Haemonchus contortus: Conventional and molecular identification and characterization via sequencing-based phylogenetic analysis in sheep

Samples collected from different stages of the tannery industry were found to be alkaline (pH 7.52 to 12.11). A good number of bacteria were found to be associated with the samples. The bacterial count ranged in between 1.34×105 to 3.44×105 cfu/ml and 1.04×105 to 6×105 cfu/ml on nutrient agar (NA) and peptone yeast extract glucose agar (PYG) medium, respectively. The maximum bacterial count was observed in bating stage while the minimum count was in the deliming stage. Primarily, 70 isolates were selected based on their different colonial morphology. After heat shock test 27 isolates were finally selected for identification and proteolytic potential. All the selected isolates were the members of different species of the genus Bacillus. The conventionally identified species were B. stearothermophilus (9), B. subtilis (4), B. brevis (3), B. pumilus (3), B. alcalophilus (2), B. badius (2), B. firmus (2) and B. lentus (2). Four important proteolytic isolates of Bacillus were selected for molecular identification. The isolates were confirmed as Bacillus subtilis strain B20 (L/P/2/1), B. subtilis strain PB18 (D/P/3/1), Bacillus sp. strain BVC38 (D/P/3′/2) and B. amyloliqefaciens strain Egy25 (B/N/3′/1). Except B/N/3′/1 all the conventional identification was in accordance with the molecular identification as the isolate B/N/3′/1 was conventionally identified as B. pumilus (B/N/3′/1). All the isolates showed positive proteolytic activities on skim milk agar and the zone ratio was in between 2.61 ± 0.44 and 6.42 ± 0.95. These isolates could be commercially utilized in the tannery and detergent industries for their proteolytic activity.

The Barber's Pole worm, Haemonchus contortus is of major concern to sheep producers, particularly in the southern hemisphere. This nematode is also commonly found in many sheep flocks in Northern hemisphere countries but is generally not associated with acute clinical pathology. As with other nematode species, the pattern of disease is changing in the United Kingdom. Changes in management practices, climate, anthelmintic resistance prevalence and parasite adaptation are possible factors thought to be responsible for this. In the present study, a combination of traditional applied parasitological and molecular species identification techniques were used to assess the capability of H. contortus infective larvae to over-winter on pasture and infect lambs in early spring. Adult and inhibited H. contortus worms were identified in previously worm-free tracer lambs that had grazed contaminated pasture in late winter/early spring (February/March). The study illustrated the benefit of using classical applied parasitology techniques in conjunction with molecular species identification methods to explore the epidemiology of gastro-intestinal nematodes of livestock. This study also demonstrated that larvae were able to survive over-winter, albeit in small numbers, and potentially contaminate pastures earlier than previously considered in northern regions of the UK.

During previous years, regarding the shifts in climate conditions in temperate region, such as occurrence of high temperatures and prolonged drought, increased occurrence frequencies of Aspergillus flavus and aflatoxins in cereal grains were recorded. A reliable and accurate identification of the fungi is of great importance for evaluating the microbiological risks of contamination. The essential point of the present investigation was molecular characterisation and identification of A. flavus isolates originating from common wheat and spelt grains collected after harvest during the period of three years (2015–2017) in Northern Serbia. A holistic approach that included PCR amplification of two DNA genomic regions and PCR-RFLP assay followed by fragment length analysis, provided complete and comprehensive characterisation of A. flavus isolated from wheat grains. The presented results indicate that there was no difference among the tested Aspergillus isolates on the molecular–genetic level. All 38 strains were identified as A. flavus by sequencing of combined ITS region and β-tubulin gene fragments (acc. no.: MH582473 to MH582510). PCR-RFLP method in combination with a Lab-on-a-chip (LoaC) electrophoresis can be successfully used to rapidly identify A. flavus isolates.

