Observations on the immune responses of sheep infected with Cytoecetes phagocytophila

Humoral and cell-mediated immune responses of sheep infected with Cytoecetes phagocytophila, the causative agent of tick-borne fever, were studied. Previous immunological studies were hampered by lack of reliable sources of antigen. Attempts, therefore, were made to improve antigen yields. Two methods were used successfully. The first was based on the properties of corticosteroids to induce granulocytosis; sheep injected with betamethasone sodium phosphate injection BP at the peak period of parasitaemia had a neutrophilia with an increase of infected cells within six hours. The second method involved the culture of infected blood overnight at 37°C which increased the number of infected cells and the number of organisms per infected cell.A relationship between complement fixing antibodies and pro¬ tective immunity was found and a threshold of protective immunity established. The kinetics of antibody responses to the organism were studied by fractionating immunoglobulins of sera from immune sheep by gel-filtration and ion-exchange chromatography and by complement fixation test. Antibody response to _C. phagocytophila was characterised by an initial production of IgM followed by IgG but the IgM persisted for long periods.The complement fixation test was also used to assess the antigenic relationships between strains of Ch phagocytophila. All strains tested showed strong antigenic relationships but quantitative differentiation was possible.A hitherto undescribed in vitro test for cell-mediated immune response of sheep was developed. The cell-mediated immune response appeared earlier than the humoral immune response

Diazoxon disrupts the expression and distribution of βIII-tubulin and MAP 1B in differentiating N2a cells

This study aimed at assessing the effects of diazoxon (DZO), a major metabolite of the insecticide diazinon (DZ), on key cytoskeletal proteins in differentiating N2a neuroblastoma cells. Initial experiments established that sub-lethal concentrations of 1, 5 and 10 μM DZO produced profound inhibition of neurite outgrowth. Densitometric scanning of probed immunoblots of N2a cell lysates demonstrated that DZO had no effect on total β-tubulin levels. However, probing with a monoclonal antibody that recognised specifically the βIII-tubulin isotype revealed that 10 μM DZO induced a significant reduction in the levels of this particular form. Levels of polyglutamylated tubulin were not altered. Exposure to 10 μM DZO also decreased the expression of microtubule associated protein 1B (MAP 1B). However, DZO had no effect on the expression of MAP tau. DZO also failed to affect the levels neurofilament light (NFL) and neurofilament medium (NFM) chain levels. Indirect immunofluorescence demonstrated that the staining of neurites in treated cells was weaker than in the controls for βIII-tubulin. In conclusion, DZO disrupts the microtubule (MT) network affecting the expression and distribution of two specific MT proteins known to be important in neuritogenesis. DZO may contribute to the developmental neurotoxicity seen following exposure to DZ

Isolation of canine Anaplasma phagocytophilum strains from clinical blood samples using the Ixodes ricinus cell line IRE/CTVM20

AbstractAnaplasma phagocytophilum is an intracellular tick-borne rickettsial pathogen, which causes granulocytic anaplasmosis in various species of livestock and companion animals and also in humans. Previously A. phagocytophilum has been isolated and propagated in cell lines derived from the tick Ixodes scapularis and in the human promyelocytic cell line HL60. In this study we used the Ixodes ricinus-derived cell line IRE/CTVM20 to isolate and propagate two new canine strains of A. phagocytophilum.Blood samples were collected by veterinarians from two dogs, one from Germany and the other from Austria. Suspicion of clinical canine granulocytic anaplasmosis was raised by the treating veterinarians and after confirmation of A. phagocytophilum infection by real-time PCR, buffy coat cells were isolated and co-cultivated with IRE/CTVM20 cells maintained at 28°C in L15/L15B medium.In the tick cells, rickettsial inclusions were first recognised after 86 days of incubation. Electron microscopic examination of tick cells infected with one of the isolates revealed cytoplasmic vacuoles containing pleomorphic organisms with individual bacteria enveloped by a bilayer membrane. Sequencing of 16S rRNA genes confirmed the isolation of A. phagocytophilum and showed the highest identity to the A. phagocytophilum human HZ strain. The two A. phagocytophilum isolates were passaged several times in IRE/CTVM20 cells and transferred to the I. scapularis cell line ISE6. This confirms for the first time the successful establishment and continuous cultivation of this pathogen in I. ricinus cells as well as infectivity of these canine strains for I. scapularis cells

