Persistence of Granulocytic EhrlichiaInfection During Wintertime in Two Sheep Flocks in Norway
© The Author(s) 2002
Received: 01 July 2000
Accepted: 14 March 2001
Published: 30 September 2001
Granulocytic Ehrlichia infection in sheep is common in Norway in areas with Ixodes ricinus. In this study, 2 sheep flocks that had been grazing on I. ricinus infested pastures the previous season, were blood sampled after being housed indoors for nearly 6 months during wintertime. Thirty animals from each flock were examined for granulocytic Ehrlichia infection in the peripheral blood by blood inoculation studies, stained blood smear evaluation, polymerase chain reaction (PCR) analysis and serology (IFAantibodies). The animals were sampled twice within a three-week period, the first time before and the second time after lambing. Two sheep in one flock were found Ehrlichia positive by both blood smear evaluation and PCR before lambing, and 3 sheep were found positive after lambing; 2 by blood smear examination and 3 by PCR. In the other flock, no sheep was found infected before lambing, but 2 ewes were found positive after lambing by both blood smear evaluation and PCR. In the first flock, 87% of the animals were found seropositive before lambing, and the mean antibody titre (log 10 ± SD) to E. equi was 2.45 ± 0.401. In the second flock, 40% were found seropositive before lambing, and the mean antibody titre was 1.93 ± 0.260. Seroprevalence and mean antibody titre in these 2 flocks were significantly different (p < 0.001). The present study indicates that sheep may be a reservoir host for granulocytic Ehrlichia infection from one grazing season to the next under natural conditions in Norway.
Persistens av granulocyttær Ehrlichia-infeksjon i to saueflokker i Norge.
Granulocyttær Ehrlichia-infeksjon (sjodogg) er vanlig i Norge i områder med Ixodes ricinus. I denne undersøkelsen ble det tatt blodprøver i 2 saueflokker som hadde beitet på I. ricinus-beite forrige sesong. Prøvene ble uttatt etter at dyrene hadde stått inne i ca. 6 måneder. Tredve dyr i hver flokk ble undersøkt for en sjodogg infeksjon ved hjelp av blodpoding, blodutstryk, PCR-analyse og serologi. Det ble tatt blod av de samme dyrene ved 2 anledninger i løpet av en 3 ukers periode; første gang før lamming og andre gang etter lamming. I den ene flokken ble 2 dyr funnet infisert før lamming, og 3 dyr ble funnet positive etter lamming. I den andre flokken ble ingen dyr funnet positive før lamming, men 2 dyr ble funnet positive etter lamming. I den første flokken var 87% av dyrene seropositive og antistoff-titret mot E. equi var 2.45 ± 0.401 (middel ± SA). I den andre flokken var 40% seropositive og titret var 1.93 ± 0.260. Både seroprevalensen og antistoff-titret mellom de 2 flokkene var signifikant forskjellige. Denne undersøkelsen indikerer at sau kan være et reservoar for sjodogg-smitte mellom 2 beitesesonger i Norge.
Granulocytic Ehrlichia infection in sheep (tick-borne fever) is endemic in coastal areas of Norway, where the tick Ixodes ricinus is abundant [28, 21]. Most cases of tick-borne fever (TBF) in sheep are diagnosed in May-June (65%) and September-October (23%) . Earlier investigations have shown that Ehrlichia phagocytophila is transmitted transtadially in I. ricinus, and that the rickettsiae may survive for several months in these ticks [14, 13]. In addition, earlier observations indicate that nearly 100% of the sheep grazing on Ixodes infested pasture may be infected with granulocytic Ehrlichia , and that the infection could persist for several months after experimental infection [7, 22].
The purpose of this study was to investigate if sheep grazing on I. ricinus infested pasture were still infected with E. phagocytophila after having been housed indoors during wintertime in Norway.
Materials and methods
Sheep from 2 flocks, A and B, from western Norway (Etne, Sunnhordland) were examined for a granulocytic Ehrlichia infection by analysis of peripheral blood. Both flocks had been grazing on I. ricinus infested pasture the previous years, verified by the local veterinarians. Sheep in flock A had grazed on tick pasture from April to November, while animals in flock B had been on tick pasture from May to July, and from September to November. When the blood samples were collected in April, the animals had been housed indoors for nearly 6 months.
Flock A consisted of 63 winterfed sheep of the old Norwegian short-tailed breed Spel, while flock B consisted of 75 sheep of the mixed breed Norwegian White Sheep. Three years earlier, all sheep in flock B had been slaughtered because of an official scrapie eradication program, and the farmer had purchased replacement lambs from Ixodes free parts of Norway. Accordingly, no animal in this flock was older than 3 years and the flock had been on Ixodes pastures for only 2 grazing seasons.
