Mysterious microsporidians: springtime outbreaks of disease in Daphnia communities in shallow pond ecosystems.

Parasites can play key roles in ecosystems, especially when they infect common hosts that play important ecological roles. Daphnia are critical grazers in many lentic freshwater ecosystems and typically reach peak densities in early spring. Daphnia have also become prominent model host organisms for the field of disease ecology, although most well-studied parasites infect them in summer or fall. Here, we report field patterns of virulent microsporidian parasites that consistently infect Daphnia in springtime, in a set of seven shallow ponds in Georgia, USA, sampled every 3-4 weeks for 18 months. We detected two distinct parasite taxa, closely matching sequences of Pseudoberwaldia daphniae and Conglomerata obtusa, both infecting all three resident species of Daphnia: D. ambigua, D. laevis, and D. parvula. To our knowledge, neither parasite has been previously reported in any of these host species or anywhere in North America. Infection prevalence peaked consistently in February-May, but the severity of these outbreaks differed substantially among ponds. Moreover, host species differed markedly in terms of their maximum infection prevalence (5% [D. parvula] to 72% [D. laevis]), mean reduction of fecundity when infected (70.6% [D. ambigua] to 99.8% [D. laevis]), mean spore yield (62,000 [D. parvula] to 377,000 [D. laevis] per host), and likelihood of being infected by each parasite. The timing and severity of the outbreaks suggests that these parasites could be impactful members of these shallow freshwater ecosystems, and that the strength of their effects is likely to hinge on the composition of ponds' zooplankton communities.

Phylogeography of the blacklegged tick (Ixodes scapularis) throughout the USA identifies candidate loci for differences in vectorial capacity.

The blacklegged tick (Ixodes scapularis (Journal of the Academy of Natural Sciences of Philadelphia, 1821, 2, 59)) is a vector of Borrelia burgdorferi sensu stricto (s.s.) (International Journal of Systematic Bacteriology, 1984, 34, 496), the causative bacterial agent of Lyme disease, part of a slow-moving epidemic of Lyme borreliosis spreading across the northern hemisphere. Well-known geographical differences in the vectorial capacity of these ticks are associated with genetic variation. Despite the need for detailed genetic information in this disease system, previous phylogeographical studies of these ticks have been restricted to relatively few populations or few genetic loci. Here we present the most comprehensive phylogeographical study of genome-wide markers in I. scapularis, conducted by using 3RAD (triple-enzyme restriction-site associated sequencing) and surveying 353 ticks from 33 counties throughout the species' range. We found limited genetic variation among populations from the Northeast and Upper Midwest, where Lyme disease is most common, and higher genetic variation among populations from the South. We identify five spatially associated genetic clusters of I. scapularis. In regions where Lyme disease is increasing in frequency, the I. scapularis populations genetically group with ticks from historically highly Lyme-endemic regions. Finally, we identify 10 variable DNA sites that contribute the most to population differentiation. These variable sites cluster on one of the chromosome-scale scaffolds for I. scapularis and are within identified genes. Our findings illuminate the need for additional research to identify loci causing variation in the vectorial capacity of I. scapularis and where additional tick sampling would be most valuable to further understand disease trends caused by pathogens transmitted by I. scapularis.

Population genetics of the Ixodes affinis (Ixodida: Ixodidae) complex in America: new findings and a host-parasite review.

Ticks are hematophagous ectoparasites associated with a wide range of vertebrate hosts. Within this group, the Ixodidae family stands out, in which the Ixodes genus contains at least 245 species worldwide, from which 55 species are present in the Neotropical region. Ixodes affinis, a tick described in 1899, has a wide distribution from the Southern Cone of America to the United States. However, since its description, morphological variability has been reported among its populations. Furthermore, attempts have been made to clarify its status as a species complex using mitochondrial markers, but mainly in restricted populations of South and Central America. Thus, information related to populations of the transition region between the Neotropical and Nearctic zones is lacking. For these reasons, the objectives of the study were to evaluate the genetic diversity and structure of I. affinis across the Americas and to compile all the published records of I. affinis in America, to elucidate the host-parasite relationships and to identify their geographical distribution. For this, a phylogeny, and AMOVA analyses were performed to assess the genetic structure of samples obtained by field work in South Carolina, USA and Yucatán, Mexico. A total of 86 sequences were retrieved from a fragment of the 16S region. Phylogeny and genetic structure analysis showed four groups that were geographically and genetically related with high branch support and Fst values, all of them statistically significant. The results obtained support the hypothesis that I. affinis it corresponds to a complex of four species, which must be validated through future morphological comparisons.

