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Verocai, G. G.

Publications and source records attributed to Verocai, G. G..

8 recordsLinked to original sources

Detection of Echinococcus multilocularis in coyotes in Washington State, USA highlights need for increased global wildlife surveillance

Echinococcus multilocularis is a zoonotic cestode that uses canids as definitive hosts and rodents as intermediate hosts. In humans, this parasite is the causative agent of alveolar echinococcosis. Recently, its range has been expanding across the Northern Hemisphere, and it is increasingly detected in wild canids, domestic dogs, and humans across Canada and the United States. While this expansion has been documented in isolated studies across the continent, a lack of routine sampling in wildlife hinders our ability to anticipate and mitigate further spread of E. multilocularis. We confirmed the presence of E. multilocularis in Washington State, USA, using a combination of morphological and molecular techniques across carcasses and field-collected scats of coyotes (Canis latrans), this regions most common wild canid. Morphological identification of adult worms was confirmed by next-generation sequencing. Over a third of all samples tested positive for E. multilocularis when all methodologies were combined. Sequencing revealed a haplotype of E. multilocularis matching a documented haplotype originally of European origin in British Columbia, Canada. Our study provides the first confirmation of E. multilocularis in a wild host on the west coast of the U.S and provides additional haplotype information crucial to tracking the geographical expansion of the parasite. We also provide a new next-generation sequencing primer targeting cestodes of canids. The difference in amplification between intestinal and fecal samples suggests that non-invasive fecal sampling using DNA metabarcoding--a popular method of helminth surveillance --may lead to underestimation of prevalence, hindering control measures. The global significance of these findings extends beyond North America; E. multilocularis is a major public health concern in Europe and Asia, where alveolar echinococcosis is increasingly diagnosed in humans. Our study highlights the urgent need for increased surveillance and improved diagnostic strategies worldwide, particularly in regions with significant human-wildlife contact. Author summaryParasites that are transmitted between wildlife, domestic animals, and people are an important part of global health. One such parasite is Echinococcus multilocularis, a small tapeworm of canids that can cause a severe, life-threatening disease in humans called alveolar echinococcosis. Many wild canid hosts of the parasite, such as coyotes, overlap significantly with domestic dogs, which facilitates transmission to humans. In Europe, Asia, and Arctic regions of North America, E. multilocularis has long been recognized as a major public health problem. In recent decades its range has expanded across the Northern Hemisphere, raising concern. In this study, we discovered E. multilocularis in coyotes in a densely populated area of Washington State, USA -- the first detection of E. multilocularis in a wild host in the region. More than one-third of our coyote samples contained E. multilocularis, confirming that it is widespread in the area. Genetic testing showed that the strain we detected matched one previously found in Canada, originally from Europe. Our findings underscore the importance of monitoring E. multilocularis and other parasites in wildlife so that emerging public health threats can be detected early, reducing risk to people and pets.

ecology↗

Comparative transcriptome profiles of first-stage larvae and adult female Dracunculus medinensis (Guinea worm)