Tick-borne fever (TBF) or other tick-associated diseases had not earlier been diagnosed in flock A, and only one lamb had been treated with synthetic pyrethroids (Coopersect vet®, Schering-Plough) the previous year. In comparison, all sheep in flock B had been treated with synthetic pyrethroids the previous year; twice in the spring and once in the autumn. Four lambs had also been treated against TBF with oxytetracycline (Terramycin vet®, Pfizer) the last spring. Post mortem examination of one of these lambs showed that it died of a bacterial septicaemia together with a granulocytic Ehrlichia infection.
Five lambs (≤ 1 year) and 25 adults (>1 year) were sampled in flock A and 30 adults (≥ 2 years) were sampled in flock B. The animals were sampled twice within a 3-week period before they were put onto pasture, the first time before and the second time after lambing. Blood samples were collected in EDTA on both occasions, while the heparinised blood and serum samples were collected before lambing only.
The heparinised blood samples were stabilised with 10% dimethyl sulphoxide (DMSO) and frozen at -70°C  in aliquots of 10 ml containing 1 ml from each of 10 animals from the same flock. These 6 stabilates were later inoculated intravenously into 2 month-old lambs, such that each aliquot was inoculated into each of 6 susceptible and not previously grazing lambs.
The susceptible lambs were followed for 21 days after inoculation. Rectal temperatures were measured daily, and EDTA-blood was sampled on days 0, 6, 8, 9, 10, and 14 post inoculation. In addition, blood samples were collected from individual lambs on days when rectal temperature ≥ 39.5°C was recorded. Serum samples were collected on days 0, 14 and 28.
Hematology and PCR analysis
Hematological values including hematocrit, hemoglobin, erythrocyte counts, and total and differential leucocyte counts were determined electronically from the EDTA-blood samples (Technicon H1®, Miles Inc., USA), and blood smears were prepared and stained with May-Grünwald Giemsa. Four hundred neutrophils were examined on each smear by microscopy and the number of cells containing Ehrlichia inclusions was recorded. The blood samples were also tested for granulocytic Ehrlichia infection by a polymerase chain reaction (PCR) technique according to . In addition, blood smears were stained with acridine orange  and tested for Eperythrozoon ovis (Ep. ovis) infection.
Serum was analysed by an indirect immunofluorescence antibody assay (IFA) to determine the antibody titre to E. equi . Briefly, 2-fold dilutions of sera were added to slides precoated with E. equi antigen (Protatek International and Organon Teknika). Bound antibodies were visualised by fluorescein-isothiocyanate (FITC)-conjugated rabbitanti-sheep immunoglobulin (Cappel, Organon Teknika). If positive, the serum was further diluted and retested. A titreof 1.6(log10reciprocal of 1:40) or more was regarded as positive. Serum was also analysed for Ep. ovis infection by an enzyme-linked immunosorbent assay (ELISA) test .
Statistical calculations were done by using Statistix®, version 4.0 (Analytical software). Statistical analyses on seroprevalence were performed using a chi-square contingency test and the antibody titres were compared using a Students t-test for independent samples. Significance was set at p < 0.05.
The animals showed no signs of disease when sampled. Except for a low number of erythrocytes, hematocrit and hemoglobin concentration in one animal, the hematological values were within normal limits .
When the 6 aliquots of DMSO-stabilated blood were inoculated into each of 6 susceptible lambs, only one reacted with fever (≥ 40°C), rickettsemia (infected neutrophils), neutropenia and seroconversion. The other 5 susceptible lambs showed no clinical or hematological signs of E. phagocytophila infection, nor any seroconversion within one month after inoculation, and Ehrlichia could not be found by either blood smear evaluation or PCR technique.
Number of granulocytic Ehrlichia infected animals in 2 sheep flocks. Thirty animals in each flock were examined by both blood smear evaluation and polymerase chain reaction (PCR) analysis.
Before lambing (n = 30)
The indirect immunofluorescence antibody titre to Ehrlichia equi in various age groups in 2 sheep flocks, after the sheep had been housed indoors for nearly 6 months. A titre below 1:40 (log10 = 1.6) was considered negative.
Number of animals
Number of seropositive animals
Mean antibody titre of seropositive animals (log10) ± SD
2.58 ± 0.250
2.41 ± 0.401
2.44 ± 0.428
2.20 ± 0.301
1.87 ± 0.211
Mean antibody titre in persistent infected animals (log10) ± SD*
2.58 ± 0.250
2.35 ± 0.151
In addition, an Ep. ovis infection was found in all animals by blood smear investigation, and all animals were also found seropositive for Ep. ovis infection.
