Gdh Gene Research Articles

Gastrointestinal protozoa in pet cats from Anhui province: prevalence and molecular characterization

IntroductionTo investigate the prevalence of Tritrichomonas foetus, Pentatrichomonas hominis, Giardia intestinalis, Cryptosporidium, Microsporidium, and Sarcocystis in domestic cats in Anhui Province, China, and their potential role as zoonotic hosts for human infection, a total of 304 fecal samples from two different sources were screened for the presence of related pathogens.MethodsUsing microscopy, along with polymerase chain reaction (PCR) or nested PCR amplification, followed by genotyping through sequence analysis.ResultsThe infection rates of T. foetus, P. hominis, G. intestinalis, Cryptosporidium, Enterocytozoon bieneusi, and Sarcocystis were 5.6%, 0%, 1.7%, 0.7%, 2.6%, and 0%, respectively. The evolutionary relationships and genetic characteristics of G. intestinalis based on the GDH gene, Cryptosporidium based on the SSU rRNA gene, and E. bieneusi based on the ITS sequence were assessed: five cases of G. intestinalis were identified, with four belonging to assemblage F and one to zoonotic assemblage B, two Cryptosporidium cases were identified as Cryptosporidium felis, and all eight E. bieneusi cases were identified as belonging to group 1, with three cases being genotype D, three EbpA, and two EbpC.DiscussionAge, neutering status, and deworming were identified as potential risk factors. Further analysis revealed that diarrhea, as a clinical symptom, could serve as an indicator for pathogen infection. Although the pathogen infection rates detected in this study were relatively low, their zoonotic transmission potential cannot be ignored. Therefore, special attention should be paid, and it is essential to establish targeted prevention plans.

Epidemiology and transmission patterns of Cryptosporidium spp., and Giardia duodenalis within a One Health framework in rural areas of Eastern Algeria.

Gastrointestinal infections constitute a significant global health concern, particularly in tropical and subtropical regions, caused by various pathogens. Among these, Cryptosporidium spp. and Giardia duodenalis are noteworthy due to their zoonotic potential. In Algeria, molecular epidemiological data on cryptosporidiosis and giardiasis are limited. To fill this gap, the present study aimed to examine the transmission dynamics of Cryptosporidium spp., and Giardia duodenalis in various households. A total of 216 samples were collected from the rural Guelma and Souk Ahras provinces, located in the eastern part of Algeria. These included human and animal faeces, as well as water and soil samples. DNA was extracted, followed by nested PCR targeting the SSU rRNA gene to detect Cryptosporidium spp., while the gp60 gene was amplified for subtyping. Detection of G. duodenalis was performed by qPCR targeting the SSU rRNA gene, followed by amplification of tpi, bg and gdh genes for genotyping and subtyping. Several Cryptosporidium species, including C. bovis, C. ryanae, C. andersoni and C. parvum, were identified in human, animal and environmental samples. The zoonotic C. parvum subtype IIaA17G2R1 was detected in human, animal and soil samples. Giardia duodenalis assemblage B was detected in a human sample, while assemblage E was found in cattle and sheep. The current investigation underscores the importance of the One Health approach in addressing issues related to intestinal parasites, highlighting the need for improved surveillance and control measures in rural settings.

Prevalence and genetic diversity of Giardia duodenalis in sheep from large-scale farms in Southern Xinjiang, China

Giardia duodenalis is a significant zoonotic parasite. In this study, 767 fresh fecal samples were collected randomly from six large-scale sheep farms in Southern Xinjiang, China. Initially, G. duodenalis was screened using PCR at the SSU rRNA gene. Positive samples then underwent PCR amplification at the bg, gdh, and tpi genes. The prevalence of G. duodenalis in sheep was 17.5% (134/767), with the highest prevalence observed in the 3-6 months age group at 26.8% (56/209) and the lowest in the over 12 months age group at 6.8% (14/205). Among the 134 positive samples, only Assemblage E was identified. A total of 106, 92, and 98 sequences of G. duodenalis were obtained at the gdh, tpi, and bg genes, respectively. Fourteen isolates of G. duodenalis were successfully amplified at all three genes, resulting in nine G. duodenalis multilocus genotypes (MLG) named MLG E1-MLG E14, indicating high genetic diversity. In conclusion, G. duodenalis infection in sheep from large-scale farms is common in Southern Xinjiang, China, showing geographical regional distributions and genetic diversity.

