Small Subunit Ribosomal RNA Research Articles

Prevalence and molecular characterization of Giardia duodenalis in dairy cattle in Central Inner Mongolia, Northern China

Giardia duodenalis is a gastrointestinal protozoan ubiquitous in nature. It is a confirmed zoonotic pathogen, and cattle are considered a source of giardiasis outbreaks in humans. This study aimed to evaluate the prevalence and multilocus genotype (MLG) of G. duodenalis in dairy cattle in Central Inner Mongolia. This study was based on the small subunit ribosomal RNA (SSU rRNA), glutamate dehydrogenase (gdh), triosephosphate isomerase (tpi), and beta-giardin (bg) genes of G. duodenalis. DNA extraction, polymerase chain reaction (PCR), and sequence analysis were performed on 505 dairy cattle fecal samples collected in 2021 from six sampling sites and four age groups in Central Inner Mongolia to determine the prevalence and MLG distribution of G. duodenalis. The PCR results of SSU rRNA revealed that the overall prevalence of G. duodenalis was 29.5% (149/505) and that the overall prevalence of the diarrhea and nondiarrhea samples was 31.5% (46/146) and 28.5% (103/359), respectively; the difference was not significant (p > 0.05). SSU rRNA sequence analysis revealed that G. duodenalis assemblage E (91.1%, 133/146) was primarily detected and that assemblage A (8.9%, 13/146) was detected in 13 samples. The G. duodenalis—positive samples were PCR amplified and sequenced for gdh, tpi, and bg, from which 38, 47, and 70 amplified sequences were obtained, respectively. A combination of G. duodenalis assemblages A and E were detected in seven samples. Multilocus genotyping yielded 25 different assemblage E MLGs, which formed six subgroups. To the best of our knowledge, this is the first report regarding G. duodenalis infection in dairy cattle in Inner Mongolia, China. This study revealed that Inner Mongolian cattle pose a risk of giardiasis transmission to humans and that the distribution of local cattle G. duodenalis assemblage E MLGs is diverse. The findings of this study can bridge the knowledge gap in the molecular epidemiological investigation of giardiasis in Central Inner Mongolia.

Exploring diversity rock-inhabiting fungi from northern Thailand: a new genus and three new species belonged to the family Herpotrichiellaceae.

Members of the family Herpotrichiellaceae are distributed worldwide and can be found in various habitats including on insects, plants, rocks, and in the soil. They are also known to be opportunistic human pathogens. In this study, 12 strains of rock-inhabiting fungi that belong to Herpotrichiellaceae were isolated from rock samples collected from forests located in Lamphun and Sukhothai provinces of northern Thailand during the period from 2021 to 2022. On the basis of the morphological characteristics, growth temperature, and multi-gene phylogenetic analyses of a combination of the internal transcribed spacer, the large subunit, and the small subunit of ribosomal RNA, beta tubulin and the translation elongation factor 1-a genes, the new genus, Petriomyces gen. nov., has been established to accommodate the single species, Pe. obovoidisporus sp. nov. In addition, three new species of Cladophialophora have also been introduced, namely, Cl. rupestricola, Cl. sribuabanensis, and Cl. thailandensis. Descriptions, illustrations, and a phylogenetic trees indicating the placement of these new taxa are provided. Here, we provide updates and discussions on the phylogenetic placement of other fungal genera within Herpotrichiellaceae.

Prevalence and molecular characterization of Cryptosporidium spp. in dairy cattle in Central Inner Mongolia, Northern China

BackgroundCryptosporidium is a gastrointestinal protozoan that widely exists in nature, it is an established zoonotic pathogen. Infected cattle are considered to be associated with cryptosporidiosis outbreaks in humans. In the present study, we aimed to assess the prevalence and species distribution of Cryptosporidium in dairy cattle in Central Inner Mongolia.MethodsWe focused on the small subunit ribosomal RNA gene (SSU rRNA) of Cryptosporidium and 60-kDa glycoprotein gene (gp60) of Cryptosporidium parvum. We collected 505 dairy cattle manure samples from 6 sampling sites in Inner Mongolia in 2021; the samples were divided into 4 groups based on age. DNA extraction, polymerase chain reaction (PCR), sequence analysis, and restriction fragment length polymorphism (RFLP) using SspI and MboII restriction endonucleases were performed. RFLP analysis was performed to determine the prevalence and species distribution of Cryptosporidium.ResultsSSU rRNA PCR revealed that the overall prevalence of Cryptosporidium infection was 29.90% (151/505), with a prevalence of 37.67% (55/146) and 26.74% (96/359) in diarrheal and nondiarrheal samples, respectively; these differences were significant. The overall prevalence of Cryptosporidium infection at the 6 sampling sites ranged from 0 to 47.06% and that among the 4 age groups ranged from 18.50 to 43.81%. SSU rRNA sequence analysis and RFLP analysis revealed the presence of 4 Cryptosporidium species, namely, C. bovis (44.37%), C. andersoni (35.10%), C. ryanae (21.85%), and C. parvum (11.92%), along with a mixed infection involving two or three Cryptosporidium species. Cryptosporidium bovis or C. andersoni was the most common cause of infection in the four age groups. The subtype of C. parvum was successfully identified as IIdA via gp60 analysis; all isolates were identified as the subtype IIdA19G1.ConclusionsTo the best of our knowledge, this is the first report of dairy cattle infected with four Cryptosporidium species in Inner Mongolia, China, along with a mixed infection involving two or three Cryptosporidium species, with C. bovis and C. andersoni as the dominant species. Moreover, this is the first study to identify C. parvum subtype IIdA19G1 in cattle in Inner Mongolia. Our study findings provide detailed information on molecular epidemiological investigation of bovine cryptosporidiosis in Inner Mongolia, suggesting that dairy cattle in this region are at risk of transmitting cryptosporidiosis to humans.

