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Comprehending the phylogeography of invasive organisms enhances our insight into their distribution dynamics, which is instrumental for the development of effective prevention and management strategies. In China, Pomacea canaliculata and Pomacea maculata are the two most widespread and damaging species of the non-native Pomacea spp.. Given this species’ rapid spread throughout country, it is urgent to investigate the genetic diversity and structure of its different geographic populations, a task undertaken in the current study using the COI and ITS1 mitochondrial and ribosomal DNA genes, respectively. The result of this study, based on a nationwide systematic survey, a collection of Pomacea spp., and the identification of cryptic species, showed that there is a degree of genetic diversity and differentiation in P. canaliculata, and that all of its variations are mainly due to differences between individuals within different geographical populations. Indeed, this species contains multiple haplotypes, but none of them form a systematic geographical population structure. Furthermore, the COI gene exhibits higher genetic diversity than the ITS1 gene. Our study further clarifies the invasive pathways and dispersal patterns of P. canaliculata in China to provide a theoretical basis. [ABSTRACT FROM AUTHOR]

Neospora caninum is a type of coccidian protozoan parasite that causes a parasitic disease (neosporosis) affecting multiple systems and is prevalent worldwide. Scant information is available on the spread of this parasite in snakes, and this research seeks to assess the prevalence of snake infection with PCR techniques, representing the inaugural effort of its kind. Using a molecular method, the brains of 100 deceased snakes from six distinct species were analyzed to determine the presence of a parasite. These snakes had been housed in captivity for the purpose of collecting poisons and were obtained from various regions of Iran between July 2019 and December 2021. Four pairs of primers were utilized in two semi-nested PCR to amplify the NC-5 gene sequence. To ensure accuracy, all samples that tested positive for NC-5 were evaluated for the existence of the ITS1 gene. Five specimens that were affirmed by both Nc-5 and ITS1 genes were sequenced in the forward and reverse orientations. The NC-5 gene was amplified from the 82 (82%) brain tissue specimens. Eight tissue specimens that were considered positive for the existence of the NC-5 gene were also verified for the existence of the N. caninum ITS1 gene. This study aimed to report for the first time the molecular characterization of N. caninum infecting Iranian terrestrial venomous snakes, and our results indicate a high frequency of this organism among them. [ABSTRACT FROM AUTHOR]

Neospora caninum is a protozoan coccidian parasite that can act as a cause of abortion in sheep. The aim of this study was to investigate the presence of this parasitic agent and its role in causing abortion in sheep of Iran. Between June 2019 and February 2022, 100 samples [brain (n = 39), placenta (n = 8), embryonic membrane (n = 7), cotyledon (n = 7), umbilical cord (n = 2), homogenate mixture of tissues (heart, liver, spleen and digestive track) (n = 37)] that were collected following the necropsies of 39 aborted ovine fetuses from different parts of the Alborz and Qazvin provinces, the north of the central region of Iran were employed for DNA extraction. Nc-5 was selected as the target gene sequence for amplification of DNA by using four pairs of primers in two semi-nested PCR. Samples considered positive for the presence of the NC-5 gene were examined to further confirm the presence of the ITS1 gene. Sequence of NC-5 gene was detected from the 27 tissue samples of 23 aborted ovine fetuses. The ITS1 gene sequence was detected in all of the 27 tissue samples that were positive for the NC-5 gene analysis. Brain tissue was the most studied tissue, and the highest number of positive cases was observed in this tissue. The present study updated the situation of ovine neosporosis in the central region of Iran and confirmed the presence of the N. caninum among sheep flocks' abortion. [ABSTRACT FROM AUTHOR]

The aim of this study was to characterize the Tunisian Fasciola spp. flukes by morphometric and molecular analyses. Flukes were collected from livers of sheep slaughtered in Sejnane slaughterhouses (Bizerte gouvernorate, Northwest Tunisia) between January and March 2021. Five morphometric parameters were determined for all the liver flukes, as follows: (i) total body length (BL), (ii) distance between ventral sucker and the tail (VS-T), (iii) distance between oral sucker and ventral sucker (OS-VS), (iv) abdomen diameter (AD), (v) tail diameter (TD) and the body length to width ratio (BL/BW). Molecular identification of the fluke specimens was carried out by polymerase chain reaction, restriction fragment polymorphism (PCR-RFLP) of a 680 bp sequence of the internal transcribes spacer 1 (ITS1) gene and by amplification, sequencing, and phylogenetic analysis of a 500 bp sequence of the ITS2 gene. Morphometric measurements showed that the mean of the total body length of the adult flukes was 21.1 ± 2.7 mm with minimum and maximum lengths of 13 and 31 mm, respectively. The PCR-RFLP analysis revealed a single profile consisting of three bands of approximately 370, 100, and 60 bp. Fasciola sequences described in the present study (GenBank numbers: OQ457027 and OQ457028) showed 99.58–100% identity to Fasciola hepatica. In conclusion, the results of this study show that molecular and phylogenetic analyses confirm the presence of a single species of F. hepatica in the Sejnane region Northwest of Tunisia. However, further studies are needed to identify the occurrence of Fasciola species in other Tunisian regions. [ABSTRACT FROM AUTHOR]

Summary: The aim of this study was to estimate the molecular infection prevalence of Toxoplasma gondii in sheep liver tissues destined for human consumption. A total number of 224 liver tissues were collected from slaughtered sheep in Sejnane slaughterhouse (Northwest Tunisia). PCR was used to detect T. gondii DNA in liver tissues followed by phylogenetic analysis of amplicons. The phylogenetic tree was then constructed to compare the partial sequences of the ITS1 gene with GenBank sequences. The overall molecular prevalence of T. gondii in sheep livers was 25% (56/224). The highest molecular prevalence of T. gondii was recorded in sheep aged of less than one year old (27.3%; 52/190). Infection prevalence was significantly higher in Noire de Thibar breed (33%; 17/51) compared to other breeds (p = 0.023). There were no differences depicted according to sheep's gender. The T. gondii sequences obtained in the present study (GenBank accession numbers: OR509829 and OR509830) were 98.40–100% homologous to T. gondii sequences published in the GenBank. These results highlight a high level of T. gondii contamination of tissues destined for human consumption. Further studies are needed to improve our knowledge on different genotypes of T. gondii that infect Tunisian sheep population. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a zoonotic infectious disease caused by Leishmania species. The identification of parasite species and the type of disease is beneficial for treatment and preventive modalities. Leishmania tropica and L. major have been reported as the main etiological agents of cutaneous leishmaniasis (CL) in Iran. The incidence of zoonotic CL has increased and different in distinct loci of Iran. Hence, we perused the Leishmania species and its genetic traits in the North East of Iran. The investigation was conducted on 200 positive smears prepared from patients' lesions suffering from CL referred to the health care centers of northeastern provinces in Iran from 2013 to 2019. The obtained positive microscopy samples were divided to score the ranges from + 1 to + 6, of them 40 smears exhibited low-parasitemia. Leishmania species analyzed using PCR–RFLP, genetic diversity indices evaluation, phylogenetic analysis, and sequencing comparison with other species in the GeneBank based on ITS1 gene. The isolated L. major strains were similar to other Iranian isolates in this region. Pairwise fixation index (FST) index was statistically significant in different L. major populations and showed the genetic differences in pairwise population of different geographical locations of Iran. The current study confirmed an old pattern endemicity of zoonotic CL in North-east of Iran. Therefore, in order to assess the hybrid formation, more epidemiological, ecological, and gene polymorphism studies are needed to understand the pathogenic role of Leishmania species in Iran. [ABSTRACT FROM AUTHOR]

