Molecular Identification Techniques Research Articles

Pathogenetic identification in ticks and yaks from Zoige County, China

Background:Ticks represent a significant vector for the transmission of infectious diseases, with the prevalence of tick-borne diseases becoming a prominent global health concern in recent decades. Anaplasma spp., Rickettsia spp., and Piroplasma have been identified as significant pathogens with the potential to impact human and animal health. However, there is a dearth of data concerning the prevalence of these pathogens in the eastern Tibetan Plateau, China.MethodsIn this study, a total of 643 Dermacentor silvarum and 314 Haemaphysalis longicornis were identified through the application of morphological and molecular identification techniques on 957 ticks collected from yaks in Zoige County. The assessed of Anaplasma spp., Rickettsia spp., Theileria spp., and Babesia spp. was assessed in 957 ticks and 96 blood samples collected from yaks.ResultsSignificant discrepancies were observed in the positivity rates for the four pathogens among the tick species and sampling sites. The identification of different species within the four pathogens was based on the analysis of the 16S rRNA of Anaplasma spp., the ompA and ompB genes of Rickettsia spp., and the 18S rRNA of Theileria spp. and Babesia spp. The prevalence ranges of the four pathogens are 9.9-50.2%, 29.5-100%, 16.2-46.4%, and 14.5-58.4%, respectively.ConclusionIn view of the growing zoonotic risks, further investigations into the prevalence of additional pathogens in ticks and animals, including livestock, in the eastern Tibetan Plateau, China, are essential.

A molecular approach to identify parrotfish (Sparisoma) species during early ontogeny.

Sparisoma species (parrotfish) comprise an important functional group contributing to coral-reef resilience. The morphological diagnostic characteristics for species identification are clearly described for adult forms but not for the early stages. Consequently, many taxonomical listings of Sparisoma larvae are restricted to the genus level. The aims of this study are to determine whether the morphological and molecular identification techniques are useful to assign the species taxonomic level to Sparisoma larvae occurring in the Gulf of Mexico and whether there is a set of diagnostic features that could be used to discriminate between species in larvae of different developmental stages. Morphological assignment of Sparisoma was performed based on morphological and meristic features for 30 larvae collected in the Gulf of Mexico from late August to mid-September 2015. To corroborate and complement the morphological assignments, molecular identification was carried out using DNA sequences from regions of two mitochondrial genes, mitochondrial cytochrome oxidase I (mtDNA COI) and mitochondrial 16S rRNA (mtDNA 16S rRNA). COI and 16S gene trees for Sparisoma and related fish taxa were constructed using sequences available in the NCBI (National Center for Biotechnology Information) GenBank and BOLD (Barcode of Life Data) databases. Two morphotypes were identified based on morphology, but no diagnostic characteristics for species discrimination were found. Molecular identification, in contrast, successfully discriminated four early development stages of Sparisoma atomarium, three stages of Sparisoma radians, and two stages of Sparisoma chrysopterum and Sparisoma aurofrenatum, therefore demonstrating the successful and necessary application of molecular taxonomic approaches for species-level identifications of Sparisoma larvae.

Commercial Trade and Conservation: DNA Barcoding of Wild Reticulated Python (Malayopython reticulatus) (Schneider, 1801) for Sustainable Management

Abstract The reticulated python, scientifically known as Malayopython reticulatus, is a prominent member of the Pythonidae family, exhibiting a wide geographic distribution across Southeast Asia. Like most python species, it is listed on Appendix II of CITES, allowing for regulated harvest and commercial export. However, inadequate monitoring of snakeskin origins within Malaysia has facilitated illegal trade, exacerbating concerns over population declines. Thus, this study aims to document the DNA barcoding of M. reticulatus in Pantai Penyabong, Johor, utilizing the COI gene marker of mitochondrial DNA (mtDNA). Observations revealed 11 reticulated pythons, with two successfully captured for blood sample extraction and amplification. Sequences obtained exhibited a high identity percentage in the NCBI GenBank database, supported by similar topologies in phylogenetic trees. These findings demonstrate the efficacy of molecular identification techniques in combating illegal wildlife trade. Governments and law enforcement agencies can enforce conservation laws and protect wild populations by verifying mislabeled python species. This study highlights the crucial role of DNA barcoding in wildlife forensics and conservation management, offering a rapid and reliable method for species identification and monitoring in the face of escalating threats.