Experimental comparison of Baylisascaris procyonis definitive host competence between domestic dogs and raccoons (Procyon lotor).

Domestic dogs can function as either paratenic or definitive hosts for the zoonotic raccoon roundworm Baylisascaris procyonis. However, factors leading to development of patent infections in dogs are under-studied. Here we compared infection dynamics of B. procyonis in dogs vs the natural raccoon host. Dogs and raccoons were inoculated 5000 or 500 B. procyonis eggs (n = 3 per dose) or were fed B. procyonis-infected laboratory mice (n = 3 per dose; mice inoculated with 1000 or 250 eggs). Fecal samples were analysed via flotation and a commercial coproantigen ELISA designed for detection of Toxocara spp. Two of 12 dogs (both received low dose larvae) developed patent infections; all 12 raccoons became infected with 10 developing patent infections. Compared with dogs, prepatent periods were shorter in raccoons and maximum egg outputs were much greater. Baylisascaris procyonis coproantigens were detectable via ELISA in all raccoons and the patently infected dogs. Finally, dogs spontaneously lost infections while all patently infected raccoons shed eggs until conclusion of the study. Our results demonstrate that dogs are clearly suboptimal hosts showing limited parasite establishment and fecundity vs raccoons. Despite the low competence, patently infected dogs still pose a risk for human exposure, emphasizing the importance of control measures.

Rickettsiales in Ticks Removed from Outdoor Workers, Southwest Georgia and Northwest Florida, USA.

We determined the prevalence of selected Rickettsiales in 362 ticks removed from outdoor workers in southwest Georgia and northwest Florida, USA. Persons submitted an average of 1.1 ticks/month. We found Ehrlichia chaffeensis in an Amblyomma maculatum tick, and Panola Mountain Ehrlichia sp. in 2 A. maculatum ticks and 1 Dermacentor variabilis tick.

Theileria orientalis Ikeda Genotype in Cattle, Virginia, USA.

Theileria orientalis Ikeda genotype is a parasite that causes a disease in cattle that results in major economic issues in Asia, New Zealand, and Australia. The parasite is transmitted by Haemaphysalis longicornis ticks, which have recently been reported in numerous states throughout the eastern United States. Concurrently, cattle in Virginia showed clinical signs consistent with a hemoprotozoan infection. We used amplicons specific for the major piroplasm surface protein and small subunit rDNA of piroplasms to test blood samples from the cattle by PCR. Bidirectional Sanger sequencing showed sequences with 100% identity with T. orientalis Ikeda genotype 2 sequences. We detected the parasite in 3 unrelated herds and from various animals sampled at 2 time points. Although other benign T. orientalis genotypes are endemic to the United States, detection of T. orientalis Ikeda genotype might represent a risk for the cattle industry in Virginia.

Antibodies to Borrelia turicatae in Experimentally Infected Dogs Cross-React with Borrelia burgdorferi Serologic Assays.

Tick-borne relapsing fever (TBRF) is caused by several Borrelia spp. (including Borrelia turicatae), which are primarily transmitted by Ornithodoros ticks. Relapsing fever group species are found worldwide, except for Antarctica. Approximately 500 human cases were reported between 1990 and 2011 in the United States (likely an underestimate), while cases in domestic and wild dogs were reported from Florida, Texas, and Washington. TBRF spirochetes are related to Borrelia burgdorferi, the agent of Lyme borreliosis. Dogs are routinely screened for B. burgdorferi, but it is unknown if infection with TBRF agents produces antibodies cross-reactive with B. burgdorferi assays. These data are critical for accurate surveillance of TBRF and Lyme borreliosis in dogs. In this study, B. burgdorferi-negative dogs were inoculated with B. turicatae, and seroconversion was confirmed by the rBipA (recombinant Borrelia immunogenic protein A) Western blot. Seropositive samples were tested with commercial and veterinary diagnostic laboratory B. burgdorferi-based tests. Borrelia turicatae-seroreactive samples cross-reacted with a whole-cell indirect fluorescent antibody (IFA) test and two multiantigen tests, but not with single-antigen tests using C6. Cross-reactivity with TBRF can confound epidemiology and surveillance efforts and confuse recommendations made by veterinarians for prevention and control. These findings demonstrate the need to critically evaluate results from B. burgdorferi diagnostic tests in the context of the assay type and the animal's geographical location and history of travel, as well as highlighting the need for commercially available specific diagnostic tests for TBRF spirochetes.

