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The guinea pig was the original animal model developed for investigating spotted fever rickettsiosis (SFR). This model system has persisted on account of the guinea pig's conduciveness to tick transmission of SFR agents and ability to recapitulate SFR in humans through clinical signs that include fever, unthriftiness, and in some cases the development of an eschar. The guinea pig is the smallest animal model for SFR that allows the collection of multiple blood and skin samples antemortem for longitudinal studies. This unit provides the basic protocols necessary to establish, maintain, and utilize a guinea pig-tick-Rickettsia model for monitoring the course of infection and immune response to an infection by spotted fever group Rickettsia (SFGR) that can be studied at biosafety level 2 (BSL-2) and arthropod containment level 2 (ACL-2); adaptations must be made for BSL-3 agents. The protocols cover methods for tick feeding and colony development, laboratory infection of ticks, tick transmission of Rickettsia to guinea pigs, and monitoring of the course of infection through clinical signs, rickettsial burden, and immune response. It should be feasible to adapt these methods to study other tick-borne pathogens. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tick transmission of SFGR to guinea pigs Support Protocol 1: Laboratory infection of ticks by injection Alternate Protocol 1: Needle inoculation of SFGR to guinea pigs Basic Protocol 2: Monitoring the course of guinea pig rickettsial infection: clinical signs Basic Protocol 3: Monitoring the course of guinea pig rickettsial infection: collection of biological specimens Support Protocol 2: Guinea pig anesthesia Basic Protocol 4: Monitoring rickettsial burden in guinea pigs by multiplex qPCR Basic Protocol 5: Monitoring guinea pig immune response to infection: blood leukocytes by flow cytometry Basic Protocol 6: Monitoring immune response to guinea pig rickettsial infection: leukocyte infiltration of skin at the tick bite site by flow cytometry Basic Protocol 7: Monitoring the immune response to guinea pig rickettsial infection: antibody titer by ELISA Support Protocol 4: Coating ELISA Plates Alternate Protocol 2: Monitoring immune response to guinea pig rickettsial infection: antibody titer by immunofluorescence assay.

Three subspecies of Northern Bahamian Rock Iguanas, Cyclura cychlura, are currently recognized: C. c. cychlura, restricted to Andros Island, and C. c. figginsi and C. c. inornata, native to the Exuma Island chain. Populations on Andros are genetically distinct from Exuma Island populations, yet genetic divergence among populations in the Exumas is inconsistent with the 2 currently recognized subspecies from those islands. The potential consequences of this discrepancy might include the recognition of a single subspecies throughout the Exumas rather than 2. That inference also ignores evidence that populations of C. cychlura are potentially adaptively divergent. We compared patterns of population relatedness in a three-tiered host-parasite system: C. cychlura iguanas, their ticks (genus Amblyomma, preferentially parasitizing these reptiles), and Rickettsia spp. endosymbionts (within tick ectoparasites). Our results indicate that while C. c. cychlura on Andros is consistently supported as a separate clade, patterns of relatedness among populations of C. c. figginsi and C. c. inornata within the Exuma Island chain are more complex. The distribution of the hosts, different tick species, and Rickettsia spp., supports the evolutionary independence of C. c. inornata. Further, these patterns are also consistent with two independent evolutionarily significant units within C. c. figginsi. Our findings suggest coevolutionary relationships between the reptile hosts, their ectoparasites, and rickettsial organisms, suggesting local adaptation. This work also speaks to the limitations of using neutral molecular markers from a single focal taxon as the sole currency for recognizing evolutionary novelty in populations of endangered species.

Amblyomma maculatum Koch (Acari: Ixodidae), the primary vector for Rickettsia parkeri, may also be infected with a rickettsia of unknown pathogenicity, "Candidatus Rickettsia andeanae." Infection rates with these rickettsiae vary geographically, and coinfected ticks have been reported. In this study, infection rates of R. parkeri and "Ca. R. andeanae" were evaluated, and rickettsial DNA levels quantified, in 335 questing adult A. maculatum collected in 2013 (n = 95), 2014 (n = 139), and 2015 (n = 101) from Oktibbeha County, MS. Overall infection rates of R. parkeri and "Ca. R. andeanae" were 28.7% and 9.3%, respectively, with three additional A. maculatum (0.9%) coinfected. While R. parkeri-infected ticks were detected all three years (34.7% in 2013; 13.7% in 2014; 43.6% in 2015), "Ca. R. andeanae" was not detected in 2013, and was detected at rates of 10.8% in 2014, and 15.8% in 2015. Interestingly, rickettsial DNA levels in singly-infected ticks were significantly lower in "Ca. R. andeanae"-infected ticks compared to R. parkeri-infected ticks (P 0.0001). Thus, both infection rates and rickettsial DNA levels were higher for R. parkeri than "Ca. R. andeanae." Infection rates of R. parkeri were also higher, and "Ca. R. andeanae" lower, here compared to A. maculatum reported previously in Kansas and Oklahoma. As we continue to monitor infection rates and levels, we anticipate that understanding temporal changes will improve our awareness of human risk for spotted fever rickettsioses. Further, these data may lead to additional studies to evaluate potential interactions among sympatric Rickettsia species in A. maculatum at the population level.

