The first molecular detection of benzimidazole resistance in Haemonchus contortus from sheep in Hejing and Minfeng counties of Southern Xinjiang.

To understand the benzimidazole (BZ) resistance of Haemonchus contortus in Southern Xinjiang, three single nucleotide polymorphisms (SNPs) designated as F167Y, E198A, and F200Y, in the isotype-1 ß-tubulin gene which are associated with BZ resistance, were investigated for H. contortus populations from sheep in Hejing and Minfeng counties of Southern Xinjiang. In brief, a total of 190 H. contortus adults were collected from 52 out of 70 slaughtered sheep in city abattoirs across two regions in Southern Xinjiang. The species identity of each adult worm was confirmed by PCR amplification of ITS-2 using H. contortus-specific primers targeting the ITS-2. The samples were then investigated for BZ-related SNPs at locus 167, 198, and 200, by PCR-sequencing of the isotype-1 ß-tubulin gene. The results showed that only E198A and F200Y mutations were detected in the investigated H. contortus populations. The E198A mutation (homozygous and heterozygote resistant: found in 40% and 30% of sequenced samples from Minfeng and Hejing counties, respectively) was predominant compared with the F200Y mutation (homozygous and heterozygote resistant: found in 14% and 13.3% of sequenced samples from Minfeng and Hejing counties, respectively). The results indicate a high prevalence of BZ resistance in H. contortus populations from certain areas of Southern Xinjiang. Our findings provide valuable information for the prevention and control of H. contortus in areas with similar conditions.

Deciphering ß-tubulin gene of carbendazim resistant Fusarium solani isolate and its comparison with other Fusarium species.

Exploration of molecular structure of ß-tubulin is key to understand mechanism of action of carbendazim since its activity depends on strong binding to ß-tubulin. Resistance against the fungicide is often associated with mutation in ß-tubulin gene. A full-length (1619 bp) ß-tubulin gene has been cloned and sequenced from a carbendazim resistant and a sensitive isolates of F. solani isolated from agricultural fields of Murshidabad (24.23 °N, 88.25 °E), West Bengal, India. Phylogenetic position of the isolates was confirmed using internal transcribed spacer and ß-tubulin gene sequences. In the ß-tubulin based phylogenetic tree, Fusarium species with available data were clustered in nine species complexes and members of both F. solani species complex and F. fujikuroi species complex were distributed into three clades each. The ß-tubulin gene of F. solani was found to be shortest due to least number of non-coding sequences indicating its primitiveness among the Fusarium species. The coding region (G + C 58.54%) was organized into five exons. The protein has 446 amino acid, 49.834 KD molecular weight and 4.64 isoelectric point. Amino acid sequence of the resistant and the sensitive isolates were identical, suggesting that the mechanism of carbendazim resistance in the F. solani isolate was not due to point mutation in ß-tubulin gene. The secondary and tertiary structure of ß-tubulin were similar in all the species except F. oxysporum f.sp. cubense. The identification of binding sites for GDP, carbendazim and α-tubulin would resolve how carbendazim prevents tubulin polymerization. All the data are useful to design tubulin-targeted fungicide with better performance.

Feline sporotrichosis caused by Sporothrix schenckii sensu stricto in Southern Thailand: phenotypic characterization, molecular identification, and antifungal susceptibility.

Feline sporotrichosis caused by the Sporothrix schenckii complex is a global subcutaneous mycosis, having higher prevalence in Latin America and Malaysia. However, its etiological agents have not been elucidated in Thailand, a neighboring country of Malaysia, where the cases are increasing. This study identified 38 feline isolates of S. schenckii from Southern Thailand, collected between 2018 and 2021, using phenotypic characterization and molecular identification using polymerase chain reaction (PCR)-sequencing of partial calmodulin (CAL) and ß-tubulin (Bt2) genes. Phenotypic characteristics proved that the isolates were S. schenckii sensu lato, with low thermotolerance. Based on partial CAL and Bt2-PCR sequencing, all isolates were identified as S. schenckii sensu stricto. Phylogenetic analyses revealed that the isolates were clustered with S. schenckii sensu stricto isolated from the cats in Malaysia. A low degree of genetic diversity was observed among the Thai feline isolates. The antifungal susceptibility of these isolates to antifungal agents, including itraconazole (ITC), ketoconazole (KTC), fluconazole (FLC), and amphotericin B (AMB), was investigated according to the M27-A3 protocol of the Clinical and Laboratory Standards Institute. Results showed low ITC, KTC, and AMB activities against S. schenckii sensu stricto isolates, with high minimum inhibitory concentration (MIC) ranges of 1-8, 1-8, and 2-16 µg/ml, respectively, whereas FLC exhibited MICs of 64 and > 64 µg/ml. This study indicated that S. schenckii sensu stricto is the causative agent responsible for feline sporotrichosis in Southern Thailand. Their phenotypic characteristics and in vitro antifungal susceptibility profiles will help to improve our understanding of this mycosis in Thailand.