Molecular Identification of Planococcus citri (Hemiptera: Pseudococcidae)

Planococcus citri is an important phytophagous pest in different agroecosystems. The species is very difficult to distinguish by morphological features. The goal of this research was to present basic data on the molecular data to confirm P. citri. The primers used in the molecular identification of P. citri consisted of mitochondrial cytochrome c oxidase subunit 1 (COI), ribosomal small subunit RNA (18S rDNA) gene, and internal transcribed spacer 2 (ITS2) gene. The 1% (w/v) agarose gel showed the presence of successful amplification of the COI, 18S rDNA, and ITS2 genes indicated with positive PCR products. The Internal transcribed spacer (ITS2), 18S ribosomal and Cytochrome c oxidase I (COI) mitochondrial genes used in this study confirmed the identity of mealybugs as P. citri. The 18S ribosomal gene showed 98% nucleotide sequence homology to the P. citri USA isolate while the COI gene and ITS2 gene showed less than 95% nucleotide sequence homology to the P. citri USA isolate and P. citri Israel isolate, respectively. At present, the nucleotide sequence data of mealybugs is still lacking; consequently, mealybug species must be correctly identified for the proper management of this pest.

First Molecular Evidence of Babesia vogeli, Babesia vulpes, and Theileria ovis in Dogs from Kyrgyzstan.

Tick-borne parasitic diseases cause mild to severe infections among vertebrate hosts, including dogs. Species in the genus Babesia are important tick-borne pathogens and have worldwide distributions. Although there are data on the prevalence and distribution of Babesia species among dogs around the world, there is no information available in Kyrgyzstan, according to a literature review. In this study, 337 dogs were screened by nested PCR for the presence of the 18S small subunit ribosomal RNA (18S SSU rRNA) gene of piroplasm species. Overall prevalence was 6.23% (21/337) for Babesia/Theileria spp. DNA sequencing of positively tested samples revealed that eighteen samples were infected with Babesia vogeli (B. vogeli) (5.34%), two samples with B. vulpes (0.59%), and one sample with Theileria ovis (T. ovis) (0.29%). The phylogenetic analyses and nucleotide sequences in contrast with those present in GenBank revealed that two nucleotide substitutions (594th and 627th) were found between B. vogeli isolates, including ours, indicating that the mutation is relatively rare. The sequences of other pathogens obtained in this study confirmed 100% nucleotide identity with B. vulpes and T. ovis sequences in GenBank. To the best of our knowledge, B. vogeli, B. vulpes, and T. ovis were detected for the first time in dogs from Kyrgyzstan, and it is thought that results will contribute to the understanding of the epidemiology of canine tick-borne pathogens in the country.

FIRST REPORT IN CANADA OF CERATONOVA GASTEROSTEA (CNIDARIA: MYXOZOA) IN THREESPINE STICKLEBACK GASTEROSTEUS ACULEATUS FROM THE FRASER RIVER, BRITISH COLUMBIA.

A survey of threespine stickleback (Gasterosteus aculeatus L.) and juvenile Chinook salmon (Oncorhynchus tshawytscha (Walbaum)) from the lower Fraser River, British Columbia, Canada and adjacent marine waters was conducted for the presence of myxozoan parasites of the genus Ceratonova Atkinson, Foott, and Bartholomew, 2014 (Bivalvulida, Ceratomyxidae). Quantitative polymerase chain reaction (PCR; small subunit ribosomal RNA gene) combined with conventional PCR and sequencing (ribosomal internal transcribed spacer region 1) detected Ceratonova gasterostea Atkinson, Foott, and Bartholomew, 2014 in 89 of 269 (33.1%) sticklebacks from the Fraser River and in 1 of 51 (2%) from Howe Sound. In contrast, Ceratonova shasta (Atkinson, Foott, and Bartholomew, 2014) was detected in 2 of 26 (7.7%) Chinook salmon from the Fraser River. Reciprocal infections were not detected. Light microscopic examination of the stickleback intestine revealed the presence of an intense infection with large numbers of pseudoplasmodia which interacted with enterocytes through pseudopodia-like projections. Myxospores with characteristic elongate valve cells were visible within mature pseudoplasmodia and free in the intestinal lumen. This is the first report of C. gasterostea in Canada.

