Small wild mammals and arboviruses in Italy

In 1980 and 1981, sera of 256 small wild mammals (rodentia, insectivora, carnivora) were collected in Piemonte and Southern Italy. They were then tested for antibody against 12 arboviruses by haemagglutination inhibition and complement fixation tests. In the North of the country, 42.8% of sera were found positive against Tahyna or Sicilian Sandfly fever viruses. In the Southern provinces, 44.3% of sera reacted with Bhanja, West Nile or other flaviviruses, Tahyna, Sicilian Sandfly fever and Arumowot viruses. These results lead to suspect the possible role of some small mammals (muridae, Clethrionomys glareolus, Talpa caeca, Talpa romana) in circulating these arboviruses in Italy.     

Serological prevalence of Coxiella burnetii in captive wild ruminants in Saudi Arabia

Serum samples from sand gazelles (n = 227), mountain gazelles (n = 232), and Arabian oryx (n = 96) reared in captivity in Riyadh, Saudi Arabia were tested for the presence of Coxiella burnetii antibodies using an indirect enzyme immunoassay. C. burnetii antibodies were present in 18.3%, 7.3%, and 46.9% of these animals, respectively. The difference in serological prevalence between the three species was statistically significant. Age- and sex-related differences in prevalence were also observed. This study is the first record of C. burnetii antibodies in Arabian gazelles.     

Prevalence of Coxiella burnetii in bovine bulk milk samples in southern Iran

One hundred (100) bovine bulk milk samples were tested for the presence of Coxiella burnetii and the detection of associated risk factors. Of the tested samples, 11% tested positive for C. burnetii. There was no correlation between the presence of C. burnetii in bulk milk samples and the kind of dairy herd the samples were taken from the number of cows in each site, and the variety of water and food. The only association that was found corresponded with seasonal samples.     

Molecular characterization of cloned variants of Coxiella burnetii isolated in China.

To study the molecular properties of Coxiella burnetii phase variants we cloned the phase variants of C. burnetii Qiyi (CBQY) strain by the red plaque technique. Three cloned strains, CBQYIC3 (phase I), CBQYIIC7 (phase II) and CBQYIIC5 (semirough-phase) were analysed by SDS-PAGE, immunoblot assay, plasmid isolation and agarose gel electrophoresis of DNA restriction fragments. The results suggest that the unique phase-dependent substance is a lipopolysaccharide and that most protein components of phase I and phase II cells are shared. No significant differences of DNA restriction fragments were found between clonal isolates of phase I and phase II C. burnetii CBQY strains. A plasmid of approximately 56 Kb was isolated from both phase I and phase II variants indicating that phase variation probably could not be attributed to its presence or absence.     

Comparison of serological detection methods for diagnosis of Ehrlichia canis infections in dogs

We determined the value of four serological assays for the diagnosis of canine monocytic ehrlichiosis by comparing them to the indirect fluorescent-antibody assay "gold standard." The specificity of Dip-S-Ticks was significantly lower than that of all of the other tests evaluated. The sensitivity of Dip-S-Ticks was significantly higher than that of Snap3Dx or the Snap Canine Combo. The sensitivity of the rMAP2 enzyme-linked immunosorbent assay (ELISA) was significantly higher than that of the Snap Canine Combo. The accuracy levels of the rMAP2 ELISA, Snap3Dx, Dip-S-Ticks, and Snap Canine Combo were 97.0, 89.8, 85.1, and 82.9%, respectively.     

Molecular evidence of a new strain of Ehrlichia canis from South America

Blood samples from dogs with clinical signs compatible with ehrlichiosis were examined for infection of Ehrlichia canis using PCR, multiplex real-time PCR, and DNA sequencing analysis. Eleven of 25 samples were positive for a new strain of E. canis. This is the first molecular identification of E. canis infection in dogs from Peru.     

Analyses of Ehrlichia canis and a canine granulocytic Ehrlichia infection.

