Molecular detection of Coxiella burnetii in ticks infesting wild and domestic animals in the Eastern region of Punjab, Pakistan.

Tick-borne pathogens are significant for human, veterinary, and wildlife health. Coxiella burnetii is an example that is widely distributed across various hosts and can cross species boundaries. In Pakistan, there is a scarcity of data regarding C. burnetii at the intersection of wildlife and livestock. Ticks were collected from ruminants and wildlife from the districts of Kasur, Pakpattan, and Okara in Pakistan. Five tick species totaling 571 ticks were collected, with the following distribution: 56.4% Hyalomma anatolicum, 22.4% Rhipicephalus microplus, 10.5% Hyalomma marginatum, 7.9% Rhipicephalus sanguineus, and 2.8% Rhipicephalus turanicus. Fifty tick pools were screened for C. burnetii to amplify a segment of the IS1111 using real-time PCR assays. Ticks collected from sheep and goats had a greater rate of positivity for C. burnetii (40% and 38%, respectively) compared to Indian long-eared hedgehogs with a prevalence of 2%. Coxiella burnetii was prominent in Rhipicephalus microplus (92.3%) and Hyalomma anatolicum (88.9%), followed by Rhipicephalus turanicus (66.6%), Rhipicephalus sanguineus (33.3%), and Hyalomma marginatum (25.0%). Ticks from Pakpattan district displayed the highest prevalence of C. burnetii (88.9%), whereas the lowest was observed in ticks from Kasur district (77.3%). There was no significant association between tick gender and C. burnetii infection. Female host animals were more likely to harbor ticks containing C. burnetii, with a prevalence rate of 81.8%. The research underscores the urgent need for comprehensive studies on C. burnetii in Pakistan, especially at the interface of wildlife and livestock. The high prevalence rates observed in certain tick species and geographic regions emphasize the importance of targeted public health interventions. Future research should focus on elucidating the transmission dynamics and implementing effective control measures to mitigate the impact of these pathogens on human, veterinary, and wildlife health in the region.

Molecular detection of Coxiella burnetii in ticks collected from animals and the environment in Uganda.

AIMS: Coxiella burnetii is a highly infectious organism that is easily spread through aerosols causing Q fever in humans. Ticks can harbour and transmit C. burnetii to animals, contributing to disease maintenance. Our aim was to examine the presence of C. burnetii in ticks in Uganda. METHODS AND RESULTS: In this study, ticks were collected from five Ugandan districts and tested by real-time PCR for C. burnetii (Coxiella outer membrane protein 1 gene). A total of 859 tick pools (9602 individual ticks) were tested, and pool positivity for C. burnetii was 5.5% (n = 47). Pooled prevalence differed by district; the highest was Luwero (7.3%), then Gulu (6.6%), and Kasese had the lowest (1.3%). However, district variation was not statistically significant (Fisher's exact = 0.07). Ticks collected from dogs and cats had the highest positivity rates [23/47, (48.9%)] followed by livestock (cattle, goats, sheep, and pigs) [18/47, (38.3%)] and vegetation [6/47, (12.8%)]. Haemaphysalis elliptica had the highest infection rates, followed by Rhipicephalus appendiculatus, Amblyomma variegatum and Rhipicephalus decoloratus had similar prevalence. CONCLUSIONS: Although ticks are not the primary transmitters of C. burnetii to humans, pathogen detection in ticks can be an indirect indicator of risk among animal hosts. Vulnerable populations, including occupations with close animal contact such as farming, butchery, and veterinary practice, have an increased risk of C. burnetii exposure. Veterinarians and clinicians should be aware that C. burnetii may cause human and animal illness in these regions.

The expanding range of emerging tick-borne viruses in Eastern Europe and the Black Sea Region.

We analysed both pooled and individual tick samples collected from four countries in Eastern Europe and the Black Sea region, using metagenome-based nanopore sequencing (NS) and targeted amplification. Initially, 1337 ticks, belonging to 11 species, were screened in 217 pools. Viruses (21 taxa) and human pathogens were detected in 46.5% and 7.3%, respectively. Tick-borne viral pathogens comprised Tacheng Tick Virus 2 (TTV2, 5.9%), Jingmen Tick Virus (JMTV, 0.9%) and Tacheng Tick Virus 1 (TTV1, 0.4%). An association of tick species with individual virus taxa was observed, with the exception of TTV2, which was observed in both Dermacentor and Haemaphysalis species. Individual ticks from pools with pathogen detection were then further screened by targeted amplification and then NS, which provided extensive genome data and revealed probable pathogen Haseki Tick Virus (HTV, 10.2%). Two distinct TTV2 clades were observed in phylogenetic analysis, one of which included closely related Dermacentor reticulatus Uukuviruses. JMTV detection indicated integrated virus sequences. Overall, we observed an expansion of newly documented pathogenic tick-borne viruses into Europe, with TTV1 being identified on the continent for the first time. These viruses should be included in the diagnostic assessment of symptomatic cases associated with tick bites and vector surveillance efforts. NS is shown as a useful tool for monitoring tick-associated pathogens in pooled or individual samples.

