Bartonella vinsonii subsp. arupensis infection in animals of veterinary importance, ticks and biopsy samples.

Testing for vector-borne pathogens in livestock is largely reliant upon blood and tissue. The role of biopsy samples remains poorly explored for detecting tick-borne bacteria in animals. In a 2-year survey, animals of veterinary importance from farms throughout the northern part of Greece were routinely checked for the presence of biopsy samples. Where detected, either a portion or a biopsy was collected together with whole blood samples and any ticks at the site of the biopsy sample. Molecular testing was carried out by real-time PCR targeting the internal transcribed spacer gene of Bartonella species. A total of 68 samples (28 blood samples, 28 biopsy samples and 12 ticks (nine Rhipicephalus bursa and three Rhipicephalus turanicus)) were collected from goats (64 samples) and cattle (four samples). Eight (11.8%) of the 68 samples were positive for Bartonella species. Of the biopsy and whole blood samples, four (14.3%) of each type were positive for Bartonella species. None of the ticks tested positive for Bartonella species. All pairs of positive biopsy samples/whole blood samples originated from the same animals. Positive samples were identified as Bartonella vinsonii subsp. arupensis. Although many more samples from a much wider spectrum of animal species is required before concluding upon the merit of biopsy samples in the study of tick-borne diseases, the significance of our finding warrants further study, both for clinical consequences in small ruminants and for those humans who are farming infected animals.

Mediterranean spotted fever in crete, Greece: clinical and therapeutic data of 15 consecutive patients.

The clinical, epidemiological, and therapeutic aspects of 15 patients with Mediterranean spotted fever (MSF), admitted to the Internal Medicine Department of the General Hospital of Sitia (southeastern Crete, Greece) between December 2000 and July 2003, were studied. Diagnosis was made on the basis of clinical signs and symptoms and was confirmed by serology. Of the patients studied, 67% were men and 33% women, with a median age of 52 years (range of 23-76 years). Ten cases (67%) were diagnosed between May and July. Of all the patients, 93% had a history of contact with animals, mainly with sheep (11 patients, 73%), while 53% of them had a history of tick-bite (33%), or reported the presence of ticks in their environment (20%). The typical eschar lesion (tache noir) at the tick-bite site was present in 53% of the patients, while the rash was present in 87% of them. Laboratory findings included leukopenia (47%), thrombocytopenia (54%), elevation of transaminases (80%), hyponatremia (33%), and microscopic hematuria (80%). Four patients (27%) displayed pulmonary infiltrates on chest radiography. All patients were treated with doxycycline (200 mg daily) and recovered rapidly. Renal function deteriorated in one patient with chronic renal failure, but he recovered thereafter.

Ticks, tick-borne rickettsiae, and Coxiella burnetii in the Greek Island of Cephalonia.

Domestic animals are the hosts of several tick species and the reservoirs of some tick-borne pathogens; hence, they play an important role in the circulation of these arthropods and their pathogens in nature. They may act as vectors, but, also, as reservoirs of spotted fever group (SFG) rickettsiae, which are the causative agents of SFG rickettsioses. Q fever is a worldwide zoonosis caused by Coxiella burnetii (C. burnetii), which can be isolated from ticks. A total of 1,848 ticks (954 female, 853 male, and 41 nymph) were collected from dogs, goats, sheep, cattle, and horses in 32 different localities of the Greek island of Cephalonia. Rhipicephalus (Rh.) bursa, Rh. turanicus, Rh. sanguineus, Dermacentor marginatus (D. marginatus), Ixodes gibbosus (I. gibbosus), Haemaphysalis (Ha.) punctata, Ha. sulcata, Hyalomma (Hy.) anatolicum excavatum and Hy. marginatum marginatum were the species identified. C. burnetii and four different SFG rickettsiae, including Rickettsia (R.) conorii, R. massiliae, R. rhipicephali, and R. aeschlimannii were detected using molecular methods. Double infection with R. massiliae and C. burnetii was found in one of the positive ticks.

Distribution of the ompA-types among ruminant and swine pneumonic strains of Pasteurella multocida exhibiting various cap-locus and toxA patterns.