Tsetse flies are major arthropod vectors of trypanosomes that cause debilitating African animal trypanosomiasis. The emergence of drug-resistant trypanosomes is a common problem in sub-Saharan Africa. This study aimed to identify tsetse flies’ seasonal variation in apparent densities and their infection rates and the occurrence of drug-resistant trypanosomes. Tsetse flies were collected from Lambwe, Kenya, during May and September 2021. Genomic DNA was extracted from them, and the ITS1 gene was amplified to detect Trypanosoma infection with subsequent species determination. Transporter genes DMT, E6M6, TbAT/P2, and TcoAde2 were targeted to detect polymorphisms associated with drug-resistance, using sequencing and comparison to drug-sensitive trypanosome species referenced in Genbank. A total of 498 tsetse flies and 29 non-tsetse flies were collected. The apparent density of flies was higher in wet season 6.2 fly per trap per density (FTD) than in the dry season 2.3 FTD (P = 0.001), with n = 386 and n = 141 flies caught in each season, respectively. Male tsetse flies (n = 311) were more numerous than females (n = 187) (P = 0.001). Non-tsetse flies included Tabanids and Stomoxys spp. Overall, Trypanosoma infection rate in tsetse was 5% (25/498) whereby Trypanosoma vivax was 4% (11/25), Trypanosoma congolense 36% (9/25), and Trypanosoma brucei 20% (5/25) (P = 0.186 for the distribution of the species), with infections being higher in females (P = 0.019) and during the wet season (P < 0.001). Numerous polymorphisms and insertions associated with drug resistance were detected in DMT and E6M6 genes in two T. congolense isolates while some isolates lacked these genes. T. brucei lacked TbAT/P2 genes. TcoAde2 sequences in three T. congolense isolates were related to those observed in trypanosomes from cattle blood in our previous study, supporting tsetse fly involvement in transmission in the region. We report Trypanosoma associated with trypanocidal drug-resistance in tsetse flies from Lambwe, Kenya. Female tsetse flies harbored more Trypanosoma infections than males. Tsetse transmission of trypanosomes is common in Lambwe. Risk of trypanosome infection would seem higher in the wet season, when tsetse flies and Trypanosoma infections are more prevalent than during the dry season. More efforts to control animal trypanosome vectors in the region are needed, with particular focus on wet seasons. [ABSTRACT FROM AUTHOR]

Despite the general consensus that fertilizer is the most important driver of the evolution of soil microbial communities, the specific effects of long-term fertilizer use on microbial communities remain unclear. Here, we collected soil samples from fertilized (NPK) and unfertilized (NF) plots in a subtropical farmland in southwestern China. NPK plots were consistently treated with chemical fertilizer (nitrogen, phosphorous, and potassium) for the 20 years; NF plots were left unfertilized for the same period. To explore the effects of long-term fertilizer use on soil microbial community structure, microbial community composition in the topsoil was assessed using the bacterial 16S rRNA gene and the full-length fungal ITS1 gene. In conjunction, we measured various soil chemical properties. We found that metrics associated with soil fertility (i.e., total nitrogen, total phosphorus, total potassium, available nitrogen, available phosphorus, and available potassium) were significantly greater in the NPK samples as compared to the NF samples, but that soil pH was significantly lower. We also found that long-term fertilizer use reshaped soil microbial community composition but did not alter community α-diversity. Notably, the bacterial phyla Nitrospirae and Planctomycetes and the fungal phylum Ascomycota were closely associated with the NPK plot, influenced by soil organic matter, total nitrogen, available potassium, and available phosphorus, while the bacterial phyla Bacteroidetes and Chloroflexi and the fungal phyla Glomeromycota and Basidiomycota were closely associated with the NF plot, influenced by soil pH. Our results provide long-term data clarifying the microbial regulation mechanisms underlying the response of farmland soil to long-term fertilizer use in subtropical China. [ABSTRACT FROM AUTHOR]

Purpose: The aim of this study was to estimate the seroprevalence and molecular prevalence and perform a molecular identification of Neospora caninum in semen of Tunisian rams. Methods: A total of 92 blood samples were collected from four farms located in four Tunisian governorates (Jendouba, Kairouan, Zaghouan and Ben Arous) and samples were screened with a commercial ELISA kit for N. caninum antibodies. For the same rams, semen samples were collected and tested for the presence of N. caninum ITS1 gene using PCR. Five amplicons were randomly selected for sequencing. A phylogenetic tree was constructed to compare the partial sequences of the ITS1 gene with sequences deposited in GenBank. Results: The seroprevalence of N. caninum infection was 25% (23/92) and PCR revealed that the molecular infection prevalence in semen was 11.95% (11/92). Kappa test showed an average agreement between seroprevalence and parasite prevalence in semen (κ = 0.44). The highest molecular prevalence was for rams that accomplished more than two mating seasons (21.0 ± 12.1%) compared to those performed less than two mating seasons and yearling individuals (4.0 ± 5.5%) (P = 0.01). There were no differences in N. caninum molecular prevalence according to either breed or locality. Comparison of the partial sequences of the ITS1 gene revealed 99–100% similarity with those deposited in GenBank. Conclusion: To the best of our knowledge, this is the first detection and molecular identification of N. caninum in semen from rams in North Africa. Our findings indicate that N. caninum infection rate was high in rams. [ABSTRACT FROM AUTHOR]

Six Trypanosoma evansi isolates collected from ponies (PH1 and PK6), camel (CB2), donkeys (DJ3 and DH4) and cattle (CK5) from Haryana, Rajasthan, Uttar Pradesh and Gujarat states of India were used for molecular characterization of internal transcribed spacer 1 (ITS 1). The DNA was isolated from purified trypanosomes of these six isolates after propagation in mice model. ITS1-PCR of purified parasite DNA yielded an amplification product approximately 540 bp in size. Nucleotide sequence of ITS1 gene of CB2 isolate had 530 bp while CK5, DH4, DJ3, and PH1 isolates had 532 bp, whereas, PK6 isolates had 533 bp size. Blast data of the Indian isolates revealed 99 % homology with other available sequences of T. evansi. Multiple alignment of nucleotide sequence of ITS1 gene variants from Indian T. evansi isolates with selected homologous sequences from GenBank revealed that nucleotide substitution mostly occurred at the position of 101-103, 218-223, 243-244, 301-396 and 470-480. The isolates PH1, CK5, DH4 and DJ3 were found more associated with T. evansi isolates from the Philippines, Thailand, Iran, Egypt and China, whereas, PK6 and CB2 isolates were related to each other and were phylogenetically distant from rest of the Indian isolates used in this study. Based on the ITS1 rDNA sequence, the Neighbour-Joining consensus tree indicated clear evidence of existence of genetic diversity among T. evansi isolates from India. [ABSTRACT FROM AUTHOR]

Natural mortality of the almond bark beetle, Scolytus amygdali Guerin-Meneville (Coleoptera: Curculionidae: Scolytinae) was studied during its spring generation under the bark of infested almond tree branches in Tunisia. Dead larvae and adults of the beetle were collected from the field and analyzed under the laboratory conditions. Two main fungal isolates were obtained from the cadavers of S. amygdali individuals, identified using morphological and molecular tools, as Aspergillus flavus and Fusarium oxysporum. In preliminary studies conducted under laboratory conditions, species of Aspergillus and Fusarium fungi were reported virulent at the concentrations of (0.4 x 107 conidia/ml) to S. amygdali. In order to test the pathogencity of the two isolated fungal species, similar concentrations were applied on larvae and adults of healthy S. amygdale individuals. Both fungi were found significantly virulent immediately after 24h under laboratory conditions. Further investigations for their suitability as biocontrol agents in the field are required. [ABSTRACT FROM AUTHOR]

Neospora caninum is an intracellular protozoan parasite from the phylum Apicomplexa, mainly associated with abortions and causing enormous economic losses. We aimed, by the present study, to estimate the molecular prevalence and phylogenetic analyses of natural infection with N. caninum in Tunisian goats. A total number of 121 meat samples were collected from slaughtered goats in the regional slaughterhouse of Béja (Northwest Tunisia) and tested from N. caninum ITS1 gene using PCR followed by sequencing of PCR products. Phylogenetic analyses were used to identify this parasite infecting goats in Nortwest Tunisia. The overall molecular prevalence was 19% (23/121). The highest molecular prevalence of N. caninum was observed in goats aged between 2 and 4 years (31.9 ± 13.27%) (P = 0.004). There was no difference in the overall molecular prevalence of N. caninum according to both localities and animal breeds. Comparison of the partial sequences of the ITS1 gene revealed 99-100% similarity with GenBank sequences. A high similarity with all the blasted genotypes was reported for N. caninum sequences. This is the first molecular study and genetic characterisation of N. caninum in North African goats. [ABSTRACT FROM AUTHOR]