American canine hepatozoonosis (ACH) is a debilitating tick-borne disease characterized by pyrexia, body wasting, myopathy, mucopurulent ocular discharge, and periosteal proliferation. The causative agent, Hepatozoon americanum, is an apicomplexan that utilizes the Gulf Coast tick, Amblyomma maculatum, as its definitive host and vector. Unlike most tick-borne disease agents, H. americanum is not transmitted via a tick bite, but is transmitted when canids ingest a tick vector that contains sporulated oocysts within the tick hemocoel or paratenic hosts with cystozoites. Our understanding of H. americanum prevalence is based on its detection in the intermediate host, wild or domestic canids, with domestic canids often showing clinical signs at the time of diagnosis. The frequency of H. americanum in A. maculatum, on the other hand, is unknown; this gap in our knowledge hinders our understanding of transmission risk. Furthermore, current diagnostic assays are limited in efficacy, and serologic assays are not widely available. To begin to address gaps in our knowledge, we developed a TaqMan® multiplex qPCR assay for H. americanum detection in A. maculatum tick extracts and evaluated infection rates in questing adult A. maculatum. Additionally, we used a co-culture system to expose H. americanum stages to host cells for in vitro development. Results from qPCR analysis of over 500 tick extracts revealed no positive samples; this suggests both low transmission risk by adult Gulf Coast tick ingestion in the sampled areas, and that surveillance should be focused in areas where ACH has been diagnosed at higher frequencies. Hepatozoon americanum was detectable by qPCR in co-culture of an infected canine buffy coat with ISE6 (Ixodes scapularis embryonic) tick cells, and microscopic examination of samples from those days revealed some structures that were suspicious for developing stages. These data are a starting point for future work to advance our understanding of H. americanum transmission and mechanisms of disease in canids with ACH.

Amblyomma maculatum is the primary vector for the spotted fever group rickettsiae, Rickettsia parkeri, a known pathogen, and "Candidatus Rickettsia andeanae," currently considered nonpathogenic. Spotted fever group rickettsiae are typically endothelial cell associated and rarely circulate in the blood. Horizontal transmission to naïve ticks through blood feeding from an infected host is likely rare. Cofeeding provides an opportunity for rickettsial transmission to naïve ticks in the absence of circulating rickettsiae. We evaluated R. parkeri transmission through cofeeding between A. maculatum adults and nymphs on beef calves. Six calves in each of two trials were infested with A. maculatum that had been capillary fed R. parkeri. Four days later, calves each received recipient A. maculatum that were either capillary fed "Ca. R. andeanae" or not capillary fed before infestation. Trials differed by whether we included a barrier to minimize adjacent feeding between recipient and donor ticks. After cofeeding, we detected R. parkeri in 27% of "Ca. R. andeanae"-free recipient ticks, whereas R. parkeri was not detected in any recipient ticks that were capillary fed "Ca. R. andeanae." Rickettsia parkeri transmission efficiency to naïve ticks was greater when ticks freely cofed in proximity. No rickettsial DNA was detected in calf blood. Results confirm cofeeding as a method of horizontal transmission of R. parkeri in the absence of host rickettsemia and suggest no evidence of transmission by cofeeding when recipient ticks are first exposed to "Ca. R. andeanae" through capillary feeding. While cofeeding may provide an opportunity for maintaining the pathogen, R. parkeri, the mechanisms driving any potential effect of "Ca. R. andeanae" on R. parkeri transmission are unclear.

We reviewed 62 new cases and 18 published reports of Dracunculus infections in domestic dogs and cats to describe the epidemiology of this parasite in dogs and cats in North America. We collected host and parasite data when available, including age, sex, and breed of dog, nematode location in the host, and any clinical signs at presentation and/or description of the apparent lesion. For dogs, infections were noted in six of the AKC breed groups, but none was reported from the toy group or the miscellaneous breed class. Age of infected dogs ranged from 7 months to 19 years (median 4 years; average 5.3 years), and infection rates were similar in male and female dogs. Most nematodes were associated with the distal extremities, but worms were also found in the chest/thorax, abdomen, head, and flank. Although most infected dogs had a single worm, three dogs had two or more worms that were collected from multiple lesions. Three new cat cases, with similar lesions, presentations and seasonality, were detected in Alabama, North Carolina and Texas. Cases were reported from a wide geographic range throughout eastern North America, during every month of the year, but 72% of infections were diagnosed in the late winter to early spring (December to May). All collected worms were larvigerous females which cannot be identified to species based on morphologic characters. Thus, we attempted to amplify and sequence a portion of the cytochrome c oxidase subunit I (COI) gene for specific identification. Although 13 worms from 12 cases were available, sequences were obtained for only eight worms from seven cases. These eight worms were D. insignis, a common parasite of raccoons (Procyon lotor) and other primarily carnivorous mammals. Female worms are the most likely to be detected in dogs and cats because male worms do not emerge, parasites should be preserved in ethanol for molecular identification. Although this study used convenience sampling of available data, we found that the parasite is widespread throughout the eastern US and Canada and that Dracunculus infections in dogs are more common than is revealed in published literature. However, more research is needed to understand the epidemiology, including transmission route(s), prevalence, and distribution of this parasite.

Understanding diagnosis of heartworm disease in the context of animal shelters' needs and expectations is crucial for developing guidelines that specifically address the unique shelter population. Accurate and economical heartworm testing strategies are essential. However, current comprehensive guidelines for the management of heartworm disease in dogs are directed toward client-owned animals and do not address needs of animal shelters and other resource-scarce facilities. Additionally, testing recommendations do not take into account regional and local differences in heartworm prevalence across the United States that occur due to abiotic and biotic factors. The objective of this study was to determine the apparent prevalence of Dirofilaria immitis microfilaremia and antigenemia in dogs from Mississippi animal shelters. Further, we compare agreement between microfilaria and antigen testing in this population. We performed a cross-sectional study using canine serum and blood bank samples representative of the Mississippi shelter population. Microfilaria testing of whole blood included a blood smear and modified Knott test. Antigen testing of serum was performed using the DiroCHEK® antigen ELISA test. Analyses included descriptive and analytic statistics as well as Cohen's kappa for test agreement. A total of 283 whole blood samples and 363 serum samples, representing 363 dogs from 18 shelters in 17 Mississippi counties, were utilized in this study. Sixty-four (22.6%) whole blood samples demonstrated D. immitis microfilariae on the modified Knott test and 125 (34.4%) serum samples had detectable D. immitis antigen. Increasing age and low body condition were associated with antigen-positive test results. Only age was associated with microfilaria-positive test results. There was moderate agreement between the antigen ELISA test and the modified Knott microfilaria test and poor agreement between the antigen ELISA and the blood smear. This study provides the first known report of the prevalence of D. immitis microfilaremia and antigenemia in Mississippi shelter dogs. We observed that prevalence of both microfilaremia and antigenemia was significantly higher in these sampled dogs compared to previous reports for the owned canine population in Mississippi. Heartworm infection presents unique management challenges for animal shelters. Knowledge of the expected prevalence in the area can be utilized for management decisions related to prevention, testing, and treatment of dogs in shelter populations.