Taxonomic revision of Cantharellus cuticulatus Corner (Cantharellaceae, Basidiomycota) from Sabah (North Borneo), Malaysia

Cantharellus cuticulatus, first described by E.J.H. Corner in 1966, is an edible mushroom distinguished by its vibrant yellow-orange cap, lamellae-like hymenium and the absence of clamp connections. This study reveals a significant shift in its distribution from the high-altitude regions of Mount Kinabalu to lowland areas, raising interesting questions about its habitat preferences and associated host plants. The research was conducted between 2021 and 2023, involving comprehensive macro- and micro-morphological analyses, as well as molecular identification techniques. After a hiatus of over half a century without any new records in Sabah, the study has successfully collected three Cantharellus cuticulatus specimens from three different districts in Sabah. To date, this species has only been documented in Peninsular Malaysia and Northern Borneo (Sabah, Sarawak and Brunei).

Tinea capitis caused by Microsporum canis: A case study of three family members in India, a non-endemic region

Tinea capitis, a common scalp infection primarily affecting children, is caused by keratinophilic dermatophytic fungi, notably Microsporum and Trichophyton species. Microsporum canis, primarily transmitted from cats and dogs to humans, is rarely reported in non-endemic regions like India. We report a cases involving three family members from Delhi, India, diagnosed with tinea capitis caused by Microsporum canis. The index case, a five-year-old boy, contracted the infection through contact with a cat, while his younger brother and sister acquired it through human-to-human transmission within the family. Clinical examination, microscopic analysis, and molecular identification techniques confirmed the diagnosis. Antifungal susceptibility testing revealed sensitivity to itraconazole and terbinafine but resistance to griseofulvin. Treatment with oral terbinafine and topical ketoconazole cream led to successful outcomes for all three patients. Molecular typing confirmed clonality of the isolates, indicating human-to-human transmission. This case study underscores the significance of considering atypical sources of infection and human-to-human transmission in the diagnosis and management of tinea capitis caused by Microsporum canis in non-endemic regions. It emphasizes the necessity of thorough contact history assessment and appropriate antifungal therapy for effective control of the infection.

Morphological Development and DNA Barcoding Identification of Pholis fangi Larvae and Juveniles in the Yellow Sea

Pholis fangi is a small, bottom-dwelling fish species endemic to the Yellow Sea and Bohai Sea in China. While previous research has focused on its population biology and molecular structure, little is known about the early developmental stages of this species. In this study, larval and juvenile P. fangi specimens were collected from the Sheyang Sea Area, Jiangsu Province, in 2017. Morphological features were examined using microscopy, and DNA barcoding was conducted to confirm species identification. The research documented detailed changes in yolk sac, fin development, and melanophore distribution patterns across larval and juvenile stages of P. fangi. Comparative analysis with other Pholis species revealed that melanophore distribution is a key distinguishing characteristic, allowing effective differentiation between larval and juvenile stages, as well as between Pholis species. This study provides valuable insights into the early life history of P. fangi, contributing to a better understanding of the genus Pholis. The findings demonstrate the utility of combining traditional morphological observation and molecular techniques for accurate species identification, particularly during the critical larval and juvenile developmental phases.

Multiplex PCR specific for genus Phytophthora and P. nicotianae with an internal plant DNA control for effective quarantine of Phytophthora species in Japan

To prevent threats from pathogens such as Phytophthora species from international plant trade, molecular identification techniques are needed for rapid, accurate quarantine inspection. Here, for quarantine control in Japan, we developed a simple DNA extraction for plants and a practical detection method that combines multiplexed PCR using primers specific for Phytophthora species, for P. nicotianae, which is the only non-quarantine Phytophthora species, and as internal controls, for plants. For the new genus-level primer set, we modified previously reported genus-specific primers to improve detectability. The new primers were able to detect mycelial DNA of 155 taxa among Phytophthora clades 1–10, with a sensitivity of 100 fg/µL for three representative species, P. ramorum, P. kernoviae and P. nicotianae. In the PCRs using DNA from non-target species, amplification was observed for only three taxa, and for some strains, four taxa in a closely related genus. Duplex and triplex PCR of the genus-specific primers combined with previously reported plant primers verified the success of DNA extraction and PCR detection from diseased plant samples, and in the triplex PCR, whether the pathogen was diagnosed as P. nicotianae or not by the species-specific primer. The new method detected the pathogen in naturally infected and inoculated plants. The amplicons using the genus-specific primer have enough variation to be sequenced to identify the species. This new method can be used immediately for detecting Phytophthora species and for quarantine control in Japan.