Microscopic and Molecular Investigation of Intestinal Microsporidia in HIV + /AIDS and Cancer Patients Undergoing Chemotherapy in Mazandaran Province, North of Iran.

The aim of this study was to identify Enterocytozoon bieneusi and Encephalitozoon spp. in fecal samples of HIV + /AIDS and cancer patients undergoing chemotherapy, and comparing the results to healthy individuals in Mazandaran province, north of Iran. Stool samples were collected from 50 HIV + /AIDS patients, 50 cancer patients, and 50 healthy samples referred to medical centers in north of Iran. Stool samples were kept in 2.5% potassium dichromate at 4°C, and stained by modified trichrome for light microscopy examination. The multiplex/nested-PCR targeted the small subunit ribosomal RNA (SSU rRNA) gene. To characterize genotypes, the nested PCR products sequencedby Bioneer Companyand was subjected to phylogenetic analyses. Ten of 50 samples (20%) of HIV + /AIDS patients, 5of50 samples (10%) of cancer patients, and 1 of healthy individuals (2%) were microscopically positive. From 50 HIV + / AIDS patients, E. bieneusi and Encephalitozoon spp. were detected in 10 (20%) and 6 (12%) cases, respectively. Furthermore, among cancer patients, 7 (14%) and 2 (4%) cases were E. bieneusi and Encephalitozoon spp., respectively. Out of 50 samples of healthy individuals, only 3 (6%)cases of E. bieneusi were observed. The genotypes D and M were detected among positive samples of E. bieneusi. E. bieneusi and then Encephalitozoon spp. are common intestinal microsporidia in HIV + /AIDS patients and cancer patients undergoing chemotherapy in Mazandaran province. E. bieneusi genotype D seems to be the predominant genotype in Mazandaran province.Due to the considerable prevalence of intestinal microsporidia, physicians are advised to pay more attention to this opportunistic infection in high-risk groups.

Molecular detection of Babesia infection in cattle in Yogyakarta, Indonesia

Abstract. Nugraheni YR, Ariyadi B, Rochmadiyanto, Kesumaningrum N, Imran K, Kartiko BP, Farhani NR, Nurani S, Sahara A, Awaludin A. 2023. Molecular detection of Babesia infection in cattle in Yogyakarta, Indonesia. Biodiversitas 24: 4192-4198. Babesiosis is a tick-borne disease caused by hemoprotozoa that poses a significant threat to livestock production worldwide, including in Indonesia. This study aimed to assess the prevalence and molecular characterization of Babesia sp., the causative agent of babesiosis, in cattle from multiple regions in Central Java, Indonesia. The disease has had substantial negative economic impacts, highlighting the need for accurate prevalence data and effective disease control measures. A total of 13 blood samples were collected from cattle exhibiting symptoms of hematuria and babesiosis. The samples were obtained from smallholder farmers who reported these cases to local veterinarians in various regions of Yogyakarta, Indonesia. The farmers were selected based on their proximity to veterinary clinics and willingness to participate in the study. Upon sample collection, each blood sample was subjected to microscopic examination using Giemsa-stained blood smears. The examination aimed to identify the presence of Babesia parasites within the red blood cells. Positive samples, indicating the presence of Babesia infection, were further analyzed by molecular assay. Molecular tests were performed using Polymerase Chain Reaction (PCR) to detect the DNA of Babesia sp. Two specific genes were targeted: cytochrome b oxidase (cytb) and 18S small subunit ribosomal RNA (18S rRNA). PCR amplification was carried out following established protocols, and the resulting products were visualized using gel electrophoresis to confirm the presence of Babesia DNA. Statistical analysis was conducted to compare the sensitivity and efficacy of the two PCR methods (cytb and 18S rRNA). The data obtained from this study contributes to our understanding of the occurrence of babesiosis in Central Java's cattle population. The findings underscore the need for comprehensive babesiosis disease surveys to obtain accurate prevalence estimates and facilitate the development of effective disease control strategies. Moreover, the more sensitive amplification targeting the cytb gene holds promise for improved diagnostic and surveillance efforts. These insights are crucial for combating babesiosis and mitigating its economic impact on livestock production.