Dermacentor variabilis, a common human-biting tick found throughout the eastern half and along the west coast of the United States, is a vector of multiple bacterial pathogens. Historically, D. variabilis has been considered a primary vector of Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever. A total of 883 adult D. variabilis, collected between 2012 and 2017 from various locations in 12 states across the United States, were screened for rickettsial DNA. Tick extracts were evaluated using three real-time PCR assays; an R. rickettsii-specific assay, a Rickettsia bellii-specific assay, and a Rickettsia genus-specific assay. Sequencing of ompA gene amplicons generated using a seminested PCR assay was used to determine the rickettsial species present in positive samples not already identified by species-specific real-time assays. A total of 87 (9.9%) tick extracts contained R. bellii DNA and 203 (23%) contained DNA of other rickettsial species, including 47 (5.3%) with Rickettsia montanensis, 11 (1.2%) with Rickettsia amblyommatis, 2 (0.2%) with Rickettsia rhipicephali, and 3 (0.3%) with Rickettsia parkeri. Only 1 (0.1%) tick extract contained DNA of R. rickettsii. These data support multiple other contemporary studies that indicate infrequent detection of R. rickettsii in D. variabilis in North America.

Parasitism of domestic cats impacts feline health and public health, when zoonotic parasites are present. Our objective was to evaluate endoparasite prevalence in cats from northern Mississippi animal shelters. Feline cadavers (n = 56) were collected from seven shelters from August 2017 to January 2018. Data included shelter, sex, reproductive status, intake date, originating source, and treatment records. Cadavers were processed to isolate stomach, and small and large intestines. Contents were strained and examined using stereomicroscopes for helminth collection and identification. Centrifugal flotation using Sheather's solution was performed on feces; urine sediments were also examined. Descriptive statistics in SAS was performed using the Frequency procedure. Kappa agreement statistics were obtained to determine agreement between fecal flotation and necropsy results. Separate logistic regression models were developed to test effects of risk factors on the probability for cats to test positive for outcomes of interest. Helminths were recovered in 82% of cats (46/56); specifically, Ancylostoma spp. (52%), Toxocara cati (43%), Taenia taeniaeformis (36%), Dipylidium caninum (29%), and Spirometra spp. (4%) were identified. Thirty-seven of 56 cats (66%) had parasite eggs or oocysts on fecal examination, including T. cati (39%), Ancylostoma spp. (34%), Cystoisospora spp. (23%), Spirometra spp. (9%), T. taeniaeformis (9%), and capillarid-type eggs (5%). Feline originating source was associated with presence of T. cati eggs in feces and presence of D. caninum in the gastrointestinal tract. Feral cats were more likely to have T. cati eggs in feces than owner surrender cats (OR 28; 95% CI: 1.9, 423), or stray cats (OR 8, 95% CI: 1.1, 57.0). Owner surrender cats were more likely to have D. caninum helminths in the gastrointestinal tract than stray cats (OR = 19.5; 95% CI: 2.0, 190). Toxocara cati exhibited strong agreement (κ = 0.70, 95% CI: 0.52, 0.89), Ancylostoma spp. exhibited moderate agreement (κ = 0.44, 95% CI: 0.22, 0.65), and cestodes exhibited poor agreement (κ = 0.02, 95% CI: −0.12, 0.15) between presence of eggs and gross helminths. Capillarid eggs (Pearsonema feliscati) were recovered in urine sediment of 6% (3/48) of cats. Overall, our study demonstrates a high level of parasitism in cats that entered Mississippi animal shelters. Parasites with zoonotic potential, such as Alaria spp., Ancylostoma spp., D. caninum, Physaloptera spp., T. taeniaeformis, T. cati, and Spirometra spp. were identified. Our results support the need for effective antiparasitic treatment of cats entering animal shelters in order to improve feline health and prevent environmental contamination with zoonotic parasites.

The guinea pig (Cavia porcellus) has an established track record as an animal model, with its utility in rickettsial research documented as early as the turn of the 20(th) century. From identifying Rickettsia rickettsii as the agent of Rocky Mountain spotted fever and ticks as the natural transmission route to evaluating protective immunity and treatment for tick-borne rickettsiae, guinea pigs have been essential for advances in our understanding of spotted fever rickettsioses (SFR). Tick feeding on guinea pigs is feasible and results in transmission of tick-borne rickettsiae. The resulting infection leads to the recapitulation of SFR as defined by clinical signs that include fever, unthrift, and in the case of transmission by a Rickettsia parkeri-infected Amblyomma maculatum tick, a characteristic eschar at the site of the bite. No other small animal model recapitulates SFR, is large enough to collect multiple blood and skin samples for longitudinal studies, and has an immune system as similar to the human immune system. In the 1980s, the use of the guinea pig was significantly reduced due to advances made to the more reproductively prolific and inexpensive murine model. These advances included the development of genetically modified murine strains, which resulted in the expansion of murine-specific reagents and assays. Still, the advantages of the guinea pig as a model for SFR persist, novel assays are being developed to better monitor guinea pig immune responses, and tools, like CRISPR/Cas9, are now available. These technical advances allow guinea pigs to again contribute to our understanding of SFR. Importantly, returning to the guinea pig model with enhanced tools will enable rickettsial researchers to corroborate and potentially refine results acquired using mice. This minireview summarizes Cavia porcellus as an animal model for human tick-borne rickettsial diseases.

