Local infection with A. phagocytophilum
in tick attachment sites of lambs were characterized by hyperplastic skin changes and inflammatory infiltrates, similar to what is described for tick bite reactions even in the absence of A. phagocytophilum
infection. Immunohistochemistry showed the presence of A. phagocytophilum
in approximately 45% of the lambs. These lambs were also positive for A. phagocytophilum
by PCR on blood and skin biopsies. A. phagocytophilum
organisms were mainly found in inflammatory cell infiltrates, particularly in PMNs and macrophages of the dermis and subcutis. In the present study, microorganisms were rarely observed in leucocytes in the blood stream of the skin biopsies, whereas Lepidi and coworkers reported that approximately 90% of the infected neutrophils in deep tissues from sheep, humans and horses were seen within vessel lumens [9
]. IHC positive organisms were sometimes observed in the mid- and peripheral part of the vessel walls, but rarely in the intimal layer in the present study. The endothelium has earlier been suggested to function as a transition site for transfer of A. phagocytophilum
to neutrophils that are loosely bound and then released into the blood stream [15
]. The present study however indicates that endothelium infection is a rare finding and it does not support the role of endothelium in the pathogenesis of A. phagocytophilum
infection in lambs, at least at the earliest phases of tick bite inoculation. This stands in contrast to studies reported in mice, for which no morphological images are available [10
]. The current study is however, limited in that it is not an experimental study. The field conditions did neither allow control with attached ticks and infectious organisms, nor a longitudinal study of skin lesions, which is best estimated, based upon serum antibody titer, and at this point, endothelium could have played a role.
The very low number (7.9 %) of sero-negative animals (titre < 40), all which were IHC-negative, indicated that most lambs were sampled after seroconversion. Most of the IHC positive skin biopsies were collected from animals with serum titres between 40 and 1280, which may indicate that these had acute infections. However, presence of maternal immunity cannot be neglected, since the half life of maternal antibodies has been estimated to be 17.5 days [27
]. Animals with titres between 2560 to 5120, were likely to have seroconverted. The IHC-positive organisms, observed in biopsies from this latter group, may therefore have been associated with an acute infection.
PCR detection of msp2
) in blood samples showed that at least 86.8 % of the lambs, had A. phagocytophilum
bacteraemia at the time of sampling. Five rrs
(16S rRNA gene) variants were encountered. Organisms in biopsies with sequences obtained from variants U02521
were detected by IHC. Differences in local inflammatory responses to these variants have never been described, but previous studies have shown that different rrs
(16S rRNA gene) variants of A. phagocytophilum
can result in different immunological responses and clinical reactions [28
]. However, in the present study, no direct relationship between gene variants, serum titre or inflammatory changes were observed. Similar histopathologic findings and inflammatory infiltrates with monocytes and neutrophils were associated with all rrs
(16S rRNA gene) variants.
Nineteen of 38 sequences (50.0 %) obtained from tick bite sites corresponded to the sequences obtained from the respective ticks. All variants, except GU459257 (isolated from the skin and blood) have previously been isolated from the blood of infected sheep. The sequences obtained from control biopsies and the blood samples however were identical in all lambs, where both sequences were obtained, indicating a haematogenous spread of organisms to the skin. In addition, two PCR positive control biopsies were collected from animals having PCR negative blood, indicating that the organisms may have originated from nearby tick bites or from another infected area due to local dissemination. However, no tick bites were observed within a 20 cm distance from the control biopsies. The reason for these PCR positive control biopsies should be further elucidated as this could comprise a nidus among animals, capable of sustaining persistent infection. Future studies should investigate the extent of cell-to-cell infection in the skin, and how far the infected cells may migrate locally away from tick bites.
Long-term survival in the skin could function as a reservoir during persistent infection and could be a source of transmission to other feeding ticks even in the absence of sustained bacteremia. A previous study reported that the presence of A. phagocytophilum
in the peripheral blood of small mammals may be short lived and that tissue samples from spleen and ear seemed to be more often infected than blood [29
]. The majority of lambs (60.5%) had hyperplastic dermatitis with perivascular to diffuse infiltration of leukocytes. The results also show that the IHC-positive organisms were associated with the leukocytes of the infiltrate at the tick bite site, indicating that PMNs and other inflammatory cells are attracted to the area and may provide a possibility for survival of A. phagocytophilum
beneath the skin surface. However, the occurrence of persistent skin infections and its role in the transmission of A. phagocytophilum
to ticks has to be further investigated.
A positive relationship between the degree of inflammation observed by histology and the number of A. phagocytophilum
organisms detected by IHC, in the same tissue sections was present. However, quantitative studies should be performed to further elucidate this, as control biopsies, also PCR positive for msp2 (p44)
, did not show inflammatory changes by histology. Neutrophil infiltration can be triggered by tick salivary components and chemotaxins produced by infected neutrophils and other cell types [30
]. In addition, the presence of pyogenic bacteria such as Staphylococcus aureus
is commonly associated with tick bites [32
], and will also favor the attraction of neutrophil granulocytes.
The present study shows that the majority of IHC positive organisms were present in the inflammatory cell aggregates. In one animal, rrs
(16S rRNA) genotyping of organisms revealed identical gene variants in the blood and an IHC-positive biopsy from the same animal that differed from the gene variant detected in the attached tick. This indicates that the tick was not the source of infection, or that the infecting variant selectively survived in the lamb. In sections where IHC-positive organisms were detected within the vessel walls, they were usually observed intracellular in leukocytes. A previous study reported that more than 95% of infected cells in tissues were mature neutrophils, based on IHC analysis and that association of A. phagocytophilum
with vessel walls was rarely observed [9
]. However, the direction of migration of organisms needs further investigation. Lesions such as vasculitis and thromboses are reported to be rare findings of A. phagocytophilum
], but were present in the tick bite wounds of some lambs in the present study. This may however be caused by tick salivary components or other pathogens like S. aureus
or streptococci that are likely to be associated with tick bites [32
In conclusion, whether the endothelium plays a role in the pathogenesis of and establishment of A. phagocytophilum infection at the site of tick bite could not be documented by the present study. Other factors or cell types, such as dendritic cells, might be involved, but this was not examined. Controlled experimental studies with serial sampling of infected skin are suggested in order to further elucidate the pathogenesis of this infection during tick attachment.