Improved molecular detection of Neorickettsia risticii with a duplex real-time PCR assay in the diagnosis of Potomac horse fever.

Neorickettsia risticii, an obligate intracellular bacterium, is the causative agent of Potomac horse fever (PHF). Diagnosis of PHF is based on demonstration of serum antibodies, isolation of N. risticii, and/or detection of nucleic acid by a PCR assay. An existing real-time PCR assay targeting the N. risticii 16S rRNA has been validated using blood samples from horses with colitis, and snails; to our knowledge, the performance of the assay for other sample types has not been reported. We describe here a modification of the 16S rRNA gene assay by the addition of a set of primers and probe targeting the N. risticii p51 gene to form a duplex assay. We validated the new assay using diagnostic specimens from 56 horses with suspected PHF. The assay consistently detected down to 5 copies of synthetic targets, and did not show any cross-reaction with common equine enteric pathogens. Although we did not establish the diagnostic sensitivity and specificity of the duplex assay, results for both gene targets were in complete agreement, with the exception of 4 fecal samples that tested positive for the 16S rRNA gene only. Further analysis indicated that testing of fecal samples using our 16S rRNA gene assay alone can produce a false-positive result.

Prevalence of Anaplasma phagocytophilum and Borrelia burgdorferi infection in horses from Pennsylvania (2017-2019) using antibody and organism-based detection.

OBJECTIVE: To determine the prevalence of Anaplasma phagocytophilum and Borrelia burgdorferi infections in Pennsylvania horses. ANIMALS: 271 horses. PROCEDURES: A survey was conducted with PCR and serology to evaluate anaplasmosis and Lyme disease infections in horses from Pennsylvania that were suspected for tick-borne infection. RESULTS: A phagocytophilum was detected in 19/271 (7.0%) Pennsylvania horses tested by the duplex PCR. B burgdorferi was not detected in any horse blood tested by PCR. Overall, 120/271 (44.3%) horses tested positive for presence of A phagocytophilum antibodies by at least the IDEXX SNAP 4Dx Plus lateral flow immunosorbent (SNAP) or indirect fluorescent antibody (IFA) assay, with 69 (25.5%) testing positive by both SNAP and IFA; 43 (15.9%) tested positive by IFA only, and 8 (3.0%) tested positive by SNAP only. Similarly, 209/271 (77.1%) horses tested positive for the presence of B burgdorferi antibodies by at least 1 test, with 139 (51.3%) testing positive by both SNAP and IFA; 45 (16.6%) tested positive by SNAP only, and 25 (9.2%) tested positive by IFA. CLINICAL RELEVANCE: Both A phagocytophilum and B burgdorferi are important tick-borne infections. The study provides prevalence data for both A phagocytophilum and B burgdorferi and compares test performance. For serologic detection, IFA detected antibodies to A phagocytophilum in a higher proportion (41.3%) of horses compared to SNAP (28.4%), while SNAP detected antibodies to B burgdorferi in a higher proportion (67.9%) of horses compared to IFA (60.5%). Both diseases showed a high seroprevalence in all areas surveyed.

An unusual outbreak of erysipelas on a goat farm in Pennsylvania.

Erysipelothrix rhusiopathiae infection and septicemia occurred in a 5-d old Boer goat found dead on a farm in western Pennsylvania. On autopsy, there was moderate, focally extensive hemorrhage along the remnants of the urachus and umbilical arteries and the apex of the urinary bladder. Microscopic examination of immunohistochemical stained tissues revealed abundant intracellular and extracellular E. rhusiopathiae antigen-positive bacilli in all tissues stained, including lung, heart, liver, skeletal muscle, kidney, and thymus. Bacteria isolated from liver and urachus were identified as E. rhusiopathiae by MALDI-TOF mass spectrometry and further confirmed by a PCR assay. An epidemiologic investigation was conducted via an on-farm questionnaire after the owners noted a 70% mortality rate from the 2019 kidding season. The epidemiologic investigation showed that E. rhusiopathiae, an opportunistic zoonotic organism, was introduced to the farm through a breach in biosecurity and was likely perpetuated among the resident poultry species.

Evaluation of a high-throughput nucleic acid extraction method for the detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples by PCR.

Johne's disease (paratuberculosis) is an economically important disease of cattle worldwide. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious gram-positive bacterium. PCR is increasingly used in diagnostic laboratories for the detection of MAP in fecal samples given the rapid test turnaround time and sensitivity and specificity comparable to fecal culture. However, efficient extraction of DNA for sensitive detection of MAP by PCR is affected by the complex lipid-rich cell wall of MAP and the presence of PCR inhibitors in feces. We evaluated a high-throughput nucleic acid extraction method (MagMAX core nucleic acid purification kit with mechanical lysis module) in conjunction with an hspX gene PCR for the detection of MAP from bovine fecal samples, which resulted in correct identification of all negative (13 of 13) and positive (35 of 35) proficiency test samples obtained from the National Veterinary Services Laboratories. In addition, all 6 negative and 50 of 51 positive diagnostic specimens tested were categorized correctly.

