Abstract
Five-month-old lambs were experimentally infected with two 16S rRNA genetic variants of Anaplasma phagocytophilum, variants 1 (GenBank accession no. M73220) and 2 (GenBank accession no. AF336220). Additional sequencing of the groESL heat shock operon gene indicated that these variants differ in three nucleotides at positions 782, 824, and 890. The variants were obtained by blood sampling of A. phagocytophilum-infected lambs from one sheep flock in Norway and were stored at −70°C with 10% dimethyl sulfoxide as a cryoprotectant before being inoculated intravenously into susceptible lambs. The infectious blood contained, per ml, approximately 0.5 × 106 neutrophils infected with either of the variants. Six weeks after the primary inoculation, the lambs were challenged with the same infectious dose of the heterologous variant. The results of the study indicate a marked difference in clinical manifestation, neutropenia, antibody response, and cross-protection after experimental infection with the two variants of A. phagocytophilum.
Tick-borne fever (TBF), caused by the bacterium Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila), is a disease of sheep that is endemic in tick (Ixodes ricinus)-infested areas in Norway. Natural infection with A. phagocytophilum has been reported in humans and a variety of domestic and wild animal species (6). A serological survey of sheep indicated that A. phagocytophilum infection is widespread along the coast of southern Norway. However, TBF had been diagnosed in only half of these seropositive sheep flocks (15). The reason for this diagnostic deficit may be the existence of different variants of the agent that cause different clinical symptoms and immunological reactions (4, 18).
Although A. phagocytophilum variants are discriminated in the laboratory on the basis of 1 to 2 nucleotide differences in the 16S rRNA gene, biological and ecological differences, including variations in host pathogenicity, vectors, and geographical distribution, clearly exist (11). Sequences from the groESL operon have been shown to more clearly delineate genetic variants of A. phagocytophilum than have sequences of the 16S rRNA gene (17). Based on a 16S rRNA gene sequence study, it has recently been shown that different variants of A. phagocytophilum may exist simultaneously in the same sheep flock (16). The aim of the present study was to investigate further the clinical, hematological, and serological responses and the cross-protective abilities of two variants of A. phagocytophilum identified in the same sheep flock in Norway.
MATERIALS AND METHODS
Source of A. phagocytophilum and DNA sequencing.
Blood samples were collected from a flock of Norwegian sheep with TBF. Both blood samples collected in EDTA tubes (EDTA-blood samples) and heparinized blood samples were taken from infected lambs. The EDTA-blood samples were used to measure hematological values and to prepare blood smears. The absolute number of infected cells per unit volume was determined by multiplying the total number of neutrophils per unit volume by the percentage of infected neutrophils counted on a May-Grünwald-Giemsa-stained blood smear. The heparinized blood samples were stored at −70°C with 10% dimethyl sulfoxide as a cryoprotectant without any propagation in cell culture or sequence passage through other sheep.
Two variants of A. phagocytophilum were identified based on sequences of the 16S rRNA gene, i.e., A. phagocytophilum variant 1 (GenBank accession no. M73220) and variant 2 (GenBank accession no. AF336220) (16). These variants differ only in a single nucleotide at position 80 in the 16S rRNA gene.
In the present study, the 1,256-bp sequences of the groESL heat shock operon of these two variants were determined as described by Sumner et al. (17).
Animals, experimental design, and hematology.
Twelve lambs, 5 months old, of the Dala breed were used in this trial. The lambs were unrelated and belonged to the experimental sheep flock at the Department of Sheep and Goat Research, Norwegian School of Veterinary Science. None of the lambs had previously been in I. ricinus-infested pastures, and they were kept indoors during the whole experimental period of 3 months. Four lambs were inoculated intravenously with 1 ml of a whole-blood dimethyl sulfoxide stabilate of A. phagocytophilum variant 1, and on the same day four lambs were inoculated with 1 ml of a whole-blood dimethyl sulfoxide stabilate of A. phagocytophilum variant 2. The infectious blood contained, per ml, approximately 0.5 × 106 neutrophils infected with either of the variants. Four lambs left uninfected were used as controls. Six weeks after the primary inoculation, the lambs were challenged with the same infectious dose of the heterologous variant.
