Correlated Population Genetic Structure in a Three-Tiered Host-Parasite System: The Potential for Coevolution and Adaptive Divergence.

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.

Correction to: Azithromycin has enhanced effects on lung fibroblasts from idiopathic pulmonary fibrosis (IPF) patients compared to controls.

After publication of our article [1], we have been notified that an extra alpha symbol (α) was mistakenly added at the beginning of the title.

Azithromycin has enhanced effects on lung fibroblasts from idiopathic pulmonary fibrosis (IPF) patients compared to controls [corrected].

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic fatal lung disease without a cure and new drug strategies are urgently needed. Differences in behavior between diseased and healthy cells are well known and drug response can be different between cells isolated from IPF patients and controls. The macrolide Azithromycin (AZT) has anti-inflammatory and immunomodulatory properties. Recently anti-fibrotic effects have been described. However, the anti-fibrotic effects on primary IPF-fibroblasts (FB) directly compared to control-FB are unknown. We hypothesized that IPF-FB react differently to AZT in terms of anti-fibrotic effects. METHODS: Primary normal human lung and IPF-FB were exposed to TGF-ß (5 ng/ml), Azithromycin (50 µM) alone or in combination prior to gene expression analysis. Pro-collagen Iα1 secretion was assessed by ELISA and protein expression by western blot (αSMA, Fibronectin, ATP6V1B2, LC3 AB (II/I), p62, Bcl-xL). Microarray analysis was performed to screen involved genes and pathways after Azithromycin treatment in control-FB. Apoptosis and intraluminal lysosomal pH were analyzed by flow cytometry. RESULTS: AZT significantly reduced collagen secretion in TGF-ß treated IPF-FB compared to TGF-ß treatment alone, but not in control-FB. Pro-fibrotic gene expression was similarly reduced after AZT treatment in IPF and control-FB. P62 and LC3II/I western blot revealed impaired autophagic flux after AZT in both control and IPF-FB with significant increase of LC3II/I after AZT in control and IPF-FB, indicating enhanced autophagy inhibition. Early apoptosis was significantly higher in TGF-ß treated IPF-FB compared to controls after AZT. Microarray analysis of control-FB treated with AZT revealed impaired lysosomal pathways. The ATPase and lysosomal pH regulator ATP6V0D2 was significantly less increased after additional AZT in IPF-FB compared to controls. Lysosomal function was impaired in both IPF and control FB, but pH was significantly more increased in TGF-ß treated IPF-FB. CONCLUSION: We report different treatment responses after AZT with enhanced anti-fibrotic and pro-apoptotic effects in IPF compared to control-FB. Possibly impaired lysosomal function contributes towards these effects. In summary, different baseline cell phenotype and behavior of IPF and control cells contribute to enhanced anti-fibrotic and pro-apoptotic effects in IPF-FB after AZT treatment and strengthen its role as a new potential anti-fibrotic compound, that should further be evaluated in clinical studies.

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.

Investigations into Outbreaks of Black Fly Attacks and Subsequent Avian Haemosporidians in Backyard-Type Poultry and Other Exposed Avian Species.

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.

Comparison of three different brushing techniques to isolate and culture primary nasal epithelial cells from human subjects.

PURPOSE: Primary nasal epithelial cells are used for diagnostic purposes in clinical routine and have been shown to be good surrogate models for bronchial epithelial cells in studies of airway inflammation and remodeling. We aimed at comparing different instruments allowing isolation of nasal epithelial cells. METHODS: Primary airway epithelial cell cultures were established using cells acquired from the inferior surface of the middle turbinate of both nostrils. Three different instruments to isolate nasal cells were used: homemade cytology brush, nasal swab, and curette. Cell count, viability, time until a confluent cell layer was reached, and success rate in establishing cell cultures were evaluated. A standard numeric pain intensity scale was used to assess the acceptability of each instrument. RESULTS: Sixty healthy adults (median with interquartile range [IQR] age of 31 [26-37] years) participated in the study. Higher number of cells (×10(5) cells/ml) was obtained using brushes (9.8 [5.9-33.5]) compared to swabs (2.4 [1.5-3.9], p < 0.0001) and curettes (5.5 [4.4-6.9], p < 0.01). Cell viability was similar between groups. Cells obtained by brushes had the fastest growth rate, and the success rate in establishing primary cell cultures was highest with brushes (90% vs. 65% for swabs and 70% for curettes). Pain was highest with curettes (VAS score 4.0 [3.0-5.0] out of 10). The epithelial phenotype of the cultures was confirmed through cytokeratin and E-cadherin staining. CONCLUSIONS: All three types of instruments allow collection and growth of human nasal epithelial cells with good acceptability to study participants. The most efficient instrument is the nasal brush.

