Species-specific PCR Research Articles

Widespread establishment of adventive populations of Leptopilina japonica (Hymenoptera, Figitidae) in North America and development of a multiplex PCR assay to identify key parasitoids of Drosophila suzukii (Diptera, Drosophilidae)

In recent years, there has been an increase in the adventive establishment and spread of parasitoid wasps outside of their native range. However, lack of taxonomic tools can hinder the efficient screening of field-collected samples to document the establishment and range expansion of parasitoids on continent-wide geographic scales. Here we report that Leptopilina japonica (Hymenoptera, Figitidae), a parasitoid of the globally invasive fruit pest Drosophila suzukii (Diptera, Drosophilidae), is now widespread in much of North America despite not having been intentionally introduced. Surveys in 2022 using a variety of methods detected L. japonica in 10 of 11 surveyed USA States and one Canadian Province where it was not previously known to occur. In most surveys, L. japonica was the most common species of D. suzukii parasitoid found. The surveys also resulted in the detection of Ganaspis cf. brasiliensis (Hymenoptera, Figitidae), the recently-released biological control agent of D. suzukii, in six USA States where it had not previously been found. These new detections are likely a result of intentional biological control introductions rather than spread of adventive populations. A species-specific multiplex PCR assay was developed as a rapid, accurate and cost-effective method to distinguish L. japonica, G. cf. brasiliensis, the closely-related cosmopolitan parasitoid Leptopilina heterotoma (Hymenoptera, Figitidae) and other native parasitoid species. This dataset and the associated molecular tools will facilitate future studies of the spread and ecological impacts of these introduced parasitoids on multiple continents.

Discovery of Fusarium pseudograminearum Causing Crown Rot of Winter Wheat in Xinjiang Uygur Autonomous Region, China.

Fusarium pseudograminearum is an important plant pathogen that invades many crops (Zhang et al. 2018). Since it was first discovered in Australia in 1951, F. pseudograminearum has been reported in many countries and regions and caused huge economic losses (Burgess et al. 2001). In 2012, crown rot of wheat caused by F. pseudograminearum was discovered for the first time in Henan Province, China (Li et al. 2012). Wheat (Triticum aestivum L.) is one of the most important food crops in Xinjiang Uygur Autonomous Region (XUAR), with 1.07 million hectares cultivated in 2020. In June 2023, a survey of crown rot disease was carried out in winter wheat cv. Xindong 20 in Hotan area, XUAR, China (80.148907°E, 37.051474°N). About 5% of wheat plants showed symptoms of crown rot such as browning of the stem base and white head. The disease was observed in 85% of wheat fields. In order to identify the pathogens, 36 pieces of diseased stem basal tissue, 0.5 cm in length, were collected and sterilized with 75% alcohol for 30s and 5% NaOCl solution for 2 min, then rinsed three times with sterile water and placed on potato dextrose agar (PDA) medium at 25°C. A total of 27 isolates with consistent morphological characteristics were obtained using single-spore technique (Leslie and Summerell. 2006), and the isolation rate was 75%. The isolates grew rapidly on PDA, produced large numbers of fluffy white hyphae, and pink pigment accumulated in the medium. The isolates were grown on 2% mung bean flour medium and identified by morphological and molecular methods. Macroconidia were abundant, relatively slender, curved to almost straight, commonly two to seven septate, and averaged 22 to 72 × 1.8 to 4.9 μm. Microconidia were not observed. The morphological characters are consistent with Fusarium (Aoki and O'Donnell. 1999). Two isolates (LP-1 and LP-3) were selected for molecular identification. Primers EF1/EF2 (5'-ATGGGTAAGGARGACAAGAC-3'/5'-GGARGTACCAGTSATCATG-3') were used to amplify a portion of the EF-1α gene (O'Donnell et al. 1998). The two 696 bp PCR products were sequenced and submitted to GenBank. The EF-1α gene sequences (GenBank Accession No: PP062794 and PP062795) shared 99.9% identity (695/696) with published F.pseudograminearum sequences (e.g., OP105187, OP105184, OP105179, OP105173). The identification was further confirmed by F. pseudograminearum species-specific PCR primers Fp1-1/Fp1-2 (Aoki and O'Donnell. 1999). The expected PCR products of 518 bp were produced only in F. pseudograminearum. Pathogenicity tests of LP-1 and LP-3 isolates were performed on 7-day-old seedlings of winter wheat cv. Xindong 20 using the drip inoculation method with a 10-μl of a 106 macroconidia ml-1 suspension near the stem base (Xu et al. 2017). The experiment was repeated five times in a 20 to 25°C greenhouse. Control seedlings were treated with sterile water. After 4 weeks, wheat seedling death and crown browning occurred in the inoculated plants with over 90% incidence. No symptoms were observed in the control plants. The pathogen was reisolated from the inoculated plants by the method described above and identified by morphological and PCR amplification using F. pseudograminearum species-specific primers Fp1-1/Fp1-2. No F. pseudograminearum was isolated from the control plants, fulfilling Koch's postulates. To our knowledge, this is the first report of F. pseudograminearum causing crown rot of winter wheat in XUAR of China. Since F. pseudograminearum can cause great damage to wheat, one of the most important food crops in China, necessary measures should be taken to prevent the spread of F. pseudograminearum to other regions.

