Kansas beef bulls with chronic anaplasmosis demonstrate satisfactory breeding soundness outcomes at breeding soundness examination.

OBJECTIVE: The objectives of this study were to evaluate the prevalence of chronic Anaplasma marginale infection in beef bulls from eastern Kansas and compare breeding soundness parameters between A marginale-infected and uninfected bulls. We hypothesized that bulls with chronic anaplasmosis would have inferior breeding soundness exam (BSE) outcomes as a result of persistent A marginale infection or the consequence of initial clinical disease compared to uninfected bulls. ANIMALS: 535 client-owned beef bulls from eastern Kansas undergoing routine BSE. METHODS: Complete BSEs were conducted by participating veterinarians according to the second edition of the Society for Theriogenology Manual for Breeding Soundness Examination of Bulls. Blood samples were collected for PCV determination and analysis of A marginale infection status via quantitative PCR and cELISA. Logistic and linear regression methods were used to evaluate factors associated with A marginale infection status and BSE parameters. RESULTS: Prevalence of chronic A marginale infection was 46% (245/535) among bulls. Unsatisfactory BSE outcome was not statistically associated with chronic anaplasmosis in this study population, although more bulls with chronic anaplasmosis had unsatisfactory BSE outcomes (15.0 ± 2.4% vs 12.0 ± 2.2%). CLINICAL RELEVANCE: Chronic anaplasmosis is prevalent among eastern Kansas breeding bulls; however, no negative association between chronic anaplasmosis and breeding soundness at time of BSE was observed.

Correction: A time-course comparative clinical and immune response evaluation study between the human pathogenic Orientia tsutsugamushi strains: Karp and Gilliam in a rhesus macaque (Macaca mulatta) model.

[This corrects the article DOI: 10.1371/journal.pntd.0010611.].

Satisfactory breeding potential is transiently eliminated in beef bulls with clinical anaplasmosis.

BACKGROUND: Natural service breeding is common in U.S. cow-calf operations. Diseases impacting bull reproductive performance have significant economic consequences for producers. Anaplasmosis may be an underappreciated cause of poor reproductive performance in bulls. The primary systemic effects of bovine anaplasmosis including anemia, fever, and weight loss, can all result in unsatisfactory reproductive performance. The objective of this pilot study was to evaluate breeding soundness examination (BSE) outcomes and clinical changes in bulls during and upon resolution of clinical anaplasmosis. Anaplasma marginale-challenged bulls were observed for clinical disease and infection progression and changes in breeding soundness compared to uninfected control bulls for 16 weeks. RESULTS: All Anaplasma marginale-challenged bulls were PCR-positive, seropositive, and showed clinical signs by 3-, 17-, and 24-days post-challenge, respectively. Clinical signs of anaplasmosis included pallor, icterus, fever (≥ 40.2 °C), and weight loss. Acute anemia was observed in all challenged bulls with PCV nadirs ≤ 18% and peak percent parasitized erythrocyte ≥ 50%. Decreased scrotal circumference and poor semen quality (e.g., increased percentage of abnormal spermatozoa, decreased progressively motile sperm), were initially observed within days after onset of clinical anaplasmosis signs and continued weeks beyond disease resolution. Control bulls remained negative for A. marginale. CONCLUSION: This pilot study demonstrates that clinical anaplasmosis reduces breeding soundness in beef bulls. Anaplasmosis should be considered as a differential for bulls with decreased semen quality, especially within endemic areas. A 90 day or greater retest window is recommended for bulls of unsatisfactory breeding potential recently recovered from clinical anaplasmosis.

A time-course comparative clinical and immune response evaluation study between the human pathogenic Orientia tsutsugamushi strains: Karp and Gilliam in a rhesus macaque (Macaca mulatta) model.

