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Babesia microti (Apicomplexa: Piroplasmida) causes a medically important tick-borne zoonotic protozoan disease. Egyptian camels are susceptible to Babesia infection; however, just a few cases have been documented. This study aimed to identify Babesia species, specifically Babesia microti, and their genetic diversity in dromedary camels in Egypt and associated hard ticks. Blood and hard tick samples were taken from 133 infested dromedary camels slaughtered in Cairo and Giza abattoirs. The study was conducted from February to November 2021. The 18S rRNA gene was amplified by polymerase chain reaction (PCR) to identify Babesia species. Nested PCR targeting the ß-tubulin gene was used to identify B. microti. The PCR results were confirmed by DNA sequencing. Phylogenetic analysis based on the ß-tubulin gene was used to detect and genotype B. microti. Three tick genera were identified in infested camels (Hyalomma, Rhipicephalus, and Amblyomma). Babesia species were detected in 3 out of 133 blood samples (2.3%), while Babesia spp. were not detected in hard ticks by using the 18S rRNA gene. B. microti was identified in 9 out of 133 blood samples (6.8%) and isolated from Rhipicephalus annulatus and Amblyomma cohaerens by the ß-tubulin gene. The phylogenetic analysis of the ß-tubulin gene revealed that USA-type B. microti was prevalent in Egyptian camels. The results of this study suggested that the Egyptian camels may be infected with Babesia spp. and the zoonotic B. microti strains, which pose a potential risk to public health.

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Microorganisms produce secondary metabolites to survive under stressful conditions. The effect of drought and heat stress on fungi isolated from Arabian desert soil during the hot (ca 40°C) and cool (ca 10°C) seasons was studied using the genome mining approach. The presence of three stress-related genes (calmodulin, polyketide synthase and beta tubulin) was analyzed molecularly using specific primers. The presence of the genes in desert fungi was compared to their antimicrobial (ten bacterial or fungal pathogens) and anticancer (liver, cervical and breast) properties and the production of thermostable enzymes (phytase and xylanase). The genes appeared to be present in the fungal sequence obtained during the summer, while none of the genes were present during winter. Appreciable differences were observed in enzyme activities, with summer activities high and winter low. The antagonistic activities of A. niger were relatively stable and varying, while those of P. chrysogenum were consistently higher in summer than in winter. The presence of the three genes seemed to correlate with the highly antagonistic activities of P. chrysogenum, while A. niger had relatively active winter isolates without any of the genes. The hot season in deserts yields fungal isolates with biological activities useful in biotechnological solutions.

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Dermatophytes are keratinophilic fungi that cause skin infections in both humans and animals. Recently, the incidence rates of fungal infections associated with Trichophyton spp. have been considered endemic in many locations. The aim of this study was to isolate and characterize Trichophyton spp. from canines and felines. In the present study, screened 442 canine (n = 386) and feline (n = 56) samples for dermatophytes. Among all the samples, ten isolates were identified as Trichophyton spp. based on micro-morphological features. For comparative analysis, we included three human strains of Trichophyton mentagrophytes complex. In vitro susceptibility of antifungal drugs indicated the highest sensitivity except for fluconazole. The canine and human strains were genetically characterized by sequencing three genes: the internal transcribed spacer region of rDNA, translation elongation factor 1- gene, and beta-tubulin. Based on sequence homology and phylogenetic analysis, the ten canine strains belonged to four different species/ genotypes such as T. mentagrophytes genotype VIII (T. indotineae) (n = 5), T. interdigitale (n = 2), T. simii (n = 2) and T. quinckeanum (n = 1). The three human strains used for comparative analysis were identified as T. mentagrophytes genotype VIII (n = 2) and T. benhamiae (n = 1). The study hence indicates that the T. mentagrophytes genotype VIII, considered as an endemic and emerging human pathogenic clone in India, is also the prevalent in animals.

