A study on the occurrence of West Nile virus in mosquitoes [Diptera: Culicidae] on the selected areas in Poland

West Nile virus (WNV), the etiologic agent of West Nile Fever (WNF), an emerging infectious disease that lately has been rapidly extending its range of occurrence from Africa to Middle East, and to Asia and Southern Europe. In Europe, cases of isolating WNV from mosquitoes representing four genera have been reported from Romania, Portugal, France, southern Russia and what is the most important, from Poland’s neighbouring countries as the Czech Republic, Slovakia and southern Ukraine. These data, as well as human and equine cases in the Czech Republic and Belarus, support hypothesis that WNV has already been present also in Poland, the more so, specific antibodies were detected in the sera of birds collected at the Kampinos and Bialowieza Primeval Forests and in human from the vicinity of Bialystok. Mosquitoes were collected in 2004–2009 at indoor and outdoor collection sites in districts: Kujawsko-Pomorskie, Mazowieckie, Podlaskie and Warmińsko-Mazurskie. In total 15400 female mosquitoes were collected in the cow sheds and overwintering in the cellars, and from human bait and CO2 traps. Mosquitoes were sorted by the collecting site, species and sex, and placed in pools of not in more than 50 specimens in special mixture of phenol and chaotropic salts and frozen in –20°C. Altogether, 15400 females were examined. Total RNA were extracted according to protocol of A&A Biotechnology. RT reaction was performed with random primers and 217-nucleotide fragment from the NS5 coding sequence was amplified by heminested PCR. PCR products were analysed on 1.5% agarose gel. The predominant species was Culex pipiens, accounting for over 42% of total insects collection. All obtained results were negative. Further investigations are needed

The occurrence of Cryptosporidium spp. in synanthropic flies in urban and rural environments

This study was carried out to determine the role of non-biting synanthropic flies as carriers of Cryptosporidium spp. oocysts in the vicinity of the city of Gdańsk (NE Poland). In 2001–2003, flies were collected from three breeding sites: cow sheds and meadows in the Bystra cattle farm and municipal landfill Szadółki using inhaustors (aspirators) and entomologic nets. A total of 2358 specimens of the families: Muscidae (n=1598), Calliphoridae (n=739) and Sarcophagidae (n=21) were collected and analysed in 249 pools consisted of 9.5 insects, in average. Microscopic examination was used to detect Cryptosporidium spp. oocysts in the fly faeces deposited on the glass microscope slides and stained by Zhiel-Nielsen method. The mean number of faecal droplets per one glass slide was 11.5. Ooocysts of Cryptosporidium spp., stained from light pink to bright red, were found in fly faeces deposited on 25 (27.5%) of 91 glass slides checked. The highest prevalence of the pathogen was observed in faecal droplets deposited by flies collected in municipal landfill (50% investigated slides). DNA of Cryptosporidium spp. was extracted from the surface eluants of flies and/or their gut homogenates and purified. Then extracts were examined by PCR using CPB-DIAGF and CPB-DIAGR primers amplifying a variable region SSU-rRNA of all Cryptosporidium species. Altogether 387 isolates, 228 from surfaces and 159 from gut homogenates, were obtained from 249 pools of flies and analyzed. A specific 435 bp fragment of DNA was obtained in 49 (12.7%) lysates tested. In 10.4% pools, DNA of the pathogen was detected only in the surface eluants while in 7.6% only in gut extracts. In the case of two pooled samples (0.8%) Cryptosporidium spp. was found in both types of lysates. In total, Cryptosporidium spp. was detected in 47/249 pools of flies (18.9%). Assumed that each positive pool contained just one infected fly, the percentage of specimens able to oocysts transmission were calculated at the minimal level 2.0% (n=47/2358). The result obtained comfirm that synanthropic flies can harbour oocysts of Cryptosporidium spp. both externally and internally, and disseminate them mechanically in the environment. Therefore, under unsanitary conditions could be involved in the transmission of human and animal cryptosporidiosis

Mechanical transmission of Cryptosporidium parvum oocysts by flies

Long term field studies and laboratory experiments demonstrated that synanthropic filth flies can mechanically transmit infectious oocysts of Cryptosporidium parvum, an anthropozoonotic protozoan parasite which significantly contributes to the mortality of immunocompromised or immunosuppressed people. C. parvum oocysts are acquired from unhygienic sources, and can pass trough fly gastrointestinal track without alteration of their infectivity and can be subsequently deposited on visited surfaces. Transmission of the oocysts by adult flies occurs via: (1) mechanical dislodgement from the exoskeleton; (2) fecal deposition; and (3) regurgitation, i.e., vomits. Filth flies can cause human or animal cryptosporidiosis via deposition of infectious oocysts on the visited foodstuf, and the biology and ecology of synanthropic filth flies indicate that their potential for mechanical transmission of C. parvum is high