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.

Spatial and risk factor analyses of vector-borne pathogens among shelter dogs in the Eastern United States.

BACKGROUND: Vector-borne infections pose significant health risks to humans, domestic animals, and wildlife. Domestic dogs (Canis lupus familiaris) in the United States may be infected with and serve as sentinel hosts for several zoonotic vector-borne pathogens. In this study, we analyzed the geographical distribution, risk factors, and co-infections associated with infection with Ehrlichia spp., Anaplasma spp., Borrelia burgdorferi, and Dirofilaria immitis in shelter dogs in the Eastern United States. METHODS: From 2016 to 2020, blood samples from 3750 shelter dogs from 19 states were examined with IDEXX SNAP® 4Dx® Plus tests to determine the seroprevalence of infection with tick-borne pathogens and infection with D. immitis. We assessed the impact of factors including age, sex, intact status, breed group, and location on infection using logistic regression. RESULTS: The overall seroprevalence of D. immitis was 11.2% (n = 419/3750), the seroprevalence of Anaplasma spp. was 2.4% (n = 90/3750), the seroprevalence of Ehrlichia spp. was 8.0% (n = 299/3750), and the seroprevalence of B. burgdorferi was 8.9% (n = 332/3750). Regional variation in seroprevalence was noted: D. immitis (17.4%, n = 355/2036) and Ehrlichia spp. (10.7%, n = 217/2036) were highest in the Southeast while seroprevalence for B. burgdorferi (19.3%, n = 143/740) and Anaplasma spp. (5.7%, n = 42/740) were highest in the Northeast. Overall, 4.8% (n = 179/3750) of dogs had co-infections, the most common of which were D. immitis/Ehrlichia spp. (1.6%, n = 59/3750), B. burgdorferi/Anaplasma spp. (1.5%, n = 55/3750), and B. burgdorferi/Ehrlichia spp. (1.2%, n = 46/3750). Risk factors significantly influenced infection across the evaluated pathogens were location and breed group. All evaluated risk factors were significant for the seroprevalence of D. immitis antigens. CONCLUSIONS: Our results demonstrate a regionally variable risk of infection with vector-borne pathogens in shelter dogs throughout the Eastern United States, likely due to varying distributions of vectors. However, as many vectors are undergoing range expansions or other changes in distribution associated with climate and landscape change, continued vector-borne pathogen surveillance is important for maintaining reliable risk assessment.

Association between vector-borne pathogen seroprevalence in shelter-housed and owned dog populations in the contiguous United States of America.

Domestic dogs are susceptible to numerous vector-borne pathogens that are of significant importance for their health. In addition to being of veterinary importance, many of these pathogens are zoonotic and thus may pose a risk to human health. In the USA, owned dogs are commonly screened for exposure to or infection with several canine vector-borne pathogens. Although the screening data are widely available to show areas where infections are being diagnosed, testing of owned dogs is expected to underestimate the actual prevalence in dogs that have no access to veterinary care. The goal of this study was to measure the association between the widely available data from a perceived low-risk population with temporally and spatially collected data from shelter-housed dog populations. These data were then used to extrapolate the prevalence in dogs that generally lack veterinary care. The focus pathogens included Dirofilaria immitis, Ehrlichia spp., Anaplasma spp., and Borrelia burgdorferi. There was a linear association between the prevalence of selected vector-borne pathogens in shelter-housed and owned dog populations and, generally, the data suggested that prevalence of heartworm (D. immitis) infection and seroprevalence of Ehrlichia spp. and B. burgdorferi are higher in shelter-housed dogs, regardless of their location, compared with the owned population. The seroprevalence of Anaplasma spp. was predicted to be higher in areas that have very low to low seroprevalence, but unexpectedly, in areas of higher seroprevalence within the owned population, the seroprevalence was expected to be lower in the shelter-housed dog population. If shelters and veterinarians make decisions to not screen dogs based on the known seroprevalence of the owned group, they are likely underestimating the risk of exposure. This is especially true for heartworm. With this new estimate of the seroprevalence in shelter-housed dogs throughout the USA, shelters and veterinarians can make evidence-based informed decisions on whether testing and screening for these pathogens is appropriate for their local dog population. This work represents an important step in understanding the relationships in the seroprevalences of vector-borne pathogens between shelter-housed and owned dogs, and provides valuable data on the risk of vector-borne diseases in dogs.

