Correction: Why Lyme disease is common in the northern US, but rare in the south: The roles of host choice, host-seeking behavior, and tick density.

[This corrects the article DOI: 10.1371/journal.pbio.3001066.].

Why Lyme disease is common in the northern US, but rare in the south: The roles of host choice, host-seeking behavior, and tick density.

Lyme disease is common in the northeastern United States, but rare in the southeast, even though the tick vector is found in both regions. Infection prevalence of Lyme spirochetes in host-seeking ticks, an important component to the risk of Lyme disease, is also high in the northeast and northern midwest, but declines sharply in the south. As ticks must acquire Lyme spirochetes from infected vertebrate hosts, the role of wildlife species composition on Lyme disease risk has been a topic of lively academic discussion. We compared tick-vertebrate host interactions using standardized sampling methods among 8 sites scattered throughout the eastern US. Geographical trends in diversity of tick hosts are gradual and do not match the sharp decline in prevalence at southern sites, but tick-host associations show a clear shift from mammals in the north to reptiles in the south. Tick infection prevalence declines north to south largely because of high tick infestation of efficient spirochete reservoir hosts (rodents and shrews) in the north but not in the south. Minimal infestation of small mammals in the south results from strong selective attachment to lizards such as skinks (which are inefficient reservoirs for Lyme spirochetes) in the southern states. Selective host choice, along with latitudinal differences in tick host-seeking behavior and variations in tick densities, explains the geographic pattern of Lyme disease in the eastern US.

A De Novo Whole Genome Assembly and Annotation of Parelaphostrongylus tenuis.

Parelaphostrongylus tenuis causes ungulate morbidity and mortality in eastern and central North America, but no reference genome sequence exists to facilitate research. Here, we present a P. tenuis genome assembly and annotation, generated with PacBio and Illumina technologies. The assembly is 491 Mbp, with 7285 scaffolds and 185 kb N50.

Possible Cross-Reactivity of Feline and White-Tailed Deer Antibodies against the SARS-CoV-2 Receptor Binding Domain.

In late 2019, a novel coronavirus began circulating within humans in central China. It was designated SARS-CoV-2 because of its genetic similarities to the 2003 SARS coronavirus (SARS-CoV). Now that SARS-CoV-2 has spread worldwide, there is a risk of it establishing new animal reservoirs and recombination with native circulating coronaviruses. To screen local animal populations in the United States for exposure to SARS-like coronaviruses, we developed a serological assay using the receptor binding domain (RBD) from SARS-CoV-2. SARS-CoV-2's RBD is antigenically distinct from common human and animal coronaviruses, allowing us to identify animals previously infected with SARS-CoV or SARS-CoV-2. Using an indirect enzyme-linked immunosorbent assay (ELISA) for SARS-CoV-2's RBD, we screened serum from wild and domestic animals for the presence of antibodies against SARS-CoV-2's RBD. Surprisingly prepandemic feline serum samples submitted to the University of Tennessee Veterinary Hospital were ∼50% positive for anti-SARS RBD antibodies. Some of these samples were serologically negative for feline coronavirus (FCoV), raising the question of the etiological agent generating anti-SARS-CoV-2 RBD cross-reactivity. We also identified several white-tailed deer from South Carolina with anti-SARS-CoV-2 antibodies. These results are intriguing, as cross-reactive antibodies toward SARS-CoV-2 RBD have not been reported to date. The etiological agent responsible for seropositivity was not readily apparent, but finding seropositive cats prior to the current SARS-CoV-2 pandemic highlights our lack of information about circulating coronaviruses in other species. IMPORTANCE We report cross-reactive antibodies from prepandemic cats and postpandemic South Carolina white-tailed deer that are specific for that SARS-CoV RBD. There are several potential explanations for this cross-reactivity, each with important implications to coronavirus disease surveillance. Perhaps the most intriguing possibility is the existence and transmission of an etiological agent (such as another coronavirus) with similarity to SARS-CoV-2's RBD region. However, we lack conclusive evidence of prepandemic transmission of a SARS-like virus. Our findings provide impetus for the adoption of a One Health Initiative focusing on infectious disease surveillance of multiple animal species to predict the next zoonotic transmission to humans and future pandemics.

