Amblyomma maculatum-associated rickettsiae in vector tissues and vertebrate hosts during tick feeding.

Rickettsia parkeri, a causative agent of spotted fever rickettsiosis, is transmitted by Amblyomma maculatum (Gulf Coast tick), a tick that may also carry a non-pathogenic spotted fever group Rickettsia, "Candidatus Rickettsia andeanae". Here, we evaluated R. parkeri and "Candidatus R. andeanae" in tissues from A. maculatum prior to, during, and after blood feeding on rabbits. Using colony-reared A. maculatum that were capillary-fed uninfected cells, R. parkeri, "Candidatus R. andeanae", or both rickettsiae, we detected higher levels of Rickettsia spp. in the respective treatment groups. Rickettsial levels increased during blood feeding for both R. parkeri and "Candidatus R. andeanae", with a greater increase in R. parkeri in co-infected ticks compared to singly-infected ticks. We detected transovarial transmission of "Candidatus R. andeanae" in egg and larval cohorts and confirmed vertical transmission of R. parkeri in one group of larvae. Rabbits from all Rickettsia-exposed groups seroconverted on immunofluorescent antibody testing using R. parkeri antigen. Visualization of "Candidatus R. andeanae" in tick salivary glands suggested potential transmission via tick feeding. Here, rickettsial levels in artificially infected ticks demonstrate changes during feeding and transovarial transmission that may be relevant for interpreting rickettsial levels detected in wild A. maculatum.

An unusual outbreak of dermatitis due to rodent mite infestation in an acute-care hospital.

We report an outbreak of dermatitis associated with Ornithonysus bacoti and Liponyssoides sanguineus infestation in an acute ambulatory care setting. Healthcare workers developed dermatitis prior to the identification of the outbreak. A collaborative team effort resulted in complete eradication.

Multiplex TaqMan® Quantitative PCR Assays for Host-Tick-Pathogen Studies Using the Guinea Pig-Tick-Rickettsia System.

Spotted Fever Rickettsiosis (SFR) is caused by spotted fever group Rickettsia spp. (SFGR), and is associated with symptoms common to other illnesses, making it challenging to diagnose before detecting SFGR-specific antibodies. The guinea pig is a valuable biomedical model for studying Spotted Fever Rickettsiosis (SFR); its immune system is more like the human immune system than that of the murine model, and guinea pigs develop characteristic clinical signs. Thus, we have a compelling interest in developing, expanding, and optimizing tools for use in our guinea pig-Amblyomma-Rickettsia system for understanding host-tick-pathogen interactions. With the design and optimization of the three multiplex TaqMan® qPCR assays described here, we can detect the two SFGR, their respective primary Amblyomma sp. vectors, and the guinea pig model as part of controlled experimental studies using tick-transmission of SFGR to guinea pigs. We developed qPCR assays that reliably detect each specific target down to 10 copies by producing plasmid standards for each assay target, optimizing the individual primer-probe sets, and optimizing the final multiplex reactions in a methodical, stepwise fashion. We anticipate that these assays, currently designed for in vivo studies, will serve as a foundation for optimal SFGR detection in other systems, including fieldwork.

Isolation of Transcriptomic-Quality Total RNA from Mouse Spinal Cords.

Assessing cells, proteins, and total RNA in the spinal cord is vital for advancing our understanding of neuroinflammation and neurodegenerative diseases. For instance, immune cells infiltrate the spinal cord in the experimental autoimmune encephalomyelitis (EAE) model, commonly used to study multiple sclerosis. Thus, it is valuable to assess total RNA to determine the neuronal and inflammatory profiles in the spinal cord. Further, RNA profiles are useful for deciphering the effects of drugs or chemicals on neuroinflammation and neurodegenerative diseases such as EAE. The purpose of this protocol and the online video illustrating it is to describe and demonstrate the expulsion of the spinal cord from the mouse spinal column and homogenization of the spinal cord using liquid nitrogen for optimal RNA isolation. Although we present this method with spinal cords from EAE mice, the technique is broadly applicable, including RNA isolation from the spinal cords of healthy mice. Proper performance of these steps is critical to achieving a sufficient yield of transcriptomic-quality spinal cord RNA when combined with final isolation using commercially available kits. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Isolation of the spinal cord from the spinal column Support Protocol: Preparation of blunt-end needle for spinal cord isolation Basic Protocol 2: Spinal cord homogenization using liquid nitrogen Basic Protocol 3: Assessment of RNA purity, quantification, and integrity.

Skin in the Game: An Assay to Monitor Leukocyte Infiltration in Dermal Lesions of a Guinea Pig Model for Tick-Borne Rickettsiosis.

Intact, the skin typically serves as an effective barrier to the external world; however, once pathogens have breached this barrier via a wound, such as a tick bite, the surrounding tissues must recruit immune cells from the blood to neutralize the pathogen. With innate and adaptive immune systems being similar between the guinea pig and human systems, the ability of guinea pigs to show clinical signs of many infectious diseases, and the large size of guinea pigs relative to a murine model, the guinea pig is a valuable model for studying tick-borne and other pathogens that invade the skin. Here, we report a novel assay for assessing guinea pig leukocyte infiltration in the skin. Briefly, we developed an optimized six-color/eight-parameter polychromatic flow cytometric panel that combines enzymatic and mechanical dissociation of skin tissue with fluorescent antibody staining to allow for the immunophenotyping of guinea pig leukocytes that have migrated into the skin, resulting in inflammation. We designed this assay using a guinea pig model for tick-borne rickettsiosis to further investigate host-pathogen interactions in the skin, with preliminary data demonstrating immunophenotyping at skin lesions from infected ticks. We anticipate that future applications will include hypothesis testing to define the primary immune cell infiltrates responding to exposure to virulent, avirulent tick-borne rickettsiae, and tick-borne rickettsiae of unknown virulence. Other relevant applications include skin lesions resulting from other vector-borne pathogens, Staphylococcus aureus infection, and Buruli ulcer caused by Mycobacterium ulcerans.