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Here we report first two cases of invasive pulmonary aspergillosis caused by Aspergillus lentulus from India, in non-neutropenic, critically ill patients with chronic obstructive pulmonary disease (COPD).

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Abstract INTRODUCTION: Benzimidazoles are commonly used for the control of veterinary nematodes. Resistance to benzimidazoles has been associated with three single nucleotide polymorphisms in the β-tubulin gene of common nematodes. However, these mutations are infrequent in the genus Ascaris spp. METHODS: In order to determine mutations associated with benzimidazole resistance in Ascaris suum, worms were collected from slaughtered pigs and a partial region of the β-tubulin gene was sequenced. RESULTS: All parasites showed the wildtype genotype for codons 167, 198, and 200 of the β-tubulin gene. CONCLUSIONS: This is the first report of genetic sequences associated with benzimidazole resistance in A. suum.

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The DNA polymorphism diffusely present in the introns of the members of the Eukaryotic beta-tubulin gene families, can be conveniently used to establish a DNA barcoding method, named tubulin-based polymorphism (TBP), that can reliably assign specific genomic fingerprintings to any plant or/and animal species. Similarly, many plant varieties can also be barcoded by TBP. The method is based on a simple cell biology concept that finds a conveniently exploitable molecular basis. It does not depend on DNA sequencing as the most classically established DNA barcode strategies. Successful applications, diversified for the different target sequences or experimental purposes, have been reported in many different plant species and, of late, a new a version applicable to animal species, including fishes, has been developed. Also, the TBP method is currently used for the genetic authentication of plant material and derived food products. Due to the use of a couple of universal primer pairs, specific for plant and animal organisms, respectively, it is effective in metabarcoding a complex matrix allowing an easy and rapid recognition of the different species present in a mixture. A simple, dedicated database made up by the genomic profile of reference materials is also part of the analytical procedure. Here we will provide some example of the TBP application and will discuss its features and uses in comparison with the DNA sequencing-based methods.

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Mecistocirrus digitatus and Toxocara vitulorum are common pathogenic nematode parasites of mithun (Bos frontalis). Species identification by morphological features was confirmed by molecular identification of M. digitatus and T. vitulorum. The internal transcribed spacer-2 (ITS-2) region and beta tubulin gene of M. digitatus were polymerase chain reaction (PCR) amplified and sequenced. ITS-2 sequence analysis showed 100% homology with other isolates of M. digitatus and 83% identity with Haemonchus contortus and H. placei, respectively. Likewise, ITS-1 and ITS-2 sequences of T. vitulorum were PCR amplified and sequenced. Sequence analysis of these internal transcribed spacers from five worms of the parasite from mithun showed no intraspecific variations with T. vitulorum isolates from domestic ruminants.

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Following our previous report on evaluation of the beta tubulin real-time PCR for detection of dermatophytosis, this study aimed to compare the real-time PCR assay with conventional methods for the clinical assessment of its diagnostic performance. Samples from a total of 853 patients with suspected dermatophyte lesions were subjected to direct examination (all samples), culture (499 samples) and real-time PCR (all samples). Fungal DNA was extracted directly from clinical samples using a conical steel bullet, followed by purification with a commercial kit and subjected to the Taq-Man probe-based real-time PCR. The study showed that among the 499 specimens for which all three methods were used, 156 (31.2%), 128 (25.6%) and 205 (41.0%) were found to be positive by direct microscopy, culture and real-time PCR respectively. Real-time PCR significantly increased the detection rate of dermatophytes compared with microscopy (288 vs 229) with 87% concordance between the two methods. The sensitivity, specificity, positive predictive value, and negative predictive value of the real-time PCR was 87.5%, 85%, 66.5% and 95.2% respectively. Although real-time PCR performed better on skin than on nail samples, it should not yet fully replace conventional diagnosis.

