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This is the first study on phylogenetic relationships in the genus Artemia Leach, 1819 using the pattern and sequence of secondary structures of internal transcribed spacer 1 (ITS1). Significant intraspecific variation in the secondary structure of ITS1 rRNA was found in Artemia tibetiana. In the phylogenetic tree based on joined primary and secondary structure sequences, Artemia urmiana and parthenogenetic populations displayed new lineages, and two New World species (Artemia franciscana and Artemia persimilis) were located in a basal clade that was not detected in previous studies. The close evolutionary relationship between A. franciscana and A. persimilis are expressively supported by the previous empirical and experimental investigation on the ability of hybridization (in natural habitats and lab conditions) and analysis on allozyme markers.

Background: Leishmaniasis is endemic in Iran. Different species of Leishmania (L.) parasites are causative agents of this disease. Correct identification of Leishmania species is important for clinical studies,prevention, and control of the diseases. Mix up of Leishmania isolates is possible in the laboratory, so there is need for verification of species for isolates of uncertain identity. Different methods may be used for this purpose including isoenzyme electrophoresis and molecular methods. The isoenzyme lectrophoresis, due to its drawbacks, is feasible only in specialized laboratories while molecular methods may be more feasible. The aim of this research was to study the application of the internal transcribedspacer 1 (ITS1) sequencing method, in comparison to isoenzyme electrophoresis method, for verification of Leishmania species.Methods: Six Leishmania isolates were received from different research institutions in Iran. The species of these isolates were known by donating institution according to their isoenzyme profile. The species of these isolates were re-identified in Pasteur Institute of Iran by PCR amplification of ITS1 followed bysequencing and comparison of these sequences with Leishmania sequences in GenBank. Isoenzyme electrophoresis was performed for confirmation of the results of ITS1.Results: ITS1 sequence showed that some isolates were mixed up or contaminated with Crithidia. Isoenzyme electrophoresis confirmed the results of ITS1 sequences.Conclusion: ITS1 sequencing is relatively more feasible than the traditional isoenzyme electrophoresismethod and is suggested for verification of Leishmania species.

Background: Trichomoniasis is a worldwide protozoan parasitic disease and metronidazole is a choice drug for its treatment. Because of disease importance in public health and its controversial ideas about the prevalence of drug resistance, this study was carried out.Methods: Fifty-two suspected vaginal samples were collected from 2006 to 2007 in Gynecology Maryam Hospital, Tehran, Iran. All isolates were examined by microscopic, culture and PCR tech­niques. The PCR products were analyzed by RFLP and CSGE methods and two suspected samples were sequenced.Results: Trichomonas vaginalis was identified from all 52 samples. Of 52 isolates, 45 samples were successfully cultured and amplified by PCR except one. Seven were positive only by PCR. Finally, ITS1 fragment was successfully amplified in 51 of 52. CSGE analysis and PCR products di­gestion by MspI followed by sequencing showed nucleotide mutation at position 209 (C209T) of the ITS1 fragment in two (3.9%) of them.Conclusion: The results showed mutation in ITS1 fragment of T. vaginalis in two (3.9%) of Ira­nian isolates which may be related to metronidazole resistance.

Background: Leishmaniasis is endemic in Iran. Different species of Leishmania (L.) parasites are causative agents of this disease. Correct identification of Leishmania species is important for clinical studies, prevention, and control of the diseases. Mix up of Leishmania isolates is possible in the laboratory, so there is need for verification of species for isolates of uncertain identity. Different methods may be used for this purpose including isoenzyme electrophoresis and molecular methods. The isoenzyme electrophoresis, due to its drawbacks, is feasible only in specialized laboratories while molecular methods may be more feasible. The aim of this research was to study the application of the internal transcribed spacer 1 (ITS1) sequencing method, in comparison to isoenzyme electrophoresis method, for verification of Leishmania species. Methods: Six Leishmania isolates were received from different research institutions in Iran. The species of these isolates were known by donating institution according to their isoenzyme profile. The species of these isolates were re-identified in Pasteur Institute of Iran by PCR amplification of ITS1 followed by sequencing and comparison of these sequences with Leishmania sequences in GenBank. Isoenzyme electrophoresis was performed for confirmation of the results of ITS1. Results: ITS1 sequence showed that some isolates were mixed up or contaminated with Crithidia. Isoenzyme electrophoresis confirmed the results of ITS1 sequences. Conclusion: ITS1 sequencing is relatively more feasible than thetraditional isoenzyme electrophoresis method and is suggested for verification of Leishmania specie.