Guinea pigs are an ideal animal model for the study of several infectious diseases, including tuberculosis, legionellosis, brucellosis, and spotted fever rickettsiosis. In comparison to the murine model, clinical signs in guinea pigs are more representative of disease in humans, the guinea pig immune system is more similar to that of the human, and their large size offers logistic advantages for sample collection while following disease progression. Unfortunately, the advantage of using guinea pigs in biomedical research, particularly in understanding the immune response to infectious agents, is limited in large part by the paucity of available reagents and lack of genetically manipulated strains. Here, we expand the utility of guinea pigs in biomedical research by establishing an optimized five-color/seven-parameter polychromatic flow cytometric assay for immunophenotyping lymphocytes. This assay fills a need for immunophenotyping peripheral blood lymphocytes and is an improvement over current published flow cytometry assays for guinea pigs. We anticipate that our approach will be an important starting point for developing new assays to evaluate the cellular immune response to infectious diseases in the guinea pig model. Importantly, we are currently using this assay for evaluating immunity to spotted fever rickettsiosis in a guinea pig-tick-Rickettsia system, where CD8+ T cells are a critical contributor to the immune response. Developing resources to utilize the guinea pig more effectively will enhance our ability to understand infectious diseases where the guinea pig would otherwise be the ideal model.

Our objective of this study was to explore the bacterial microbiome in fresh or fresh-frozen adult Amblyomma maculatum (Gulf Coast ticks) using extracts enriched for microbial DNA. We collected 100 questing adult A. maculatum, surface disinfected them, and extracted DNA from individual ticks collected the same day or after storage at −80 °C. Because only extracts with microbial DNA concentrations above 2 ng/μL were considered suitable for individual analysis, we expected fewer samples to meet these requirements. Of individual ticks extracted, 48 extracts met this minimum concentration. We pooled 20 additional extracts that had lower concentrations to obtain seven additional pools that met the minimum DNA concentration. Libraries created from these 55 samples were sequenced using an Illumina MiSeq platform, and data sets were analyzed using QIIME to identify relative abundance of microorganisms by phylum down to genus levels. Proteobacteria were in greatest abundance, followed by Actinobacteria, Firmicutes, and Bacteroidetes, at levels between 1.9% and 6.4% average relative abundance. Consistent with the Francisella-like endosymbiont known to be present in A. maculatum, the genus Francisella was detected at highest relative abundance (72.9%; SE 0.02%) for all samples. Among the top ten genera identified (relative abundance ≥ 0.5%) were potential extraction kit contaminants, Sphingomonas and Methylobacterium, the soil bacterium Actinomycetospora, and the known A. maculatum-associated genus, Rickettsia. Four samples had Rickettsia at greater than 1% relative abundance, while nine additional samples had Rickettsia at low (0.01–0.04%) relative abundance. In this study, we used the entire microbe-enriched DNA extract for whole ticks for microbiome analysis. A direct comparison of the microbiome in microbe-enriched DNA and total genomic DNA extracts from halves of the same tick would be useful to determine the utility of this extraction method in this system. We anticipate that future tick microbiome studies will be valuable to explore the influence of microbial diversity on pathogen maintenance and transmission, and to evaluate niche-specific microbiomes within individual tick tissues.

The Gulf Coast tick, Amblyomma maculatum Koch, has become increasingly important in public health for its role as a vector of the recently recognized human pathogen, Rickettsia parkeri. More recently, these ticks were also found to harbor a novel spotted fever group rickettsia, "Candidatus Rickettsia andeanae." First identified in Peru, and subsequently reported in ticks collected in the United States, Chile, and Argentina, "Ca. R. andeanae" remains largely uncharacterized, in part because of the lack of a stable isolate. Although the isolation of "Ca. R. andeanae" was recently described in DH82, Vero, and Drosophila S2 cells, its stability in these cell lines was not shown. To evaluate "Ca. R. andeanae" transmission and pathogenicity in vertebrates, as well as further describe biological characteristics of this candidate species to fulfill criteria for its establishment as a new species, availability of a stable isolate is essential. Here we describe the propagation of "Ca. R. andeanae" by using a primary culture derived from naturally infected A. maculatum embryos. Subsequent passage of the "Ca. R. andeanae" isolate to ISE6 (Ixodes scapularis embryonic) and Vero (African green monkey kidney epithelial) cell lines demonstrated limited propagation of the rickettsiae. Treatment of the infected primary cells with tetracycline resulted in cultures negative for "Ca. R. andeanae" by polymerase chain reaction and microscopy. Establishment of an isolate of "Ca. R. andeanae" will promote further investigation into the significance of this tick-associated rickettsia, including its role in spotted fever and interactions with the sympatric species, R. parkeri in A.

Although a variety of disease agents have been reported from bed bugs, the mechanical and biological disease transmission potential of bed bugs remains unelucidated. In this study we assayed survivability of the mildly pathogenic spotted fever group rickettsia, Rickettsia parkeri, in bed bugs after feeding on R. parkeri-infected chicken blood. Two groups of 15 adult bed bugs each were fed on infected or noninfected blood, and two groups of fourth-instar bed bugs also were fed on either infected or noninfected blood. One group of 15 adult bed bugs received no bloodmeal and was included as an additional control. Two weeks postfeeding, two pools of five live bed bugs from each group were surface sterilized, macerated, and placed in Vero cell cultures in an attempt to grow live organism. The remaining five individual bed bugs from each group were dissected, their salivary glands were removed for immunofluorescence assay (IFA) staining, and the remaining body parts were processed for polymerase chain reaction (PCR) analysis. Results indicated that no immature (now molted to fifth instar) bed bugs were positive for R. parkeri by IFA or PCR, indicating that organisms did not survive the molting process. After 4 wk of cell culture, no organisms were seen in cultures from any of the treatment or control groups, nor were any cultures PCR positive. However, two of the adult bed bugs were IFA positive for rickettsia-like organisms, and these two specimens were also PCR positive using R. parkeri-specific primers. These IFA and PCR results indicate that remnants of Rickettsia parkeri (possibly whole organisms) survived in the bugs for 2 wk, but the viability of the organisms in these two specimens could not be determined.