The aim of this study was to estimate the molecular prevalence of Toxoplasma gondii, Neospora caninum and the co-infection by both Apicomplexan parasites in uterus tissues of cows. PCR was used to detect T. gondii and N. caninum DNA in uterus from 140 uteri of slaughtered cows in the regional slaughterhouse of Béja (Northwest Tunisia). Positive PCR products were sequenced and used for the phylogenetic analysis. The overall molecular prevalence of T. gondii and N. caninum in cows’ uterus was 5 and 15.57%, respectively. Co-infection prevalence by the two parasites was estimated to be 2.85%. Risk factors including the age categories significantly affected the molecular prevalence of T. gondii and N. caninum in cows’ uterus. The highest molecular prevalence of T. gondii (11.5 ± 3.1) and N. caninum (21.1 ± 11.1; p = 0.038) was observed in cows aged of more than 8 years. There were no differences depicted according to cow’s breeds and localities. Comparison of the partial sequences of the ITS1 gene revealed 100% similarity among our N. caninum sequence (MW136256) and those deposited in GenBank. The T. gondii sequence described in this study (MW260335) was 99.4–100% homologous to T. gondii sequences published in the GenBank. To the best of our knowledge, this is the first molecular evidence of N. caninum and T. gondii co-infection in naturally infected cows in North Africa. This information is pertinent in designing control programmes that would reduce economic losses in the livestock industry. [ABSTRACT FROM AUTHOR]

Phytopathogenic fungi cause several diseases in the common bean (Phaseolus vulgaris), diminishing its productivity, i.e. white mold. This work aimed to isolate and characterize the causal agent of the white mold disease observed in a common bean commercial field, in Northern Mexico; as well as to identify a promising native biological control agent against this phytopathogen. Based on macro/microscopic traits (white-cottony mycelium, and sclerotia formation) and molecular identification [sequencing of the internal transcribed spacer 1 (ITS1) gene], a fungal isolate (FRM18) obtained from symptomatic common bean plants, was identified as Sclerotinia sclerotiorum. In addition, a bacterial strain (FSQ1) isolated from the common bean rhizosphere showed in vitro antagonism against S. sclerotiorum FRM18, inhibiting 2.4 ± 0.2 cm of the studied phytopathogen growth. Based on macro/microscopic traits (gram-positive bacteria, bacillary morphology, and endospore production) and molecular identification (sequencing of the 16S rRNA gene), strain FSQ1 was affiliated to the genus Bacillus. Common bean plants inoculated with S. sclerotiorum FRM18 showed a height reduction of 25% at day: 60 and white mold disease was observed (compared to un-inoculated plants), validating the Koch's postulates, which confirm that strain FRM18 is the causal agent of this disease in the field. However, common bean plants co-inoculated with S. sclerotiorum FRM18 and Bacillus sp. FSQ1 (compared to un-inoculated plants) were not affected in their development, and symptoms of the disease were not observed. Even when lipopeptides are the potential action mode of Bacillus sp. FSQ1 to inhibit S. sclerotiorum FRM18, further researches are necessary to determine the main antimicrobial compounds produced by strain FSQ1. [ABSTRACT FROM AUTHOR]

Patelloida pygmaea is one of the most abundantly distributed intertidal marine gastropods along the China coast. The mitochondrial (COI) and internal transcribed spacer 1 (ITS1) DNA of P. pygmaea, one hundred and thirty-seven samples collected from 13 localities along its distribution along the China coast, were used to investigate the phylogeographic patterns of P. pygmaea. Phylogeographic analysis revealed three distinct phylogeographic clades, the South China Sea (SCSC), the Bohai and Yellow Sea (BYSC), and the East China Sea (ECSC) clades. The results of AMOVA showed significant genetic differentiation (P < 0.001) among the three clades. The vicariance of the three lineages was created by the glacial drop in sea level, the Changjiang Estuary, and the sea surface temperature gradient. Based on the ITS1 gene, the BYSC clade was split into two subclades, the north and south clades. We speculate that the Shandong Peninsula was another possible barrier limiting gene flow in the BYSC. Ocean currents played more roles in influencing gene flow within the three clades. The mismatch analysis confirmed the demographic population expansion (P > 0.05). Phylogeographic patterns of P. pygmaea have been primarily affected by Pleistocene climatic oscillations and environmental conditions. During the glacial periods, the sea regressed and moist and warm conditions, characterizing glacial refugia, became prevalent in the region and caused isolated P. pygmaea populations to expand. [ABSTRACT FROM AUTHOR]

Foliar fungi (defined as all fungal species in leaves after surface sterilization; hereafter, 'FF') are of great importance to host plant growth and health, and can also affect ecosystem functioning. Despite this importance, few studies have explicitly examined the role of host filtering in shaping local FF communities, and we know little about the differences of FF community assembly between symptomatic (caused by fungal pathogens) and asymptomatic leaves, and whether there is phylogenetic congruence between host plants and FF. We examined FF communities from 25 host plant species (for each species, symptomatic and asymptomatic leaves, respectively) in an alpine meadow of the Tibetan Plateau using MiSeq sequencing of ITS1 gene biomarkers. We evaluated the phylogenetic congruence of FF–plant interactions based on cophylogenetic analysis, and examined α- and β-phylogenetic diversity indices of the FF communities. We found strong support for phylogenetic congruence between host plants and FF for both asymptomatic and symptomatic leaves, and a host-caused filter appears to play a major role in shaping FF communities. Most importantly, we provided independent lines of evidence that host environmental filtering (caused by fungal infections) outweighs competitive exclusion in driving FF community assembly in symptomatic leaves. Our results help strengthen the foundation of FF community assembly by demonstrating the importance of host environmental filtering in driving FF community assembly. [ABSTRACT FROM AUTHOR]

Neosporosis primarily affects cattle and dogs and is not currently considered a zoonotic disease. Toxoplasmosis is a zoonosis with a worldwide distribution that is asymptomatic in most cases, but when acquired during pregnancy, it can have serious consequences. The seropositivity rates determined by the indirect fluorescent antibody test for Neospora caninum (N. caninum) and Toxoplasma gondii (T. gondii) were 24.3% (49 samples) and 26.8% (54 samples), respectively. PCR positivity for N. caninum was observed in two samples of cord blood (1%) using the Nc5 and ITS1 gene, positivity for T. gondii was observed in 16 samples using the primer for the B1 gene (5.5% positivity in cord blood and 2.5% positivity in placental tissue). None of the samples showed structures characteristic of tissue cysts or inflammatory infiltrate on histopathology. Significant associations were observed only between N. caninum seropositivity and the presence of domestic animals (p = 0.039) and presence of dogs (p = 0.038) and between T. gondii seropositivity and basic sanitation (p = 0.04). This study obtained important findings regarding the seroprevalence and molecular detection of N. caninum and T. gondii in pregnant women; however, more studies are necessary to establish a correlation between risk factors and infection. [ABSTRACT FROM AUTHOR]

Cystoisospora suis is a pathogen that causes diarrhea in pigs and can lead to serious disease. Species identification, especially by histopathological examination, is often difficult because of morphologically similar parasites such as Eimeria species. In this study, we used histopathological, bacteriological, virological, and parasitological methods to identify the cause of the disease in two piglets with severe diarrhea. Villous atrophy, diffuse necrosis, and flattening of mucosal epithelial cells were found in the ilea of examined piglets, and coccidian parasites were found in the cytoplasm of the epithelial cells. In some merozoites in the meronts, the presence of two nuclei indicated type 1 merozoites, characteristic of C. suis. According to Cystoisospora-specific PCR targeting the rRNA internal transcribed spacer 1 (ITS1) gene, the sequences of the products were 98.5% similar to those of C. suis. Escherichia coli (O149 serogroup) exhibiting a virulence factor profile (LT, STb, and EAST1 as toxins and F4 as a colonization factor) was detected in one piglet. No other bacteria or significant enteric viruses were found. Co-infection with C. suis and E. coli could imply aggravation of the disease, although further study is needed to assess the pathogenicity of this interaction. This study is the first to clarify by molecular analysis the sequences of C. suis detected in piglets in Japan. [ABSTRACT FROM AUTHOR]

Cystoisospora suis is a pathogen that causes diarrhea in pigs and can lead to serious disease. Species identification, especially by histopathological examination, is often difficult because of morphologically similar parasites such as Eimeria species. In this study, we used histopathological, bacteriological, virological, and parasitological methods to identify the cause of the disease in two piglets with severe diarrhea. Villous atrophy, diffuse necrosis, and flattening of mucosal epithelial cells were found in the ilea of examined piglets, and coccidian parasites were found in the cytoplasm of the epithelial cells. In some merozoites in the meronts, the presence of two nuclei indicated type 1 merozoites, characteristic of C. suis. According to Cystoisospora-specific PCR targeting the rRNA internal transcribed spacer 1 (ITS1) gene, the sequences of the products were 98.5% similar to those of C. suis. Escherichia coli (O149 serogroup) exhibiting a virulence factor profile (LT, STb, and EAST1 as toxins and F4 as a colonization factor) was detected in one piglet. No other bacteria or significant enteric viruses were found. Co-infection with C. suis and E. coli could imply aggravation of the disease, although further study is needed to assess the pathogenicity of this interaction. This study is the first to clarify by molecular analysis the sequences of C. suis detected in piglets in Japan. [ABSTRACT FROM AUTHOR]