Pasteurella multocida is an important pathogen in food-producing animals and numerous virulence genes have been identified in an attempt to elucidate the pathogenesis of pasteurellosis. Currently, some of these genes including the capsule biosynthesis genes, the toxA and the OMPs-encoding genes have been suggested as epidemiological markers. However, the number of studies concerning ruminant isolates is limited, while, no attempt has ever been made to investigate the existence of ompA sequence diversity among P. multocida isolates. The aim of the present study was the comparative analysis of 144 P. multocida pneumonic isolates obtained from sheep, goats, cattle and pigs by determining the distribution of the ompA-types in conjunction with the cap-locus and toxA patterns. The ompA genotypes of the isolates were determined using both a PCR-RFLP method and DNA sequence analysis. The most prevalent capsule biosynthesis gene among the isolates was capA (86.1%); a noticeable, however, rate of capD-positive isolates (38.6%) was found among the ovine isolates that had been associated primarily with the capsule type A in the past. Moreover, an unexpectedly high percentage of toxA-positive pneumonic isolates was noticed among small ruminants (93.2% and 85.7% in sheep and goats, respectively), indicating an important epidemiological role of toxigenic P. multocida for these species. Despite their great heterogeneity, certain ompA-genotypes were associated with specific host species, showing evidence of a host preference. The OmpA-based PCR-RFLP method developed proved to be a valuable tool in typing P. multocida strains.

Isolation and identification of a rickettsial strain related to Rickettsia massiliae in Greek ticks.

Adult ticks were collected in a rural area of central Greece in order to isolate and identify rickettsiae. A hemolymph test using Gimenez staining was used for detection, while simultaneous isolation was performed using the shell-vial technique. Serologic, antigenic, and genomic characterization of the isolates was achieved by microimmunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP), and pulsed-field gel electrophoresis (PFGE), respectively. Although none of the 242 collected ticks was positive by the hemolymph test, one rickettsial isolate, designated GS, was obtained by the shell-vial technique. This isolate originated from a female Rhipicephalus sanguineus. Microimmunofluorescence serologic typing by the method of Philip and others demonstrated that GS belongs to the same serotype as the recently isolated Rickettsia massiliae (Mtu1). Protein analysis by SDS-PAGE and immunoblotting by Western blot revealed similar profiles between the two rickettsiae. Using Alu I, Rsa I, and Pst I restriction endonucleases in PCR-RFLP analysis, GS and R. massiliae were found to possess identical restriction sites. However, PFGE showed differences when the two genomes were digested with Bss HII and Sma I restriction endonucleases, in spite of their equal size. In conclusion, the first rickettsial isolation in Greece was found to be antigenically identical and genotypically close to the French isolate R. massiliae, despite small differences showed by PFGE.

Genotypic identification of murine typhus Rickettsia in rats and their fleas in an endemic area of Greece by the polymerase chain reaction and restriction fragment length polymorphism.

Forty-nine cases of murine typhus were diagnosed in recent years in residents of several communities around the city of Chalkis, the capital of the Prefecture of Evia. (Euboea) Evia is an island connected to central mainland Greece by a bridge. To investigate the endemicity of murine typhus in this area, 226 fleas (Xenopsylla cheopis) and blood samples were collected from 53 rats (Rattus norvegicus) trapped in this area. The polymerase chain reaction followed by restriction fragment length polymorphism analysis (PCR-RFLP) was used to detect and identify Rickettsia typhi, the etiologic agent murine typhus, in the rat blood samples (buffy coat cells) as well as in their fleas. An indirect immunofluorescent antibody (IFA) assay was performed to detect antibodies against R. typhi in rat serum samples. The presence of R. typhi in both fleas and rat blood samples was demonstrated. The frequency of infection for X. cheopis was 4%, while 18% of the rats had buffy coat cells infected, and 92% of the rat sera tested by IFA were positive for anti-R. typhi antibodies. The present work is the first successful application of PCR-RFLP in a field study of naturally infected rats and their fleas in Europe.

Sequence diversity of the leukotoxin (lktA) gene in caprine and ovine strains of Mannheimia haemolytica.