Molecular Detection and Epidemiology of Potentially Zoonotic Cryptosporidium spp. and Giardia duodenalis in Wild Boar (Sus scrofa) from Eastern Spain.

The protozoans Giardia duodenalis and Cryptosporidium spp. are common causes of gastrointestinal disease in humans and animals. While both are commonly documented in domestic animals, few studies have analysed their presence in wildlife. To assess the prevalence of both parasites in wild boar (Sus scrofa) in the Valencian Community (eastern Spain), 498 wild boar faecal samples were collected from 2018 to 2022. Cryptosporidium spp. was detected by performing a nested PCR targeting a 578 bp sequence of the small subunit ribosomal RNA gene (SSU rRNA), followed by sequencing and phylogenetic analysis. For G. duodenalis, a qPCR amplifying a fragment of 62 bp from the SSU rRNA was employed. Positive samples were genotyped for glutamate dehydrogenase and β-giardin genes. Different epidemiological factors were considered potential modulating variables in the transmission of both parasites. G. duodenalis prevalence was 1.20%, while Cryptosporidium spp. prevalence reached 21.7%. Coinfection was observed in 0.2%. Genotyping of G. duodenalis isolates only detected genotype E. Two species of Cryptosporidium spp. were identified: Cryptosporidium scrofarum and Cryptosporidium suis. The results of this study demonstrate that the exposure to Cryptosporidium spp. in wild boars is high, particularly among young individuals belonging to the Typical Mediterranean climate. Moreover, the probability of infection is dependent on both the season and the density of wild boars. On the other side, exposure to G. duodenalis seems scarce and is influenced, in turn, by the climate. Both Cryptosporidium species detected in the present study have been reported in humans. Due to wild boar increasing in number and their colonisation of urban and peri-urban areas, this could represent an inherent health risk for the human population.

Comparative detection efficacy of primers targeting SpeI-AvaI restriction fragment and small subunit ribosomal RNA gene of Babesia bigemina

The objective of the study is to evaluate the comparative detection efficacy of primers targeting SpeI-AvaI restriction fragment and small subunit ribosomal RNA (SSU rRNA) gene of Babesia bigemina by employing conventional polymerase chain reaction (PCR) on 783 animals (296 cattle and 487 buffaloes) of low lying (bet) area of Punjab. The detection rate of SpeI-AvaI and SSU rRNA PCR assays was 3.96% (31/783), and 6.64% (52/783), respectively. Among cattle and buffaloes, prevalence of B. bigemina was higher (P<0.01) in cattle by both the primers. The sensitivity and specificity of SSU rRNA PCR as compared to SpeI-AvaI restriction fragment PCR was 100% and 97.2%, respectively. The blast analysis of the nucleotides of the sequenced amplicons of Ludhiana isolates of SpeI-AvaI and SSU rRNA PCR assay of B. bigemina showed 83 and 100% similarity with available sequence in Genbank. The analysis of evolutionary divergence revealed that range of divergence was lying between 0.000 to 0.011 between SSU rRNA sequence with the other sequences of B. bigemina as well as Babesia species. To conclude, the primers targeting SSU rRNA gene are a better tool for amplification of the B. bigemina.

Comparative detection efficacy of primers targeting SpeI-AvaI restriction fragment and small subunit ribosomal RNA gene of Babesia bigemina

The objective of the study is to evaluate the comparative detection efficacy of primers targeting SpeI-AvaI restriction fragment and small subunit ribosomal RNA (SSU rRNA) gene of Babesia bigemina by employing conventional polymerase chain reaction (PCR) on 783 animals (296 cattle and 487 buffaloes) of low lying (bet) area of Punjab. The detection rate of SpeI-AvaI and SSU rRNA PCR assays was 3.96% (31/783), and 6.64% (52/783), respectively. Among cattle and buffaloes, prevalence of B. bigemina was higher (P<0.01) in cattle by both the primers. The sensitivity and specificity of SSU rRNA PCR as compared to SpeI-AvaI restriction fragment PCR was 100% and 97.2%, respectively. The blast analysis of the nucleotides of the sequenced amplicons of Ludhiana isolates of SpeI-AvaI and SSU rRNA PCR assay of B. bigemina showed 83 and 100% similarity with available sequence in Genbank. The analysis of evolutionary divergence revealed that range of divergence was lying between 0.000 to 0.011 between SSU rRNA sequence with the other sequences of B. bigemina as well as Babesia species. To conclude, the primers targeting SSU rRNA gene are a better tool for amplification of the B. bigemina.

First Report of Bacterial Leaf Spot of Red Maple Caused by Pseudomonas syringae pv. syringae in Tennessee.