Are artefacts misleading the diagnosis of Ascaris lumbricoides in microscopy? Observations.

Stool has multiple components, which include undigested food material, plant, animal products, normal intestinal microbiome, and parasites. Due to the existence of all the elements, stool parasite examination is cumbersome, especially with identification of the eggs of Ascaris lumbricoides. We examined 650 stool samples of pregnant women before anti-helminthic treatment. We found that the prevalence ofAscaris lumbricoideswas 5.4% (95% CI 3.8-7.4, n = 35) by a single observer in microscopy, and the majority (33/35) were identified as decorticated fertilized eggs. The prevalence of Ascaris by molecular methods was 2.6% (95% CI 1.5-4.2%, n = 17). Five samples were positive by both methods. The prevalence of structures resemblingAscariswas 4.6% (95% CI 3.1-6.5, n = 30). Three of the positive samples were confirmed with sequencing. With the subjective nature of microscopy along with the naked eye examination, errors can happen. Hence adequate training and confirmation with molecular techniques for identification of Ascaris lumbricoides are advisable.

Identifying organisms directly from BacT/Alert FAN plus blood culture bottles using Vitek MS in a state-wide laboratory network

Identifying organisms directly from positive blood culture bottles using matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF MS) has many advantages to patients, clinical services, and laboratories. However, few published methods have demonstrated good performance using the current BioMérieux culture bottles and MALDI-TOF system: BacT/Alert FAN plus and Vitek MS. The effect of transporting bottles on test performance has not been assessed for any direct-from-bottle MS method. In this study, 802 positive blood culture bottles were analysed including 234 requiring inter-laboratory transport, using a method involving protein extraction with formic acid and acetonitrile. Correct identification rates were high for Staphylococcus aureus (58/58 of new diagnostic samples), Enterococcus faecalis (27/27), Gram-negative bacilli (160/176, 90.1%), and coagulase-negative Staphylococcus species (108/132, 81.8%). Three false identifications were made, none with clinical significance. For Gram-positive cocci in pairs or chains, more correct identifications were made from bottles analysed immediately compared to transported bottles (67% vs 44%, p=0.016), and longer transport time was associated with slightly lower probability of correct identification (OR 0.984 per additional hour, p=0.040). Transportation was not associated with a difference for other organism types. This technique is a vastly more cost-effective alternative to molecular techniques for rapid identification of bacteraemia isolates, and performance is minimally affected by inter-laboratory transport of bottles at ambient temperature.

Genotyping, bionomics and host-feeding behavior of Phlebotomus spp. (Diptera: Psychodidae) in Iran

The main goal of the current study was to investigate the molecular systematics and host–vector interactions of sandfly species (Phlebotomus spp.) in Iran. Sandflies were collected using CDC light traps, sticky traps, and BG-Sentinel traps at seven trapping sites in Iran during 2019 and 2020. The source of blood meals was identified using primer pairs specific to either the 16S rRNA or the mitochondrial cytochrome b (cytb) genes. A total of 736 blood-fed female sandflies were collected. Six species of the genus Phlebotomus (Ph.) and four species of the genus Sergentomyia (Se.) were identified using molecular identification techniques. The most abundant sandfly species were Ph. papatasi (38.45 %) and Se. sintoni (36.28 %). Blood meal analysis identified 19 different host species, specifically, 15 mammalian and four bird species. The most common blood meal sources were Rattus norvegicus, followed by Homo sapiens, Bos primigenius f. taurus, Ovis orientalis f. aries, Meriones persicus, Gallus gallus f. domestica, Rhombomys opimus, and Canis lupus; other host species had incidences of less than 5 %. We observed that Ph. major complex and Se. sintoni had mostly mammalophilic blood-feeding behavior, while Ph. papatasi and Ph. sergenti had mostly anthropophilic and ornithophilic blood-feeding preferences, respectively. The current results confirmed cytb is a suitable gene for regular species level identification, and that phylogenetic analyses of cytb allowed for clear demarcation of sandfly species.