Mannheimia haemolytica is the aetiological agent of pneumonic pasteurellosis in small ruminants. The primary virulence factor of the bacterium is a leukotoxin (LktA), which induces apoptosis in susceptible cells via mitochondrial targeting. It has been previously shown that certain lktA alleles are associated either with cattle or sheep. The objective of the present study was to investigate lktA sequence variation among ovine and caprine M haemolytica strains isolated from pneumonic lungs, revealing any potential adaptation for the caprine host, for which there is no available data. Furthermore, we investigated amino acid variation in the N-terminal part of the sequences and its effect on targeting mitochondria. Data analysis showed that the prevalent caprine genotype differed at a single non-synonymous site from a previously described uncommon bovine allele, whereas the ovine sequences represented new, distinct alleles. N-terminal sequence differences did not affect the mitochondrial targeting ability of the isolates; interestingly enough in one case, mitochondrial matrix targeting was indicated rather than membrane association, suggesting an alternative LktA trafficking pattern.

Haemorrhagic diathesis in a ram with Anaplasma phagocytophilum infection.

A 10-month-old ram with fever, inappetence and haemorrhagic diathesis had petechiae and ecchymoses at various body sites and was infested by ticks. Haematological examination revealed pancytopenia, while serum biochemistry indicated hepatic dysfunction. Blood smears were negative for Ehrlichia spp. and other haemoparasites. Paired sera revealed infection by Anaplasma phagocytophilum, but testing by polymerase chain reaction was negative. Treatment with oxytetracycline was effective. This is the first reported clinical case of ovine anaplasmosis in Greece caused by A. phagocytophilum.

Murine typhus in Cyprus: 21 paediatric cases.

The aim of this article is to present the manifestations of Rickettsia typhi infection in childhood. Twenty-one children under 15 years of age were hospitalised in the Department of Paediatrics of the Archbishop Makarios Hospital in Nicosia, Cyprus, from 2000 to 2006 with Rickettsia typhi infection. Ten of them were boys and 11 were girls. The median age was eight years (range four to 13 years). The most common clinical features were fever (100%) and rash (57%). Lymphadenopathy, usually cervical, was also a frequent finding (37%). Severe headache was rather infrequent (29%). Splenomegaly or hepatomegaly were less frequent findings (24% and 10%, respectively). Mild elevation of liver enzymes (AST and ALT elevated >1-fold in 81% and 75%, respectively) was the most frequent laboratory finding. Thrombocytopenia (28%) and leucopenia (17%) were less frequent. Nearly half of the patients (10/21) came from four neighbouring villages, where most residents work in agriculture. All of the children were treated with appropriate antibiotic regimens and had complete recovery. Rickettsia typhi infection should be considered in the differential diagnosis of children who present during the summer or early autumn months with prolonged fever and rash with or without lymphadenopathy.

Epidemiological study of Q fever in humans, ruminant animals, and ticks in Cyprus using a geographical information system.

A cross-sectional study of Q fever was conducted in a representative sample of the human and animal population in Cyprus in order to assess the seroprevalence of Q fever and the prevalence of related risk factors. A total of 583 human and 974 ruminant animal serum samples were collected and tested for the detection of antibodies against Coxiella burnetii phase II antigen using an indirect immunofluorescent assay. One hundred forty-one ticks were collected from the infested animals examined; the polymerase chain reaction and the shell-vial technique were used to detect and isolate C. burnetii. Standardized questionnaires were used to obtain information concerning inhabitants and their animals. A geographical information system was used to identify high-risk regions. The prevalence of IgG antibodies against C. burnetii phase II antigen was estimated at 52.7% for humans, 48.2% for goats, 18.9% for sheep, and 24% for bovines. C. burnetii was detected in 11 (7.8%) ticks. Using the geographical information system, two villages were identified as high-risk regions on the basis of high seroprevalence rates of IgG antibodies in humans and animals. Risk factors related to Q fever seropositivity were identified by logistic regression analysis and included age, residence, occupation, use of manure in the garden, ownership of animals (especially goats), and the presence of tick-infested or aborting animals. Q fever poses an occupational hazard to humans living in close contact with sheep and/or goats. In parallel, ticks should be considered an important aspect in the epidemiology of Q fever and should be further studied to better elucidate their role.

Cat scratch disease simulating a malignant process of the chest wall with coexistent osteomyelitis.

A 6-y-old girl developed fever, soft-tissue mass in the right chest wall, osteomyelitis of the 10th rib and hepatic granuloma. Cat scratch disease was diagnosed by histological examination of the mass and serological tests. The patient was treated successfully with antibiotics and recovered completely, as shown by a 10 month follow-up.

Q fever in the Greek island of Crete: detection, isolation, and molecular identification of eight strains of Coxiella burnetii from clinical samples.