Red maple (Acer rubrum L.) is an economically important ornamental nursery plant grown for its aesthetic value. In May 2022, field and container-grown red maple 'October Glory' plants exhibited severe leaf spots in a commercial nursery in Warren Co., Tennessee. Leaf spots were brown-to-black color with a yellow halo (Fig. 1a). Disease severity was about 40% of leaf area and incidence was 60-70% of 10,000 plants. Symptomatic leaf tissues were surface sterilized with 0.525% sodium hypochlorite for 1 min and washed twice with sterilized water. Bacterial colonies, cream colored and circular with smooth margins, were obtained on King's B (KB) and nutrient agar media after 3 days of incubation at 28°C. Bacteria were gram-negative and fluorescent on KB under UV light. The biochemical and physiological test results were negative for cytochrome C oxidase, pectolytic activity on potato slices, and arginine dehydrolase, but positive for gelatin liquefaction, aesculin hydrolysis, and levan production. The BIOLOG test was positive for the utilization of D-galactose, D-galacturonic acid, D-galactonic acid lactone, D-gluconic acid, and was negative for the utilization of β-methyl-D-glucoside, N-acetyl-D-glucosamine, α-hydroxybutyric acid, D-glucose-6-phosphate, α-keto-butyric acid, and α-keto-glutaric acid. To confirm the bacterial identity, total genomic DNA was extracted using DNeasy PowerLyzer Microbial Kit directly from pure cultures (strains FBG1662 and FBG4230). The small subunit ribosomal RNA (16S rRNA), RNA polymerase sigma factor (rpoDp and rpoDs), citrate synthase (gltA), DNA gyrase (gyrB) genes were amplified and sequenced by primers 8F/1492R (Galkiewicz et al. 2008), rpoDpF/R, rpoDsF/R, gltAF/R, and gyrBF/R (Sarkar and Guttman 2004), respectively. The sequences of the two strains (GenBank accession nos. 16S: OP962145 and OP948281; rpoDp: OP998258 and OP957300; rpoDs: OP998259 and OP957299: gltA: OP998256 and OP957301; gyrB: OP998257 and OP957302) were >99% similar (100% coverage) to the complete genome of Pseudomonas syringae pv. syringae (CP026568) in the NCBI database. A phylogenetic analysis was performed and confirmed the identity using concatenated sequences of gltA, gyrB, rpoDp, rpoDs, and 16S of P. syringae pv. syringae and other closely related taxa retrieved from GenBank (Fig. 2). Based on morphological and molecular identification, both bacterial strains were identified as P. syringae pv. syringae. Pathogenicity test was conducted by spray inoculation of ten one-year-old red maple 'October Glory' with bacterial suspension (107 CFU/ml) using bacterial strain FBG4230. Ten plants were sprayed with sterilized water as control. All plants were covered with clear plastic for 24 h and incubated in a greenhouse at 21 to 23°C, 70%RH, 16-h photoperiod. At seven days after inoculation, brown-to-black leaf spots surrounded by yellow halo were developed on all inoculated plants (Fig. 1b), while the control plants remained symptomless. The bacterium was re-isolated from the inoculated plants and it was 100% identical to P. syringae pv. syringae using biochemical tests as well as sequence analysis. P. syringae has been reported pathogenic in red maple causing leaf spot in Oregon (Malvick and Moore, 1988). To our knowledge, this is the first report of bacterial leaf spot caused by P. syringae pv. syringae in red maple in Tennessee. Identification of this bacterial pathogen on red maple is crucial in developing timely management practices.

Comparison of Kato-Katz, PCR and coproantigen for the diagnosis of Taenia solium taeniasis.

Four methods were compared for the diagnosis of human taeniasis caused by Taenia solium. Fecal samples from persons living in a T. solium endemic region of Madagascar were examined for taeniid eggs by the Kato–Katz method. Subsequently, samples positive (n = 16) and negative (n = 200) for T. solium eggs were examined by (i) amplification of the fragment of small subunit of the mitochondrial ribosomal RNA (rrnS) gene using conventional polymerase chain reaction (PCR) and (ii) a nested PCR of a fragment of the T. solium Tso31 gene. Additionally, 12 egg-positive and all egg-negative samples were tested for coproantigen detection. A further 9 egg-positive fecal samples were examined using both PCRs. Of the 12 egg-positive samples tested by PCRs and coproantigen methods, 9 (75%) were positive by rrnS PCR, 3 (25%) using Tso31-nested PCR and 9 (75%) by coproantigen testing. None of the 200 egg-negative fecal samples was positive in either rrnS or Tso31-nested PCR. Twenty of the 25 egg-positive samples (80%) were positive in rrnS PCR, and DNA sequencing of PCR amplicons was obtained from 18 samples, all confirmed to be T. solium. Twelve of the 25 egg-positive samples (48%) were positive in the Tso31-nested PCR, all of which were also positive by rrnS PCR. It is suggested that species-specific diagnosis of T. solium taeniasis may be achieved by either coprological examination to detect eggs or coproantigen testing, followed by rrnS PCR and DNA sequencing to confirm the tapeworm species in egg-positive or coproantigen-positive samples.