Sporothrix schenckii sensu stricto is a causative agent of feline sporotrichosis in Southern Thailand identified by PCR-sequencing of calmodulin and ß-tubulin genes. Phenotypic tests are not recommended for species identification. All isolates showed low susceptibility to commonly used antifungals.

Identification and molecular characterization of Subramaniula asteroides causing human fungal keratitis: a case report.

BACKGROUND: Keratitis due to by filamentous fungi are not easy to diagnose thus causing a delay in correct therapy. There are many descriptions of keratitis due to Candida, Fusarium and Aspergillus genera. Subramaniula genus has only recently been reported to cause human infections and there are few descriptions of eye infections due to this filamentous fungus. Diagnosis of fungal keratitis is usually based on microscopic and cultural techniques of samples obtained by corneal swabbing or scraping. Considering the amount of time required to obtain culture results it is wise to use other diagnostic methods, such as molecular analyses. Therapeutic options against these fungi are limited by low tissue penetration in the eye due to ocular barriers. We describe the first case of S. asteroides human keratitis treated with isavuconazole. CASE PRESENTATION: We describe a rare case of fungal keratitis unresponsive to antimicrobial treatment in a 65-year-old male patient without a history of diabetes or immunological diseases. He reported that the onset of symptoms occurred during a long holiday in Cape Verde Island. Initial treatment with topical antibiotics associated to steroids were ineffective, allowing a slow clinical progression of disease to corneal perforation. On admission in our Hospital, slit-lamp examination of the left eye showed conjunctival congestion and hyperemia, a large inferior corneal ulceration with brown pigment, corneal edema, about 3 mm of hypopyon and irido-lenticular synechiae. The slow clinical progression of the disease to corneal perforation and the aspect of the ulcer were consistent with a mycotic etiology. Molecular methods used on fungal colonies isolated by Sabouraud's dextrose agar cultures allowed the identification of Subramaniula asteroids from corneal scraping. Antimicrobial test showed a good susceptibility of this filamentous fungus to voriconazole and isavuconazole. Moreover, this fungal keratitis was successfully treated with isavuconazole, without side effects, observing a progressive clinical improvement. CONCLUSIONS: Molecular methods may be useful for the identification of filamentous fungal keratitis on scraping samples thus shortening the time of diagnosis. Systemic therapy by isavuconazole could be useful to treat the filamentous fungal keratitis, reducing the possible adverse effects due to the use of voriconazole by systemic administration.

A detection of benzimidazole resistance-associated SNPs in the isotype-1 ß-tubulin gene in Haemonchus contortus from wild blue sheep (Pseudois nayaur) sympatric with sheep in Helan Mountains, China.

BACKGROUND: Benzimidazole (BZ) resistance is an increasingly serious problem due to the excessive use of this anthelmintic for controlling Haemonchus contortus, which is one of the major gastrointestinal nematodes infecting small ruminants worldwide. Three known single nucleotide polymorphisms (SNPs), F167Y (TAC), E198A (GCA) and F200Y (TAC), in the isotype-1 ß-tubulin gene of H. contortus are associated with BZ resistance. Comprehending the spread and origins of BZ resistance-associated SNPs has important implications for the control of this nematode. RESULTS: Twenty-seven adult H. contortus were harvested from wild blue sheep (Pseudois nayaur), small wild ruminants sympatric with domestic ruminants, inhabiting the Helan Mountains, China, to monitor the status of BZ resistance. In addition, 20 adult H. contortus from domestic sheep sympatric with this wild ruminant and 36 isotype-1 ß-tubulin haplotype sequences of H. contortus (two of these haplotypes, E198A3 and E198A4, possessed resistance-associated SNP E198A (GCA) from domestic ruminants in eight other geographical regions of China were used to further define the origins of BZ resistance-associated SNPs within the worms collected from blue sheep. The BZ resistance-associated SNP E198A was detected, whereas SNPs F167Y (TAC) and F200Y (TAC) were not found within the worms collected from blue sheep, and the frequency of homozygous resistant E198A (GCA) was 7.40%. The evolutionary tree and network showed consistent topologies for which there was no obvious boundary among the worms from the wild and domestic hosts, and two haplotypes (E198A1 and E198A2) possessing E198A from the wild blue sheep had two different independent origins. E198A1 had the same origin with E198A3 but E198A2 had a different origin with them. Population genetic analyses revealed a low level of Fst values (ranging from 0 to 0.19749) between all H. contortus worm groups in China. CONCLUSIONS: Results of the current study of the three BZ resistance-associated SNPs of H. contortus from wild blue sheep suggested that only E198A (GCA) was present within the worms collected from the wild ruminants and had multiple independent origins.

