Circulation and diagnostics of Puumala virus in Norway: nephropatia epidemica incidence and rodent population dynamics.

Hantaviruses pose a public health concern worldwide causing haemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Puumala virus (PUUV) is the most prevalent hantavirus in Central and Northern Europe, and causes a mild form of HFRS, also known as nephropathia epidemica (NE). In nature, the main host of PUUV is the bank vole (Myodes glareolus), and transmission to humans occurs through inhalation of aerosols from rodent excreta. Nephropathia epidemica is particularly prevalent in Nordic countries, however, few studies of PUUV have been performed in Norway. The aim of this study was to analyse the dynamics of PUUV in Norway and compare with bank vole population dynamics, and also to complement the current diagnostic methodology of NE in Norway. Our results showed a significant seasonal and geographical variation of NE, and a general parallel peak trend between bank vole population densities and human NE incidence. A real-time and a nested PCR were successfully established as an invaluable diagnostic tool, with detection and sequencing of PUUV in a human serum sample for the first time in Norway. Phylogenetic analysis showed clustering of the obtained human sample with previous Norwegian bank vole isolates.

PCR screening of tick-borne agents in sensitive conservation areas, Southeast Portugal.

The Southeast region of Portugal, particularly the Guadiana valley, is currently the reintroduction territory of Lynx pardinus (Iberian lynx), one of the most endangered felids in the world that is only found in the Iberian Peninsula. Over the last century, populations have declined, placing L. pardinus at extremely high risk of extinction in the wild and relying on reintroduction projects. Among the aspects taken into account in the establishment of new populations is the sanitary status of the selected habitats, especially concerning infectious diseases, including tick-borne pathogens (TBPs). This study presents the results of TBPs survey on ticks collected at sensitive conservation areas of Southeast Portugal. From 2012 to 2014, 231 ticks obtained from vegetation, sympatric domestic and wild animals were submitted for analysis. The presence of Babesia spp., Cytauxzoon spp., Theileria spp., Hepatozoon spp., Anaplasma spp., Ehrlichia spp., Candidatus Neoehrlichia mikurensis, among other Anaplasmataceae, and Coxiella burnetii were investigated by PCR. Six tick species were recorded, Dermacentor marginatus (n = 13/5.6%), Hyalomma lusitanicum (n = 175/75.8%), Ixodes ricinus (n = 4/1.7%), Rhipicephalus bursa (n = 7/3.0%), R. pusillus (n = 21/9.1%) and R. sanguineus sensu lato (n = 11/4.8%). The molecular screening confirmed the presence of two tick-borne pathogens, C. burnetii (N = 34) and Anaplasma platys (N = 1), and one tick-endosymbiont, Candidatus Midichloria mitochondrii (N = 45). The results obtained provide new information on the circulation of ticks and TBPs with potential veterinary importance in Iberian lynx habitat.

In vitro and in vivo comparison of transport media for detecting nasopharyngeal carriage of Streptococcus pneumoniae.

BACKGROUND: As a standard method for pneumococcal carriage studies, the World Health Organization recommends nasopharyngeal swabs be transported and stored at cool temperatures in a medium containing skim-milk, tryptone, glucose and glycerol (STGG). An enrichment broth used for transport at room temperature in three carriage studies performed in Norway may have a higher sensitivity than STGG. We therefore compared the media in vitro and in vivo. METHODS: For the in vitro component, three strains (serotype 4, 19F and 3) were suspended in STGG and enrichment broth. Recovery was compared using latex agglutination, quantification of bacterial loads by real-time PCR of the lytA gene, and counting colonies from incubated plates. For the in vivo comparison, paired swabs were obtained from 100 children and transported in STGG at cool temperatures or in enrichment broth at room temperature. Carriage was identified by latex agglutination and confirmed by Quellung reaction. RESULTS: In vitro, the cycle threshold values obtained by PCR did not differ between the two media (p = 0.853) and no clear difference in colony counts was apparent after incubation (p = 0.593). In vivo, pneumococci were recovered in 46% of swabs transported in STGG and 51% of those transported in enrichment broth (Kappa statistic 0.90, p = 0.063). DISCUSSION: Overall, no statistical differences in sensitivity were found between STGG and enrichment broth. Nevertheless, some serotype differences were observed and STGG appeared slightly less sensitive than enrichment broth for detection of nasopharyngeal carriage of pneumococci by culturing. We recommend the continued use of STGG for transport and storage of nasopharyngeal swabs in pneumococcal carriage studies for the benefit of comparability between studies and settings, including more resource-limited settings.

Rickettsia massiliae and Rickettsia conorii Israeli Spotted Fever Strain Differentially Regulate Endothelial Cell Responses.

Rickettsiae primarily target microvascular endothelial cells. However, it remains elusive how endothelial cell responses to rickettsiae play a role in the pathogenesis of rickettsial diseases. In the present study, we employed two rickettsial species with high sequence homology but differing virulence to investigate the pathological endothelial cell responses. Rickettsia massiliae is a newly documented human pathogen that causes a mild spotted fever rickettsiosis. The "Israeli spotted fever" strain of R. conorii (ISF) causes severe disease with a mortality rate up to 30% in hospitalized patients. At 48 hours post infection (HPI), R. conorii (ISF) induced a significant elevation of IL-8 and IL-6 while R. massiliae induced a statistically significant elevated amount of MCP-1 at both transcriptional and protein synthesis levels. Strikingly, R. conorii (ISF), but not R. massiliae, caused a significant level of cell death or injury in HMEC-1 cells at 72 HPI, demonstrated by live-dead cell staining, annexin V staining and lactate dehydrogenase release. Monolayers of endothelial cells infected with R. conorii (ISF) showed a statistically significant decrease in electrical resistance across the monolayer compared to both R. massiliae-infected and uninfected cells at 72 HPI, suggesting increased endothelial permeability. Interestingly, pharmacological inhibitors of caspase-1 significantly reduced the release of lactate dehydrogenase by R. conorii (ISF)-infected HMEC-1 cells, which suggests the role of caspase-1 in mediating the death of endothelial cells. Taken together, our data illustrated that a distinct proinflammatory cytokine profile and endothelial dysfunction, as evidenced by endothelial cell death/injury and increased permeability, are associated with the severity of rickettsial diseases.

