Characterization of Anaplasma phagocytophilum major surface protein 5 and the extent of its cross-reactivity with A. marginale.

Major surface protein 5 (Msp5) of Anaplasma marginale is highly conserved in the genus Anaplasma and the antigen used in a commercially available competitive enzyme-linked immunosorbent assay (cELISA) for serologic identification of cattle with anaplasmosis. This study analyzes the degrees of conservation of Msp5 among various isolates of Anaplasma phagocytophilum and the extent of serologic cross-reactivity between recombinant Msp5 (rMsp5) of Anaplasma marginale and A. phagocytophilum. The msp5 genes from various isolates of A. phagocytophilum were sequenced and compared. rMsp5 proteins of A. phagocytophilum and A. marginale were used separately in an indirect ELISA to detect cross-reactivity in serum samples from humans and dogs infected with A. phagocytophilum and cattle infected with A. marginale. Serum samples were also tested with a commercially available competitive ELISA that uses monoclonal antibody ANAF16C1. There were 100% sequence identities in the msp5 genes among all of the A. phagocytophilum isolates from the United States and a horse isolate from Sweden. Sheep isolates from Norway and dog isolates from Sweden were 99% identical to one another but differed in 17 base pairs from the United States isolates and the horse isolate. Serologic cross-reactivity was identified when serum samples from cattle infected with A. marginale were reacted with rMsp5 of A. phagocytophilum and when serum samples from humans and dogs infected with A. phagocytophilum were reacted with rMsp5 of A. marginale in an indirect-ELISA format. Serum samples from dogs or humans infected with A. phagocytophilum did not cross-react with rMsp5 of A. marginale when tested with the commercially available cELISA. These results suggest that rMsp5 of A. phagocytophilum is highly conserved among United States and European isolates and that serologic distinction between A. phagocytophilum and A. marginale infections cannot be accomplished if rMsp5 from either organism is used in an indirect ELISA.

Guidelines for the diagnosis of tick-borne bacterial diseases in Europe.

Ticks are obligate haematophagous acarines that parasitise every class of vertebrate (including man) and have a worldwide distribution. An increasing awareness of tick-borne diseases among clinicians and scientific researchers has led to the recent description of a number of emerging tick-borne bacterial diseases. Since the identification of Borrelia burgdorferi as the agent of Lyme disease in 1982, 11 tick-borne human bacterial pathogens have been described in Europe. Aetiological diagnosis of tick-transmitted diseases is often difficult and relies on specialised laboratories using very specific tools. Interpretation of laboratory data is very important in order to establish the diagnosis. These guidelines aim to help clinicians and microbiologists in diagnosing infection transmitted by tick bites and to provide the scientific and medical community with a better understanding of these infectious diseases.

Ehrlichia-infected ticks on migrating birds.

During the spring of 1996, an estimated 581,395 Ehrlichia-infected ticks were imported into Sweden by migrating birds. Ehrlichia gene sequences found in ticks collected from these migrating birds were identical to those of granulocytic ehrlichiosis found in domestic animals and humans in Sweden. These findings support the idea that birds may play a role in dispersing Ehrlichia.

First long-term study of the seroresponse to the agent of human granulocytic ehrlichiosis among residents of a tick-endemic area of Sweden.

The seroprevalence of granulocytic ehrlichiosis has been documented in several studies, but little data exists on incidence rates. Using sera stored from an earlier study on Lyme borreliosis, 290 residents of Aspö Island could be followed prospectively during two tick seasons (1992-1994). Immunoglobulin G antibodies to granulocytic ehrlichiosis were detected by an immunofluorescence assay using Ehrli- chia equi as antigen. Seroprevalence rates increased significantly over time, and at the 1994 follow-up, 28% of the residents were seropositive. Negative-to-positive seroconversion (incidence) rates were 3.9% and 11.1%, respectively, during the two seasons. A highly significant correlation was found between a positive serologic response for granulocytic ehrlichiosis and Borrelia burgdorferi. No such correlations were found for clinical Lyme borreliosis, self-reported arthralgia or number of recorded tick bites. It was concluded that granulocytic ehrlichiosis is highly endemic in this part of Sweden, with a seroconversion rate as high as 11% over a single tick season. Further studies are necessary to correlate these findings with clinical signs of human granulocytic ehrlichiosis.

[Human granulocytic ehrlichiosis in Norway]. / Human granulocytaer ehrlichiose i Norge.

BACKGROUND: The bacterium that causes human granulocytic ehrlichiosis may be transmitted by ticks. MATERIAL AND METHODS: We describe two patients with human granulocytic ehrlichiosis. During the summer of 1998, both patients were bitten by ticks. Four to 7 days later they developed influenza-like symptoms with fever, headache and myalgia. After 4 and 21 days, respectively, both patients were given doxycycline for suspected bacterial respiratory diseases, and recovered. RESULTS: Blood samples for human granulocytic ehrlichiosis antibodies showed a fourfold increase in titer in one patient and a remaining high titer in the other. Both patients had a positive polymerase chain reaction with primers specific for the Ehrlichia phagocytophilae genogroup. INTERPRETATION: The two patients fulfill the human granulocytic ehrlichiosis diagnostic criteria set by Centers for Disease Controls and Prevention, and are the first two human granulocytic ehrlichiosis cases described in Norway.