Ehrlichia canis and canine granulocytic Ehrlichia sp. (CGE) infect canine monocytes and granulocytes, respectively. E. canis has been cultured in vitro and used to develop an immunofluorescence assay. CGE has not been cultured, and a serologic assay is not available. The sera of dogs infected with CGE were reported to react with E. canis by immunofluorescence. In this study, the temporal response of immunoglobulin G (IgG) was determined by an enzyme-linked immunosorbent assay (ELISA) with purified E. canis antigen in four dogs experimentally infected with E. canis, in two dogs experimentally infected with CGE, and in one dog infected with E. canis and subsequently infected with CGE. E. canis-infected dogs developed an IgG ELISA result of 1.5 or greater for the optical density signal/noise ratio by 2 months postinfection. CGE challenge of a dog with a previous E. canis infection induced an anamnestic increase in the IgG ELISA result; however, CGE infection alone did not induce a significant IgG ELISA response. Western immunoblot analysis showed that dogs infected with E. canis developed antibodies initially that reacted with low-molecular-mass proteins (30, 24, and 21 kDa) and subsequently with higher-molecular-mass proteins (160, 100, 78, 64, 47, and 40 kDa). In contrast, CGE-infected dogs showed reactions with the same higher-molecular-mass proteins of E. canis but, unlike E. canis-infected dogs, not with the low-molecular-mass proteins of E. canis. Of 10 serum samples collected in the field of Indonesia from dogs with tropical canine pancytopenia, all had an optical density signal minus noise value of 2.54 or greater in the IgG ELISA and reacted with E. canis antigen in a pattern similar to that of serum samples from dogs experimentally infected with E. canis in Western immunoblotting. This study suggests that the IgG ELISA and Western immunoblotting with purified E. canis as the antigen are useful in distinguishing between E. canis and CGE infections in dogs.     

Glycosylation of homologous immunodominant proteins of Ehrlichia chaffeensis and Ehrlichia canis

The glycoprotein genes of Ehrlichia chaffeensis (1,644 bp) and Ehrlichia canis (2,064 bp) encode proteins of 548 to 688 amino acids with predicted molecular masses of only 61 and 73 kDa but with electrophoretic mobilities of 120 kDa (P120) and 140 kDa (P140), respectively. The 120-kDa protein gene of E. chaffeensis contains four identical 240-bp tandem repeat units, and the 140-kDa protein gene of E. canis has 14 nearly identical, tandemly arranged 108-bp repeat units. Conserved serine-rich motifs identified in the repeat units of P120 and P140 were also found in the repeat units of the human granulocytotropic ehrlichiosis agent 130-kDa protein and of the fimbria-associated adhesin protein Fap1 of Streptococcus parasanguis. Nearly the entire (99%) E. chaffeensis P120 gene (1,616 bp), the 14-repeat region (78%) of the E. canis P140 gene (1,620 bp), and a 2-repeat region from the E. chaffeensis P120 gene (520 bp) were expressed in Escherichia coli. The recombinant proteins exhibited molecular masses ranging from 1.6 to 2 times larger than those predicted by the amino acid sequences. Antibodies against the recombinant proteins reacted with E. chaffeensis P120 and E. canis P140, respectively. Carbohydrate was detected on the E. chaffeensis and E. canis recombinant proteins, including the two-repeat polypeptide region of E. chaffeensis P120. A carbohydrate compositional analysis identified glucose, galactose, and xylose on the recombinant proteins. The presence of only one site for N-linked (Asn-Xaa-Ser/Thr) glycosylation, a lack of effect of N-glycosidase F, the presence of 70 and 126 Ser/Thr glycosylation sites in the repeat regions of P120 and P140, respectively, and a high molar ratio of carbohydrate to protein suggest that the glycans may be O linked.     

Seroprevalence of Ehrlichia canis and of Canine Granulocytic Ehrlichia Infection in Dogs in Switzerland

Serum samples from 996 dogs in Switzerland were examined for antibodies to Ehrlichia canis and to the agent causing canine granulocytic ehrlichiosis (CGE). Ehrlichiosis, borreliosis, and systemic illness not associated with ticks were suspected in 75, 122, and 157 of these dogs, respectively. The remainder of the serum samples were obtained from clinically healthy dogs which resided north (n = 235) or south (n = 407) of the Alps. The serum samples were tested by an indirect immunofluorescence technique for antibodies to the two agents incriminated, E. canis and Ehrlichia phagocytophila, a surrogate marker of the agent of CGE. Twenty-two of 996 (2.2%) serum samples had antibodies to E. canis and were distributed as follows: 20 of 75 (26.7%) samples from dogs suspected of having ehrlichiosis, 1 of 122 (0.8%) from dogs suspected of having borreliosis, and 1 of 407 (0.2%) from healthy dogs which resided south of the Alps. Of the 75 (7.5%) serum samples that had antibodies to E. phagocytophila, significantly more samples were from ill dogs than from healthy dogs. Among the sera from healthy dogs, antibodies to E. phagocytophila were significantly more prevalent in the north. Because seropositive dogs had a history of travel outside Switzerland and because Rhipicephalus sanguineus is found exclusively south of the Alps, it was presumed that, in contrast to the agent of CGE, E. canis is not indigenous to Switzerland.     