Background: Trichomoniasis is a worldwide protozoan parasitic disease and metronidazole is a choice drug for its treatment. Because of disease importance in public health and its controversial ideas about the prevalence of drug resistance, this study was carried out. Methods: Fifty-two suspected vaginal samples were collected from 2006 to 2007 in Gynecology Maryam Hospital, Tehran, Iran. All isolates were examined by microscopic, culture and PCR tech­niques. The PCR products were analyzed by RFLP and CSGE methods and two suspected samples were sequenced. Results: Trichomonas vaginalis was identified from all 52 samples. Of 52 isolates, 45 samples were successfully cultured and amplified by PCR except one. Seven were positive only by PCR. Finally, ITS1 fragment was successfully amplified in 51 of 52. CSGE analysis and PCR products di­gestion by MspI followed by sequencing showed nucleotide mutation at position 209 (C209T) of the ITS1 fragment in two (3.9%) of them. Conclusion: The results showed mutation in ITS1 fragment of T. vaginalis in two (3.9%) of Ira­nian isolates which may be related to metronidazole resistance.

Background: Leishmaniasis is a major medical health problem and distributes in nearly half of 31 provinces of Iran. We aimed to identify cutaneous and visceral Leishmania spp. isolated from infected humans and domestic dogs in various regions of Iran, 2010‒2013.Methods: DNA was extracted from 108 lesion exudate samples of suspected patients to cutaneous leishmaniasis and nine liver and spleen aspirates of infected dogs cultured in RPMI-1640 and amplified using partial sequence of ITS1 gene. The PCR amplicons were digested using HaeIII endonuclease enzyme and used in restriction fragment length polymorphism (RFLP) assay. Then, 48 amplicons representing various hosts were sequenced and compared to se­quences from GenBank databases using BLAST.Results: PCR-RFLP analysis showed that 60 and 48 CL patients were infected by Leishmania tropica and L. major, respectively. From nine canine visceral leishmaniasis (CVL) isolates, eight isolates were identified as L. infantum and one as L. tropica. The greatest similarity of 95.7% in ITS1 region was seen between L. infantum and L. major. Furthermore, the lowest similarity with 65.7% was seen between L. tropica and L. major. Intra-species comparison of ITS1 region in L. infantum, L. major and L. tropica isolates were showed 100%, 98.2% and 72.4 % similarities, respectively.Conclusion: PCR-RFLP based on ITS1 region is an appropriate method to distinguish three Leishmania spp. of L. major, L. tropica, and L. infantum. In intra-species comparison of ITS1 region, genotypic variations showed that L. tropica isolates were more heterogeneous than L. major and L. infantum isolates.

Abstract Background/Objective The cestode Echinococcus granulosus causes cystic echinococcosis, a zoonotic parasitic infection that constitutes a significant public health risk. This parasite has been documented to have potential reservoirs and carriers among wild canids, namely wolves, foxes and jackals. This study aimed to determine the prevalence and molecular characteristics of E. granulosus sensu lato species/genotypes among wild canids in three northern, northeastern and north‐western Iran regions. Methods From 2019 to 2022, 93 wild canid carcasses (69 jackals), (22 foxes) and (2 wolves) were collected that were killed in car accidents or illnesses. Analyses of morphology and morphometry were performed to verify the presence of E. granulosus. To determine E. granulosus s.l. species/genotypes, polymerase chain reaction (PCR)‐RFLP (ITS1) was performed utilizing the Bsh1236I (BstUI) restriction enzyme. COX1, NADH1 and ITS1 gene sequencing were also performed to confirm the PCR‐RFLP results. Results During this study, 93 wild canids were examined, and 3.2% (95% CI: 0%–7%) of the 93 were infected with Echinococcus. The north‐western region of Iran showed two out of 30 jackals (6.6%) infected with adult Echinococcus compared to one out of 35 jackals (2.8%) in the northern region. DNA from Echinococcus was detected in these individuals by PCR. Based on PCR‐RFLP analysis of the ITS1 gene and sequencing of COX1, NADH1 and ITS1 gene, E. granulosus sensu stricto genotype was confirmed in the jackals that had been infected. Conclusion Evidence shows that E. granulosus occurs in jackals in Iran, with the E. granulosus s.s. genotype being the most common. This parasite has been identified as a zoonotic parasite with a genotype that can be transmitted to livestock and humans. Establishing effective control measures to prevent the spread of echinococcosis and ensure public health is crucial.