Human impact on the diversity and virulence of the ubiquitous zoonotic parasite Toxoplasma gondii.

A majority of emerging infectious diseases in humans are zoonoses. Understanding factors that influence the emergence and transmission of zoonoses is pivotal for their prevention and control. Toxoplasma gondii is one of the most widespread zoonotic pathogens known today. Whereas only a few genotypes of T. gondii dominate in the Northern Hemisphere, many genotypes coexist in South America. Furthermore, T. gondii strains from South America are more likely to be virulent than those from the Northern Hemisphere. However, it is not clear what factor(s) shaped modern-day genetic diversity and virulence of T. gondii Here, our analysis suggests that the rise and expansion of farming in the past 11,000 years established the domestic cat/mouse transmission cycle for T. gondii, which has undoubtedly played a significant role in the selection of certain linages of T. gondii Our mathematical simulations showed that within the domestic transmission cycle, intermediately mouse-virulent T. gondii genotypes have an adaptive advantage and eventually become dominant due to a balance between lower host mortality and the ability to superinfect mice previously infected with a less virulent T. gondii strain. Our analysis of the global type II lineage of T. gondii suggests its Old World origin but recent expansion in North America, which is likely the consequence of global human migration and trading. These results have significant implications concerning transmission and evolution of zoonotic pathogens in the rapidly expanding anthropized environment demanded by rapid growth of the human population and intensive international trading at present and in the future.

Seroprevalence, DNA isolation, and genetic characterization of Toxoplasma gondii from black bear (Ursus americanus) sera collected in Eastern Oklahoma.

Black bears (Ursus americanus) are commonly exposed to Toxoplasma gondii. However, there are no reports of exposure or infection with T. gondii in black bears from Oklahoma. The purpose of our project was to determine the seroprevalence of T. gondii antibodies in black bears collected in Oklahoma. Additionally, since only serum was available from these bears, we sought to determine if DNA extraction and PCR amplification for T. gondii was possible on serum samples from bears with positive titers. Seroprevalence was determined using modified agglutination test (MAT). Serum was collected from 44 live-trapped bears in southeastern Oklahoma; 32 (73% ± 58-84%) had antibodies against T. gondii. Seroprevalence in adult bears (85% ± 67-95%) was significantly higher (p = 0.028) than yearlings (33.0% ± 56-80%). Adult bears were 3.4 times more likely to have antibodies to T. gondii than yearlings. From the bears with positive titers, T. gondii DNA was detected in 12 of the 32 seropositive samples by PCR of the B1 gene, with two of the samples showing variation in two nucleotide positions when compared with available sequences. Multilocus PCR-RFLP genotyping of these 12 samples revealed three ToxoDB genotypes, including #2 (type III, haplogroup 3), #4 (type XII, haplogroup 12), and #74 (haplogroup 12). To the best of our knowledge, this is the first report of T. gondii seroprevalence in black bears from Oklahoma. Our results indicate that exposure and infection with T. gondii in black bears from Oklahoma is common.

GENOTYPE IDENTIFICATION OF TOXOPLASMA GONDII IN MACROPODS FROM A ZOOLOGICAL PARK IN FLORIDA, USA.

