Detection of Borrelia miyamotoi and other tick-borne pathogens in human clinical specimens and Ixodes scapularis ticks in New York State, 2012-2015.

Borrelia miyamotoi (Bm) is a recently emerging bacterial agent transmitted by several species of ixodid ticks. Diagnosis of Bm infection can be challenging, as the organism is not easily cultivable. We have developed and validated a multiplex real-time PCR to simultaneously identify Bm infection and the agents causing human granulocytic anaplasmosis and human monocytic ehrlichiosis, Anaplasma phagocytophilum and Ehrlichia chaffeensis, respectively. The assay is 100% specific; highly sensitive, detecting 11 gene copies of Bm DNA in both whole blood and cerebral spinal fluid; and provides rapid results in less than two hours. A retrospective study of 796 clinical specimens collected between the years 2012 and 2014 and a prospective study of 366 clinical specimens were performed utilizing this novel assay to evaluate the frequency of Bm infection in New York State (NYS). Eight clinical specimens (1%) were found to be positive for Bm, 216 were positive for A. phagocytophilum, and 10 were positive for E. chaffeensis. Additionally, we tested 411 I. scapularis ticks collected in NYS during 2013 and 2014 in a separate multiplex real-time PCR to determine the prevalence of Bm, A. phagocytophilum, Borrelia burgdorferi s.s., and Borrelia species. Our results indicated rates of 1.5%, 27%, 19.7%, and 8.8% respectively. The ability to monitor both the frequency and geographic distribution of Bm cases and the prevalence and geographic distribution of Bm in ticks will help create a better understanding of this emerging tick-borne pathogen.

Serum canine pancreatic lipase immunoreactivity in experimentally induced and naturally occurring canine monocytic ehrlichiosis (Ehrlichia canis).

Ehrlichia canis infection causes multisystemic disease in dogs (canine monocytic ehrlichiosis, CME) which is associated with variable morbidity and mortality. Atypical clinical manifestations, including gastrointestinal signs, may occasionally occur in CME and approximately 10-15% of dogs are presented with historical or clinical evidence of vomiting, diarrhea, and/or abdominal discomfort. The objective of this study was to investigate if there are any alterations in serum canine pancreatic lipase immunoreactivity (cPLI) in dogs with experimentally induced or naturally occurring monocytic ehrlichiosis. Serum samples from 10 Beagle dogs experimentally infected with E. canis and two healthy uninfected Beagles were serially examined; samples from 20 naturally infected dogs (10 with non-myelosuppressive [NME] and 10 with myelosuppressive [ME] ehrlichiosis) were also examined at a given point in time (cross-sectional sampling). None of the experimentally infected Beagles showed gastrointestinal signs or increased cPLI concentrations prior to or following the artificial infection. Three naturally infected dogs with NME and one with ME demonstrated serum cPLI concentrations in the diagnostic range for pancreatitis (>400 µg/L) without showing gastrointestinal signs. The results of the present study indicated that 4/20 (20%) of dogs naturally infected with E. canis demonstrated increased serum cPLI concentrations consistent with mild and clinically inapparent pancreatitis.

Influence of experimental canine ehrlichiosis on the E-ADA activity and purine levels in serum and possible functional correlations with pathogenesis.

The aim of this study was to evaluate adenosine deaminase activity and purines levels in serum of dogs experimentally infected by Ehrlichia canis. Banked serum samples of dogs divided into two groups with five animals each: healthy animals and animals infected by E. canis. The concentration of purines (adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine, inosine, hypoxanthine, xanthine and uric acid), and adenosine deaminase (E-ADA) activity in sera were evaluated. Samples were collected on days 12 and 30 post-infection (PI). The E-ADA activity showed a significant reduction on day 12 PI, and increased on day 30 PI in dogs infected with E. canis. On day 12, an increase in seric concentration of ATP, ADP and adenosine was verified, and different levels of hypoxanthine, xanthine and uric acid had a drastic reduction in infected compared healthy dogs (P<0.05). However, on day 30 PI, the levels of seric ADP and AMP decreased, unlike the concentration of xanthine and uric acid that increased significantly in infected dogs (P<0.05). Therefore, the activity of E-ADA and purine levels are altered in experimental canine ehrlichiosis, probably with the purpose of modulating the pathogenesis of the disease related to immune response, oxidative stress and coagulation disorders in acute phase.

The Anaplasma phagocytophilum PleC histidine kinase and PleD diguanylate cyclase two-component system and role of cyclic Di-GMP in host cell infection.