BACKGROUND: Scrub typhus is a vector-borne febrile illness caused by Orientia tsutsugamushi transmitted by the bite of Trombiculid mites. O. tsutsugamushi has a high genetic diversity and is increasingly recognized to have a wider global distribution than previously assumed. METHODOLOGY/PRINCIPLE FINDINGS: We evaluated the clinical outcomes and host immune responses of the two most relevant human pathogenic strains of O. tsutsugamushi; Karp (n = 4) and Gilliam (n = 4) in a time-course study over 80 days post infection (dpi) in a standardized scrub typhus non-human primate rhesus macaque model. We observed distinct features in clinical progression and immune response between the two strains; Gilliam-infected macaques developed more pronounced systemic infection characterized by an earlier onset of bacteremia, lymph node enlargement, eschar lesions and higher inflammatory markers during the acute phase of infection, when compared to the Karp strain. C-reactive protein (CRP) plasma levels, interferon gamma (IFN-γ, interleukin-1 receptor antagonist (IL-1ra), IL-15 serum concentrations, CRP/IL10- and IFN-γ/IL-10 ratios correlated positively with bacterial load in blood, implying activation of the innate immune response and preferential development of a T helper-type 1 immune response. The O. tsutsugamushi-specific immune memory responses in cells isolated from skin and lymph nodes at 80 dpi were more markedly elevated in the Gilliam-infected macaques than in the Karp-infected group. The comparative cytokine response dynamics of both strains revealed significant up-regulation of IFN-γ, tumor necrosis factor (TNF), IL-15, IL-6, IL-18, regulatory IL-1ra, IL-10, IL-8 and granulocyte-colony-stimulating factor (G-CSF). These data suggest that the clinical outcomes and host immune responses to scrub typhus could be associated with counter balancing effects of pro- and anti-inflammatory cytokine-mediated responses. Currently, no data on characterized time-course comparisons of O. tsutsugamushi strains regarding measures of disease severity and immune response is available. Our study provides evidence for the strain-specificity of host responses in scrub typhus, which supports our understanding of processes at the initial inoculation site (eschar), systemic disease progression, protective and/or pathogenic host immune mechanisms and cellular immune memory function. CONCLUSIONS/SIGNIFICANCE: This study characterised an improved intradermal rhesus macaque challenge model for scrub typhus, whereby the Gilliam strain infection associated with higher disease severity in the rhesus macaque model than the previous Karp strain infection. Difficulties associated with inoculum quantitation for obligate-intracellular bacteria were overcome by using functional inoculum titrations in outbred mice. The Gilliam-based rhesus macaque model provides improved endpoint measurements and contributes towards the identification of correlates of protection for future vaccine development.

Failure to Eliminate Persistent Anaplasma marginale Infection from Cattle Using Labeled Doses of Chlortetracycline and Oxytetracycline Antimicrobials.

Bovine anaplasmosis, caused by the intracellular rickettsial pathogen Anaplasma marginale, is the most prevalent tick-transmitted disease of cattle worldwide. In the U.S., tetracycline antimicrobials are commonly used to treat and control anaplasmosis. Oxytetracycline, administered by injection, is indicated for treatment of clinical anaplasmosis in beef and dairy cattle and calves. Chlortetracycline, administered orally, is indicated for control of active anaplasmosis infection in beef and nonlactating dairy cattle. Tetracyclines have been demonstrated to be effective for treating active anaplasmosis, but their ability to eliminate A. marginale at currently approved therapeutic doses or dosing regimens remains unclear. In the absence of approved dosing regimens for A. marginale clearance, a study was conducted to determine the effect of approved oxytetracycline and chlortetracycline indications on A. marginale bacteremia. Fifteen animals with persistent anaplasmosis were enrolled and divided into three treatment groups. Group 1 (n = 6) received oral chlortetracycline (1.1 mg/kg bodyweight) administered via hand-fed medicated feed for 60 consecutive days. Group 2 (n = 6) received injectable oxytetracycline administered subcutaneously at 19.8 mg/kg bodyweight three times in 3-week intervals. Group 3 (n = 3) served as an untreated control. After 60 days, bacteremia failed to permanently decrease in response to treatment. This result indicates that clearance of A. marginale is unlikely to be reliably achieved using currently approved tetracycline-based regimens to manage anaplasmosis.

Prevalence of Cytauxzoon felis Infection-Carriers in Eastern Kansas Domestic Cats.