Application of recombinase polymerase amplification with CRISPR/Cas12a and multienzyme isothermal rapid amplification with lateral flow dipstick assay for Bactrocera correcta.

Bactrocera correcta is a quarantine pest that negatively impacts the fruit and vegetable industry. Differentiating B. correcta from similar species, especially in non-adult stages, remains challenging. Rapid molecular identification techniques, such as recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a and multienzyme isothermal rapid amplification with lateral flow dipstick (MIRA-LFD), play a crucial role in early monitoring and safeguarding agricultural production. Our study introduces two methods for the rapid visual identification of B. correcta. Bactrocera correcta specific RPA primers, CRISPR RNA (crRNA), and the LFD probe were designed based on the cox1 genes. The RPA reaction conditions were optimized (at 37 °C for 8 min) for effective template DNA amplification. Two nucleic acid detection methods were established to visualize RPA. In the RPA-CRISPR/Cas12a system, the optimal LbCas12a/crRNA concentration ratio was 200:400 nmol L-1. Successful amplification was determined by the presence or absence of green fluorescence following 15 min incubation at 37 °C. The MIRA-LFD system achieved precise identification of the target species within 4 min at 37 °C. Both methods exhibited high specificity and sensitivity, allowing for detection from 1.0 × 10-1 ng μL-1 of DNA. Combined with rapid DNA extraction, rapid identification of individual B. correcta at different developmental stages was achieved, enhancing the practicality and convenience of the established methods. Our research findings demonstrate that both the RPA-CRISPR/Cas12a and MIRA-LFD methods for B. correcta detection was accurate and rapid (within 30 min and 10 min, respectively), at 37 °C. Our methods do not rely on expensive equipment, thus possess high practical value, providing improved identification solutions for port quarantine pests and field applications. © 2024 Society of Chemical Industry.

Isolation, characterization, and screening of yeast biodiversity for multi- hydrolytic enzymes

The current investigation was conducted with the objective of identifying yeasts that possess the ability to secrete a range of hydrolytic enzymes. Yeast isolates were collected from various samples, and the morphological characteristics of both the colony and yeast cells were examined. The capacity for enzyme production was assessed in multiple isolates by inoculating them in different media and quantifying their activities in terms of pectinase, amylase, urease, lipase, β-galactosidase, and protease. Out of a total of 42 yeast isolates, 30 displayed enzymatic activity. It is plausible that the yeast isolates obtained in this study hold potential for the production of industrial enzymes. Molecular identification techniques were employed to identify two isolates that exhibited multi-enzyme production and the highest enzyme activities. It was determined that these isolates belonged to two genera of yeasts, namely Pichia and Hanseniaspora. Specifically, isolate Y 16 was identified as Pichia kudriavizvii OL621856, while isolate Y 26 was identified as Hanseniaspora guillermondii OL621857 strains.

Occurrence of Pseudomonas cichorii Causing Midrib Rot of Lettuce Under Hydroponic Cultivation in Chile

