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.

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.

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.

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.

Serum Derived Transfer Factor Stimulates the Innate Immune System to Improve Survival Traits in High Risk Pathogen Scenarios.

Preclinical Research Transfer Factors (TFs) are low molecular weight (<5,000 daltons) biological response mediators. In the present study, a serum derived TF improved the ability of the recipient animal to survive high-risk infectious challenges (salmonellosis and canine parvoviral enteritis (CPV)) by altering the host's cytokine response profile. Mice mortally challenged with 5,000 colony-forming units of Salmonella experienced a group mortality of 73% while mice treated with a single 5 mg dose of the TF demonstrated a significant decrease in morbidity (7%, p ≤ 0.01). The splenic bacterial load in untreated mice was over 10,000 times higher than that in the TF treated mice. Twenty-four hours post-administration, the treated murine population expressed a rapid temporal increase in serum IL-6 (26-fold) and INF-γ (77-fold) concentrations. IL-6 can act as a critical signal regulating action against bacterial pathogens. A comparative double-blind study performed using dogs confirmed to be undergoing a canine parvovirus challenge showed that when conventional supportive therapy was supplemented with a single 5 mg TF dose there was a reduction (p ≤ 0.01) in group mortality (68% of the TF treated group survived versus 32% of the placebo group), an observation consistent with the observed increase in INF-γ, a cytokine associated with promoting antiviral activity. Drug Dev Res 78 : 189-195, 2017. © 2017 Wiley Periodicals, Inc.