Comparison of three DNA extraction methods for molecular confirmation of Mycobacterium avium subspecies paratuberculosis from the VersaTrek™ liquid cultures of bovine fecal samples.

We evaluated three DNA extraction methods for confirmation of Mycobacterium avium subspecies paratuberculosis from liquid cultures of bovine feces. Use of DNA Extract All Reagents Kit™ resulted in efficient extraction of amplifiable DNA from higher proportion (96.29%) of known positive samples compared to Chelex-100 resin (25.92%) and polyethylene glycol (0%).

Detection of Brucella canis infection in dogs by blood culture and bacterial identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Brucella canis was recovered from dogs that were canine brucellosis suspect by blood culture using a modified lysis method. Organism identity was established by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The instrument-provided security library identified the isolates as Brucella species. The isolates were further identified as B. canis with the help of phenotypic and genotypic characteristics. The mass spectral profiles from characterized B. canis isolates, when added to the MALDI-TOF MS standard reference library, allowed successful presumptive identification of B. canis.

An animal model of a newly emerging human ehrlichiosis.

BACKGROUND: Human ehrlichioses are emerging life-threatening diseases transmitted by ticks. Animal models have been developed to study disease development; however, there is no valid small animal model that uses a human ehrlichial pathogen. The objective of this study was to develop a mouse model for ehrlichiosis with the newly discovered human pathogen, Ehrlichia muris-like agent (EMLA). METHODS: Three strains of mice were inoculated with different doses of EMLA by the intravenous, intraperitoneal, or intradermal route and evaluated for clinical and pathologic changes during the course of infection. RESULTS: EMLA infected C57Bl/6, BALB/c, and C3H/HeN mice and induced lethal or persistent infection in a route- and dose-dependent manner. The clinical chemistry and hematologic changes were similar to those of human infection by Ehrlichia chaffeensis or EMLA. Bacterial distribution in tissues differed after intradermal infection, compared with the distribution after intravenous or intraperitoneal injection. Lethal infection did not cause remarkable pathologic changes, but it caused fluid imbalance. EMLA infection of endothelium and mononuclear cells likely plays a role in the severe outcome. CONCLUSIONS: The EMLA mouse model mimics human infection and can be used to study pathogenesis and immunity and for development of a vector transmission model of ehrlichiosis.

Early induction of interleukin-10 limits antigen-specific CD4⁺ T cell expansion, function, and secondary recall responses during persistent phagosomal infection.

Diverse pathogens have evolved to survive and replicate in the endosomes or phagosomes of the host cells and establish persistent infection. Ehrlichiae are Gram-negative, intracellular bacteria that are transmitted by ticks. Ehrlichiae reside in the endosomes of the host phagocytic or endothelial cells and establish persistent infection in their vertebrate reservoir hosts. CD4(+) T cells play a critical role in protection against phagosomal infections. In the present study, we investigated the expansion, maintenance, and functional status of antigen-specific CD4(+) T cells during persistent Ehrlichia muris infection in wild-type and interleukin-10 (IL-10)-deficient mice. Our study indicated that early induction of IL-10 led to reduced inflammatory responses and impaired bacterial clearance during persistent Ehrlichia infection. Notably, we demonstrated that the functional production of gamma interferon (IFN-γ) by antigen-specific CD4(+) T cells maintained during a persistent phagosomal infection progressively deteriorates. The functional loss of IFN-γ production by antigen-specific CD4(+) T cells was reversed in the absence of IL-10. Furthermore, we demonstrated that transient blockade of IL-10 receptor during the T cell priming phase early in infection was sufficient to enhance the magnitude and the functional capacity of antigen-specific effector and memory CD4(+) T cells, which translated into an enhanced recall response. Our findings provide new insights into the functional status of antigen-specific CD4(+) T cells maintained during persistent phagosomal infection. The study supports the concept that a better understanding of the factors that influence the priming and differentiation of CD4(+) T cells may provide a basis to induce a protective immune response against persistent infections.

Complete Genome Sequence of Ehrlichia muris Strain AS145T, a Model Monocytotropic Ehrlichia Strain.

We report here the complete genome sequence of Ehrlichia muris strain AS145(T), which was isolated from a wild mouse in 1983 in Japan. E. muris establishes persistent infections in laboratory mice and is widely used as a surrogate pathogen in a murine model of ehrlichiosis.

Recombinant Ehrlichia P29 protein induces a protective immune response in a mouse model of ehrlichiosis.