DNA barcoding is a straightforward strategy that uses short orthologous genetic sequences and standard genomes to specify species. This technique has the capability of molecular identification, detection of living species and discovery of unfamiliar species, preservation of genetic resources, identification of genetic diversity and phylogenetic characterization, and testing of differentiated existing plant species, as well as assuring the safety and efficacy of pharmaceuticals. In this experiment, the allelic diversity of 7 classes of medicinal plants viz. fenugreek, local fenugreek, waybread, cumin, flax, fixweed and sesame, from Ilam and Khuzestan provinces; west and south of Iran, respectively, was carried out employing this technique and with the aid of primers designed and established on ITS nuclear barcodes (ITS1 and ITS2 genes). The results indicated that there was a great difference between the fragments and the duplicated sequences of ITS1 and ITS2 barcodes in different samples. Moreover, the nucleotide searching of the sequences showed that there was a very high similarity (more than 90%) between the acquired sequences and their equivalents in the NCBI database. The nucleotide sequence of the ITS1 gene of fenugreek showed the highest similarity (78.2%) with native fenugreek. Regarding the ITS1 gene, more amount of G~C content than A~T was observed and, in the waybread plants the amount of C base was higher than G, and for native fenugreek, the amount of A~T content was more than G~C. In the case of ITS2 position, in all examined samples (except the fixweed plant, which had higher A~T), the values ​​of G~C content were higher than A~T. The output of the cluster analysis with the UPGMA algorithm showed the precise grouping and separation of species and the high potential of these sequences using the barcode system in the phylogenetic evaluation of medicinal plant species.

Abstract Background Neospora caninum has been documented to infect most domestic wildlife but is known to primarily infect dogs and cattle and is considered an important cause of abortion in camels. Objective The aim of this study was to estimate the molecular detection of Neospora caninum in tissues of naturally infected camelids. Methods Brain, tongue (bottom and tip) and masseter muscles from 35 slaughtered camelids from Tataouine and Médenine regions were collected (n = 140 samples). PCR was used to amplify and detect N. caninum DNA in tissues samples followed by sequencing of some PCR products. A phylogenetic tree was then constructed to compare the partial sequences of the ITS1 gene with GenBank sequences. Histopathology examination was used to detect Neospora spp. cysts, but no lesions were observed. Results The overall molecular detection of N. caninum in camelids was 34.3% (12/35). The highest molecular detection of N. caninum was recorded in animals of more than 3 years old (6/9) and in animals aged between 1 and 3 years old (4/12). Whilst, the lowest molecular detection (2/14) was observed in animals 1 year or younger (p = 0.035). There were no significant differences in molecular detection of N. caninum according to both locality and gender (p > 0.05). Similarly, there was no difference of prevalence between different anatomical locations. Comparison of the partial sequences of the ITS1 gene revealed 100–95.5% similarity among our N. caninum amplicon (MW551566) and those deposited in GenBank. Conclusion These results highlight the presence of a risk infection by N. caninum in camels. For preventing N. caninum infection further studies are needed to improve our knowledge about the epidemiology of neosporosis in North Africa.

Abstract 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.

Cystic echinococcosis is an important cosmopolitan parasitic zoonosis that causes public health and economic problems in Egypt. The present study was undertaken to identify genotypes of hydatid cyst (HC) DNA isolated from different animal isolates and to identify the genotype of secondary hydatid cysts (HCs) developed in rabbits experimentally infected with camel HC for detection of any genetic mutation. In the present study, we extracted DNA from the germinal layers of 8 HCs collected from 3 camels, 1 cattle, 1 sheep and 3 donkeys in addition to 3 secondary HCs collected from rabbits experimentally infected with camel HC. PCR amplification of the ITS1 gene of all examined samples showed an amplified DNA band at 1115 bp. The partial nucleotide sequences of the ITS1 gene of all isolates were aligned and compared with the reference sequences of the genotypes G1–G8 in GenBank. The camel and rabbit samples were identified as Echinococcus canadensis genotype 6 (G6), while the cattle and sheep samples belonged to E. granulosus sensu stricto (G1). The donkey isolates belonged to E. equines (G4). Alignment of the ITS1 partial nucleotide sequences of the camel HCs and rabbit secondary HCs isolates with the G6 partial nucleotide sequence in GenBank was performed. Both camel HCs and rabbit secondary HCs isolates exhibited the same sequence identity matrix, which indicated the absence of mutation in the rabbit secondary HCs. It can be concluded that camel and rabbit samples were identified as E. canadensis (G6), the cattle and sheep samples belonged to E. granulosus sensu stricto (G1) and donkey isolates belonged to E. equines (G4). No mutation occurred during HCs transmission from camel to rabbit.

