Dimosthenis Chochlakis

Carriage of Rickettsia spp., Coxiella burnetii and Anaplasma spp. by endemic and migratory wild birds and their ectoparasites in Cyprus

Three hundred and sixty-eight (368) species of birds (local, endemic and migratory) have been recorded in Cyprus. According to a rough estimate, nearly a quarter of a billion migratory birds fly through the island during the two periods of migration, thus supporting the role of the island as a stopover of great importance. Despite the fact that the role of migratory birds in the transmission of pathogens from the endemic source to Europe has been recognized, the connection between birds, dispersal of ticks infected with medically important pathogens and outbreaks of certain zoonoses remains a hypothesis [1].

Analysis of pathogen co-occurrence in host-seeking adult hard ticks from Serbia

Past studies in Serbia have reported concurrent infections of Ixodes ricinus ticks with Borrelia burgdorferi sensu lato genospecies, Anaplasma phagocytophilum and Francisella tularensis. As a step forward, this investigation included a broader range of microorganisms and five most common and abundant tick species in Serbia. Five tick species were identified (Dermacentor marginatus, D. reticulatus, Haemaphysalis punctata, H. concinna and I. ricinus) and analyzed for the presence of seven pathogens. Anaplasma ovis, A. phagocytophilum, Babesia canis, B. burgdorferi s.l., Coxiella burnetii, Rickettsia helvetica and R. monacensis were detected. Sequencing of samples positive for F. tularensis revealed the presence of Francisella-like endosymbionts. No Bartonella spp. DNA was amplified. Concurrent infections were present in three tick species (D. reticulatus, H. concinna and I. ricinus). The rate of co-infections was highest in I. ricinus (20/27), while this tick species harbored the broadest range of co-infection combinations, with dual, triple and a quadruple infection(s) being detected.

Rats as indicators of the presence and dispersal of six zoonotic microbial agents in Cyprus, an island ecosystem: a seroepidemiological study.

A total of 622 rats (402 Rattus norvegicus and 220 R. rattus frugivorus) were collected in 51 different areas in Cyprus during 2000-2003 and used as indicators of the presence and dispersal of six zoonotic microbial agents. IgG antibodies against Rickettsia typhi (241/496, 48.6%), R. conorii (209/500, 41.8%), Toxoplasma sp. (138/494, 27.9%), Coxiella burnetti (63/494, 12.8%), Bartonella henselae (52/494, 10.5%) and Leishmania infantum (36/494, 7.3%) were detected by indirect immunofluorescence test. There was variation in the association between the seropositivity of the six microbial agents and other factors. Rat species affected R. typhi and R. conorii seropositivity, the prefecture where the rats were caught affected R. typhi, C. burnetii, B. henselae, T. gondii and L. infantum, the sampling season impacted on R. typhi, R. conorii, T. gondii and L. infantum, and the flea species affected R. typhi, R. conorii and B. henselae. These results were analysed using geographical information system (GIS) technology and the seropositivity in rats against the pathogens tested appeared to follow the occurrence of these pathogens in humans. This suggests that rats could be used as disease sentinels and, together with GIS technology, they could be a useful tool for the identification of endemic foci and high-risk areas for each pathogen.

TICK-BORNE BACTERIA IN MOUFLONS AND THEIR ECTOPARASITES IN CYPRUS

The Cypriot mouflon (Ovis orientalis ophion), a once almost extirpated species of wild sheep, is under strict surveillance because it can be threatened by likely transmission of pathogenic bacteria, such as Anaplasma spp., Rickettsia spp., and Coxiella burnetii, primarily from domestic ungulates. We collected 77 blood samples from Cypriot mouflons and 663 of their ectoparasites (Rhipicephalus turanicus, Rhipicephalus sanguineus, Rhipicephalus bursa, Hyalomma anatolicum excavatum, Hyalomma marginatum, Haemaphysalis punctata, Haemaphysalis sulcata, and Ixodes gibossus) and tested them by polymerase chain reaction and sequencing. Twenty-three mouflon blood samples (30%) were positive for C. burnetii, 23 (30%) for Rickettsia spp., and 8 (10%) for Anaplasma ovis. Of 109 pools of ectoparasites, 32.1% were positive for C. burnetii, 28.4% for Rickettsia spp., and 10.9% for A. ovis; 11.9% were positive for both C. burnetii and Rickettsia spp., 6.4% for both Rickettsia spp. and A. ovis, and 2.8% for all three pathogens. This is the first survey that records the presence of tick-borne pathogens, both in the Cypriot mouflon and in ticks parasitizing it.

