ABSTRACT
BACKGROUND: Tsetse flies (Glossina sp.) are vectors of Trypanosoma brucei subspecies that cause human African trypanosomiasis (HAT). Capturing and screening tsetse is critical for HAT surveillance. Classically, tsetse have been microscopically analysed to identify trypanosomes, but this is increasingly replaced with molecular xenomonitoring. Nonetheless, sensitive T. brucei-detection assays, such as TBR-PCR, are vulnerable to DNA cross-contamination. This may occur at capture, when often multiple live tsetse are retained temporarily in the cage of a trap. This study set out to determine whether infected tsetse can contaminate naïve tsetse with T. brucei DNA via faeces when co-housed. METHODOLOGY/PRINCIPLE FINDINGS: Insectary-reared teneral G. morsitans morsitans were fed an infectious T. b. brucei-spiked bloodmeal. At 19 days post-infection, infected and naïve tsetse were caged together in the following ratios: (T1) 9:3, (T2) 6:6 (T3) 1:11 and a control (C0) 0:12 in triplicate. Following 24-hour incubation, DNA was extracted from each fly and screened for parasite DNA presence using PCR and qPCR. All insectary-reared infected flies were positive for T. brucei DNA using TBR-qPCR. However, naïve tsetse also tested positive. Even at a ratio of 1 infected to 11 naïve flies, 91% of naïve tsetse gave positive TBR-qPCR results. Furthermore, the quantity of T. brucei DNA detected in naïve tsetse was significantly correlated with cage infection ratio. With evidence of cross-contamination, field-caught tsetse from Tanzania were then assessed using the same screening protocol. End-point TBR-PCR predicted a sample population prevalence of 24.8%. Using qPCR and Cq cut-offs optimised on insectary-reared flies, we estimated that prevalence was 0.5% (95% confidence interval [0.36, 0.73]). CONCLUSIONS/SIGNIFICANCE: Our results show that infected tsetse can contaminate naïve flies with T. brucei DNA when co-caged, and that the level of contamination can be extensive. Whilst simple PCR may overestimate infection prevalence, quantitative PCR offers a means of eliminating false positives.
Subject(s)
Trypanosoma brucei brucei , Trypanosomiasis, African , Tsetse Flies , Animals , Tsetse Flies/parasitology , Trypanosoma brucei brucei/isolation & purification , Trypanosoma brucei brucei/genetics , Trypanosomiasis, African/transmission , Trypanosomiasis, African/epidemiology , Trypanosomiasis, African/parasitology , Trypanosomiasis, African/diagnosis , DNA, Protozoan/genetics , DNA, Protozoan/analysis , Insect Vectors/parasitology , Feces/parasitology , Female , Male , Polymerase Chain Reaction/methodsABSTRACT
The zoonotic parasite Cryptosporidium parvum is a global cause of gastrointestinal disease in humans and ruminants. Sequence analysis of the highly polymorphic gp60 gene enabled the classification of C. parvum isolates into multiple groups (e.g., IIa, IIc, Id) and a large number of subtypes. In Europe, subtype IIaA15G2R1 is largely predominant and has been associated with many water- and food-borne outbreaks. In this study, we generated new whole-genome sequence (WGS) data from 123 human- and ruminant-derived isolates collected in 13 European countries and included other available WGS data from Europe, Egypt, China, and the United States (n = 72) in the largest comparative genomics study to date. We applied rigorous filters to exclude mixed infections and analyzed a data set from 141 isolates from the zoonotic groups IIa (n = 119) and IId (n = 22). Based on 28,047 high-quality, biallelic genomic SNPs, we identified three distinct and strongly supported populations: Isolates from China (IId) and Egypt (IIa and IId) formed population 1; a minority of European isolates (IIa and IId) formed population 2; and the majority of European (IIa, including all IIaA15G2R1 isolates) and all isolates from the United States (IIa) clustered in population 3. Based on analyses of the population structure, population genetics, and recombination, we show that population 3 has recently emerged and expanded throughout Europe to then, possibly from the United Kingdom, reach the United States, where it also expanded. The reason(s) for the successful spread of population 3 remain elusive, although genes under selective pressure uniquely in this population were identified.
