Species-specific Polymerase Chain Reaction Assay Research Articles

First molecular survey of tick-borne protozoan and bacterial pathogens in the questing tick population in Bangladesh

Questing ticks carry various tick-borne pathogens (TBPs) that are responsible for causing tick-borne diseases (TBDs) in humans and animals around the globe, especially in the tropics and sub-tropics. Information on the distribution of ticks and TBPs in a specific geography is crucial for the formulation of mitigation measures against TBDs. Therefore, this study aimed to survey the TBPs in the questing tick population in Bangladesh. A total of 2748 questing hard ticks were collected from the pastures in Sylhet, Bandarban, Sirajganj, Dhaka, and Mymensingh districts through the flagging method. After morphological identification, the ticks were grouped into 142 pools based on their species, sexes, life stages, and collection sites. The genomic DNA extracted from tick specimens was screened for 14 pathogens, namely Babesia bigemina (AMA-1), Babesia bovis (RAP-1), Babesia naoakii (AMA-1), Babesia ovis (18S rRNA), Theileria luwenshuni (18S rRNA), Theileria annulata (Tams-1), Theileria orientalis (MPSP), Anaplasma marginale (groEL), Anaplasma phagocytophilum (16S rRNA), Anaplasma bovis (16S rRNA), Anaplasma platys (16S rRNA), Ehrlichia spp. (16S rRNA), Rickettsia spp. (gltA), and Borrelia (Bo.) spp. (flagellin B) using genus and species-specific polymerase chain reaction (PCR) assays. The prevalence of the detected pathogens was calculated using the maximum likelihood method (MLE) with 95 % confidence interval (CI). Among 2748 ixodid ticks, 2332 (84.86 %) and 416 (15.14 %) were identified as Haemaphysalis bispinosa and Rhipicephalus microplus, respectively. Haemaphysalis bispinosa was found to carry all the seven detected pathogens, while larvae of R. microplus were found to carry only Bo. theileri. Among the TBPs, the highest detection rate was observed in A. bovis (20/142 pools, 0.81 %, CI: 0.51–1.20), followed by T. orientalis (19/142 pools, 0.72 %, CI: 0.44–1.09), T. luwenshuni (9/142 pools, 0.34 %, CI: 0.16–0.62), B. ovis (4/142 pools, 0.15 %, CI: 0.05 – 0.34) and Bo. theileri (4/142 pools, 0.15 %, CI: 0.05–0.34), Ehrlichia ewingii (3/142 pools, 0.11 %, CI: 0.03–0.29), and Babesia bigemina (1/142, 0.04 %, CI: 0.00 – 0.16). This study reports the existence of T. luwenshuni, E. ewingii, and Bo. theileri in Bangladesh for the first time. The novel findings of this study are the foremost documentation of transovarian transmission of B. bigemina and E. ewingii in H. bispinosa and also provide primary molecular evidence on the presence of E. ewingii and Bo. theileri in H. bispinosa. Therefore, this study may shed light on the circulating TBPs in ticks in the natural environment and thereby advocate awareness among physicians and veterinarians to control and prevent TBDs in Bangladesh.

Comparison of qPCR and metabarcoding for environmental DNA surveillance of a freshwater parasite.

Analysis of environmental DNA (eDNA) has been successfully used across freshwater ecological parasitology to inform management of ecologically and economically important species. However, most studies have used species-specific quantitative polymerase chain reaction (qPCR) assays to detect target taxa. While generally effective, this approach limits the amount of community and management-supporting data that can be obtained from eDNA samples. If eDNA metabarcoding could be conducted with the same accuracy of a single species approach, researchers could simultaneously detect a target species while obtaining vast community data from eDNA samples. We sampled 38 freshwater sites on Fort McCoy, Wisconsin and compared qPCR to metabarcoding for eDNA detection of the ectoparasitic gill louse Salmincola edwardsii, an obligate parasite of Salvelinus fishes (chars). We found no evidence to suggest S. edwardsii occupancy or detection probabilities differed between qPCR and metabarcoding. Further, we found that the number of S. edwardsii reads from metabarcoding were negatively predictive of C T values from qPCR (C T value indicates cycle a significant amount of target eDNA is detected, with lower C Ts indicative of more DNA), demonstrating that our metabarcoding reads positively predicted qPCR DNA quantities. However, the number of reads was not predictive of overall qPCR score (number of positive qPCR replicates). In addition to S. edwardsii, metabarcoding led to the detection of a vast community of over 2600 invertebrate taxa. We underscore the necessity for conducting similar analyses across environments and target species, as the ecology of eDNA will vary on a per-study basis. Our results suggest that eDNA metabarcoding provides a highly sensitive and accurate method for detecting parasitic gill lice while also illuminating the broader biological community and co-occurrence of species in the environment.