Variant -and individual dependent nature of persistent Anaplasma phagocytophilum infection

<p>Abstract</p> <p>Background</p> <p><it>Anaplasma phagocytophilum </it>is the causative agent of tick-borne fever in ruminants and human granulocytotropic anaplasmosis (HGA). The bacterium is able to survive for several months in immune-competent sheep by modifying important cellular and humoral defence mechanisms. Little is known about how different strains of <it>A. phagocytophilum </it>propagate in their natural hosts during persistent infection.</p> <p>Methods</p> <p>Two groups of five lambs were infected with each of two <it>16S </it>rRNA gene variants of <it>A. phagocytophilum</it>, i.e. <it>16S </it>variant 1 which is identical to GenBank no <ext-link ext-link-id="M73220" ext-link-type="gen">M73220</ext-link> and <it>16S </it>variant 2 which is identical to GenBank no <ext-link ext-link-id="AF336220" ext-link-type="gen">AF336220</ext-link>, respectively. The lambs were infected intravenously and followed by blood sampling for six months. <it>A. phagocytophilum </it>infection in the peripheral blood was detected by absolute quantitative real-time PCR.</p> <p>Results</p> <p>Both <it>16S </it>rRNA gene variants of <it>A. phagocytophilum </it>established persistent infection for at least six months and showed cyclic bacteraemias, but variant 1 introduced more frequent periods of bacteraemia and higher number of organisms than <it>16S </it>rRNA gene variant 2 in the peripheral blood.</p> <p>Conclusion</p> <p>Organisms were available from blood more or less constantly during the persistent infection and there were individual differences in cyclic activity of <it>A. phagocytophilum </it>in the infected animals. Two <it>16S </it>rRNA gene variants of <it>A. phagocytophilum </it>show differences in cyclic activity during persistent infection in lambs.</p

Delineating Anaplasma phagocytophilum Ecotypes in Coexisting, Discrete Enzootic Cycles

Genetically distinct subpopulations have adapted to different niche

Risk factors for <em>Coxiella burnetii</em> antibodies in bulk tank milk from Danish dairy herds

The aim was to identify risk factors associated with Coxiella burnetii antibody positivity in bulk tank milk (BTM) samples from 100 randomly selected Danish dairy cattle herds. Antibody levels were measured by an enzyme-linked immuno-sorbent assay. Before testing the herds, the farm managers were interviewed about hired labour, biosecurity, housing and herd health during the 12 months prior to the study. Variables considered important for C. burnetii antibody positivity in multivariable logistic regression analysis included the sharing of machines between farms (OR = 3.6), human contacts (OR = 4.2), artificial insemination by other people than artificial insemination technicians (OR = 7.7), routine herd health contract with the veterinarian (OR = 4.3) and hygiene precautions taken by veterinarians (OR = 5). In addition, herd size, hired labour, trading of cattle between farms, quarantine and use of calving and disease pens also showed significant association in univariable analysis. This study demonstrates that strict biosecurity is important for the prevention of infections with C. burnetii

Coxiella burnetii Phagocytosis Is Regulated by GTPases of the Rho Family and the RhoA Effectors mDia1 and ROCK