Subject(s)
Cryptosporidiosis , Cryptosporidium parvum , Disease Outbreaks , Cryptosporidium parvum/genetics , United States/epidemiology , Europe/epidemiology , Humans , Cryptosporidiosis/parasitology , Cryptosporidiosis/epidemiology , Animals , Genomics/methods , Polymorphism, Single Nucleotide , Phylogeny , Whole Genome Sequencing/methods , Genome, Protozoan , China/epidemiology , Egypt/epidemiologyABSTRACT
BACKGROUND: The giant roundworm Ascaris is an intestinal nematode, causing ascariasis by infecting humans and pigs worldwide. Recent estimates suggest that Ascaris infects over half a billion people, with chronic infections leading to reduced growth and cognitive ability. Ascariasis affects innumerable pigs worldwide and is known to reduce production yields via decreased growth and condemnation of livers. The predominant anthelminthic drugs used to treat ascariasis are the benzimidazoles. Benzimidazoles interact with ß-tubulins and block their function, and several benzimidazole resistance-associated mutations have been described in the ß-tubulins of ruminant nematodes. Recent research on ascarids has shown that these canonical benzimidazole resistance-associated mutations are likely not present in the ß-tubulins of Ascaris, Ascaridia or Parascaris, even in phenotypically resistant populations. METHODS: To further determine the putative absence of key ß-tubulin polymorphisms, we screened two ß-tubulin isotypes of Ascaris, highly expressed in adult worms. Using adult and egg samples of Ascaris obtained from pigs and humans worldwide, we performed deep amplicon sequencing to look for canonical resistance-associated mutations in Ascaris ß-tubulins. Subsequently, we examined these data in closer detail to study the population dynamics of Ascaris and genetic diversity within the two isotypes and tested whether genotypes appeared to partition across human and pig hosts. RESULTS: In the 187 isolates, 69 genotypes were found, made up of eight haplotypes of ß-tubulin isotype A and 20 haplotypes of isotype B. Single nucleotide polymorphisms were seen at 14 and 37 positions for ß-tubulin isotype A and isotype B, respectively. No evidence of any canonical benzimidazole resistance-associated mutations was found in either human- or pig-derived Ascaris isolates. There was, however, a difference in the genetic diversity of each isotype and distribution of ß-tubulin genotypes between human- and pig-derived Ascaris. Statistical tests of population differentiation show significant differences (p < 0.001) between pig- and human-derived worms; however, more diversity was seen between worms from different populations than worms from different hosts. CONCLUSIONS: Our work suggests an absence of canonical ß-tubulin mutations within Ascaris, but alternative modes of anthelminthic resistance may emerge necessitating continued genetic scrutiny alongside monitoring of drug efficacy.
Subject(s)
Anthelmintics , Ascariasis , Ascaris , Benzimidazoles , Drug Resistance , Mutation , Tubulin , Tubulin/genetics , Animals , Benzimidazoles/pharmacology , Drug Resistance/genetics , Ascariasis/parasitology , Ascariasis/veterinary , Ascariasis/drug therapy , Anthelmintics/pharmacology , Swine , Ascaris/genetics , Ascaris/drug effects , Humans , Swine Diseases/parasitology , Swine Diseases/drug therapyABSTRACT
Dicrocoelium lancet flukes cause significant production loss in ruminant livestock. Although co-infection with multiple Dicrocoelium species within a host is common, techniques for studying the composition of these complex parasite communities are lacking. The pathogenicity, epidemiology, and therapeutic susceptibility of different helminth species vary, and little is known about the interactions that take place between co-infecting species and their hosts. Here, we describe the first applicationof metabarcoding deep amplicon sequencing method to studythe Dicrocoelium species in sheep and goats. First, rDNA ITS-2 sequences of four Dicrocoelium species (Dicrocoelium dendriticum, Dicrocoelium hospes, Dicrocoelium orientalis, and Dicrocoelium chinensis) were extracted from the NCBI public database. Phylogenetic analysis revealed separate clades of Dicrocoelium species; hence, molecular differentiation between each species is possible in co-infections. Second, 202 flukes belonging to seventeen host populations (morphologically verified as belonging to the Dicrocoelium genus) were evaluated to determine the deep amplicon sequencing read threshold of an individual fluke for each of the four species. The accuracy of the method in proportional quantification of samples collected from single hosts was further assessed. Overall, 198 (98.01%) flukes were confirmed as D. dendriticum and 1.98% produced no reads. The comparison of genetic distances between rDNA ITS-2 revealed 86% to 98% identity between the Dicrocoelium species. Phylogenetic analysis demonstrated a distinct clustering of species, apart from D. orientalis and D. chinensis, which sit very close to each other in a single large clade whereas D. hospes and D. dendriticum are separated into their own clade. In conclusion each sample was identified as D. dendriticum based on the proportion of MiSeq reads and validated the presence of this group of parasites in the Gilgit Baltistan and Khyber Pakhtunkhwa provinces of Pakistan. The metabarcoding deep amplicon sequencing technology and bioinformatics pathway have several potential applications, including species interactions during co-infections, identifying the host and geographical distribution of Dicrocoelium in livestock, drug therapy response evaluation and understanding of the emergence and spread of drug resistance.
