GENETIC AND TRAIT VARIABILITY OF GYRINICOLA REVEALS THE EXISTENCE OF AT LEAST FOUR SPECIES WITHIN THE UNITED STATES.

The tadpole-dwelling pinworm, Gyrinicola batrachiensis (Walton, 1929) Adamson, 1981 was recognized as the sole representative of the genus across Canada and the United States. However, evaluation of the morphology of these parasites across their range revealed considerable morphological variability that suggested diagnosable morphotypes. These morphotypes were associated with different species of anurans, several of which occurred in sympatry. Herein we use an extensive geographic sampling across the United States to obtain the morphotypes, screen their genetic diversity, and analyze this information using an integrative approach. We reconstructed their phylogeny using nuclear ribosomal partial genes 18S and 28S, ITS1, 5.8S, and ITS2, as well as 5 mitochondrial genes generated with Next-Generation sequencing technology. This phylogeny reveals 3 well-resolved lineages, which upon the use of a statistical approach (bPTP [Bayesian implementation of the Poisson tree processes]) supports the delimitation of 4 distinct groups equivalent to species. These putative species groups were tested using morphological characteristics paired with a MANOVA and canonical variate analysis. Results suggest that at least 4 species of Gyrinicola are present within North America, resulting in the resurrection of G. armatus (Walton, 1933) and the description of 2 new species.

Venipuncture and Blood Collection on Conscious Virginia Opossums (Didelphis virginiana).

We developed a venipuncture technique of the ventral caudal vein in conscious Virginia opossums (Didelphis virginiana) in cage traps, enabling blood sampling without anesthesia. Blood samples were successfully collected from all 28 opossums in this study by this technique. Draw volume of <0.1 mL occurred in only four opossums (14%).

MOLECULAR PHYLOGENETIC ANALYSIS OF GYRINICOLA BATRACHIENSIS (NEMATODA: OXYUROIDEA) LENDS SUPPORT TO THE MONOPHYLY OF THE GENUS AND THE RESURRECTION OF GYRINICOLINAE.

Gyrinicola Yamaguti, 1938, includes 6 species of oxyurid found within the intestinal tract of numerous, larval, anuran species in Europe, Asia, South America, and North America. The systematic placement and hierarchical treatment of the genus has shifted at least 5 times since its discovery; the group was first considered as its own family (Gyrinicolidae), then treated as a subfamily (Gyrinicolinae) of Cosmocercidae, then as a member of the Pharyngodonidae, followed by movement back to the Cosmocercidae, and finally a recent proposal suggested the resurrection of the Gyrinicolidae. Species in the genus vary widely in the morphology of the uterine tract, a characteristic often used to indicate membership in the genus, as it is tied to the reproductive mode. However, until recently very few genetic data were available to aid in the placement of this unique group of worms, and before this study to the best of our knowledge none existed for the North American species. To examine the monophyly and placement of the Gyrinicola we sampled populations of Gyrinicola batrachiensis across North America and screened them for genetic diversity using nuclear markers 18S and 28S. Phylogenies suggest at least 3 clades exist among the nematodes from North America and that these clades, alongside Gyrinicola japonica, form a well-supported group within Oxyuroidea. Further representation of Pharyngodonidae from other vertebrate classes may help clarify the relationship of this historical grouping to other members of the Oxyuroidea.

CLINICAL AND SUBCLINICAL CYTAUXZOON FELIS INFECTIONS IN DOMESTIC CATS FROM A RECENTLY IDENTIFIED ENDEMIC REGION.

Cytauxzoon felis is a tick-transmitted intraerythrocytic apicomplexan infecting felids in the southeastern and midwestern United States. Bobcats (Lynx rufus) are the natural wildlife reservoir of C. felis, where in enzootic areas prevalence can reach 100%. Domestic cats can be subclinically infected with C. felis or can develop cytauxzoonosis. Two studies have documented the presence of C. felis in domestic cats in Illinois; these studies have shown a limited number of cases submitted to specialty labs. During 2014-2018, we obtained blood samples collected by veterinary staff from 514 cats that were apparently healthy and 74 cats that were suspected of cytauxzoonosis. These samples were screened using a sensitive, nested PCR to detect the presence of C. felis DNA. We herein document frequent occurrences of cytauxzoonosis (8-15 cases/year from 4 veterinary clinics) and 12.5% prevalence of subclinical infections in southern Illinois, a locality showing a sharp increase in cases of cytauxzoonosis. Our results suggest a high risk of cytauxzoonosis in southern Illinois, despite only recently being recognized in the area. We found no specific risk factors for cytauxzoonosis or subclinical infections in this location. In addition, cases of cytauxzoonosis occur every month of the year (with the highest frequency in summer) and therefore tick prevention should be used in domestic cats in enzootic regions throughout the year.

Three new species of Cytauxzoon in European wild felids.

Protists of the genus Cytauxzoon infect a wide variety of wild and domestic felids worldwide. While the American Cytauxzoon felis has been well described, data on the European isolates of Cytauxzoon are still scant. The aim of the current study was to determine the genetic diversity of European Cytauxzoon spp. in wild felids across Europe by analyzing one nuclear and two mitochondrial genes, along with representative complete mitochondrial genomes. Overall, 106 biological samples from wild felids (92 from Felis silvestris and 14 from Lynx lynx) from Germany, Romania, Czech Republic, and Luxembourg were collected and screened for the presence of Cytauxzoon spp. using nested PCR protocols, targeting the highly conserved 18S rDNA, mitochondrial cytochrome b (CytB) and cytochrome c oxidase subunit I (COI) genes. Furthermore, 18 previously confirmed wild felid biological samples from Europe, and comparative material from USA positive for C. felis, were included in the study. In 18S rDNA sequences analyses, Cytauxzoon spp. from felids formed two separate clades of New World and Old World isolates, with a low inner diversity of the European clade. In contrast to 18S rDNA, the phylogenetic analyses of CytB and COI genes affirmatively revealed three highly supported clades, resulting in three defined genotypes. Similar intra- and interspecific variability of CytB and COI genes was observed in the case of different Babesia spp. Considering geography, host species and analyses of three genes, we conclude that the three detected genotypes of Cytauxzoon in European wild felids represent three new species, which we herein describe.