Cytauxzoon felis is a hemoprotozoal tick-transmitted pathogen of felids. Felids that survive acute disease often remain infected and serve as reservoirs for subsequent tick transmission to other susceptible felines. States adjacent to Kansas have identified C. felis-domestic cat carriers while statewide awareness and concern of cytauxzoonosis have increased. The objective of this study was to determine the prevalence of C. felis-carriers in the eastern Kansas domestic cat population using a sensitive quantitative PCR assay targeting the C. felis Cox3 mitochondrial gene. An overall C. felis infection prevalence of 25.8% was determined for asymptomatic domestic cats in eastern Kansas. Significantly more C. felis-carrier cats were identified in spring and fall, suggesting a seasonal fluctuation of survivors. Additionally, a greater percentage of feral and owned cats were positive for C. felis compared to rescue/rescinded cats. This study demonstrates that C. felis-domestic cat carriers are common among cats that spend at least a portion of time outdoors in eastern Kansas, and that more cats likely survive cytauxzoonosis than expected. Understanding the role of domestic cat carriers of C. felis is essential in developing cytauxzoonosis mitigation strategies, including recommending year-round use of acaricide products for all cats that spend any time outdoors.

Development of a subcutaneous ear implant to deliver an anaplasmosis vaccine to dairy steers.

Bovine anaplasmosis is the most prevalent tick-transmitted disease of cattle worldwide and a major obstacle to profitable beef production. Use of chlortetracycline-medicated feed to control active anaplasmosis infections during the vector season has raised concerns about the potential emergence of antimicrobial resistance in bacteria that may pose a risk to human health. Furthermore, the absence of effectiveness data for a commercially available, conditionally licensed anaplasmosis vaccine is a major impediment to implementing anaplasmosis control programs. The primary objective of this study was to develop a single-dose vaccine delivery platform to produce long-lasting protective immunity against anaplasmosis infections. Twelve Holstein steers, aged 11 to 12 wk, were administered a novel 3-stage, single-dose vaccine against Anaplasma marginale, a major surface protein 1a. The vaccine consisted of a soluble vaccine administered subcutaneously (s.c.) for immune priming, a vaccine depot of a biodegradable polyanhydride rod with intermediate slow release of the vaccine for boosting immune response, and an immune-isolated vaccine platform for extended antigen release (VPEAR implant) deposited s.c. in the ear. Six calves were randomly assigned to 2 vaccine constructs (n = 3) that featured rods and implants containing a combination of 2 different adjuvants, diethylaminoethyl (DEAE)-Dextran and Quil-A (Group A). The remaining 6 calves were randomly assigned to 2 vaccine constructs (n = 3) that featured rods and implants containing the same adjuvant (either DEAE-Dextran or Quil A) (Group B). Twenty-one months post-implantation, calves were challenged intravenously with A. marginale stabilate and were monitored weekly for signs of fever, decreased packed cell volume (PCV) and bacteremia. Data were analyzed using a mixed-effects model and chi-squared tests (SAS v9.04.01, SAS Institute, Cary, NC). Calves in Group A had higher PCV than calves in Group B (P = 0.006) at day 35 post-infection. Calves in Group A were less likely to require antibiotic intervention compared with calves in Group B (P = 0.014). Results indicate that calves exhibited diminished clinical signs of anaplasmosis when antigen was delivered with a combination of adjuvants as opposed to a single adjuvant. This demonstrates the feasibility of providing long-lasting protection against clinical bovine anaplasmosis infections using a subcutaneous ear implant vaccine construct.

Rickettsial Illnesses as Important Causes of Febrile Illness in Chittagong, Bangladesh.

We conducted a yearlong prospective study of febrile patients admitted to a tertiary referral hospital in Chittagong, Bangladesh, to assess the proportion of patients with rickettsial illnesses and identify the causative pathogens, strain genotypes, and associated seasonality patterns. We diagnosed scrub typhus in 16.8% (70/416) and murine typhus in 5.8% (24/416) of patients; 2 patients had infections attributable to undifferentiated Rickettsia spp. and 2 had DNA sequence-confirmed R. felis infection. Orientia tsutsugamushi genotypes included Karp, Gilliam, Kato, and TA763-like strains, with a prominence of Karp-like strains. Scrub typhus admissions peaked in a biphasic pattern before and after the rainy season, whereas murine typhus more frequently occurred before the rainy season. Death occurred in 4% (18/416) of cases; case-fatality rates were 4% each for scrub typhus (3/70) and murine typhus (1/28). Overall, 23.1% (96/416) of patients had evidence of treatable rickettsial illnesses, providing important evidence toward optimizing empirical treatment strategies.