Subject(s)
Dicrocoeliasis , Dicrocoelium , Goat Diseases , Goats , High-Throughput Nucleotide Sequencing , Phylogeny , Sheep Diseases , Animals , Dicrocoelium/genetics , Dicrocoelium/isolation & purification , Sheep/parasitology , Goats/parasitology , Dicrocoeliasis/parasitology , Dicrocoeliasis/veterinary , Dicrocoeliasis/epidemiology , Pakistan/epidemiology , Sheep Diseases/parasitology , Sheep Diseases/epidemiology , Goat Diseases/parasitology , Goat Diseases/epidemiology , DNA, Helminth/genetics , DNA Barcoding, Taxonomic/methods , Ruminants/parasitology , Coinfection/parasitology , Coinfection/epidemiologyABSTRACT
Toxocara canis and Toxocara cati are globally distributed, zoonotic roundworm parasites. Human infection can have serious clinical consequences including blindness and brain disorders. In addition to ingesting environmental eggs, humans can become infected by eating infective larvae in raw or undercooked meat products. To date, no studies have assessed the prevalence of Toxocara spp. larvae in meat from animals consumed as food in the UK or assessed tissue exudates for the presence of anti-Toxocara antibodies. This study aimed to assess the potential risk to consumers eating meat products from animals infected with Toxocara spp. Tissue samples were obtained from 155 different food producing animals in the south, southwest and east of England, UK. Tissue samples (n = 226), either muscle or liver, were processed by artificial digestion followed by microscopic sediment evaluation for Toxocara spp. larvae, and tissue exudate samples (n = 141) were tested for the presence of anti-Toxocara antibodies using a commercial ELISA kit. A logistic regression model was used to compare anti-Toxocara antibody prevalence by host species, tissue type and source. While no larvae were found by microscopic examination after tissue digestion, the overall prevalence of anti-Toxocara antibodies in tissue exudates was 27.7%. By species, 35.3% of cattle (n = 34), 15.0% of sheep (n = 60), 54.6% of goats (n = 11) and 61.1% of pigs (n = 18) had anti-Toxocara antibodies. Logistic regression analysis found pigs were more likely to be positive for anti-Toxocara antibodies (odds ration (OR) = 2.89, P = 0.0786) compared with the other species sampled but only at a 10% significance level. The high prevalence of anti-Toxocara antibodies in tissue exudates suggests that exposure of food animals to this parasite is common in England. Tissue exudate serology on meat products within the human food chain could be applied in support of food safety and to identify practices that increase risks of foodborne transmission of zoonotic toxocariasis.
Subject(s)
Antibodies, Helminth , Toxocara , Toxocariasis , Animals , Toxocariasis/epidemiology , Toxocariasis/parasitology , Toxocara/immunology , Toxocara/isolation & purification , Antibodies, Helminth/blood , Antibodies, Helminth/analysis , Sheep , Swine , Cattle , Enzyme-Linked Immunosorbent Assay/veterinary , England/epidemiology , Meat/parasitology , Liver/parasitology , Goats , Exudates and Transudates/parasitology , Swine Diseases/parasitology , Humans , Muscles/parasitology , Sheep Diseases/parasitology , Sheep Diseases/epidemiology , Food ParasitologyABSTRACT
Animal African trypanosomiasis (AAT) is a significant food security and economic burden in sub-Saharan Africa. Current AAT empirical and immunodiagnostic surveillance tools suffer from poor sensitivity and specificity, with blood sampling requiring animal restraint and trained personnel. Faecal sampling could increase sampling accessibility, scale, and species range. Therefore, this study assessed feasibility of detecting Trypanosoma DNA in the faeces of experimentally-infected cattle. Holstein-Friesian calves were inoculated with Trypanosoma brucei brucei AnTat 1.1 (n = 5) or T. congolense Savannah IL3000 (n = 6) in separate studies. Faecal and blood samples were collected concurrently over 10 weeks and screened using species-specific PCR and qPCR assays. T. brucei DNA was detected in 85% of post-inoculation (PI) faecal samples (n = 114/134) by qPCR and 50% by PCR between 4 and 66 days PI. However, T. congolense DNA was detected in just 3.4% (n = 5/145) of PI faecal samples by qPCR, and none by PCR. These results confirm the ability to consistently detect T. brucei DNA, but not T. congolense DNA, in infected cattle faeces. This disparity may derive from the differences in Trypanosoma species tissue distribution and/or extravasation. Therefore, whilst faeces are a promising substrate to screen for T. brucei infection, blood sampling is required to detect T. congolense in cattle.