Thiophanate-methyl resistance in Sclerotinia homoeocarpa from golf courses in China.

Sclerotinia homoeocarpa causes dollar spot disease on many turfgrass species and is a significant problem worldwide. Thiophanate-methyl (TM), a methyl benzimidazole carbamate (MBC) fungicide, has been used for over forty years to manage dollar spot. Here we describe genetic mutations linked to three distinct TM fungicide resistance phenotypes: sensitive (S), moderately resistant (MR) and highly resistant (HR). These were established using multiple doses of TM, compared to previous studies using single discriminatory doses. In total, 19 S, 3 MR and 22 HR isolates were detected. Analysis of the ß-tubulin gene revealed the MR isolates had a point mutation from T to A at codon 200 changing phenylalanine (TTC) to tyrosine (TAC). Twenty HR isolates had a mutation at codon 198 changing glutamic acid (GAG) to alanine (GCG) and two HR isolates had a mutation at codon 198 changing glutamic acid (GAG) to lysine (AAG). Allele-specific PCR assays were developed for rapid detection of these mutations in isolates of S. homoeocarpa. In addition, our results suggest a two-dose system for in vitro screening provides useful information for monitoring the development of resistance.

The First Case of Total Dystrophic Onychomycosis Caused by Aspergillus clavatus Resistant to Antifungal Drugs.

Onychomycosis is a common fungal infection of nails which is mainly caused by dermatophyte species and less often by yeasts and non-dermatophyte molds. We present a case of onychomycosis due to Aspergillus clavatus for the first time worldwide. The patient was an immunocompetent 32-year-old woman who identified with Psoriasis of the nail. The presence of A. clavatus in a nail sample was confirmed using microscopic and culture analysis followed by PCR of the ß-tubulin gene. After antifungal susceptibility test, it is revealed that the isolate was resistant to the majority of common antifungal drugs, but finally the patient was treated with itraconazole 200 mg daily. A. clavatus and drug-resistant A. clavatus have not previously been reported from onychomycosis.

Restriction analysis of ß-tubulin gene for differentiation of the common pathogenic dermatophytes.

BACKGROUND: Identification of dermatophytes at the species level, relying on macro- and microscopic properties of the colonies is time-consuming, questioned in many circumstances, and requires considerable expertise. In this study, we examined the potency of a new genetic marker, ß-tubulin (BT2) gene, for differentiation of dermatophytes in an in silico and experimental restriction fragment length polymorphism (RFLP) profile. METHODS: The BT2 sequences of dermatophyte species were retrieved from GenBank and analyzed using bioinformatics softwares to choose suitable restriction enzyme(s). Forty reference culture collections and 100 clinical isolates were PCR-amplified using the primers T1 and Bt2b and consequently subjected to virtual RFLP analysis. The dermatophytes were identified according to specific lengths of bands in agarose gel electrophoresis. RESULTS: After digestion of partially amplified ß-tubulin gene with the restriction enzyme FatI, three dermatophyte species, that is, Microsporum gypseum, M. canis, and Trichophyton verrucosum yielded unique restriction maps while the remaining species including T. interdigitale, T. rubrum, T. tonsurans, T. schoenleinii, and T. violaceum, were identified by further restriction digestion by Alw21I, MwoI, and HpyCH4V endonucleases. The length of RFLP products was same as of those expected by computer analysis. CONCLUSION: The two-step BT2 restriction mapping used in this study is an effective tool for reliable differentiation of the clinically relevant species of dermatophytes.

