Analysis of genetic identity of North American Anaplasma phagocytophilum strains by pulsed-field gel electrophoresis.

Biological and geographic heterogeneity of anthropozoonosis caused by Anaplasma phagocytophilum is poorly understood. Seven North American A. phagocytophilum strains were compared by PFGE. The average genome size was 1.58 Mbp, and restriction patterns were identical. New World strains of A. phagocytophilum have a large genome and a high degree of genetic uniformity.

ankA: an Ehrlichia phagocytophila group gene encoding a cytoplasmic protein antigen with ankyrin repeats.

Human granulocytic ehrlichiosis (HGE) is a potentially fatal, tick-borne disease caused by a bacterium related or identical to Ehrlichia phagocytophila. To identify and characterize E. phagocytophila group-specific protein antigen genes, we prepared and screened HGE agent and Ehrlichia equi genomic DNA expression libraries using polyclonal equine E. equi antibodies. Two clones, one each from HGE agent and E. equi, that were recognized specifically by antibodies to the E. phagocytophila group ehrlichiae had complete open reading frames of 3,693 and 3,615 nucleotides, respectively. The two clones were 96.6% identical and predicted a protein with at least 11 tandemly repeated ankyrin motifs. Thus, the gene was named ank (for ankyrin). When the encoded protein, named AnkA, was expressed in Escherichia coli, it was recognized by antibodies from rabbits and mice immunized with the HGE agent, sera from humans convalescent from HGE, and sera from horses convalescent from HGE and E. equi infection. Monospecific AnkA antibodies reacted with proteins in HGE agent immunoblots, and AnkA monoclonal antibodies detected cytoplasmic antigen in E. phagocytophila group bacteria and also detected antigen associated with chromatin in infected but not uninfected HL-60 cell cultures. These results suggest that this Ehrlichia protein may influence host cell gene expression.

Experimental inoculation with human granulocytic Ehrlichia agent derived from high- and low-passage cell culture in horses.

We report the successful infection throughout intravenous inoculation with low and high passage of in vitro-grown human granulocytic ehrlichiosis (HGE) agent in horses. Differences in disease severity but not in incubation time, hematological changes, PCR detection, ehrlichial load, seroconversion time, and titer range were noted between horses infected with a low and a high passage of in vitro-grown HGE agent.

Improved sensitivity of PCR for diagnosis of human granulocytic ehrlichiosis using epank1 genes of Ehrlichia phagocytophila-group ehrlichiae.

The agent of human granulocytic ehrlichiosis (HGE), Ehrlichia phagocytophila, and Ehrlichia equi probably comprise variants of a single Ehrlichia species now called the Ehrlichia phagocytophila genogroup. These variants share a unique 153-kDa protein antigen with ankyrin repeat motifs encoded by the epank1 gene. The epank1 gene was investigated as an improved target for PCR diagnosis of HGE compared with the currently used 16S rRNA gene target. Primers for epank1 flanking a region that spans part of the 5' ankyrin repeat coding region and part of the unique 3' region were synthesized. Blood samples from 31 patients with suspected HGE who were previously tested by 16S rRNA gene (16S) PCR and indirect immunofluorescent antibody test (IFA) were retrospectively tested with the epank1 primers. Eleven patients were 16S PCR positive and had a seroconversion detected by IFA (group A), 10 patients were 16S PCR negative but had a seroconversion detected by IFA (group B), and 10 patients were 16S PCR negative and seronegative (group C). Ten of the 11 group A patients were epank1 PCR positive, all 10 of the group B patients were epank1 PCR positive, and all of the PCR-negative and seronegative patients (group C) were epank1 PCR negative. The epank1 primers are more sensitive than the previously used 16S rRNA gene primers and therefore may be more useful in diagnostic testing for HGE.

Seroprevalence of human granulocytic ehrlichiosis among permanent residents of northwestern Wisconsin.

Four-hundred seventy-five permanent residents of Wisconsin were tested for antibodies to the agent of human granulocytic ehrlichiosis (HGE) by indirect immunofluorescent antibody (IFA) testing with Ehrlichia equi as antigen marker. Each resident completed a standard survey questionnaire about outdoor activities, animal and tick exposure, and any febrile illness during the preceding 12 months. Seventy-one serum samples (14.9%) contained E. equi antibodies. The mean IFA titer for seropositive residents was 250 (range, 80-10,240). Seropositive residents were older than seronegative ones (62 vs. 56 years; P = .019). None of the seropositive residents had a history suggestive of ehrlichiosis. There was no association between the IFA test outcome and specific demographic variables or history of tick bites. HGE appears to be a common subclinical or mild infection among residents in northwestern Wisconsin.