Effects of temperature on the survival of Sarcoptes scabiei of black bear (Ursus americanus) origin.

For two decades, the incidence and range of sarcoptic mange in black bears (Ursus americanus) in Pennsylvania has increased. The causative agent, Sarcoptes scabiei, can be directly or indirectly transmitted; therefore, data on environmental persistence is important for guiding management and public communications. The objective of this study was to determine the survival of S. scabiei at different temperatures. Full section skin samples and superficial skin scrapes were collected from bears immediately after euthanasia due to severe mange. After ~ 24 h on ice packs (shipment to lab), samples were placed in dishes at 0, 4, 18, or 30 °C and 60, 20, 12, and 25% relative humidity, respectively, and the percentage of mites alive, by life stage, was periodically determined. Humidity was recorded but not controlled. Temperature significantly affected mite survival, which was shortest at 0 °C (mostly ≤ 4 h) and longest at 4 °C (up to 13 days). No mites survived beyond 8 days at 18 °C or 6 days at 30 °C. Mites from full-thickness skin sections survived significantly longer than those from superficial skin scrapes. Adults typically survived longer than nymphs and larvae except at 30 °C where adults survived the shortest time. These data indicate that at cooler temperatures, S. scabiei can survive for days to over a week in the environment, especially if on host skin. However, these data also indicate that the environment is unlikely to be a long-term source of S. scabiei infection to bears, other wildlife, or domestic animals.

Abnormal Helminth Egg Development, Strange Morphology, and the Identification of Intestinal Helminth Infections.

Occasionally, abnormal forms of parasitic helminth eggs are detected during routine diagnostics. This finding can prove problematic in diagnosis because morphologic analysis based on tightly defined measurements is the primary method used to identify the infecting species and molecular confirmation of species is not always feasible. We describe instances of malformed nematode eggs (primarily from members of the superfamily Ascaridoidea) from human clinical practice and experimental trials on animals. On the basis of our observations and historical literature, we propose that unusual development and morphology of nematode and trematode eggs are associated with early infection. Further observational studies and experimentation are needed to identify additional factors that might cause abnormalities in egg morphology and production. Abnormal egg morphology can be observed early in the course of infection and can confound accurate diagnosis of intestinal helminthiases.

Rat Lungworm Infection Associated with Central Nervous System Disease - Eight U.S. States, January 2011-January 2017.

Angiostrongyliasis is caused by infection and migration to the brain of larvae of the parasitic nematode Angiostrongylus cantonensis, or rat lungworm. Adult A. cantonensis reside in the lungs of the definitive wild rodent host, where they produce larvae passed in feces, which are then ingested by snails and slugs (gastropods). Human infection typically occurs when gastropods containing mature larvae are inadvertently ingested by humans. Although human infection often is asymptomatic or involves transient mild symptoms, larval migration to the brain can lead to eosinophilic meningitis, focal neurologic deficits, coma, and death. The majority of cases of human angiostrongyliasis occur in Asia and the Pacific Islands, including Hawaii, but autochthonous and imported cases have been reported in the continental United States (1,2), underscoring the importance of provider recognition to ensure prompt identification and treatment. The epidemiologic and clinical features of 12 angiostrongyliasis cases in the continental United States were analyzed. These cases were identified through A. cantonensis polymerase chain reaction (PCR) testing (3) of cerebrospinal fluid (CSF) submitted to CDC from within the continental United States. Six cases were likely a result of autochthonous transmission in the southern United States. All 12 patients had CSF pleocytosis and eosinophilia, consistent with eosinophilic meningitis. Health care providers need to be aware of the possibility of angiostrongyliasis in patients with eosinophilic meningitis, especially in residents in the southern United States or persons who have traveled outside the continental United States and have a history of ingestion of gastropods or contaminated raw vegetables.