Continuous cultivation of tobacco could cause serious soil health problems, which could cause bacterial soil to change to fungal soil. In order to study the diversity and richness of fungal community in tobacco-growing soil under different crop rotation, three treatments were set up in this study: CK (tobacco continuous cropping); B (barley-tobacco rotation cropping) and R (oilseed rape-tobacco rotation cropping). The results of this study showed that rotation with other crops significantly decreased the soil fungal OTUs, and also decreased the community richness, evenness, diversity and coverage of fungal communities. Among them, B decreased the most. In the analysis of the composition and structure of the fungal community, it was found that the proportion of plant pathogens Nectriaceae decreased from 19.67% in CK to 5.63% in B, which greatly reduced the possibility of soil-borne diseases. In the analysis of the correlation between soil environmental factors and fungal communities, it was found that Filobasidiaceae had a strong correlation with TP and AP, and Erysiphaceae had a strong correlation with TK and AK. NO3−-N and NH4+-N were the two environmental factors with the strongest correlation with fungal communities. The results of this study showed that rotation with other crops slowed down the process of soil fungi in tobacco-growing soil and changed the dominant species of soil fungi community. At the same time, crop rotation changed the diversity and richness of soil fungal community by changing the physical and chemical properties of soil. [ABSTRACT FROM AUTHOR]

Fungi belonging to the genus Neosartorya (teleomorph of Aspergillus spp.) are of great concern in the production and storage of berries and fruit-based products, mainly due to the production of thermoresistant ascospores that cause food spoilage and possible secretion of mycotoxins. We initially tested the antifungal effect of six natural extracts against 20 isolates of Neosartorya spp. using a traditional inhibition test on Petri dishes. Tested isolates did not respond uniformly, creating 5 groups of descending sensitivity. Ten isolates best representing of the established sensitivity clusters were chosen for further investigation using a Biolog™ MT2 microplate assay with the same 6 natural extracts. Additionally, to test for metabolic profile changes, we used a Biolog™ FF microplate assay after pre-incubation with marigold extract. All natural extracts had an inhibitory effect on Neosartorya spp. growth and impacted its metabolism. Lavender and tea tree oil extracts at a concentration of 1000 µg mL−1 presented the strongest antifungal effect during the inhibition test, however all extracts exhibited inhibitory properties at even the lowest dose (5 µg mL−1). The fungal stress response in the presence of marigold extract was characterized by a decrease of amino acids and carbohydrates consumption and an uptake of carboxylic acids on the FF microplates, where the 10 studied isolates also presented differences in their innate resilience, creating 3 distinctive sensitivity groups of high, average and low sensitivity. The results confirm that natural plant extracts and essential oils inhibit and alter the growth and metabolism of Neosartorya spp. suggesting a possible future use in sustainable agriculture as an alternative to chemical fungicides used in traditional crop protection. [ABSTRACT FROM AUTHOR]

Anaerobic lignocellulosic microbial consortia are known to be prodigiously efficient at converting lignocellulosic biomass to methane. In this study, the efficacy of anaerobic fungal consortia (AFC) from five different inocula, including Bubalus bubalis rumen fluid (RU), in degrading filter paper, microcrystalline cellulose, and rice straw (RS), was screened. The AFC from RU performed best in lignocellulosic material degradation and methane production; thus, RU was selected for further experiments. Consecutive batch subculturing (CBS) was performed in RU to enrich and stabilize the dominant and key microorganisms categorized as anaerobic fungi, using the addition of antibacterial agents to suppress the growth of untargeted bacteria. After the CBS, subculture E19 proved the most efficient, with RS degradation of 84% and a methane yield of 310 mL/g VSadded, representing 1.83- and 2.25-fold increases compared to the initial seed, respectively. The microbial community of E19 consisted of anaerobic fungi (uncultured Neocallimastigales, Anaeromyces sp., Orpinomyces sp., and Feramyces sp.) coexisting with anaerobic bacteria (streptomycin resistant Proteiniphilum acetatigenes), and methanogens. The E19 consortium was able to use various carbon sources (87.5%) and contained potential genes encoding enzymes involved in RS degradation. The microbial community of E19 was highly stable, making it a promising inoculum for biomass degradation, especially for anaerobic digestion to produce biogas. [ABSTRACT FROM AUTHOR]

Purpose: Strongyloides stercoralis is a parasite with special characteristics presenting it as a unique nematode. Iran is an endemic area for S.stercoralis. In this study, nested-qPCR-high resolution melting (HRM) technology was applied on some human isolates of S.stercoralis from this country by focusing on evolutionary genetics analysis. Methods: Twelve human isolates of S. stercoralis were collected from four endemic provinces of Iran. Genomic DNA was extracted from a single filariform larva for every isolate. Using specific primers targeting partial regions in cox1 gene, nested-qPCR-HRM was performed and melting-curve profiles were analyzed alongside the evaluation of genetic proximity and phylogenetic analysis using MEGA7 and DnaSP5 software. Results: The melting temperature (Tm) values of the isolates were 77.9 °C−78.3 °C. All isolates from Guilan, Mazandaran, and Khouzestan Provinces shared Tm values of 78.2 °C to 78.3 °C, while the isolates from Hormozgan Province showed Tm values of 77.9 °C, 78.0 °C, and 78.1 °C. The phylogenetic tree illustrated that the sequences of the current study included nine haplotypes. Tajima's D index analyses showed that cox1 gene in S. stercoralis isolates was negative (Tajima's D = − 0.27). Conclusion: The isolates were divided into five temperature groups. Although HRM assay compared to PCR sequencing identified more limited genetic changes, it revealed that the mean of Tm of the isolates from Hormozgan Province was lower than those of other provinces and represented specific haplotypes for this geographical region on the phylogenetic tree. [ABSTRACT FROM AUTHOR]

Purpose: Coccidiosis of domestic chicken is an important disease caused by any of seven species of Eimeria which, by developing within the epithelial cells of the intestine, cause lesions therein. We carried out a study on poultry farms located in various regions of Iran to determine the incidence and spread of Eimeria species by employing a single PCR test. Methods: A total of 64 fully confirmed clinically intestinal tracts were collected from different parts of Iran. From these 64 intestinal tracts, 82 samples were prepared from the different sites involved in the digestive tract. In morphological assessment, 23 samples could not be isolated and its information was not evaluated. Results: Using morphological methods, the following seven species of Eimeria were identified: E. acervulina (15/59; 25.42%), E. tenella (30/59; 50.84%), E. maxima (12/59; 20.33%), E. praecox (1/59; 1.69%), E. necatrix (2/59; 3.38%), E. mitis (5/59; 8.47%), and E. mivati (2/59; 3.38%). Mixed infections were found in eight (13.55%) samples. In molecular assessment, 31 samples could not be isolated and its information was not evaluated. Totally, the following five species were identified using molecular methods: E. acervulina (35/51; 68.62%), E. tenella (33/51; 64.70%), E. maxima (6/51; 11.76%), E. brunetti (5/51; 9.80%), and E. necatrix (2/51; 3.92%). Mixed infections were found in 23 (45.09%) samples. Conclusions: The present study is an update on the situation of poultry coccidiosis in Iran and provides the first data on the molecular detection, identification, and characterization of Eimeria spp. in the poultry population of this country and confirmed the presence of different species of this parasite in this area. According to the results, E. acervulina and E. tenella, as the main disease-causing species, should be considered in control programs such as treatment and vaccination strategies. [ABSTRACT FROM AUTHOR]