Determination of Technological Properties and CRISPR Profiles of Streptococcus thermophilus Isolates Obtained from Local Yogurt Samples.

The aim of this study was to obtain data on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) profiles of Streptococcus thermophilus (S. thermophilus) isolates resulting from acquired immune memory in addition to their technological starter properties for the selection of potential starter cultures from local yogurt samples. A total of 24 S. thermophilus isolates were collected from six local yogurt samples including Afyon/Dinar, Uşak, Konya/Karapınar, and Tokat provinces of Türkiye. Strain-specific CRISPR I-II-III and IV primers were used to determine the CRISPR profiles of the isolates. The isolates commonly had CRISPR II and IV profiles, while only one isolate had a CRISPR III profile. Polymerase chain reaction (PCR)-based and culture-based analyses were also carried out to obtain data on the technological properties of the isolates. The PCR analyses were performed for the prtS gene for protease activity, the ureC gene for urease enzyme, the gdh gene for glutamate dehydrogenase, the cox gene for competence frequency, the csp gene involved in heat-shock stress resistance of the isolates with specific primers. Culture-based analyses including antimicrobial activity and acid-production ability of the isolates were completed, and proteolytic and lipolytic properties were also screened. Native spacer sequences resulting from acquired immune memory were obtained for CRISPR IV profiles of yogurt samples from the Konya-Karapınar and Tokat provinces and CRISPR III profiles of yogurt samples from the Uşak province. In conclusion, our study results suggest that it is possible to select the isolates with the desired level of technological characteristics, prioritizing the ones with the most diverse CRISPR profiles and with native spacers for potential industrial application as starter cultures. We believe that this study provides data for further biological studies on the impact of centuries of human domestication on evolutionary adaptations and how these microorganisms manage survival and symbiosis.

Antagonistic Effect of Microplastic Polyvinyl Chloride and Nitrification Inhibitor on Soil Nitrous Oxide Emission: An Overlooked Risk of Microplastic to the Agrochemical Effectiveness.

Microplastics are widely persistent in agricultural ecosystems and may affect soil nitrous oxide (N2O) emissions. Nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) is applied to decelerate nitrification and reduce soil N2O emission. Nevertheless, the interactive effects of nitrification inhibitors and microplastics on soil N2O emissions have not been investigated. Sole DMPP, polyvinyl chloride (PVC), and polystyrene (PS) substantially reduced agricultural soil N2O emission rates by 25.93%, 69.04%, and 73.89%, respectively. Nevertheless, PVC and DMPP had antagonistic effects on the N2O emission rates. The observed reductions in N2O emissions could be attributed to variations in soil oxygen availability, electron transport system activities, and Firmicutes, nap, and GDH genes. Moreover, the DMPP, PVC, and PS alone or copresences significantly enhanced the soil ecosystem multifunctionality (EMF). The findings shed light on the role of microplastics in soil N2O emission, EMF, and the microbial community, expanding the understanding of microplastics' effects on agrochemical effectiveness.

Development and application of a quadruplex TaqMan fluorescence quantitative PCR typing method for Streptococcus suis generalis, type 2, type 7 and type 9.