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Otitis externa caused by fungi (otomycosis) occurs more commonly in tropical areas with high moisture than in temperate regions. Bilateral otomycosis is, however, rarely reported. In a case of bilateral otitis externa in a 56-year-old male patient in Taiwan, direct microscopic examination of the cerumen as well as isolation of strains indicated the presence of two Aspergillus species being different in each of both ears. The species were identified by DNA sequence comparisons and additional morphological confirmation of diagnostic characteristics as Aspergillus flavus and Aspergillus terreus. The rarely reported occurrence of two Aspergillus species in otitis of the same patient deserves attention in other cases of otomycosis, particularly with respect to potentially different resistances of different species against antifungals. Treatment with nystatin/neomycin was not successful, but with clotrimazole was effective.

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Haemonchus contortus isolates were evaluated for benzimidazole (BZ) resistance or susceptibility by allele-specific PCR based on ß-tubulin isotype 1 gene polymorphisms at the F167Y, E198A, and F200Y sites. Two isolates, one presumed susceptible from wild pronghorn antelope (PH) and one known to be resistant from goats (VM), were also assayed phenotypically for BZ resistance or susceptibility in the larval development assay (Drenchrite®). The BZ EC50 was 0.198 µM (intermediate between susceptible and weak resistant) for PH with critical well 5 (intermediate between susceptible and weak resistant) and 1.456 µM (intermediate weak resistant and resistant) for VM with critical well 8.5 (resistant). Genotypically, DNA extracted from pooled VM L3 larvae in the Drenchrite® wells with the highest BZ concentration was homozygous susceptible (SS) at the F167Y and E198A sites and homozygous resistant (RR) at the F200Y site by PCR, and sequence analysis bore this out. PH L3 larvae DNA from a control well (no BZ) was SS at all three sites by PCR, confirmed by sequence analysis. All single adult worm samples (N = 21) from PH, VM, Egypt goat (EG), and a Texas llama were SS at F167Y and E198A by PCR; however, only 3 PH worms and 1 EG worm were SS at F200Y. Three additional PH worms were RS and upon cloning two clones were identified as resistant by sequencing and two as susceptible. Clones from single adult worms VM, llama, and EG samples that were RR by PCR at F200Y were sequence verified as resistant. In this study, F200Y was the most frequently found genotypic marker for BZ resistance or susceptibility in the different Haemonchus isolates.

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Early differentiation of dermatophytosis from other cutaneous mycoses is essential to avoid inaccurate therapy. DNA-based techniques including real-time PCR have increasingly been considered for detection of fungal elements in clinical specimens. In this study, after partial sequence analysis of beta tubulin (BT2) gene in 13 common and rare pathogenic dermatophyte species, a pan-dermatophyte primer and probe set was designed in a TaqMan probe-based PCR format. The sensitivity and specificity of the system was tested with 22 reference strains of dermatophytes, 234 positive clinical specimens, 32 DNA samples extracted from normal nails, several fungi other than dermatophytes and human DNAs. Analytical detection limit of the designed PCR on serially diluted DNAs of prepared recombinant plasmid indicated that only five molecules per sample are the minimum number for reliable detection by the assay. A total of 226 out of 234 (96.5%) DNAs extracted from clinical samples, but none of the 32 nail samples, from healthy volunteers were positive in PCR. The real-time PCR targeted beta tubulin gene established in this study could be a sensitive diagnostic tool which is significantly faster than the conventional culture method and should be useful in the clinical settings, in large-scale epidemiological studies and in clinical trials of antifungal therapy.