Subject(s)
Trypanosoma brucei brucei , Trypanosoma congolense , Trypanosoma , Trypanosomiasis, African , Humans , Cattle , Animals , Trypanosoma brucei brucei/genetics , Trypanosoma congolense/genetics , Trypanosomiasis, African/diagnosis , Trypanosomiasis, African/veterinary , Trypanosomiasis, African/epidemiology , Trypanosoma/genetics , DNA , FecesABSTRACT
Gastrointestinal parasites are among the most economically important pathogens of small ruminants causing serious economic losses and animal welfare problems for the livestock industry worldwide. The emergence of anthelmintic resistant H. contortus in small ruminants is a serious problem because it undermines effective helminth control and results in reduced productivity. Little is known about resistance to benzimidazoles (BZ) in Haemonchus in goats and sheep in Uganda. The objective of this study was to determine the prevalence of gastrointestinal parasites and to identify the presence of benzimidazole resistance associated mutations in the ß-tubulin isotype 1 gene of Haemonchus contortus in goats from selected districts of Uganda. A total of 200 goats from 10 districts of Uganda slaughtered at Kalerwe abattoir in Kampala were sampled for H. contortus adult worms. Faecal samples were also collected to detect other intestinal parasites. Faecal microscopy and analysis were performed using flotation and sedimentation techniques. DNA was extracted from adult worms and PCR and sequencing of the ITS- 2 region and ß-tubulin isotype 1 gene performed to identify H. contortus species and to determine the presence of mutations associated with anthelmintic resistance respectively. Faecal microscopy showed that the most prevalent intestinal parasites were coccidia (98%), strongyles (97.5%), Strongyloides (82%), Paramphistomum (74.5%), Moniezia (46%), Fasciola (1.5%) and Trichuris (1%). Most goats had a high intestinal burden of coccidia (≥ 5000 oocyst per gram) and strongyles (≥ 1000 egg per gram), 65% and 67.5%, respectively. The prevalence of H. contortus adult worms was 63% (126/200). Sequencing of the partial ß-tubulin isotype 1 gene of 54 Haemonchus contortus adult male isolates revealed the presence of mutations associated with anthelmintic resistance. The F200Y mutation was the most common mutation (13% of samples with good beta-tubulin sequences) followed by the E198A and E198K mutations, both found in 9% of sequenced samples. Mutation F167Y was not identified in any of the samples and there were no heterozygous individuals for any of the SNPS associated with BZ resistance that were identified in this study. These findings highlight the need for controlled use of anthelmintics especially benzimidazoles, to enable sustainable control of H. contortus in Uganda, and a need for further investigation to understand the resistance of other parasites identified in this study.
Subject(s)
Anthelmintics , Haemonchus , Parasites , Male , Sheep , Animals , Haemonchus/genetics , Tubulin/genetics , Goats , Prevalence , Uganda/epidemiology , Benzimidazoles/pharmacology , Anthelmintics/pharmacology , MutationABSTRACT
Ascariasis is the most prevalent zoonotic helminthic disease worldwide, and is responsible for nutritional deficiencies, particularly hindering the physical and neurological development of children. The appearance of anthelmintic resistance in Ascaris is a risk for the target of eliminating ascariasis as a public health problem by 2030 set by the World Health Organisation. The development of a vaccine could be key to achieving this target. Here we have applied an in silico approach to design a multi-epitope polypeptide that contains T-cell and B-cell epitopes of reported novel potential vaccination targets, alongside epitopes from established vaccination candidates. An artificial toll-like receptor-4 (TLR4) adjuvant (RS09) was added to improve immunogenicity. The constructed peptide was found to be non-allergic, non-toxic, with adequate antigenic and physicochemical characteristics, such as solubility and potential expression in Escherichia coli. A tertiary structure of the polypeptide was used to predict the presence of discontinuous B-cell epitopes and to confirm the molecular binding stability with TLR2 and TLR4 molecules. Immune simulations predicted an increase in B-cell and T-cell immune response after injection. This polypeptide can now be validated experimentally and compared to other vaccine candidates to assess its possible impact in human health.