Dracunculus medinensis, also called the Guinea worm, is a nematode that causes dracunculiasis, a debilitating neglected tropical disease in humans. The parasite is currently targeted by the global Guinea Worm Eradication Program (GWEP). Historically, GWEP in endemic countries have focused on interrupting transmission of the disease through intervention such as isolation and management of patients, health education, provision of improved water sources and promotion of filtering drinking water to avoid ingestion of the copepod intermediate host (IH) that may contain infectious third-stage larvae. The recent shift of Guinea worm infections in animals - particularly domestic dogs - has introduced an additional challenge to the eradication program, underscoring the urgent need for diagnostics and therapeutics. Understanding the parasite biology and survival strategies in the mammalian host, the copepod IH, and fresh water is pivotal to identifying new control measures. Comparative transcriptomic analysis provides a powerful tool to uncover the molecular mechanisms underlying parasite survival and adaptations. Here, we compared the transcriptome of adult gravid female and first-stage larvae (L1), the stage infective for the copepod IH. Comparative transcriptomic analysis of two adult females and their L1 revealed an upregulation of genes involved in translation, transcription, and DNA repair in L1, likely reflecting adaptations essential for survival in freshwater and subsequent infection of copepods. Additionally, genes involved in cuticle formation were upregulated in adult females highlighting the role of cuticle integrity in retaining millions of L1 until the gravid female worm emerges. We identified highly expressed genes in the adult female that may represent promising candidates for diagnostic markers. This study provides novel insights into the biology of the Guinea worm by examining the transcriptome of L1 and adult female stages. These findings could support the development of novel diagnostics and therapeutics to advance the ongoing eradication effort. Authors summaryGuinea worm disease is caused by the nematode Dracunculus medinensis, a parasitic worm responsible for a debilitating neglected tropical disease in humans and targeted for global eradication. Infection occurs through the consumption of contaminated drinking water with the infective larval stage harbored within fresh water crustacean copepods - the intermediate host of the parasite. The high number of Guinea worm infections in animals specially dogs poses a significant challenge to eradication, as infected animals act as reservoirs, contributing to the parasites persistence in the environment. Furthermore, the absence of early diagnostic tools and effective therapeutics complicates disease control. In this study, we performed a comparative transcriptome analysis of adult female Guinea worms and their first-stage larvae. We identified highly expressed genes in the adult female that may represent promising candidates for diagnostic markers. Additionally, we found genes and pathways upregulated in first-stage larvae which are likely essential for survival in freshwater and subsequent infection of copepods. Our study provides insight into the molecular mechanisms underlying Guinea worm survival across life stages and environments. These findings aim to support the development of novel diagnostics and therapeutics to advance ongoing eradication efforts of this neglected tropical disease.

genomics↗

Using deep amplicon sequencing as a molecular xenomonitoring approach for detecting filarial nematodes in biting arthropod vectors

Filarial nematodes are an important group of parasites that impact public, veterinary, and wildlife health globally. In order to understand these impacts and minimize their effects, scientists use molecular xenomonitoring techniques to understand their distribution and track elimination efforts. However, these molecular techniques can have narrow diagnostic capacity due to their species-specific approach which limits our understanding of important co-endemic filarial nematodes. Next-generation sequencing offers the ability to detect multiple species of coinfecting filarial nematodes and thus improve are ability to monitor, treat, and eliminate these pathogens. In this paper, we have developed a deep amplicon sequencing approach using filarial nematode primers targeting the cytochrome oxidase c subunit 1 (coxI) gene. To replicate molecular xenomonitoring conditions, third stage larvae (L3) of three species of filarioid nematodes (Brugia malayi, Brugia pahangi, Dirofilaria immitis) were spiked in different proportions to pools comprising various amounts of female Aedes aegypti mosquitoes (0, 10, 50, 100). Each pool was subjected to DNA extraction and Oxford Nanopore Technologies (ONT) deep amplicon sequencing protocols. Two sets of demultiplexing pipelines were utilized to optimize this novel approach, each reaching, 92.71% and 97.92% accuracy in identification of species composition across mock pools. However, in heterogenous pools, filarial species D. immitis exhibited an overrepresentation of reads and B. pahangi an underrepresentation of reads. We discuss reasons for recount biases and how this new molecular xenomonitoring tool could be implemented to serve public health, veterinary medicine, and scientific advancement. Note: Supplementary data associated with this article Author summaryHuman and animal diseases caused by filarial nematodes affect millions of people worldwide, particularly in low-income countries. These parasites are transmitted by blood-feeding arthropod vectors, such as mosquitoes and black flies. Thus, a major sector of public health research focuses on how to monitor, treat, and eliminate these harmful pathogens. An effective way is to capture these arthropod vectors and molecularly test these for filarial DNA in large sample pools. However, these pools can comprise multiple filarial species which targeted genetic analysis can miss. This proof-of-concept study seeks to circumvent these issues by using new next-generation sequencing approaches to capture the wider filarial diversity that may be contained in a single vector pool. We believe this tool could largely be beneficial to governments and organizations seeking to eliminate these filarial nematodes and become certified as a region free of certain devastating filarial species. Furthermore, we know very little about filarial diversity and this could be an integral tool to define their geographic distribution and future emerging threats to both human and animal health.

microbiology↗

An open source MALDI-TOF mass spectrometry database for the identification of North American tick species of medical and veterinary importance.

Matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI-TOF MS) is a fast emerging and robust method for rapid and accurate characterization of arthropod vectors. Correct identification of ticks is key in monitoring medically or veterinary relevant species and associated pathogens. It has been extensively used for tick identification based on leg proteins; however, in North America, MALDI-TOF MS has not been explored for comprehensive identification of ticks, except for a few studies focusing on a limited number of species. Herein, we have generated proteomic spectral profiles of nine North American tick species belonging to five genera obtained from laboratory-maintained colonies. From the 407 high-quality MS spectra generated in this study, 44 were deposited in our newly made database and used as reference. All remaining MS spectra were used to query and validate the database. All specimens were identified correctly to the species level using MALDI-TOF MS, with reliable Log Score Values (LSVs) ranging from 1.72 to 2.86, and median and mean values of 2.41 and 2.40, respectively. A single exception noted: an Amblyomma mixtum was misidentified as Amblyomma tenellum. As a cost-effective, user-friendly, and high-throughput method, MALDI-TOF MS can serve as an alternative for accurately identifying tick species of veterinary and public health importance in North America, thereby supporting tick-borne disease surveillance efforts.

microbiology↗

Population genomics reveals an ancient origin of heartworms in canids

Heartworms (Dirofilaria immitis) are parasitic nematodes that cause significant cardiopulmonary-associated morbidity and mortality in canids worldwide. The global spread of heartworms is believed to have occurred alongside the dispersal of domesticated dogs. To test this theory, we analysed the genomes of 127 specimens collected from mammalian carnivore hosts across four continents. Here we show distinct genetic differences between heartworms from different continents, indicating a more ancient dispersal in canid hosts than previously recognised. Using admixture analyses, we find an Asian origin for Australian heartworms, consistent with the arrival of dingoes thousands of years ago. Finally, the genetic relatedness between European and Central American heartworms suggests that modern dispersal, likely associated with human colonisation of the Americas by Europeans, occurred with domesticated dogs. This work sheds light on the population dynamics and deep evolutionary history of a globally widespread parasite of veterinary significance.

evolutionary biology↗

Assessing the performance of TRX and DUF148 antigens for detection of prepatent Guinea worm (Dracunculus medinensis) infection in dogs

Guinea worm (GW, Dracunculus medinensis) is a nematode that causes a painful and debilitating neglected tropical disease in humans. The GW Eradication Program has decreased human infections by >99% over the last 40 years. However, GW emergence in animal hosts, particularly dogs, has hampered eradication efforts. Currently, there is no method for diagnosing GW infection in animals during the prepatent period, before the adult female worms emerge. Previous works have identified two GW proteins, TRX and DUF148, as immunoreactive antigens with GW-positive human and dog sera. This study developed and validated indirect enzyme-linked immunosorbent assays (ELISA) using each antigen alone or in a combination of both antigens. Using serum samples from experimentally exposed dogs, TRX and DUF148 showed reactivity at 9- and 11-weeks post-exposure, respectively. In an experimentally infected ferret, TRX and DUF148 showed reactivity at 13- and 15-weeks post-exposure, respectively. These antigens were further validated using sera of dogs from endemic villages in Chad (n=47) and shelter dogs from the non-endemic United States (n=492). DUF148 showed better reactivity and sensitivity of 76.6.% in detecting GW infection in prepatent sera compared to TRX. However, DUF148 cross-reacted with one serum sample from Brugia pahangi experimental infection and several shelter dog sera. The anti-DUF148 titer was significantly higher in the shelter dogs positive for gastrointestinal nematodes than in negative dogs. To mitigate this cross-reaction, we produced 3 peptides of DUF148. Peptide 3 from the C-terminal was more reactive with prepatent sera and had a sensitivity of 83%; however, the specificity was not superior to DUF148 whole antigen. The antibody response to DUF148 in Chad dogs with the history of GW emergence waned overtime but was detectable until two years post-GW-emergence. Our findings could facilitate the development of diagnostic methods for early detection of GW infection in dogs in endemic countries. Authors summaryDracunculiasis or Guinea worm (GW) disease is a neglected tropical disease caused by the nematode Dracunculus medinensis and is targeted for eradication by the World Health Organization. The main challenge for the eradication program is the emergence of animal infections, especially dogs. Diagnostic tests are needed to find infected dogs during the prepatent period to better control infection and prevent the spread of GW. Previous studies have found two immunoreactive GW proteins, TRX and DUF148. In this study, we validated these antigens to detect infection in before GW emergence. Using sera of dogs from endemic areas of Chad, we found that DUF148 was more reactive and had promising sensitivity to detect the prepatent infection in an indirect ELISA assay. However, DUF148 also showed cross-reaction with some sera of dogs from the Unites States, a non-endemic area for GW. To mitigate this cross-reactivity, we performed ELISA using shorter peptides. We found peptide 3 that covers the C-terminal of the protein is the immunogenic part of DUF148. However, peptide 3 ELISA did not outperform whole antigen ELISA. This study confirms the applicability of DUF148 ELISA in detecting prepatent infections in dogs and could assist the GW Eradication Program.