There are limited reports of the genetic characterization of Toxoplasma gondii infecting captive macropods in North America. A novel genotype, ToxoDB PCR-RFLP genotype 263, was reported from six wallabies at a zoological facility in Virginia, USA, prompting an investigation into the genotypes from T. gondii strains infecting macropods at a zoological park in Florida, USA. Cardiac muscle and/or lung samples from an agile wallaby (Macropus agilis, n = 1), red kangaroos (Macropus rufus, n = 8), red-necked wallaby (Macropus rufogriseus, n = 1), and a tammar wallaby (Macropus eugenii, n = 1) that died between 2014 and 2018 were collected. All 11 cases were confirmed to have died from systemic toxoplasmosis by histopathology and immunohistochemical staining. Multilocus PCR-RFLP genotyping of T. gondii was performed directly on tissue samples or on parasites isolated from myocardium by mouse bioassay. Two cases of toxoplasmosis were identified as the reported novel genotype, ToxoDB PCR-RFLP genotype 263, but no common source of exposure could be identified. Five cases were identified as genotype 2 (type III strain, haplogroup 3), and four cases were identified as genotype 216, which has been previously reported in North American wildlife. There were no overt differences in lesion severity or distribution related to genotype. These results suggest that the premise was contaminated with at least three genotypes of T. gondii causing systemic toxoplasmosis in macropods. The largest cluster of fatal toxoplasmosis in macropods in the study period occurred following severe rainfall flooding of the exhibit, suggesting the transmission of T. gondii by water and pointing out the importance of this transmission mechanism. In summary, our study revealed three T. gondii outbreaks that caused significant loss of macropods within 5 yr in a zoological facility in Florida. More studies are needed to understand transmission and prevention of toxoplasmosis in sensitive zoo animals.

SEROSURVEY, HEMATOLOGY, AND CAUSES OF MORTALITY OF FREE-RANGING AMERICAN MARTENS ( MARTES AMERICANA) IN MICHIGAN.

To better understand the clinical pathology, diseases, and causes of mortality of reintroduced American martens ( Martes americana) in Michigan, a study was conducted from 2011 to 2015 in the Upper and Lower Peninsulas of Michigan. Samples obtained from live trapping ( n = 58) or harvested carcasses ( n = 34) were serologically tested for select pathogens. Antibodies against Toxoplasma gondii and canine distemper virus were detected in 58 and 3.4% of samples, respectively. All samples were seronegative for Leptospira spp. and negative for Dirofilaria immitis antigen. Urine samples tested for Leptospira spp. via immunofluorescent antibody assay ( n = 7), polymerase chain reaction ( n = 6) , or both ( n = 3) were all negative. Parvovirus DNA was detected in 9.1% of small intestine samples ( n = 22) collected from carcasses and in 3.7% of fecal samples ( n = 27) collected during live trapping. Complete blood counts ( n = 64) and serum biochemistries ( n = 63) were obtained from 49 live-trapped martens. Biochemical parameters found to be significantly different ( P < 0.05) between genders were calcium, creatinine, glucose, and phosphorus. There was no significant difference between genders for any hematologic parameter. Significant differences ( P < 0.05) between summer and winter seasons were found in total estimated white blood cell count, neutrophils, lymphocytes, monocytes, alkaline phosphatase, bicarbonate, calcium, creatinine, globulin, glucose, phosphorus, potassium, sodium, and total protein. There was no significant difference in blood cell count or serum biochemistry values between radio-collared ( n = 17) and noncollared ( n = 47) martens. Animals seropositive for T. gondii were found to have significantly higher ( P < 0.05) eosinophil and globulin levels than seronegative animals. The primary natural cause for mortality of radio-collared American martens was predation. Histologic examinations revealed a high percentage (60%) of martens with verminous or granulomatous pneumonia.

Investigating seagrass in Toxoplasma gondii transmission in Florida (Trichechus manatus latirostris) and Antillean (T. m. manatus) manatees.

Toxoplasma gondii is a feline protozoan reported to cause morbidity and mortality in manatees and other marine mammals. Given the herbivorous nature of manatees, ingestion of oocysts from contaminated water or seagrass is presumed to be their primary mode of infection. The objectives of this study were to investigate oocyst contamination of seagrass beds in Puerto Rico and determine the seroprevalence of T. gondii in Antillean (Trichechus manatus manatus) and Florida (T. m. latirostris) manatees. Sera or plasma from Antillean (n = 5) and Florida (n = 351) manatees were tested for T. gondii antibodies using the modified agglutination test. No T. gondii DNA was detected via PCR in seagrass samples (n = 33) collected from Puerto Rico. Seroprevalence was 0%, suggesting a lower prevalence of T. gondii in these manatee populations than previously reported. This was the first study to investigate the potential oocyst contamination of the manatee diet, and similar studies are important for understanding the epidemiology of T. gondii in herbivorous marine mammals.