Multigene phylogenies of Ophiostomataceae associated with Monterey pine bark beetles in Spain reveal three new fungal species.

Ophiostoma species, some of which cause sapstain in timber and/or are mild pathogens, are common fungal associates of bark beetles (Coleoptera: Scolytinae). Three new Ophiostomataceae from Spain are recognized in the present study based on comparisons of sequence data for three gene regions as well as morphological characteristics. The new taxa are described as Ophiostoma nebulare sp. nov., Ophiostoma euskadiense sp. nov. and Graphilbum crescericum sp. nov.

New Insights on Tools for Detecting ß-Tubulin Polymorphisms in Trichuris trichiura Using rhAmpTM SNP Genotyping.

Soil-transmitted helminth (STH) infections, commonly treated with benzimidazoles, are linked to resistance through single nucleotide polymorphisms (SNPs) at position 167, 198, or 200 in the ß-tubulin isotype 1 gene. The aim of this study was to establish a novel genotyping assay characterized by its rapidity and specificity. This assay was designed to detect the presence of SNPs within the partial ß-tubulin gene of Trichuris trichiura. This was achieved through the biallelic discrimination at codons 167, 198, and 200 by employing the competitive binding of two allele-specific forward primers. The specificity and reliability of this assay were subsequently confirmed using Trichuris samples isolated from captive primates. Furthermore, a molecular study was conducted to substantiate the utility of the ß-tubulin gene as a molecular marker. The assays showed high sensitivity and specificity when applied to field samples. Nevertheless, none of the SNPs within the ß-tubulin gene were detected in any of the adult worms or eggs from the analyzed populations. All specimens consistently displayed an SS genotype. The examination of the ß-tubulin gene further validated the established close relationships between the T. trichiura clade and Trichuris suis clade. This reaffirms its utility as a marker for phylogenetic analysis.

Isolation and Molecular Characterization of Indigenous Penicillium chrysogenum/rubens Strain Portfolio for Penicillin V Production.

Beta (ß)-lactam antibiotic is an industrially important molecule produced by Penicillium chrysogenum/rubens. Penicillin is a building block for 6-aminopenicillanic acid (6-APA), an important active pharmaceutical intermediate (API) used for semi-synthetic antibiotics biosynthesis. In this investigation, we isolated and identified Penicillium chrysogenum, P. rubens, P. brocae, P. citrinum, Aspergillus fumigatus, A. sydowii, Talaromyces tratensis, Scopulariopsis brevicaulis, P. oxalicum, and P. dipodomyicola using the internal transcribed spacer (ITS) region and the ß-tubulin (BenA) gene for precise species identification from Indian origin. Furthermore, the BenA gene distinguished between complex species of P. chrysogenum and P. rubens to a certain extent which partially failed by the ITS region. In addition, these species were distinguished by metabolic markers profiled by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Secalonic acid, Meleagrin, and Roquefortine C were absent in P. rubens. The crude extract evaluated for PenV production by antibacterial activities by well diffusion method against Staphylococcus aureus NCIM-2079. A high-performance liquid chromatography (HPLC) method was developed for simultaneous detection of 6-APA, phenoxymethyl penicillin (PenV), and phenoxyacetic acid (POA). The pivotal objective was the development of an indigenous strain portfolio for PenV production. Here, a library of 80 strains of P. chrysogenum/rubens was screened for PenV production. Results showed 28 strains capable of producing PenV in a range from 10 to 120 mg/L when 80 strains were screened for its production. In addition, fermentation parameters, precursor concentration, incubation period, inoculum size, pH, and temperature were monitored for the improved PenV production using promising P. rubens strain BIONCL P45. In conclusion, P. chrysogenum/rubens strains can be explored for the industrial-scale PenV production.

Detection of Colletotrichum spp. Resistant to Benomyl by Using Molecular Techniques.

