Comparison of a multiplex PCR with DNA barcoding for identification of container breeding mosquito species.

BACKGROUND: Identification of mosquitoes greatly relies on morphological specification. Since some species cannot be distinguished reliably by morphological methods, it is important to incorporate molecular techniques into the diagnostic pipeline. DNA barcoding using Sanger sequencing is currently widely used for identification of mosquito species. However, this method does not allow detection of multiple species in one sample, which would be important when analysing mosquito eggs. Detection of container breeding Aedes is typically performed by collecting eggs using ovitraps. These traps consist of a black container filled with water and a wooden spatula inserted for oviposition support. Aedes mosquitoes of different species might lay single or multiple eggs on the spatula. In contrast to Sanger sequencing of specific polymerase chain reaction (PCR) products, multiplex PCR protocols targeting specific species of interest can be of advantage for detection of multiple species in the same sample. METHODS: For this purpose, we adapted a previously published PCR protocol for simultaneous detection of four different Aedes species that are relevant for Austrian monitoring programmes, as they can be found in ovitraps: Aedes albopictus, Aedes japonicus, Aedes koreicus, and Aedes geniculatus. For evaluation of the multiplex PCR protocol, we analysed 2271 ovitrap mosquito samples from the years 2021 and 2022, which were collected within the scope of an Austrian nationwide monitoring programme. We compared the results of the multiplex PCR to the results of DNA barcoding. RESULTS: Of 2271 samples, the multiplex PCR could identify 1990 samples, while species determination using DNA barcoding of the mitochondrial cytochrome c oxidase subunit I gene was possible in 1722 samples. The multiplex PCR showed a mixture of different species in 47 samples, which could not be detected with DNA barcoding. CONCLUSIONS: In conclusion, identification of Aedes species in ovitrap samples was more successful when using the multiplex PCR protocol as opposed to the DNA barcoding protocol. Additionally, the multiplex PCR allowed us to detect multiple species in the same sample, while those species might have been missed when using DNA barcoding with Sanger sequencing alone. Therefore, we propose that the multiplex PCR protocol is highly suitable and of great advantage when analysing mosquito eggs from ovitraps.

Demodicosis in a Kunekune pig and molecular characterisation of porcine demodectic mites involved: a case report.

BACKGROUND: In January 2021, a female 1-year-old Kunekune was presented at the University Clinic for Swine with severe reduction of the field of vision resulting in prolonged reaction time when targeting barriers, due to moderate to severe thickening of the skin around both orbits also affecting the eyelids. METHODS: Clinical examination revealed skin hyperplasia, nodular enlargement of the skin pores of the axillar and inguinal region. Ophthalmologists decided to remove parts of the thickened periocular skin, followed by histopathological examination. RESULTS: Once large amounts of demodectic mites were detected by histopathology, demodicosis could be diagnosed and treatment of the pig was started using sarolaner. Morphological and molecular analyses were performed. Histopathological and parasitological exams led to the aetiological diagnosis of demodicosis in the affected Kunekune pig. Severe skin lesions were revealed to be the consequence of an infestation with Demodex sp. Morphological analyses confirmed the involvement of D. phylloides. Molecular characterization indicated a Demodex species closely related to mites documented in wild boar - most probably D. phylloides for which no explicit sequences are available in GenBank yet. Treatment with sarolaner (2.6 mg/kg) resulted in a substantial regression of skin lesions, already detectable 1 month after first treatment. CONCLUSIONS: Demodicosis is a very rare disease in pigs that is most probably related to an impaired immune response to the mites. Demodectic mange should be included in the list of differential diagnoses in cases of periocular alterations of the skin of pigs.

A citizen science report-Tiger mosquitoes (Aedes albopictus) in allotment gardens in Graz, Styria, Austria.

Aedes albopictus, the Asian tiger mosquito, is an invasive species not native to Europe. Due to its ability to transmit pathogens, such as dengue, chikungunya and Zika viruses, Ae. albopictus is considered a major health threat. In Austria, it was first reported in 2012 in the Western province of Tyrol and was documented in the metropolitan area of Vienna in 2020, demonstrating its ability to colonize urban areas. In July 2021, a garden owner from Graz, Styria, Austria, contacted experts because of the possible presence of tiger mosquitoes in an allotment garden complex. Accordingly, citizen scientists collected adult mosquitoes and set up ovitraps. Adults and eggs were sent to the laboratory for morphological examination and molecular DNA barcoding within the mitochondrial cytochrome c oxidase subunit I gene. In total, 217 eggs of Ae. albopictus were found at the allotment garden as well as at a second location in the city of Graz. In addition, 14 adult Ae. albopictus specimens, of which 7 were molecularly identified as an identical haplotype, were collected at the allotment garden. With its mild climate and numerous parks and gardens, Graz provides the perfect environment for reproduction of tropical/subtropical alien Aedes mosquitoes. The presence of eggs and adult specimens in the current study period indicates that Ae. albopictus is already breeding in Graz. However, monitoring efforts need to be continued to determine whether stable populations of Ae. albopictus can survive there.