European ground squirrels Spermophilus citellus (Linnaeus) do not share identical Cryptosporidium spp. with North American ground squirrels.

Cryptosporidium Tyzzer, 1910 is one of the most common protistan parasites of vertebrates. The results of this study provide the first data on Cryptosporidium diversity in the European ground squirrel Spermophilus citellus (Linnaeus). A total of 128 faecal samples of European ground squirrels from 39 localities in the Czech Republic were analysed for the presence of Cryptosporidium spp. by microscopy and PCR/sequence analysis of small subunit ribosomal RNA (SSU) and the actin gene. While the microscopical examination did not reveal the presence of any Cryptosporidium oocysts, eight samples from six localities were PCR-positive. Phylogenetic analyses revealed the presence of five different Cryptosporidium spp. isolates. Four isolates, designated as Cryptosporidium sp. isolate Sc01-04, detected in wild populations and never recorded before, clustered closely to Cryptosporidium genotypes that have previously been found in North American ground squirrels' species. Cryptosporidium sciurinum Prediger, Ježková, Holubová, Sak, Konečný, Rost, McEvoy, Rajský et Kváč, 2021 was found in an animal sanctuary. Because C. sciurinum had previously been detected in Eurasian red squirrels Sciurus vulgaris Linnaeus at the same facility, it can be concluded that this Cryptosporidium was transmitted from tree squirrels to ground squirrels within the animal sanctuary. The results indicate that populations of European and North American ground squirrels are parasitised by different Cryptosporidium spp. At the same time, this is the first description of the occurrence of C. sciurinum in ground squirrels.

Description of lipase producing novel yeast species Debaryomyces apis f.a., sp. nov. and a modified pH indicator dye-based method for the screening of lipase producing microorganisms

Four yeast strains were isolated from the gut of stingless bee, collected in Churdhar, Himachal Pradesh, India. Physiological characterization, morphological examination, and sequence analysis of small subunit ribosomal RNA (18S rRNA) genes, internal transcribed spacer (ITS) region, and D1/D2 domain of the large subunit rRNA gene revealed that the four strains isolated from the gut of stingless bee belonged to the Debaryomyces clade. Strain CIG-23HT showed sequence divergence of 7.5% from Debaryomyces nepalensis JCM 2095T, 7.8% from Debaryomyces udenii JCM 7855T, and Debaryomyces coudertii JCM 2387T in the D1/D2 domain. In the ITS region sequences, strain CIG-23HT showed a 15% sequence divergence from Debaryomyces nepalensis JCM 2095T and Debaryomyces coudertii JCM 2387T. In 18S rRNA gene sequence, the strain CIG-23HT showed 1.14% sequence divergence from Debaryomyces nepalensis JCM 2095 and and Debaryomyces coudertii JCM 2387, and 0.83% sequence divergence from Debaryomyces hansenii NRRL Y-7426. Strain CIG-23HT can utilize more carbon sources than closely related species. The findings suggest that strain CIG-23HT is a novel species of the genus Debaryomyces, and we propose to name it as Debaryomyces apis f.a., sp. nov. The holotype is CBS 16297T, and the isotypes are MTCC 12914T and KCTC 37024T. The MycoBank number of Debaryomyces apis f.a., sp. nov. is MB836065. Additionally, a method using cresol red and Bromothymol blue pH indicator dyes was developed to screen for lipase producers, which is more sensitive and efficient than the currently used phenol red and rhodamine B dye-based screening methods, and avoids the problem of less differentiable zone of hydrolysis.

Molecular characterization of Moniezia denticulata (Rudolphi, 1810) and its distinction from M. expansa infecting sheep and goats raised in the north and north-western regions of India.

The tapeworms of Moniezia spp. are heteroxenous parasites and their adult forms occur in ruminants' alimentary tract. They steal a significant portion of hosts' nourishment initiating monieziasis, thereby inflicting economic losses in animal rearing. Despite their high economic importance, the molecular characterization and taxonomic status of these parasites have remained poorly understood. In the present study, cestodes were isolated from the sheep and goats' intestines and were stained with Gower's carmine. Upon careful evaluation of morphological characters, 2 species Moniezia denticulata and Moniezia expansa were identified. The genomic DNA was extracted and polymerase chain reaction (PCR) amplified targeting regions of mitochondrial cytochrome c oxidase subunit 1 (cox1), small subunit ribosomal RNA (SSU rRNA) and internal transcribed spacer 1–5.8S rRNA (ITS1–5.8S rRNA) genes followed by sequencing. The partial sequences of cox1, SSU rRNA and ITS1–5.8S rRNA genes of M. denticulata generated in the present study revealed that even though they share high similarities with M. benedeni (93.2% cox1; 92.6% SSU rRNA; 84.70% ITS1–5.8S rRNA) and M. expansa (88.85% cox1; 92.27% SSU rRNA; 81.70% ITS1–5.8S rRNA), they are not identical to them. In the maximum likelihood phylogenetic trees, M. denticulata and M. expansa consistently appeared as distinct species from each other. The high values of pairwise divergence between these 2 species collected in the present study confirmed their separate identity. The present study reports the first molecular characterization of M. denticulata with reference to M. expansa infecting sheep and goats in India.