Human granulocytic ehrlichiosis--a clinical case in Scandinavia.

A clinical case of human granulocytic ehrlichiosis in Scandinavia is presented. The patient developed high fever, myalgia, headache and dyspnoea. Doxycycline treatment resulted in a dramatic improvement. Laboratory confirmation included a fourfold change in anti-Ehrlichia equi IFA titre and a positive PCR confirmed by gene sequence analysis.

Sequence analysis of the ank gene of granulocytic ehrlichiae.

The ank gene of the agent of human granulocytic ehrlichiosis (HGE) codes for a protein with a predicted molecular size of 131.2 kDa that is recognized by serum from both dogs and humans infected with granulocytic ehrlichiae. As part of an effort to assess the phylogenetic relatedness of granulocytic ehrlichiae from different geographic regions and in different host species, the ank gene was PCR amplified and sequenced from a variety of sources. These included 10 blood specimens from patients with confirmed human granulocytic ehrlichiosis (three from New York, four from Wisconsin, two from Slovenia, and one from Sweden). Also examined was a canine granulocytic ehrlichia sample obtained from Minnesota, Ehrlichia equi from California, Ehrlichia phagocytophila from Sweden, and the granulocytic ehrlichia isolate USG3. The sequences showed a high level of homology (>95.5% identity), with the lowest homology occurring between a New York HGE agent and the Swedish E. phagocytophila. Several 3-bp deletions and a variable number of 51- and 81-bp direct repeats were noted. Although the North American HGE sequences showed the highest conservation (>98.1% identity), phylogenetic analyses indicated that these samples represent two separate clades, one including the three New York HGE samples and the USG3 strain and another with the Wisconsin HGE and Minnesota canine sequences. Two of the New York samples and the USG3 strain showed 100% identity over the entire 3,696-bp product. Likewise, three of the Wisconsin human samples and the Minnesota dog sample were identical (3,693 bp). Whereas phylogenetic analysis showed that the E. equi sequence was most closely related to the Upper Midwest samples, analysis of the repeat structures showed it to be more similar to the European samples. Overall, the genetic analysis based on the ank gene showed that the granulocytic ehrlichiae are closely related, appear to infect multiple species, and can be grouped into at least three different clades, two North American and one European.

Detection of granulocytic Ehrlichia species DNA by PCR in persistently infected dogs.

Three female beagle dogs inoculated with granulocytic Ehrlichia species were monitored for four to six months to determine whether there was evidence that the organisms persisted. The dogs were inoculated intravenously with blood containing an Ehrlichia species closely related to Ehrlichia equi and Ehrlichia phagocytophila, and identical to the human granulocytic ehrlichiosis agent with respect to its 16S rRNA gene sequence. The clinical signs were evaluated, and blood samples were collected for haematology, serum biochemistry and serology. Ehrlichial inclusions in the blood were monitored by microscopy, and ehrlichial DNA was detected by the polymerase chain reaction (PCR). Two of the dogs were injected with prednisolone on days 54 to 56 and days 152 to 154 after infection, and the other was injected with prednisolone on days 95 to 97 after infection. The dogs were euthanased and examined postmortem. Ehrlichial inclusions were demonstrated in the neutrophils and seroconversion occurred shortly after inoculation. Two of the dogs developed acute disease with rectal temperatures above 39.0 degrees C, after which no further clinical signs were observed. The administration of corticosteroids seemed to facilitate the detection of ehrlichial inclusions. Ehrlichial DNA was detected intermittently by PCR in blood samples from two of the dogs throughout the study. Persistent infection was demonstrated up to five-and-a-half months after inoculation.

[Varying clinical picture and course of human granulocytic ehrlichiosis. Twelve Scandinavian cases of the new tick-borne zoonosis are presented]. / Varierande klinisk bild och förlopp vid human granulocytär ehrlichios. Tolv skandinaviska fall av den nya fästingburna zoonosen redovisas.

In the twelve clinical cases of human granulocytic ehrlichiosis (HGE) so far identified in Scandinavia (ten in Sweden, two in Norway), clinical presentation varied from a mild febrile illness to a severe septic condition with such systemic complications as acute respiratory distress syndrome (ARDS). Laboratory verification was based on PCR (polymerase chain reaction) in ten cases, and on serology in two cases. Sequence analysis of 16S rDNA showed the infectious agents to belong to the Ehrlichia phagocytophila genogroup. Seroprevalence data indicate widespread human exposure to granulocytic Ehrlichia; mean seroprevalence, 15-20% of 1,000 clinical sera from tick-exposed patients (mainly from Sweden and Norway). Proposals for diagnostic criteria and procedures, and case management are presented in the article.

Serological evidence of Ehrlichia infection in Swedish Lyme borreliosis patients.