Molecular epidemiology of parasitic protozoa and Ehrlichia canis in wildlife in Madrid (central Spain)

Parasitology Research - Wildlife species are involved in the transmission of diverse pathogens. This study aimed to monitor raccoons (Procyon lotor), American minks (Neovison vison), and red foxes...     

Role of antibody in Coxiella burnetii infection.

BALB/c mice infected with Coxiella burnetii phase I developed a state of acquired resistance which could be detected during week 2 postinfection. Immune serum, administered to normal mice 24 h before challenge with C. burnetii, appeared to accelerate the development of resistance. An increased clearance rate could be measured in these serum recipients 1 week postinfection. Simultaneous administration of immune serum and C. burnetii did not affect the normal clearance rate of rickettsiae from the spleens of infected mice during week 1, but enhanced clearance of the organism by 14 days postchallenge. Passive transfer of immune serum 24 h after challenge of normal mice with viable C. burnetii had no effect on rickettsial growth within the spleens of animals treated in this fashion. Treatment of athymic mice with immune serum 24 h before challenge with C. burnetii had no effect on rickettsial multiplication within the spleens of these T-cell-deficient animals.     

Prevalence of antibodies to Coxiella burnetii in Japan.

We evaluated the prevalence of Coxiella burnetii antibodies in 626 human serum samples (275 from veterinarians, 107 from meat-processing workers, 184 from respiratory-disorder patients, and 60 from healthy humans) by the indirect immunofluorescence test. Of the serum samples examined, 54 (8.6%) and 103 (16.5%) reacted positively to phase I and II antigens, respectively, of C. burnetii. The rates differed for healthy humans and respiratory-disorder patients. Antibody prevalence was high for healthy humans living in close contact with animals (e.g., veterinarians and meat-processing workers).     

Identification and cloning of immunodominant antigens of Coxiella burnetii

A sublethal-challenge model was established in BALB/c mice by using protection from the development of severe splenomegaly as an indicator of vaccinogenic activity for evaluation of the protective efficacies of vaccine candidates. To determine the immunodominant antigens as defined by reaction to an infection-derived antibody, mouse sera from different stages of experimental infection with various doses of Coxiella burnetii were tested by immunoblotting. Proteins with molecular masses of 14, 16, 21, 28, 32, 45 to 50, 57, and 60 kDa were recognized as immunodominant antigens. Antibody responses in whole-cell antigen (WCA)-vaccinated mice were compared with those in unvaccinated mice by immunoblotting using two-dimensional gel-separated C. burnetii antigens. The results indicated that there were significantly different antibody responses during different stages of vaccination and challenge, suggesting that several specific immunogenic antigens may play critical roles in the protection of mice against challenge. To clone these immunogenic antigens, a genomic DNA library of Nine Mile phase I was screened with convalescent-phase antisera from mice. Eighteen novel immunoreactive proteins with molecular masses ranging from approximately 14 to 67 kDa were cloned and identified. Interestingly, several recombinant proteins reacted with sera from both early-stage infected and WCA-vaccinated prechallenged mice. These results suggest that these proteins may play critical roles in the development of protective immunity and that they are logical candidates for vaccine and serodiagnostic reagents.     

Molecular detection of Ehrlichia canis,Anaplasma bovis,Anaplasma platys,Candidatus Midichloria mitochondrii and Babesia canis vogeli in ticks from Israel

Ticks are vectors of important pathogens of human and animals. Therefore, their microbial carriage capacity is constantly being investigated. The aim of this study was to characterize the diversity of domestic animal pathogens in ticks collected from vegetation and the ground, from different parts of Israel. Non-engorged questing adult ticks were collected from 13 localities. A total of 1196 ticks in 131 pools—83 pools of Rhipicephalus turanicus and 48 of Rhipicephalus sanguineus (with two to ten ticks per pool)—were included in this study. In addition, 13 single free-roaming Hyalomma spp. ticks were collected. Screening by molecular techniques revealed the presence of Ehrlichia canis, Anaplasma platys, Anaplasma bovis and Babesia canis vogeli DNA in R. turanicus ticks. E. canis, A. bovis, B. canis vogeli and Candidatus Midichloria mitochondrii DNA sequences were detected in R. sanguineus ticks. Candidatus Midichloria mitochondrii DNA was also detected in Hyalomma spp. ticks. Neither Hepatozoon spp. nor Bartonella spp. DNA was detected in any of the ticks examined. This study describes the first detection of E. canis in the tick R. turanicus, which may serve as a vector of this canine pathogen; E. canis was the most common pathogen detected in the collected questing ticks. It also describes the first detection of A. bovis and Candidatus Midichloria mitochondrii in Israel. To the best of the author's knowledge, this is the first report describing the detection of DNA of the latter two pathogens in R. sanguineus, and of A. bovis in R. turanicus.     