Subject(s)
Ascariasis , Vaccines , Child , Humans , Ascariasis/prevention & control , Toll-Like Receptor 4 , Epitopes, B-Lymphocyte , Escherichia coli , PeptidesABSTRACT
BACKGROUND: Toxocara canis and Toxocara cati are intestinal parasites of dogs, cats and foxes, with infected animals shedding eggs of the parasite in their faeces. If humans accidentally ingest embryonated Toxocara spp. eggs from the environment, severe clinical consequences, including blindness and brain damage, can occur. Previous work has demonstrated the presence of Toxocara spp. eggs on vegetable produce grown in the UK, but only in small-scale community gardens. The aim of this study was to determine whether Toxocara spp. eggs are also present on vegetables grown on commercial farms in the UK, which supply produce to a greater number of people. METHODS: A total of 120 samples (300 g each) of spinach (Spinacia oleracea) were collected across four farms in the south of England, UK. The samples were processed using a sieving approach followed by multiplex quantitative polymerase chain reaction analysis. RESULTS: Overall, 23.0% of samples were positive for T. canis (28/120; 95% confidence interval 16.7-31.7%) and 1.7% for T. cati (2/120; 95% confidence interval 0.5-5.9%). There was a statistically significant difference in the number of positive samples between farms (P = 0.0064). To our knowledge, this is the first report of the isolation of Toxocara spp. from vegetables grown on commercial farms in the UK. CONCLUSIONS: The results of this study highlight the requirement for the thorough washing of vegetables prior to their consumption, especially those such as spinach which may be eaten without first peeling or cooking, and effective farm biosecurity measures to minimise access to farmland by definitive host species of Toxocara spp.
Subject(s)
Spinacia oleracea , Toxocara , Animals , Humans , Foxes/parasitology , Spinacia oleracea/parasitology , Toxocara/isolation & purification , United KingdomABSTRACT
Toxoplasma gondii is a zoonotic parasite of importance to both human and animal health. The parasite has various transmission routes, and the meat of infected animals appears to be a major source of human infections in Europe. We aimed to estimate T. gondii prevalence in a selection of animal host species. A systematic literature review resulting in 226 eligible publications was carried out, and serological data were analyzed using an age-dependent Bayesian hierarchical model to obtain estimates for the regional T. gondii seroprevalence in livestock, wildlife, and felids. Prevalence estimates varied between species, regions, indoor/outdoor rearing, and types of detection methods applied. The lowest estimated seroprevalence was observed for indoor-kept lagomorphs at 4.8% (95% CI: 1.8-7.5%) and the highest for outdoor-kept sheep at 63.3% (95% CI: 53.0-79.3%). Overall, T. gondii seroprevalence estimates were highest within Eastern Europe, whilst being lowest in Northern Europe. Prevalence data based on direct detection methods were scarce and were not modelled but rather directly summarized by species. The outcomes of the meta-analysis can be used to extrapolate data to areas with a lack of data and provide valuable inputs for future source attribution approaches aiming to estimate the relative contribution of different sources of T. gondii human infection.
ABSTRACT
PURPOSE: Dicrocoeliosis can be an important cause of production loss in ruminants due to the cost of liver condemnation at slaughter. The aim of the present study was to determine the prevalence of Dicrocoelium infection and to predict the ecological niches and climatic variables that support dicrocoeliosis in the Himalayan ranges of Pakistan. METHODS AND RESULTS: Dicrocoelium was detected in 33 of 381 liver samples and 238 of 6060 blood samples taken from sheep and goat herds in the area. The prevalence of dicrocoeliosis was higher in sheep than in goats and highest in females aged more than 3 years. An environmental risk map was created to predict active zones of transmission and showed the highest probability values in central parts of the Chitral district in the northwest of Pakistan. Climatic variables of the mean monthly diurnal temperature range (Bio2), annual precipitation (Bio12), and normalised difference vegetation index (NDVI) were found to be significantly (p < 0.05) associated with the presence of Dicrocoelium infection. CONCLUSION: Together, the findings of this study demonstrate the most suitable ecological niches and climatic variables influencing the risk of dicrocoeliosis in the Himalayan ranges of Pakistan. The methods and results could be used as a reference to inform the control of dicrocoeliosis in the region.