An emerging pathogen found in farm-cultured fourfinger threadfin (Eleutheronema tetradactylum), Edwardsiella piscicida, its genetic profile, virulent genes, and pathogenicity

The diseased fourfinger threadfin (Eleutheronema tetradactylum) exhibited clinical symptoms, such as hemorrhaging in the buccal area and skin. Gross pathological examination revealed hepatic hemorrhage, spleen enlargement, and brain congestion. Histopathological examination revealed multifocal necrosis and the presence of bacteria in various organs, including the heart, liver, spleen, and head kidney. Severe blood vessel congestion was also observed. All bacterial isolates obtained from the liver, spleen, head kidney, and brain were identified as gram-negative, motile, rod-shaped bacilli that displayed α-hemolysis. Utilizing a combination of the API 20 E system and species-specific primer-based polymerase chain reaction assays (EDtA, EDtD, and EDi primer sets), as well as sequencing of the 16S ribosomal DNA (16S rDNA) and iron-cofactor superoxide dismutase (sodB), the findings unequivocally established the identity of all isolates as E. piscicida. Examination of the E. piscicida isolates using transmission electron microscopy revealed the presence of flagella. Furthermore, genetic fingerprinting using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) demonstrated that the E. piscicida strains isolated from the fourfinger threadfin constituted a distinct genotype separate from the previously identified strains associated with Edwardsiellosis in different fish hosts. The pathogenicity of the representative E. piscicida isolates (E8–E12) was evaluated by subjecting the fourfinger threadfin to intraperitoneal injections. The gross lesions and histopathological alterations observed in experimentally infected fourfinger threadfins closely mirrored those observed in naturally infected fish. This study confirmed that E. piscicida-induced Edwardsiellosis could lead to disease outbreaks in farm-cultured fourfinger threadfin.

First report of dog ticks and tick-borne pathogens they are carrying in Malawi.

Ticks are vectors for transmitting tick-borne pathogens (TBPs) in animals and humans. Therefore, tick identification is necessary to understand the distribution of tick species and the pathogens they carry. Unfortunately, data on dog ticks and the TBPs they harbor in Malawi are incomplete. This study aimed to identify dog ticks and the TBPs they transmit in Malawi. One hundred thirty-two ticks were collected from 87 apparently healthy but infested domestic dogs in four districts of Malawi, which were pooled into 128 tick samples. The ticks were morphologically identified under a stereomicroscope using identification keys, and species identification was authenticated by polymerase chain reaction (PCR) through the amplification and sequencing of 12S rRNA and cytochrome c oxidase subunit I (CO1) genes. The tick species identified were Rhipicephalus sanguineus sensu lato (58.3%), Haemaphysalis elliptica (32.6%), and Hyalomma truncatum (9.1%). Screening for TBPs using species-specific PCR assays revealed that 48.4% of the ticks were infected with at least one TBP. The TBP detection rates were 13.3% for Anaplasma platys, 10.2% for Babesia rossi, 8.6% for B. vogeli, 6.3% for Ehrlichia canis, 3.9% for A. phagocytophilum, 3.1% for B. gibsoni, 2.3% for B. canis and 0.8% for Hepatozoon canis. Co-infections of up to three pathogens were observed in 48.4% of the positive samples. This is the first study to identify dog ticks and the TBPs they harbor in Malawi. These findings provide the basis for understanding dog tick distribution and pathogens they carry in Malawi. This study necessitates the examination of ticks from more study locations to have a better picture of tick challenge, and the development of ticks and tick-borne disease control methods in Malawi.

Sibling Species Composition and Susceptibility Status of Anopheles gambiae s.l. to Insecticides Used for Indoor Residual Spraying in Eastern Uganda.