The GTPases belonging to the Rho family control the actin cytoskeleton rearrangements needed for particle internalization during phagocytosis. ROCK and mDia1 are downstream effectors of RhoA, a GTPase involved in that process. Coxiella burnetii, the etiologic agent of Q fever, is internalized by the host´s cells in an actin-dependent manner. Nevertheless, the molecular mechanism involved in this process has been poorly characterized. This work analyzes the role of different GTPases of the Rho family and some downstream effectors in the internalization of C. burnetii by phagocytic and non-phagocytic cells. The internalization of C. burnetii into HeLa and RAW cells was significantly inhibited when the cells were treated with Clostridium difficile Toxin B which irreversibly inactivates members of the Rho family. In addition, the internalization was reduced in HeLa cells that overexpressed the dominant negative mutants of RhoA, Rac1 or Cdc42 or that were knocked down for the Rho GTPases. The pharmacological inhibition or the knocking down of ROCK diminished bacterium internalization. Moreover, C. burnetii was less efficiently internalized in HeLa cells overexpressing mDia1-N1, a dominant negative mutant of mDia1, while the overexpression of the constitutively active mutant mDia1-ΔN3 increased bacteria uptake. Interestingly, when HeLa and RAW cells were infected, RhoA, Rac1 and mDia1 were recruited to membrane cell fractions. Our results suggest that the GTPases of the Rho family play an important role in C. burnetii phagocytosis in both HeLa and RAW cells. Additionally, we present evidence that ROCK and mDia1, which are downstream effectors of RhoA, are involved in that processFil: Salinas Ojeda, Romina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Ortiz Flores, Rodolfo Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Distel, Jesús Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Aguilera, Milton Osmar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Colombo, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Beron, Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentin

Diverse tick-borne microorganisms identified in free-living ungulates in Slovakia

Background: Free-living ungulates are hosts of ixodid ticks and reservoirs of tick-borne microorganisms in central Europe and many regions around the world. Tissue samples and engorged ticks were obtained from roe deer, red deer, fallow deer, mouflon, and wild boar hunted in deciduous forests of south-western Slovakia. DNA isolated from these samples was screened for the presence of tick-borne microorganisms by PCR-based methods. Results: Ticks were found to infest all examined ungulate species. The principal infesting tick was Ixodes ricinus, identified on 90.4% of wildlife, and included all developmental stages. Larvae and nymphs of Haemaphysalis concinna were feeding on 9.6% of wildlife. Two specimens of Dermacentor reticulatus were also identified. Ungulates were positive for A. phagocytophilum and Theileria spp. Anaplasma phagocytophilum was found to infect 96.1% of cervids, 88.9% of mouflon, and 28.2% of wild boar, whereas Theileria spp. was detected only in cervids (94.6%). Importantly, a high rate of cervids (89%) showed mixed infections with both these microorganisms. In addition to A. phagocytophilum and Theileria spp., Rickettsia helvetica, R. monacensis, unidentified Rickettsia sp., Coxiella burnetii, "Candidatus Neoehrlichia mikurensis", Borrelia burgdorferi (s.l.) and Babesia venatorum were identified in engorged I. ricinus. Furthermore, A. phagocytophilum, Babesia spp. and Theileria spp. were detected in engorged H. concinna. Analysis of 16S rRNA and groEL gene sequences revealed the presence of five and two A. phagocytophilum variants, respectively, among which sequences identified in wild boar showed identity to the sequence of the causative agent of human granulocytic anaplasmosis (HGA). Phylogenetic analysis of Theileria 18S rRNA gene sequences amplified from cervids and engorged I. ricinus ticks segregated jointly with sequences of T. capreoli isolates into a moderately supported monophyletic clade. Conclusions: The findings indicate that free-living ungulates are reservoirs for A. phagocytophilum and Theileria spp. and engorged ixodid ticks attached to ungulates are good sentinels for the presence of agents of public and veterinary concern. Further analyses of the A. phagocytophilum genetic variants and Theileria species and their associations with vector ticks and free-living ungulates are required.Fil: Kazimírová, Mária. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Hamšíková, Zuzana. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Spitalská, Eva. Slovak Academy of Sciences. Institute of Virology. Biomedical Research Center,; EslovaquiaFil: Minichová, Lenka. Slovak Academy of Sciences. Institute of Virology. Biomedical Research Center,; EslovaquiaFil: Mahríková, Lenka. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Caban, Radoslav. Široká ; EslovaquiaFil: Sprong, Hein. National Institute for Public Health and Environment.Laboratory for Zoonoses and Environmental Microbiology; Países BajosFil: Fonville, Manoj. National Institute for Public Health and Environment.Laboratory for Zoonoses and Environmental Microbiology; Países BajosFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Kocianová, Elena. Slovak Academy of Sciences. Institute of Virology. Biomedical Research Center,; Eslovaqui