Subject(s)
Dicrocoeliasis , Dicrocoelium , Sheep Diseases , Sheep , Animals , Sheep Diseases/epidemiology , Dicrocoeliasis/epidemiology , Dicrocoeliasis/veterinary , Liver , Ruminants , Goats , EcosystemABSTRACT
BACKGROUND: Human, animal, and environmental health are increasingly threatened by the emergence and spread of antibiotic resistance. Inappropriate use of antibiotic treatments commonly contributes to this threat, but it is also becoming apparent that multiple, interconnected environmental factors can play a significant role. Thus, a One Health approach is required for a comprehensive understanding of the environmental dimensions of antibiotic resistance and inform science-based decisions and actions. The broad and multidisciplinary nature of the problem poses several open questions drawing upon a wide heterogeneous range of studies. OBJECTIVE: This study seeks to collect and catalogue the evidence of the potential effects of environmental factors on the abundance or detection of antibiotic resistance determinants in the outdoor environment, i.e., antibiotic resistant bacteria and mobile genetic elements carrying antibiotic resistance genes, and the effect on those caused by local environmental conditions of either natural or anthropogenic origin. METHODS: Here, we describe the protocol for a systematic evidence map to address this, which will be performed in adherence to best practice guidelines. We will search the literature from 1990 to present, using the following electronic databases: MEDLINE, Embase, and the Web of Science Core Collection as well as the grey literature. We shall include full-text, scientific articles published in English. Reviewers will work in pairs to screen title, abstract and keywords first and then full-text documents. Data extraction will adhere to a code book purposely designed. Risk of bias assessment will not be conducted as part of this SEM. We will combine tables, graphs, and other suitable visualisation techniques to compile a database i) of studies investigating the factors associated with the prevalence of antibiotic resistance in the environment and ii) map the distribution, network, cross-disciplinarity, impact and trends in the literature.
Subject(s)
Anti-Bacterial Agents , Bacteria , Animals , Humans , Prevalence , Drug Resistance, Microbial/genetics , Bacteria/genetics , Bias , Anti-Bacterial Agents/pharmacologyABSTRACT
BACKGROUND: Despite considerable recent reductions in antimicrobial use, the UK gamebird industry continues to struggle with production diseases during the rearing season, necessitating significant antibiotic use. This observational study investigated the presence of genes conferring resistance to ß-lactam antibiotics within industry-reared pheasants and red-legged partridges in the UK. METHODS: DNA was extracted from 60 pooled caecal samples collected from gamebirds at routine postmortem examinations during the rearing season. Genes encoding extended-spectrum ß-lactamases (ESBL) were detected by PCR and the corresponding alleles were determined. RESULTS: Over half (53%) of the samples harboured genes encoding blaTEM resistance, with blaSHV identified in 20% of samples. The blaTEM gene was more common on sites with higher antibiotic use, whereas blaSHV was predominantly found in birds younger than 5 weeks. Genotyping of the identified resistance genes revealed the presence of blaTEM-1 , blaSHV-1 and blaSHV-11 alleles. LIMITATIONS: This was a small-scale study conducted at four sites in southern England. CONCLUSION: This is the first report of the presence of ESBL genes in gamebirds, highlighting the need for further research into antimicrobial resistance in UK gamebirds.
Subject(s)
Galliformes , beta-Lactamases , Animals , beta-Lactamases/genetics , beta-Lactam Resistance/genetics , Anti-Bacterial Agents/pharmacology , Quail , United KingdomABSTRACT
BACKGROUND: The flagellated parasite Giardia duodenalis is a major and global cause of diarrhoeal disease. Eight genetically very distinct groups, known as assemblages A to H, have been recognized in the G. duodenalis species complex, two of which (assemblages A and B) infect humans and other mammalian hosts. Informative typing schemes are essential to understand transmission pathways, characterize outbreaks and trace zoonotic transmission. In this study, we evaluated a published multi-locus sequence typing (MLST) scheme for G. duodenalis assemblage A, which is based on six polymorphic markers. METHODS: We genotyped 60 human-derived and 11 animal-derived G. duodenalis isolates collected in Europe and on other continents based on the published protocol. After retrieving previously published genotyping data and excluding isolates whose sequences showed allelic sequence heterozygosity, we analysed a dataset comprising 146 isolates. RESULTS: We identified novel variants at five of the six markers and identified 78 distinct MLST types in the overall dataset. Phylogenetic interpretation of typing data confirmed that sub-assemblage AII only comprises human-derived isolates, whereas sub-assemblage AI comprises all animal-derived isolates and a few human-derived isolates, suggesting limited zoonotic transmission. Within sub-assemblage AII, isolates from two outbreaks, which occurred in Sweden and Italy, respectively, had unique and distinct MLST types. Population genetic analysis showed a lack of clustering by geographical origin of the isolates. CONCLUSION: The MLST scheme evaluated provides sufficient discriminatory power for epidemiological studies of G. duodenalis assemblage A.