Malaria remains one of the most critical disease causing morbidity and mortality in Uganda. Indoor residual spraying (IRS) and the use of insecticide-treated bed nets are currently the predominant malaria vector control interventions. However, the emergence and spread of insecticide resistance among malaria vectors threaten the continued effectiveness of these interventions to control the disease, particularly in high transmission areas. To inform decisions on vector control, the current study evaluated the Anopheles malaria vector species and their susceptibility levels to 0.1% bendiocarb and 0.25% pirimiphos-methyl insecticides used in IRS intervention program in Namutumba district, Eastern Uganda. Anopheles larvae were collected between March and May 2017 from different breeding sites in the parishes of Nsinze and Nawaikona in Nsinze sub-county and reared to adults to assess the susceptibility status of populations in the study area. Mosquitoes were identified using morphological keys and species-specific polymerase chain reaction (PCR) assays. Susceptibility tests were conducted on 2- to 5-day-old non-blood-fed adult female Anopheles that emerged using insecticide-impregnated papers with 0.1% bendiocarb and 0.25% pirimiphos-methyl following standard World Health Organization (WHO) insecticide susceptibility bioassays. A Log-probit regression model was used to derive the knock-down rates for 50% and 95% of exposed mosquitoes. A total of 700 mosquito larvae were collected from different breeding sites. Morphological identification showed that 500 individuals that emerged belonged to Anopheles gambiae sensu lato (s.l.), the main malaria vector. The PCR results showed that the dominant sibling species under the A. gambiae complex was Anopheles arabiensis 99.5% (395/397). WHO bioassay tests revealed that the population of mosquitoes exhibited high levels of susceptibility (24-hour post-exposure mortality 98-100%) to both insecticides tested. The median knock-down time, KDT50, ranged from 6.6 to 81.4 minutes, while the KDT95 ranged from 21.6 to 118.9 minutes for 0.25% pirimiphos-methyl. The KDT50 for 0.1% bendiocarb ranged from 2.8 to 62.9 minutes, whereas the KDT95 ranged from 36.0 to 88.5 minutes. These findings indicate that bendiocarb and pirimiphos-methyl are still effective against the major malaria vector, A. arabiensis in Nsinze sub-county, Namutumba district, Uganda and can be effectively used for IRS. The study has provided baseline information on the insecticide susceptibility status on malaria vectors in the study area. However, routine continuous monitoring program of insecticide susceptibility and malaria vector composition is required so as to guide future decisions on insecticide use for IRS intervention toward malaria elimination and to track future changes in vector population.

Two species-specific TaqMan-based quantitative polymerase chain reaction assays for the detection in soil of Paenibacillus polymyxa inocula.

The increasingly widespread use of beneficial microbial inocula in agriculture gives rise to two primary needs: i) the assessment of the environmental risk, i.e. their impact on local soil microbiome and soil properties; ii) being able to track them and monitor their persistence and fate to both optimize their formulation and application method. In previous years, PCR-based methods have detected bacterial or fungal bioinoculant at the species or strain level. However, the selective detection, quantification, and monitoring of target microbial species in a complex ecosystem such as soil require that the tests possess high specificity and sensitivity. The work proposes a quantitative real-time PCR detection method using TaqMan chemistry, showing high specificity and sensitivity for the Paenibacillus polymyxa K16 strain. The primer and probe sets were designed using the polymyxin gene cluster targeting pmxC and pmxE sequences. Validation tests showed that these assays allowed a discriminant and specific detection of P. polymyxa K16 in soil. The TaqMan-assay developed could thus ensure the necessary level of discrimination required by commercial and regulatory purposes to detect and monitor the bioinoculant in soil.