In the present study, the number of sheep that is actually infected with TBF at the time of sampling is unknown. Both flocks had been grazing in areas were I. ricinius is abundant. In flock A, 4 sheep (13%) out of 30 were found infected in blood smears and by PCR, i.e. 15% of the seropositive sheep. In comparison, only 2 animals (7%) in flock B were found infected, i.e. 16.7% of the seropositive animals. The sensitivity of the tests used may have been increased either by examination of more neutrophils or by use of a nested PCR technique .
An earlier experimental study showed that at least 4 out of 5 E. phagocytophila infected lambs were still infectious by experimental inoculation 6 months after the initial infection . However, in the present investigation, the sheep could have been infected with granulocytic Ehrlichia up to 12 months before the blood was collected.
In the present study, more animals were found infected after lambing than before. It is difficult to judge whether the immunosuppression occurring in ewes around lambing may have caused a relapse of the infection. These results indicate, however, that granulocytic Ehrlichia infection in peripheral blood varies in infected sheep, as earlier observed in experimental TBF infection [7, 22]. More sheep might therefore have been found positive by increasing the number of samples per animal.
The hematological values of the animals were within normal limits, except for a low number of erythrocytes in one lamb. Since a subclinical Ep. ovis infection was found in all sheep, the low number of red blood cells in that animal might have been caused by this infection .
E. equi was used as antigen in the serological analysis. Strong serological cross-reactions between E. equi, E. phagocytophila and the agent causing human granulocytic ehrlichiosis (HGE) have been reported [6, 15, 18]. It is therefore possible to use any of these closely related antigens to obtain acceptable results in serosurvey, but the IgG titres may differ noticeably depending on the source of the antigen [4, 26].
In flock A, 87% of the sheep were found seropositive 6 months after last exposure to ticks. In contrast, after a stable period of 6 months, the seroprevalence in cattle was reduced by more than 40% and no animals were found infected . One explanation of this difference could be that sheep are a more competent and natural host for E. phagocytophila than cattle.
While most of the animals in flock A were found to be seropositive against E. equi, less than half of the sheep in flock B were seropositive. In addition, the antibody titres were highest in flock A. This result is in accordance with earlier observations on TBF infection in cattle grazing on tick pasture, where the increase in titre was parallel to an increase in seroprevalence .
The present study indicates that most problems associated with TBF based on herd history seem to occur in the flock with the lowest seroprevalence and titres. No obvious reasons for this observation were found, other than flock A may have been more thoroughly infected and therefore better immunized than flock B. Sheep in flock A were annually grazing for 3 months longer on Ixodes pastures than those in flock B. In addition, animals in flock B had only been on tick pasture for 2 seasons, and lack of sufficient immunity against TBF after restocking may be one explanation for disease problems the previous spring and low seroprevalence.
Another explanation of the difference in seroprevalence could be that the flocks were infected with Ehrlichia at different periods of the grazing season. The sheep had been housed indoors for nearly half a year, but the antibody titres in E. phagocytophila infected lambs seem to last for at least 6 months in experimentally infected lambs [17, 22].
Four lambs in flock B were treated against TBF in spring the previous year, and if the sheep in this flock were infected mainly at that time, the actual titres may reflect antibody levels developed about 12 months earlier. Unfortunately, no blood samples were collected from the sheep in the autumn to verify this assumption. In one study in humans, the antibodies remained detectable in about half of the HGE patients one year after onset of symptoms .
No difference in seroprevalence and antibody titres was observed between lambs and adults in flock A. This is in accordance with similar observations in cattle . However, in flock B, only 20% of the 2-year-old sheep were seropositive, compared with 50% of the 3-year-old sheep. According to the farmer, the youngest animals had the previous year a shorter period on Ixodes infested pasture when compared with the oldest animals.
Different strains of granulocytic Ehrlichia could be involved in these 2 flocks. Earlier observations indicate that different strains of E. phagocytophila may cause differences in both clinical and immunological responses [7, 25]. In addition, sheep breed variation in susceptibility to a TBF infection has also been reported . However, to the authors knowledge, no difference in clinical reaction to an E. phagocytophila infection has been observed between sheep breeds in Norway.
All sheep in flock B had been treated 3 times with synthetic pyrethroids the previous year, while only one lamb in flock A was treated. The difference in treatment régime may also have had some effect on the seroprevalence. However, earlier studies indicate that lambs treated with synthetic pyrethroids may become infected with TBF within 3 weeks on Ixodes infested pasture . In addition, the antibody titres in synthetic pyrethroid treated and untreated animals were not significantly different at the end of the grazing season .
In conclusion, the present study indicates that sheep under natural conditions in Norway may be a reservoir host for granulocytic Ehrlichia infection from one grazing season to the next. However, to analyse the epidemiological importance of this, a more detailed study during tick infestation is needed.
The authors wish to thank the 2 farmers Egil Selland and Sigve Sørheim for all collaboration, and Etne sau- og geitalslag and Pfizer AS for economic support of the study.
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