Streptococcus suis (SS) is one of the most important pathogens causing major economic losses in the global pig farming industry and is a serious threat to public health safety. It has multiple serotypes, with poor cross-protection between serotypes, and effective typing methods are lacking. In this study, a quadruplex TaqMan fluorescence quantitative PCR assay that can differentiate between Streptococcus suis types 2, 7 and 9 was developed using the gdh gene, a generic gene for Streptococcus suis, and cps2J, cps7H and cps9J, genes encoding podocarp-associated genes for types 2, 7 and 9, respectively, as targets. The method is specific enough to accurately type Streptococcus suis pigmentosus without detecting non-target pathogens (Escherichia coli, Pasteurella multocida, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae and etal). The sensitivity was high, with a minimum lower detection line of 10 copies for P-SS and P-SS9, and 100 copies for P-SS2 and P-SS7. The standard curves generated showed good linearity with R2 of 0.999, 0.999, 0.997 and 0.998 respectively. The repeatability was good, with coefficients of variation between batch to batch and batch to batch tests ranging from 0.21% to 1.10%. Testing of 156 samples yielded 68 positive and 88 negative samples, of which the positive rate of SS was 5.77% (9/156), SS2 was 20.51% (32/156), SS7 was 8.33% (13/156) and SS9 was 9.6% (15/156), which was in line with the existing fluorescent quantitative PCR assay of 93.75%~100%, which was higher than the detection rate of conventional PCR. The quadruplex TaqMan fluorescence quantitative PCR method of Streptococcus suis generic, type 2, 7 and 9 established in this study can accurately differentiate the three serotypes of Streptococcus suis that currently have high prevalence and pathogenicity, which is of great importance for accurate clinical prevention and treatment, epidemiological investigation and vaccine development.

Current status of intestinal parasite infections in fecal samples of dogs in Korea.

This study surveyed the current status of intestinal parasite infections in Korean dog fecal samples. A total of 367 fecal samples were collected from the northern (Seoul and Gyeonggi-do), central (Chungcheong-do), and southern (Gyeongsang-do) regions and analyzed using the saturated sodium nitrate flotation technique and the nucleotide sequences of 18S rRNA. Six species of intestinal parasites were detected using the flotation technique. Among them, helminth eggs detected included Toxocara canis (6.0%), Toxascaris leonina (1.1%), Trichuris vulpis (6.8%), Ancylostoma caninum (2.7%), and Spirometra sp. (1.1%). Additionally, Cystoisospora sp. (7.6%) oocysts were also detected. The prevalence of intestinal parasite infections was higher in shelter dogs than in pet dogs. Molecular genetic assays revealed the gdh and 18S rRNA genes of Giardia duodenalis (type D) in 4.9% of fecal samples. To the best of our knowledge, 18S rRNA genes of Cryptosporidium canis were identified in 1.9% of fecal samples for the first time in Korea. These findings provide an overview of the current status of intestinal parasite infections in fecal samples of dogs from Korea and can be helpful in the surveillance of zoonotic parasite infections related to dogs.

Environmental surveillance of Cryptosporidium and Giardia in surface supply water and treated sewage intended for reuse from an urban area in Brazil

Environmental monitoring of protozoa, with the potential to trigger diseases, is essential for decision-making by managing authorities and for the control of water surveillance. This study aimed to detect and quantify Cryptosporidium oocysts and Giardia cysts in surface water for drinking water supply and treated sewage for reuse in the city of São Paulo. Samples collected bimonthly for one year were concentrated using the USEPA 1623.1 and 1693 methods for surface water and treated effluents, respectively. Immunofluorescence and nucleic acid amplification techniques were used to detect and quantify (oo)cysts. The cloning technique followed by sequencing and phylogenetic analyses were performed to characterize species and genotypes. The immunofluorescence detected Cryptosporidium spp. and Giardia spp. in 69.2% (9/13) and 100% (13/13) of the surface water samples (0.1–41 oocysts/L and 7.2–354 cysts/L, respectively). In the reuse samples, 85.7% (12/14) were positive for both protozoa and the concentrations varied from 0.4 to 100.6 oocysts/L and 1.2 and 93.5 cysts/L. qPCR assays showed that 100% of surface water (0.1–14.6 oocysts/L and 0.3–639.8 cysts/L) and reused samples (0.1–26.6 oocysts/L and 0.3–92.5 cysts/L) were positive for both protozoa. Species C. parvum, C. hominis, and C. muris were identified using the 18S rRNA gene, demonstrating anthroponotic and zoonotic species in the samples. Multilocus SSU rRNAanalyses of the SSU rRNA, tpi, and gdh genes from Giardia intestinalis identified the AII, BII, and BIV assemblages, revealing that contamination in the different matrices comes from human isolates. The study showed the circulation of these protozoa in the São Paulo city area and the impairment of surface water supply in metropolitan regions impacted by the discharge of untreated or inadequately treated sewage regarding the removal of protozoa, emphasizing the need to implement policies for water safety, to prevent the spread of these protozoa in the population.