New Species-Specific Real-Time PCR Assays for Colletotrichum Species Causing Bitter Rot of Apple.

Bitter rot of apple is an economically important worldwide disease caused by different Colletotrichum species, depending on many factors such as climate, geography, other hosts, and crop management practices. Culture, morphology, and single-locus sequencing-based methods for identifying the Colletotrichum species are severely limited in effectiveness, while the multilocus sequence typing methods available for delineating species are costly, time-intensive, and require high expertise. We developed species-specific hydrolysis probe real-time PCR assays for the following nine Colletotrichum species causing bitter rot in the Mid-Atlantic U.S.A.: C. fructicola, C. chrysophilum, C. noveboracense, C. gloeosporioides s.s., C. henanense, C. siamense and C. theobromicola from the C. gloeosporioides species complex, and C. fioriniae and C. nymphaeae from the C. acutatum species complex. After searching 14 gene regions, we designed primers and probes in 5 of them for the nine target species. Four primer-probe set pairs were able to be duplexed. Sensitivity tests showed as little as 0.5 pg DNA were detectable. These real-time PCR assays will provide rapid and reliable identification of these key Colletotrichum species and will be critically important for studies aiming to elucidate their biology, epidemiology, and management on apples as the number one produced and consumed tree fruit in the U.S.A.

Co-resistance to methicillin and clindamycin among coagulase-negative staphylococci isolates recovered from pet food in Brazil

Pet food have been considered as possible vehicles of bacterial pathogens. The sudden boom of the pet food industry due to the worldwide increase in companion animal ownership calls for pet food investigations. Herein, this study aimed to determine the frequency, antimicrobial susceptibility profile, and molecular characteristics of coagulase-negative staphylococci (CoNS) in different pet food brands in Brazil. Eighty-six pet food packages were screened for CoNS. All isolates were identified at species level by MALDI-TOF MS and species-specific PCR. Antimicrobial susceptibility testing was performed by disc diffusion and broth microdilution (vancomycin and teicoplanin only) methods. The D-test was used to screen for inducible clindamycin phenotype (MLS-B). SCCmec typing and detection of mecA, vanA, vanB, and virulence-encoding genes were done by PCR. A total of 16 (18.6 %) CoNS isolates were recovered from pet food samples. Isolates were generally multidrug-resistant (MDR). All isolates were completely resistant (100 %) to penicillin. Resistances (12.5 % - 75 %) were also observed for fluoroquinolones, sulfamethoxazole-trimethoprim, tetracycline, rifampicin, erythromycin, and tobramycin. Isolates were susceptible to vancomycin (MICs <0.25–1 μg/mL) and teicoplanin (MICs <0.25–4 μg/mL). Intriguingly, 3/8 (37.5 %) CoNS isolates with the ERYRCLIS antibiotype expressed MLS-B phenotype. All isolates harboured blaZ gene. Seven (43.8 %) isolates carried mecA; and among them, the SCCmec Type III was the most frequent (n = 5/7; 71.4 %). Isolates also harboured seb, see, seg, sej, sem, etb, tsst, pvl, and hla toxin virulence-encoding genes (6.3 % - 25 %). A total of 12/16 (75 %) isolates were biofilm producers, while the icaAB gene was detected in an S. pasteuri isolate. Herein, it is shown that pet food is a potential source of clinically important Gram-positive bacterial pathogens. To the best of our knowledge, this is the first report of MLS-B phenotype and MR-CoNS in pet food in Latin America.

