Jacqueline O. Borges

Hematophagous arthropods and associated pathogens are becoming more prevalent globally. With limited treatment options, prevention is the top priority to reduce disease. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) has emerged as a standard detection method for many laboratories, but it has several drawbacks. Degraded sample quality, failed RNA preparations, and the presence of reaction inhibitors can lead to false negative results, introducing the need for a positive control. Existing control methods rely on adding exogenous nucleotides during amplification reactions, which may not reflect the quality of the sample. Incorporating assays to detect endogenous mosquito mRNA transcripts provides assurance that sample quality is sufficient for the downstream amplification of pathogen RNA. In the present study, we applied the Rapid Amplification of cDNA Ends (RACE) technique to obtain a full-length calreticulin cDNA sequence from the Culex restuans mosquito, collected in Massachusetts, USA. We then used the newly discovered cDNA sequence to design primers and probes spanning the first exon–exon junction of the calreticulin gene, which allowed for specific and accurate RNA transcript detection in an RT-qPCR assay. This assay detected the presence of calreticulin transcripts in extracted pools of mosquito RNA. This approach may be further developed to include other relevant mosquito species and, with extensive validation, may have potential for use as an endogenous control in arbovirus surveillance.

Pathogen surveillance contributes to the prediction of outbreaks and implementation of strategies to minimize disease transmission. White-tailed deer (Odocoileus virginianus) host important disease vectors and often support the proliferation of associated pathogens. Such vectors include ticks: hematophagous acarids that facilitate pathogen transmission between animals through blood feeding. Piroplasmida, an order of blood-borne protozoa including Babesia and Theileria, exploits this feeding to infect definitive hosts. Accordingly, we investigated the prevalence of Theileria cervi infections in Massachusetts white-tailed deer. Conventional polymerase chain reaction (PCR) was used to detect parasite gene fragments encoding the small ribosomal subunit (18S rRNA). Confirmation of species identity was determined by Oxford nanopore sequencing and Sequencher analysis. This revealed a 32.6% prevalence of T. cervi across Massachusetts. Whilst surveillance of deer blood uncovers parasite abundance and distribution, inversely, this allows us to also monitor vector populations. As Amblyomma americanum is the only vector implicated in the transmission of T. cervi, the detection of T. cervi in regions without anticipated Amblyomma presence may indicate expansion of vector populations. These findings provide valuable insights into the prevalence and ecology of this piroplasm and its vector, with possible implications for veterinary and public health.