In Chile, lettuce is the vegetable that has increased in cultivated area the most in recent years, reaching 8,309 ha. The Coquimbo Region contributes the most to this growth in production with 3,284 ha in 2022 (ODEPA 2023). Most lettuce is grown under open field conditions, but there is significant production in greenhouses and an increase in hydroponic production systems (INIA 2017). During April to June 2021 and 2022 in the Coquimbo Region, butterhead-type lettuce seedlings (Lactuca sativa) cv. Neil, cultivated under a hydroponic system, showed severe brown to black lesions in the leaves and midrib (Figure S1). To determine the etiology of this problem, samples of diseased plants were taken. Pieces of symptomatic tissue were macerated, and the extract was spread on nutrient agar (NA) and on King's B medium (KB) and incubated at 23°C for 48 h. The bacterial colonies observed were predominantly circular, creamy-white in color with irregular margins and fluorescent in KB medium. Isolates were gram-negative strictly aerobic. LOPAT test (Lelliot et al. 1966) results of two selected isolates were: levan production (-), oxidase reaction (+), potato soft rot (-), arginine dihydrolase production (-), and tobacco hypersensitivity (+), which corresponds to the profile of Pseudomonas cichorii. Molecular identification was performed through amplification and sequencing of the 16S rRNA (GenBank Accessions No. OR540674 to OR540675), gyrB and rpoD genes (Hwang et al. 2005; Sarkar and Guttmann 2004) (GenBank Accessions No. OR558279 to OR558282). BLAST analysis of the 16S rRNA gene of the isolates resulted in a match with a 99.86% identity with P. cichorii type strain ATCC 10857 (NR_112070.1). BLAST analysis of gyrB and rpoD resulted in a match with a 100% (630/630 bp) and >99% (546/550 bp) identity respectively, with strains of P. cichorii. Five six-month-old lettuce plants cv. Desert Storm were pricked in the midrib with a toothpick smeared with a fresh colony grown on KB medium. Seven days after inoculation, the plants showed dark brown, watery lesions, characteristic of damage caused by P. cichorii (Figure S1). Bacteria were isolated again from the inoculated plants and were identified as P. cichorii using LOPAT and molecular identification techniques. Midrib rot caused by P. cichorii was reported as an emerging disease of greenhouse-grown lettuce by Cottyn et al. (2009). In Chile, P. cichorii was previously described affecting nectarine fruits (Pinto de Torres and Carreño Ibañez 1983) and reported as a pathogen of lettuce among others horticultural crops by Servicio Agrícola y Ganadero of the Government of Chile (Acuña 2008), but this is the first report of P. cichorii affecting hydroponic lettuce plants in Chile. These results will be the basis of future studies to evaluate the origin of the infection, the potential dissemination, and the implementation of disease management to avoid the damage caused by this bacterium in hydroponic systems in this crop of growing importance in Chile.

Environmental influence on abundance and infection patterns of snail intermediate hosts of liver and intestinal flukes in North and Central Vietnam.

Liver and intestinal flukes (LIF) are important groups of foodborne zoonotic trematodes (FZTs) in Southeast Asia, including Vietnam. Their complex life cycles require specific freshwater snail species as the obligatory first intermediate hosts. In 2019, we conducted a longitudinal study in Yen Bai and Thanh Hoa provinces in North and Central Vietnam, respectively, to investigate the diversity of LIF and their infection prevalence in relation to snail host abundance and environmental factors. Using a combination of morphological and molecular identification techniques, we identified 10 LIF species infecting 11 snail host species. We observed significant seasonal variation in the mean abundance of several snail host species, with the majority of snails collected during the spring. We also detected seasonal changes in LIF species composition, with the highest species richness reported in the spring. Clonorchis sinensis and Fasciola gigantica, two medically important human liver flukes in Asia, were found only in the spring in Yen Bai. Our study revealed that not all snail host species have the same probability of becoming infected, and we recorded seasonal variations in the prevalence of LIF infection in different snail species in relation to water parameters.

When ice and sea are not barriers for flies: First report of Trichocera maculipennis (Diptera) in South America

Abstract During the 2022/2023 austral winter, large swarms of Trichocera (Saltrichocera) maculipennis Meigen, 1818 (Diptera: Trichoceridae), were observed around house roofs in Puerto Williams (Navarino Island, southern Chile, 54° S). In January 2023, the presence of this species was also confirmed in Punta Arenas (53° S), with specimens collected within the Chilean Antarctic Institute warehouse facilities. Trichocera maculipennis is native to the Holarctic region and became established on King George Island (South Shetland Islands, Maritime Antarctic) over 15 years ago. However, the species has not previously been reported from South America. Taxonomic and molecular identification techniques were used to confirm the identity of the new specimens obtained from Navarino Island and Punta Arenas. Preliminary observations of the fly's habitat preferences and swarming behaviour in Puerto Williams are provided, as well as comparisons in wing morphometrics between flies from Puerto Williams and King George Island. The fly's extensive distribution and high abundance in Puerto Williams are consistent with human‐facilitated introduction and several years' establishment. These findings emphasise the importance of implementing robust management strategies and biosecurity measures, particularly during transportation between logistical hubs in southern South America and the remote areas of the Cape Horn Biosphere Reserve and Antarctica.