Multiplex PCR Assay for Detection and Identification of Gnomoniopsis idaeicola, the Causal Agent of Blackberry Collapse

Blackberry collapse is an emerging disease in Oregon caused by Gnomoniopsis idaeicola. Symptoms associated with the disease include cane lesions, reduced primocane production, wilting, and plant death. Symptomatic plants are removed to reduce disease spread in the field. Unfortunately, blackberry collapse symptoms can be difficult to differentiate from those of other non-lethal cane diseases. Diagnostic procedures for blackberry collapse currently require several weeks to complete, necessitating development of a species-specific PCR assay to identify G. idaeicola in culture and in planta. The assay targets tef-1α and β -tubulin and includes primers as internal controls for PCR reactions of plant tissue or fungal cultures. This species-specific assay will provide rapid diagnosis of blackberry collapse and aid with epidemiological studies.

Characterization of β-lactamase and virulence genes in Pseudomonas aeruginosa isolated from clinical, environmental and poultry sources in Bangladesh.

The emergence and spread of multidrug-resistant pathogens like Pseudomonas aeruginosa are major concerns for public health worldwide. This study aimed to assess the prevalence of P. aeruginosa in clinical, environmental, and poultry sources in Bangladesh, along with their antibiotic susceptibility and the profiling of β-lactamase and virulence genes using standard molecular and microbiology techniques. We collected 110 samples from five different locations, viz., BAU residential area (BAURA; n = 15), BAU Healthcare Center (BAUHCC; n = 20), BAU Veterinary Teaching Hospital (BAUVTH; n = 22), Poultry Market (PM; n = 30) and Mymensingh Medical College Hospital (MCCH; n = 23). After overnight enrichment in nutrient broth, 89 probable Pseudomonas isolates (80.90%) were screened through selective culture, gram-staining and biochemical tests. Using genus- and species-specific PCR, we confirmed 22 isolates (20.0%) as P. aeruginosa from these samples. Antibiogram profiling revealed that 100.0% P. aeruginosa isolates (n = 22) were multidrug-resistant isolates, showing resistance against Doripenem, Penicillin, Ceftazidime, Cefepime, and Imipenem. Furthermore, resistance to aztreonam was observed in 95.45% isolates. However, P. aeruginosa isolates showed a varying degree of sensitivity against Amikacin, Gentamicin, and Ciprofloxacin. The blaTEM gene was detected in 86.0% isolates, while blaCMY, blaSHV and blaOXA, were detected in 27.0%, 18.0% and 5.0% of the P. aeruginosa isolates, respectively. The algD gene was detected in 32.0% isolates, whereas lasB and exoA genes were identified in 9.0% and 5.0% P. aeruginosa isolates. However, none of the P. aeruginosa isolates harbored exoS gene. Hence, this study provides valuable and novel insights on the resistance and virulence of circulating P. aeruginosa within the clinical, environmental, and poultry environments of Bangladesh. These findings are crucial for understanding the emergence of β-lactamase resistance in P. aeruginosa, highlighting its usefulness in the treatment and control of P. aeruginosa infections in both human and animal populations.

Adulteration detection of Gastrodia elata Blume and its commercial products using chloroplast DNA marker-based species-specific PCR assays

Gastrodia elata Blume has been recognized as a precious herbal medicine for thousands years due to its abundant bioactive ingredients and health-promoting features. The inadvertent or intentional adulteration of G. elata poses a severe crisis for consumers and significantly disrupts the market order. Therefore, accurate discrimination of G. elata from its adulterants is crucial to ensure its therapeutic effects and protect consumers’ interests. In this study, two species-specific PCR assays for molecular discrimination of G. elata from its five adulterants were established by targeting the chloroplast rps12 and clpP regions, respectively. Both of the assays can reliably identify as little as 0.1 % of deliberate adulteration with a detection threshold of only 0.01 ng of template DNA, and the assays are effective in detecting adulteration within G. elata as well as its commercial extracts. The present study provide a highly sensitive and reliable method for ascertaining the authenticity of G. elata and its commercial products, and the methodology can be employed in the authenticity and adulteration detection of other herbal products.