Prevalence of Anaplasma sp. in goats and sheep in Cyprus.

A seroprevalence study of Anaplasma infection was conducted in a stratified random sample of goats and sheep in Cyprus in which the sample locations were recorded using a geographical information system (GIS). The aim of the study was to estimate the prevalence of Anaplasma phagocytophilum and other Anaplasma species in sheep and goats, and to identify high-risk regions. A total of 689 serum samples (343 from sheep and 346 from goats) were randomly collected and tested for the detection of antibodies against A. phagocytophilum antigen using an indirect immunofluorescent assay. The polymerase chain reaction followed by sequencing analysis was used for the detection and molecular characterization of Anaplasma sp DNA in the blood samples. The prevalence of IgG antibodies against A. phagocytophilum antigen was 18% for goats, and 31% for sheep. Six new genotypes were detected in goats and sheep; by sequence analysis one was identified as A. phagocytophilum, one as Anaplasma platys and the remaining four as Anaplasma species. The results provide evidence for the presence of A. phagocytophilum and Anaplasma species in sheep and goats in Cyprus.

Rickettsia typhi and Rickettsia felis in Xenopsylla cheopis and Leptopsylla segnis Parasitizing Rats in Cyprus

Fleas collected from rats during a three-year period (2000-2003) in 51 areas of all provinces of Cyprus were tested by molecular analysis to characterize the prevalence and identity of fleaborne rickettsiae. Rickettsia typhi, the causative agent of murine typhus, was detected in Xenopsylla cheopis (4%) and in Leptopsylla segnis (6.6%). Rickettsia felis was detected in X. cheopis (1%). This is the first report of R. typhi in X. cheopis and L. segnis from rats, in Cyprus, and the first report of R. felis in X. cheopis in Europe. The role of fleas (mainly X. cheopis) was confirmed in the epidemiologic cycle of murine typhus in Cyprus by interrelation of current results with those of previous studies. The geographic distribution of fleas coincided with the geographic distribution of the pathogen they can harbor, which emphasizes the potential risk of flea-transmitted infections in Cyprus.

First Detection of Spotted Fever Group Rickettsiae in Ticks in Serbia

Ticks can transmit multiple pathogenic bacteria responsible for diseases in animals and humans such as Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, and spotted fever group Rickettsia sp. The current study aimed to investigate the presence of Rickettsiae in ticks collected from seven localities in Serbia. One hundred thirty-one (131) questing ticks belonging to 5 tick species (Dermacentor marginatus, Dermacentor reticulatus, Haemaphysalis punctata, Haemaphysalis Concinna, and Ixodes ricinus) were collected in 2007 and 2009. Ticks were tested by polymerase chain reaction, amplifying gltA, ompA, and 17-kd genes, and sequencing analysis, revealing the presence of Rickettsia helvetica and Rickettsia monacensis in I. ricinus ticks only (infection rates 7.7% and 15.4% for R. helvetica and R. monacensis, respectively). R. helvetica has been isolated from I. ricinus ticks and has been implicated in fatal perimyocarditis. R. monacensis was first identified in I. ricinus samples collected in Germany and has recently been implicated in human infection. The results of the current study make fundamental the need to evaluate the incidence of infection with R. helvetica and R. monacensis among the resident population.