Raman signal optimization based on residual network adaptive focusing

Due to its high sensitivity and specificity, Micro-Raman spectroscopy has emerged as a vital technique for molecular recognition and identification. As a weakly scattered signal, ensuring the accurate focus of the sample is essential for acquiring high quality Raman spectral signal and its analysis, especially in some complex microenvironments such as intracellular settings. Traditional autofocus methods are often time consuming or necessitate additional hardware, limiting real-time sample observation and device compatibility. Here, we propose an adaptive focusing method based on residual network to realize rapid and accurate focusing on Micro-Raman measurements. Using only a bright field image of the sample acquired on any image plane, we can predict the defocus distance with a residual network trained by Resnet50, in which the focus position is determined by combining the gradient and discrete cosine transform. Further, detailed regional division of the bright field map used for characterizing the height variation of actual sample surface is performed. As a result, a focus prediction map with 1μm accuracy is obtained from a bright field image in 120 ms. Based on this method, we successfully realize Raman signal optimization and the necessary correction of spectral information. This adaptive focusing method based on residual network is beneficial to further enhance the sensitivity and accuracy of Micro-Raman spectroscopy technology, which is of great significance in promoting the wide application of Raman spectroscopy.

Green extraction of puromycin-based antibiotics from Streptomyces albofaciens (MS38) for sustainable biopharmaceutical applications.

Background: Microbial secondary metabolites have shown promise as a source of novel antimicrobial agents. In this study, we aimed to isolate, characterize, and evaluate the antimicrobial activity of compound from a novel Streptomyces albofaciens strain MS38. The objective was to identify a potential bioactive compound with broad-spectrum antimicrobial properties. Methods: The isolated strain MS38 on starch casein agar was characterized using morphological, physiological, and molecular identification techniques. The compound was obtained from the fermented broth through extraction with n-butanol and further purification using silica gel column chromatography and high-performance liquid chromatography (HPLC). Structural elucidation was conducted using Ultraviolet (UV), Infrared (IR), nuclear magnetic resonance (NMR), and mass spectrometry (MS) techniques. The antimicrobial activity was evaluated using the agar well diffusion method and the microplate Alamar blue assay (MABA). Results: The isolated strain MS38 was identified as novel S. albofaciens based on morphological characteristics and confirmed by 16S sequences analysis and MALDI-TOF MS. The compound obtained from the fermented broth exhibited substantial antimicrobial activity against a variety of pathogenic bacteria and fungi. Structural analysis revealed a complex chemical structure with characteristic functional groups indicative of potential antimicrobial properties. The compound demonstrated strong activity against both Gram-positive (Staphylococcus Spp.) and Gram-negative (Klebsiella pneumoniae and Escherichia coli) bacteria, as well as fungi, including Candida albicans and Trichophyton rubrum. Conclusion: This study successfully isolated and characterized a bioactive compound from a novel S. albofaciens MS38. The compound exhibited significant antimicrobial activity against a range of pathogenic microorganisms. These findings underscore the importance of exploring microbial biodiversity for the discovery of novel antimicrobial agents. This study contributes to the growing knowledge of microbial secondary metabolites with potential therapeutic value.

Microbial characterization of Sichuan Baoning vinegar: lactic acid bacteria, acetic acid bacteria and yeasts.