Multistate Infestation with the Exotic Disease-Vector Tick Haemaphysalis longicornis - United States, August 2017-September 2018.

Haemaphysalis longicornis is a tick indigenous to eastern Asia and an important vector of human and animal disease agents, resulting in such outcomes as human hemorrhagic fever and reduction of production in dairy cattle by 25%. H. longicornis was discovered on a sheep in New Jersey in August 2017 (1). This was the first detection in the United States outside of quarantine. In the spring of 2018, the tick was again detected at the index site, and later, in other counties in New Jersey, in seven other states in the eastern United States, and in Arkansas. The hosts included six species of domestic animals, six species of wildlife, and humans. To forestall adverse consequences in humans, pets, livestock, and wildlife, several critical actions are indicated, including expanded surveillance to determine the evolving distribution of H. longicornis, detection of pathogens that H. longicornis currently harbors, determination of the capacity of H. longicornis to serve as a vector for a range of potential pathogens, and evaluation of effective agents and methods for the control of H. longicornis.

Parasitaemia data and molecular characterization of Haemoproteus catharti from New World vultures (Cathartidae) reveals a novel clade of Haemosporida.

BACKGROUND: New World vultures (Cathartiformes: Cathartidae) are obligate scavengers comprised of seven species in five genera throughout the Americas. Of these, turkey vultures (Cathartes aura) and black vultures (Coragyps atratus) are the most widespread and, although ecologically similar, have evolved differences in morphology, physiology, and behaviour. Three species of haemosporidians have been reported in New World vultures to date: Haemoproteus catharti, Leucocytozoon toddi and Plasmodium elongatum, although few studies have investigated haemosporidian parasites in this important group of species. In this study, morphological and molecular methods were used to investigate the epidemiology and molecular biology of haemosporidian parasites of New World vultures in North America. METHODS: Blood and/or tissue samples were obtained from 162 turkey vultures and 95 black vultures in six states of the USA. Parasites were identified based on their morphology in blood smears, and sequences of the mitochondrial cytochrome b and nuclear adenylosuccinate lyase genes were obtained for molecular characterization. RESULTS: No parasites were detected in black vultures, whereas 24% of turkey vultures across all sampling locations were positive for H. catharti by blood smear analysis and/or PCR testing. The phylogenetic analysis of cytochrome b gene sequences revealed that H. catharti is closely related to MYCAMH1, a yet unidentified haemosporidian from wood storks (Mycteria americana) in southeastern USA and northern Brazil. Haemoproteus catharti and MYCAMH1 represent a clade that is unmistakably separate from all other Haemoproteus spp., being most closely related to Haemocystidium spp. from reptiles and to Plasmodium spp. from birds and reptiles. CONCLUSIONS: Haemoproteus catharti is a widely-distributed parasite of turkey vultures in North America that is evolutionarily distinct from other haemosporidian parasites. These results reveal that the genetic diversity and evolutionary relationships of avian haemosporidians are still being uncovered, and future studies combining a comprehensive evaluation of morphological and life cycle characteristics with the analysis of multiple nuclear and mitochondrial genes will be useful to redefine the genus boundaries of these parasites and to re-evaluate the relationships amongst haemosporidians of birds, reptiles and mammals.

Cryptic infection of a broad taxonomic and geographic diversity of tadpoles by Perkinsea protists.

