Otterly diverse - A high diversity of Dracunculus species (Spirurida: Dracunculoidea) in North American river otters (Lontra canadensis).

The genus Dracunculus contains numerous species of subcutaneous parasites of mammals and reptiles. In North America, there are at least three mammal-infecting species of Dracunculus. Reports of Dracunculus infections have been reported from river otters (Lontra canadensis) since the early 1900s; however, little is known about the species infecting otters or their ecology. Most reports of Dracunculus do not have a definitive species identified because females, the most common sex found due to their larger size and location in the extremities of the host, lack distinguishing morphological characteristics, and few studies have used molecular methods to confirm identifications. Thus, outside of Ontario, Canada, where both D. insignis and D. lutrae have been confirmed in otters, the species of Dracunculus in river otters is unknown. In the current study, molecular characterization of nematodes from river otters revealed a high diversity of Dracunculus species. In addition to confirming D. insignis infections, two new clades were detected. One clade was a novel species in any host and the other was a clade previously detected in Virginia opossums (Didelphis virginiana) from the USA and a domestic dog from Spain. No infections with D. lutrae were detected and neither new lineage was genetically similar to D. jaguape, which was recently described from a neotropical otter (Lontra longicaudis) from Argentina. These data also indicate that Dracunculus spp. infections in otters are widespread throughout Eastern North America. Currently the life cycles for most of the Dracunculus spp. infecting otters are unknown. Studies on the diversity, life cycle, and natural history of Dracunculidae parasites in wildlife are important because the related parasite, D. medinensis (human Guinea worm) is the subject of an international eradication campaign and there are increasing reports of these parasites in new geographic locations and new hosts, including new species in humans and domestic dogs.

Development and validation of a quantitative PCR for the detection of Guinea worm (Dracunculus medinensis).

Dracunculus medinensis (Guinea worm) is a parasitic nematode that can cause the debilitating disease dracunculiasis (Guinea worm disease) in humans. The global Guinea Worm Eradication Program has led intervention and eradication efforts since the 1980s, and Guinea worm infections in people have decreased >99.99%. With the final goal of eradication drawing nearer, reports of animal infections from some remaining endemic countries pose unique challenges. Currently, confirmation of suspected Guinea worm infection relies on conventional molecular techniques such as polymerase chain reaction (PCR), which is not specific to Guinea worm and, therefore, requires sequencing of the PCR products to confirm the identity of suspect samples, a process that often takes a few weeks. To decrease the time required for species confirmation, we developed a quantitative PCR assay targeting the mitochondrial cytochrome b (cytb) gene of Guinea worm. Our assay has a limit of detection of 10 copies per reaction. The mean analytical parameters (± SE) were as follows: efficiency = 93.4 ± 7.7%, y-intercept = 40.93 ± 1.11, slope = -3.4896 ± 0.12, and the R2 = 0.999 ± 0.004. The assay did not amplify other nematodes found in Guinea worm-endemic regions and demonstrated 100% diagnostic sensitivity and specificity. Implementation of this quantitative PCR assay for Guinea worm identification could eliminate the need for DNA sequencing to confirm species. Thus, this approach can be implemented to provide more rapid confirmation of Guinea worm infections, leading to faster execution of Guinea worm interventions while increasing our understanding of infection patterns.

Investigating Flubendazole as an Anthelmintic Treatment for Guinea Worm (Dracunculus medinensis): Clinical Trials in Laboratory-Reared Ferrets and Domestic Dogs in Chad.

Dracunculus medinensis (Guinea worm [GW]), a zoonotic nematode targeted for eradication, has been managed using interventions aimed at humans; however, increases in domestic dog GW infections highlight the need for novel approaches. We conducted two clinical trials evaluating the efficacy of subcutaneously injected flubendazole (FBZ) as a treatment of GW infection. The first trial was conducted administering FBZ to experimentally infected ferrets; the second trial involved administering FBZ or a placebo to domestic dogs in the Republic of Tchad (Chad). We found contrasting results between the two trials. When adult gravid female GW were recovered from ferrets treated with FBZ, larvae presented in poor condition, with low to no motility, and an inability to infect copepods. Histopathology results indicated a disruption to morulae development within uteri of worms from treated animals. Results from the trial in Chadian dogs failed to indicate significant treatment of or prevention against GW infection. However, the difference in treatment intervals (1 month for ferrets and 6 months for dogs) or the timing of treatment (ferrets were treated later in the GW life-cycle than dogs) could explain different responses to the subcutaneous FBZ injections. Both trials provided valuable data guiding the use of FBZ in future trials (such as decreasing treatment intervals or increasing the dose of FBZ in dogs to increase exposure), and highlighted important lessons learned during the implementation of a field-based, double-blinded randomized control trial in Chadian dogs.

Effects of Temephos (Abate®), Spinosad (Natular®), and Diflubenzuron on the Survival of Cyclopoid Copepods.