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Objective Based on our previous established cohort of myelodysplastic syndrome (MDS), we investigated the potential effect of beta-tubulin (TUBB) gene in the transformation of MDS into acute leukemia Methods From our nested case-control study cohort of MDS patients, we chose 11 paired transformed and nontransformed MDS patients.TUBB gene expression was tested by quantitative real-time PCR.TUBB-siRNA transfection was used to down-regulate TUBB gene expression in SKM-1 cell line.The function of TUBB gene in SKM-1 cell line was evaluated by cell proliferation, soft agar clone formation and electron microscope.Results TUBB gene expression in MDS patients in transformed group were significantly higher than that in control group (2.91 ± 0.41 vs 0.90 ± 0.23, P ＜0.01).After TUBB-siRNA transfection, A450/630nm of SKM-1 cells at 24 h, 48 h and 72 h were 0.299 ± 0.045, 0.526 ± 0.034 and 0.652 ± 0.035, respectively, which were significantly decreased than those in negative-siRNA group (0.438 ±0.074, 0.858 ±0.064 and 0.974 ±0.044) (P ＜0.05).Soft agar clone formation in TUBB-siRNA group was (7.0 ±0.2)％, which was significantly reduced than that of negative-siRNA group (25.0 ± 0.2)％ (P ＜ 0.01).Electron microscope showed significant apoptotic signs in TUBB-siRNA group, including vacuoles in cytoplasm and karyorrhexis.Conclusion Our results indicate that TUBB gene may play a role in the transformation of MDS into acute leukemia by affecting the proliferation of malignant clones.

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Azole resistance among clinical isolates of Aspergillus fumigatus is becoming a serious problem in Europe, but the status in Japan is not yet known in detail. The aim of this study was to determine the present status of azole resistance in A. fumigatus in Japan. We employed 171 clinical isolates of A. fumigatus sensu stricto collected from 1987 to 2008 at the Medical Mycology Research Center, Chiba University, Japan for azole resistance determination. Identification of all isolates were re-examined both from the aspect of morphology and molecular phylogeny. The antifungal susceptibility of these isolates was tested based on the CLSI M38-A2 broth microdilution method. In our collection, only 1 (0.6%) and 2 isolates (1.2%) showed elevated MIC to voriconazole and itraconazole, respectively. Our study disclosed that the frequency of azole resistance in A. fumigatus still remains low in this collection.

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Members of the genus Malassezia are lipophilic basidiomycetous yeasts, which are part of the normal cutaneous microbiota of humans and other warm-blooded animals. Currently, this genus consists of 14 species that have been characterized by phenetic and molecular methods. Although several molecular methods have been used to identify and/or differentiate Malassezia species, the sequencing of the rRNA genes and the chitin synthase-2 gene (CHS2) are the most widely employed. There is little information about the ß-tubulin gene in the genus Malassezia, a gene has been used for the analysis of complex species groups. The aim of the present study was to sequence a fragment of the ß-tubulin gene of Malassezia species and analyze their phylogenetic relationship using a multilocus sequence approach based on two rRNA genes (ITS including 5.8S rRNA and D1/D2 region of 26S rRNA) together with two protein encoding genes (CHS2 and ß-tubulin). The phylogenetic study of the partial ß-tubulin gene sequences indicated that this molecular marker can be used to assess diversity and identify new species. The multilocus sequence analysis of the four loci provides robust support to delineate species at the terminal nodes and could help to estimate divergence times for the origin and diversification of Malassezia species.

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A puromycin-N-acetyltransferase gene (pac) is widely used as a selection marker for eukaryotic gene manipulation. However, it has never been utilized for molecular studies in the ciliate Tetrahymena thermophila, in spite of the limited number of selection markers available for this organism. To utilize pac as a maker gene for T. thermophila, the nucleotide sequence of the pac gene was altered to accord with the most preferred codon-usage in T. thermophila. This codon-optimized pac gene expressed in T. thermophila conferred a resistance to transformed cells against 2,000 µg/ml of puromycin dihydrochloride, whereas the growth of wild-type cells was completely inhibited by 200 µg/ml. Furthermore, an expression cassette constructed with the codon-optimized pac and an MTT1 promoter was effectively utilized for experiments to tag endogenous proteins of interest by fusing the cassette into the target gene locus. These results indicate that pac can be used as a selection marker in molecular studies of T. thermophila.