Development of a Triplex qPCR Assay Based on the TaqMan Probe for the Detection of Haemophilus parasuis, Streptococcus suis Serotype 2 and Pasteurella multocida

Porcine respiratory disease is a significant economic problem for the global swine industry. Haemophilus parasuis (H. parasuis), Streptococcus suis (S. suis), and Pasteurella multocida (P. multocida) are three important pathogenic bacteria of the swine respiratory tract. Notably, the three pathogens not only frequently manifest as mixed infections, but their striking clinical similarities also present difficulties for pig populations in terms of disease prevention and treatment. Thus, we developed a triplex real-time quantitative polymerase chain reaction (qPCR) assay based on a TaqMan probe for the detection of H. parasuis, S. suis serotype 2, and P. multocida. Primers and probes were designed to target the conserved regions of the H. parasuis OmpP2 gene, the S. suis serotype 2 gdh gene, and the P. multocida Kmt1 gene. By optimizing the reaction system and conditions, a triplex qPCR method for simultaneous detection of H. parasuis, S. suis serotype 2, and P. multocida was successfully established. The amplification efficiencies of the standard curves for all three pathogens were found to be highly similar, with values of 102.105% for H. parasuis, 105.297% for S. suis serotype 2, and 104.829% for P. multocida, and all R2 values achieving 0.999. The specificity analysis results showed that the triplex qPCR method had a strong specificity. The sensitivity test results indicated that the limit of detection can reach 50 copies/μL for all three pathogens. Both intra- and inter-assay coefficients of variation for repeatability were below 1%. This triplex qPCR method was shown to have good specificity, sensitivity, and reproducibility. Finally, the triplex qPCR method established in this study was compared with the nested PCR as recommended by the Chinese national standard (GB/T34750-2017) for H. parasuis, the PCR as recommended by the Chinese national standard (GB/T 19915.9-2005) for S. suis serotype 2, and the PCR as recommended by the Chinese agricultural industry standard (NY/T 564-2016) for P. multocida by detecting the same clinical samples. Both methods are reasonably consistent, while the triplex qPCR assay was more sensitive. In summary, triplex qPCR serves not only as a rapid and accurate detection and early prevention method for these pathogens but also constitutes a robust tool for microbial quality control in specific pathogen-free pigs.

Pyruvate stimulates transamination of leucine into α-ketoisocaproic acid and supports 3-methylbutanal production by Lactococcus lactis.

To investigate the effect of pyruvate and glucose on leucine transamination and 3-methylbutanal production by Lactococcus lactis, including the comparison with cells possessing glutamate dehydrogenase (GDH) activity. Lactococcus lactis cells were incubated in chemically defined medium (CDM) with the pH controlled at 5.2 to mimic cheese conditions. Pyruvate supplementation stimulated the production of the key flavour compound 3-methylbutanal by 3-4 times after 72 h of incubation. Concurrently, alanine production increased, demonstrating the involvement of pyruvate in transamination reactions. Glucose-metabolizing cells excreted α-ketoisocaproic acid and produced even 3 times more 3-methylbutanal after 24 h than pyruvate-supplemented cells. Conjugal transfer technique was used to transfer the plasmid pGdh442 carrying the gdh gene encoding for GDH to L. lactis. Introducing GDH did not stimulate the excretion of α-ketoisocaproic acid and the production of 3-methylbutanal. These results demonstrate that Lactococcus uses pyruvate to transaminate leucine into α-ketoisocaproic acid which supports 3-methylbutanal production. Surprisingly, GDH activity did not stimulate leucine transamination and 3-methylbutanal production.

Molecular characterization of Giardia duodenalis, Cryptosporidium spp., and Entamoeba spp. infecting domestic and feral/stray cats in Jordan.