Autofluorescence microscopy for paired-matched morphological and molecular identification of individual chigger mites (Acari: Trombiculidae), the vectors of scrub typhus.

BACKGROUND: Conventional gold standard characterization of chigger mites involves chemical preparation procedures (i.e. specimen clearing) for visualization of morphological features, which however contributes to destruction of the arthropod host DNA and any endosymbiont or pathogen DNA harbored within the specimen. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a novel work flow based on autofluorescence microscopy was developed to enable identification of trombiculid mites to the species level on the basis of morphological traits without any special preparation, while preserving the mite DNA for subsequent genotyping. A panel of 16 specifically selected fluorescence microscopy images of mite features from available identification keys served for complete chigger morphological identification to the species level, and was paired with corresponding genotype data. We evaluated and validated this method for paired chigger morphological and genotypic ID using the mitochondrial cytochrome c oxidase subunit I gene (coi) in 113 chigger specimens representing 12 species and 7 genera (Leptotrombidium, Ascoschoengastia, Gahrliepia, Walchia, Blankaartia, Schoengastia and Schoutedenichia) from the Lao People's Democratic Republic (Lao PDR) to the species level (complete characterization), and 153 chiggers from 5 genera (Leptotrombidium, Ascoschoengastia, Helenicula, Schoengastiella and Walchia) from Thailand, Cambodia and Lao PDR to the genus level. A phylogenetic tree constructed from 77 coi gene sequences (approximately 640 bp length, n = 52 new coi sequences and n = 25 downloaded from GenBank), demonstrated clear grouping of assigned morphotypes at the genus levels, although evidence of both genetic polymorphism and morphological plasticity was found. CONCLUSIONS/SIGNIFICANCE: With this new methodology, we provided the largest collection of characterized coi gene sequences for trombiculid mites to date, and almost doubled the number of available characterized coi gene sequences with a single study. The ability to provide paired phenotypic-genotypic data is of central importance for future characterization of mites and dissecting the molecular epidemiology of mites transmitting diseases like scrub typhus.

Characterization of the rhesus macaque (Macaca mulatta) scrub typhus model: Susceptibility to intradermal challenge with the human pathogen Orientia tsutsugamushi Karp.

BACKGROUND: Scrub typhus is an important endemic disease in tropical Asia caused by Orientia tsutsugamushi for which no effective broadly protective vaccine is available. The successful evaluation of vaccine candidates requires well-characterized animal models and a better understanding of the immune response against O. tsutsugamushi. While many animal species have been used to study host immunity and vaccine responses in scrub typhus, only limited data exists in non-human primate (NHP) models. METHODOLOGY/PRINCIPLE FINDINGS: In this study we evaluated a NHP scrub typhus disease model based on intradermal inoculation of O. tsutsugamushi Karp strain in rhesus macaques (n = 7). After an intradermal inoculation with 106 murine LD50 of O. tsutsugamushi at the anterior thigh (n = 4) or mock inoculum (n = 3), a series of time course investigations involving hematological, biochemical, molecular and immunological assays were performed, until day 28, when tissues were collected for pathology and immunohistochemistry. In all NHPs with O. tsutsugamushi inoculation, but not with mock inoculation, the development of a classic eschar with central necrosis, regional lymphadenopathy, and elevation of body temperature was observed on days 7-21 post inoculation (pi); bacteremia was detected by qPCR on days 6-18 pi; and alteration of liver enzyme function and increase of white blood cells on day 14 pi. Immune assays demonstrated raised serum levels of soluble cell adhesion molecules, anti-O. tsutsugamushi-specific antibody responses (IgM and IgG) and pathogen-specific cell-mediated immune responses in inoculated macaques. The qPCR assays detected O. tsutsugamushi in eschar, spleen, draining and non-draining lymph nodes, and immuno-double staining demonstrated intracellular O. tsutsugamushi in antigen presenting cells of eschars and lymph nodes. CONCLUSIONS/SIGNIFICANCE: These data show the potential of using rhesus macaques as a scrub typhus model, for evaluation of correlates of protection in both natural and vaccine induced immunity, and support the evaluation of future vaccine candidates against scrub typhus.