We detected Rickettsia parkeri in 20%-33% of Amblyomma maculatum ticks sampled in North Carolina. Results highlight the high frequencies of R. parkeri-infected ticks in the state with the highest annual incidence of Rocky Mountain spotted fever. Epidemiologic studies are needed to definitively link R. parkeri to cases of spotted fever rickettsiosis.

Rickettsia parkeri, a recently identified cause of spotted fever rickettsiosis in the United States, has been found in Amblyomma triste ticks in several countries of South America, including Argentina, where it is believed to cause disease in humans. We describe the clinical and epidemiologic characteristics of 2 patients in Argentina with confirmed R. parkeri infection and 7 additional patients with suspected R. parkeri rickettsiosis identified at 1 hospital during 2004-2009. The frequency and character of clinical signs and symptoms among these 9 patients closely resembled those described for patients in the United States (presence of an inoculation eschar, maculopapular rash often associated with pustules or vesicles, infrequent gastrointestinal manifestations, and relatively benign clinical course). Many R. parkeri infections in South America are likely to be misdiagnosed as other infectious diseases, including Rocky Mountain spotted fever, dengue, or leptospirosis.

Cattle have been recognized as hosts for Amblyomma maculatum, the Gulf Coast tick, for over 100 years. For nearly as long, A. maculatum have been known to harbor the spotted fever group Rickettsia (SFGR), now known as Rickettsia parkeri. However, human infection with R. parkeri was not documented until 2004. Results presented herein describe a laboratory and a field study evaluating cattle and the natural history of A. maculatum and R. parkeri in Mississippi. In the laboratory study, seroconversion to R. parkeri antigen occurred in calves exposed to R. parkeri by injection or by feeding R. parkeri-infected A. maculatum, and two out of six animals were transiently rickettsemic. All calves remained clinically normal during the study, except for gotch ear-like lesions in all tick-infested calves, regardless of infection status of ticks, suggesting that R. parkeri is not involved in the condition. In the field study, A. maculatum (n=34) removed from Mississippi sale barn cattle (n=183) and the cattle hosts were tested for R. parkeri. Cattle were not rickettsemic by polymerase chain reaction, but 49.7% demonstrated low titers to R. parkeri antigen when tested by indirect fluorescent antibody for SFGR. Of ticks removed from cattle, 11.8% were hemolymph positive and 8.7% were indirect fluorescent antibody positive. Approximately 22% (5/23) and 4% (1/23) of harvested tick extracts were positive for R. parkeri by polymerase chain reaction of the 17 kDa antigen gene and ompA gene, respectively. An amplicon for the ompA gene from one tick was successfully sequenced and showed 100% similarity with the homologous sequence of R. parkeri. Thus, cattle may harbor R. parkeri-infected A. maculatum and produce antibodies to SFGR. Cattle may play a role in the natural history of R. parkeri infection by expanding populations of A. maculatum and transporting R. parkeri-infected ticks to various locations, rather than as a reservoir for R. parkeri.

Salivary glands, midgut, Malpighian tubules, and ovaries were dissected from infected, colony-derived Amblyomma maculatum (Gulf Coast ticks) injected as nymphs with either Rickettsia parkeri (a spotted fever group rickettsia [SFGR]; treatment) or phosphate-buffered saline (negative control). For comparison, similar tissues were dissected from hemolymph-positive, field-collected ticks. Tissues were analyzed by indirect fluorescent antibody (IFA) tests. All phosphate-buffered saline-injected ticks were IFA negative, whereas SFGR were detected by IFA in 100% of the salivary glands and ovaries and 78 and 75% of midgut and Malpighian tubule samples, respectively, of R. parkeri-injected ticks. Nearly 22% (10/46) of the field-collected ticks were hemolymph positive. Of those, SFGR were detected by IFA in 80% of the salivary glands, 67% of the ovaries, and 60% in the midgut and Malpighian tubules. This is the first study to assess the distribution of SFGR in select tissues of A. maculatum ticks.

Because tick-borne diseases are becoming increasingly important throughout the world, monitoring their causative agents in wildlife may serve as a useful indicator of potential human exposure. We assessed the presence of known and putative zoonotic, tick-borne agents in four wildlife species in Mississippi. Animals were tested for exposure to or infection with Ehrlichia chaffeensis, Ehrlichia ewingii, Borrelia lonestari, Rickettsia spp., Anaplasma phagocytophilum, and Francisella tularensis. Whole blood and serum were tested from white-tailed deer (WTD; Odocoileus virginianus) and feral swine (Sus scrofa); serum was tested from raccoons (Procyon lotor) and opossums (Didelphis virginiana). We used polymerase chain reaction to detect all agents in blood, whereas an indirect fluorescent antibody assay was used to detect antibodies to E. chaffeensis, B. lonestari, and Rickettsia parkeri (spotted fever group rickettsiae) antigens in serum. Molecular evidence of infection with E. chaffeensis, B. lonestari, and An. phagocytophilum was detected only in WTD. Antibodies to E. chaffeensis antigen were detected in 43.9% of WTD, 32.8% of swine, 42.1% of raccoons, and 15.8% of opossums. Serologic evidence of exposure to B. lonestari antigen was found in 19.3% of WTD, 6.9% of swine, and 5.3% of raccoons, but not in opossums. Interestingly, the percent of animals with antibodies reactive to spotted fever group rickettsiae (R. parkeri antigen) was highest in raccoons (73.7%) and opossums (57.9%). These results support the role of WTD as reservoirs for E. chaffeensis, B. lonestari, and An. phagocytophilum, as well as provide additional evidence for exposure of raccoons and opossums to E. chaffeensis. Finally, we provide new data that feral swine may have antibodies to these agents. Thus, in general, these four wildlife species are exposed to tick-borne disease agents in Mississippi, suggesting that ticks carry and have the potential to transmit the agents to humans in the state.