Two species-specific TaqMan-based quantitative polymerase chain reaction assays for the detection in soil of Paenibacillus polymyxa inocula

Two species-specific TaqMan-based quantitative polymerase chain reaction assays for the detection in soil of Paenibacillus polymyxa inocula

Geographical distribution, molecular and toxin diversity of the dinoflagellate species Gambierdiscus honu in the Pacific region

An increase in cases of ciguatera poisoning (CP) and expansion of the causative species in the South Pacific region highlight the need for baseline data on toxic microalgal species to help identify new areas of risk and manage known hot spots. Gambierdiscus honu is a toxin producing and potential CP causing dinoflagellate species, first described in 2017. Currently no high-resolution geographical distribution, intraspecific genetic variation or toxin production diversity data is available for G. honu. This research aimed to further characterize G. honu by investigating its distribution using species-specific real-time polymerase chain reaction assays at 25 sites in an area spanning ∼8000 km of the Coral Sea/Pacific Ocean, and assessing intraspecific genetic variation, toxicity and toxin production of isolated strains. Assessment of genetic variation of the partial rRNA operon of isolates demonstrated no significant intraspecific population structure, in addition to a lack of adherence to isolation by distance (IBD) model of evolution. The detected distribution of G. honu in the Pacific region was within the expected tropical to temperate latitudinal ranges of 10° to -30° and extended from Australia to French Polynesia. In the lipophilic fractions, the neuroblastoma cell-based assay (CBA-N2a) showed no ciguatoxin (CTX)-like activity for nine of the 10 isolates, and an atypical pattern for CAWD233 isolate which showed cytotoxic activity in OV- and OV+ conditions. In the same way, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis confirmed no Pacific-CTXs (CTX-3B, CTX-3C, CTX-4A, CTX-4B) were produced by the ten strains. The CBA-N2a assessment of the hydrophilic fractions showed moderate to high cytotoxicity in both OV- and OV+ condition for all the strains showing a cytotoxic profile similar to that of gambierone. Indeed, this study is the first to show the cytotoxic activity of gambierone on mouse neuroblastoma cells while no cytotoxicity was observed when 44-MG was analysed at the same concentrations using the CBA-N2a. Analysis of the hydrophilic via LC-MS/MS confirmed production of gambierone in all isolates, ranging from 2.1 to 38.1 pg/cell, with 44-methylgambierone (44-MG) also produced by eight of the isolates, ranging from 0.3 to 42.9 pg/cell. No maitotoxin-1 was detected in any of the isolates. Classification of the G. honu strains according to the quantities of gambierone produced aligned with the classification of their cytotoxicity using the CBA-N2a. Finally, no maitotoxin-1 (MTX) was detected in any of the isolates. This study shows G. honu is widely distributed within the Pacific region with no significant intraspecific population structure present. This aligns with the view of microalgal populations as global metapopulations, however more in-depth assessment with other genetic markers could detect further structure. Toxicity diversity across 10 isolates assessed did not display any geographical patterns.

Widespread presence of a highly virulent Edwardsiella ictaluri strain in farmed tilapia, Oreochromis spp.

Edwardsiella ictaluri is an emerging bacterial pathogen that affects farmed tilapia (Oreochromis spp.). This study reports the widespread presence of E. ictaluri in farmed tilapia in Vietnam. Among 26 disease outbreaks from nine provinces in Northern Vietnam during 2019-2021, 19 outbreaks originated from imported seeds, while outbreaks in seven farms were from domestic sources. Clinically sick fish showed the appearance of numerous white spots in visceral organs, and accumulative mortality reached 30%-65%. A total of 26 representative bacterial isolates recovered from 26 disease outbreaks were identified as E. ictaluri based on a combination of phenotypic tests, genus- and species-specific polymerase chain reaction assays, 16S rRNA and gyrB sequencing, and phylogenetic analysis. All isolates harboured the same virulence gene profiles esrC+ , evpC+ , ureA-C+ , eseI- , escD- and virD4- . Antimicrobial susceptibility tests revealed that 80.8%-100% of isolates were multidrug resistant, with resistance to 4-8 antimicrobials in the groups of penicillin, macrolides, sulfonamides, amphenicols and glycopeptides. The experimental challenge successfully induced disease that mimicked natural infection. The median lethal doses (LD50 ) of the tested isolates (n = 4) were 42-61 colony forming units/fish, indicating their extremely high virulence. This emerging pathogen is established and has spread to various geographical locations, causing serious impacts on farmed tilapia in northern Vietnam. It is likely that this pathogen will continue to spread through contaminated stocks (both imported and domestic sources) and persist. Thus, increased awareness, combined with biosecurity measures and emergent vaccination programs is essential to mitigate the negative impact of this emerging disease on the tilapia farming industry.