Strong interferon-gamma mediated cellular immunity to scrub typhus demonstrated using a novel whole cell antigen ELISpot assay in rhesus macaques and humans.

Scrub typhus is a febrile infection caused by the obligate intracellular bacterium Orientia tsutsugamushi, which causes significant morbidity and mortality across the Asia-Pacific region. The control of this vector-borne disease is challenging due to humans being dead-end hosts, vertical maintenance of the pathogen in the vector itself, and a potentially large rodent reservoir of unclear significance, coupled with a lack of accurate diagnostic tests. Development of an effective vaccine is highly desirable. This however requires better characterization of the natural immune response of this neglected but important disease. Here we implement a novel IFN-γ ELISpot assay as a tool for studying O. tsutsugamushi induced cellular immune responses in an experimental scrub typhus rhesus macaque model and human populations. Whole cell antigen for O. tsutsugamushi (OT-WCA) was prepared by heat inactivation of Karp-strain bacteria. Rhesus macaques were infected intradermally with O. tsutsugamushi. Freshly isolated peripheral blood mononuclear cells (PBMC) from infected (n = 10) and uninfected animals (n = 5) were stimulated with OT-WCA, and IFN-γ secreting cells quantitated by ELISpot assay at five time points over 28 days. PBMC were then assayed from people in a scrub typhus-endemic region of Thailand (n = 105) and responses compared to those from a partially exposed population in a non-endemic region (n = 14), and to a naïve population in UK (n = 12). Mean results at Day 0 prior to O. tsutsugamushi infection were 12 (95% CI 0-25) and 15 (2-27) spot-forming cells (SFC)/106 PBMC for infected and control macaques respectively. Strong O. tsutsugamushi-specific IFN-γ responses were seen post infection, with ELISpot responses 20-fold higher than baseline at Day 7 (mean 235, 95% CI 200-270 SFC/106 PBMC), 105-fold higher at Day 14 (mean 1261, 95% CI 1,097-1,425 SFC/106 PBMC), 125-fold higher at Day 21 (mean 1,498, 95% CI 1,496-1,500 SFC/106 PBMC) and 118-fold higher at Day 28 (mean 1,416, 95% CI 1,306-1,527 SFC/106 PBMC). No significant change was found in the control group at any time point compared to baseline. Humans from a scrub typhus endemic region of Thailand had mean responses of 189 (95% CI 88-290) SFC/106 PBMC compared to mean responses of 40 (95% CI 9-71) SFC/106 PBMC in people from a non-endemic region and 3 (95% CI 0-7) SFC/106 PBMC in naïve controls. In summary, this highly sensitive assay will enable field immunogenicity studies and further characterization of the host response to O. tsutsugamushi, and provides a link between human and animal models to accelerate vaccine development.

Blood-Brain Barrier Function and Biomarkers of Central Nervous System Injury in Rickettsial Versus Other Neurological Infections in Laos.

Blood-brain barrier (BBB) function and cerebrospinal fluid (CSF) biomarkers were measured in patients admitted to hospital with severe neurological infections in the Lao People's Democratic Republic (N = 66), including bacterial meningitis (BM; N = 9) or tuberculosis meningitis (TBM; N = 11), Japanese encephalitis virus (JEV; N = 25), and rickettsial infections (N = 21) including murine and scrub typhus patients. The albumin index (AI) and glial fibrillary acidic protein (GFAP) levels were significantly higher in BM and TBM than other diseases but were also raised in individual rickettsial patients. Total tau protein was significantly raised in the CSF of JEV patients. No differences were found between clinical or neurological symptoms, AI, or biomarker levels that allowed distinction between severe neurological involvement by Orientia tsutsugamushi compared with Rickettsia species.