Colletotrichum species is known as the major causal pathogen of red pepper anthracnose in Korea and various groups of fungicides are registered for the management of the disease. However, the consistent use of fungicides has resulted in the development of resistance in many red pepper-growing areas of Korea. Effective management of the occurrence of fungicide resistance depends on constant monitoring and early detection. Thus, in this study, various methods such as agar dilution method (ADM), gene sequencing, allele-specific polymerase chain reaction (PCR), and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were applied for the detection of benzimidazole resistance among 24 isolates of Colletotrichum acutatum s. lat. and Colletotrichum gloeosporioides s. lat. The result of the ADM showed that C. gloeosporioides s. lat. was classified into sensitive and resistant isolates to benomyl while C. acutatum s. lat. was insensitive at ≥1 µg/ml of benomyl. The sequence analysis of the ß-tubulin gene showed the presence of a single nucleotide mutation at the 198th amino acid position of five isolates (16CACY14, 16CAYY19, 15HN5, 15KJ1, and 16CAYY7) of C. gloeosporioides s. lat. Allele-specific PCR and PCR-RFLP were used to detect point mutation at 198th amino acid position and this was done within a day unlike ADM which usually takes more than one week and thus saving time and resources that are essential in the fungicide resistance management in the field. Therefore, the molecular techniques established in this study can warrant early detection of benzimidazole fungicide resistance for the adoption of management strategies that can prevent yield losses among farmers.

Identification of fungi of the genus Aspergillus section nigri using polyphasic taxonomy.

In spite of the taxonomy of the Aspergillus species of the Nigri Section being regarded as troublesome, a number of methods have been proposed to aid in the classification of this Section. This work aimed to distinguish Aspergillus species of the Nigri Section from foods, grains and caves on the basis in Polyphasic Taxonomy by utilizing morphologic and physiologic characters, and sequencing of ß-tubulin and calmodulin genes. The morphologic identification proved useful for some species, such as A. carbonarius and Aspergillus sp UFLA DCA 01, despite not having been totally effective in elucidating species related to A. niger. The isolation of the species of the Nigri Section on Creatine Sucrose Agar (CREA) enabled to distinguish the Aspergillus sp species, which was characterized by the lack of sporulation and by the production of sclerotia. Scanning Electron microscopy (SEM) allowed distinguishing the species into two distinct groups. The production of Ochratoxin A (OTA) was only found in the A. carbonarius and A. niger species. The sequencing of ß-tubulin gene was efficient in differing most of the Aspergillus species from the Nigri Section with the exception of Aspergillus UFLA DCA 01, which could not be distinguished from A. costaricaensis. This species is morphologically similar to A. costaricaencis for its low sporulation capacity and high sclerotia production, but it differs morphologically from A. costaricaensis for its conidial ornamentation and size of vesicles. Equally, based on partial calmodulin gene sequence data Aspergillus UFLA DCA 01 differs from A. costaricaensis.

Teladorsagia circumcincta beta tubulin: the presence of the E198L polymorphism on its own is associated with benzimidazole resistance.

BACKGROUND: Benzimidazole resistance is associated with isotype-1 ß-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta. METHODS: The benzimidazole phenotypic resistance was measured by the faecal egg count reduction test (FECRT) and the egg hatch test (EHT) using a discriminating dose (DD) in 39 sheep flocks. Around 1000 larvae collected before and after treatment were used for DNA extraction. The resistant species identified in all flocks was T. circumcincta. The resistance alleles frequencies were measured for F200Y and E198A. A 371-bp fragment of the isotype-1 ß-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L. RESULTS: The percentage of resistant flocks was 35% by FECRT or 26% by EHT; however, F200Y and E198A SNPs were absent in T. circumcincta. The amplification of a 371-bp fragment confirmed the absence of F167Y and F200Y in 6 resistant flocks. Regarding codon 198, all samples after treatment carried a leucine (CTA). A pyrosequencing assay analysed the allele frequencies for the first two bases at codon 198 independently, G/C and A/T. The correlation between C and T frequencies was almost 1 (r = 0.929, P < 0.0001) and the mean value of both was calculated to measure the leucine frequency; this value ranged between 10.4-80.7% before treatment, and 82.3-92.8% after treatment. High and similar correlations were reported between the genotypic variables (C frequency, T frequency or mean of both frequencies) and phenotypic resistance (r > 0.720, P < 0.0001), although negatively associated with the FECRT and positively with the EHT. According to multivariate linear regression analysis, the T frequency was the most significant variable influencing the phenotypic resistance (FECRT or EHT; P < 0.0001). In the EHT, 67.1% of the phenotypic variability is associated with the T frequency but in the FECRT only 33.4%; therefore, the EHT using a DD seems to detect the genotypic resistance more accurately than the FECRT. CONCLUSIONS: The E198L polymorphism can confer BZ resistance on its own in T. circumcincta.