Rectal temperatures were measured daily in all of the lambs throughout the experimental period. The incubation period was defined as the period between inoculation and the first day of fever (≥40.0°C). The duration of fever was recorded as the number of days with elevated body temperature (≥40.0°C). The magnitude of fever of each lamb was estimated from the area of plots of daily temperature on 5-mm2 grids and calculated according to the trapezium rule (20). For this purpose, 40°C was taken as the baseline.
Blood samples were collected daily into EDTA tubes during the fever period following the inoculation of infected blood and on a weekly basis after the fever had subsided. In addition, EDTA-blood samples were later collected from individual lambs when rectal temperatures above 40.0°C were recorded. With these blood samples, hematological values, including total and differential leukocyte counts, were determined electronically (Technicon H1; Miles Inc., Tarrytown, N.Y.) and blood smears were prepared and stained with May-Grünwald-Giemsa stains. Four hundred neutrophils were examined on each smear by microscopy, and the number of cells containing Anaplasma inclusions was recorded. All of the lambs were weighed weekly during the whole experimental period.
Serology.
Sera were collected on days 0, 7, 14, and 28 after each inoculation and analyzed by using an indirect immunofluorescence antibody assay to determine the titer of antibody to Ehrlichia equi antigen (1, 15). Briefly, twofold dilutions of sera were added to slides precoated with E. equi antigen (Protatec, St. Paul, Minn.). Bound antibodies were visualized by fluorescein isothiocyanate-conjugated rabbit anti-sheep immunoglobulin (Cappel; Organon Teknika, West Chester, Pa.). Sera were screened for antibodies at a dilution of 1:40. If positive, the serum was further diluted and retested. A titer of 1.6 (log10 reciprocal of 1:40) or more was regarded as positive.
Statistics.
Statistical calculations were done by use of Statistix, version 4.0 (Analytical Software), and a two-sample t test was used to determine the hematological variables. A P value of <0.05 was considered significant.
Nucleotide sequence accession number.
The groESL sequences found in the present study are available in the GenBank database under accession numbers AF548385 and AF548386.
RESULTS
DNA sequence analysis.
The variants differ at positions 782, 824, and 890 in the groESL heat shock operon gene. Comparison with the GenBank database revealed that variant 1 (AF548385) and variant 2 (AF548386) each differ in a single nucleotide from A. phagocytophilum groESL sequences amplified from roe deer (Capreolus capreolus) in Slovenia (GenBank accession no. AF478558) and from sheep in Scotland (GenBank accession no. EPU96730), respectively. Furthermore, they were 99.6 and 99.4% similar to A. phagocytophilum groESL sequences amplified from I. ricinus ticks and from the blood of human patients in Slovenia (GenBank accession no. AF033101).
Clinical parameters, hematology, and serology.
All lambs inoculated with A. phagocytophilum developed fever. However, significant differences in clinical signs and hematological reactions were observed (Table 1). A 1- to 4-day period with reduced appetite was observed in lambs infected with A. phagocytophilum variant 1, while lambs infected with the other variant showed no signs of clinical illness. No hematological reactions were observed in the control lambs. A marked difference in onset of infection of neutrophils and percentage of infected neutrophils was observed between lambs infected with the two variants (Table 2). After the primary inoculation, the titers of antibody to E. equi antigen were absent in three of the four sheep infected with A. phagocytophilum variant 2 and only a single lamb seroconverted. In contrast, all of the lambs infected with variant 1 mounted high antibody titers (Fig. 1). Neither clinical symptoms nor seroconversion was detected in the control lambs.
TABLE 1.