Development of PCR-based assays for the detection of the evident and latent infection with Stilbocrea banihashemiana, the causal agent of fruit tree cankers

The fig Bionectria canker, caused by Stilbocrea banihashemiana, is rapidly spreading in fruit trees, posing a significant threat to the horticultural industry. Early and specific detection of this pathogen is crucial for effective management. We developed a PCR-based assay using species-specific primers designed by exploiting sequence differences in the translation elongation factor 1-alpha (tef1) gene for early, rapid, and reliable diagnostics of this pathogen. The primers successfully amplified the target pathogen and did not react with other non-target fig canker-causing agents in direct and nested PCR. The designated primer pair, TEF-Sb3, with a 577 bp amplicon, demonstrated its efficacy by successfully detecting the pathogen with the lowest detectable concentration of 1 × 10−15 and 1 × 10−17 g.L−1 in direct and nested PCR, respectively. Furthermore, the selected primer pair had high performance in detecting the target pathogen in naturally infected samples with evident canker symptoms in nested PCR. Moreover, our molecular tool has effectively identified latent infection of S. banihashemiana in asymptomatic samples from fig and walnut trees from three different regions of Fars Province. This is a notable advancement, as traditional culture-based methods have failed to detect these infections. Our results suggested that the designed species-specific PCR assays were sufficiently sensitive, specific, and reliable for identifying S. banihashemiana from pure cultures and detecting the pathogen in symptomatic and asymptomatic trees. This assay can be a rapid and cost-effective tool for monitoring and managing Bionectria canker caused by S. banihashemiana in natural and potential host trees.

Early detection of rare and elusive endangered species using environmental DNA: a case study for the Eurasian otter and the white-clawed crayfish in northwestern Italy

AbstractMonitoring, management and conservation of rare and elusive species often requires early detection of individuals, especially for re-introduced and endangered taxa. Environmental DNA (eDNA) approaches can enhance the detection power of traditional biomonitoring methods for low-density, newly-established populations. In this study, we used species-specific Real Time PCR TaqMan assays to assess the presence of two endangered freshwater species, the white-clawed crayfish Austropotamobius pallipes and the Eurasian otter Lutra lutra at eight sites in four river catchments in Liguria (northwestern Italy). The Eurasian otter was considered extinct in the study area since the 1980s. However, recent, although scattered sightings indicated a recolonisation by a few individuals. The white-clawed crayfish populations declined drastically and became increasingly dispersed in the western part of Liguria. Our eDNA analysis confirmed the presence of both species in some of the selected rivers and detected Eurasian otter DNA where the species was not recorded through traditional monitoring methods. This study confirms eDNA-based monitoring approaches as valuable tools to assess the presence of rare and elusive species and help implement protection plans at a local scale.

Cultivable microbial diversity, peptide profiles, and bio-functional properties in Parmigiano Reggiano cheese.

Lactic acid bacteria (LAB) communities shape the sensorial and functional properties of artisanal hard-cooked and long-ripened cheeses made with raw bovine milk like Parmigiano Reggiano (PR) cheese. While patterns of microbial evolution have been well studied in PR cheese, there is a lack of information about how this microbial diversity affects the metabolic and functional properties of PR cheese. To fill this information gap, we characterized the cultivable fraction of natural whey starter (NWS) and PR cheeses at different ripening times, both at the species and strain level, and investigated the possible correlation between microbial composition and the evolution of peptide profiles over cheese ripening. The results showed that NWS was a complex community of several biotypes belonging to a few species, namely, Streptococcus thermophilus, Lactobacillus helveticus, and Lactobacillus delbrueckii subsp. lactis. A new species-specific PCR assay was successful in discriminating the cheese-associated species Lacticaseibacillus casei, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, and Lacticaseibacillus zeae. Based on the resolved patterns of species and biotype distribution, Lcb. paracasei and Lcb. zeae were most frequently isolated after 24 and 30 months of ripening, while the number of biotypes was inversely related to the ripening time. Peptidomics analysis revealed more than 520 peptides in cheese samples. To the best of our knowledge, this is the most comprehensive survey of peptides in PR cheese. Most of them were from β-caseins, which represent the best substrate for LAB cell-envelope proteases. The abundance of peptides from β-casein 38-88 region continuously increased during ripening. Remarkably, this region contains precursors for the anti-hypertensive lactotripeptides VPP and IPP, as well as for β-casomorphins. We found that the ripening time strongly affects bioactive peptide profiles and that the occurrence of Lcb. zeae species is positively linked to the incidence of eight anti-hypertensive peptides. This result highlighted how the presence of specific LAB species is likely a pivotal factor in determining PR functional properties.