Serologic evidence of a natural infection of white-tailed deer with the agent of human granulocytic ehrlichiosis in Wisconsin and Maryland.

White-tailed deer participate in the maintenance of the Ixodes tick life cycle and are reservoirs for some tick-borne infectious agents. Deer may be useful sentinels for tick-transmitted agents, such as ehrlichiae. In order to determine whether white-tailed deer are markers of natural transmission or are reservoirs for the human granulocytic ehrlichiosis (HGE) agent, we performed indirect immunofluorescent-antibody (IFA) tests and immunoblotting with the HGE agent and Ehrlichia chaffeensis on sera from 43 and 294 deer captured in northwest Wisconsin during 1994 and 1995, respectively, and 12 deer from southern Maryland. According to IFA testing, 47% of 1994 Wisconsin sera, 60% of 1995 Wisconsin sera, and 25% of Maryland sera contained HGE agent antibodies. All IFA-positive deer sera tested reacted with the 44-kDa band which is unique to the Ehrlichia phagocytophila group. Serologic reactions to E. chaffeensis were detected by IFA testing in 15 of 337 (4%) Wisconsin deer and in 10 of 12 (83%) Maryland deer, while 60 and 80% of E. chaffeensis IFA-positive Wisconsin and Maryland deer sera, respectively, reacted with the E. chaffeensis 28- to 29-kDa antigens by immunoblotting. A total of 4% of deer from Wisconsin and 25% of deer from Maryland were found by IFA testing to have antibodies to both the HGE agent and E. chaffeensis; 75% of these were confirmed to contain E. chaffeensis antibodies by immunoblotting. These results suggest that white-tailed deer in diverse geographical regions of the United States are naturally infected with the HGE agent, E. chaffeensis, or both and that these animals, and potentially humans, are exposed to infected ticks at a high frequency in nature.

Delay of estradiol-induced surge secretion of LH in gilts by intracerebroventricular injection of morphine.

In Experiment 1, ovariectomized (OVX) gilts, 143 d old and 58.5 +/- 1.8 kg BW, received 10 micrograms estradiol benzoate (EB)/kg BW i.m. and either 500 micrograms of the endogenous opioid peptide (EOP) agonist, morphine (MOR), in saline (SAL; n = 5), or SAL (n = 4) intracerebro-ventricularly at 40 and 48 hr after EB. With the exception of one MOR-treated gilt, which was deleted from Experiment 1, LH secretion was suppressed for at least 50 hr in all gilts. Timing of the LH surge was similar among gilts. However, total LH secreted was greater (P < 0.05) after SAL than MOR. The experiment was repeated at 179 d of age and 78.6 +/- 1.2 kg BW, except that treatments were reversed among gilts. Emergence of the LH surge was delayed (P < 0.005) by 10.8 hr and time to maximum LH concentration (P < 0.05) by 6.8 hr after MOR than after SAL. Magnitude and total LH secreted were not different among gilts. In Experiment 2, gilts which had displayed estrous cycles of 18-22 d were OVX and treated as in Experiment 1, except MOR (n = 3) or SAL (n = 4) were injected 10 hr later than in Experiment 1, i.e., at 50 and 58 hr after EB. Secretion of LH was suppressed for at least 60 hr in both groups. Time to emergence of the LH surge was delayed by 27 hr (P < 0.05) after MOR compared to after SAL. However, other parameters of the surge were not different among gilts. Thus, EOP modulate LH surge secretion negatively in the pig.

Antigenic diversity of granulocytic Ehrlichia isolates from humans in Wisconsin and New York and a horse in California.

The agent of human granulocytic ehrlichiosis (HGE), Ehrlichia phagocytophila, and Ehrlichia equi are very similar. HGE is of variable severity. Genetic and antigenic differences among 3 human isolates (Webster, Spooner, and NY-8) and 1 horse isolate (MRK) were evaluated. The 16S rRNA gene sequences were identical in all human isolates. By use of 5 homologous antisera from these 3 humans and 1 horse and an additional 5 antisera in heterologous reactions, the immunodominant antigens of each isolate were noted to differ in molecular size: 43 kDa in the Webster (Wisconsin) isolate, 46 kDa in the Spooner (Wisconsin) isolate, 42 and 45 kDa in the NY-8 (New York State) isolate, and a 42 kDa doublet in the E. equi MRK isolate from California. Two sera from a Wisconsin patient reacted weakly or not at all with the NY-8 isolate. Antigenic structural diversity exists among otherwise indistinguishable granulocytic ehrlichial isolates.