In vitro-selected resistance to fluoroquinolones in two Brucella strains associated with mutational changes in gyrA

In the present study, Brucella melitensis biovar Abortus 2308 and Brucella abortus 3196 biotype 5 reference strains, which are susceptible to fluoroquinolones, became in vitro-resistant to fluoroquinolones by culture in trypticase soy agar. The quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes of the two reference strains were analysed by polymerase chain reaction sequencing analysis to obtain the wild-type sequence. These sequences were then compared with the corresponding sequences of four in vitro-selected fluoroquinolone-resistant mutants to characterise mutations associated with resistance. Sequencing of the ofloxacin-selected resistant mutant 2308 revealed a transition of GAT to AAT (corresponding to position 87 of Escherichia coli gyrA), leading to substitution of Asp91-->Asn, whilst at the same position the ciprofloxacin-selected resistant mutant 2308 revealed a transition of GAT to TAT (corresponding to the same position of E. coli as above), leading to substitution of Asp91-->Tyr. The ofloxacin-selected resistant mutant 3196 had a transition of GCT to GTT, generating an amino acid change of Ala87-->Val. Amino acid changes were detected in the portion of the Brucella gyrA gene (Ala71 to Gln110) corresponding to the E. coli gyrA QRDR region (Ala67 to Gln110). Amino acid changes were also detected in Ser83, corresponding to the region where fluoroquinolone-associated amino acid changes are most commonly found in other bacterial species.

Use of MALDI-TOF mass spectrometry in the battle against bacterial infectious diseases: recent achievements and future perspectives.

ABSTRACT Introduction: Advancements in microbial identification occur increasingly faster as more laboratories explore, refine and extend the use of mass spectrometry in the field of microbiology. Areas covered: This review covers the latest knowledge found in the literature for quick identification of various classes of bacterial pathogens known to cause human infection by the use of MALDI-TOF MS technology. Except for identification of bacterial strains, more researchers try to ‘battle time’ in favor of the patient. These novel approaches to identify bacteria directly from clinical samples and even determine antibiotic resistance are extensively revised and discussed. Expert commentary: Mass spectrometry is the future of bacterial identification and creates a new era in modern microbiology. Its incorporation in routine practice seems to be not too far, providing a valuable alternative, especially in terms of time, to conventional techniques. If the technology further advances, quick bacterial identification and probable identification of common antibiotic resistance might guide patient decision-making regarding bacterial infectious diseases in the near future.

Coxiella burnetii in wildlife and ticks in an endemic area

BACKGROUND Ticks are considered to be a natural reservoir of Coxiella burnetii and are responsible for the spread of infection in wild animals and for the transmission to domestic animals. More than 40 tick species are naturally infected with C. burnetii. In Cyprus, few studies have been carried out on the distribution and incidence of C. burnetii infection in wildlife and the threat that infected wild animals pose to humans and domestic animals remains uncertain. METHODS During 3 studies in Cyprus, lasting 7 years (2000-2006), ticks were collected from rats (98 Rattus norvegicus and 38 R. rattus), 74 mouflons (Ovis orientalis ophion), 32 foxes (Vulpes vulpes indutus), 247 hares (Lepus europaeus), 557 birds (endemic and migratory) and 10 different tick species. All samples were tested for the presence of Coxiella burnetii using molecular assays. RESULTS In total, 31% (23/74) of mouflons, 28% (9/32) of foxes, 48% (15/31) of hares and 31% (41/131) of birds were positive for C. burnetii. We tested 1315 ticks (195 pools) and C. burnetii was detected in 28.9% (56/195) of them. Forty percent (24/60) of ticks collected from hares and 25.2% (27/107) of tick pools collected from mouflons were positive for C. burnetii. However, C. burnetii was detected in only one tick from foxes and one from birds. Positive samples were prevalent all over the island and did not show a specific geographic distribution pattern. CONCLUSIONS Several animal and tick species collected from wildlife are potential sources of C. burnetii in Cyprus. These species are abundant in the area and may represent a risk for domestic livestock that share grazing environments.