Emerging and well-characterized chlamydial infections detected in a wide range of wild Australian birds.

Birds can act as successful long-distance vectors and reservoirs for numerous zoonotic bacterial, parasitic and viral pathogens, which can be a concern given the interconnectedness of animal, human and environmental health. Examples of such avian pathogens are members of the genus Chlamydia. Presently, there is a lack of research investigating chlamydial infections in Australian wild and captive birds and the subsequent risks to humans and other animals. In our current study, we investigated the prevalence and genetic diversity of chlamydial organisms infecting wild birds from Queensland and the rate of co-infections with beak and feather disease virus (BFDV). We screened 1114 samples collected from 564 different birds from 16 orders admitted to the Australia Zoo Wildlife Hospital from May 2019 to February 2021 for Chlamydia and BFDV. Utilizing species-specific quantitative polymerase chain reaction (qPCR) assays, we revealed an overall Chlamydiaceae prevalence of 29.26% (165/564; 95% confidence interval (CI) 25.65-33.14), including 3.19% (18/564; 95% CI 2.03-4.99%) prevalence of the zoonotic Chlamydia psittaci. Chlamydiaceae co-infection with BFDV was detected in 9.75% (55/564; 95% CI 7.57-12.48%) of the birds. Molecular characterization of the chlamydial 16S rRNA and ompA genes identified C. psittaci, in addition to novel and other genetically diverse Chlamydia species: avian Chlamydia abortus, Ca. Chlamydia ibidis and Chlamydia pneumoniae, all detected for the first time in Australia within a novel avian host range (crows, figbirds, herons, kookaburras, lapwings and shearwaters). This study shows that C. psittaci and other emerging Chlamydia species are prevalent in a wider range of avian hosts than previously anticipated, potentially increasing the risk of spill-over to Australian wildlife, livestock and humans. Going forward, we need to further characterize C. psittaci and other emerging Chlamydia species to determine their exact genetic identity, potential reservoirs, and factors influencing infection spill-over.

Development of droplet digital Polymerase Chain Reaction assays for the detection of long-finned (Anguilla dieffenbachii) and short-finned (Anguilla australis) eels in environmental samples.

Freshwater eels are ecologically, and culturally important worldwide. The New Zealand long-finned eel (Anguilla dieffenbachii) and short-finned eel (Anguilla australis) are apex predators, playing an important role in ecosystem functioning of rivers and lakes. Recently, there has been a national decline in their populations due to habitat destruction and commercial harvest. The emergence of targeted environmental DNA detection methodologies provides an opportunity to enhance information about their past and present distributions. In this study we successfully developed species-specific droplet digital Polymerase Chain Reaction (ddPCR) assays to detect A. dieffenbachii and A. australis DNA in water and sediment samples. Assays utilized primers and probes designed for regions of the mitochondrial cytochrome b and 16S ribosomal RNA genes in A. dieffenbachii and A. australis, respectively. River water samples (n = 27) were analyzed using metabarcoding of fish taxa and were compared with the ddPCR assays. The presence of A. dieffenbachii and A. australis DNA was detected in a greater number of water samples using ddPCR in comparison to metabarcoding. There was a strong and positive correlation between gene copies (ddPCR analyses) and relative eel sequence reads (metabarcoding analyses) when compared to eel biomass. These ddPCR assays provide a new method for assessing spatial distributions of A. dieffenbachii and A. australis in a range of environments and sample types.

The development of genus-specific and species-specific real-time PCR assays for the authentication of Patagonian toothfish and Antarctic toothfish in commercial seafood products.

The substitution or mislabeling of toothfish is an issue of significant concern for seafood authorities; it also reduces the effectiveness of marine conservation and management programs for its over-exploitation and illegal trafficking, boosting the need for identification methods. Two species-specific real-time polymerase chain reaction (PCR) assays for the identification of Patagonian toothfish (Dissostichus eleginoides) and Antarctic toothfish (Dissostichus mawsoni) and a genus-specific real-time PCR assay for Dissostichus spp. identification were developed based on fragments of the 16S rRNA and COI (cytochrome c oxidase subunit I) genes. These methods were confirmed to be rapid, simple, and sensitive (absolute sensitivity of 0.0002 ng μL-1 and relative sensitivity of 0.1g kg-1 with good specificity). These methods can be applied to processed and commercial fish products. These approaches can be beneficial for protecting both consumers and producers from economic fraud and might also help protect toothfish from over-exploitation as well as combat illegal, unreported, and unregulated (IUU) fisheries. © 2021 Society of Chemical Industry.