A female tuna of odd appearance, with the size of a Pacific bluefin tuna (Thunnus orientalis Temminck and Schlegel, 1844), anal fin count of bigeye tuna (Thunnus obesus Lowe, 1839), pectoral fins of yellowfin tuna (Thunnus albacares Bonnaterre, 1788), caught in waters west of the island of Xiaoliuqiu, Pingtung, Taiwan, received considerable media attention after its landing at Tungkang fishing port on 14 December 2020. This tuna has now been identified by molecular systematic means. Fishermen and fish merchants suggested it was a hybrid form, unique in living memory; however, it had matured ovaries and developing ova. To confirm whether this fish was a hybrid, we determined its parental species by analysing the cytochrome b gene (cyt b) and cytochrome c oxidase subunit 1 (COI) sequences of the mitochondrial DNA for maternal inheritance, and the internal transcribed spacer 1 (ITS1) gene sequence from the nuclear DNA to confirm both parents’ lineages. Genomic DNA was isolated from fast-skeletal muscle, and primers were designed based on the known sequences of conserved regions among tunas. According to cyt b and COI, the mother of the peculiar tuna was a Pacific bluefin tuna (T. orientalis), and the ITS1 sequence showed that both parents were of this species. We therefore conclude that despite the mixed morphological appearance, this abnormal tuna was a Pacific bluefin tuna, not a hybrid.

Fatal hepatic sarcocystosis was diagnosed as the cause of death in four pinnipeds: two captive Hawaiian monk seals (Monachus schauinslandi), a captive, and a free-ranging California sea lion (Zalophus californianus). Based on necropsy, histopathology, electron microscopy and DNA sequencing, intralesional protozoal schizonts were determined to have caused the necrotizing hepatitis observed. Transmission Electron Microscopy (TEM) revealed schizonts similar to Sarcocystis canis in hepatocytes. PCR-DNA sequencing and phylogenetic analysis at the conserved 18S rRNA and variable ITS1 gene markers within the nuclear rRNA gene array from schizont-laden tissue established that the parasites were indistinguishable from Sarcocystis canis at the 18S rRNA locus. However, six distinct single nucleotide polymorphisms (SNPs) were resolved at ITS1 suggesting that the parasites infecting pinnipeds were distinct from S. canis, which commonly infects bears and dogs. We hypothesize that the parasite represents a novel Sarcocystis variant that we refer to as S. canis-like that infects pinnipeds. The definitive host of S. canis is enigmatic and its life cycle incomplete. These findings document a critical need to identify the life cycle(s), definitive host(s), and all susceptible marine and terrestrial intermediate hosts of S. canis and the S. canis-like variant infecting pinnipeds.

Humans can be infected with anthroponotic (Ancylostoma duodenale and Necator americanus) and with zoonotic (Ancylostoma ceylanicum, A. caninum, A. braziliense, and Uncinaria stenocephala) hookworms from dogs. Anthroponotic species are usually thought not to infect dogs. We used the internal transcribed spacer–1 (ITS1) gene in a quantitative PCR to detect anthroponotic and zoonotic hookworm species in fecal samples from 54 children and 79 dogs living in an indigenous community in tropical Northwestern Ecuador. Hookworm DNA was detected in 59.3% of children and 92.4% of dogs. Among samples from children, zoonotic hookworms were detected in 24.1% (A. ceylanicum 14.8%, A. caninum 11.1%, and A. braziliense 1.9%), whilst in dog samples, anthroponotic species were detected in 19.0% (N. americanus 12.4% and A. duodenale 6.3%). Sanger sequencing was performed successfully on 60 qPCR-positive samples (16 from children and 44 from dogs), and consensus sequences were obtained with >98% homology to GenBank references for hookworm spp. Phylogenetic analysis showed a close relationship between anthroponotic and zoonotic Ancylostoma species and no heterogeneity between A. duodenale and A. caninum; in human samples, we found A. ceylanicum but not A. braziliense sequences and we were unable to identify N. americanus in the dog samples. No infections with U. stenocephala were detected. Our data provide evidence for high rates of hookworm infections in indigenous children and dogs in a marginalized rural setting in coastal Ecuador. We also found evidence for potential cross-transmission of hookworm spp. between humans and dogs that represent a potential domestic reservoir for zoonotic and anthroponotic hookworms.