The decline of amphibian populations, particularly frogs, is often cited as an example in support of the claim that Earth is undergoing its sixth mass extinction event. Amphibians seem to be particularly sensitive to emerging diseases (e.g., fungal and viral pathogens), yet the diversity and geographic distribution of infectious agents are only starting to be investigated. Recent work has linked a previously undescribed protist with mass-mortality events in the United States, in which infected frog tadpoles have an abnormally enlarged yellowish liver filled with protist cells of a presumed parasite. Phylogenetic analyses revealed that this infectious agent was affiliated with the Perkinsea: a parasitic group within the alveolates exemplified by Perkinsus sp., a "marine" protist responsible for mass-mortality events in commercial shellfish populations. Using small subunit (SSU) ribosomal DNA (rDNA) sequencing, we developed a targeted PCR protocol for preferentially sampling a clade of the Perkinsea. We tested this protocol on freshwater environmental DNA, revealing a wide diversity of Perkinsea lineages in these environments. Then, we used the same protocol to test for Perkinsea-like lineages in livers of 182 tadpoles from multiple families of frogs. We identified a distinct Perkinsea clade, encompassing a low level of SSU rDNA variation different from the lineage previously associated with tadpole mass-mortality events. Members of this clade were present in 38 tadpoles sampled from 14 distinct genera/phylogroups, from five countries across three continents. These data provide, to our knowledge, the first evidence that Perkinsea-like protists infect tadpoles across a wide taxonomic range of frogs in tropical and temperate environments, including oceanic islands.

Possible Role of Fish as Transport Hosts for Dracunculus spp. Larvae.

To inform Dracunculus medinensis (Guinea worm) eradication efforts, we evaluated the role of fish as transport hosts for Dracunculus worms. Ferrets fed fish that had ingested infected copepods became infected, highlighting the importance of recommendations to cook fish, bury entrails, and prevent dogs from consuming raw fish and entrails.

BAYLISASCARIS PROCYONIS LARVA MIGRANS IN TWO CAPTIVE NORTH AMERICAN BEAVERS (CASTOR CANADENSIS).

Baylisascaris procyonis larva migrans was diagnosed in two North American beavers ( Castor canadensis ) belonging to a zoological park in Clarke County, Georgia. Both beavers presented with neurological signs. One beaver died naturally and despite attempted treatment, the other beaver was euthanatized because of severe clinical signs and poor prognosis. Histologic evaluation of the beavers revealed evidence of parasitic migration characterized by several lesions, including eosinophilic granulomas in various organs, as well as necrotizing eosinophilic and lymphoplasmacytic to granulomatous polioencephalitis, leukoencephalitis and cervical leukomyelitis. This represents the first confirmed case of B. procyonis larva migrans in beaver and the first non-raccoon ( Procyon lotor ) host in the southeastern United States. This report highlights the need for clinicians and diagnosticians to consider baylisascariasis in animals with compatible clinical signs. Preventative measures should be considered for captive animals, because early diagnosis of B. procyonis is challenging, and treatment is often unrewarding.

Guinea Worm (Dracunculus medinensis) Infection in a Wild-Caught Frog, Chad.

A third-stage (infective) larva of Dracunculus medinensis, the causative agent of Guinea worm disease, was recovered from a wild-caught Phrynobatrachus francisci frog in Chad. Although green frogs (Lithobates clamitans) have been experimentally infected with D. medinensis worms, our findings prove that frogs can serve as natural paratenic hosts.

Possible Role of Fish and Frogs as Paratenic Hosts of Dracunculus medinensis, Chad.

Copepods infected with Dracunculus medinensis larvae collected from infected dogs in Chad were fed to 2 species of fish and tadpoles. Although they readily ingested copepods, neither species of fish, Nile tilapia (Oreochromis niloticus) nor fathead minnow (Pimephalis promelas), were found to harbor Dracunculus larvae when examined 2-3 weeks later. Tadpoles ingested copepods much more slowly; however, upon examination at the same time interval, tadpoles of green frogs (Lithobates [Rana] clamitans) were found to harbor small numbers of Dracunculus larvae. Two ferrets (Mustela putorius furo) were fed fish or tadpoles that had been exposed to infected copepods. Only the ferret fed tadpoles harbored developing Dracunculus larvae at necropsy 70-80 days postexposure. These observations confirm that D. medinensis, like other species in the genus Dracunculus, can readily survive and remain infective in potential paratenic hosts, especially tadpoles.

Baylisascaris procyonis Roundworm Seroprevalence among Wildlife Rehabilitators, United States and Canada, 2012-2015.

Baylisascaris procyonis roundworms can cause potentially fatal neural larva migrans in many species, including humans. However, the clinical spectrum of baylisascariasis is not completely understood. We tested 347 asymptomatic adult wildlife rehabilitators for B. procyonis antibodies; 24 were positive, suggesting that subclinical baylisascariasis is occurring among this population.