Over a period of 6 years (1989 to 1995), serum samples from 3,300 patients suspected to be infected by Coxiella burnetii were assayed for the presence of antibodies against antigen phase II of the microorganism by the indirect immunofluorescence antibody technique (IFAT). One hundred fifty-two cases were recorded, and blood samples from 17 patients were cultured for the isolation of the pathogen. By a centrifugation shell vial technique, eight strains were isolated from patients suffering from acute Q fever. The microorganism was detected in the cultures by IFAT, by Gimenez staining, and by the cytopathogenic effect on Vero and human embryonic lung (HEL) cells. PCR followed by restriction fragment length polymorphism analysis was used to confirm the diagnosis and identify the Coxiella burnetii strains within the cell cultures as well as to compare them with reference strains. In order to avoid time-consuming cultures, to achieve direct detection of Coxiella burnetii in clinical samples (blood, buffy coat, etc.), and to increase the specificity and sensitivity of the detection, nested PCR was performed. The first step of DNA extraction was performed with the QIAamp blood kit 250. For the second step of the PCR assays, the conditions of temperature and times of recycling were properly modified, and the microorganism was detected within 4 h. Our study demonstrates that Q fever is an endemic disease in Crete and that the diagnosis of Coxiella burnetii infection can be rapidly achieved by the detection of the microorganism in buffy coat samples by nested PCR. Although the presenting symptoms of the disease in this study differed from those in other studies, the Cretan strains do not differ genotypically from the reference strains (Nine Mile and Q212).

In vitro susceptibility of Coxiella burnetii to linezolid in comparison with its susceptibilities to quinolones, doxycycline, and clarithromycin.

The in vitro susceptibility to linezolid shown by nine Greek isolates of Coxiella burnetii derived from patients with acute Q fever was investigated. MICs of linezolid were compared with those of pefloxacin, ciprofloxacin, ofloxacin, trovafloxacin, doxycycline, and clarithromycin using the shell vial assay. MICs of linezolid and clarithromycin ranged from 2 to 4 microg/ml; those of doxycycline, trovafloxacin, and ofloxacin ranged from 1 to 2 microg/ml; those of pefloxacin ranged from 1 to 4 microg/ml; and those of ciprofloxacin ranged from 4 to 8 microg/ml. Linezolid was effective in controlling intracellular parasites in cultures of Vero cells infected by C. burnetii. No bactericidal activity by linezolid was obtained against C. burnetii at 8 microg/ml.

Diagnosis of quinolone-resistant Coxiella burnetii strains by PCR-RFLP.

A total of 12 strains of Coxiella burnetii (8 Greek isolates from acute Q-fever patients, two reference strains-Nine Mile and Q212-and two pefloxacin-resistant laboratory strains) were examined for the presence of point mutations in the quinolone resistance determining region (QRDR) of gyrA gene by direct DNA sequencing of the polymerase chain reaction (PCR)-amplified fragments. The gene sequences of all eight Greek isolates and the two reference strains Nine Mile and Q212 [minimal inhibitory concentration (MIC)A) at the corresponding codon 87 of E. coli. This mutation lead to the substitution of Glu (codon GAG) by Lys (codon AAG ). Restriction maps of amplified gyrA gene sequences were determined by GCG Wisconsin PACKAGE, and the MnlI restriction enzyme was found to cut only the sensitive strains sequences and not the resistant ones. The present PCR-RFLP analysis has proved to be a simple, rapid, and useful method for the detection of Coxiella burnetii and, at the same time, for the diagnosis of quinolone-resistant Coxiella burnetii strains.

In vitro susceptibility of Coxiella burnetii to trovafloxacin in comparison with susceptibilities to pefloxacin, ciprofloxacin, ofloxacin, doxycycline, and clarithromycin.

The antibiotic susceptibilities of eight Greek isolates of Coxiella burnetii to trovafloxacin were determined by the shell vial assay. MICs of trovafloxacin and ofloxacin ranged from 1 to 2 microg/ml, those of pefloxacin ranged from 1 to 4 microg/ml, those of ciprofloxacin ranged from 4 to 8 microg/ml, those of doxycycline ranged from 1 to 2 microg/ml, and those of clarithromycin ranged from 2 to 4 microg/ml. Trovafloxacin exhibited no activity against C. burnetii at 4 microg/ml.