Leishmaniasis is a group of diseases caused by protozoa of the genus Leishmania and is transmitted by the bite female sand fly. The present work is characterized as a descriptive study in two areas: a forest area located in the Parque Estadual do Rio Doce, and another urban area located in the municipality of Timóteo-MG, with the objective of identifying the presence of Leishmania spp. and the blood source of the collected female sand flies. Part of the females were obtained from the Parque Estadual do Rio Doce, and part was collected using 19 ligth traps distributed in residences of Timóteo. For molecular studies of Leishmania spp. DNA, the ITS1 gene was used, and in the search for blood source, the CytB gene was used and positive samples were sequenced. The study demonstrated that there are at least three species of Leishmania circulating in the study areas: Leishmania (Viannia) braziliensis, Leishmania (Leishmania) amazonensis, and Leishmania (V.) guyanensis. Nyssomyia whitmani was the predominant sand fly species in the urban area of Timóteo with a positive diagnosis for the presence of Leishmania braziliensis DNA. We found the presence of blood from Gallus gallus (Chicken) and Sus scrofa (Pig) in sand flies. The present study demonstrates that Leishmania braziliensis is the main agent of cutaneous leishmaniasis in the study area, with the effective participation of Nyssomyia whitmani as the vector and both Gallus gallus and Sus scrofa acting as a food source for female sand flies, and helping maintaining the sand fly life.

Abstract Background Cutaneous leishmaniasis is a disease transmitted by sand fly bites. This disease is highly prevalent in Syria where Leishmania major and Leishmania tropica are the known aetiological agents. In 2011, more than 58,000 cases were reported in the country by the Ministry of Health. The central region of the country harbors 20 % of the reported cases. However, the epidemiology of the disease in this area is not well understood. An epidemiological survey was conducted in 2010 to identity the circulating parasite and the sand fly vector in the central provinces of Edlib and Hama. Methods Sand fly specimens were collected using CDC light traps and identified morphologically. Total DNA was extracted from the abdomens of female specimens and from Giemsa-stained skin lesion smears of 80 patients. Leishmania parasites were first identified by sequencing the ITS1 gene amplicons. Then polymorphism analysis was performed using the RFLP technique. Results A total of 2142 sand flies were collected. They belonged to eight species, among which Phlebotomus sergenti and Phlebotomus papatasi were the most predominant. L. tropica ITS1 gene was amplified from two pools of P. sergenti specimens and from skin smears of cutaneous leishmaniasis patients. This suggests that P. sergenti is the potential vector species in the study area. The digestion profiles of the obtained amplicons by TaqI restriction enzyme were identical for all analysed L. tropica parasites. Moreover, L. infantum ITS1 gene was amplified from two pools of Phlebotomus tobbi in the relatively humid zone of Edlib. Conclusions L. tropica is confirmed to be the aetiological agent of cutaneous leishmaniasis cases in the central provinces. RFLP technique failed to show any genetic heterogeneity in the ITS1 gene among the tested parasites. The molecular detection of this parasite in human skin smears and in P. sergenti supports the vector status of this species in the study area. The detection of L. infantum in P. tobbi specimens indicates a potential circulation of this parasite in the humid zone of Edlib. Further epidemiological studies are needed to evaluate the burden of this visceral parasite in the study region.

