Pathogen Detection and Genotyping With Trace Sample by A Phi29 Based Unbiased Exponential Amplification

Ticks are vectors for many infectious diseases such as spotted fever group (SFG) rickettsioses and borrelioses. Ticks are valuable material for pathogen ecology study. Ticks have several growth stages with significant varying size, and therefore, in most cases, the collected ticks cannot provide sufficient DNA for subsequent studies, particularly for multiple pathogen screening and genotyping. Unbiased pretreatment of the tick samples for subsequent analysis is an urgent need for subsequent ecological survey and other studies. Phi29 DNA polymerase, an enzyme with strand displacement activity, could exponentially amplify DNA randomly and non-biasedly, generating large quantities of DNA. In the present study, we developed a Phi29 based unbiased exponential amplification (PEA) assay for unbiased treatment of sample nucleic acid to provide sufficient DNA for genetic analysis. By using tick borne pathogen detection and genotype as a model, we tested and evaluated the feasibility of the assay. Nucleic acid were extracted from single ticks and subjected to PEA. The results showed that tick DNA could be amplified up to 10 5 folds. The amplified products were successfully used for pathogen screening and genotyping. With the amplified DNA from single tick, Rickettsia was successfully detected and genotyped. A new genotype of Rickettsia was identified from ticks collected from Dandong city, Liaoning province, Northeast China. This PEA assay is universal and can also be extended to other applications where samples are greatly limited.

Geographic Partitioning of Dengue Virus Transmission Risk in Florida

Dengue viruses (DENVs) cause the greatest public health burden globally among the arthropod-borne viruses. DENV transmission risk has also expanded from tropical to subtropical regions due to the increasing range of its principal mosquito vector, Aedes aegypti. Focal outbreaks of dengue fever (dengue) in the state of Florida (FL) in the USA have increased since 2009. However, little is known about the competence of Ae. aegypti populations across different regions of FL to transmit DENVs. To understand the effects of DENV genotype and serotype variations on vector susceptibility and transmission potential in FL, we orally infected a colony of Ae. aegypti (Orlando/ORL) with low passage or laboratory DENV-1 through -4. Low passage DENVs were more infectious to and had higher transmission potential by ORL mosquitoes. We used these same DENVs to examine natural Ae. aegypti populations to determine whether spatial distributions correlated with differential vector competence. Vector competence across all DENV serotypes was greater for mosquitoes from areas with the highest dengue incidence in south FL compared to north FL. Vector competence for low passage DENVs was significantly higher, revealing that transmission risk is influenced by virus/vector combinations. These data support a targeted mosquito-plus-pathogen screening approach to more accurately estimate DENV transmission risk.

Geographic partitioning of dengue virus transmission risk in Florida

Dengue viruses (DENVs) cause the greatest public health burden globally among the arthropod-borne viruses. DENV transmission risk has also expanded from tropical to subtropical regions due to the increasing range of its principal mosquito vector, Aedes aegypti. Focal outbreaks of dengue fever (dengue) in the state of Florida (FL) in the USA have increased since 2009. However, little is known about the competence of Ae. aegypti populations across different regions of FL to transmit DENVs. To understand the effects of DENV genotype and serotype variations on vector susceptibility and transmission potential in FL, we orally infected a colony of Ae. aegypti (Orlando/ORL) with laboratory and wild type DENV-1 through -4. Wild type DENVs were more infectious to and transmissible by ORL mosquitoes. We used these same DENVs to examine natural Ae. aegypti populations to determine whether spatial distributions correlated with differential vector competence. Vector competence across all DENV serotypes was greater for mosquitoes from areas with the highest dengue incidence in south FL compared to north FL. Vector competence for wild type DENVs was significantly higher, revealing that transmission risk is influenced by virus/vector combinations. These data support a targeted mosquito-plus-pathogen screening approach to more accurately estimate DENV transmission risk.

Seasonal and Age-Associated Pathogen Distribution in Newborn Calves with Diarrhea Admitted to ICU

Calf mortality constitutes a substantial loss for agriculture economy-based countries and is also a significant herd problem in developed countries. However, the occurrence and frequency of responsible gastro-intestinal (GI) pathogens in severe newborn diarrhea is still not well known. We aimed to determine the seasonal and age-associated pathogen distribution of severe diarrhea in newborn calves admitted to the intensive care unit (ICU) of Erciyes University animal hospital over a year. Fecal samples were collected during the ICU admissions, and specimens were subjected to a diarrheal pathogen screening panel that included bovine coronavirus (BCoV), Cryptosporidium spp., ETEC K99+, and bovine rotavirus, using RT-PCR and conventional PCR methods. Further isolation experiments were performed with permissive cell cultures and bacterial enrichment methods to identify the clinical importance of infectious pathogen shedding in the ICU. Among the hospitalized calves aged less than 45 days old, the majority of calves originated from small farms (85.9%). The pathogen that most frequently occurred was Cryptosporidium spp. (61.5%) followed by rotavirus (56.4%). The frequency of animal admission to ICU and GI pathogen identification was higher during the winter season (44.9%) when compared to other seasons. Most calves included in the study were 1–6 days old (44.9%). Lastly, co-infection with rotavirus and Cryptosporidium spp. occurred more frequently than other dual or multi-infection events. This study was the first to define severe diarrhea—causing GI pathogens from ICU admitted newborn calves in Turkey.