Ehrlichioses are emerging tick-borne bacterial diseases of humans and animals for which no vaccines are available. The diseases are caused by obligately intracellular bacteria belonging to the genus Ehrlichia. Several immunoreactive proteins of ehrlichiae have been identified based on their reactivity with immune sera from human patients and animals. These include the major outer membrane proteins, ankyrin repeat proteins and tandem repeat proteins (TRP). Polyclonal antibodies directed against the tandem repeats (TRs) of Ehrlichia chaffeensis TRP32, TRP47 and TRP120 have been shown to provide protection in mice. In the present study, we evaluated E. muris P29, which is the ortholog of E. chaffeensis TRP47 and E. canis TRP36, as a subunit vaccine in a mouse model of ehrlichiosis. Our study indicated that unlike E. chaffeensis TRP47 and E. canis TRP36, orthologs of E. muris (P29) and E. muris-like agent (EMLA) do not contain tandem repeats. Immunization of mice with recombinant E. muris P29 induced significant protection against a challenge infection. The protection induced by E. muris P29 was associated with induction of strong antibody responses. In contrast to development of P29-specific IgG antibodies following immunization, development of P29-specific IgG antibodies, but not IgM antibodies, was impaired during persistent E. muris infection. Furthermore, our study indicated that CD4+ T cells target P29 during E. muris infection and differentiate into IFN-γ-producing Th1 effector/memory cells. In conclusion, our study indicated that orthologs of E. muris P29 showed considerable variation in the central tandem repeat region among different species, induction of P29-specific IgG antibody response was impaired during persistent E. muris infection, and rP29 induced protective immune responses.

Structure-based vaccines provide protection in a mouse model of ehrlichiosis.

BACKGROUND: Recent advances in bioinformatics have made it possible to predict the B cell and T cell epitopes of antigenic proteins. This has led to design of peptide based vaccines that are more specific, safe, and easy to produce. The obligately intracellular gram negative bacteria Ehrlichia cause ehrlichioses in humans and animals. As yet there are no vaccines to protect against Ehrlichia infection. METHODOLOGY/PRINCIPAL FINDINGS: We applied the principle of structural vaccinology to design peptides to the epitopes of Ehrlichia muris outer membrane P28-19 (OMP-1/P28) and Ehrlichia Heat shock protein 60 (Hsp60/GroEL) antigenic proteins. Both P28-19 and Ehrlichia Hsp60 peptides reacted with polyclonal antibodies against E. canis and E. chaffeensis and could be used as a diagnostic tool for ehrlichiosis. In addition, we demonstrated that mice vaccinated with Ehrlichia P28-19 and Hsp60 peptides and later challenged with E. muris were protected against the pathogen. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate the power of structural vaccines and could be a new strategy in the development of vaccines to provide protection against pathogenic microorganisms.

Immunization with Ehrlichia P28 outer membrane proteins confers protection in a mouse model of ehrlichiosis.

The obligately intracellular bacterium Ehrlichia chaffeensis that resides in mononuclear phagocytes is the etiologic agent of human monocytotropic ehrlichiosis (HME). HME is an emerging and often life-threatening, tick-transmitted infectious disease in the United States. Effective primary immune responses against Ehrlichia infection involve generation of Ehrlichia-specific gamma interferon (IFN-γ)-producing CD4(+) T cells and cytotoxic CD8(+) T cells, activation of macrophages by IFN-γ, and production of Ehrlichia-specific antibodies of the Th1 isotype. Currently, there are no vaccines available against HME. We evaluated the ability of 28-kDa outer membrane proteins (P28-OMP-1) of the closely related Ehrlichia muris to stimulate long-term protective memory T and B cell responses and confer protection in mice. The spleens of mice vaccinated with E. muris P28-9, P28-12, P28-19, or a mixture of these three P28 proteins (P28s) using a DNA prime-protein boost regimen and challenged with E. muris had significantly lower bacterial loads than the spleens of mock-vaccinated mice. Mice immunized with P28-9, P28-12, P28-19, or the mixture induced Ehrlichia-specific CD4(+) Th1 cells. Interestingly, mice immunized with P28-14, orthologs of which in E. chaffeensis and E. canis are primarily expressed in tick cells, failed to lower the ehrlichial burden in the spleen. Immunization with the recombinant P28-19 protein alone also significantly decreased the bacterial load in the spleen and liver compared to those of the controls. Our study reports, for the first time, the protective roles of the Ehrlichia P28-9 and P28-12 proteins in addition to confirming previous reports of the protective ability of P28-19. Partial protection induced by immunization with P28-9, P28-12, and P28-19 against Ehrlichia was associated with the generation of Ehrlichia-specific cell-mediated and humoral immune responses.

Natural killer cells promote tissue injury and systemic inflammatory responses during fatal Ehrlichia-induced toxic shock-like syndrome.