Abstract Background The fungal microbiome, or mycobiome, is a poorly described component of the gut ecosystem and little is known about its structure and development in children. In South Africa, there have been no culture-independent evaluations of the child gut mycobiota. This study aimed to characterise the gut mycobiota and explore the relationships between fungi and bacteria in the gut microbiome of children from Cape Town communities. Methods Stool samples were collected from children enrolled in the TB-CHAMP clinical trial. Internal transcribed spacer 1 (ITS1) gene sequencing was performed on a total of 115 stool samples using the Illumina MiSeq platform. Differences in fungal diversity and composition in relation to demographic, clinical, and environmental factors were investigated, and correlations between fungi and previously described bacterial populations in the same samples were described. Results Taxa from the genera Candida and Saccharomyces were detected in all participants. Differential abundance analysis showed that Candida spp. were significantly more abundant in children younger than 2 years compared to older children. The gut mycobiota was less diverse than the bacterial microbiota of the same participants, consistent with the findings of other human microbiome studies. The variation in richness and evenness of fungi was substantial, even between individuals of the same age. There was significant association between vitamin A supplementation and higher fungal alpha diversity (p = 0.047), and girls were shown to have lower fungal alpha diversity (p = 0.003). Co-occurrence between several bacterial taxa and Candida albicans was observed. Conclusions The dominant fungal taxa in our study population were similar to those reported in other paediatric studies; however, it remains difficult to identify the true core gut mycobiota due to the challenges set by the low abundance of gut fungi and the lack of true gut colonising species. The connection between the microbiota, vitamin A supplementation, and growth and immunity warrants exploration, especially in populations at risk for micronutrient deficiencies. While we were able to provide insight into the gut mycobiota of young South African children, further functional studies are necessary to explain the role of the mycobiota and the correlations between bacteria and fungi in human health.

The rapid reduction in coral reefs worldwide has led to increasing attention toward protecting and restoring coral reef ecosystems. Coral reefs not only have a rich diversity of coral species, but they can also provide important products and services for human beings. One type of coral, Echinogorgia, has important scientific research value and application prospects. To understand the diversity of coral species, diving surveys were conducted in Xiamen Bay in 2017 and 2021, and a total of 928 samples were collected. Taxonomic research was conducted using methods such as morphological identification through electron microscopy. Specific phylogenetic trees of the COI gene, mtMuts gene, and ITS1 gene were analyzed. There were 47 specimens of Echinogorgia coral included among 928 samples. Fifteen species of Echinogorgia were identified, including Echinogorgia ramosa, Echinogorgia flexilis, Echinogorgia russelli, Echinogorgia ramulosa, and Echinogorgia gracilima (which represent the newly recorded species in the waters of China). This study increases the species diversity records in China and contributes to new geographical distribution information of Echinogorgia worldwide. The primary data also serve as the baseline data for long-term biomonitoring programs to estimate the status of octocorals in Xiamen Bay.

Abstract Background Tsetse flies can transmit various Trypanosoma spp. that cause trypanosomiasis in humans, wild animals, and domestic animals. Amplicon deep sequencing of the 12S ribosomal RNA (rRNA) gene can be used to detect mammalian tsetse hosts, and the 18S rRNA gene can be used to detect all associated eukaryotic pathogens, including Trypanosoma spp. Methods Tsetse flies were collected from the Serengeti National Park (n = 48), Maswa Game Reserve (n = 42), and Tarangire National Park (n = 49) in Tanzania in 2012–13. Amplicon deep sequencing targeting mammal-specific 12S rRNA and 18S rRNA genes was performed to screen the blood-feeding sources of tsetse flies and eukaryotic parasites in tsetse flies, respectively. Results 12S rRNA gene deep sequencing revealed that various mammals were blood-feeding sources of the tsetse flies, including humans, common warthogs, African buffalos, mice, giraffes, African elephants, waterbucks, and lions. Genes of humans were less frequently detected in Serengeti (P = 0.0024), whereas African buffaloes were detected more frequently as a blood-feeding source (P = 0.0010). 18S rRNA gene deep sequencing showed that six tsetse samples harbored the Trypanosoma gene, which was identified as Trypanosoma godfreyi and Trypanosoma simiae in subsequent ITS1 gene sequencing. Conclusions Through amplicon deep sequencing targeting the 12S rRNA and 18S rRNA genes, various mammalian animals were identified as blood-meal sources, and two Trypanosoma species were detected in tsetse flies collected from the Maswa Game Reserve, Serengeti National Park, and Tarangire National Park in Tanzania. This study illustrates the patterns of parasitism of tsetse fly, wild animals targeted by the fly, and Trypanosoma spp. carried by the fly in Tanzania. It may provide essential data for formulating better strategies to control African trypanosomes. Graphical Abstract