Within-Season Changes in Cytauxzoon felis Parasitemia in Bobcats.

Cytauxzoon felis is a pathogen responsible for cytauxzoonosis, a highly fatal disease in domestic cats. Although most studies of C. felis have focused on this parasite in domestic cats, bobcats are the reservoir host. In stark contrast, there is little information relative to the progression of C. felis infections in bobcats. We studied bobcats in southern Illinois during 2014-2017 to evaluate which environmental factors (i.e., ambient temperature; number of daylight hours; trapping year, month, and day) influenced C. felis parasitemia levels. Mean ambient temperature at 1 wk and 2 wk prior to sampling was associated with increased parasitemia levels. Vector activity intensifies with higher temperatures, suggesting that increased parasitemia levels are an adaptation to facilitate transmission.

Documentation of the Expansion of the Gulf Coast Tick (Amblyomma maculatum) and Rickettsia parkeri: First Report in Illinois.

The Gulf Coast tick, Amblyomma maculatum, is of public and veterinary health concern, as it is the primary vector of Rickettsia parkeri and Hepatozoon americanum, causative agents of Rickettsiosis and American canine hepatozoonosis. The Gulf Coast tick's range has expanded over the last 50 yr into the mid-Atlantic states, and its expansion is expected to continue northward. We are reporting the presence of A. maculatum for the first time in Illinois, including a total of 18 specimens collected at 6 different sites during surveys in 2013 and 2019. Fourteen of these specimens were screened for Rickettsia parkeri, which resulted in the detection of this bacteria in 8 samples from 4 counties. By depositing these specimens in scientific collections, we provide materialistic evidence of their establishment in 2 counties. We urge health officials to rely on and use scientific collections to document the expansion of these and other vectors across the country. Additionally, we recommend that health practitioners become aware of the clinical similarities between Rocky Mountain Spotted Fever (caused by Rickettsia rickettsii) and "tidewater" fever (caused by R. parkeri).

Chronic Cytauxzoon felis infections in wild-caught bobcats (Lynx rufus).

Cytauxzoon felis, and the resulting disease, cytauxzoonosis, is an emerging threat to domestic cats in the Midwest and Southeastern United States. Domestic cats that survive cytauxzoonosis (or are subclinically infected) are chronically infected with C. felis, yet to date, there is no information relative to chronic infections in bobcats, the natural reservoir. Over a period of 3.5 years (2014-2017), we captured and re-captured 5 bobcats in southern Illinois. One bobcat was captured each year of trapping, 1 was caught in the first and third year and 3 were recaptured approximately 1 year apart. We screened bobcats for the presence of C. felis using a nested PCR that amplified the nuclear small subunit (SSU) 18S rRNA. In addition, we amplified and sequenced the internal transcribed spacers 1 and 2 (ITS1 and ITS2) to detect if the strains of C. felis in each bobcat were consistent over time. All bobcats were positive for C. felis at the initial and subsequent capture(s). Bobcats that were PCR-positive for C. felis had blood smears screened for the presence of C. felis; all PCR-positive bobcats had detectable parasites in blood smears. The strains of C. felis present were consistent each year in 4 of 5 bobcats indicating these bobcats remained infected during this period. One bobcat appeared to be infected with a different strain based on a polymorphism at a nucleotide in ITS1. Our study provides important details of the epizootiology of C. felis: bobcats are chronically infected and are not immune to reinfection with new strains of C. felis.

Concurrent Examination of Bobcats and Ticks Reveals High Prevalence of Cytauxzoon felis in Southern Illinois.

Cytauxzoon felis is an intraerythrocytic apicomplexan of felids enzootic in the southeastern United States. In domestic cats (Felis catus), this parasite can result in the highly fatal disease cytauxzoonosis or bobcat fever. Bobcats (Lynx rufus) are the wild animal reservoir host. To date, the characterization of prevalence of C. felis in bobcats is mostly based on broad-scale surveys from hunter-harvested specimens collected across large geographic areas, usually consisting of multiple states. Detailed studies on the development, transmission, distribution, effects, and prevalence of C. felis in the tick vectors are scarce. To fill some of these gaps in the literature, such as prevalence in ticks and bobcats in a discrete region, we examined bobcats and ticks in an 8,000-km2 portion of southern Illinois. We screened for C. felis using a nested polymerase chain reaction that amplifies a fragment of the nuclear small subunit (SSU) 18S rRNA. We screened 125 individual bobcats collected in southern Illinois from 2003 to 2015; of these, 70.6% were positive for C. felis. In addition, we screened 214 ticks of both vector species (Amblyomma americanum and Dermacentor variabilis) and detected C. felis in 15.6% of them; this prevalence is higher than reported by previous surveys. Our study reports the prevalence of C. felis in ticks and bobcats from south Illinois. We found that 70.6% of bobcats and 15.6% of ticks were infected with C. felis, which suggests risk of transmission to domestic cats.