This study aimed to carry out a molecular screening for the presence of Giardia, Cryptosporidium, and/or Entamoeba in the feces of pet and stray/feral cats in Jordan. G. duodenalis was found in 27.9% (95% CI, 23.2-32.9) of the 348 sampled cats overall; E. histolytica was found in only 0.6% (95% CI, 0.1-2.1) of the cats, while none of the sampled cats had Cryptosporidium infections. The infection rate of G. duodenalis among indoor cats (32.3%) did not differ significantly from that among outdoor cats (24.1%). There were significantly more infections (p = 0.0004) geographically in the cold semiarid areas (67%) than in the cold desert areas (24%). Multilocus sequence typing analysis of amplicons based on the bg, tpi, and gdh genes revealed that the majority of G. duodenalis infections were zoonotic assemblage B (65.9%; 64 of 97 positive samples); followed by feline-specific assemblage F (18.5%, 18/97); cattle-specific assemblage E (5.2%, 5/97); and then assemblage C that was shared with canids (1.0%; 1/97). Within Giardia isolates, a substitution mutation (A/G) was found at position 297 of the complete protein coding sequence (cds) of tpi-assemblage B, which may represent a new spreading mutation within this gene among the cat population in Jordan. The results of the present study suggest that close human-cat interactions could play a role in zoonotic transmission of Giardia, but further research is needed to determine the possible contribution of cats to the transmission of other protozoa to humans.

Integrating loop-mediated isothermal amplification with lateral flow assay to achieve a highly sensitive method for detecting Streptococcus suis Genome in raw pork

Streptococcus suis (S.suis), a zoonotic foodborne pathogen prevalent in Southeast Asia, poses a substantial threat to human and animal health because of its ability to cause severe and life-threatening illnesses. To address this challenge, a rapid and highly sensitive detection platform for S. suis in raw pork was developed by integrating loop-mediated isothermal amplification (LAMP) and a lateral flow assay (LFA), S. suis LAMP-LFA. LAMP reactions targeting the S. suis glutamate dehydrogenase (gdh) gene were optimized for specific detection of S. suis within 45 min at an isothermal temperature of 65 °C. The assay exhibited marked sensitivity, with a detection limit of 100 fg for genomic DNA extracted from S. suis cultures. Notably, this method showed no cross-reactivity with other bacterial contaminants commonly found in raw pork. The resulting LAMP amplicons were effectively detected using LFA, with a test limit of 101 CFU per 25 g of raw pork. S. suis LAMP-LFA proved to be highly specific and reliable, with no false-positives detected in spiked pork samples or pork samples containing other bacterial contaminants. Due to its high sensitivity, specificity, and rapid turnaround time, the proposed technique has immense potential as a field-deployable screening test for S. suis detection in raw pork, contributing to enhanced food safety and public health protection.

Assessment of Plant Growth Promotion Potential of Endophytic Bacterium B. subtilis KU21 Isolated from Rosmarinus officinalis.

The current study aimed to evaluate the plant growth-promoting (PGP) potential of endophytic strain Bacillus subtilis KU21 isolated from the roots of Rosmarinus officinalis. The strain exhibited multiple traits of plant growth promotion viz., phosphate (P) solubilization, nitrogen fixation, indole-3-acetic acid (IAA), siderophore, hydrogen cyanide (HCN), lytic enzymes production, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. The isolate also exhibited antagonistic activity against phytopathogenic fungi, i.e., Fusarium oxysporum, Fusarium graminiarum, and Rhizoctonia solani. The P-solubilization activity of B. subtilis KU21 was further elucidated via detection of glucose dehydrogenase (gdh) gene involved in the production of gluconic acid which is responsible for P-solubilization. Further, B. subtilis KU21 was evaluated for in vivo growth promotion studies of tomato (test crop) under net house conditions. A remarkable increase in seed germination, plant growth parameters, nutrient acquisition, and soil quality parameters (NPK) was observed in B. subtilis KU21-treated plants over untreated control. Hence, the proposed module could be recommended for sustainable tomato production in the Northwest Himalayan region without compromising soil health and fertility.

Genomic insight of phosphate solubilization and plant growth promotion of two taxonomically distinct winter crops by Enterobacter sp. DRP3.