Molecular identification and phylogenetic analysis of gastrointestinal nematodes in different populations of Kazakh sheep

This study investigates the diversity and distribution of marine yeasts in southeastern Arabian Sea, focusing on coastal and offshore regions. Yeast strains were identified through ITS and D1/D2 domain rRNA gene sequencing and phylogenetic analysis. Microscopic analysis was performed to examine budding patterns and cell morphology. A total of 45 yeast strains were isolated from water samples collected at various depths from locations, including Kochi, Kollam, Trivandrum, Rameswaram, and the coastal waters of the Lakshadweep Islands (Minicoy, Kalpeni, and Kavaratti). Morphologically, the yeast strains were predominantly oval to rod-shaped and clustered into six clades: Debaryomyces, Kodamaea, Meyerozyma, Starmerella (Ascomycota), Rhodotorula and Sterigmatomyces (Basidiomycota). Notably, Starmerella strains were the most abundant, particularly in coastal waters. Among them, S. etchellsii and M. caribbica exhibited significant dry weight and protein concentrations, ranging from 13.9% to 65.1%. This study expands the limited knowledge of marine yeast diversity in Indian waters and provides insights into their molecular identification and potential biotechnological applications.

BackgroundHaemonchosis, due to infection with Haemonchus contributes to livestock morbidity globally, resulting in economic losses. Hybrids between Haemonchus contortus and H. placei have been evidenced, where sharing of hosts, geographical sympatry, and farming practices allow opportunities for hybridization. In Thailand, high prevelances of H. contortus infecting goats have been documented, and high levels of drug resistance are strongly suspected, due to unregulated and widespread use of anthelmintics (e.g. benzimidazoles and imidazothiazoles). Moreover, the exchange of genetic material facilitates the spread of anthelmintic resistance. Here, we aim to identify the Haemonchus species infecting goats, investigate their population genetic structure, and assess anthelmintic resistance to albendazole and levamisole.ResultsUsing 188 Haemonchus adults obtained from goats across six provinces in Thailand, molecular identification was performed using the nuclear ITS2 region. The population genetic structure was investigated by amplifying the mitochondrial COI gene of representative H. contortus specimens. Genotypic resistance to albendazole and levamisole resistance status were assessed via the single-nucleotide polymorphisms in the β-tubulin and hco-acr-8 gene, respectively. Of the specimens, 97.3% were molecularly identified as H. contortus, while 2.7% were potential hybrids between H. contortus and H. placei. Hybrids were identified in Nakhon Pathom, Kanchanaburi, Ratchaburi, and Suphanburi provinces. The population genetic structure of H. contortus revealed high genetic diversity, high gene flow, and low genetic differentiation between populations. High levels of albendazole resistance were detected, with an overall frequency of 0.56 and 0.44 for the susceptible and resistant alleles, respectively. Compared to albendazole, lower levels of levamisole resistance were obtained, with an overall frequency of 0.87 and 0.13 for the susceptible and resistant alleles, respectively.ConclusionsThis study revealed the hybrid form of H. contortus and H. placei in goats, high genetic diversity of H. contortus populations, and the presence of albendazole and levamisole resistance. The growing challenge of drug resistance and hybridization in Haemonchus populations demonstrates the urgent need for regulated drug use and the implementation of sustainable management practices.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12917-025-05133-9.

To understand the biocontrol ability of Arthrobotrys oligospora isolated from the Northern Tianshan Mountain slope pasture, Xinjiang region, China, on livestock gastrointestinal nematode diseases, we acquired 68 soil samples and isolated 26 nematode-trapping fungi using Haemonchus contortus L3. Eight isolates were identified based on their morphological and molecular identification. The predacious activity against H. contortus was detected before and after passage through the sheep gastrointestinal tract. As a result, these eight isolates were identified as A. oligospora. They displayed predacious activities ranging from 90% to 97%. Six of the isolates could pass through the sheep gastrointestinal tract significantly reducing the number of H. contortus larvae by 76–79%. This study shows that A. oligospora isolated from the northern slope pasture of Tianshan Mountain has high predacious activity against H. contortus larvae and partly passing through the sheep gastrointestinal tract. This study also shows that conidial suspensions have no toxic side-effects on the sheep, indicating that they have the potential for the development of oral biological agents which prevent and control livestock gastrointestinal nematode diseases.