Artificial infection of the bed bug with Rickettsia parkeri.

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.

Heterakis isolonche Infection Associated with Severe Nodular Typhlitis and Suspect Aberrant Pulmonary Migration in a Golden Pheasant (Chrysolopus pictus).

This case report describes the clinical, parasitologic, pathologic, and histologic characteristics of a golden pheasant (Chrysolopus pictus) with an infection of Heterakis isolonche in Mississippi. An approximately 2-yr-old golden pheasant from a flock of 8 to 10 birds was submitted to the Poultry Research and Diagnostic Laboratory in Pearl, MS, for necropsy. Clinical history indicated that three flock mates had died of unknown causes in the past. At necropsy, the submitted pheasant showed severe nodular typhlitis associated with the presence of numerous whitish small nematodes inside the cecal walls and lumen with morphologic features consistent with H. isolonche. The histologic examination showed multifocal to coalescing, nodular, granulomatous, and lymphocytic typhlitis with fibroplasia, and multiple intralesional nematodes. Furthermore, the presence of similar nematodes in the lung indicated a possible aberrant migration of Heterakis sp. to this organ. The flock was subsequently treated with an oxfendazole-containing dewormer and suffered no further losses.

Reporte de Caso- Infección por Heterakis isolonche asociada a tiflitis nodular severa y posible migración pulmonar aberrante en un faisán dorado (Chrysolopus pictus). Este informe de caso describe las características clínicas, parasitológicas, patológicas e histológicas de un faisán dorado (Chrysolopus pictus) con una infección por Heterakis isolonche en Mississippi. Un faisán dorado de aproximadamente dos años de edad de una parvada de ocho a diez aves fue remitido al Laboratorio de Investigación y Diagnóstico Avícolas en Pearl, Mississippi, para su necropsia. La historia clínica indicó que tres aves de la misma parvada habían muerto previamente por causas desconocidas. En la necropsia se observó tiflitis nodular grave asociada con la presencia de numerosos nematodos pequeños blanquecinos dentro de las paredes cecales y en el lumen con características morfológicas compatibles con H. isolonche. El examen histológico mostró tiflitis multifocal nodular coalescente, granulomatosa y linfocítica con fibroplasia y múltiples nematodos intralesionales. Además, la presencia de nematodos similares en el pulmón indicó una posible migración aberrante de Heterakis sp. a este órgano. Posteriormente, la parvada fue tratada con un antiparasitario que contenía oxfendazol y no presentó más pérdidas por mortalidad.

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.

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.

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.

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.

Rickettsia parkeri Rickettsiosis, Argentina.

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.

Distribution of spotted fever group rickettsiae in select tissues of experimentally infected and field-collected Gulf Coast ticks.

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.

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.

A Comprehensive Approach for the Diagnosis of Primary Ciliary Dyskinesia-Experiences from the First 100 Patients of the PCD-UNIBE Diagnostic Center.

Primary ciliary dyskinesia (PCD) is a rare genetic disease characterized by dyskinetic cilia. Respiratory symptoms usually start at birth. The lack of diagnostic gold standard tests is challenging, as PCD diagnostics requires different methods with high expertise. We founded PCD-UNIBE as the first comprehensive PCD diagnostic center in Switzerland. Our diagnostic approach includes nasal brushing and cell culture with analysis of ciliary motility via high-speed-videomicroscopy (HSVM) and immunofluorescence labeling (IF) of structural proteins. Selected patients undergo electron microscopy (TEM) of ciliary ultrastructure and genetics. We report here on the first 100 patients assessed by PCD-UNIBE. All patients received HSVM fresh, IF, and cell culture (success rate of 90%). We repeated the HSVM with cell cultures and conducted TEM in 30 patients and genetics in 31 patients. Results from cell cultures were much clearer compared to fresh samples. For 80 patients, we found no evidence of PCD, 17 were diagnosed with PCD, two remained inconclusive, and one case is ongoing. HSVM was diagnostic in 12, IF in 14, TEM in five and genetics in 11 cases. None of the methods was able to diagnose all 17 PCD cases, highlighting that a comprehensive approach is essential for an accurate diagnosis of PCD.