Flatfish may be underestimated in the diet of gray seals (Halichoerus grypus)

The United States east coast population of the gray seal (Halichoerus grypus (Fabricius, 1791)) was once hunted to near extirpation, but the population has since rebounded due to protection by the Marine Mammal Protection Act of 1972. Although this population growth is seen as a success by conservationists, others are concerned about the economic and ecological impact of gray seals on New England fisheries. The study objective was to quantify flatfish presence in the diet of gray seals using an analysis of prey DNA in seal scat (molecular scatology). This may reduce a potential bias in other diet analysis methods since flatfish may not be swallowed whole, and therefore, their identifying otoliths may not be present. Scats were collected from Muskeget and Monomoy islands off Cape Cod, Massachusetts, USA, in 2016, 2017, and 2018 and analyzed for flatfish presence using species-specific polymerase chain reaction (PCR) assays and a hard parts analysis using otoliths. Frequency of occurrence for flatfish species from DNA was higher than estimated in previous literature on gray seal diet and a concurrent otolith analysis of the same samples, suggesting that previous analyses may have potentially underestimated the importance of flatfish in the diet.

Characterization and Antimicrobial Resistance of Escherichia coli and Its Serotypes Isolated from Poultry Feed in Relation to Seasons in Karachi, Pakistan

Background: The increasing prevalence of Escherichia coli as a foodborne pathogen in poultry poses a high risk to food safety. The resistant strains of E. coli may contribute resistant genes to human endogenous flora, causing fatal diseases. Moreover, these pathogens are serious threats to poultry farming. Objectives: The current study aimed to investigate the effects of seasonal variations on the bacterial load of E. coli and to evaluate its antibiogram profile. Methods: All feed samples were evaluated for the identification of E. coli and its serotypes, using conventional culture methods and biochemical characterization. Positive samples were confirmed by polymerase chain reaction (PCR) assay. The bacterial load of E. coli was estimated by measuring the total viable count, and the antibiogram data were calculated using two methods, that is, disc diffusion method and minimum inhibitory concentration (MIC) measurement. Results: Of 204 feed samples investigated, 38 isolates were positive for E. coli. All positive samples were also confirmed via universal and species-specific PCR assays, and 8/38 were documented as E. coli 0157:H7 strains. The bacterial load of E. coli was also determined by measuring the total viable count, and the results revealed the highest ratio (6.44×108 CFU/g) from June to August and the lowest ratio (2.06×108 CFU/g) from December to February. The multidrug resistance of E. coli O157:H7 was validated by antimicrobial susceptibility tests since all isolates showed high resistance to chloramphenicol, penicillin derivatives, fluoroquinolones, and oxytetracycline, respectively, and were only susceptible to aminoglycosides. Conclusions: Considering the high bacterial load of E. coli from June to August, the poultry industry needs to establish appropriate and effective hygienic and storage policies, especially during these alarming months. Moreover, surfacing and propagation of resistant strains of these pathogens may obscure future assessments for treatment purposes.

Comparative evaluation of three agar media-based methods for presumptive identification of seafood-originated Vibrio parahaemolyticus strains

Vibrio parahaemolyticus has been one of the major bacterial species responsible for summer food poisoning. However, the limitations of current medium-based strategies for its detection necessitates a more effective selective or discriminatory agar media-based method. In this study, we first developed ChromoVP agar for more specific differentiation of V. parahaemolyticus from other bacterial species. Secondly, we compared this new medium with the commercially-available CHROMagar™ Vibrio medium and thiosulfate–citrate–bile salts–sucrose (TCBS) agar medium for the identification of V. parahaemolyticus strains isolated from seafood. The sensitivity, specificity, accuracy, and positive and negative predictive values of each of these agar media-based methods in the identification of V. parahaemolyticus were determined and compared using 38 V. parahaemolyticus strains and 102 other Vibrio and non-Vibrio strains. As a benchmark, a species-specific polymerase chain reaction assay was performed to detect V. parahaemolyticus and four other Vibrio species in seafood samples based on their genetic differences. Our results showed that ChromoVP agar was more sensitive (89.5%), specific (89.2%), and accurate (89.3%) than CHROMagar™ Vibrio and TCBS agar media in differentiating V. parahaemolyticus strains from other organisms. Therefore, ChromoVP agar is an effective medium for the isolation and enumeration of V. parahaemolyticus from seafood samples.