Aggressive Alternaria brassicicola with reduced fungicide sensitivity can be associated with naturally infested broccoli seeds.

Alternaria brassicicola is a part of Alternaria complex that causes leaf blight and head rot (ABHR) in brassica crops. Infested broccoli seeds can play an important role in introducing A. brassicicola in transplant houses and production fields. However, characterization of natural seed infestation and seed-to-seedling transmission of A. brassicicola in broccoli is yet to be demonstrated. In this research we characterized Alternaria spp. isolates from commercial broccoli seedlots for their species identity, pathogenicity and aggressiveness on broccoli and their sensitivity to Quinone-outside inhibitor (QoI) fungicide (azoxystrobin). Two hundred commercial seedlots from two broccoli cultivars; Cultivar 1 (EC; n=100 seedlots) and Cultivar 2 (ED; n=100 seedlots) were evaluated for the presence of A. brassicicola under in-vitro conditions using a seedling grow-out assay. Alternaria spp. was detected in 31 and 28% of the commercial seedlots of Cultivar 1 and Cultivar 2, respectively. The seed-to-seedling transmission (%) varied considerably within each positive infested seedlot, which ranged from 1.3 to 17.3%. Subsequent molecular identification of single spore cultures (n=138) was made by sequencing four housekeeping genes; actin, the major allergen (Alta1), plasma membrane ATPase and Glyceraldehyde-3-phosphate dehydrogenase (GPD), and later the sequences were concatenated and compared for the phylogenetic distance with diverse Alternaria species. Ninety-six percent (n=133) of the isolates formed a cluster with a known A. brassicicola based on multigene phylogeny, which were later confirmed as A. brassicicola using a species-specific PCR assay. One hundred percent of the A. brassicicola seed isolates (n=133) were either highly- or moderately- aggressive on broccoli (cv. Emerald Crown) based on a detached leaf assay. Sensitivity of representative A. brassicicola isolates (n=58) to azoxystrobin was evaluated using a spore germination assay and the EC50 values (effective fungicide concentration (ppm) at which germination of conidia of isolates were reduced by 50% compared to control) for each isolate was determined. A. brassicicola isolates from naturally infested commercial broccoli seeds were sensitive to azoxystrobin with considerably low EC50 values in the range of <0.0001 ppm to 0.33 ppm; however, there were a few isolates (14%), which showed 100-fold reduced sensitivity from the most sensitive isolate (EC50 =0.0001 ppm). Our results confirm that commercial broccoli seedlots can be naturally contaminated with pathogenic and aggressive A. brassicicola. We also provide evidence for potential presence of A. brassicicola isolates with reduced azoxystrobin-sensitivity in naturally infested commercial broccoli seedlots, which has never been reported before. Together, these findings may have implications in considerations for seed-health testing, seed treatments and greenhouse scouting to limit introduction of infested seedlots in commercial broccoli fields.

Ticks (Acari: Ixodidae) and rickettsiae associated with wild boars in a rural area of Minas Gerais, Brazil

Wild boars or feral pigs are classified by the Brazilian Institute for the Environment and Renewable Resources (IBAMA) in “Category I of invasive exotic species”. They cause economic losses, harm the environment, serve as hosts and reservoirs for several zoonotic disease agents, and provide a blood meal for tick species that act as vectors for zoonotic diseases. The objective of this study was to identify tick species on wild boars, assess host-seeking ticks in the related environment, and identify other potential tick hosts coexisting with wild boars on a farm located in the state of Minas Gerais, southeastern Brazil. Additionally, the study aimed to determine the presence of rickettsiae in these arthropods and assess the exposure of wild boars to rickettsiae species from the Spotted Fever Group and Rickettsia bellii through serology. A total of 3585 host-seeking ticks from three species (Amblyomma sculptum – 41.58%; Amblyomma dubitatum – 0.39% and Rhipicephalus microplus – 0.05%) were collected in the environment and A. sculptum was the most abundant species. Thirty-one wild boars were evaluated, resulting in the collection of 415 ticks, all of which were A. sculptum. Rickettsia DNA was not detected in samples of A. sculptum and R. microplus from the environment or in A. sculptum ticks from wild boars. However, all A. dubitatum ticks (n = 14) had Rickettsia bellii DNA confirmed by the species-specific PCR protocol. Out of the 31 serum samples from wild boars, 24 reacted with at least one Rickettsia antigen. Among these, seven individuals exhibited a reaction to a probable homologous antigen (PHA) of three rickettsiae species: R. rickettsii (n = 3), R. amblyommatis (n = 3) and R. rhipicephali (n = 1). Despite the high prevalence of seroreactivity, titers were low, indicating limited exposure to Rickettsia spp. Camera traps generated 874 animal records, capturing a total of 1688 individuals. At least 11 species of birds and 14 species of mammals (12 wild and two domestic) shared the environment with wild boars and potentially shared ticks with them. These findings provide baseline information for understanding the sharing of ticks and tick-borne pathogens between wild boars and other animals within the Cerrado biome. Further studies are necessary to monitor the potential and actual risk of wild boars to harbor infected ticks and their role in the transmission and maintenance cycle of Rickettsia spp.