Geographic, clinical, serologic, and molecular evidence of granulocytic ehrlichiosis, a likely zoonotic disease, in Minnesota and Wisconsin dogs.

Seventeen Minnesota and Wisconsin dogs with granulocytic ehrlichosis were studied. The diagnoses were made by finding ehrlichia morulae in peripheral blood neutrophils. Eight dogs were studied retrospectively, and nine dogs were studied prospectively. The medical records of all dogs were reviewed. Eighty-eight percent of the dogs were purebred and 76% were spayed females. The median age was 8 years. Sixty-five percent of the cases were diagnosed in October and November. Fever and lethargy were the most common clinical signs. The most frequent laboratory findings were lymphopenia, thrombocytopenia, elevated activities of serum alkaline phosphatase and amylase, and hypoalbuminemia. No dogs seroreacted to Ehrlichia canis or Ehrlichia chaffeensis antigens, which are cross-reactive. Seventy-five percent of the dogs tested during the acute phase of disease and 100% of the dogs tested during convalescence were seropositive for E. equi antigens. Granulocytic ehrlichial 16S rRNA gene DNAs from six dogs were amplified by PCR. Sequence analysis of a 919-bp sequence of the ehrlichial 16S rRNA gene amplified by PCR from the blood of two dogs revealed the agent to be identical to the agent of human granulocytic ehrlichiosis in Minnesota and Wisconsin and to be very similar to E. equi and Ehrlichia phagocytophila and less similar to E. canis, Ehrlichia ewingii, and E. chaffeensis. The geographic, clinical, serologic, and molecular evidence indicates that granulocytic ehrlichiosis in Minnesota and Wisconsin dogs is not caused by E. ewingii, but suggests that it is a zoonotic disease caused by an agent closely related to E. equi and that dogs likely contribute to the enzootic cycle and human infection.

Clinical and laboratory characteristics of human granulocytic ehrlichiosis.

OBJECTIVE: To characterize the clinical and laboratory features observed in patients with human granulocytic ehrlichiosis (HGE) and evaluate the utility of the diagnostic tools used to confirm the diagnosis. DESIGN: Retrospective case study of 41 patients with laboratory-diagnosed HGE. SETTING: A total of 228 patients from Minnesota and Wisconsin were evaluated between June 1990 and May 1995. METHODS: Cases were presumptively identified by a history of an influenzalike illness acquired in an area known to be endemic for ticks. Diagnostic laboratory testing included microscopic examination of Wright-stained peripheral blood smears for presence of neutrophilic morulae, polymerase chain reaction (PCR) analysis of acute-phase blood samples for the Ehrlichia phagocytophila/Ehrlichia equi group DNA, and evaluation of serological responses by indirect immunofluorescent antibody assay (IFA), using E equi as antigen. RESULTS: All patients presented with a temperature of at least 37.6 degrees C, and most had headache, myalgias, chills, and varying combinations of leukopenia, anemia, and thrombocytopenia. Eighty percent of the patients tested demonstrated morulae in the cytoplasm of peripheral blood neutrophils. Only 16 of 37 patients tested by PCR were positive for HGE, whereas serum IFA assays of acute or convalescent blood samples detected antibodies against E equi in 38 of 40 patients tested. Two patients died, and the calculated case fatality rate was 4.9%. CONCLUSIONS: Human granulocytic ehrlichiosis is being increasingly recognized in Wisconsin and Minnesota. A more severe illness is associated with increased age, anemia, increased percentage of neutrophils and decreased percentage of lymphocytes in peripheral blood, and presence of morulae in neutrophils. The differential diagnosis for patients who develop an influenzalike illness following a tick bite should include HGE. Microscopic examination of the acute-phase blood smear to detect neutrophilic morulae is currently the quickest and most practical screening method for diagnosing HGE in the upper Midwest.

Isolation and characterization of a new strain of Ehrlichia chaffeensis from a patient with nearly fatal monocytic ehrlichiosis.