The intracellular bacteria Wolbachia pipientis infects arthropods and filarial nematodes and is able to manipulate host reproduction. It has been reported an association between parthenogenesis and Wolbachia infection in weevils from the tribe Naupactini. A curing experiment suggested that a threshold density of Wolbachia is required for parthenogenetic reproduction to occur. The aim of this study was to analyze Wolbachia infection status and density in two sexually reproducing species from the tribe Naupactini, Naupactus xanthographus and Naupactus dissimulator. Wolbachia infection was detected in individuals from both species in several geographic locations, not being fixed. Quantification through real time PCR confirmed that Wolbachia loads in sexual species were significantly lower than in parthenogenetic ones; these results support the hypothesis of a threshold level for parthenogenetic reproduction to occur in Naupactini weevils. Strain typing showed that both sexual species carry wNau1, the most frequent strain in parthenogenetic Naupactini weevils. In addition, the presence of the WO phage, which might be an important factor regulating infection density in some hosts, was detected in this strain. Finally, Wolbachia wNau1 was located throughout the whole insect body, which is in agreement with the idea of a recent acquisition by horizontal transfer of wNau1 across the tribe Naupactini. [ABSTRACT FROM AUTHOR]

Tapeworms of the genus Taenia include several species of important parasites with considerable medical and veterinary significance. Accurate identification of these species in dogs is the prerequisite of any prevention and control program. Here, we have applied an efficient method for differentiating four major Taeniid species in dogs, i.e., Taenia hydatigena, T. multiceps, T. ovis, and Echinococcus granulosus sensu stricto. High-resolution melting (HRM) analysis is simpler, less expensive, and faster technique than conventional DNA-based assays and enables us to detect PCR amplicons in a closed system. Metacestode samples were collected from local abattoirs from sheep. All the isolates had already been identified by PCR-sequencing, and their sequence data were deposited in the GenBank. Real-time PCR coupled with HRM analysis targeting mitochondrial cox1 and ITS1 genes was used to differentiate taeniid species. Distinct melting curves were obtained from ITS1 region enabling accurate differentiation of three Taenia species and E. granulosus in dogs. The HRM curves of Taenia species and E .granulosus were clearly separated at Tm of 85 to 87 °C. In addition, double-pick melting curves were produced in mixed infections. Cox1 melting curves were not decisive enough to distinguish four taeniids. In this work, the efficiency of HRM analysis to differentiate four major taeniid species in dogs has been demonstrated using ITS1 gene. [ABSTRACT FROM AUTHOR]

Since 1999, the number of asymptomatic leishmaniasis cases has increased continuously in Thailand, particularly among patients with HIV who are prone to develop symptoms of cutaneous and visceral leishmaniasis further. The asymptomatic infection could play a key role in Leishmania transmission and distribution. Understanding population structure and phylogeographic patterns could be crucially needed to develop effective diagnoses and appropriate guidelines for therapy. In this study, genetic variation and geographic distribution of the Leishmania/HIV co-infected population were investigated in endemic northern and southern Thailand. Interestingly, Leishmania orientalis was common and predominant in these two regions with common regional haplotype distribution but not for the others. Recent population expansion was estimated, probably due to the movement and migration of asymptomatic individuals; therefore, the transmission and prevalence of Leishmania infection could be underestimated. These findings of imbalanced population structure and phylogeographic distribution patterns provide valuable, insightful population structure and geographic distribution of Leishmania/HIV co-infection to empower prevention and control of transmission and expansion of asymptomatic leishmaniasis. [ABSTRACT FROM AUTHOR]

The Proteaceae, a diverse family of woody flowering plants in the Southern Hemisphere, contains many species known to be susceptible to Phytophthora cinnamomi, both in the natural environment and in cut-flower orchards. Very little is known about the prevalence of P. cinnamomi and other oomycetes across these landscapes. To address this knowledge gap, we used a double ITS1 and RPS10 gene metabarcoding approach and traditional isolation protocols to investigate oomycetes in orchards and natural stands of Proteaceae across South Africa, South Africa (eastern and western), Australia, and Europe. The RPS10 primers amplified more samples, including various Pythium species, while the ITS primers detected more Phytophthora phylotypes. Both datasets showed that geographic regions influenced oomycete species richness and community composition, while they did not show any variation between orchards and natural vegetation. RPS10 metabarcoding detected the largest number of species and provided greater statistical confidence than ITS1 when considering oomycete species composition. Metabarcoding also showed that orchards had a higher abundance of P. cinnamomi compared to native stands, although this was not found when isolating through baiting of roots and rhizosphere soil. Direct isolation and metabarcoding are complementary, with metabarcoding serving as an early detection tool. However, it cannot distinguish living viable propagules from residual DNA of dead propagules, limiting its use for diagnostic purposes related to Phytophthora management and control. These results, along with those of other recent studies, show that metabarcoding offers an effective tool to describe the dynamics of soil oomycetes in different ecosystems. [ABSTRACT FROM AUTHOR]

Australian species make up seventeen of the world’s twenty-five recognised species ofSorghum, with the genus separated into five sections:Eu-sorghum, Chaetosorghum, Heterosorghum, Para-sorghumandStiposorghum. Whereas the genetic relationships within sectionEu-sorghumare well known, little is known about the genetic relationships and crossabilities outside the primary genepool. We made a detailed investigation of phylogenetic relationships withinSorghumto identify wild species most closely related to cultivated sorghum (with outgroupsZea maysandSaccharum officinarum). The ribosomal ITS1 gene of ten species and the chloroplastndhF gene from nineteen species were sequenced. Independent and combined analyses of the ITS1 andndhF data sets were undertaken. TheEu-sorghumspecies were resolved into a strongly supported lineage by all three analyses, and included the Australian nativesS. laxiflorumandS. macrospermumin the ITS1 and combined analyses. All remaining sorghum species were resolved into a second well-supported lineage in the combined analyses, although some internal relationships within this second lineage remain unresolved. We identifiedS. laxiflorumandS. macrospermumas the Australian species most closely related to cultivated sorghum. Our data support a reduction in the number of subgeneric sections from five to three:Eu-sorghum(unchanged); a combinedChaetosorghum/Heterosorghumto reflect the very close relationship between these two species; and a combinedPara-sorghum/Stiposorghumsection, thereby removing the unclear taxonomic and genetic boundaries between these species. [ABSTRACT FROM AUTHOR]

Rapid development in industrialization and urbanization causes serious environmental issues, of which acid rain is one of the quintessential hazards, negatively affecting soil ecology. Liming has been investigated for a long time as the most effective amendment to alter the adverse effects of soil acidity resulting from acid rain. Herein, this study tested the biochar produced from invasive plants as an alternative amendment and hypothesized that biochar can maintain better availability of macronutrients under acid rain than liming by improving soil chemical and biological properties. Therefore, a pot experiment was conducted to compare the effects of lime and biochar at two rates (1% and 3%) on soil available nitrogen (N), phosphorous (P) and potassium (K) under simulated acid rain of two pH levels (4.5: pH4.5 and 2.5: pH2.5) as compared with tap water (pH7.1) as a control treatment. Biochar was produced using different invasive plants, including Blackjack (Biden Pilosa), Wedelia (Wedelia trilobata) and Bitter Vine (Mikania micrantha Kunth). Liming decreased the availability of soil N, P, and K by 36.3% as compared with the control due to the great increment in soil pH and exchangeable calcium (Ca2+) by 59% and 16-fold, respectively. Moreover, liming reduced the alpha diversity of soil bacteria and fungi by 27% and 11%, respectively. In contrast, biochar at different types and rates resulted in a fourfold increment in the available N, P, and K as an average under acid rain (pH4.5 and pH2.5) owing to maintaining a neutral pH (6.5–7), which is the most favorable level for soil microbial and enzymatic activites, and the bioavailability of soil nutrients. Furthermore, biochar caused balanced increments in Ca2+ by threefold, cation exchange capacity by 45%, urease activity by 16%, and fungal diversity by 10%, while having a slight reduction in bacterial diversity by 2.5%. Based on the path, correlation, and principal component analyses, the exchangeable aluminum was a moderator for the reductions in macronutrients' availability under acid rain, which decreased by 40% and 35% under liming and biochar, respectively. This study strongly recommended the use of biochar from invasive plants instead of lime for sustainable improvements in soil properties under acid rain. Highlights: Biochar of invasive plants maintained a neutral soil pH under acid rain, while liming alkalinized the soil pH to 9.5. Biochar caused a four-fold increment in soil available N, P higher than liming. BJ-biochar increased soil available P and K higher than other two types, while BV-biochar maximized soil available N. [ABSTRACT FROM AUTHOR]