Morphology and genetic characterization of Physaloptera sibirica Petrow & Gorbunov, 1931 (Spirurida: Physalopteridae), from the hog-badger Arctonyx collaris Cuvier (Carnivora: Mustelidae), with molecular phylogeny of Physalopteridae

BackgroundNematodes of the family Physalopteridae (Spirurida: Physalopteroidea) commonly parasitize the alimentary canal of all major vertebrate groups. However, many physalopterid species are not adequately described, especially regarding the detailed morphology of the cephalic end. The current genetic database for Physaloptera species is still very limited, which seriously hampers molecular-based species identification. Additionally, the systematic status of some genera and the evolutionary relationships of the subfamilies in the Physalopteridae remain under debate.MethodsNew morphological data for Physaloptera sibirica was gathered using light and scanning electron microscopy based on newly collected specimens from the hog badger Arctonyx collaris Cuvier (Carnivora: Mustelidae) in China. Six different genetic markers, including nuclear small ribosomal DNA (18S), large ribosomal DNA (28S) and internal transcribed spacer (ITS), mitochondrial cytochrome c oxidase subunit 1 (cox1) and subunit 2 (cox2), and the 12S small subunit ribosomal RNA gene of P. sibirica were sequenced and analyzed for the first time to our knowledge. Additionally, to construct a basic molecular phylogenetic framework for the Physalopteridae, phylogenetic analyses were performed based on the cox1 and 18S + cox1 genes using maximum likelihood (ML) and Bayesian inference (BI) methods.ResultsScanning electron microscopy (SEM) observation displayed the details of the cephalic structures, deirids, excretory pore, caudal papillae, vulva, phasmids and egg of P. sibirica for the first time to our knowledge. Pairwise comparison of the sequences obtained for P. sibirica did not reveal intraspecific divergence regarding the 18S, 28S, cox1 and 12S genetic markers and a low level of divergence in the ITS (0.16%) and cox2 (2.39%) regions. Maximum likelihood and Bayesian inference analyses showed that the representatives of Physalopteridae formed two major clades (species of Physalopterinae + Thubunaeinae parasitic in terrestrial vertebrates and Proleptinae only occurring in marine or freshwater fishes). Turgida turgida was found nested among representatives of Physaloptera. Physaloptera sibirica clustered together with P. rara. Physalopteroides sp. (Thubunaeinae) formed a sister relationship to the physalopterine Abbreviata caucasica.ConclusionsPhysaloptera sibirica was redescribed, which is the fourth nematode parasite reported from the hog badger A. collaris, and A. collaris represents a new host for P. sibirica. The phylogenetic results challenged the validity of the subfamily Thubunaeinae and of the genus Turgida and supported dividing the family Physalopteridae into two subfamilies, Physalopterinae and Proleptinae. However, we do not make any immediate systematic changes in the Physalopteridae, because a more rigorous study with broader representation of the Physalopteridae is required. These present findings contribute to morphologically identifying P. sibirica more accurately and provide new insights into the systematics of the Physalopteridae.Graphical

Dominant infection of Cryptosporidium baileyi in broiler chickens in Zhejiang Province, China.

Cryptosporidium is a common enteric parasite in chickens. A total of 812 fecal specimens were collected from 11 broiler farms in Zhejiang Province, China, and analyzed by nested PCR amplification based on the small subunit ribosomal RNA (SSU rRNA) gene. The overall infection rate of Cryptosporidium was 6.3% (51/812), and five of 11 farms were Cryptosporidium positive. Broilers aged > 90 days accounted for the highest infection rate of 16.1% (6/56), followed by those aged 30-60 days (10.6%, 38/358) and 60-90 days (4/378, 1.1%). Two Cryptosporidium species were identified by sequence analysis, with the predominant species being C. baileyi (96.1%, 49/51) and the minor infection being C. meleagridis (3.9%, 2/51). Based on the 60-kDa glycoprotein (gp60) gene, two C. meleagridis-positive isolates were identified as one known subtype, IIIbA24G1R1. This study indicated the common occurrence of C. baileyi in broiler chickens in this region and low zoonotic transmission potential of Cryptosporidium to humans.