Until recently,Amblyomma maculatum(the Gulf Coast tick) had garnered little attention compared to other species of human-biting ticks in the United States.A. maculatumis now recognized as the principal vector ofRickettsia parkeri, a pathogenic spotted fever group rickettsia (SFGR) that causes an eschar-associated illness in humans that resembles Rocky Mountain spotted fever. A novel SFGR, distinct from other recognizedRickettsiaspp., has also been detected recently inA. maculatumspecimens collected in several regions of the southeastern United States. In this study, 198 questing adult Gulf Coast ticks were collected at 4 locations in Florida and Mississippi; 28% of these ticks were infected withR. parkeri, and 2% of these were infected with a novel SFGR. Seventeen isolates ofR. parkerifrom individual specimens ofA. maculatumwere cultivated in Vero E6 cells; however, all attempts to isolate the novel SFGR were unsuccessful. Partial genetic characterization of the novel SFGR revealed identity with several recently described, incompletely characterized, and noncultivated SFGR, including “CandidatusRickettsia andeanae” andRickettsiasp. Argentina detected in several species of Neotropical ticks from Argentina and Peru. These findings suggest that each of these “novel” rickettsiae represent the same species. This study considerably expanded the number of low-passage,A. maculatum-derived isolates ofR. parkeriand characterized a second, sympatricRickettsiasp. found in Gulf Coast ticks.

Amblyomma americanum is an aggressive ixodid tick that has been implicated as a vector for several bacterial agents. Among these is Ehrlichia chaffeensis, which causes human monocytic (or monocytotropic) ehrlichiosis. In this study, experimental tick transmission of E. chaffeensis from infected lone star ticks to deer was revisited, and the question of whether it would be possible to re-isolate the organism from deer was asked, because this had not been done previously. Here, we were able to transmit a wild strain of E. chaffeensis from acquisition-fed lone star ticks to white-tailed deer. Ehrlichia chaffeensis was re-isolated from one white-tailed deer on multiple days during the infection and from another deer on one day during the infection. Peak rickettsemias for E. chaffeensis-infected deer were 17 DPI with acquisition-fed ticks and 14 DPI with needle-inoculated deer. This study supports the role of the lone star tick and white-tailed deer as vector and reservoir host for E. chaffeensis, demonstrating culture re-isolation of E. chaffeensis in deer infected by experimental tick transmission for the first time.

In late spring of 2009 and 2010, there were reports of severe black fly (Simulium spp., shown in Fig. 1) outbreaks in various counties in Mississippi, especially those in and around the Mississippi River Delta. Complaints were of black flies attacking multiple species of backyard poultry and causing high morbidity and mortality in affected flocks. At several affected locations, black flies were readily observed swarming around and feeding on birds. A large number of these parasites were easily trapped on fly strips (Fig. 2). Multifocal to coalescing cutaneous hemorrhagic lesions, consistent with fly bites, were seen on the birds. Upon necropsy examination, a large number of black flies were also observed in the digestive tract (Fig. 3). Although black flies may cause disease directly, such as cardiopulmonary collapse and anaphylactoid reactions, detection of Leucocytozoon in blood smears (Fig. 4) of affected birds prompted further investigations of this protozoan as a cause of disease. Leucocytozoon spp. are known to be transmitted by black flies and may be associated with morbidity and mortality in birds such as poultry. From June 2009 through July 2012, the investigation included a total collection of 1068 individual blood samples, representing 371 individual premises in 89 counties/parishes across Mississippi (59), Alabama (10), Louisiana (4), and Tennessee (16). Of the 371 premises where blood samples were collected, 96 (26%) were either positive or highly suspected to be positive for Leucocytozoon spp. by blood smear analysis, and 5 (1.2%) were positive for Haemoproteus spp. by blood smear analysis. Attempts to diagnose Leucocytozoon spp. by PCR analysis and sequencing were complicated by coinfections with two closely related haemosporidians (Haemoproteus spp. and Plasmodium spp.). A novel technique involving flow cytometry was also explored. This study discusses the black fly field outbreak, the involvement of haemosporidians, molecular methods for detection of both the black flies and blood parasites, and initial attempts at flow cytometry.

Latent infection with Toxoplasma gondii is common in humans (approximately 30% of the global population) and is a significant risk factor for schizophrenia. Since prevalence of T. gondii infection is far greater than prevalence of schizophrenia (0.5-1%), genetic risk factors are likely also necessary to contribute to schizophrenia. To test this concept in an animal model, Nurr1-null heterozygous (+/-) mice and wild-type (+/+) mice were evaluate using an emergence test, activity in an open field and with a novel object, response to bobcat urine and prepulse inhibition of the acoustic startle response (PPI) prior to and 6 weeks after infection with T. gondii. In the emergence test, T. gondii infection significantly decreased the amount of time spent in the cylinder. Toxoplasma gondii infection significantly elevated open field activity in both +/+ and +/- mice but this increase was significantly exacerbated in +/- mice. T. gondii infection reduced PPI in male +/- mice but this was not statistically significant. Aversion to bobcat urine was abolished by T. gondii infection in +/+ mice. In female +/- mice, aversion to bobcat urine remained after T. gondii infection while the male +/- mice showed no aversion to bobcat urine. Antibody titers of infected mice were a critical variable associated with changes in open field activity, such that an inverted U shaped relationship existed between antibody titers and the percent change in open field activity with a significant increase in activity at low and medium antibody titers but no effect at high antibody titers. These data demonstrate that the Nurr1 +/- genotype predisposes mice to T. gondii-induced alterations in behaviors that involve dopamine neurotransmission and are associated with symptoms of schizophrenia. We propose that these alterations in murine behavior were due to further exacerbation of the altered dopamine neurotransmission in Nurr1 +/- mice.