Standards for Methods Utilizing Environmental DNA for Detection of Fish Species.

Environmental DNA (eDNA) techniques are gaining attention as cost-effective, non-invasive strategies for acquiring information on fish and other aquatic organisms from water samples. Currently, eDNA approaches are used to detect specific fish species and determine fish community diversity. Various protocols used with eDNA methods for aquatic organism detection have been reported in different eDNA studies, but there are no general recommendations for fish detection. Herein, we reviewed 168 papers to supplement and highlight the key criteria for each step of eDNA technology in fish detection and provide general suggestions for eliminating detection errors. Although there is no unified recommendation for the application of diverse eDNA in detecting fish species, in most cases, 1 or 2 L surface water collection and eDNA capture on 0.7-μm glass fiber filters followed by extraction with a DNeasy Blood and Tissue Kit or PowerWater DNA Isolation Kit are useful for obtaining high-quality eDNA. Subsequently, species-specific quantitative polymerase chain reaction (qPCR) assays based on mitochondrial cytochrome b gene markers or eDNA metabarcoding based on both 12S and 16S rRNA markers via high-throughput sequencing can effectively detect target DNA or estimate species richness. Furthermore, detection errors can be minimized by mitigating contamination, negative control, PCR replication, and using multiple genetic markers. Our aim is to provide a useful strategy for fish eDNA technology that can be applied by researchers, advisors, and managers.

Gardnerella vaginalis Clade Distribution Is Associated With Behavioral Practices and Nugent Score in Women Who Have Sex With Women.

Gardnerella vaginalis is detected in women with and without bacterial vaginosis (BV). Identification of 4 G. vaginalis clades raised the possibility that pathogenic and commensal clades exist. We investigated the association of behavioral practices and Nugent Score with G. vaginalis clade distribution in women who have sex with women (WSW). Longitudinal self-collected vaginal specimens were analyzed using established G. vaginalis species-specific and clade-typing polymerase chain reaction assays. Logistic regression assessed factors associated with detection of G. vaginalis clades, and multinomial regression assessed factors associated with number of clades. Clades 1, 2, and 3 and multiclade communities (<2 clades) were associated with Nugent-BV. Clade 1 (odds ratio [OR], 3.36; 95% confidence interval [CI], 1.65-6.84) and multiclade communities (relative risk ratio [RRR], 9.51; 95% CI, 4.36-20.73) were also associated with Lactobacillus-deficient vaginal microbiota. Clade 4 was neither associated with Nugent-BV nor Lactobacillus-deficient microbiota (OR, 1.49; 95% CI, 0.67-3.33). Specific clades were associated with differing behavioral practices. Clade 1 was associated with increasing number of recent sexual partners and smoking, whereas clade 2 was associated with penile-vaginal sex and sharing of sex toys with female partners. Our results suggest that G. vaginalis clades have varying levels of pathogenicity in WSW, with acquisition occurring through sexual activity. These findings suggest that partner treatment may be an appropriate strategy to improve BV cure.

Two for the price of one: Co-infection with Rickettsia bellii and spotted fever group Rickettsia in Amblyomma (Acari: Ixodidae) ticks recovered from wild birds in Brazil