Proteome and antigen profiling of Coxiella burnetii developmental forms

A biphasic developmental cycle whereby highly resistant small-cell variants (SCVs) are generated from large-cell variants (LCVs) is considered fundamental to the virulence of Coxiella burnetii, the causative agent of human Q fever. In this study a proteome analysis of C. burnetii developmental forms was conducted to provide insight into their unique biological and immunological properties. Silver-stained gels of SCV and LCV lysates separated by two-dimensional (2-D) gel electrophoresis resolved over 675 proteins in both developmental forms. Forty-eight proteins were greater than twofold more abundant in LCVs than in SCVs, with six proteins greater than twofold more abundant in SCVs than in LCVs. Four and 15 upregulated proteins of SCVs and LCVs, respectively, were identified by mass spectrometry, and their predicted functional roles are consistent with a metabolically active LCV and a structurally resistant SCV. One-dimensional and 2-D immunoblots of cell form lysates probed with sera from infected/vaccinated guinea pigs and convalescent-phase serum from human patients who had recovered from acute Q fever, respectively, revealed both unique SCV/LCV antigens and common SCV/LCV antigens that were often differentially synthesized. Antigens recognized during human infection were identified by mass spectroscopy and included both previously described immunodominant proteins of C. burnetii and novel immunogenic proteins that may be important in the pathophysiology of clinical Q fever and/or the induction of protective immunity.     

Antigenic characterization of ehrlichiae: protein immunoblotting of Ehrlichia canis, Ehrlichia sennetsu, and Ehrlichia risticii.

In recent years a febrile illness apparently associated with tick bite in patients in the United States has been attributed to infection by an Ehrlichia species. This implication is based on serologic responses to E. canis, morphologic demonstration of ehrlichiae in clinical materials, and a single isolate distinct from E. canis which was obtained from a human patient by the Centers for Disease Control. Little is known about the antigens of the ehrlichiae. This report expands the breadth of available knowledge concerning the antigenic components and serologic responses to component antigens of E. canis, E. sennetsu, and E. risticii. Protein immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by using density gradient-purified ehrlichiae and homologous antisera demonstrated reproducible and characteristic antigens within each species (for E. sennetsu, 91, 64, 54, 44, 36, 34, 28, 25, and 24 kDa; for E. risticii, 70, 52, 48, 44, 35, 28, 24, 23, and 20 kDa; for E. canis, 110, 64, 52, 42, 33, 28, 24, 23, and 20 kDa). When antisera were reacted with heterologous antigens, cross-reactivity among these species was virtually restricted to the 70-kDa antigen. Furthermore, when serum samples obtained from 10 patients who were convalescing from ehrlichiosis were tested against each antigen, only three serum samples had any reactivities, and these serum samples reacted with only a few of the antigenic bands. These results documented the molecular sizes of electrophoretically separated antigens of the three Ehrlichia species, confirm their serologic relationships, and support the novel nature of the agent(s) of human ehrlichiosis in the United States.     

Q热立克次体单克隆抗体的产生及其特性的研究

Q热立克次体存在相变异的现象。随着相变异的出现,Q热立克次体的生物学特性、理化性质和抗原性等方面均有变化。这种变化的发生与Q热立克次体的表面结构和抗原组分的改变有密切关系。为了在分子水平上研究Q热立克次体,阐明相变异的机制或研究Q热立克次体的致病性、血清免疫学     

Interaction between Dermacentor reticulatus cells and Coxiella burnetii in vivo

By electron microscopy the distribution of Coxiella burnetii was followed in females of Dermacentor reticulatus injected intracoelomally in a dose of about 10(3) EID50 per tick. The heaviest infestation with coxiellae was noticed in the cells of haemolymph, fat body, Malpighian tubulus and tracheal complex. No rickettsiae were found in Gene's organ. Unexpected was the propagation of rickettsiae in muscle fibres. C. burnetii multiplied in all organs affected. Heavy infection resulted at the marked damage of cell components. Coxiellae were evident in the haemocytes; in organs they formed small and large cell variants; endospore formations were observed free in the haemolymph.