iSeq 100 for metagenomic pathogen screening in ticks

Background Ticks are blood-sucking ectoparasites that play a pivotal role in the transmission of various pathogens to humans and animals. In Korea, Haemaphysalis longicornis is the predominant tick species and is recognized as the vector of pathogens causing various diseases such as babesiosis, borreliosis, rickettsiosis, and severe fever with thrombocytopenia syndrome. Methods In this study, the targeted high-throughput sequencing of the 16S rRNA V4 region was performed using the state-of-the-art sequencing instrument, iSeq 100, to screen bacterial pathogens in H. longicornis, and the findings were compared with those using conventional PCR with specific primers. Microbiome analyses were performed with EzBioCloud, a commercially available ChunLab bioinformatics cloud platform. ANOVA-Like Differential Expression tool (ALDEx2) was used for differential abundance analysis. Results Rickettsia spp. were detected in 16 out of 37 samples using iSeq 100, and this was confirmed using a PCR assay. In the phylogenetic analysis using gltA and ompA sequences of the detected Rickettsia, the highest sequence similarity was found with ‘Candidatus Rickettsia jingxinensis’ isolate Xian-Hl-79, ‘Ca. R. jingxinensis’ isolate F18, and ‘Ca. R. longicornii‘ isolate ROK-HL727. In the microbiome study, Coxiella AB001519, a known tick symbiont, was detected in all 37 tick samples. Actinomycetospora chiangmaiensis was more abundant in Rickettsia-positive samples than in Rickettsia-negative samples. Conclusions In this study, iSeq 100 was used to investigate the microbiome of H. longicornis, and the potentially pathogenic Rickettsia strain was detected in 16 out of 37 ticks. We believe that this approach will aid in large-scale pathogen screening of arthropods to be used in vector-borne disease control programs. Graphical Abstract

Patterns of infection, origins and transmission of ranaviruses among the ectothermic vertebrates of Asia

Ranaviral infections, a malady of ectothermic vertebrates, are becoming frequent, severe, and widespread, causing mortality among both native and cultured species, raising odds of species extinctions and economic losses. This turn of events is possibly due to the broad host range of ranaviruses and the transmission of these pathogens through regional and international trade in Asia, where outbreaks have been increasingly reported over the past decade. Here we focus attention on the origins, means of transmission, and patterns of spread of this infection within the region. Infections have been recorded in both cultured and wild populations in at least twelve countries/administrative regions, together with mass die-offs in some regions. Despite the imminent seriousness of the disease in Asia, surveillance efforts are still incipient. Some of the infections transmitted within Asia may transmit across host-taxon barriers, posing a significant risk to native species. Factors such as rising temperatures due to global climate change seem to exacerbate ranaviral activity, as most known outbreaks have been recorded during summer; however, data are still inadequate to verify this for Asia. Import risk analysis, using protocols such as Pandora+, pre-border pathogen screening, and effective biosecurity measures, can be used to mitigate introduction to uninfected areas and curb transmission within Asia. Comprehensive surveillance using molecular diagnostic tools for ranavirus species and variants will help in understanding the prevalence and disease burden in the region. This is an important step towards conserving native biodiversity and safeguarding the aquaculture industry.

Hedgehogs and Squirrels as Hosts of Zoonotic Bartonella Species

Free-living animals frequently play a key role in the circulation of various zoonotic vector-borne pathogens. Bacteria of the genus Bartonella are transmitted by blood-feeding arthropods and infect a large range of mammals. Although only several species have been identified as causative agents of human disease, it has been proposed that any Bartonella species found in animals may be capable of infecting humans. Within a wide-ranging survey in various geographical regions of the Czech Republic, cadavers of accidentally killed synurbic mammalian species, namely Eurasian red squirrel (Sciurus vulgaris), European hedgehog (Erinaceus europaeus) and Northern white-breasted hedgehog (Erinaceus roumanicus), were sampled and tested for Bartonella presence using multiple PCR reaction approach targeting several DNA loci. We demonstrate that cadavers constitute an available and highly useful source of biological material for pathogen screening. High infection rates of Bartonella spp., ranging from 24% to 76%, were confirmed for all three tested mammalian species, and spleen, ear, lung and liver tissues were demonstrated as the most suitable for Bartonella DNA detection. The wide spectrum of Bartonella spp. that were identified includes three species with previously validated zoonotic potential, B. grahamii, B. melophagi and B. washoensis, accompanied by ‘Candidatus B. rudakovii’ and two putative novel species, Bartonella sp. ERIN and Bartonella sp. SCIER.

iSeq100 for metagenomic pathogen screening in ticks

Background: Ticks are blood sucking ectoparasites that play a pivotal role in the transmission of various pathogens to humans and animals. In Korea, Haemaphysalis longicornis is the predominant tick species and is recognized as the vector of pathogens causing various diseases such as babesiosis, borreliosis, rickettsiosis, and severe fever with thrombocytopenia syndrome. Methods: In this study, we developed a method to screen bacterial pathogens in H. longicornis using targeted high-throughput sequencing of the 16S rRNA V4 region using the state-of-the-art sequencing instrument, iSeq 100, and compared the findings with those of conventional PCR with specific primers. Results: Rickettsia spp. were detected in 18 out of 39 samples using iSeq 100 but in only 14 samples using conventional PCR. In the phylogenetic analysis using gltA and ompA sequences of the detected Rickettsia, the highest sequence similarity was found with Candidatus Rickettsia jingxinensis isolate Xian-Hl-79, Ca. R. jingxinensis isolate F18, and Ca. R. longicornii isolate ROK-HL727. In the microbiome study, Coxiella AB001519, a known tick symbiont, was detected in all 39 tick samples. The Actinomycetospora chiangmaiensis group was more abundant in Rickettsia spp.-positive samples than in Rickettsia spp.-negative samples. Conclusions: Thus, we demonstrated that iSeq100 can rapidly and economically screen potential pathogens in ticks and can be applied to large-scale pathogen screening in arthropods for vector-borne disease control programs.