We studied sera from patients who had participated in a prospective study of borreliosis in Sweden and had acquired tick bites in areas of the country with a high prevalence of granulocytic ehrlichial infections in animals. The sera were examined for IgG anti Ehrlichia antibodies by an indirect immunofluorescence assay using a locally isolated bovine Ehrlichia antigen. Confirmation of the serological results was done at the Unité des Rickettsies, Marseille, France. Three out of 37 of the investigated patients and 1 out of 100 investigated healthy blood donors had significant antibody titres to granulocytotropic Ehrlichiae. No patient or blood donor had specific antibody titres to Ehrlichia chaffeensis. These data suggest that Scandinavian Ehrlichia species can infect and evoke immunological response in tick-exposed humans.

Feline granulocytic ehrlichiosis--a report of a new clinical entity and characterisation of the infectious agent.

A 14-month-old shorthaired cat was presented to the Animal Hospital in Skara, Sweden, with a two-day history of lethargy, anorexia and tachypnoea. Clinical examination and laboratory investigations revealed fever, dehydration, tick infestation, neutrophilia with left shift, lymphopenia, hyperglycaemia and intracytoplasmic neutrophilic Ehrlichia inclusions. After treatment with intravenous doxycycline and lactated Ringer's solution the temperature returned to normal. Oral treatment with doxycycline continued for 20 days. The ehrlichiosis diagnosis was confirmed by serology, polymerase chain reaction and DNA sequencing. No relapse was observed during the eight-month follow-up period. The granulocytotropic Ehrlichia strain found in the cat was later characterised by analysis of the 16S rRNA gene sequence which showed 100 per cent identity to DNA sequences found in Swedish canine and equine granulocytotropic Ehrlichia strains. This is, to the best of the authors' knowledge, the first reported case of granulocytic ehrlichiosis in a cat.

Early manifestations of granulocytic ehrlichiosis in dogs inoculated experimentally with a Swedish Ehrlichia species isolate.

Seven beagles were inoculated experimentally with a Swedish canine Ehrlichia species isolate to study its pathogenicity. With respect to the 16S rRNA gene sequence, the isolate was identical to the human granulocytic ehrlichiosis (HGE) agent and closely related to both Ehrlichia equi and E phagocytophila. After an incubation period of four to 11 days, the most prominent clinical signs were high fever for two to five days and depression. All the dogs developed profound thrombocytopenia, moderate leukopenia and a strong serological antibody response. Ehrlichial inclusions were detected in blood neutrophils from four to 14 days after inoculation for four to eight days. Ehrlichial DNA could be detected by polymerase chain reaction during the parasitaemic stage and a few days before and after microscopic inclusions were visible. Postmortem, the dogs showed reactive splenic hyperplasia and non-specific mononuclear reactive hepatitis.

[Does the infectious disease, human ehrlichiosis, exist in Sweden? Ticks get a hold of new zoonoses]. / Finns infektionssjukdomen human ehrlichios i Sverige? Fästingar fäste för nya zoonoser.

Human ehrlichiosis diseases, decently recognised as emerging human infections in the USA, are caused by vector-borne, strictly intracellular bacteria of the family Rickettsiaceae. Human monocytic ehrlichiosis is caused by Ehrlichia schaffeensis, whereas the agent causing human granulocytic ehrlichiosis (HGE) has yet to be identified [1]. The putative increase in the occurrence of these primary zoonoses is dependent on the complex relationship between the infectious agents, the vectors, and the hosts (rodents, deer) which constitute the wild-life reservoir. In Scandinavia, granulocytic ehrlichiosis is well known in veterinary medicine. Pasture fever in cattle and sheep is caused by Ehrlichia phagocytophila, whereas granulocytic ehrlichiosis in horses and dogs is caused by a new, recently characterised Ehrlichia species [2]. All species of Ehrlichia causing granulocytic ehrlichiosis are closely related both genetically and antigenically, and are all transmitted by ticks of the genus Ixodes. In Sweden, veterinary cases of granulocytic ehrlichiosis are characterised by a geographical distribution corresponding well with that of Ixodes ricinus, and a seasonal distribution similar to that of Lyme borreliosis. In Europe, clinical cases of HGE have so far been reported only from Slovenia [5], though seroprevalence figures of 8-17 per cent have been reported for tick-exposed populations [6, 7]. As most cases are probably subclinical, and as clinical symptoms, when present, are non-specific, clinical diagnosis is dependent on the clinician's awareness of the existence of the disease. Laboratory diagnostic tests are now available at Kalmar.

Clinical features and serology of 14 dogs affected by granulocytic ehrlichiosis in Sweden.

The clinical features and the titres to Ehrlichia equi, E canis, E risticii, Rickettsia rickettsii and Borrelia afzelii in 14 Swedish dogs, in which ehrlichiosis was diagnosed on the basis of the presence of inclusions in granulocytes, are reported. Most of the dogs were moderately ill but made a rapid recovery after treatment with doxycycline. The dogs with inclusions were thrombocytopenic. Analysis of the antibody titres indicates that serology to E equi will remain the most appropriate serological test for granulocytic ehrlichiosis in Swedish dogs, until a specific test is developed for detecting the recently identified subspecies of Ehrlichia.