NEWLY DESCRIBED TOXOPLASMA GONDII STRAIN CAUSES HIGH MORTALITY IN RED NECKED WALLABIES (MACROPUS RUFOGRISEUS) IN A ZOO.

This manuscript describes an outbreak of fatal toxoplasmosis in wallabies. Ten adult red necked wallabies (Macropus rufogriseus) were imported from New Zealand to the Virginia Zoo. Agglutination testing upon admission into quarantine showed all animals to be negative for antibodies to Toxoplasma gondii. Nine of these wallabies died from acute toxoplasmosis within 59-565 (average 224) days after being moved onto exhibit. Clinical signs included lethargy, diarrhea, tachypnea, and ataxia that progressed rapidly; death without premonitory signs occurred in one case. Histopathologic examination revealed interstitial pneumonia, encephalomyelitis, myositis, enteritis, and myocarditis. The diagnosis was confirmed through serologic, histopathologic, and polymerase chain reaction (PCR) testing. Multilocus PCR-RFLP (restriction fragment length polymorphism) genotyping revealed that the first six animals were infected by a previously undiscovered Toxoplasma gondii genotype, designated as ToxoDB PCR-RFLP genotype No. 263. These six cases survived for an average of 118 days on exhibit before succumbing to toxoplasmosis. The other three wallabies were infected with a Toxoplasma gondii strain of ToxoDB PCR-RFLP genotype No. 4, which is a common strain type circulating in wild animals in North America. These three cases survived for an average of 435 days on exhibit before succumbing to toxoplasmosis. The outbreaks of toxoplasmosis in these wallabies are likely from two different sources. Furthermore, the results highlight Toxoplasma gondii PCR-RFLP genotyping in parasite diagnosis and understanding parasite transmission and potential mitigation procedures.

Persistence of Two Isolates of Trichomonas gallinae in Simulated Bird Baths With and Without Organic Material.

Trichomonas gallinae, a well-documented protozoan parasite of avian hosts, has been implicated in major passerine mortality events recently and historically throughout the literature. It has been suggested that bird baths and artificial water sources could serve as a source of infection for naive birds; however, trichomonad persistence in water is not well understood. We measured the persistence of T. gallinae isolates from two avian hosts in distilled water and distilled water with the addition of organic material. We inoculated plastic containers in a laboratory setting with 1 × 10(6) trichomonads and then sampled 500 µl from each container at various time points postinoculation (0-20 hr). The 500-µl aliquots were inoculated into flasks with 5 ml of modified Diamond media at each time point. Flasks were incubated at 37 C and examined by light microscopy for five consecutive days for the characteristic movements of live trichomonads. The maximum persistence was 16 hr with a Cooper's hawk (Accipiter cooperii) isolate in the organic material treatment, far longer than the 1 hr persistence previously reported. We show that T. gallinae isolates are capable of persisting for long periods of time in water, illustrating that bird baths may be validated as a potential source of transmission in epidemics.

Frequent cross-species transmission of parvoviruses among diverse carnivore hosts.

Although parvoviruses are commonly described in domestic carnivores, little is known about their biodiversity in nondomestic species. A phylogenetic analysis of VP2 gene sequences from puma, coyote, gray wolf, bobcat, raccoon, and striped skunk revealed two major groups related to either feline panleukopenia virus ("FPV-like") or canine parvovirus ("CPV-like"). Cross-species transmission was commonplace, with multiple introductions into each host species but, with the exception of raccoons, relatively little evidence for onward transmission in nondomestic species.

Serologic survey of antibodies to Trypanosoma cruzi in coyotes and red foxes from Pennsylvania and Tennessee.