Study of rhizospheric microbiome-mediated plant growth promotional attributes currently highlighted as a key tool for the development of suitable bio-inoculants for sustainable agriculture purposes. In this context, we have conducted a detailed study regarding the characterization of phosphate solubilizing potential by plant growth-promoting bacteria that have been isolated from the rhizosphere of a pteridophyte Dicranopteris sp., growing on the lateritic belt of West Bengal. We have isolated three potent bacterial strains, namely DRP1, DRP2, and DRP3 from the rhizoids-region of Dicranopteris sp. Among the isolated strains, DRP3 is found to have the highest phosphate solubilizing potentiality and is able to produce 655.89 and 627.58µg ml-1 soluble phosphate by solubilizing tricalcium phosphate (TCP) and Jordan rock phosphate, respectively. This strain is also able to solubilize Purulia rock phosphate moderately (133.51µg ml-1). Whole-genome sequencing and further analysis of the studied strain revealed the presence of pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase gdh gene along with several others that were well known for their role in phosphate solubilization. Further downstream, quantitative reverse transcriptase PCR-based expression study revealed 1.59-fold upregulation of PQQ-dependent gdh gene during the solubilization of TCP. Root colonization potential of the studied strain on two taxonomically distinct winter crops viz. Cicer arietinum and Triticum aestivum has been checked by using scanning electron microscopy. Other biochemical analyses for plant growth promotion traits including indole acetic acid production (132.02µg ml-1), potassium solubilization (3mg l-1), biofilm formation, and exopolymeric substances productions (1.88-2.03µg ml-1) also has been performed. This study highlighted the active involvement of PQQ-dependent gdh gene during phosphate solubilization from any Enterobacter group. Moreover, our study explored different roadmaps for sustainable farming methods and the preservation of food security without endangering soil health in the future.

Molecular epidemiology and multilocus genotyping of Giardia duodenalis in individuals attending major public hospitals in Shiraz, southwestern Iran: A public health concern

Giardia duodenalis is one of the most common causes of waterborne disease worldwide, and is often associated with outbreaks of diarrhea in areas with poor sanitation and hygiene. This study aimed to assess the prevalence and genetic diversity of G. duodenalis assemblages in individuals attending major public hospitals in Shiraz, southwestern Iran. From August 2022 to May 2023, a total of 614 stool samples from individuals were collected and initially examined for G. duodenalis cysts using parasitological techniques, sucrose flotation, and microscopy. Microscopy-positive samples were validated by SSU-PCR amplification of the parasite DNA. A multilocus genotyping (MLG) scheme, which focused on the triose phosphate isomerase (tpi) and the glutamate dehydrogenase (gdh) genes, was employed for genotyping purposes. G. duodenalis cysts were found in 7.5% (46/614) and 8.5% (52/614) of samples through microscopy and SSU-PCR, respectively. Successful amplification and sequencing results were obtained for 77.3% (17/22) and 45.5% (10/22) of the infected samples at the tpi and gdh loci, respectively. MLG data for the two loci were available for only five samples. Out of the 22 samples genotyped at any loci, 54.5% (12/22) were identified as assemblage A, while 45.5% (10/22) were identified as assemblage B. AII was the most predominant sub-assemblage identified [54.5% (12/22)], followed by BIII [27% (6/22)], discordant BIII/BIV [13.6% (3/22)], and BIV [4.5% (1/22)]. In the present study, no assemblages suited for non-human animal hosts (e.g., CF) were detected. This suggests that the transmission of human giardiasis in Shiraz is primarily anthroponotic. Further molecular-based analyses are necessary to confirm and expand upon these findings. These analyses will also help determine the presence and public health importance of the parasite in environmental samples, such as drinking water.

In vitro Culture and Multilocus Genotyping of Giardia duodenalis Trophozoites Obtained from Human Fecal Samples in Southwest Iran.