Diversity of haemoparasites in migratory small ruminants of Himalayas, India

Vector-borne haemoparasitic diseases of migratory small ruminants have been less explored to date. Therefore, transhumant migratory sheep and goats of the Himalayas were molecularly screened for various vector-borne pathogens like Anaplasma, Theileria, Babesia, Anaplasma marginale, Theileria luwenshuni, Theileria orientalis, and Trypanosoma evansi using their specific primers. Out of the 171 animals screened, 73.09% of the animals were infected with Anaplasma spp. and 74.85% of the animals with Theileria spp., while no animals were found infected with Babesia spp. and Trypanosoma evansi. In a quest to identify the species, a few positive PCR amplicons representing Anaplasma spp., Theileria spp., and Theileria/Babesia spp. were sequenced. The sequences obtained were further BLAST analyzed to reveal maximum identity with Anaplasma capra (100%), Theileria luwenshuni (99.71%), and Theileria sinensis (98.73%). Among them Theileria luwenshuni was further confirmed with their species-specific PCR and the positive amplicons were sequenced and BLAST analysed to reveal their maximumsimilarity with Chinese isolates rather than Indian isolates. Further, phylogenetic analyses of the resulting sequences were conducted to identify their evolutionary history. It revealed that 1100 bp- amplicons of Theileria spp. were showing maximum likeness to Theileria sp. MK, a non-transforming Theileria. This is the first report of zoonotic Anaplasma capra and Theileria sp. MK in small ruminants from India and it could be mostly due to their transboundary migration history through the Indo-Tibetan border during summer seasons as a part of the traditional migratory system of livestock rearing practice in the Himalayas. The high prevalence of haemoparasites in migratory small ruminants calls for the adoption of effective control measures.

Histopathology, Isolation and Molecular Detection of Pasturella multocida from Sheep in Gadag Region of Karnataka, India

Bacterial pneumonia is the most common respiratory problem in small ruminants. The heart blood and lung tissue samples from two dead sheep suffering from pneumonia at the age of six month were subjected for histopathology, bacterial isolation &amp; identification and antibiogram. Acute fibrinous bronchopneumonia was observed at necropsy and histopatholgy. The isolates were identified as P. multocida based on cultural and biochemical properties, and additionally confirmed by species-specific PCR amplification of KMT1 (Multocida toxin hydrolase gene) gene with the amplicon of 460 bp. The antibiogram showed susceptibility to chloramphenicol, β-lactams, quinolones, fluoroquinolones and aminoglycosides. In this study, the occurrence of ovine pasteurellosis caused by P. multocida with acute fibrinous bronchopneumonia was recorded.

Detection of Chlamydia psittaci in the Genital Tract of Horses and in Environmental Samples: A Pilot Study in Sardinia.

