalatum, S. haematobium and S. mansoni in the African sub-group while the East Asian species are more distant. S. incognitum lies basal to the combined African/Indian clade. The mtDNA analysis strongly supports the hypothesis that S. nasale is closely affiliated with the African/Indian schistosome group rather than the East Asian Schistosoma species.

An approach to revealing blood fluke life cycles, taxonomy, and diversity: provision of key reference data including DNA sequence from single life cycle stages.

Revealing diversity among extant blood flukes, and the patterns of relationships among them, has been hindered by the difficulty of determining if specimens described from different life cycle stages, hosts, geographic localities, and times represent the same or different species. Persistent collection of all available life cycle stages and provision of exact collection localities, host identification, reference DNA sequences for the parasite, and voucher specimens eventually will provide the framework needed to piece together individual life cycles and facilitate reconciliation with classical taxonomic descriptions, including those based on single life cycle stages. It also provides a means to document unique or rare species that might only ever be recovered from a single life cycle stage. With an emphasis on the value of new information from field collections of any available life cycle stages, here we provide data for several blood fluke cercariae from freshwater snails from Kenya, Uganda, and Australia. Similar data are provided for adult worms of Macrobilharzia macrobilharzia and miracidia of Bivitellobilharzia nairi. Some schistosome and sanguinicolid cercariae that we recovered have peculiar morphological features, and our phylogenetic analyses (18S and 28S rDNA and mtDNA CO1) suggest that 2 of the new schistosome specimens likely represent previously unknown lineages. Our results also provide new insights into 2 of the 4 remaining schistosome genera yet to be extensively characterized with respect to their position in molecular phylogenies, Macrobilharzia and Bivitellobilharzia. The accessibility of each life cycle stage is likely to vary dramatically from one parasite species to the next, and our examples validate the potential usefulness of information gleaned from even one such stage, whatever it might be.