Trypanosoma cruzi is a zoonotic parasite of humans and other mammalian hosts with distribution throughout the Americas. Domestic and wild canine species are reservoirs for human T. cruzi infections. The present study examined the prevalence of antibodies to T. cruzi in wild canids from the United States. Sera from 13 red foxes (Vulpes vulpes) and 263 coyotes (Canis latrans), originating in Pennsylvania and Tennessee, were assayed for antibodies to T. cruzi with immunochromatographic tests. Antibodies to T. cruzi were found in 2 of 276 (0.72%) of all wild canids tested. Both T. cruzi-positive wild canids were coyotes and represented 2 of 21 (9.52%) wild canids assayed from Tennessee. Antibodies to T. cruzi were not detected in red fox. Anti-T. cruzi antibodies were not found in any wild canids from Pennsylvania. These results suggest that coyotes are exposed to T. cruzi in Tennessee but not in Pennsylvania.

RAPID POINT-OF-CARE TESTING FOR DETECTION OF ANTIBODIES TO TOXOPLASMA GONDII IN BLACK VULTURES AND RING-BILLED GULLS FROM PENNSYLVANIA.

Toxoplasma gondii is a zoonotic protozoan parasite that infects most warm-blooded animals, including birds. Scavenging birds are epidemiologically important hosts because they can serve as indicators of environmental T. gondii levels. A rapid point-of-care (POC) test that detects antibodies to T. gondii in humans is commercially available. In this research, we assessed the ability of the human POC test to detect anti-T. gondii antibodies in 106 black vultures (Coragyps atratus) and 23 ring-billed gulls (Larus delawarensis) from Pennsylvania, USA. Serum samples were tested with the POC test and compared to the modified agglutination test (MAT) in a blinded study. Overall, anti-T. gondii antibodies were detected in 2.8% (3/106) of black vultures and 60.9% (14/23) of ring-billed gulls by the POC test. One false-positive POC test occurred in a black vulture that was negative by MAT. False-negative results were obtained in 2 black vultures and 4 ring-billed gulls that had MAT titers of 1:25 or 1:50. The sensitivity and specificity of the POC for both black vultures and ring-billed gulls combined were 95.7% and 95.5%, respectively. This is the first study using human POC tests to detect antibodies to T. gondii in birds. Further study of the rapid test as a screening tool for serological surveillance of T. gondii in birds is warranted.

Toxoplasma gondii Survey in Waterfowl and Gulls from Eight USA States.

Sera from 391 waterbirds from eight USA states were tested for Toxoplasma gondii antibodies using the modified agglutination test. Fifteen different waterbird species (26.6%; n=104) were seropositive. Of the adults, 25.4% (n=52) showed a significantly higher T. gondii seroprevalence compared with juveniles (13.4%; n=17); however, sex was not a significant factor.

Isolation of Toxoplasma gondii in cell culture: an alternative to bioassay.

Toxoplasma gondii is an apicomplexan protozoan parasite that can infect mammals and birds. The infection can cause acute toxoplasmosis and death in susceptible hosts. Bioassay using cats and mice has been the standard for the isolation of T. gondii from infected hosts for the past several decades. However, bioassay is labor-intensive, expensive, and involves using laboratory animals. To search alternative approaches and o work towards replacement of animal experiments, we summarized the key literature and conducted four experiments to isolate T. gondii in vitro by cell culture. A few heart tissue samples from animals with the highest antibody titers in a given collection were used for T. gondii isolation. These experiments included samples from five out of 51 wild ducks, four of 46 wild turkeys, six of 24 white-tailed deer, as well as from six kangaroos that had died with acute toxoplasmosis in a zoo. These experiments resulted in three isolates from five chronically infected wild ducks (60%), four isolates from four chronically infected wild turkeys (100%), one isolate from six chronically infected white-tailed deer (17%), and four isolates from six kangaroos with acute toxoplasmosis (67%). In addition, five isolates from the five chronically infected wild ducks were obtained by bioassay in mice, showing a 100% success rate, which is higher than the 60% rate by direct cell culture. These T. gondii isolates were successfully propagated in human foreskin fibroblast (HFF) or Vero cells, and genotyped by multilocus PCR-RFLP markers. The results showed that it is practical to isolate T. gondii directly in cell culture. Although the cell culture approach may not be as sensitive as the bioassay, it does provide an alternative that is simple, cost-effective, ethically more acceptable, and less time-sensitive to isolate T. gondii. In this paper we propose a procedure that may be applied and further optimized for isolation of T. gondii.