Clinical variables for A. phagocytophilum variant 1- and 2-infected lambsa
| A. phagocytophilum variant(s) | Incubation period (days) | Maximum temp (°C) | Duration of fever (days) | Magnitude of fever (mm2)e | Nadir of neutropenia (<0.7 × 109 liter−1) | Duration of neutropenia (days) | Wt reduction (kg)b |
|---|---|---|---|---|---|---|---|
| 1 | 3.3 ± 0.43* | 41.95 ± 0.087* | 8.8 ± 1.92** | 1,677 ± 278*** | 0.34 ± 0.081 | 9.3 ± 1.30 | 3.5 ± 0.50** |
| 2 | 5.0 ± 0.71 | 40.98 ± 0.415 | 3.3 ± 1.09 | 364 ± 199 | 0.59c | 1 | 1.0 ± 0.71 |
| 1 and then 2 | 6.0d | 40.0 | 1.0 | 0 | 0.051 | 1 | 0.5 ± 0.50 |
| 2 and then 1 | 3.8 ± 0.43 | 41.28 ± 0.083 | 4.5 ± 1.80 | 554 ± 195 | 0.33 ± 0.061 | 7.3 ± 2.05 | 3.3 ± 1.79 |
Values are means ± standard deviations. Groups of four 5-month-old lambs were infected with one of the two variants. Six weeks after the primary inoculation, the lambs were challenged with the heterologous variant. Statistical differences between A. phagocytophilum variant 1- and variant 2-infected lambs are shown. *P < 0.05; **, P < 0.01; ***, P < 0.001.
Weight reduction was measured 1 week after inoculation.
Only one lamb developed neutropenia (this lamb did not scroconvert [Fig. 1]).
Only one lamb reacted with fever and rickettsemia.
Measurements are based on plots of daily temperature on 5-mm2 grids (see text for details).
TABLE 2.
Infected neutrophils observed in A. phagocytophilum variant 1- and 2-infected lambsa
| A. phagocytophilum variant(s) | % Infected neutrophils (mean) on indicated day after inoculation or challenge |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| 2 | 3 | 4 | 5 | 6 | 7 | 8 | 10 | 12 | 14 | |
| 1 | <1 | 22.3 | 51.3*** | 51.0** | 44.5** | 37.5** | 38.3** | 8.5 | 1.9 | <1 |
| 2 | 0 | 0 | 0.9 | 24.0 | 23.8 | 13.0 | 6.9 | 1.3 | 0.13 | <1 |
| 1 and then 2 | 0 | <1 | 1.0 | 2.3 | 3.8 | 1.8 | 1.0 | <1 | 0 | 0 |
| 2 and then 1 | <1 | 28.0 | 62.3 | 38.8 | 22.3 | 14.5 | 10.0 | 4.0 | 1.3 | <1 |
Groups of four 5-month-old lambs were infected with one of the two variants. Six weeks after the primary inoculation, the lambs were challenged with the heterologous variant. No inclusions were observed before day 2. Statistical differences between A. phagocytophilum variant 1- and variant 2-infected lambs are shown. **, P < 0.01; ***, P < 0.001.
FIG. 1.
Mean titers (+ standard deviations) of antibody to E. equi antigen in lambs infected with two variants of A. phagocytophilum. Four lambs in each group were inoculated with either A. phagocytophilum variant 1 or A. phagocytophilum variant 2 on day 0 and challenged with the heterologous variant after 6 weeks. A titer below 1:40 (log10, 1.6) was considered negative. Four lambs were uninfected controls. The arrows indicate the times of inoculation. Symbols: ⧫, A. phagocytophilum variant 1 (day 0) and A. phagocytophilum variant 2 (day 42); ▪, A. phagocytophilum variant 2 (day 0) and A. phagocytophilum variant 1 (day 42); , uninfected controls; *, only lamb that was found seropositive (titer, 1:40).