Ehrlichia chaffeensis is the causative agent of human monocytic ehrlichiosis, a disease that ranges in severity from asymptomatic infection to death. Only one isolate of E. chaffeensis has been made, the Arkansas strain, upon which all characterizations of the agent of human monocytic ehrlichiosis have been based. We report the isolation and characterization of a new strain of E. chaffeensis, the 91HE17 strain, which was cultivated from a patient with a nearly fatal illness. The new isolate grows best in culture with careful control of pH. The two isolates are nearly identical as determined by light and electron microscopy and have significant antigenic identity in fluorescent-antibody and immunoblot assays using polyclonal antisera and the E. chaffeensis-specific monoclonal antibody 1A9. Isolate 91HE17 had 99.9% nucleotide sequence identity with the Arkansas strain in the 16S rRNA gene. Parts of the Escherichia coli GroE operon homologs had identical restriction enzyme digestion patterns, and a 425-bp region of the GroEL gene had at least 99.8% sequence identity between the E. chaffeensis Arkansas and 91HE17 strains. Isolate 91HE17 lacked an epitope identified in E. chaffeensis Arkansas by the monoclonal antibody 6A1. This new E. chaffeensis isolate is very similar to the Arkansas strain and provides the opportunity to substantiate the existence of diversity among ehrlichiae which infect humans. Specific factors which differ among strains may then be compared to assess their potential contributions toward cellular pathogenicity and ultimately toward the development of disease in humans.

Serologic cross-reactions among Ehrlichia equi, Ehrlichia phagocytophila, and human granulocytic Ehrlichia.

Homology in the 16S rDNAs shows that the agent of human granulocytic ehrlichiosis (HGE) is closely related to the veterinary pathogens Erlichia equi and Erlichia phagocytophila. After HGE, patients develop antibodies reactive with E. equi and E. phagocytophila; thus, we hypothesized that these species are closely related and share significant antigenicity. Antisera from humans, horses, dogs, and cattle were tested by indirect fluorescent-antibody assay (IFA) for antibodies reactive with E. equi and other ehrlichiae and tested by immunoblot to identify the specific reactions with E. equi. All convalescent-phase sera from human patients with HGE and from animals infected or immunized with E. equi or E. phagocytophila had antibodies reactive with E. equi by IFA; no reactions with Ehrlichia chaffeensis occurred with these sera, and only one horse naturally infected with E. equi had a serologic reaction against Ehrlichia sennetsu. Human and animal sera obtained after infection or immunization with other Ehrlichia, Rickettsia, and Bartonella species did not react with E. equi by IFA. E. equi immunoblots revealed as many as 19 bands with equine anti-E. equi serum. All HGE agent, E. equi, and E. phagocytophila antisera tested reacted with a 44-kDa antigen of E. equi, while other anti-Ehrlichia spp. sera reacted with this antigen rarely or not at all. HGE agent, E. equi, and E. phagocytophila antisera but not other sera also reacted occasionally with 25-, 42-, and 100-kDa antigens. Most sera reacted with antigens between approximately 56 and 75 kDa, probably heat shock proteins. The HGE agent, E. equi, and E. phagocytophila share significant antigenicity by IFA and immunoblot. Coupled with the nearly identical nucleotide sequences of 16S rRNA genes, these data indicate that E. equi, E. phagocytophila, and the human granulocytic ehrlichia are closely related or identical species.

N-methyl-d,l-aspartate modulation of pituitary hormone secretion in the pig: role of opioid peptides.

Sixteen ovariectomized (OVX) mature gilts, averaging 139.6 +/- 3.1 kg body weight (BW) were assigned randomly to receive either progesterone (P, 0.85 mg/kg BW, n = 8), or corn oil vehicle (OIL, n = 8) injections im twice daily for 10 d. On the day of experiment, all gilts received either the EAA agonist, N-methyl-d,l-aspartate (NMA; 10 mg/kg BW, iv) alone or NMA plus the EOP antagonist, naloxone (NAL, 1 mg/kg BW, iv), resulting in the following groups of 4 gilts each: OIL-NMA, OIL-NMA-NAL, P-NMA and P-NMA-NAL. Blood samples were collected via jugular cannula every 15 min for 6 hr. All pigs received NMA 5 min following pretreatment with either 0.9% saline or NAL 2 hr after blood collection began and a GnRH challenge 3 hr after NMA. Administration of NMA suppressed (P < 0.03) LH secretion in OIL-NMA gilts and treatment with NAL failed to reverse the suppressive effect of NMA on LH secretion in OIL-NMA-NAL gilts. Similar to OIL-NMA gilts, NMA decreased (P < 0.03) mean serum LH concentrations in P-NMA gilts. However, in P-NMA-NAL gilts, serum LH concentrations were not changed following treatment. All gilts responded to GnRH with increased (P < 0.01) LH secretion. Additionally, administration of NMA increased (P < 0.01) growth hormone (GH) and prolactin (PRL) secretion in both OIL-NMA and P-NMA gilts, but this increase in GH and PRL secretion was attenuated (P < 0.01) by pretreatment with NAL in OIL-NMA-NAL and P-NMA-NAL gilts.(ABSTRACT TRUNCATED AT 250 WORDS)