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Human monocytic ehrlichiosis (HME) is a zoonotic emerging tick-borne disease with clinical signs that range from mild symptoms to multiple organ failure and death.Ehrlichia chaffeensis, the aetiologic agent of HME, is reported to infect a divergent range of mammals. Although cattle are common hosts of the primary vector of this pathogen, the susceptibility of this host toE. chaffeensishas not been reported to date. This study was undertaken to determine if cattle could provide a useful infection model ofE. chaffeensis. Dairy calves were injected with DH82 cells infected with the Arkansas, St Vincent or 91HE17 strain ofE. chaffeensis, and monitored for signs of clinical ehrlichiosis and for infection of peripheral blood and ticks by PCR assay. Splenectomized and spleen-intact calves were injected with cryopreserved stabilates ofE. chaffeensis-infected DH82 cells for the first experiment. Mild clinical signs were occasionally observed among these calves, and only two blood samples were PCR-positive, while several ticks fed on each calf tested PCR-positive. The second experiment involved injection of normal calves with active cultures of the sameE. chaffeensisstrains. Interestingly, three of six calves inoculated with active cultures became recumbent and died or had to be euthanized. All of the surviving calves in this experiment tested PCR-positive on multiple dates, but fewer ticks fed on these calves were PCR-positive. These results suggest that a bovine disease model could facilitate the understanding of factors that affect the severity of HME.

The cellular stress response is characterized by the production of heat shock proteins (HSP) which serve important cytoprotective functions. Paradoxically, in vitro induction of the stress response promotes cytopathic effect mediated by infection with canine distemper virus (CDV). The stress-mediated increase in cytopathic effect is correlated to the formation of complexes between the viral nucleocapsid (NC) and the major inducible member of the approximately 70 kDa family of HSP (hsp72). The objective of the present study was to document the functional significance of CDV NC-HSP interaction. Cytoplasmic NC was purified from Vero cells lytically infected with the Onderstepoort strain of CDV. Both ultrastructural variants of CDV NC interacted with both hsp72 and the constitutively expressed member of the approximately 70 kDa family of HSP (hsp73) in a reversible and ATP-dependent manner. An effect of hsp72/73 on NC polymerase activity was demonstrated using cell-free assays derived from either Vero or HeLa cell lines. Antibody specific to hsp72 suppressed both basal and stress-enhanced polymerase activity whereas hsp73-specific antibody had no affect. Supplementation of purified hsp72/73, but not hsp73 alone, enhanced basal polymerase activity in a dosage-dependent manner. Using purified NC variants, polymerase activity was demonstrated in pre-formed hsp72/73-NC complexes but not in NC devoid of HSP. These results suggest that the stimulatory effect of the stress response upon CDV gene expression may, in part, be mediated by a reversible and direct interaction between hsp72 and the viral core particle.

Nucleocapsid (NC) variants expressed by the Onderstepoort strain of canine distemper virus (CDV) were ultrastructurally and biochemically characterized. Three isolated variants were defined which corresponded to the three variants observed within the cytoplasm of infected cells. Dense NC (D-NC), isolated on discontinuous caesium chloride (CsCl) isopycnic gradients, had an average density of 1.2971 +/- 0.0042 g/ml. Ultrastructurally, D-NC were 1620.0 +/- 112.1 nm in length with a 20.1 +/- 1.3 nm outer and a 5.8 +/- 0.7 nm inner core diameter. The D-NC protein composition was 89.7% of a 61K protein (N), 8.4% of a 75K protein (P) and 1.9% of a 160K to 200K protein (L). A single species of nucleic acid, 15 kb in length, was isolated from D-NC. Light NC (L-NC), similarly isolated, had an average density of 1.2894 +/- 0.0040 g/ml. L-NC differed ultrastructurally from D-NC in that poor resolution of NC subunits, a larger outer diameter (32.0 +/- 2.8 nm), and a greater inner core diameter (10.4 +/- 0.6 nm) were observed. The average L-NC strand length was 1574.4 +/- 115.8 nm. The protein composition was the same as D-NC with the exception of an additional 70K protein, representing 4.0 to 7.7% of the total L-NC protein mass. A 15 kb nucleic acid was also identified in L-NC, although heightened sensitivity of encapsidated L-NC nucleic acid to non-specific nuclease degradation was observed. The ratio of D-NC to L-NC isolated from individual virus preparations varied and was independent of viral infectivity. A third NC variant, defective-NC (Df-NC), was also identified. This had the lowest density on CsCl gradients (1.2460 +/- 0.0046 g/ml). The Df-NC structures were truncated to a uniform length of 87.0 +/- 5.8 nm. Diameter measurements were between those of D-NC and L-NC, being 24.4 +/- 1.4 nm (outer) and 6.9 +/- 0.4 nm (inner core). Like L-NC, the 70K protein was present but in greater amounts, representing as much as 43.7% of the total Df-NC protein mass. RNase A-sensitive nucleic acid was isolated from Df-NC which ranged in size from 1.16 to 0.67 kb with a majority of the material being 0.86 kb in length. For both L-NC and Df-NC, canine CDV convalescent serum reacted with viral N and P proteins in Western blot analyses but not with the 70K protein, suggesting a host cellular origin for the latter.