Purification, molecular characterization and gene expression analysis of an aspartic protease (Sc-ASP113) from the nematode Steinernema carpocapsae during the parasitic stage

Acute bacterial infection of the central nervous system requires rapid and adequate management. Etiological diagnosis is hence crucial. Moreover, the epidemic threat of certain bacteria necessitates a reliable characterization of the involved bacterial strains to follow the spread of epidemic strains. Conventional identification and characterization of etiological agents are basically based on culture and identification of bacterial markers most frequently by serological assays. Molecular identification and characterization of bacteria have been employed. They provide more reliable analysis of bacterial isolates. Molecular methods for non-culture diagnosis of bacterial infections have recently been developed. In many cases, the molecular assays have decreased the identification time of positive cultures and rescued detection of pathogens in culture-negative clinical samples.

Salmonella enterica is a major zoonotic pathogen responsible for foodborne illnesses worldwide. In this study, we aimed to identify S. enterica isolates from 200 clinical and environmental samples using both traditional microbiological techniques and molecular methods. Of the 100 bacterial isolates, 20 were confirmed as S. enterica through biochemical tests and molecular identification. Polymerase chain reaction (PCR) was employed to amplify the 16S rRNA, B-scDNAp, and Hd genes for species confirmation and characterization. All 20 isolates tested positive for the 16S rRNA gene, while 85% and 75% of the isolates were positive for B-scDNAp and Hd genes, respectively. The results highlight the effectiveness of using multiple molecular markers for accurate detection and strain differentiation in S. enterica. This approach enhances diagnostic precision and contributes to the understanding of the genetic diversity and potential virulence factors of S. enterica, offering valuable insights for public health surveillance and control strategies.

Identification of Ectomycorrhiza Fungi (ECM) based on morphological characters has been laborious and time consuming, especially samples collected from the environment. Additionally, due to its microscopic nature and limited morphological characters, intraspecies variation is difficult to detect. In view of this, this study aimed at confirming the earlier identification of these fungi, which was based on morphological characters, and also to find suitable molecular restriction fragment length polymorphism (RFLP) markers for the identification of ECM fungi up to the level of species or isolates as part of an expandable database of RFLP patterns of the internal transcribed spacers (ITS) region of ECM fungi. Mycelia of 14 species of ectomycorrhizal fungi representing five genera were isolated in pure culture and characterized by morphological and molecular methods. Molecular identification was performed by analysis of the internal transcribed spacers of the nuclear encoded ribosomal RNA (n-RNA) gene region using restriction fragment length polymorphism (RFLP). The region was first amplified by polymerase chain reaction with specific primers and then cleaved with different restriction enzymes. The degree of polymorphism, although extensive, proved inadequate for proper identification of most of the isolates. Depending on the restriction enzymes used, the genera or species could be grouped on the basis of common fragment patterns, thereby confirming the potential of the small subunit (SSU)-ITS region in PCR-RFLP in molecular characterization and identification of ectomycorrhizal fungi. Based on the results of this study, congruent of morphology and molecular RFLP analysis is recommended for characterization of species/strain of ectomycorrhizal fungi. Keywords: Classification, diversity, ectomycorrhizal fungi, ecosystem, ribosomal DNA African Journal of Biotechnology Vol. 12(22), pp. 3390-3398