To the Editor: Rickettsia parkeri, a spotted fever group Rickettsia (SFGR) bacterium, is transmitted by Amblyomma maculatum, the Gulf Coast tick (1). The prevalence of R. parkeri in Gulf Coast ticks has been reported as

A 1-year-old castrated male Saanen goat was observed to have drooping and edema of the left ear consistent with published accounts of gotch ear in cattle associated with a tick bite. The goat's left ear was edematous from the tip of the pinna to the base of the ear. No signs of trauma or infectious processes were observed. Three engorged Gulf Coast ticks (Amblyomma maculatum) were observed attached inside the ear. Ticks were removed and the ear biopsied at tick attachment sites. The affected ear was treated topically with betadine after removal of the ticks. No other treatment was administered. The goat remained free of clinical signs and the edema of the ear resolved within 3 days after tick removal. No clinical adverse effects of the condition were evident. All three ticks were positive for spotted fever group rickettsia by polymerase chain reaction analysis and showed 100% similarity with the homologous sequence of Rickettsia parkeri. There was no immunohistochemical evidence of spotted fever group rickettsia in the ear samples, supporting the hypothesis that gotch ear is not due to rickettsial infection. This report represents the first apparent case of gotch ear in a goat.

The Companion Animal Parasite Council hosted a meeting to identify quantifiable factors that can influence the prevalence of tick-borne disease agents among dogs in North America. This report summarizes the approach used and the factors identified for further analysis with mathematical models of canine exposure to tick-borne pathogens.

Background Relapsing fever spirochetes are global yet neglected pathogens causing recurrent febrile episodes, chills, nausea, vomiting, and pregnancy complications. Given these nonspecific clinical manifestations, improving diagnostic assays for relapsing fever spirochetes will allow for identification of endemic foci and expedite proper treatment. Previously, an antigen designated the Borrelia immunogenic protein A (BipA) was identified in the North American species Borrelia hermsii. Thus far, BipA appears unique to relapsing fever spirochetes. The antigen remains unidentified outside of these pathogens, while interspecies amino acid identity for BipA in relapsing fever spirochetes is only 24–36%. The current study investigated the immunogenicity of BipA in Borrelia turicatae, a species distributed in the southern United States and Latin America. Methodology/Principal Findings bipA was amplified from six isolates of Borrelia turicatae, and sequence analysis demonstrated that the gene is conserved among isolates. A tick transmission system was developed for B. turicatae in mice and a canine, two likely vertebrate hosts, which enabled the evaluation of serological responses against recombinant BipA (rBipA). These studies indicated that BipA is antigenic in both animal systems after infection by tick bite, yet serum antibodies failed to bind to B. hermsii rBipA at a detectable level. Moreover, mice continued to generate an antibody response against BipA one year after the initial infection, further demonstrating the protein's potential toward identifying endemic foci for B. turicatae. Conclusions/Significance These initial studies support the hypothesis that BipA is a spirochete antigen unique to a relapsing fever Borrelia species, and could be used to improve efforts for identifying B. turicatae endemic regions., Author Summary Undiagnosed febrile illnesses continue to afflict those in resource poor countries. Relapsing fever spirochetes are one such pathogen causing a significant health burden, yet the pathogenesis, ecology, and distribution of B. turicatae is understudied. To address these shortcomings, we analyzed the amino acid sequence of the Borrelia immunogenic protein A (BipA) in isolates of B. turicatae. Mice and a canine were also infected by tick bite and transmission and serological responses were evaluated in these two likely mammalian hosts. B. turicatae was visualized within the blood of both animals and antibody responses generated against recombinant BipA indicated that the antigen that may be unique to infections caused by B. turicatae. Moreover, mice continued to generate antibodies a year after tick bite, suggesting a persistent infection. Our results indicate that the immune responses generated against BipA could identify additional vertebrate hosts, define endemic foci for B. turicatae, and increase the awareness of the disease to improve healthcare.

Rickettsia parkeri is a recently recognized human pathogen primarily associated with the Gulf Coast tick Amblyomma maculatum, with immature stages of this tick reported from wild vertebrates. To better understand the role of vertebrates in the natural history of this bacterium, we evaluated small mammals and ground-dwelling birds for evidence of infection with R. parkeri or exposure to the organism. We sampled small mammals (n=39) and passerines (n=47) in both north-central and southeast Mississippi, while northern bobwhite (Colinus virginianus) samples (n=31) were obtained from farms in central Mississippi. Blood from all sampled animals was tested using polymerase chain reaction (PCR) for spotted fever group rickettsiae (SFGR), and for antibodies to SFGR using R. parkeri antigen. Ectoparasite samples were removed from animals and included mites, lice, fleas, and immature ticks. Of 39 small mammal samples collected, 7 were positive for antibodies to SFGR; none tested positive by PCR for DNA of SFGR. Of 47 passerine blood samples collected, none were positive for DNA of SFGR by PCR, nor did any show serological evidence of exposure. Finally, none of 31 northern bobwhite samples tested were positive for SFGR DNA, while 7 were seropositive for rickettsial antibodies. Detection of seropositive rodents and quail suggests a role for these host species in the natural history of SFGR, possibly including R. parkeri, but the extent of their role has not yet been elucidated.

