Insight into the underlying immune interaction of Rickettsia infection in the vector-pathogen-host interface

Ixodid ticks are second only to mosquitos in their notorious role as vectors of pathogens to both animals and humans. Rickettsioses are among the most important tick-borne diseases in Europe, Mediterranean spotted fever in particular. To date, many studies have been performed in order to uncover the underlying mechanisms of this disease, in terms of the interactions among its constituents, i.e., the pathogen, Rickettsia conorii, its vector, Rhipicephalus sanguineus tick, and a vertebrate host. However, important gaps remain in this knowledge, among them studies of the relationship of its vector and pathogen, and also the role of tick saliva in vector-host interactions. Thus, in order to approach these limitations, in the first part of this dissertation three studies were performed in order to analyze the vector-pathogen-host interface in natural settings (chapter 2). As a result of the first of these studies, co-infections of Borrelia lusitaniae with R. helvetica and R. slovaca were found in ticks collected from a natural safari park in the south of Portugal; new host-pathogen associations were found in the second study described, performed in Madeira Island, namely lizards infected with R. monacensis, as well as detection of R. helvetica in ticks, which was a first occurrence in this island; and in the last study a new species of Rickettsia was isolated from soft ticks collected from pig pens from Alentejo, Portugal. In chapter 3 experimental studies on the vector were performed through analysis of R. massiliae in Rh. sanguineus tick organs. We performed a quantitative analysis of R. massiliae in the salivary glands of feeding Rh. sanguineus ticks, and observed a statistically significant increase in bacterial load during the first two days, followed by a plateau up to day 6 of feeding. An ultrastructural study was also performed on the salivary glands, ovaries and midgut of R. massiliae infected-Rh. sanguineus, where we observed the reactivation phenomenon of Rickettsia in the salivary glands of fed ticks as a result of tick feeding. In the final part of this dissertation the role of tick saliva was ascertained in terms of bacterial burden and immune responses in a murine susceptible host, using uninfected Rh. sanguineus ticks and C3H/HeJ mice. No statistically significant differences in bacterial load were observed between the two groups of R. conorii-infected animals, one of which infested with ticks. However, host cytokine analysis of both groups of animals revealed statistically significant differences, suggesting an inhibitory effect of tick saliva on host pro-inflammatory responses (chapter 4); RESUMO: Estudo das interacções imunes do hospedeiro e vector resultantes da infecção por Rickettsia spp. Os ixodídeos desempenham um papel fundamental como vectores de agentes patogénicos tanto em animais como em humanos. As rickettsioses, com destaque para a febre escaronodular, encontram-se entre as doenças transmitidas por carraças mais importantes na Europa. Até à data muitos estudos foram efectuados de modo a descortinar os mecanismos subjacentes a esta doença, em termos das interacções entre os seus constituintes, i.e., agente patogénico, Rickettsia conorii , o seu vector, o ixodídeo Rhipicephalus sanguineus, e um hospedeiro vertebrado. Todavia, existem ainda lacunas neste conhecimento a nível da relação vector-agente patogénico, para além do papel da saliva do vector nas interacções agente patogénico-hospedeiro. Assim, numa tentativa de colmatar estas limitações, na primeira parte desta dissertação foram realizados três estudos para análise dos fenómenos que ocorrem naturalmente na interface vector-agente patogénico-hospedeiro ocorrentes na natureza (capítulo 2). Como resultado do primeiro destes estudos, foram encontradas co-infecções de Borrelia lusitaniae com R. helvetica e R. slovaca em ixodídeos capturados num parque safari no sul de Portugal. No segundo estudo descrito, efectuado na ilha da Madeira, foram encontradas novas associações hospedeiro-agente patogénico, nomeadamente lagartixas infectadas com R. monacensis, detectou-se R. helvetica em carraças, pela primeira vez nesta ilha, e foi possível isolar uma nova espécie de Rickettsia a partir de carraças de corpo mole capturados em pocilgas no Alentejo, Portugal. O capítulo 3 aborda os resultados de estudos experimentais efetuados no vector, através da análise de R. massiliae em orgãos de Rh. sanguineus. Realizámos uma análise quantitativa da R. massiliae em glândulas salivares de Rh. sanguineus durante a sua refeição sanguínea, tendo-se observado um aumento estatisticamente significativo da carga bacteriana nos dois primeiros dias de alimentação, seguido de um patamar até ao dia 6 da alimentação sanguínea. Foi também efectuado um estudo ultraestrutural nas glândulas salivares, ovários e intestino médio de Rh. sanguineus infectados com R. massiliae, onde observámos o fenómeno de reactivação da Rickettsia nas glândulas salivares dos ixodídeos alimentados, resultante do processo de alimentação. Na parte final desta dissertação analisámos o papel da saliva de ixodídeos em termos da carga bacteriana e respostas imunes num hospedeiro murino susceptível, usando Rh. sanguineus não infectados e ratinhos C3H/HeJ. Não verificámos diferenças estatisticamente significativas entre as cargas bacterianas de dois grupos de animais infectados com R. conorii, em que apenas um dos grupos estava infestado com carraças. Todavia, a análise de citoquinas no hospedeiro em ambos os grupos experimentais revelou diferenças estatisticamente significativas, sugerindo um efeito inibitório da saliva dos ixodídeos nas respostas pro-inflamatórias do hospedeiro (capítulo 4)

Management strategies of enterovirus D68 outbreaks: current perspectives.

Following its discovery in California in 1962, enterovirus D68 (EV-D68) was reported only sporadically around the world. In August 2014, a marked increase of EV-D68 cases in young children with severe respiratory infections was reported in the USA and Canada and later in Europe and Asia. Some of these cases were also found to be associated with acute flaccid paralysis, which exacerbated public health concern, and has since triggered international efforts to strengthen both EV-D68 and acute flaccid paralysis surveillance systems. This review summarizes the current knowledge on EV-D68, offering an overview of EV-D68 epidemiology, clinical presentations, diagnostic methodologies, and treatment strategies, as well as surveillance and outbreak management

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, pneurnococci 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