Objectives: The purpose of this study was to determine the species composition of Eimeria circu¬lating in Mymensingh district, Bangladesh, using Internal Transcribed Spacer 1 (ITS1) sequences in polymerase chain reaction (PCR) assay. Materials and Methods: Coccidian oocysts were isolated and sporulated in a solution containing 2% potassium dichromate from litter slurry collected from 13 commercially active broiler farms in the research region. Genomic DNA was isolated from sporulated oocysts and used to amplify the Eimeria species-specific ITS1 gene by PCR amplification. Electrophoresis of 1.5% agarose gel was used to visualize the amplified PCR products. Results: In the study samples from Mymensingh district, Bangladesh, the presence of Eimeria brunetti, Eimeria acervulina, Eimeria necatrix, Eimeria mitis, and Eimeria tenella was identified. Conclusions: The findings of this study may shed light on the zonal approach to chicken coccidio¬sis control. Additionally, it suggests that ITS1-based PCR might be used in the field to accurately identify Eimeria species. [J Adv Vet Anim Res 2021; 8(3.000): 489-493]

This article describes the dataset of the eukaryotic and fungal microbiome in bulk soil and root of wild Brassica napus at five different grassland sites in South Korea. The microbiome datasets were obtained using Illumina MiSeq sequencing of the 18S rRNA gene and ITS1 gene. The raw sequences and metadata used for analysis are available at the National Center for Biotechnology Information (NCBI) (BioProject ID: PRJNA821335). Raw data were clustered into amplicon sequence variants (ASVs) using the DADA2 pipeline and aligned against the SILVA 132 reference database and UNITE database. A total of 5702 eukaryotic ASVs (1,913,372 reads) and 4565 fungal ASVs (9,032,969 reads) were extracted after quality-filtering. Rhizaria was the most dominant eukaryote at the class level, and Olpidiomycetes was the dominant fungal class in this dataset. As unintended releases of transgenic B. napus have been reported in South Korea [1], the microbiome datasets produced in this work will be used as the foundation for environmental risk assessment to understand the potential effect of released transgenic B. napus on the natural ecosystem.

Primary amebic meningoencephalitis (PAM) is a rare and fatal central nervous system infection caused by Naegleria fowleri, a free-living amoeba found in the environment. To date, eight pathogenic N. fowleri genotypes have been reported worldwide. We aimed to explore the genotypes of N. fowleri that cause primary amebic meningoencephalitis in Thailand. In 2021, the 17th PAM case was reported, and a retrospective literature search of PAM cases in Thailand from 1982 through April 2021 was performed. Phylogenetic and genotyping analyses of the two mitochondrial (12S rRNA and 16S rRNA) and nuclear (ITS1 and 5.8s rRNA) genes of N. fowleri were performed on four available clinical isolates. Based on the mitochondrial and nuclear genes, N. fowleri genotype T3 was found to cause PAM in three out of four cases. However, disagreement between the genotype based on the mitochondrial and nuclear genes was found in one of the PAM cases, in which the 12S rRNA locus suggested the causative genotype as T1, while the ITS1 implied genotype T4. The discrepancy between the mitochondrial and nuclear genome was previously observed, which suggests the possible horizontal gene transfer among N. fowleri species. Based on the ITS1 gene, two N. fowleri genotypes, T3 and T4, were found to be the genotypes causing PAM in this study. In addition, N. fowleri genotype T2 was previously reported in a traveler who was infected in Thailand. Thus, at least three genotypes (T2, T3, and T4) of N. fowleri are found to be associated with PAM in Thailand.

Wetland drainage has been intensively implemented globally, and it has exerted significant effects on wetland ecosystems. The effects of wetland drainage on the soil fungal community remain to be clarified. Soil samples were collected at depths of 0–5 and 5–10 cm in freshwater Phragmites australis wetlands to investigate changes in the fungal community before and after drainage (termed FW and DFW, respectively) using high-throughput sequencing of the fungal-specific internal transcribed spacer 1 (ITS1) gene region. No significant differences in the α diversity of the soil fungal community were found in 0–10 cm soils between FW and DFW (p > 0.05), except for the abundance-based coverage estimator (ACE) and Chao1 indices in 5–10 cm soils. Significantly higher values of ACE and Chao1 in 5–10 cm soils in FW than in DFW indicated that wetland drainage may reduce fungal community richness in 5–10 cm soils. Ascomycota, Sordariomycetes, and Cephalothecaceae were the dominant fungal phylum, class, and family, respectively, in 0–5 and 5–10 cm soils of both FW and DFW, representing as high as 76.17, 58.22, and 45.21% of the fungal community in 5–10 FW soils, respectively. Saprotrophic fungi predominated in both FW and DFW. Drainage altered both the fungal community structure and some edaphic factors. Mantel tests and Spearman correlation analyses implied that edaphic factors [i.e., soil organic matter (SOM), electronic conductivity (EC), pH, and clay] also affected soil fungal community structure. Overall, wetland drainage altered the community structure of the fungal community in the freshwater wetlands.