Molecular characterization of ß-tubulin gene associated with benzimidazole resistance in larvae of field isolates of Parascaris (Nematoda: Ascarididae).

Since the past 2 decades, an increasing number of resistance to the Benzimidazoles (BZs) have been reported in nematode parasites of livestock. More recently, detection of single-nucleotide polymorphisms (SNPs) at codons of 167, 198 or 200 of the ß-tubulin gene has been attributed to the occurrence of resistance. In the present study, we investigated the presence of those SNPs in the ß-tubulin isotype-1 gene in different isolates of Parascaris in horse. Also, the mitochondrial (mt) and ribosomal genes were sequenced for species confirmation of the isolates. The analysis of sequences inferred from COII gene confirmed that those isolates were P. equorum. The distance between mt genes obtained here and several ascarid species in equids and other hosts suggests the need for the combination of more genetic data with morphologic and other diagnostic measures. The analysis on ß-tubulin isotype-1 gene revealed no resistance-related SNPs or substitutions at the expected codon positions and selection pressure with BZs has not occurred for Parascaris worms. Although the molecular data showed the susceptibility of Parascaris isolates against BZs, other mechanisms of resistance should be also investigated to confirm the validity of molecular results.

Osteomyelitis of the rib cage by Aspergillus flavus.

BACKGROUND: Aspergillus osteomyelitis of the ribs is relatively uncommon. It is a debilitating and severe form of invasive aspergillosis. CASE REPORT: A 61year-old female presented with spontaneous chest pain on the right side of the rib cage and a palpable soft-tissue mass. FDG-PET/CT scan identified activity in the infected site. The lesion was punctured, and purulent material was sent to the laboratory. Aspergillus complex Flavi was isolated. An antifungal treatment with voriconazole was started. The lesion healed, and no recurrence was observed at 8-month follow-up. Molecular identification of the isolate was based on PCR amplification and sequencing of ß-tubulin gene. Aspergillus flavus was identified. CONCLUSIONS: Our case highlights the relevance of microbiological studies in patients with osteomyelitis and the involvement of soft tissue. The FDG-PET/CT scan was found to be a useful tool for revealing the extent of the disease and evaluating the response to the antifungal therapy.

Genetic and Toxigenic Variability within Aspergillus flavus Population Isolated from Maize in Two Diverse Environments in Kenya.

Aspergillus flavus is the main producer of carcinogenic aflatoxins in agricultural commodities such as maize. This fungus occurs naturally on crops, and produces aflatoxins when environmental conditions are favorable. The aim of this study is to analyse the genetic variability among 109 A. flavus isolates previously recovered from maize sampled from a known aflatoxin-hotspot (Eastern region, Kenya) and the major maize-growing area in the Rift Valley (Kenya), and to determine their toxigenic potential. DNA analyses of internal transcribed spacer (ITS) regions of ribosomal DNA, partial ß-tubulin gene (benA) and calmodulin gene (CaM) sequences were used. The strains were further analyzed for the presence of four aflatoxin-biosynthesis genes in relation to their capability to produce aflatoxins and other metabolites, targeting the regulatory gene aflR and the structural genes aflP, aflD, and aflQ. In addition, the metabolic profile of the fungal strains was unraveled using state-of-the-art LC-MS/MS instrumentation. The three gene-sequence data grouped the isolates into two major clades, A. minisclerotigenes and A. flavus. A. minisclerotigenes was most prevalent in Eastern Kenya, while A. flavus was common in both regions. A. parasiticus was represented by a single isolate collected from Rift Valley. Diversity existed within the A. flavus population, which formed several subclades. An inconsistency in identification of some isolates using the three markers was observed. The calmodulin gene sequences showed wider variation of polymorphisms. The aflatoxin production pattern was not consistent with the presence of aflatoxigenic genes, suggesting an inability of the primers to always detect the genes or presence of genetic mutations. Significant variation was observed in toxin profiles of the isolates. This is the first time that a profound metabolic profiling of A. flavus isolates was done in Kenya. Positive associations were evident for some metabolites, while for others no associations were found and for a few metabolite-pairs negative associations were seen. Additionally, the growth medium influenced the mycotoxin metabolite production. These results confirm the wide variation that exists among the group A. flavus and the need for more insight in clustering the group.