Dracunculus medinensis, also known as the African Guinea worm, is the causative agent of dracunculiasis and the focus of the global Guinea Worm Eradication Program (GWEP). Transmission of D. medinensis to humans occurs primarily by drinking water containing cyclopoid copepods infected with third-stage D. medinensis larvae. A common intervention to interrupt transmission and decrease the number of copepods in infected water bodies is the application of the organophosphate larvicide Abate® (temephos). However, the use of alternative compounds to help decrease copepod populations would be beneficial to the GWEP. We compared the immobilization of copepods by three compounds: Abate, Natular® (spinosad), and diflubenzuron. Our results confirm that neither diflubenzuron nor Natular immobilized copepods as quickly or as effectively as Abate. However, doubling or tripling the suggested concentration of Natular resulted in immobilization rates similar to Abate over 72 hours of continuous exposure. Further research on the possible effects of higher concentrations of Natular on the environment and nontarget organisms is necessary to determine whether this compound can be used safely to control the copepod population.

Molecular Confirmation of Ranavirus Infection in Amphibians From Chad, Africa.

Ranaviruses are DNA viruses (Family Iridoviridae; Subfamily Alphairidovirinae) and ranaviral disease is considered an emerging infectious disease of ectothermic vertebrates. Ranavirus infection can have varying pathological effects on infected amphibians, reptiles, and fish, most notably causing significant mortality events and population declines. Despite having a broad global range with reports from six continents, only a single incidental finding in Xenopus longipes from mainland Africa (Cameroon) is known and lacks molecular confirmation. Thus, there is a considerable knowledge gap concerning ranaviruses in Africa. We opportunistically obtained tissue samples from 160 amphibians representing five genera (Hoplobatrachus, Hylarana, Ptychadena, Pyxicephalus, and Xenopus) and two turtles (Pelomedusa sp.) from Chad, Africa. Samples were tested for ranavirus infection using a conventional PCR assay targeting the major capsid protein (MCP). A total of 25/160 (16%) frogs tested positive including 15/87 (17%) Hoplobatrachus occipitalis, 10/58 (17%) Ptychadena spp., 0/3 Pyxicephalus spp., 0/9 Xenopus spp., and 0/3 Hylarana spp. One of two turtles tested positive. Partial MCP gene sequences indicated all samples were >98% similar to several frog virus 3 (FV3)-like sequences. Additional gene targets (DNA polymerase [DNApol], ribonucleotide reductase alpha [RNR- α], ribonucleotide reductase beta subunit [RNR- ß]) were sequenced to provide further detailed classification of the virus. Sequences of individual gene targets indicate that the ranavirus detected in frogs in Chad is most similar to tiger frog virus (TFV), a FV3-like virus previously isolated from diseased amphibians cultured in China and Thailand. Full genome sequencing of one sample indicates that the Chad frog virus (CFV) is a well-supported sister group to the TFVs previously determined from Asia. This work represents the first molecular confirmation of ranaviruses from Africa and is a first step in comparing ranavirus phylogeography on a local and global scale.

Susceptibility of anurans, lizards, and fish to infection with Dracunculus species larvae and implications for their roles as paratenic hosts.

Dracunculus spp. are parasitic nematodes that infect numerous species of mammals and reptiles. The life cycles of Dracunculus species are complex, and unknowns remain regarding the role of paratenic and transport hosts in transmission to definitive hosts. We had two primary objectives: to assess the susceptibility of several species of anurans, lizards, and fish as paratenic hosts for Dracunculus species, and to determine the long-term persistence of Dracunculus infections in African clawed frogs (Xenopus laevis). Animals were orally exposed to copepods infected with infectious third-stage larvae (L3s) of either Dracunculus insignis or D. medinensis. Dracunculus L3s were recovered from four anuran species, two lizard species, and one fish species, demonstrating that Dracunculus can infect tissues of a diversity of species. In long-term persistence trials, D. medinensis L3s were recovered from African clawed frogs tissues up to 58 days post-infection, and D. insignis L3s were recovered up to 244 days post-infection. Our findings regarding the susceptibility of novel species of frogs, lizards, and fish to infection with Dracunculus nematodes, and long-term persistence of L3s in paratenic hosts, address pressing knowledge gaps regarding Dracunculus infection in paratenic hosts and may guide future research regarding the transmission of Dracunculus to definitive mammalian hosts.

Copepod consumption by amphibians and fish with implications for transmission of Dracunculus species.