Evaluating the Clinical and Immune Responses to Spotted Fever Rickettsioses in the Guinea Pig-Tick-Rickettsia System.

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.

Rickettsia parkeri in Amblyomma maculatum ticks, North Carolina, USA, 2009-2010.

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.

Beyond the IFA: Revisiting the ELISA as a More Sensitive, Objective, and Quantitative Evaluation of Spotted Fever Group Rickettsia Exposure.

Based on limited serological studies, at least 10% of the US population has been exposed to spotted fever group Rickettsia (SFGR) species. The immunofluorescence antibody assay (IFA) has been the gold standard for the serodiagnosis of rickettsial infections such as spotted fever rickettsiosis (SFR). However, the IFA is semi-quantitative and subjective, requiring a high level of expertise to interpret it correctly. Here, we developed an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Rickettsia parkeri infection in the guinea pig. Our ELISA is an objective, quantitative, and high-throughput assay that shows greater sensitivity and resolution in observed titers than the IFA. We methodically optimized relevant parameters in sequence for optimal signal-to-noise ratio and low coefficient of variation% values. We used a guinea pig model as it is a part of our overall research efforts to understand the immunological and clinical response to SFGR species after tick transmission. Guinea pigs are a useful model to study SFR and show clinical signs of SFR, such as fever and eschars. We anticipate that this assay will be easily adapted to other hosts, including humans and other SFGR species.

The guinea pig model for tick-borne spotted fever rickettsioses: A second look.

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 20th 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.

Scarcity of Hepatozoon americanum in Gulf Coast tick vectors and potential for cultivating the protozoan.

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.

An optimized five-color/seven-parameter flow cytometry panel for immunophenotyping guinea pig peripheral blood lymphocytes.

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.

Role of the lone star tick, Amblyomma americanum (L.), in human and animal diseases.

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.

Prevalence of endoparasites in northern Mississippi shelter cats.

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.

Multistate Survey of American Dog Ticks (Dermacentor variabilis) for Rickettsia Species.

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.

Transmission of Amblyomma maculatum-Associated Rickettsia spp. During Cofeeding on Cattle.

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.

Transmission of bacterial agents from lone star ticks to white-tailed deer.

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.

Microbial Communities in North American Ixodid Ticks of Veterinary and Medical Importance.

Interest in microbial communities, or microbiota, of blood-feeding arthropods such as ticks (order Parasitiformes, suborder Ixodida) is increasing. Studies on tick microorganisms historically emphasized pathogens of high medical or veterinary importance. Current techniques allow for simultaneous detection of pathogens of interest, non-pathogenic symbionts, like Coxiella-LE and Francisella-LE, and microorganisms of unknown pathogenic potential. While each generation of ticks begins with a maternally acquired repertoire of microorganisms, microhabitats off and on vertebrate hosts can alter the microbiome during the life cycle. Further, blood-feeding may allow for horizontal exchange of various pathogenic microbiota that may or may not also be capable of vertical transmission. Thus, the tick microbiome may be in constant flux. The geographical spread of tick vector populations has resulted in a broader appreciation of tick-borne diseases and tick-associated microorganisms. Over the last decade, next-generation sequencing technology targeting the 16S rRNA gene led to documented snapshots of bacterial communities among life stages of laboratory and field-collected ticks, ticks in various feeding states, and tick tissues. Characterizing tick bacterial communities at population and individual tissue levels may lead to identification of markers for pathogen maintenance, and thus, indicators of disease "potential" rather than disease state. Defining the role of microbiota within the tick may lead to novel control measures targeting tick-bacterial interactions. Here, we review our current understanding of microbial communities for some vectors in the family Ixodidae (hard ticks) in North America, and interpret published findings for audiences in veterinary and medical fields with an appreciation of tick-borne disease.

Co-infection of white-tailed deer with multiple strains of Ehrlichia chaffeensis.

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.

Aspidoderidae from North America, with the description of a new species of Aspidodera (Nematoda: Heterakoidea).

Aspidodera sogandaresi n. sp. (Heterakoidea: Aspidoderidae) from Dasypus novemcinctus Linnaeus, 1758 is herein described. This nematode occurs in armadillos from as far south as the canal zone of Panama, north through central Mexico, and into the southern United States. Previously identified as Aspidodera fasciata (Schneider, 1866), this new species has blunt projections on the lips and lateral expansions at the distal tips of the spicules, whereas A. fasciata has conspicuous digitiform projections on the lips, and a terminal round expansion at the tips of the spicules. Other species of the family present in North America include Aspidodera binansata Railliet and Henry, 1913; Aspidodera vazi Proença, 1937; and Lauroia trinidadensis Cameron, 1939.