The enteric protozoa, Giardia duodenalis (G. duodenalis), consists of eight distinct assemblages (A-H) with identical morphological characteristics and a direct life cycle. Successful axenic cultivation of this parasite is an important preliminary step for biological, drug resistance and phylogenetic studies. Moreover, G. duodenalis exhibits great genetic and biotypic diversity. The current study aimed to evaluate In vitro culture and multilocus genotyping of G. duodenalis trophozoites obtained from human fecal samples in southwest Iran. Thirty human fecal specimens containing G. duodenalis cysts were collected from Ahvaz city (southwest of Iran). The purification of cysts was carried out by the sucrose flotation technique. The cysts were inoculated in a modified TYI-S-33 medium and was daily monitored for the development and viability of trophozoites. After extracting DNA, gdh, bg and tpi genes were evaluated using molecular techniques (the semi-nested PCR for gdh gene and the nested PCR for tpi and bg genes). Eventually, the amplified fragments were sequenced and then, the phylogenetic tree was drawn. Of 30, the trophozoites were encysted from five samples. All three genes were detected in two cases of five samples using molecular techniques. The multilocus phylogenetic analysis demonstrated that all the two samples belonged to assemblage A and sub-assemblage AІІ. Our findings indicated the presence of different numbers of trophozoites with variable development and survival rates in modified TYI-S-33 medium. Furthermore, the multilocus genotyping showed that these trophozoites belonged to assemblage A and sub-assemblage AІІ.

Molecular characterization and zoonotic potential of Cryptosporidium spp. and Giardia duodenalis in humans and domestic animals in Heilongjiang Province, China

BackgroundCryptosporidiosis and giardiasis are significant parasitic diseases shared between humans and domestic animals. Due to the close contact between humans and domestic animals in rural areas, it is important to consider the potential transmission of zoonotic parasites from infected domestic animals to humans. This investigation aimed to determine the prevalence and molecular characteristics of Cryptosporidium spp. and Giardia duodenalis in domestic animals and villagers.MethodsA total of 116 fecal samples from villagers and 686 fecal samples from domestic animals in Heilongjiang Province, China, were analyzed for two parasites using nested polymerase chain reaction (PCR) targeting various genetic loci and DNA sequence analysis of the PCR products.ResultsBy sequence analysis of the SSU rRNA gene, the prevalence of Cryptosporidium in humans was 0.9% (1/116), with one species of C. parvum (n = 1) detected; among domestic animals, the prevalence was 2.6% (18/686), with five species identified: C. suis (n = 7) and C. scrofarum (n = 7) in pigs, C. meleagridis (n = 1) in chickens, C. andersoni (n = 1) in cattle, and C. canis (n = 2) in foxes. C. parvum and C. canis were further subtyped as IIdA19G1 and XXa4 on the basis of gp60 gene. Regarding G. duodenalis, based on the SSU rRNA, bg, gdh, and tpi genes, the prevalence in domestic animals was 5.1% (31/608), with three assemblages identified: A (n = 1) in pigs, D (n = 1) in foxes, and E (n = 27) in geese, cattle, pigs, ducks, and sheep, along with mixed infection of A + E (n = 1) in one pig and B + E (n = 1) in one sheep. No G. duodenalis was detected in humans (0/116).ConclusionsThe present results show that no overlap of subtypes between animals and villagers was found in Cryptosporidium spp. and G. duodenalis, indicating a minor role of domestic animals in infecting humans in this population. However, the presence of zoonotic protozoa in domestic animals highlights the need for special attention to high-risk individuals during close contact with domestic animals.Graphical

Expression of the synthetic CYP102A1-LG23 gene and functional analysis of recombinant P450 BM3-LG23 cytochrome in actinobacteria <i>Mycolicibacterium smegmatis</i>

