Spotted fever group (SFG) rickettsioses are increasingly recognized worldwide as threats to public health. Rickettsia rickettsii, Rickettsia parkeri, and Rickettsia rickettsii subspecies californica cause spotted fever rickettsioses, including Rocky Mountain spotted fever. These disease agents are transmitted to humans by various tick vectors in the United States. There is growing concern that other tick species, such as Haemaphysalis leporispalustris, may also transmit new and potentially unrecognized SFG rickettsial pathogens. In this study, we found that 6.1 % of 296 questing H. leporispalustris ticks (21 larvae, 260 nymphs, 9 males, and 6 females) collected from 38 towns across nine counties in Maine, USA, were positive for Rickettsia spp. Further multilocus sequence typing and phylogenetic analysis revealed that this is a new Rickettsia genotype (Rickettsia sp. ME2023) belonging to the SFG group and close to Candidatus Rickettsia lanei. Tick vectors and rickettsial species associated with SFG rickettsioses in New England warrant further investigation. Additionally, the role of H. leporispalustris in pathogen enzootic cycles and transmission requires further study.

Humulus lupulus L., the common hop plant, is a commercially cultivated crop used as a beer flavoring agent that has been grown in Massachusetts since the 1600s (Machado et al., 2019 ; Rumney, 1998). In 2018, Diaporthe leaf spot, caused by the fungal pathogen Diaporthe humulicola, was found at hopyard research plots in Connecticut, followed by reports in multiple New York counties in 2023 (Allan-Perkins et al., 2020; Sharma et al., 2023). Additionally, Diaporthe halo blight, caused by the same fungal pathogen, was reported in Prince Edward Island (PE), Canada (Foster et al., 2024). In August of 2021, severe stem blight symptoms with visible fungal fruiting bodies were observed on >50% of plants at a hopyard in Franklin County, MA. Brown spots were observed on stems. Severe infection presented as widespread dark browning on stems, leaves, and cones. Samples were collected from the hop cultivars Mt Rainier (N=5), Magnum (N=1), and Teamaker (N=6). Under brightfield microscopy (400x) conidia congruent with Diaporthe spp. were observed on stem samples. Diseased stems from all cultivars were peeled and cut into 2 to 4 mm2 pieces, then surface sterilized in 70% ethanol for 60s, and 1% NaOCl for 60s. Samples were rinsed in sterile water, dried for 3 min and placed on potato dextrose agar (PDA). Plates were incubated at 25℃ for 5 days. 11 isolates were obtained from stem tissues of 11 plants. Isolates were cultured on PDA. 10 isolates had morphological traits consistent with the pycnidium of holotype UAMH 12076 (Allans-Perkins et al, 2020). Isolate sequences were identical, using one reinfection tests were performed on detached stem segments obtained from field grown mature plants (≥ 1 year) of cvs. Cascade and Magnum.3 wounding methods and an untreated control were used for pathogenicity testing to simulate field conditions. Each treatment had 6 replicates per cultivar and all treatments had an agar plug of inoculum applied to them, then were incubated at room temperature. Wounding with a knife involved peeling off the outer tissue layer, insect damage was simulated by using a needle to puncture the stem 3 times, and friction damage from hop support ropes was mimicked by rubbing rope against the stem. Plugs were removed 5 days post-application. Pathogenicity was observed as fungal growth and necrosis of plant tissue on all non-control methods in all samples 5 days post plug removal, fulfilling Koch's postulates. The pathogenic organism was reisolated from all inoculated tissues, except the control and identified using morphological characteristics and ITS sequence analysis. DNA amplification of the ITS region was performed according to the methods of White et al. (1990). A maximum likelihood phylogenetic tree based on the region was constructed using the MEGA10 program (Saitou & Nei, 1987). Sequence data was collected from NCBI BlastN, then aligned and trimmed. The ITS sequences (NCBI Acc. Nos. PQ555192, PQ555193, PQ555194) from the 3 isolates (UMASS001, UMASS002, UMASS003) matched 100% (543/543bp) with those of the type specimen of D. humulicola, which causes Diaporthe leaf spot and halo blight, collected in CT (NCBI Acc. No. MN152929) and 2 ex-type sequences from CT and Canada respectively (NCBI Acc. Nos. MN152927, NR172855).This is the first report of D. humulicola associated with stem blight disease. Our findings indicate that the same pathogen that infects stems can also infect cones and leaves. Therefore, Diaporthe disease management strategies should target all plant parts.

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.