Genetic Variation of the ß-tubulin Gene of Babesia caballi Strains.

BACKGROUND: Equine piroplasmosis is caused by two haemoprotozoan parasites: Babesia caballi and Theileria equi. Negative economic impact on international trade has been associated to endemic sites. This is the reason why carrier detection requires reliable diagnostic methods. Various diagnostic modalities can be used alone or in combination including PCR. However, genetic variation of commonly used genes is still of debate. The aim of this research was to sequence the ß-tubulin gene of a B. caballi strain from Spain and to compare it with known ß-tubulin sequences. METHODS: DNA was isolated from a cryopreserved strain from Spain and acute and chronic carrier horses. Firstly, degenerated primer pairs were designed based on GenBank sequences of different Babesia and Theileria species for sequencing. The primers were redesigned to amplify both parasites, simultaneously. Finally, a species-specific primer pair for B. caballi was designed and a Restriction Fragment Length Polymorphism-PCR (PCR-RFLP) assay performed to know the difference of known B. caballi strains. RESULTS: We provided new insights of the ß-tubulin gene and a good molecular coverage of this gene, contributing with a number of useful primers to amplify T. equi and B. caballi. Moreover, PCR-RFLP assays based on the exon II of this gene confirmed the causative B. caballi strain in Spanish horses. CONCLUSION: We reported useful primer pairs for diagnostic and a new sequence of the ß-tubulin gene of B. caballi, which will facilitate the development of future assays and the detection of infected horses, preventing thus the spread of this disease worldwide.

Isothermal diagnostic assays for the detection of soil-transmitted helminths based on the SmartAmp2 method.

BACKGROUND: Diagnosis of soil-transmitted helminths (STHs) has traditionally relied on stool microscopy, which has a number of critical deficiencies. Molecular diagnostics are powerful tools to identify closely related species, but the requirement for costly equipment makes their implementation difficult in low-resource or field settings. Rapid, sensitive and cost-effective diagnostic tools are crucial for accurate estimation of STH infection intensity in MDA programmes in which the goal is to reduce morbidity following repeated rounds of chemotherapy. RESULTS: In this study, colourimetric isothermal assays were developed using SmartAmp2 primer sets and reagents in loop-mediated amplification (LAMP) assays. Species-specific primer sets, designed on a specific target sequence in the ß-tubulin gene, were used to identify Necator americanus, Trichuris trichiura and Ascaris lumbricoides. After initial optimization on control plasmids and genomic DNA from adult worms, assays were evaluated on field samples. Assays showed high sensitivity and demonstrated high tolerance to inhibitors in spiked faecal samples. Rapid and sensitive colourimetric assays were successfully developed to identify the STHs in field samples using hydroxy napthol blue (HNB) dye. CONCLUSIONS: Rapid and simple colourimetric diagnostic assays, using the SmartAmp2 method, were developed, with the potential to be applied in the field for detection of STH infections and the estimation of response to treatment. However, further validation on large numbers of field samples is needed.

Toxin-producing Epichloë bromicola strains symbiotic with the forage grass Elymus dahuricus in China.

Cool-season grasses (Poaceae subfamily Poöideae) are an important forage component for livestock in western China, and many have seed-transmitted symbionts of the genus Epichloë, fungal endophytes that are broadly distributed geographically and in many tribes of the Poöideae. Epichloë strains can produce any of several classes of alkaloids, of which ergot alkaloids and indole-diterpenes can be toxic to mammalian and invertebrate herbivores, whereas lolines and peramine are more selective against invertebrates. The authors characterized genotypes and alkaloid profiles of Epichloë bromicola isolates symbiotic with Elymus dahuricus, an important forage grass in rangelands of China. The endophyte was seed-transmitted and occasionally produced fruiting bodies (stromata), but its sexual state was not observed on this host. The genome sequence of E. bromicola isolate E7626 from El. dahuricus in Xinjiang Province revealed gene sets for peramine, ergot alkaloids, and indole-diterpenes. In multiplex polymerase chain reaction (PCR) screens of El. dahuricus-endophyte isolates from Beijing and two locations in Shanxi Province, most were also positive for these genes. Ergovaline and other ergot alkaloids, terpendoles and other indole-diterpenes, and peramine were confirmed in El. dahuricus plants with E. bromicola. The presence of ergot alkaloids and indole-diterpenes in this grass is a potential concern for managers of grazing livestock.