Pregnancy outcome in relation to treatment of murine typhus and scrub typhus infection: a fever cohort and a case series analysis.

BACKGROUND: There is a paucity of published reports on pregnancy outcome following scrub and murine typhus despite these infections being leading causes of undifferentiated fever in Asia. This study aimed to relate pregnancy outcome with treatment of typhus. METHODOLOGY/PRINCIPAL FINDINGS: Data were analyzed from: i) pregnant women with a diagnosis of scrub and/or murine typhus from a fever cohort studies; ii) case series of published studies in PubMed using the search terms "scrub typhus" (ST), "murine typhus" (MT), "Orientia tsutsugamushi", "Rickettsia tsutsugamushi", "Rickettsia typhi", "rickettsiae", "typhus", or "rickettsiosis"; and "pregnancy", until February 2014 and iii) an unpublished case series. Fever clearance time (FCT) and pregnancy outcome (miscarriage and delivery) were compared to treatment. Poor neonatal outcome was a composite measure for pregnancies sustained to 28 weeks or more of gestation ending in stillbirth, preterm birth, or delivery of a growth restricted or low birth weight newborn. RESULTS: There were 26 women in the fever cohort. MT and ST were clinically indistinguishable apart from two ST patients with eschars. FCTs (median [range] hours) were 25 [16-42] for azithromycin (n=5), 34 [20-53] for antimalarials (n=5) and 92 [6-260] for other antibiotics/supportive therapy (n=16). There were 36.4% (8/22) with a poor neonatal outcome. In 18 years, 97 pregnancies were collated, 82 with known outcomes, including two maternal deaths. Proportions of miscarriage 17.3% (14/81) and poor neonatal outcomes 41.8% (28/67) were high, increasing with longer FCTs (p=0.050, linear trend). Use of azithromycin was not significantly associated with improved neonatal outcomes (p=0.610). CONCLUSION: The published ST and MT world literature amounts to less than 100 pregnancies due to under recognition and under diagnosis. Evidence supporting the most commonly used treatment, azithromycin, is weak. Collaborative, prospective clinical trials in pregnant women are urgently required to reduce the burden of adverse maternal and newborn outcomes and to determine the safety and efficacy of antimicrobial treatment.

An intradermal inoculation model of scrub typhus in Swiss CD-1 mice demonstrates more rapid dissemination of virulent strains of Orientia tsutsugamushi.

Scrub typhus is an important endemic disease of the Asia-Pacific region caused by Orientia tsutsugamushi. To develop an effective vaccine to prevent scrub typhus infection, a better understanding of the initial host-pathogen interaction is needed. The objective of this study was to investigate early bacterial dissemination in a CD-1 Swiss outbred mouse model after intradermal injection of O. tsutsugamushi. Three human pathogenic strains of O. tsutsugamushi (Karp, Gilliam, and Woods) were chosen to investigate the early infection characteristics associated with bacterial virulence. Tissue biopsies of the intradermal injection site and draining lymph nodes were examined using histology and immunohistochemistry to characterize bacterial dissemination, and correlated with quantitative real-time PCR for O. tsutsugamushi in blood and tissue from major organs. Soluble adhesion molecules were measured to examine cellular activation in response to infection. No eschar formation was seen at the inoculation site and no clinical disease developed within the 7 day period of observation. However, O. tsutsugamushi was localized at the injection site and in the draining lymph nodes by day 7 post inoculation. Evidence of leukocyte and endothelial activation was present by day 7 with significantly raised levels of sL-selectin, sICAM-1 and sVCAM-1. Infection with the Karp strain was associated with earlier and higher bacterial loads and more extensive dissemination in various tissues than the less pathogenic Gilliam and Woods strains. The bacterial loads of O. tsutsugamushi were highest in the lungs and spleens of mice inoculated with Karp and Gilliam, but not Woods strains. Strains of higher virulence resulted in more rapid systemic infection and dissemination in this model. The CD-1 mouse intradermal inoculation model demonstrates features relevant to early scrub typhus infection in humans, including the development of regional lymphadenopathy, leukocyte activation and distant organ dissemination after low-dose intradermal injection with O. tsutsugamushi.