Quantitative study of Rickettsia massiliae in Rhipicephalus sanguineus organs.

Rickettsia massiliae, belonging to the spotted fever group of Rickettsia, is a human pathogen causing a similar course of disease to that caused by R. conorii, the originally recognized etiologic agent of Mediterranean spotted fever. In view of this similarity, we performed an ultrastructural study of R. massiliae in organs of Rhipicephalus sanguineus ticks, in order to advance knowledge of the complex dynamics at the tick-pathogen interface in rickettsioses. Adult R. massiliae-infected Rh. sanguineus ticks were fed on uninfected Hartley strain guinea pigs, and five females were collected daily throughout their feeding period up to day 6, and analyzed by quantitative real-time PCR and electron microscopy. An increase in rickettsial content was observed in the salivary glands, particularly in the first two days of feeding, and a plateau was observed between days 3 and 6. Rickettsial organisms were observed in all tick organs analyzed, in higher numbers in the fed state, and statistically significant differences were observed in measurements of the periplasmic layer of R. massiliae in salivary glands of fed and unfed Rh. sanguineus ticks, with increased thickness in the former case. This study provides insight into the interface between R. massiliae and Rh. sanguineus ticks, highlighting the need for analysis of R. massiliae to fully ascertain its place as an important pathogenic agent of a spotted fever rickettsiosis.

Rickettsia lusitaniae sp. nov. isolated from the soft tick Ornithodoros erraticus (Acarina: Argasidae).

In this study a novel Rickettsia from the spotted fever group, isolated from Ornithodoros erraticus soft ticks collected from pigpens in the south of Portugal, is described. After initial screening revealed Rickettsia-positive ticks, isolation attempts were then performed. Successful isolates were achieved by shell-vial technique using Vero E6 cells at 28°C. Molecular characterization of the isolate was performed based on analysis of five rickettsial genes gltA, ompA, ompB, sca1 and htr with their subsequent concatenation along with other rickettsial species resulting in a clustering of the new isolate with Rickettsia felis and Rickettsia hoogstraalii. The degree of nucleotide sequence similarity with other rickettsiae fulfills the criteria for classification of our isolate as a novel species. The name Rickettsia lusitaniae sp. nov. (=CEVDI PoTiRo) is proposed for this new species found in O. erraticus.

Coinfections of Rickettsia slovaca and Rickettsia helvetica with Borrelia lusitaniae in ticks collected in a Safari Park, Portugal.

Borrelia and Rickettsia bacteria are the most important tick-borne agents causing disease in Portugal. Identification and characterization of these circulating agents, mainly in recreational areas, is crucial for the development of preventive measures in response to the gradually increasing exposure of humans to tick vectors. A total of 677 questing ticks including Dermacentor marginatus, Rhipicephalus sanguineus, Ixodes ricinus, Hyalomma lusitanicum, H. marginatum, and Haemaphysalis punctata were collected in a Safari Park in Alentejo, Portugal, to investigate the prevalences of infection and characterize Borrelia and Rickettsia species. From a total of 371 ticks tested by PCR for Borrelia burgdorferi sensu lato (s.l.), of which 247 were tested for Rickettsia, an infection prevalence of 18.3% was found for B. lusitaniae and 55.1% for Rickettsia spp. Sequence analysis of positive amplicons identified the presence of B. lusitaniae (18.3%), R. monacensis strain IRS3 (51.7%), and R. helvetica (48.3%) in I. ricinus. R. slovaca (41.5%), R. raoultii (58.5%), and also B. lusitaniae (21%) were identified in D. marginatus ticks. One (5.9%) H. lusitanicum was infected with B. lusitaniae, and R. massiliae was found in one Rhipicephalus sanguineus. Coinfection was found in 7 (20%) I. ricinus and 34 (23.3%) D. marginatus ticks. We report, for the first time, simultaneous infection with R. helvetica and B. lusitaniae and also R. slovaca, the agent of TIBOLA/DEBONEL, with B. lusitaniae. Additionally, 6 isolates of B. lusitaniae were established, and isolates of Rickettsia were also obtained for the detected species using tick macerates cultured in mammalian and mosquito cell lines. This report describes the detection and isolation of tick-borne agents from a Portuguese Safari Park, highlighting the increased likelihood of infection with multiple agents to potential visitors or staff.

Detection of Borrelia lusitaniae, Rickettsia sp. IRS3, Rickettsia monacensis, and Anaplasma phagocytophilum in Ixodes ricinus collected in Madeira Island, Portugal.

A total of 300 Ixodes ricinus ticks were tested by polymerase chain reaction (PCR) for the presence of Borrelia spp., Rickettsia spp., and Anaplasma phagocytophilum. Sequence analysis demonstrated 8 (2.7%) ticks infected with B. lusitaniae, 60 (20%) with Rickettsia spp., and 1 (0.3%) with A. phagocytophilum. Seven (2.3%) ticks were coinfected with B. lusitaniae and Rickettsia spp., 2 (0.6%) with R. monacensis, and 5 (1.7%) with Rickettsia sp. IRS3. The results of this study suggest simultaneous transmission of multiple tick-borne agents on Madeira Island, Portugal.