The members of the Chlamydiaceae family are important pathogens that infect a wide range of vertebrate hosts, including humans. Among them, Chlamydia psittaci, historically considered as an avian agent, has recently been identified in livestock, primarily sheep and cattle, but also in horses, with the infection being linked to reproductive disorders, such as abortion, absorption of embryos, stillbirth, and the birth of weak foals. Much less is known about chlamydial infections in the Sardinian equine population. This study aimed to identify the chlamydial diversity in genital samples from asymptomatic Sardinian horses. However, some horses had a previous history of reproductive disorders, i.e., abortion and infertility. A total of 60 horses (39 mares and 21 stallions) were opportunistically recruited from 17 equine farms in central-northern Sardinia. Vaginal and uterine swabs from mares and urethral swabs and seminal fluid from stallions were sampled for the presence of chlamydial DNA. Samples from environments where the horses lived were also tested for the detection of Chlamydia spp. Eight vaginal swabs (8/39; 20%), two uterine swabs (2/27; 7%), two seminal fluid samples (2/20; 10%), and one urethral swab (1/21; 4.7%) were found to be positive for Chlamydia spp. by PCR analysis. In addition, results from environmental samples showed the presence of Chlamydia spp. in three environmental swabs (3/8; 37.5%) and five water samples (5/16; 31.2%). Sequencing results revealed that strains here identified were 99-100% similar to members belonging to the Chlamydiaceae family, including C. abortus, C. psittaci, and uncultured Chlamydia genotypes. ompA species-specific PCR performed on samples was found to be positive after 16S rRNA amplification gave positive results for C. psittaci. These results reveal the first presence of C. psittaci in the genital tract of horses and in the environment in Sardinia and indicate that this pathogen could be the prevailing cause of infertility and abortion in the tested equines. However, these findings need further proof and highlight the importance of adopting a 'One Health' approach to control the presence of this zoonotic bacteria in domestic animals in order to understand its impact on people exposed to the infection risk.

Exploring the potential of extreme citizen science with Danish high school students using environmental DNA for marine monitoring

Citizen science involves non-professionals assisting with scientific research, contributing data, and conducting experiments under professional guidance. In this paper, we describe the citizen science project DNA &amp;amp; life, based at the Natural History Museum of Denmark, which actively engages high school students in both fieldwork and advanced laboratory analyses by collecting and analyzing eDNA samples from marine environments across Denmark. The analysis is performed via species-specific real-time PCR and in a case study we present data gathered in the project from 2017-2023 with focus on the potential of eDNA detecting in use for species monitoring. The results include seasonal occurrence of Mnemiopsis leidyi and national distributions of selected species of particular ecological interest Perca fluviatilis, Anguilla anguilla, Neogobius melanostomus and Alexandrium ostenfeldii. In addition to the eDNA case study, we present a new way of conducting citizen science and eDNA analysis, as a development of the established DNA &amp;amp; life project. Local DNA laboratories are created to shorten the path from sampling site to laboratory facility, creating the possibility for Danish high schools to participate in a new version of the project. In close collaboration with the academic staff of the DNA &amp;amp; life project both teachers and students help develop facilities, protocols and laboratory analyses in an extreme citizen science and co-creation approach, where the participants are involved in a higher level of laboratory work and data analysis. Allowing high school students to perform intricate molecular lab analyzes through an extreme citizen science approach has demonstrated encouraging outcomes and potential for data of high quality in terms of sampling and analyzing eDNA with the purpose of species monitoring and conservation.

Efficient and Direct Identification of Ditylenchus destructor and D. dipsaci in Soil and Plant Tissues Using a Species-Specific PCR Assay

Ditylenchus destructor and D. dipsaci are important nematodes that have a significant economic impact on agronomic and horticultural plants worldwide. Microscopic observation alone may not distinguish between D. destructor and D. dipsaci. Accurate and rapid identification of these two species is essential for effective pest management. In the present study, a species-specific PCR assay was developed to detect and differentiate D. destructor and D. dipsaci based on the rDNA-ITS sequences. The primers developed in this study can specifically amplify fragments of DNA from D. destructor and D. dipsaci in the target population, without amplifying DNA from other non-target nematodes within the genus Ditylenchus. The sensitivity test revealed that this procedure has the ability to detect single second-stage juveniles (J2) of D. dipsaci at a dilution of 1/128 and D. destructor at a dilution of 1/64. Additionally, it can detect genomic DNA (gDNA) at concentrations of 10 pg/µL for D. dipsaci and 1 ng/µL for D. destructor. These results align with previously reported results obtained through RPA and LAMP methods. Furthermore, the primers developed in this study for D. destructor not only were able to amplify six different haplotypes of nematodes but also successfully detected it in infested plant roots and soil samples, thereby shortening the time and reducing the number of steps required for detection. Thus, this assay, which does not necessitate taxonomic or morphological expertise, significantly enhances the diagnosis of D. destructor and D. dipsaci in infested fields. This advancement aids in the early control of these nematodes.