Microorganisms play an important role in the tobacco aging process. Before the aging process, raw tobacco leaves must be threshed and redried. In order to explore the differences of microbial community structure of threshed and redried tobacco leaves from different origins at home and abroad, 14 groups of tobacco leaves from 8 different countries were tested by high-throughput DNA sequencing and microbiology analysis. Then, through amplicon sequence variants (ASV) cluster analysis, Venn diagram and species labeling and other microbial diversity analysis, the dominant bacteria and fungi on the surface of threshed and redried tobacco leaves were obtained. The results showed that there were significant differences in the composition of tobacco bacteria and fungi after threshing and redrying from different geographical areas. The relative abundance of Microbacterium and Sphingomonas in domestic tobacco leaves was significantly higher than that of foreign tobacco leaves. The relative abundance of Pseudomonas in foreign tobacco bacterial colonies was significantly higher than that of domestic tobacco leaves. In terms of fungi, the relative abundance of Aspergillus and Alternaria in domestic tobacco leaves was significantly higher than that of foreign tobacco leaves. Septoria, Sampaiozyma, Cladosporium and Phoma account for significantly higher proportions of foreign tobacco leaves. These microorganisms may be indispensable in aging process to form different flavors of tobacco leaves. It provides an important theoretical basis for the further use of microorganisms to promote tobacco leaf aging. Key points: Different growth locations of tobacco at home and abroad affect bacterial and fungal communities. Proteobacteria and Actinomycetes were the dominant phylum in redrying tobacco leaves. [ABSTRACT FROM AUTHOR]

Cutaneous leishmaniasis cases have increased dramatically in recent years in Nepal. The study offers molecular identification of the Leishmania species using 40 patient's aspiration biopsy samples, targeting markers kinetoplast minicircle DNA (kDNA) and internal transcribed spacer-1 (ITS1). Among molecularly diagnosed 22 cutaneous leishmaniasis cases, L. donovani complex was identified in 13 instances and L. major in 9 cases. The ITS1 PCR was positive in 12 of the positive nested- kDNA PCR cases (12/22), confirming L. donovani complex in seven of the cases and L. major in five of the cases. In addition, the study conclude that concurrent occurrence of atypical cutaneous infections caused by L. donovani parasite in 59.1% of cases and typical cutaneous infections caused by L. major parasite in 40.9% of cases. A Phylogentic analaysis showed that the detected L. donovani species present null genetic distances from seven references of L. donovani, but slight differences between ITS1 sequences and not grouped into a significant monophyletic cluster. [ABSTRACT FROM AUTHOR]

More Phytophthora species are detected from environmental samples through DNA metabarcoding than are isolated by baiting. We investigated whether bulking soil samples and baiting ~ 300 g samples in standard 1 L tubs resulted in the isolation of fewer Phytophthora and Phytophythium species compared with using 15 – 20 g samples of non-bulked soil in individual 200 ml tubs. At each of four sites with dying vegetation, 50 soil samples were collected and baited separately in small tubs, followed by plating of lesioned baits over 7 days. The number of Phytophthora species obtained was compared with those obtained from bulking the 50 samples and baiting subsamples in large tubs. Half of the asymptomatic baits were plated on day 7 and the remaining were assessed for the presence of Phytophthora using metabarcoding. Root samples with rhizosphere soil from the bulked soil in each site were also assessed using metabarcoding. A higher number of Phytophthora species was recovered from each site from baiting small volumes of non-bulked soil than large volumes of bulked soils. Metabarcoding of the asymptomatic baits revealed species not isolated from lesioned baits. More species were detected from the roots using metabarcoding than were isolated from baits. Metabarcoding did not reveal any species from the rhizosphere soil and roots that were not also detected from metabarcoding of the asymptomatic baits and/or plating. The numbers of Phytopythium species detected using the different methods followed the same trend as for Phytophthora. It was concluded that baiting small samples from across a site in separate small tubs results in the isolation of a higher number of Phytophthora species than the standard technique of baiting large samples of bulked soils, and that this, together with metabarcoding of asymptomatic baits, detects the highest total number of species. [ABSTRACT FROM AUTHOR]

A new method for extracting DNA from plants is proposed, using the example of wild grapes Vitis amurensis Rupr. for further preparation of libraries for metagenomic analysis. The method is based on the isolation of DNA by an inexpensive CTAB method with an additional stage of DNA purification using silica spin columns (CTAB-spin method). A comparative analysis of the results of metagenomic analysis of endophytes on DNA isolated using the proposed CTAB-spin method and using the commercial kit ZymoBIOMICS DNA Miniprep (Zymo Research) was performed. It was found that when using the CTAB-spin method, the number of sequences of the 16S rRNA site and the diversity of bacterial genera were 2.8 and 1.2 times greater, respectively, than when using the ZymoBIOMICS kit. At the same time, the number of sequences of the internal transcribed spacer 1 (ITS1) and the biodiversity of endophytic fungi did not differ significantly during DNA extraction by two methods. Thus, the proposed method of DNA isolation for metagenomic analysis is an available and effective alternative to commercial kits for the isolation of plant DNA for new-generation sequencing methods. [ABSTRACT FROM AUTHOR]

Background and aims: In semiarid ecosystems, changes in plant communities are promoted under shrub canopies during restoration, but the link between shrub community restoration dynamics and changes in soil microbe communities is still unclear. Methods: We characterized the community structure and plant interactions of soil microbes by combining different methodological approaches (including high-throughput sequencing of the 16S rRNA gene and ITS gene, analysis of phospholipid fatty acids (PLFAs) and chloroform fumigation) and the key driving factors along a successional gradient of Sophora moorcroftiana shrub community in the middle reaches of the Yarlung Zangbo River. Results: Soil microbial biomass carbon (MBC) and nitrogen (MBN), total PLFAs, and alpha diversity increased significantly as the successional stage advanced, and MBC and MBN were positively correlated with the carbon and nitrogen contents in the soil. Mantel test showed that successional stage-induced changes in soil microbial beta diversity were mainly associated with shrub coverage and soil physicochemical properties. The relative abundances of bacterial PLFAs, particularly those of gram-negative bacteria, such as Bacteroidetes, Alphaproteobacteria and Betaproteobacteria, significantly decreased with succession; the opposite was true for Acidobacteria, Planctomycetes, Gemmatimonadetes Deltaproteobacteria and Gammaproteobacteria. However, the proportion of fungi did not significantly differ among the four successional stages; the dominant phyla were Ascomycota and Basidiomycota. Conclusion: We suggest that shrubs directly shape soil microbial communities or indirectly affect such communities by altering soil substrates. Our findings advance the current understanding of sand-stabilizing plant–soil interactions during natural restoration and the reversal of desertification in stressful desert ecosystems. [ABSTRACT FROM AUTHOR]

Background and Aims: Soil net nitrogen mineralization (Nmin) rate and its temperature sensitivity (Q10) are critical parameters for predicting nitrogen availability under global warming. However, the controlling factors of the net Nmin rate and its Q10 at the regional scale remain highly uncertain, especially in different ecosystem types. Methods: An incubation experiment at three different temperatures was conducted to measure the net Nmin rate and to calculate its Q10. High-throughput sequencing was applied to explore the microbial community diversity and composition. Results: Results showed that the overall net Nmin rate was highest in paddy fields, followed by forest and dryland fields, whereas its Q10 was not different among ecosystems. Structural equation models revealed that the net Nmin rate was mainly affected by soil properties in dryland habitats, whereas it was mainly influenced by soil microbial communities and mean annual temperature (MAT) in paddy habitats. In forest habitats, the net Nmin rate was mainly affected by bacterial communities rather than fungal communities. The Q10 of net Nmin was close to 2.0, indicating that the net Nmin rate is sensitive to climatic warming. The Q10 was impacted by the bacterial community composition in dryland soils, whereas it was influenced by soil properties and bacterial diversity in paddy and forest soils. Conclusion: This study provides a novel quantification on the contribution of soil microbial diversity and community composition to soil net Nmin process in three ecosystem types, with implications for microbial-mediated N mineralization process under climatic change scenarios. [ABSTRACT FROM AUTHOR]

Background: The number of nucleotide sequences in public repositories has exploded recently. However, the data contain errors, leading to incorrect species identification. Several fighting fish (Betta spp.) are poorly described, with unresolved cryptic species complexes masking undescribed species. Here, DNA barcoding was used to detect erroneous sequences in public repositories. Objective: This study reflects the current quantitative and qualitative status of DNA barcoding in fighting fish and provides a rapid and reliable identification tool. Methods: A total of 1034 barcode sequences were analyzed from mitochondrial cytochrome c oxidase I (COI) and cytochrome b (Cytb) genes from 71 fighting fish species. Results: The nearest neighbor test showed the highest percentage of intraspecific nearest neighbors at 93.41% for COI and 91.67% for Cytb, which can be used as reference barcodes for certain taxa. Intraspecific variation was usually less than 13%, while most species differed by more than 54%. The barcoding gap, calculated from the difference between inter- and intraspecific sequence divergences, was negative in the COI data set indicating overlapping intra- and interspecific sequence divergence. Sequence saturation was observed in the Cytb data set but not in the COI data set. Conclusion: The COI gene should thus be used as the main barcoding marker for fighting fish. [ABSTRACT FROM AUTHOR]