Origanum L. (Lamiaceae) is an important genus of medicinal and aromatic plants used since ancient times as culinary herbs and remedies in traditional medicine. Although it is a relatively small genus, intra-generic species delineation, as well as its inter-generic relationships within tribe Mentheae, are still poorly understood. High resolution melting (HRM) analysis, coupled with microsatellite markers (SSRs), could facilitate the molecular identification and characterization of certain genotypes more efficiently and relatively faster when compared to other analytical methods. In this study, 38 Origanum samples corresponding to six Origanum taxa (O. dictamnus, O. majorana, O. onites, O. scabrum, O. sipyleum, and O. vulgare subsp. hirtum) were analyzed, using six microsatellite loci. Our goal was to molecularly identify and discriminate among the selected samples and to evaluate the ability of the HRM technique as an analytical tool for the discrimination of Origanum species from Greece. The temperature-shifted melting curves produced by the HRM analysis, resulted in 98 unique HRM profiles, which enabled the discrimination of the Origanum genotypes studied. According to the similarity dendrogram based on the HRM profiles, six unique clusters were formed, each one corresponding to a single taxon. In conclusion, HRM genotyping provided a fast, cost-effective method, well suited for the molecular characterization and identification of Origanum taxa and for the authentication of the original genetic material.

An integrated approach, based on the analysis of both molecular and morphological characters, has led to the unambiguous detection and identification of the rust hyperparasite Cladosporium tenuissimum from aeciospores of the two-needle pine rust fungi Cronartium flaccidum and Peridermium pini. Cladosporium tenuissimum was first detected from contaminated field-collected rust spores using the polymerase chain reaction (PCR) method. The similar-sized amplified DNA of the parasite was then separated from rust DNA using electrophoretic migration, reamplified separately with the nested PCR, and sequenced. Sequence comparison in the data banks enabled the hyperparasite to be recognised as a species of Cladosporium. Molecular detection was followed by conventional identification, obtained by plating rust spores on potato dextrose agar, a selective medium for rusts, since they are unable to grow on such a common substrate, and isolating the hyperparasite in pure culture. It was subsequently identified as C. tenuissimum. Traditional identification would not have been possible without guidance from the molecular data, which focused attention on the mycoparasite. Macro- and micro-scopic features of colonies are also given to help with future identification on spore sources from other geographical areas and, if this should occur, future identification on other rusts.Key words: mycoparasitism, PCR detection, traditional detection, Cladosporium tenuissimum, Cronartium flaccidum, Peridermium pini.

Molecular identification and characterization of Anaplasma platys and Ehrlichia canis in dogs in Mexico

Aim: To demonstrate the importance of molecular identification of G. lucidum basidiomata, used as nutraceutics, in Abuja - Nigeria.
 Study Design: Molecular characterization via comparative genomics and vitro regeneration of selected specimens of local G. lucidum from Abuja, Nigeria.
 Place and Duration: Department of Biological Sciences, Nigerian Defence Academy, Kaduna Nigeria and Biotechnology Advanced Research Center, Sheda Science and Technology Complex, Abuja between February 2018 to June 2019.
 Methodology: Genomic DNAs of twelve (12) selected specimens were isolate in good quantities and qualities that were amenable to sharp and distinct PCR amplifications and Sanger’s sequence analyses. The molecular identification was performed using the internal transcribed spacers (ITS) sequences amplified from the samples to run similarity search with reference database sequences in the gene bank. In-vitro regeneration of the samples using tissue culture techniques in the laboratory was carried out following optimization of the surface sterilization, regeneration of pure mycelia and pure spawn formation.
 Results: Nucleotide sequence data mining of the national centre for biotechnology information (NCBI) with the query sequences using basic local alignment search tool (BLAST) showed that 25% of the samples are not G. lucidum. This implies a significant difference between the morphological and molecular identification at (n-1) degree of freedom with (p= .01).
 Conclusion: The molecular identification and in-vitro regeneration of local G. lucidum is indeed a necessity for proper and effective utilization of the mushroom because there is a significant potential margin of error in the use of morphological characteristics for G. lucidum identification as observed through this molecular analysis.