Identification of Lactic Acid Bacteria Strains Isolated from Sourdoughs Prepared with Different Flour Types

Species identification is the first step in the examination of newly isolated microorganism strains, including the cases when they are intended for application in the development of probiotic preparations or starters for different food products. The thorough identification process of newly isolated strains combines the application of different physiological, biochemical, and molecular genetic methods. The aim of the present study was to identify the species-level lactic acid bacteria (LAB) strains isolated from spontaneously fermented sourdoughs prepared from different flour types: Khorosan wheat, wheat, barley, buckwheat, spelled, spelt, and corn. Both classical phenotypic (cellular morphology characterization, and API 50 CHL) and molecular genetic methods (RAPD, ARDRA-analysis, 16S rDNA sequencing, and species-specific PCR) were applied. It was found that cultures with a short-rod morphology predominated among the 30 sourdough isolates. According to the RAPD profiles obtained, the isolates were divided into nine genotypes corresponding to nine genetically distinct strains. It was determined that individual sourdoughs made with different flour types shared cultures with a common genotype. The analysis of the physiological and biochemical profiles of the LAB isolates performed with the API 50 CHL system divided them into two groups according to their identification: Lactiplantibacillus plantarum (Lp. plantarum) 1 and Levilactobacillus brevis (Lv. brevis) 3. According to the 16S rDNA restriction profile, the LAB isolates showed two profiles corresponding to the Lp. plantarum and Lv. brevis groups. 16S rDNA sequencing and a comparison of the partially read 16S rDNA sequences of the studied isolates confirmed that some of them belonged to the Lv. Brevis species, but did not provide sufficient evidence that the rest of the cultures belonged to the Lp. Plantarum species. The species-specific PCR clearly separated the isolates from the Lp. plantarum group into two groups: isolates of the Lp. plantarum species and isolates of the Lp. paraplantarum species. The summary of the results of the conducted polyphasic taxonomic study determined the investigated LAB strains isolated from spontaneously fermented sourdoughs as representatives of the Lv. brevis, Lp. plantarum ssp. paraplantarum, and Lp. paraplantarum species.

Novel rpsK / rpsD primer-probe assay improves detection of Campylobacter jejuni and Campylobacter coli in human stool.

Campylobacter causes bacterial enteritis, dysentery, and growth faltering in children in low- and middle-income countries (LMICs). Campylobacter spp. are fastidious organisms, and their detection often relies on culture independent diagnostic technologies, especially in LMICs. Campylobacter jejuni and Campylobacter coli are most often the infectious agents and in high income settings together account for 95% of Campylobacter infections. Several other Campylobacter species have been detected in LMIC children at an increased prevalence relative to high income settings. After doing extensive whole genome sequencing of isolates of C. jejuni and C. coli in Peru, we observed heterogeneity in the binding sites for the main species-specific PCR assay (cadF) and designed an alternative rpsKD-based qPCR assay to detect both C. jejuni and C. coli. The rpsKD-based qPCR assay identified 23% more C.jejuni/ C.coli samples than the cadF assay among 47 Campylobacter genus positive cadF negative samples verified to have C. jejuni and or C. coli with shotgun metagenomics. This assay can be expected to be useful in diagnostic studies of enteric infectious diseases and be useful in revising the attribution estimates of Campylobacter in LMICs.

The Distribution of Sarcocsytis Species Described by Ungulates-Canids Life Cycle in Intestines of Small Predators of the Family Mustelidae.

Using molecular techniques, we have previously shown that carnivorous mammals of the family Mustelidae might be common definitive hosts for various protozoan Sarcocystis species. In the present study we aimed to unravel whether Sarcocystis species using ungulates as intermediate hosts and canids or felids as definitive hosts can be found in intestine of mustelids. Small intestine samples of 93 individual mustelids of five different species from Lithuania were examined. Sarcocystis species were identified based on species-specific PCR and subsequent cox1 sequencing. Six Sarcocystis species (S. arieticanis, S. bertrami, S. capracanis, S. capreolicanis, S. linearis and S. morae) defined by ungulate-canid life cycle were detected for the first time in small intestines of mustelids. By contrast, the prevalence of Sarcocystis characterised by ungulate-felid life cycle was low (3.2%). Overall, 76% of the examined animals were positive for at least one of the studied Sarcocystis species. Four species, S. arieticanis, S. bertrami, S. capracanis and S. morae were most commonly found, with the detection rate of about 40%. The current finding, in addition to our previous studies, suggests that mustelids play an important role in the spread of various Sarcocystis species.