There was also a clear difference in clinical reactions between the groups of lambs infected with the two variants after challenge (Tables 1 and 2). No signs of clinical illness except fever were observed. The lambs infected with A. phagocytophilum variant 1 were almost fully protected against variant 2, since only one of the four lambs reacted with an elevated temperature. Furthermore, there was only a very modest and late infection of neutrophils in this group of lambs. In contrast, when the lambs infected with A. phagocytophilum variant 2 were challenged with variant 1, all of the lambs developed high temperatures and displayed hematological reactions similar to those seen following primary infection. In addition, the antibody titers in the two lamb groups were similar 1 week after challenge.
DISCUSSION
In the present study, a significant difference in clinical reactions between sheep infected with two variants of A. phagocytophilum was observed. Earlier investigations have shown that lambs in the bacteremic and neutropenic periods of TBF are more susceptible to secondary infections (2, 5, 7, 19). The present study may indicate, therefore, that general resistance to other infectious agents is less affected in lambs infected with A. phagocytophilum variant 2. This assumption is supported by the fact that this variant has been found mainly in a flock with no known disease problem due to tick-borne infections (16). However, 16S rRNA gene sequence analyses have shown that several variants of A. phagocytophilum, including variant 2, are involved in fatal cases of TBF, and this indicates that other factors, such as climate, health condition, management, and other microorganisms, may be important in this context (S. Stuen, S. Nevland, and T. Moum, Abstr. Int. Conf. Rickettsiae Rickettsial Dis., p. 147, 2002.)
Only the 16S rRNA and groESL heat shock operon genes of the Anaplasma variants have been sequenced in this study. Whether sequence variations in these or other conserved genes can be used as molecular markers for pathogenicity has yet to be determined. Variations in genes coding for surface proteins are more likely to affect qualities such as virulence, host range, and interaction with arthropod vectors. Recently, it has been shown that antibodies specific for 44-kDa proteins may play a role in immunity against Anaplasma infection and that the genes encoding the 44-kDa outer membrane proteins may be involved in the pathogenesis of, and the immunoresponse to, A. phagocytophilum in mice (8, 9, 22). However, a recent study of mice indicates that the histopathological lesions observed in A. phagocytophilum-infected animals may have an immunopathological basis (10). In order to evaluate the differences in pathogenicity observed in this study, virulence-related genes will have to be studied.
Only one of the four lambs inoculated with A. phagocytophilum variant 2 reacted with a detectable antibody titer within the first 6 weeks of the infection. The sensitivity of the present antibody test might have been increased by the use of a more appropriate antigen. Strong serological cross-reactions between all members of the A. phagocytophilum group have been reported, but the titer of the antibody to a heterologous variant is normally less than that to a homologous variant (3, 12). Unfortunately, E. equi antigen was the only antigen that was available for use in the present study.
A clear difference in clinical reactions between the two variants was also observed after challenge. Similar results were found in an earlier cross-protection study of lambs that involved A. phagocytophilum variant 1 and the A. phagocytophilum prototype, earlier classified as the human granulocytic ehrlichiosis agent (GenBank accession no. U02521) (14). Earlier experimental studies have shown that full protective immunity to challenge with homologous A. phagocytophilum variants lasts from a few months to more than 1 year (4, 21). Resistance to reinfection increases with increasing frequency of challenge (13). Unfortunately, no challenge with the homologous variants was done in the present work.
In conclusion, the results of the present study indicate marked differences in clinical manifestation, neutropenia, antibody response, and cross-protection after infection with variants of A. phagocytophilum. Although the prevalences of Anaplasma variants in sheep flocks are not known, the existence of such differences between two A. phagocytophilum variants in sheep on the same pastures may explain the earlier observation that lambs on neighboring pastures may suffer differently from tick-borne infections. However, further analyses of fatal cases of TBF must be performed in order to elucidate differences in pathogenicity.
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