The occurrence and zoonotic potential of Cryptosporidium species in freshwater biota

BackgroundProtozoan pathogens from the genus Cryptosporidium cause the diarrhoeal disease cryptosporidiosis in humans and animals globally. Freshwater biota could act as potential reservoirs or zoonotic sources of Cryptosporidium infections for livestock and people, but Cryptosporidium occurrence in aquatic biota is largely unexplored. The aim of this study was to investigate the occurrence of Cryptosporidium in a range of freshwater organisms in upland rivers across England and Wales.MethodsFish were sampled by electrofishing, invertebrate larvae by kick sampling and the otter Lutra lutra and mink Mustela vison through faecal samples collected opportunistically as part of a nation-wide study. PCR targeting the small subunit ribosomal RNA gene was used to detect Cryptosporidium species.ResultsCryptosporidium occurred in just 0.8% of all the samples and in none of 73 samples from nine invertebrate genera. Cryptosporidium was detected in two of 2/74 fish samples (2.7%), both salmonids, and in 2/92 otter faecal samples (2.17%), but there were no positive samples in mink (0/24) or the bullhead Cottus gobio (0/16).ConclusionsLow detection rate of human-infective Cryptosporidium species in aquatic fauna indicates they may present a low risk of contamination of some upland freshwaters.Graphical

First Report of Rusty Root of Panax quinquefolius Caused by Pseudomonas marginalis in Tennessee and the United States.

American ginseng (Panax quinquefolius L.) is one of the most valuable medicinal plants that is native to the U.S. This plant is naturally grown under hardwood canopies or artificially cultivated in fields covered with shade. Bacterial infections were observed on 5-year-old cultivated American ginseng roots in Rutherford Co., TN, in March 2022. Infected roots were exhibiting brown lesions in varying sizes. Under severe infection, the periderm of the root was ruptured, leaving a scabbed appearance on the root. The disease severity (percentage root area diseased) was nearly 20% and the disease incidence was nearly 10% out of 20 plants. Bacterial streaming from the infected tissue was observed under the microscope. Bacteria were isolated from surface-sterilized infected root tissue (0.525% NaOCl; 1 min) by plating 10-fold serial dilutions onto yeast dextrose carbonate and King's B (KB) media. Gram-negative, fluorescent bacterial colonies of the isolates FBG1141A and FBG1141B were milky white and translucent on KB at 28 °C. The biochemical and physiological tests including oxidase, levan, arginine dihydrolase, catalase, esculin, mobility test, and growth at 35°C were positive but gelatine and starch hydrolasis were negative. Bacterial suspension prepared with sterile distilled water (1×108 CFU/ml) resulted in soft rot on potato slices. The BIOLOG test showed positive results for the utilization of D-gluconic acid, D-glucuronic acid, D-galactose, D-glucose, L-serine and citric acid but negative results for the utilization of cellobiose and L-rhamnose. Bacterial identity was further confirmed by extracting the total genomic DNA using DNeasy PowerLyzer Microbial Kit directly from the two pure cultures. The small subunit ribosomal RNA (16S rRNA) and RNA polymerase sigma factor (rpoD) genes were amplified and sequenced by the primers 8F/1492R (Galkiewicz et al. 2008) and PsEG30F/PsEG790R (Mulet et al. 2009), respectively. The sequences (GenBank accession nos. OP549779, OP550133: 16S; OP554814, OP554815: rpoD) were 99.26% similar to 16S rRNA and 100% to rpoD genes of Pseudomonas marginalis (LC507983: 16S and MH49410: rpoD) from several hosts in multiple countries in the NCBI database. A phylogenetic analysis was performed by adding the concatenated sequences of 16S and rpoD from other closely related taxa obtained from GenBank (Fig. 1). Pathogenicity test was performed by spraying a suspension of the P. marginalis FBG1141A strain (108 CFU/ml) on six 2-year-old American ginseng roots wounded with a sterilized needle. Plants were covered with clear plastic for 24 h and maintained inside a greenhouse at 21 to 23°C, 70% RH, 16-h photoperiod. Six wounded roots were sprayed with sterilized water as controls and kept in the same condition. Inoculated roots showed rusty root symptoms after 4 weeks (Fig. 2a), while controls remained asymptomatic (Fig. 2b). The bacterium was re-isolated from the infected tissue and confirmed as P. marginalis using physiological and biochemical tests as well as sequencing. P. marginalis has been previously reported causing rusty-colored roots on Korean Ginseng (P. ginseng C.A. Mey)(Choi et al. 2005; Farh et al. 2018; Lee et al. 2011) but to our knowledge, this is the first report of rusty root caused by P. marginalis on American ginseng (P. quinquefolius) in Tennessee and the U.S. Identification of bacterial pathogen impacting the economic yield of American ginseng can be effective for developing correct disease management strategies.