Human monocytotropic ehrlichiosis is caused by Ehrlichia chaffeensis, a Gram-negative bacterium lacking lipopolysaccharide. We have shown that fatal murine ehrlichiosis is associated with CD8(+)T cell-mediated tissue damage, tumor necrosis factor-alpha, and interleukin (IL)-10 overproduction, and CD4(+)Th1 hyporesponsiveness. In this study, we examined the relative contributions of natural killer (NK) and NKT cells in Ehrlichia-induced toxic shock. Lethal ehrlichial infection in wild-type mice induced a decline in NKT cell numbers, and late expansion and migration of activated NK cells to the liver, a main infection site that coincided with development of hepatic injury. The spatial and temporal changes in NK and NKT cells in lethally infected mice correlated with higher NK cell cytotoxic activity, higher expression of cytotoxic molecules such as granzyme B, higher production of interferon-gamma and tumor necrosis factor-alpha, increased hepatic infiltration with CD8alphaCD11c(+) dendritic cells and CD8(+)T cells, decreased splenic CD4(+)T cells, increased serum concentrations of IL-12p40, IL-18, RANTES, and monocyte chemotactic protein-1, and elevated production of IL-18 by liver mononuclear cells compared with nonlethally infected mice. Depletion of NK cells prevented development of severe liver injury, decreased serum levels of interferon-gamma, tumor necrosis factor-alpha, and IL-10, and enhanced bacterial elimination. These data indicate that NK cells promote immunopathology and defective anti-ehrlichial immunity, possibly via decreasing the protective immune response mediated by interferon-gamma producing CD4(+)Th1 and NKT cells.

Persistent infection contributes to heterologous protective immunity against fatal ehrlichiosis.

Human monocytotropic ehrlichiosis (HME), an emerging and often life-threatening tick-transmitted disease, is caused by the obligately intracellular bacterium Ehrlichia chaffeensis. HME is modeled in C57BL/6 mice using Ehrlichia muris, which causes persistent infection, and Ixodes ovatus Ehrlichia (IOE), which is either acutely lethal or sublethal depending on the dose and route of inoculation. A persistent primary E. muris infection, but not a sublethal IOE infection, protects mice against an ordinarily lethal secondary IOE challenge. In the present study, we determined the role of persistent infection in maintenance of protective memory immune responses. E. muris-infected mice were treated with doxycycline or left untreated and then challenged with an ordinarily lethal dose of IOE. Compared to E. muris-primed mice treated with doxycycline, untreated mice persistently infected with E. muris had significantly greater numbers of antigen-specific gamma interferon-producing splenic memory T cells, significant expansion of CD4(+) CD25(+) T regulatory cells, and production of transforming growth factor beta1 in the spleen. Importantly, E. muris-primed mice treated with doxycycline showed significantly greater susceptibility to challenge infection with IOE compared to untreated mice persistently infected with E. muris. The study indicated that persistent ehrlichial infection contributes to heterologous protection by stimulating the maintenance of memory T-cell responses.

Protective heterologous immunity against fatal ehrlichiosis and lack of protection following homologous challenge.

The roles of antibodies and memory T cells in protection against virulent Ehrlichia have not been completely investigated. In this study, we addressed these issues by using murine models of mild and fatal ehrlichiosis caused by related monocytotropic Ehrlichia strains. Mice were primed with either Ehrlichia muris or closely related virulent ehrlichiae transmitted by Ixodes ovatus (IOE) ticks given intraperitoneally or intradermally. All groups were reinfected intraperitoneally, 30 days later, with a lethal high dose of IOE. Priming with E. muris, but not IOE, induced strong CD4+ and CD8+ memory type 1 T-cell responses, Ehrlichia-specific immunoglobulin G (IgG) antibodies, and persistent infection. Compared to IOE-primed mice, subsequent lethal IOE challenge of E. muris-primed mice, resulted in (i) 100% protection against lethal infection, (ii) strong Ehrlichia-specific secondary gamma interferon (IFN-gamma)-producing effector/effector memory CD4+ and CD8+ T-cell responses, (iii) enhanced secondary anti-ehrlichial antibody response, (iv) accelerated bacterial clearance, and (v) the formation of granulomas in the liver and lung. E. muris-primed mice challenged with IOE had lower levels of serum interleukin-1alpha (IL-1alpha), IL-6, and IL-10 compared to unprimed mice challenged with IOE. Interestingly, the fatal secondary response in IOE-primed mice correlated with (i) decline in the Ehrlichia-specific CD4+ and CD8+ type 1 responses, (ii) marked hepatic apoptosis and necrosis, and (iii) substantial bacterial clearance, suggesting that fatal secondary response is due to immune-mediated tissue damage. In conclusion, protection against fatal ehrlichial infection correlates with strong expansion of IFN-gamma-producing CD4+ and CD8+ effector memory type 1 T cells, which appear to be maintained in the presence of IgG antibodies and persistent infection.