The gastrointestinal tracts of 32 free-ranging, 9-banded armadillos (Dasypus novemcinctus) from north-central Florida were examined for the presence of helminths during July 1991 to November 1993. Aspidodera sp. (Nematoda: Aspidoderidae), most closely resembling Aspidodera sogandaresi, were recovered from 20 of 32 armadillos examined. Total numbers of A. sogandaresi ranged from 1 to 1,021 per infected animal, and followed an inverse correlation to body condition index for those animals. The cystacanth stage of 1 acanthocephalan, Macracanthorhynchus ingens, was present in 1 armadillo, and is the first report of M. ingens in the 9-banded armadillo. The present study contributes to the known natural history of the 9-banded armadillo, an important animal research model.

We reviewed scientific literature pertaining to known and putative disease agents associated with the lone star tick, Amblyomma americanum. Reports in the literature concerning the role of the lone star tick in the transmission of pathogens of human and animal diseases have sometimes been unclear and even contradictory. This overview has indicated that A. americanum is involved in the ecology of several disease agents of humans and other animals, and the role of this tick as a vector of these diseases ranges from incidental to significant. Probably the clearest relationship is that of Ehrlichia chaffeensis and A. americanum. Also, there is a definite association between A. americanum and tularemia, as well as between the lone star tick and Theileria cervi to white-tailed deer. Evidence of Babesia cervi (= odocoilei) being transmitted to deer by A. americanum is largely circumstantial at this time. The role of A. americanum in cases of southern tick-associated rash illness (STARI) is currently a subject of intensive investigations with important implications. The lone star tick has been historically reported to be a vector of Rocky Mountain spotted fever rickettsiae, but current opinions are to the contrary. Evidence incriminated A. americanum as the vector of Bullis fever in the 1940s, but the disease apparently has disappeared. Q fever virus has been found in unfed A. americanum, but the vector potential, if any, is poorly understood at this time. Typhus fever and toxoplasmosis have been studied in the lone star tick, and several non-pathogenic organisms have been recovered. Implications of these tick-disease relationships are discussed.

Amblyomma americanum (L.), the lone star tick, is an aggressive ixodid tick that has been implicated as a vector for several bacteria. Among these bacteria are the disease agents Ehrlichia chaffeensis and Ehrlichia ewingii, and the putative disease agent "Borrelia lonestari." The hypothesis in this study was that wild lone star ticks from northeastern Georgia are capable of transmitting all three agents to white-tailed deer, Odocoileus virginianus, a known reservoir host for E. chaffeensis. In this study, transmission of all three agents from wild caught lone star ticks to captive reared white-tailed deer was demonstrated by polymerase chain reaction (PCR), culture, or serology. Two of three deer showed evidence of E. chaffeensis and E. ewingii infection by polymerase chain reaction assay; all three deer showed evidence of B. lonestari by PCR assay. E. chaffeensis was isolated in culture from both PCR-positive deer on multiple days. All three deer seroconverted to E. chaffeensis, whereas one deer seroconverted to B. lonestari. This study supports the role of lone star ticks and white-tailed deer as a vector and reservoir host for E. chaffeensis and E. ewingii and suggests for the first time, transmission of B. lonestari from lone star ticks to white-tailed deer.

We investigated the effect of exposing deer to multiple strains of Ehrlichia chaffeensis that differed in number of tandem repeats in either the variable-length PCR target (VLPT) gene or 120 kDa antigen gene. We hypothesized that infection with one strain would provide immunity to infection with other strains of E. chaffeensis. All deer initially exposed to strain A (604-2) became PCR and culture positive by 10 days post-infection (DPI). Three deer infected with strain A and subsequently inoculated with strain B (623-4) became infected with strain B. Two deer infected with strain A and subsequently inoculated with strain C (125B) became infected with strain C. Of three deer, each infected with strain B and subsequently inoculated with strain C, one was PCR positive for strain C. Of three deer previously inoculated with both strains A and B, and subsequently inoculated with strain C, one showed delayed evidence of strain C. Western blot analysis demonstrated that deer sera reacted differently to antigens from each exposed strain. A complementary in vitro study demonstrated that exposure to two strains differing in VLPT repeats may lead to co-infection of DH82 cells. These results complement a previous study and further show that deer can become sequentially infected with up to three strains of E. chaffeensis. This suggests that competitive exclusion, a phenomenon described in related organisms such as Anaplasma marginale whereby infection with one strain precludes subsequent infection by a second, distinct strain of the same species, may not occur with E. chaffeensis.

Background Amblyomma maculatum is the primary vector for Rickettsia parkeri, a spotted fever group rickettsia (SFGR) and human pathogen. Cotton rats and quail are known hosts for larval and nymphal A. maculatum; however, the role of these hosts in the ecology of R. parkeri is unknown. Methods Cotton rats and quail were inoculated with low or high doses of R. parkeri (strain Portsmouth) grown in Vero cells to evaluate infection by R. parkeri in these two hosts species. Animals were euthanized 2, 4, 7, 10, and 14 days post-injection (dpi) and blood, skin, and spleen samples were collected to analyze by Vero cell culture and polymerase chain reaction (PCR). In a second trial, cotton rats and quail were inoculated with R. parkeri and nymphal A. maculatum ticks were allowed to feed on animals. Animals were euthanized on 14, 20, 28, 31, and 38 dpi and blood and tissues were collected for serology and PCR assays. Fed ticks were tested for R. parkeri by PCR and Vero cell culture. Results Rickettsia parkeri was isolated in cell culture and detected by PCR in skin, blood, and spleen tissues of cotton rats in the initial trial 2, 4, and 7 dpi, but not in quail tissues. In the second trial, no ticks tested positive for R. parkeri by PCR or cell culture. Conclusions These studies demonstrate that viable R. parkeri rickettsiae can persist in the tissues of cotton rats for at least 7 days following subcutaneous inoculation of these bacteria; however, quail are apparently resistant to infection. Rickettsia parkeri was not detected in nymphal ticks that fed on R. parkeri-inoculated cotton rats or quail, suggesting an alternate route of transmission to naïve ticks.