Search

Anaplasma phagocytophilum is the aetiological agent of equine granulocytic anaplasmosis (EGA). The aim of this survey was to assess the prevalence of anti-A. phagocytophilum antibodies in the horse population of the Czech Republic (CZ) and to investigate possible links between seropositivity and the geographic origin, age, and/or sex of the tested horses. Antibodies against A. phagocytophilum were screened using an indirect fluorescent antibody method (IFA). Serum samples from 96 healthy horses from 8 localities (7 within the CZ and one control upland/tick-free locality in Ukraine) were examined. While the controls tested negative, the seroprevalence in the Czech localities was estimated at 73%, which suggests that subclinical EGA is common among horses in the CZ. Significant differences between seropositivity rates in individual stables were demonstrated (explicable by the different environment and/or management styles of the horses stabled therein). Horses in stables from which previous manifest cases had been reported tended to have higher average titres. Mares displayed a somewhat higher seropositivity rate than did stallions; age had no demonstrable effect upon the serological status of the examined horses., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Anaplasma phagocytophilum is a tick-borne rickettsial pathogen that has measurable genetic heterogeneity across its geographical range and reservoir spectrum. In the present study, publicly available sequences of the genes that have prevailingly been used for typing A. phagocytophilum were analysed to identify the segments giving the highest resolution with respect to the predictability of host and geographical provenances of the strains. Selected partial sequences of 16S rRNA, groL, msp4 and ank genes were then employed in a tentative multilocus typing scheme used to characterize the strains causing equine granulocytic anaplasmosis (EGA). We were able to both identify alleles characteristic for equine strains of A. phagocytophilum and distinguish two unique genetic variants infecting horses in the Czech Republic. This resolution far exceeded the discriminatory potential of any of the four sequenced genes when used singly. The two novel A. phagocytophilum variants appeared to be phylogenetically closer to the strains reported as causing human disease in Slovenia than to strains thus far isolated from other European EGA cases. A decline in the quality of recently deposited A. phagocytophilum sequences was also demonstrated.

During the period 2000-2003, patients hospitalized for suspected tick-borne encephalitis in the Czech Republic were screened for possible A. phagocytophilum co-infection. Blood samples taken at admission were tested for the presence of A. phagocytophilum DNA by nested PCR using a modified target sequence as an internal control, and sera were tested for the presence of antibodies by indirect immunofluorescence and western blotting methods using cell-culture-derived antigens. To verify the assay specificity, a set of 45 sera of Patagonian residents served as a non-tick-exposed control group, and a set of 14 B. henselae-positive sera was used to check cross-reactivity. Of 809 patients hospitalized, 80 (9.9%) showed IgG antibodies reactive to A. phagocytophilum at > or =80 (reciprocal dilution factor) and 50 (6.2%) at > or =160; two (0.2%) patients showed elevated IgM titers of 40. No full blood obtained from 162 patients tested positive in PCR when false negativity was excluded. During hospitalization, the diagnosis of tick-borne encephalitis was confirmed in 536 patients, 57 (10.6%) of whom had anti-A. phagocytophilum IgG antibodies reactive at > or =80 and 41 (7.6%) at > or =160, which did not differ significantly from the whole set (P = 0.66/0.30), the maximum IgG titer registered was 5120, and no IgM titer reached the 40 cut-off. Available paired sera from 189 tick-borne encephalitis patients showed no significant shifts, but one case of slight seroconversion (IgG shift from < 80 to 320) was detected in one of the non-tick-borne encephalitis patients. The sex of the patient showed no significance for the prevalence of A. phagocytophilum antibodies; however, the seropositive patients were older on average than those who were seronegative (43.5 +/- 15.9 vs. 37.9 +/- 18.3 years, P = 0.05). Clinical manifestation of the disease did not differ noticeably between patients with and without A. phagocytophilumreactive antibodies, except for fever duration, which was significantly longer in patients with titers > or =1280. Overall, A. phagocytophilum co-infection did not seem to be a frequent and/or significant complication of tick-borne encephalitis acquired in the Czech Republic.

A sheep herd from which contaminated cheese was produced, causing 21 cases of alimentary tick-borne encephalitis (TBE) in human beings, was tested serologically for the presence of specific antibodies against both the TBE virus (TBEV) and Anaplasma phagocytophilum, the cause of tick-borne fever (TBF) in ruminants, and compared with three other herds variously exposed to tick bites but without any TBE history. Virus-neutralisation (VN) with the TBEV strain Hypr and CV-1 cells was used in TBE tests, and indirect immunofluorescence assay (IFA) with neutrophils from goats experimentally infected with A. phagocytophilum was used for TBF testing. In 13 sheep from the incriminated herd (N =41), VN titres ranging from 1/4 to 1/128 traced previous TBE infection and all sheep had elevated titres of A. phagocytophilum antibodies ranging from 1/80 to 1/5120 in IFA, whereas two other herds (N = 8 and 9) were seronegative for TBEV and had significantly lower levels of A. phagocytophilum antibodies, corresponding to a lesser challenge from TBF. A control herd (N = 10) that was grazed on tick-free meadows in north Norway was completely seronegative. The respective distributions of positive titres of A. phagocytophilum and TBEV antibodies in the incriminated herd were not mutually random; the animals with higher anti-A. phagocytophilum titres tended to have lower anti-TBEV titres and vice versa (Spearman correlation coeff. =-0.86, p< or =0.01). The authors hypothesize that the immunosuppressive effect of TBF co-infection in sheep could be a contributory cause of TBE-virus contamination of milk, an aspect of TBE epidemiology that has not been considered thus far.

Altogether 112 sera from 67 patients hospitalised for tick-borne encephalitis (TBE) were tested for the presence of antibodies against human granulocytic ehrlichiosis (HGE). Indirect immunofluorescence assay and western blotting were used for the screening and confirmation respectively. A laboratory culture of HGE agent in HL-60 cells served as an antigen supply. Commercially available human sera were used as positive controls; the respective IgG and IgM cut-offs of 1/80 and 1/40 were applied. Six patients (8.9%) exhibited specific IgG antibodies against HGE at or above the cut-off level, while no serum sample was shown to be IgM-reactive at the cut-off. The mean age of the subset of positive patients was 42 (+/- 16.7) years which exceeded that of the total TBE set averaging 37.5 (+/- 17.3) years. One patient, a woman aged 32, exhibited an elevated HGE titre of 1/80 on admission 15 days after TBE onset, that still slightly increased up to 1/160 by the end of the following week. Corresponding WB-pattern showed significant MSP, p30, and p28 bands. These findings suggest a case of concurrent TBE with HGE. Overall, this study demonstrates that TBE/HGE co-infections could be encountered in central Europe.