To establish sustainable resources and founder populations for genetic improvement of the Siamese fighting fish Betta splendens, genetic diversity in wild and hatchery stocks was examined using mitochondrial (mt) DNA genes cytochrome b (cytb), 16S ribosomal DNA (16S rDNA), and cytochrome oxidase subunit I (COI), and eight microsatellite loci. Based on mtDNA sequences, restrictive levels of polymorphism (0, 3, and 1 substitutions) were observed in this study. For analysis of microsatellites, fluorescent multiplex PCR was developed, and subsequently identifying moderate levels of observed (Ho = 0.4488) and expected (He = 0.6627) heterozygosities and a high number of alleles per locus (15.125 alleles) for overall samples. Comparison of Siamese fighting fish from different sources revealed large genetic differences between pairs of farmed fish (eight groups) and between wild (three geographic locations) and farmed fish (P < 0.0031 following Bonferroni correction). This suggested limited exchanges of genetic resources between commercial farms. When different color varieties of B. splendens were compared, large genetic distances and significant FST estimates and genetic heterogeneity were found (P < 0.0031). Effective population sizes (Ne) were estimated and two farms (NP2-2BS and BK1-4BS) showed Ne greater than 10. Among color varieties, Multi-colors and Blue revealed reasonable Ne (large and 27.9), but lower Ne values (3.6–8.4) were found for the remaining color varieties. These results indicate an urgent need for the establishment of gene pool resources of B. splendens for effective genetic improvement of Siamese fighting fish in Thailand. [ABSTRACT FROM AUTHOR]

African animal trypanosomosis (nagana) is becoming prevalent beyond its traditionally defined geographical boundaries in African tsetse belts. However, knowledge of clinically important trypanosomes and infection rate in non-tsetse hematophagous flies and domestic animals are limited. This study characterized the potential mechanical vectors, their host feeding patterns, and trypanosome infection in them and domestic animals outside the tsetse belt in northern Kenya. Field-trapped flies and blood from camels, cattle, donkeys, goats, and sheep were screened for trypanosome infection by microscopy and polymerase chain reaction (PCR) of the internal transcribed spacer 1 region. Blood-fed specimens were analysed using PCR-HRM and/or sequencing of 16S rRNA gene to identify vertebrate blood-meal host sources. Hippobosca camelina, Stomoxys calcitrans, Tabanus spp., and Pangonia rueppellii were identified as potential vectors of trypanosomes outside the tsetse belt in Marsabit County. The trypanosome species, Trypanosoma vivax, T. evansi, T. brucei, and T. congolense were recovered in biting flies as well as in camels (Camelus dromedarius). The diversity of parasites in the biting flies was similar to that detected in the tsetse fly Glossina pallidipes collected from the tsetse-infested Shimba Hills, in coastal Kenya, suggesting a wide geographic distribution of the trypanosomes in Kenya. The biting flies fed on camels, cattle, goats, and sheep. Furthermore, we identified diverse clinical outcomes based on PCV (anemia), heamorrhagia) associated with infection with disparate Trypanosoma species. Thus, infection of flies and camels by diverse Trypanosoma species could contribute to the complex epidemiology of observed trypanosomosis in camels. [ABSTRACT FROM AUTHOR]

The Coryneliaceae is a relatively small family of mainly pathogenic fungi occurring on a diversity of hosts with a wide global distribution. Members of the family are recognized by their black, upright and elongated ascomata. Historically, the taxonomy of this group was mainly based on morphological characters, but in more recent years, DNA sequence data have resulted in new revisions. The genus Pewenomyces was recently described based on P. kutranfy, a canker pathogen on Araucaria araucana in Chile. Morphologically, this fungus resembles species in Caliciopsis and Hypsotheca. During the study in which Pewenomyces was described, three putative species were identified from the same host, two of which were observed only from cultures obtained by isolating from plant tissues. At the time of describing P. kutranfy, there was uncertainty regarding its novelty because two species of Caliciopsis (C. brevipes and C. cochlearis), a closely related genus in the Coryneliaceae, had previously also been described from the same host and location, but for which DNA sequence data were not available. In this study, phylogenetic analyses that were carried out for the three putative Pewenomyces species using sequences for seven gene regions confirmed that they were distinct species. Herbarium specimens for the two Caliciopsis species were obtained for morphological comparisons and phylogenetic analyses. Although the holotypes for the two Caliciopsis species did not yield adequate DNA for a phylogenetic analysis, a detailed morphological study established that these species were clearly different from any of the Pewenomyces taxa. The three putative species are consequently described here as Pewenomyces lalenivora sp. nov., P. tapulicola sp. nov., and P. kalosus sp. nov. [ABSTRACT FROM AUTHOR]

Purpose: Cutaneous leishmaniasis (CL) is a major vector-borne disease that affects people globally, including Iran. Different factors are associated with leishmaniasis pathogenicity; recently, a link of the possible relationship between Leishmania RNA Virus (LRV) and disease severity was proposed, especially in the New World leishmaniasis (NWL). This study was aimed to investigate the presence of LRV2 in Leishmania isolates in Aran o Bidgol, Isfahan province. Methods: Samples were collected from 110 CL-suspected patients referred to the health center. In this study, we aimed to investigate CL cases (parasitologically and clinically), identify Leishmania species (by ITS1-PCR–RFLP), and finally detection of LRV2 (by RdRp-semi-nested PCR). Results: Parasitological methods showed 60 positive cases, based on the HaeIII enzyme restriction profile, 59 cases were caused by L. major and 1 case by L. tropica. Our project is the first study on LRV2 isolation in Aran o Bidgol city and the LRV was successfully detected from a single L. major isolated in a women's hand lesion. Using BLAST, 94.8–100% similarity was observed in the RdRp sequence of current LRV isolate with those available in GenBank from Iran or overseas. Conclusion: L. major was the main cause of CL in Aran o Bidgol, although L. tropica is also present in a much lower proportion in the area. This is the first report on the presence of LRV2 in Aran o Bidgol and the fifth in Iran. [ABSTRACT FROM AUTHOR]

Purpose: Rare-earth elements (REEs) have been listed as emerging pollutants, and REEs often occur together with heavy metals (HMs) in the environment. Large amounts of REEs and their coexisting HMs enter into the surrounding soils through dust, surface runoff, and leachate, causing serious REE and HM co-contamination and resulting in ecological crisis. The ecological effects caused by REEs have been gradually concerned, but ignore the synergistic effect of coexistence with HMs. Soil microorganisms are closely related to the soil ecosystem stability. At present, under long-term REE–HM disturbance, the response of bacterial and fungal communities and the effects of community functions remain unclear. In this study, the response of bacterial and fungal communities to different REE and HM co-contamination levels and the community predicted function were analyzed. Methods: 16S rRNA and ITS1 high-throughput sequencing were performed for bacteria and fungi, respectively. The bacteria community functions were predicted using the PICRUSt2 method. Results: The co-contamination caused decreases in bacterial and fungal community richness and diversity, with significant changes in community structure and composition, especially in the most serious co-contaminated soils. With the increase in the pollution levels, the bacterial communities became reorganize, whereas fungal communities had a certain buffer capacity. The microbial symbiotic pattern changed under severe co-contamination conditions, and microorganisms enhanced interactions to advance the dominant taxa adaptability or resistance. Bacterial communities developed more competitive relationships, whereas fungal communities developed more symbiotic relationships. Thus, bacteria are more sensitive than fungi. PICRUSt2 prediction results showed that bacterial community could resist cell damage caused by exotic REEs and HMs by strengthening the efflux transport system, DNA repair function, cell defense mechanism, and detoxification mechanism. The co-contamination might enhance bacterial antibiotic resistance; dissimilatory and assimilatory nitrate reduction and denitrification might be the nitrogen metabolic predominant processes. Conclusion: For the first time, we systematically confirmed that bacteria and fungi respond differently under long-term REE and HM exposure: bacterial are more sensitive than fungi. The ecological functions of bacterial community were changed, and the ecological risk of desert steppe soil environment might be increased. Our results have important significance for ecological risk assessments in co-contamination environment. [ABSTRACT FROM AUTHOR]