Sichuan Baoning vinegar, a typical representative of Sichuan bran vinegar, is a famous traditional fermented food made from cereals in China. At present, there are few studies on microbial characterization of culturable microorganisms in solid-state fermentation of Sichuan bran vinegar. To comprehensively understand the diversity of lactic acid bacteria, acetic acid bacteria and yeasts, which play an important role in the fermentation of Sichuan bran vinegar, traditional culture-dependent methods combined with morphological, biochemical, and molecular identification techniques were employed to screen and identify these isolates. A total of 34 lactic acid bacteria isolates, 39 acetic acid bacteria isolates, and 48 yeast isolates were obtained. Lactic acid bacteria were dominated by Enterococcus durans, Leuconostoc citreum, Lactococcus lactis, and Lactiplantibacillus plantarum, respectively. Latilactobacillus sakei was the first discovery in cereal vinegar. Acetic acid bacteria were mainly Acetobacter pomorum and A. pasteurianus. The dominant yeast isolates were Saccharomyces cerevisiae, in addition to four non-Saccharomyces yeasts. DNA fingerprinting revealed that isolates belonging to the same species exhibited intraspecific diversity, and there were differences between phenotypic and genotypic classification results. This study further enriches studies on cereal vinegar and lays a foundation for the development of vinegar starters.

Evidence of zoonotic rickettsiae in ixodid ticks of domestic animals in some communal farms in the Eastern Cape Province, South Africa.

The abundance of tick populations in South Africa represents a probable risk for both animal and human health. Rickettsia spp. and Borrelia spp. are well-known agents of emerging human tick-borne infectious diseases worldwide. Nevertheless, the epidemiology of their infections has been underreported in South Africa. Therefore, this study aimed to profile zoonotic Rickettsia and Borrelia species from ticks infesting domesticated animals in the Eastern Cape, South Africa. Morphological and molecular identification techniques were conducted on 1,200 tick samples collected from domestic animals before screening for the target bacterial pathogens. The molecular identification of the tick samples was based on the amplification of the 12S rRNA mitochondrial Deoxyribonucleic acid. At the same time, those of Rickettsia and Borrelia species were carried out by amplifying fragments of gltA and ompB genes for Rickettsia and flaB gene for Borrelia spp. Thereafter, the positive amplicons for Rickettsia ompB were sequenced and further analyzed. Borrelia PCRs were negative; therefore, sequencing could not be performed. Eight species of ticks belonging to three genera; Rhipicephalus, Amblyomma, and Haemaphysalis, were identified. A total of 27% (320/1,200) samples were confirmed positive for Rickettsia, of which 23% (74/320) were positive for ompB genes. Phylogenetic analysis of ompB revealed a high homology to rickettsial reference strains from GenBank, with no positive result for Borrelia. The generated sequences showed homology with R. africae-KX227790 (100%), R. parkeri-KY113111 (99.8%), R. peacockii (99.3%), and R. slovaca-JX683122 (99.1%) representative sequences in GenBank. The findings from this study revealed that ticks harbored Rickettsia species with possible zoonotic potential.

Investigation of aspergillosis outbreak in young ducklings: Unraveling the role of hatcheries in Aspergillus fumigatus transmission.

Aspergillosis is a disease that affects several species of birds and causes substantial losses in the poultry business. The purpose of the investigation was to identify the pathogen responsible for a respiratory outbreak among juvenile ducklings. An epidemic of Aspergillosis infected a total of 800 Muscovy ducks that were being reared in El-Beheira Governorate. Tissue samples were obtained to isolate suspected fungi from diseased birds and the hatchery environment. In addition, identification and molecular characterization were performed on the obtained fungal isolates. Affected birds displayed acute respiratory manifestations such as difficulty breathing, gasping for air, nasal discharge, and a mortality rate of up to 28.1%. Postmortem examination revealed bronchitis, tracheitis, congested lungs, air sacculitis, severe multifocal granulomatous pneumonia, a congested, enlarged liver, and a congested kidney with nephritis. Mycological examination revealed seven Aspergillus (A.) spp. isolates from ducklings and six from hatcheries. Isolate colonial morphology and microscopical examination were as follows: A. fumigatus, A. niger, Syncephalastrum racemosum, and four untypable isolates. These isolates were further identified by polymerase chain reaction (PCR), and the internal transcribed spacers (ITSs) gene was detected. Four representative isolates were submitted for sequencing and further phylogenetic analysis. The source of duckling infection might be linked to the hatchery environment due to the observed similarity of isolates from both affected birds and the hatchery, as evidenced by phylogenetic analysis. Our findings demonstrated the significance of appropriate hatchery control in preventing infection in young ducklings. Furthermore, the use of molecular identification techniques would be helpful for tracing the source of infection and rapid diagnosis of Aspergillus in the field.