Subject(s)
Giardia lamblia , Giardiasis , Animals , Humans , Giardiasis/parasitology , Multilocus Sequence Typing , Phylogeny , Genotype , Feces/parasitology , Mammals/geneticsABSTRACT
Ascariasis is the most prevalent helminthic disease affecting both humans and pigs and is caused by the roundworms Ascaris lumbricoides and Ascaris suum. While preventive chemotherapy continues to be the most common control method, recent reports of anthelminthic resistance highlight the need for development of a vaccine against ascariasis. The aim of this study was to use a reverse vaccinology approach to identify potential vaccine candidates for Ascaris. Three Ascaris proteomes predicted from whole-genome sequences were analyzed. Candidate proteins were identified using open-access bioinformatic tools (e.g., Vacceed, VaxiJen, Bepipred 2.0) which test for different characteristics such as sub-cellular location, T-cell and B-cell molecular binding, antigenicity, allergenicity and phylogenetic relationship with other nematode proteins. From over 100,000 protein sequences analyzed, four transmembrane proteins were predicted to be non-allergen antigens and potential vaccine candidates. The four proteins are a Piezo protein, two voltage-dependent calcium channels and a protocadherin-like protein, are all expressed in either the muscle or ovaries of both Ascaris species, and all contained high affinity epitopes for T-cells and B-cells. The use of a reverse vaccinology approach allowed the prediction of four new potential vaccination targets against ascariasis in humans and pigs. These targets can now be further tested in in vitro and in vivo assays to prove efficacy in both pigs and humans.
ABSTRACT
Control of tropical theileriosis, caused by the apicomplexan Theileria annulata, depends on the use of a single drug, buparvaquone, the efficacy of which is compromised by the emergence of resistance. The present study was undertaken to improve understanding of the role of mutations conferring buparvaquone resistance in T. annulata, and the effects of selection pressures on their emergence and spread. First, we investigated genetic characteristics of the cytochrome b locus associated with buparvaquone resistance in 10 susceptible and 7 resistant T. annulata isolates. The 129G (GGC) mutation was found in the Q01 binding pocket and 253S (TCT) and 262S (TCA) mutations were identified within the Q02 binding pocket. Next, we examined field isolates and identified cytochrome b mutations 129G (GGC), 253S (TCT) and 262S (TCA) in 21/75 buffalo-derived and 19/119 cattle-derived T. annulata isolates, providing evidence of positive selection pressure. Both hard and soft selective sweeps were identified, with striking differences between isolates. For example, 19 buffalo-derived and 7 cattle-derived isolates contained 129G (GGC) and 253S (TCT) resistance haplotypes at a high frequency, implying the emergence of resistance by a single mutation. Two buffalo-derived and 12 cattle-derived isolates contained equally high frequencies of 129G (GGC), 253S (TCT), 129G (GGC)/253S (TCT) and 262S (TCA) resistance haplotypes, implying the emergence of resistance by pre-existing or recurrent mutations. Phylogenetic analysis further revealed that 9 and 21 unique haplotypes in buffalo and cattle-derived isolates were present in a single lineage, suggesting a single origin. We propose that animal migration between farms is an important factor in the spread of buparvaquone resistance in endemic regions of Pakistan. The overall outcomes will be useful in understanding how drug resistance emerges and spreads, and this information will help design strategies to optimise the use and lifespan of the single most drug use to control tropical theileriosis.
Subject(s)
Theileria annulata , Theileriasis , Cattle , Animals , Theileria annulata/genetics , Cytochromes b/genetics , Phylogeny , Theileriasis/drug therapyABSTRACT
Ascaris species are soil-transmitted helminths that infect humans and livestock mainly in low and middle-income countries. Benzimidazole (BZ) class drugs have predominated for many years in the treatment of Ascaris infections, but persistent use of BZs has already led to widespread resistance in other nematodes, and treatment failure is emerging for Ascaris. Benzimidazoles act by binding to ß-tubulin proteins and destabilising microtubules. Three mutations in the ß-tubulin protein family are associated with BZ resistance. Seven shared ß-tubulin isotypes were identified in Ascaris lumbricoides and A. suum genomes. Benzimidazoles were predicted to bind to all ß-tubulin isotypes using in silico docking, demonstrating that the selectivity of BZs to interact with one or two ß-tubulin isotypes is likely the result of isotype expression levels affecting the frequency of interaction. Ascaris ß-tubulin isotype A clusters with helminth ß-tubulins previously shown to interact with BZ. Molecular dynamics simulations using ß-tubulin isotype A highlighted the key role of amino acid E198 in BZ-ß-tubulin interactions. Simulations indicated that mutations at amino acids E198A and F200Y alter binding of BZ, whereas there was no obvious effect of the F167Y mutation. In conclusion, the key interactions vital for BZ binding with ß-tubulins have been identified and show how mutations can lead to resistance in nematodes.