Cutaneous leishmaniasis (CL) is one of the most neglected tropical diseases and an important health problem in many countries. It is an endemic disease in most regions of Iraq, while being non-endemic in the Kurdistan Region. The techniques frequently used for detection of CL are not very sensitive. Therefore, this study aimed to identify a sensitive method for diagnosis of CL in clinical samples. The present study was performed in December 2019 to December 2020 in Kalar General Hospital. Clinical samples were collected from 85 suspected CL cases. Sixty-four (75.29%), 71 (83.53%) and 84 (98.82%) cases were detected as positive for CL by microscopy, PCR, and nested PCR, respectively. Of the 84 nested PCR-confirmed CL patients, 46 (54.8%) were female and 38 (45.2%) were male. The most predominate rate of infection was in the 30–39-year age group (29.76%) and the lowest was in the ≥ 60-year group (3.57%). Forty (47.62%) patients had a single lesion. The statistical analysis showed significant differences between age groups and between the number of lesions. The sensitivities of microscopy, conventional PCR, and nested PCR were 80.77%, 86.6% and 100%, respectively, while all three methods showed 100% specificity. Furthermore, PCR-ITS1 followed by a simple restriction fragment length polymorphism (RFLP) analysis using HaeIII endonuclease indicated that Leishmania major was responsible for all CL infections in the study area. [ABSTRACT FROM AUTHOR]

The housefly (Musca domestica) is a wide-ranging insect, often associated with decaying matter from livestock and humans. The septic environments in which houseflies live are believed to be a rich source for microbial acquisition. Although the housefly can harbor a wide range of microorganisms, it is not yet well known which microbes are always recurrent, which are dispensable and which environmentally dependent. In the present study, we aim at identifying which microbes are recurrently associated with the housefly gut throughout the species' life cycle and whether their acquisition relies on the fly's living environment. We surveyed three housefly strains—two of them kept under standard laboratory conditions for a long time and one wild-caught. To track any shifts happening throughout the lifecycle of the housefly and to test the consistency of the revealed microbial communities, we sampled houseflies at five developmental stages over the course of four consecutive generations. Both the bacterial and fungal microbiota of five developmental stages were studied for all samples, using amplicon sequencing for the 16S and ITS1 rRNA gene, respectively. Results revealed diverse microbial communities yet consistent for each of the two distinct sampling environments. The wild-caught population showed a more diverse and more distinct gut microbiota than the two laboratory strains, even though the strain was phylogenetically similar and shared geographic origin with one of them. Two bacterial genera, Myroides and Providencia, and two yeasts, Trichosporon and Candida tropicalis, were present in all sampled larvae and pupae, regardless of the strain. Analysis of the provided diet revealed that the flies acquired the yeasts through feeding. Our main findings show that houseflies might lose microbial diversity when reared in controlled environments, however they can maintain a consistent set of bacteria. We conclude that although the environment can facilitate certain microbial transmission routes for the housefly, and despite the fungal microbiota being largely acquired through diet, the larval bacterial gut microbiome remains relatively consistent within the same developmental stage. [ABSTRACT FROM AUTHOR]

Trichomoniasis is a sexually transmitted infection (STI) caused by the flagellated protozoan Trichomonas vaginalis. Little information is available on the epidemiology and genetic diversity of T. vaginalis in Ilam City, southwestern Iran. A descriptive cross-sectional investigation was carried out between July 2017 and December 2018 on the suspected women patients referred to eight gynecology clinics of Ilam City for probable Trichomonas infection. They were undergone a set of clinical, parasitological, and molecular examinations. During clinical consultation, posterior vaginal fornix secretions and urine samples were gathered from the participants. For the reasons such as physical conditions and cultural and religious constraints, most of participating women, especially young girls due to their virginity, preferred to give urine samples instead of vaginal discharge. The presence of Trichomonas was diagnosed by microscopic examination and molecular detection using conventional PCR targeting ITS1-rDNA. A total of 1765 suspected individuals were examined clinically via vaginal secretions (495 specimens) and urine samples (1270 specimens). Of them, 21 (1.18%) cases, including 13 vaginal secretions and 8 urine samples, were positive for Trichomonas infection by microscopy. Slightly more than half of the patients (11/21, 52.4%) complained of vulvar itching, burning, and frequent urination. Cervical lesions, patchy erythema, and vaginal discharge were recorded in 28.6%, 23.8%, and 19% of the patients respectively. All patients with positive microscopic identification were confirmed by amplification of 450-bp fragment of ITS1-rDNA. Phylogenetic analysis revealed a high rate of genetic homogeneity in which all our isolates together with homologous sequences from China, Philippines, Austria, and USA were clustered within the same clade. A statistically significant relationship was recorded between the patients positive for trichomoniasis and the presence of chronic disease (e.g., diabetes, immune system deficiency). [ABSTRACT FROM AUTHOR]

The gut mycobiome exhibits major influence on the gastrointestinal health and disease but received less attention due to low abundance. This study characterizes the fungal community and compares the microbial diversity between indoor and outdoor cats. Genomic DNA was extracted and sequenced by targeting the Internal Transcribed Spacer 1 (ITS1) region using Flongle flow cell on MinION™ sequencing platform. Results show the phylum Ascomycota and genus Peniophorella were numerous in indoor cats, whereas the Basidiomycota and Pichia were abundant in outdoor cats. Peniophorella formed the core mycobiome in both feline populations. Furthermore, alpha (p value = 0.0207) and beta diversities (p value = 0.009) results showed significant differences between the two groups. Overall, indoor cats have greater amounts of Peniophorella, whereas outdoor cats have higher Trichosporon and unclassified Sordariaceae. The study also suggests that keeping a cat indoors or left as a stray will affect their respective gut mycobiome. [ABSTRACT FROM AUTHOR]

Spiders prevalent in cattle sheds were observed predating on adult Culicoides sp. Even juveniles of Neoscona mukerjei (Tikader 1980) predated on small insects which included blood engorged Culicoides oxystoma; whereas adults preferred larger one. Adults and juveniles of Menemerus bivittatus (Dufour 1831) and juveniles of Marpissa sp. also feed on engorged adults which were identified as C. peregrinus (Kieffer 1910) and C. oxystoma (Kieffer 1910) collected within the animal sheds. Predations on Culicoides midges by M. bivittatus in field condition were supplemented by gut content analysis using Culicoides sp. specific primer pairs. Diet analysis of M. bivittatus (both sexes) diet collected from two different sheds demonstrated the presence of biting midges as one of the prey. Laboratory experiments on time intervals for prey choice decision by M. bivittatus depicted inter-individual differences, however, no variation was observed in prey selection (different age groups of midges). [ABSTRACT FROM AUTHOR]

Soil microorganisms could affect the quality of tobacco leaves, however, little is known about the association of tobacco chemical components and soil fungal communities. In the present study, the relationship between soil fungi and tobacco quality based on chemical components in Bijie was investigated. The results showed that the total harmony scores (THS) of the analyzed tobacco leaves ranged from 46.55 ± 3.5 to 91.55 ± 2.25. Analyses of chemical components revealed that high contents of nicotine (≥ 1.06%) and sugar (total sugar: ≥ 22.96%, reducing sugar: ≥ 19.62%), as well as low potassium level (≤ 2.68%) were the main factors limiting the quality of flue-cured tobacco leaves. Pearson correlation analysis indicated that soil nitrate, available potassium/phosphorous, and organic matter significantly correlated with tobacco nicotine, potassium, and chloride levels (p < 0.05). Besides, the analysis of alpha- and beta-diversity of soil fungal communities implied that fungal structure rather than the richness affected the chemical quality of tobacco. In detail, the relative abundance of Humicola olivacea species in soils was positively correlated with the THS of tobaccos (r = 0.52, p < 0.05). Moreover, the species including Mortierella alpina, Mortierella hyalina, Tausonia pullulan, and Humicola olivacea were negatively correlated with tobacco sugar (r ≤ − 0.45, p < 0.05) while, Codinaea acaciae and Saitozyma podzolica species were negatively correlated with tobacco nicotine (r ≤ − 0.51, p < 0.05). The present study provides a preliminary basis for utilizing fungal species in soils to improve the chemical quality of tobacco in the studied area. [ABSTRACT FROM AUTHOR]