The bacterium Rickettsia bellii has been detected in 25 species of ticks in the American continents, but its pathogenic potential is considered as undetermined. A possible role for this species in the phenomenon of transovarial exclusion of pathogenic members of the spotted fever group (SFG) of Rickettsia has been suggested and co-infections with pathogenic species have been reported infrequently in both North and South America. Traditional methods for the molecular detection of rickettsial agents in ticks focus largely on the identification of sequences found in SFG Rickettsia, an approach that may overlook the presence of co-infections with R. bellii. Two novel, species-specific polymerase chain reaction (PCR) assays, targeting the genes encoding the surface cell antigen (Sca), autotransporter proteins sca9 and sca14, were developed and validated for the detection of R. bellii using 150 Amblyomma ticks collected from wild birds in Brazil. Co-infection of R. bellii infected ticks was evaluated using a novel PCR assay targeting the ompA sequence characteristic of SFG Rickettsia. Preliminary species-level identification was achieved by restriction fragment length polymorphism (RFLP) analysis and subsequently confirmed by sequencing of amplicons. Nine out of seventy-three Amblyomma longirostre and one of two Amblyomma calcaratum ticks were shown to be co-infected with R. bellii and Rickettsia amblyommatis, while two out of sixty-seven Amblyomma sp. haplotype Nazaré ticks were recorded as co-infected with R. bellii and the Rickettsia parkeri-like bacterium, strain ApPR. Interestingly, our data represent the first records of R. bellii in association with A. calcaratum and Amblyomma sp. haplotype Nazaré. The novel PCR-RFLP systems reported herein, provide an alternative, rapid and cost-efficient (relative to strategies based on sequencing or real-time PCR), approach to evaluate rickettsial co-infection of ticks, a potentially significant phenomenon that has most likely been underestimated to date.

Species-specific PCR assays for Gambierdiscus excentricus and Gambierdiscus silvae (Gonyaulacales, Dinophyceae).

The two most toxic Gambierdiscus species identified from the Caribbean are G.excentricus and G.silvae. These species are the primary causes of ciguatera fish poisoning and likely contribute disproportionately to the toxicity of marine food webs. While Gambierdiscus species are difficult to distinguish using light or scanning electron microscopy, reliable species-specific molecular identification methods have been developed and used successfully to identify a number of other Gambierdiscus species. Corresponding species-specific assays are not yet available for G.excentricus and G.silvae, which imposes limitations on species identification and related ecological studies. The following note describes species-specific polymerase chain reaction assays for G.excentricus and G.silvae that can be used for these purposes.

Potential for Seed Transmission of Verticillium longisporum in Oilseed Rape (Brassica napus).

Verticillium longisporum is a soilborne vascular fungal pathogen that has spread throughout the European oilseed rape cultivation area since the 1980s and was detected in canola fields in Canada in 2014. In a series of greenhouse and field inoculation experiments using V. longisporum-resistant and susceptible cultivars of winter and spring types of oilseed rape, the present study investigated the potential of V. longisporum dissemination by seeds of Brassica napus. Greenhouse inoculation studies with a DsRed-labeled isolate of V. longisporum confirmed the systemic growth of the pathogen from roots to seeds. Further monitoring of plant colonization in the greenhouse with a species-specific real-time polymerase chain reaction assay verified the pathogen growth from roots to stem bases, pods, and seeds in root-inoculated plants. The frequency of recovery of viable colonies of V. longisporum from seeds harvested from greenhouse-grown inoculated plants ranged from 0.08 to 13.3%. The frequency of seed transmission in the greenhouse differed in oilseed rape cultivars varying in susceptibility to V. longisporum. Subsequent studies on transmission of the disease into the offspring revealed that only 1.7 to 2.3% of plants showed disease symptoms as confirmed by the formation of microsclerotia in the stems. Results from field-grown plants differed from the greenhouse studies. The degree of seed transmission in the field was dependent on the crop type. Although only low concentrations of DNA of V. longisporum were detectable in seeds harvested from severely infected winter oilseed rape, significantly greater concentrations of fungal DNA were found in seeds of spring-type oilseed rape, at similar soil conditions and inoculum densities. Correspondingly, plating seeds that were harvested from infected plants on agar yielded viable V. longisporum colonies only from seeds of the spring-type but not of the winter-type plants. Lack of seed infection in the winter-type crop was confirmed in two seasons. Equally, none of the offspring grown from seeds from severely diseased winter oilseed rape plants developed symptoms of Verticillium stem striping. The results suggest that the rate of seed transmission of V. longisporum depends on the degree of plant colonization, which is significantly faster under greenhouse than field conditions and in a spring-sown crop compared with an autumn-sown oilseed rape crop. According to our studies, disease transmission by seeds from European winter oilseed rape production cannot be confirmed.