Subject(s)
Anthelmintics , Helminths , Amino Acids/genetics , Animals , Anthelmintics/pharmacology , Anthelmintics/therapeutic use , Ascaris , Benzimidazoles/pharmacology , Benzimidazoles/therapeutic use , Drug Resistance/genetics , Humans , Molecular Docking Simulation , Mutation , Tubulin/geneticsABSTRACT
The neglected tropical disease trichuriasis is caused by the whipworm Trichuris trichiura, a soil-transmitted helminth that has infected humans for millennia. Today, T. trichiura infects as many as 500 million people, predominantly in communities with poor sanitary infrastructure enabling sustained faecal-oral transmission. Using whole-genome sequencing of geographically distributed worms collected from human and other primate hosts, together with ancient samples preserved in archaeologically-defined latrines and deposits dated up to one thousand years old, we present the first population genomics study of T. trichiura. We describe the continent-scale genetic structure between whipworms infecting humans and baboons relative to those infecting other primates. Admixture and population demographic analyses support a stepwise distribution of genetic variation that is highest in Uganda, consistent with an African origin and subsequent translocation with human migration. Finally, genome-wide analyses between human samples and between human and non-human primate samples reveal local regions of genetic differentiation between geographically distinct populations. These data provide insight into zoonotic reservoirs of human-infective T. trichiura and will support future efforts toward the implementation of genomic epidemiology of this globally important helminth.
Subject(s)
Trichuriasis , Trichuris , Animals , Genome-Wide Association Study , Humans , Metagenomics , Phylogeny , Primates/genetics , Trichuriasis/epidemiology , Trichuris/geneticsABSTRACT
Toxocara canis and T. cati are zoonotic roundworm parasites of dogs, cats and foxes. These definitive hosts pass eggs in their faeces, which contaminate the environment and can subsequently be ingested via soil or contaminated vegetables. In humans, infection with Toxocara can have serious health implications. This proof-of-concept study aimed to investigate the presence of Toxocara spp. eggs on 'ready-to-eat' vegetables (lettuce, spinach, spring onion and celery) sampled from community gardens in southern England. The contamination of vegetables with Toxocara eggs has never been investigated in the UK before, and more widely, this is the first time vegetables grown in community gardens in Europe have been assessed for Toxocara egg contamination. Sixteen community gardens participated in the study, providing 82 vegetable samples fit for analysis. Study participants also completed an anonymous questionnaire on observed visits to the sites by definitive hosts of Toxocara. Comparison of egg recovery methods was performed using lettuce samples spiked with a series of Toxocara spp. egg concentrations, with sedimentation and centrifugal concentration retrieving the highest number of eggs. A sample (100 g) of each vegetable type obtained from participating community gardens was tested for the presence of Toxocara eggs using the optimised method. Two lettuce samples tested positive for Toxocara spp. eggs, giving a prevalence of 2.4% (95% CI =1.3-3.5%) for vegetable samples overall, and 6.5% (95% CI = 4.7-8.3%; n = 31) specifically for lettuce. Questionnaire data revealed that foxes, cats and dogs frequently visited the community gardens in the study, with 88% (68/77) of respondents reporting seeing a definitive host species or the faeces of a definitive host at their site. This proof-of-concept study showed for the first time the presence of Toxocara spp. eggs on vegetables grown in the UK, as well as within the soil where these vegetables originated, and highlights biosecurity and zoonotic risks in community gardens. This study establishes a method for assessment of Toxocara spp. eggs on vegetable produce and paves the way for larger-scale investigations of Toxocara spp. egg contamination on field-grown vegetables.
ABSTRACT
Toxoplasma gondii is a major foodborne pathogen capable of infecting all warm-blooded animals, including humans. Although oocyst-associated toxoplasmosis outbreaks have been documented, the relevance of the environmental transmission route remains poorly investigated. Thus, we carried out an extensive systematic review on T. gondii oocyst contamination of soil, water, fresh produce, and mollusk bivalves, following the PRISMA guidelines. Studies published up to the end of 2020 were searched for in public databases and screened. The reference sections of the selected articles were examined to identify additional studies. A total of 102 out of 3201 articles were selected: 34 articles focused on soil, 40 focused on water, 23 focused on fresh produce (vegetables/fruits), and 21 focused on bivalve mollusks. Toxoplasma gondii oocysts were found in all matrices worldwide, with detection rates ranging from 0.09% (1/1109) to 100% (8/8) using bioassay or PCR-based detection methods. There was a high heterogeneity (I2 = 98.9%), which was influenced by both the sampling strategy (e.g., sampling site and sample type, sample composition, sample origin, season, number of samples, cat presence) and methodology (recovery and detection methods). Harmonized approaches are needed for the detection of T. gondii in different environmental matrices in order to obtain robust and comparable results.