Wide distribution of Mediterranean and African spotted fever agents and the first identification of Israeli spotted fever agent in ticks in Uganda.

Rickettsia microorganisms are causative agents of several neglected emerging infectious diseases in humans transmitted by arthropods including ticks. In this study, ticks were collected from four geographical regions of Uganda and pooled in sizes of 1-179 ticks based on location, tick species, life stage, host, and time of collection. Then, they were tested by real-time PCR for Rickettsia species with primers targeting gltA, 17kDa and ompA genes, followed by Sanger sequencing of the 17kDa and ompA genes. Of the 471 tick pools tested, 116 (24.6%) were positive for Rickettsia spp. by the gltA primers. The prevalence of Rickettsia varied by district with Gulu recording the highest (30.1%) followed by Luwero (28.1%) and Kasese had the lowest (14%). Tick pools from livestock (cattle, goats, sheep, and pigs) had the highest positivity rate, 26.9%, followed by vegetation, 23.1%, and pets (dogs and cats), 19.7%. Of 116 gltA-positive tick pools, 86 pools were positive using 17kDa primers of which 48 purified PCR products were successfully sequenced. The predominant Rickettsia spp. identified was R. africae (n = 15) in four tick species, followed by R. conorii (n = 5) in three tick species (Haemaphysalis elliptica, Rhipicephalus appendiculatus, and Rh. decoloratus). Rickettsia conorii subsp. israelensis was detected in one tick pool. These findings indicate that multiple Rickettsia spp. capable of causing human illness are circulating in the four diverse geographical regions of Uganda including new strains previously known to occur in the Mediterranean region. Physicians should be informed about Rickettsia spp. as potential causes of acute febrile illnesses in these regions. Continued and expanded surveillance is essential to further identify and locate potential hotspots with Rickettsia spp. of concern.

Applying next generation sequencing to detect tick-pathogens in Dermacentor nuttalli, Ixodes persulcatus, and Hyalomma asiaticum collected from Mongolia.

Ticks and tick-borne diseases represent major threats to the public health of the Mongolian population, of which an estimated 26% live a traditional nomadic pastoralist lifestyle that puts them at increased risk for exposure. Ticks were collected by dragging and removal from livestock in Khentii, Selenge, Tuv, and Umnugovi aimags (provinces) during March-May 2020. Using next-generation sequencing (NGS) with confirmatory PCR and DNA sequencing, we sought to characterize the microbial species present in Dermacentor nuttalli (n = 98), Hyalomma asiaticum (n = 38), and Ixodes persulcatus (n = 72) tick pools. Rickettsia spp. were detected in 90.4% of tick pools, with Khentii, Selenge, and Tuv tick pools all having 100% pool positivity. Coxiella spp. were detected at an overall pool positivity rate of 60%, while Francisella spp. were detected in 20% of pools and Borrelia spp. detected in 13% of pools. Additional confirmatory testing for Rickettsia-positive pools demonstrated Rickettsia raoultii (n = 105), Candidatus Rickettsia tarasevichiae (n = 65) and R. slovaca/R. sibirica (n = 2), as well as the first report of Candidatus Rickettsia jingxinensis (n = 1) in Mongolia. For Coxiella spp. reads, most samples were identified as a Coxiella endosymbiont (n = 117), although Coxiella burnetii was detected in eight pools collected in Umnugovi. Borrelia species that were identified include Borrelia burgdorferi sensu lato (n = 3), B. garinii (n = 2), B. miyamotoi (n = 16), and B. afzelii (n = 3). All Francisella spp. reads were identified as Francisella endosymbiont species. Our findings emphasize the utility of NGS to provide baseline data across multiple tick-borne pathogen groups, which in turn can be used to inform health policy, determine regions for expanded surveillance, and guide risk mitigation strategies.

A custom hepatitis A virus assay for whole-genome sequencing.

Since 2016, the United States has experienced a resurgence in the number of hepatitis A virus (HAV) cases and outbreaks. These outbreaks have been sustained by person-to-person transmission with cases occurring predominantly in high-risk populations including intravenous drug users, individuals experiencing homelessness, and men who have sex with men. To investigate HAV transmission, a molecular-surveillance system consisting of real-time RT-PCR (rRT-PCR) for detection, and a conventional RT-PCR assay for genotyping of HAV, was established in New York State (NYS) in 2019. Since then, a total of 271 HAV-positive serum samples collected from cases across NYS between 2019 and 2021 were identified by rRT-PCR. To rapidly and efficiently generate HAV whole-genome sequences, a custom AmpliSeq™ panel was designed in collaboration with Thermo Fisher. To streamline the process, sample preparation was performed on an Ion Chef and sequencing on an Ion S5XL. Of the 271 HAV-positive samples, the whole-genome sequencing (WGS) assay successfully generated 134 near-complete, high-quality HAV sequences. Phylogenetic analysis of the VP1-2A region identified 216 IB, 48 IA, and 2 IIIA genotypes, while 5 were unable to be typed due to poor sequence in this key region. The HAV whole-genome sequencing approach provided a more efficient and streamlined approach for genotyping HAV compared to previous methods and resulted in phylogenetic trees with enhanced resolution compared to the HAV VP1-2A region alone.