Persistence of two Trichomonas gallinae isolates in chlorinated and distilled water with or without organic material.

Trichomonas gallinae is a protozoan parasite commonly found in columbids, passerines, and raptors. In passerines and columbids, trichomonosis causes significant morbidity and mortality associated with contaminated bird feeders and waters. However, there has been little work on the persistence of T. gallinae in water to determine if artificial waters are a likely source of infection for naive birds. To examine drinking water as a source of T. gallinae transmission, we inoculated 1 x 10(6) trichomonads into containers with 500 ml of either distilled or chlorinated water. In addition, we inoculated the same number of trichomonads in distilled or chlorinated water contaminated with 15 g organic matter. Aliquots of 0.5 ml were collected from each container at 0, 0.5, 1, 5, 10, or 20 min; inoculated into a Trichomonas culture packet; and incubated at 37 C for 6 days. Survival was best in the presence of organic matter, with either distilled or chlorinated water. Uncontaminated chlorinated water did not allow survival at any sampling period.

Survey of antibodies to Leishmania spp. in wild canids from Pennsylvania and Tennessee.

Visceral leishmaniasis (VL) is a zoonosis with worldwide distribution. Infections with the Leishmania donovani complex, including Leishmania infantum, cause the VL. Domestic dogs are the most important reservoir host for human VL, and wild canids are also susceptible. In the United States, infections with L. infantum are common in the foxhound dog breed. Little information is available regarding L. infantum in wild canids in the Unites States. Sera from 11 foxes and 256 coyotes originating in Pennsylvania and Tennessee (USA) were tested for antibodies to visceralizing Leishmania spp. with rapid immunochromatographic dipstick assays, which utilize recombinant antigen K39. Anti-Leishmania spp. antibodies were found in 5 of 267 (1.9%) of wild canids from Pennsylvania, including four coyotes and one red fox. These results suggest that wild canids are exposed to Leishmania spp. at a low level in the United States.

Case report: Halicephalobus gingivalis in a Tennessee pony.

A 17-year-old female grade pony presented to University of Tennessee Veterinary Medical Center in May of 2021 for evaluation of multifocal, firm, sessile, circular lesions of various diameters on the ventrum and flank. The lesions had been present for two weeks at presentation. An excisional biopsy found numerous adult and larval rhabditid nematodes most consistent with Halicephalobus gingivalis. PCR targeting a portion of the large ribosomal subunit confirmed this diagnosis. The patient was treated with a high dose course of ivermectin followed by fenbendazole. The patient began showing neurologic signs five months after initial diagnosis. Due to the poor prognosis, euthanasia was elected. PCR of CNS tissues confirmed the presence of H. gingivalis in the brain, and one adult worm and several larvae were found on histologic sections of the cerebellum. H. gingivalis is a rare but lethal disease of horses and people.

Novel methods of immunogenic antigen selection for serological diagnosis of Parelaphostrongylus tenuis infection.

This paper outlines methods used to identify novel antigens for use in the development of serological assays. Specifically, we applied these methods to a neurogenic parasitic nematode of cervids called Parelaphostrongylus tenuis. This parasite is of particular concern in both wild and domestic ungulates as it causes significant neurological signs and definitive diagnosis is only possible post-mortem, necessitating the development of serologic assays for antemortem diagnosis. Proteins extracted from P. tenuis organisms were affinity isolated using antibodies enriched from seropositive moose (Alces alces). The proteins were analyzed using mass spectrometry and liquid chromatography to obtain amino acid sequences that were then cross-referenced to open reading frames predicted from an assembled transcriptome. An antigen of interest was assessed for immunogenic epitopes and subsequently synthesized into 10-mer synthetic overlapping peptides representing these regions. These synthetic peptides were then assessed for reactivity against positive and negative moose sera and demonstrated potential use as a serological assay in diagnostic laboratories. Known negative moose sera revealed significantly lower optical density when compared to the positive samples (p < 0.05). This method serves as a pipeline for the construction of diagnostic assays of pathogens in both human and veterinary medicine.