Cytochrome CYP102A1 (P450 BM3) from Priestia megaterium (bas. Bacillus megaterium) has a number of specific features making it an ideal target for directed evolution and other synthetic applications. Previously, the CYP102A1-LG23 mutant with 14 mutations in the heme was obtained providing 7β-hydroxylation of steroid substrates of the androstane series with the formation of products possessing anti-inflammatory and neuroprotective activity. In this study, the synthetic cyp102A1-LG23 gene encoding the P450 BM3 mutant variant was expressed in Mycolicibacterium smegmatis cells as part of mono- and bicistronic operons together with the synthetic gdh or zwf2 genes encoding glucose dehydrogenase (GDH) and glucose-6-phosphate dehydrogenase (G6PD), respectively. The functional activity of the recombinant enzymes was shown in vivo by the example of hydroxylation of androst-4-ene-3,17-dione (AD) to 7β-OH-AD in growing cultures of mycolicibacteria. Biocatalytic activity was doubled by increasing the CYP102A1-LG23 protein solubility in the cell and organizing the cofactor regeneration additional system by introducing GDH and G6PD. The maximum level of 7β-OH-AD amounting 37,68 mol % was achieved by co-expression the cyp102A1-LG23 and gdh genes in M. smegmatis. The results evidence to the perspective of using synthetic genes to obtain recombinant enzymes, expand the understanding of the hydroxylation of steroid compounds by bacterial cytochromes and can be demand for the methods of microbiological production of 7β-hydroxylated steroids by genetically modified mycolicibacteria.

The detection and phylogenetic characterization of Cryptosporidium, Cystoisospora, and Giardia duodenalis of cats in South Korea.

Cryptosporidium, Cystoisospora, and Giardia duodenalis are gastrointestinal protozoa parasites that cause diarrhea in various animals. However, information regarding the detection and phylogenetic characterization of gastrointestinal protozoa parasites in cats is limited throughout South Korea. Therefore, this study aimed to determine the detection and identify subspecies of gastrointestinal protozoa parasites in cats from South Korea. A total of 290 fecal samples were collected from stray, companion, and shelter cats in six provinces. Cryptosporidium, Cystoisospora, and G. duodenalis were identified by PCR. All positive samples were subtyped by PCR and sequencing of gp60, ITS-1, tpi, bg, and gdh. The overall detection of gastrointestinal protozoan parasitic infection was 17.93%. G. duodenalis was the most prevalent, with 7.93%, followed by Cystoisospora spp. (7.24%) and Cryptosporidium spp. (4.48%). In addition, C. felis (n=10), C. parvum (n=2), C. ryanae (n=1), Cystoisospora felis (n=14), Cystoisospora suis (n=5), Cystoisospora ohioensis (n=1), Cystoisospora spp. were identified in subspecies analysis of positive samples. C. felis showed a significant association with diarrhea (7.81%) and living condition (6.04%), and Cystoisospora felis in diarreha (9.38%) according to detection. Through phylogenetic analysis of the tpi, bg, and gdh genes from 23 G. duodenalispositive samples, it was confirmed that the samples of present study belonged to assemblage A, B, C, and D. South Korean cats have a high rate of gastrointestinal protozoan parasites infection with cat-specific Cryptosporidium and Cystoisospora, which are associated with living conditions and diarrhea symptoms. Moreover, zoonotic and other animal-specific subtype of protozoan parasites have been detected in cat feces.

Genotyping of G. duodenalis in the People Referred to Health Centers of Semnan City.

Giardia duodenalis (G. duodenalis), is one of the major causes of gastrointestinal disorders worldwide, infecting the small intestine of humans and animals. Based on the genetic characteristics of the parasite, eight genotypes (A to H) have been identified in clinical samples. The main purpose of the present study was to find the genetic diversity of Giardia in people referred to health centers in Semnan, Iran, using PCR. Totally, 300 stool samples were collected from people referred to health centers in Semnan. The stool samples were first examined using the microscopic method (direct method and Lugol staining), and the samples were checked with trichrome staining. After DNA extraction, the GDH gene of positive samples was amplified by the semi-nested PCR method. The genotype of positive samples was determined by the sequencing method. Out of 300 samples, only 20 (6.66%) samples were found to be positive in the microscopic examination of the stool. In the PCR test, only 13 (4.33%) of the samples were positive. According to the multiple alignment results, it was found that the isolates belonged to AII, BIII, and BIV genotypes. Most of which are related to people without clinical symptoms of diarrhea. Identification of AII, BIV, and BIII genotypes indicates the anthroponotic and anthropozoonotic transmission cycle of Giardia infection in Semnan.