microbiology↗

ves1α genes expression is the major determinant of Babesia bovis-infected erythrocytes cytoadhesion to endothelial cells

Babesia bovis causes the most pathogenic form of babesiosis in cattle, resulting in high mortality in naive adults. This parasite invades red blood cells (RBCs) within the bovine hosts where they multiply and produce clinical disease. Babesia bovis exports numerous proteins into invaded RBCs changing its properties. Thus, the infected RBCs (iRBCs) are capable to cytoadhere in the microvasculature of internal organs and brain, leading to respiratory distress, neurologic signs, and mortality. Variant Erythrocyte Surface Antigen 1 (VESA1) is one of those exported proteins by B. bovis which represents a major virulence factor due to its central role in immune evasion by antigenic variation and intravascular parasite sequestration. VESA1 is a heterodimer protein encoded by ves1 and ves1{beta} multigene family and localized on the ridges, the focal point for cytoadhesion. To gain further insights into the molecular mechanisms of cytoadhesion of B. bovis, we panned the parasites with bovine brain microvasculature endothelial cells, which resulted in obtaining several clones with different cytoadherence abilities. The transcriptome analysis of 2 high and 2 low cytoadherent clones revealed that ves1 sequences were diversified, likely resulting from genomic recombination. On the other hand, ves1{beta} sequences were almost identical among these 4 clones. Insertion and expression of ves1 of a clone with high binding into ef-1 locus of a low binging clone increased cytoadherence confirming the role of ves1 suggested by our transcriptome data. Whole genome sequencing of cytoadherent clones revealed active locus of ves1 on chromosome 2. These results suggest that VESA1a proteins encoded by ves1 genes determine the cytoadherence specificity and/or cytoadherence strength of B. bovis and they are in the active site for recombination. Author summaryBabesia bovis is an apicomplexan intraerythrocytic protozoan parasite which causes the most pathogenic form of babesiosis in cattle. This pathogenicity is the result of parasite multiplication and cytoadherence of infected red blood cells (iRBCs) in the microvasculature of brain and internal organs and is mediated by B. bovis surface exposed ligand, Variant Erythrocyte Surface Antigen 1 (VESA1). Here using parasite panning assay, transcriptomics, and genetic tools, we showed that VESA1a is the main determinant of B. bovis cytoadhesion. VESA1 are large hypervariable proteins (>100kDa) consisting of VESA1a and VESA1b subunits encoded by ves1 and ves1{beta} multigene family. Panning B. bovis with bovine brain endothelial cells resulted in obtaining cytoadherent parasite clones with different binding abilities. Comparative transcriptome analysis revealed diversification of ves1 sequences. Insertion and expression of ves1 of a clone with high-binding ability in the genome of a low-binding clone increased cytoadherence confirming the role of ves1. Mapping RNA-seq on the genome of cytoadherent clones revealed the locus of active transcription and this locus was suggested to be the active site for recombination which promoted the production of variants of ves1 with different binding abilities. Altogether, our results provide new insights into B. bovis cytoadhesion and VESA1 biology.

microbiology↗

Evaluation of a novel Tc-24 recombinant antigen ELISA for serologic testing for Trypanosoma cruzi in dogs

Chagas disease is a parasitic infection caused by Trypanosoma cruzi. Diagnosis of chroni Chagas disease in dogs relies on limited serological test options. This study used a new Tc-24 recombinant antigen ELISA on an archival set of 70 dog serum samples from multi-dog kennel environments in Texas subjected to three existing Chagas serological tests. Tc-24 ELISA produced a quantitative result and could detect anti-T. cruzi antibodies in dogs with high sensitivity and specificity. Comparing individual tests to Tc-24 ELISA resulted in strong associations and correlations, which suggest that Tc-24 ELISA is a reliable and accurate diagnostic tool for dogs with a single test.

zoology↗