Parasitic nematodes in the genus Dracunculus have a complex life cycle that requires more than one host species in both aquatic and terrestrial habitats. The most well-studied species, Dracunculus medinensis, is the causative agent of human Guinea worm disease (dracunculiasis). There are several other Dracunculus species that infect non-human animals, primarily wildlife (reptiles and mammals). The classic route of D. medinensis transmission to humans is through the ingestion of water containing the intermediate host, a cyclopoid copepod, infected with third-stage larvae (L3s). However, many animal hosts (e.g., terrestrial snakes, dogs) of other Dracunculus sp. appear unlikely to ingest a large number of copepods while drinking. Therefore, alternative routes of infection (e.g., paratenic or transport hosts) may facilitate Dracunculus transmission to these species. To better understand the role of paratenic and transport hosts in Dracunculus transmission to animal definitive hosts, we compared copepod ingestion rates for aquatic species (fish, frogs [tadpoles and adults], and newts) which may serve as paratenic or transport hosts. We hypothesized that fish would consume more copepods than amphibians. Our findings confirm that African clawed frogs (Xenopus laevis) and fish consume copepods, but that fish ingest, on average, significantly higher numbers (68% [34/50]) than adult African clawed frogs (36% [18/50]) during a 24-h time period. Our results suggest that amphibians and fish may play a role in the transmission of Dracunculus to definitive hosts. Still, additional research is required to determine whether, in the wild, fish or frogs are serving as paratenic or transport hosts. If so, they may facilitate Dracunculus transmission. However, if these animals simply act as dead-end hosts or as means of copepod population control, they may decrease Dracunculus transmission.

Cooking copepods: The survival of cyclopoid copepods (Crustacea: Copepoda) in simulated provisioned water containers and implications for the Guinea Worm Eradication Program in Chad, Africa.

INTRODUCTION: The global Guinea Worm Eradication Program has reduced numbers of human infections of Guinea worm disease (dracunculiasis) to 49 cases in four countries. However, infections of domestic animals (dogs and cats) have recently been recognized and are increasing. Typically, Guinea worm (Dracunculus medinensis) transmission occurs via the ingestion of copepods from water. Despite several interventions, including tethering of dogs while worms emerge, the number of infected dogs continue to increase. One hypothesis is that dogs could be infected through the ingestion of copepods in provisioned water. OBJECTIVES: The purpose of this study was to determine whether copepods can survive in water containers under typical Chadian temperatures. METHODS: Four container types (plastic, glass, gourd, and metal) were seeded with copepods and exposed to simulated Chadian temperatures. RESULTS: All copepods in the metal containers died within 4 h. Conversely, after 8 h live copepods were still present in plastic, glass, and gourd containers. CONCLUSIONS: If provisioned water is provided to potential hosts of D. medinensis, metal containers create the most inhospitable environment for copepods. Plastic containers have little effect on copepod mortality. The use of metal containers for water provisions could be a useful tool assisting with the interruption of D. medinensis transmission among dogs.

Dogs and the classic route of Guinea Worm transmission: an evaluation of copepod ingestion.

Dracunculus medinensis, the causative agent of Guinea worm disease in humans, is being reported with increasing frequency in dogs. However, the route(s) of transmission to dogs is still poorly understood. Classical transmission to humans occurs via drinking water that contains cyclopoid copepods infected with third stage larvae of D. medinensis, but due to the method of dog drinking (lapping) compared to humans (suction and/or retrieval of water into containers), it seems unlikely that dogs would ingest copepods readily through drinking. We exposed lab raised beagles to varying densities of uninfected copepods in 2 liters of water to evaluate the number of copepods ingested during a drinking event. We confirmed dogs can ingest copepod intermediate hosts while drinking; however, low numbers were ingested at the densities that are typically observed in Chad suggesting this transmission route may be unlikely. Overall, the relative importance of the classic transmission route and alternate transmission routes, such as paratenic and transport hosts, needs investigation in order to further clarify the epidemiology of guinea worm infections in dogs.

Rickettsia species in ticks collected from wild pigs (Sus scrofa) and Philippine deer (Rusa marianna) on Guam, Marianna Islands, USA.

The prevalence and diversity of ticks on wildlife species on Guam is understudied, as to date no work has been conducted on the infection of these ticks with Rickettsia (obligate intracellular pathogens that use a variety of ectoparasites as vectors and can cause disease in humans, domestic animals, and wildlife species). The goal of our study was to investigate the presence of Rickettsia species on the island of Guam by testing ticks found on Philippine deer (Rusa marianna) and wild pigs (Sus scrofa). Increasing numbers of these species have led to increased interactions with humans, including hunting, highlighting the importance of studies on vector prevalence and associated zoonotic pathogens. In this study, ticks were removed from Philippine deer and wild pigs in March and April of 2015 and tested for Rickettsia spp. using nested PCR. Overall, a low prevalence of Rickettsia spp. was detected (5.4% (6/112 ticks)). Ticks removed from wild pigs were identified as Amblyomma breviscutatum, one of which was positive for Rickettsia ambylommatis. Ticks recovered from Philippine deer were identified as Rhipicephalus microplus, and five were positive for Rickettsia; two with R. amblyommatis and one with 'Candidatus Rickettsia senegalensis', a recently proposed species in the R. felis cluster. The remaining two sequences were short and species classification was not possible. Rickettsia felis is a known zoonotic pathogen in the spotted fever group of Rickettsia and there is evidence that 'C. R. senegalensis' can also cause illness in people. This study confirms the occurrence of Rickettsia in ticks on Guam and highlights the presence of potential human pathogenic species in the R. felis cluster.