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BACKGROUND: In order to deworm the ruminants especially of sheep in Iran, consumption of benzimidazoles has more than 2 decades history and today farmers are using imidazothiazoles, macrocyclic lactones and mostly benzimidazole compounds (BZs) to treat infected farm animals. It has been demonstrated that the most common molecular mechanism leading to BZsresistance in Haemonchus contortus is a single mutation at amino acid 200 (phenylalanine to tyrosine) of the isotype 1 of beta tubulin gene. According to the report of such mutations in Iranian Teladorsagia circumcincta isolates with Restriction Site Created PCR-RFLP, we decided to evaluate the frequency of such mutations in H. contortus in three different geographical areas of Iran. METHODS: A total of 102 collected adult male H. contortus were evaluated with PCR-RFLP (using PSP1406I as restriction enzyme). By means of a second step to compare function of different methods and to increase sensitivity of detection mechanism, a third of samples were examined by another PCR-RFLP method (using TaaI as restriction enzyme) and finally beta tubulin gene of two samples was sequenced. RESULTS: All of samples were detected as BZss homozygote. Finally, beta tubulin gene sequencing of two samples showed no point mutation at codon 200. CONCLUSION: It seems that BZresistance of H. contortus in Iran is not a serious problem as anticipated before.

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An Ophiostoma fungus was isolated from a stump of Pinus thunbergii in a forest on the West coast of Korea. Microscopic analysis using a light microscope, a stereo microscope, and a scanning electron microscope revealed that it had morphological features of Pesotum and Sporothix synanarmorphs. Based on the beta-tubulin gene sequence analysis, the fungus was identified as the anamorph of Ophiostoma floccosum. Mycological properties of the species including its growth properties on different culture media were described.

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We isolated and identified a strain of Eurotium rubrum from Meju that has not been reported in Korea. This fungus is yellowish brown; reverse dark brown on CYA and PDA while yellow on 2% MEA at 25degrees C. Cleistothecia are first bright yellow and gradually turned brown. Mycerial growth on CYA attained a diameter of 30 mm at 20degrees C, 37 mm at 25degrees C and 32 mm at 30degrees C after 15 days. The isolate grew slower on 2% MEA (< 20 mm 15 days at 25degrees C) compared to CYA and PDA (< 40 mm 15 days at 25degrees C). Cleistothecia are superficial, yellow to light brown, globose to subglobose, 40~75 microm in diameter. Asci are 8-spored and globose to subglobose 8~11 microm. Ascospores are disciform, 4.0~5.0 microm in length and 4.2~4.5 microm in width. Conidia are ovate or bacillar, finely roughened to densely spinulose, 4.6~6.0 microm in length and 3.0~4.3 microm in width. Compared to known Eurotium rubrum, the Korean isolate showed 99% sequence similarity in ITS rDNA (554 bp) and calmodulin (750 bp) gene and 100% in beta-tubulin (1016 bp) gene. The E. rubrum isolate also had weak beta-glucosidase and protease activities.

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During 2007 survey of post-harvest diseases of yam performed in May and June, severe tuber loss caused by blue mold was observed in Iksan, Cheonbuk Province. Two species of Penicillium were isolated from the infected tubers. Based on beta-tubulin gene sequence analysis, and cultural and morphological characteristics, the isolates were identified as Penicillium sclerotigenum and P. polonicum. P. sclerotigenum, which is a novel to Korea, is presently described and illustrated.

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Grape fruits with blue mold symptoms were collected from house storages in different locations in Korea and were investigated for their association with Penicillium species. A total of 12 isolates of Penicillium were isolated from the collected fruits. Based on morphological and cultural characteristics and beta-tublin gene sequence data analysis, they were identified as P. bialowiezense, P. citrinum, P. echinulatum, P. expansum, P. solitum and unidentified Penicillium species. P. solitum was the predominant followed by P. expansum. P. bialowiezense and P. echinulatum were newly recorded in Korea. beta-Tubulin gene sequences could be used to distinguish each species of Penicillium and the molecular groups were correlated well with the morphological species. The unidentified species was supposed to be a new species, not previously reported in literature.

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