Anaplasma phagocytophilum, the etiologic agent of human granulocytic anaplasmosis (HGA), has genes predicted to encode three sensor kinases, one of which is annotated PleC, and three response regulators, one of which is PleD. Prior to this study, the roles of PleC and PleD in the obligatory intracellular parasitism of A. phagocytophilum and their biochemical activities were unknown. The present study illustrates the relevance of these factors by demonstrating that both pleC and pleD were expressed in an HGA patient. During A. phagocytophilum development in human promyelocytic HL-60 cells, PleC and PleD were synchronously upregulated at the exponential growth stage and downregulated prior to extracellular release. A recombinant PleC kinase domain (rPleCHKD) has histidine kinase activity; no activity was observed when the conserved site of phosphorylation was replaced with alanine. A recombinant PleD (rPleD) has autokinase activity using phosphorylated rPleCHKD as the phosphoryl donor but not with two other recombinant histidine kinases. rPleCHKD could not serve as the phosphoryl donor for a mutant rPleD (with a conserved aspartic acid, the site of phosphorylation, replaced by alanine) or two other A. phagocytophilum recombinant response regulators. rPleD had diguanylate cyclase activity to generate cyclic (c) di-GMP from GTP in vitro. UV cross-linking of A. phagocytophilum lysate with c-di-[(32)P]GMP detected an approximately 47-kDa endogenous protein, presumably c-di-GMP downstream receptor. A new hydrophobic c-di-GMP derivative, 2'-O-di(tert-butyldimethylsilyl)-c-di-GMP, inhibited A. phagocytophilum infection in HL-60 cells. Our results suggest that the two-component PleC-PleD system is a diguanylate cyclase and that a c-di-GMP-receptor complex regulates A. phagocytophilum intracellular infection.

c-Jun NH2-terminal kinase 2 inhibits gamma interferon production during Anaplasma phagocytophilum infection.

Gamma interferon (IFN-gamma) plays a critical role in the early eradication of Anaplasma phagocytophilum. However, the mechanisms that regulate IFN-gamma production upon infection remain poorly understood. Here we show that c-Jun NH2-terminal kinase 2 (JNK2) inhibits IFN-gamma production during A. phagocytophilum infection. jnk2-null mice were more refractory to infection with A. phagocytophilum and produced increased levels of IFN-gamma after challenge with the pathogen. The resistance of jnk2-null mice to A. phagocytophilum infection was due to elevated levels of IFN-gamma secreted by conventional and natural killer (NK) T cells. The administration of alpha-galactosylceramide, a strong NK T-cell agonist, increased IFN-gamma release and protected mice from A. phagocytophilum, further demonstrating the inhibitory effect of JNK2 on IFN-gamma production. Collectively, these findings provide strong evidence that JNK2 is an important regulatory protein for IFN-gamma secretion upon challenge with A. phagocytophilum.

Frontline: control of Anaplasma phagocytophilum, an obligate intracellular pathogen, in the absence of inducible nitric oxide synthase, phagocyte NADPH oxidase, tumor necrosis factor, Toll-like receptor (TLR)2 and TLR4, or the TLR adaptor molecule MyD88.

Anaplasma phagocytophilum is an obligate intracellular bacterium that is related to rickettsial organisms and replicates in the hostile environment of neutrophils. Previous studies with SCID mice suggested that T and/or B cells are required for its control in vivo. Here, we used mice deficient for Toll-like receptor (TLR)2 and TLR4, MyD88, tumor necrosis factor, inducible nitric oxide synthase, or phagocyte NADPH oxidase (gp91(phox-/-)) to define the pathways that are critical for the recognition and the killing of this pathogen. Whereas SCID mice developed a 60-fold higher bacterial load in the blood compared to wild-type mice and succumbed to infection, all other gene-deficient mouse strains were fully capable in overcoming a systemic infection with A. phagocytophilum. From these data we conclude that effector mechanisms that are crucial to the defense against numerous other intracellular pathogens are dispensable for the control of A. phagocytophilum.

Granulocytic ehrlichiosis in mice deficient in phagocyte oxidase or inducible nitric oxide synthase.

Mice deficient in phox (gp91(phox-/-)) or NOS2 (NOS2(-/-)) were infected with the agent of human granulocytic ehrlichiosis (HGE) to evaluate the importance of these pathways in the eradication of HGE bacteria. NOS2(-/-) mice had delayed clearance of the HGE agent in comparison to control or gp91(phox-/-) mice, suggesting that reactive nitrogen intermediates play a role in the early control of HGE.