Background: We aimed to compare semi-nested PCR with indirect ELISA to diagnose human fasciolosis. Methods: Overall, 70 serum samples were collected from different areas in Iran suspected for fascioliasis. Individuals were classified based on diagnostic of fascioliasis and habitat in endemic areas. Finally, all serum samples were tested by indirect ELISA (using secretory excretory antigen) and semi-nested PCR (using ITS1 gene). The study was conducted in the School of Publish Health, Tehran University of Medical Sciences, Iran in 2021. Results: Significant differences were found between agreement and similarity of patients' results of indirect ELISA and semi-nested PCR 94.46% and 98.4% respectively (Cohen's kappa ≥0.6; P-value≤0.05). No cross-reactions were observed with other parasitic diseases (toxocariasis, hydatidosis, strongyloidiasis, toxoplasmosis, cutaneous leishmaniasis, taeniasis and trichinosis). 69.84% of samples were positive by both techniques. In addition, the percentage of agreement and similarity between the results of the two techniques based on habitat in endemic areas was 88.9-100% and 97.7-100%, respectively (Cohen's kappa ≥0.6; P-value≤0.05). Conclusion: Semi-nested PCR could be a suitable method for following up on patients' treatment and a confirmatory method for ELISA as for diagnosis of human fascioliasis.

Background & objectives: Cutaneous leishmaniasis (CL) in Marrakesh-Safi region located in the central-south part of Morocco is a public health problem. This study assessed the efficiency of a microscopic examination method in establishing the diagnosis of CL and PCR for the characterization and identification of the circulating Leishmania strains in different CL foci of the study area. Methods: A total of 297 smears obtained from cutaneous lesions of suspected patients with CL were stained with May-Grünwald Giemsa (MGG) for microscopic examination. For each positive smear, genomic DNA was extracted and PCR-analysed, targeting the small subunit ribosomal ribonucleic acid (ssu rRNA) gene to detect Leishmania DNA. Then, the internal transcribed spacer 1 (ITS1) was amplified and sequenced in order to identify the Leishmania species. The sensitivity and specificity of the conventional microscopy with ssu rRNA gene were compared by Leishmania nested PCR (LnPCR) and ITS1 gene by ITS-PCR. Results: A total of 257 smears were positive in the microscopic examination, i.e. the detection rate of amastigotes by optical microscopy was 86.53% (257/297). The LnPCR was found to have a specificity and a sensitivity of 100%, each. Interestingly, the sequencing results showed that 99.61% (256/257) of the isolates had Leishmania tropica and 0.39% (1/257) had L. infantum infection. Interpretation & conclusion: Though, classical microscopic examination is useful and economical, it is not sensitive enough, especially in endemic regions where several Leishmania species coexist. In such situations, PCR constitutes a complementary method for the identification of the causal species. The results indicate that both the L. tropica (dominant) and L. infantum are the causative agents of CL in the Marrakesh-Safi region. The rate of CL infection is high in Imintanout, and Chichaoua provinces. Hence, early diagnosis and prompt treatment of CL patients is necessary to prevent its extension to neighboring localities.

The family Paramphistomidae including Gigantocotyle explanatum regularly infects ruminants and causes immense economic losses to the livestock industry by decreasing dairy products and growth rates. The present study was aimed to determine the novel molecular data of G. explanatum in Pakistan using ribosomal DNA (ITS1-5.8S, ITS2) regions. Adult flukes, G. explanatum, were collected from bile ducts of infected buffaloes. The most relevant sequences from the other parts of the world were downloaded from the GenBank. High intraspecific variations were obtained at 5 end region of ITS1 gene. The 3 end of ITS1 was conserved and showed 96% similarity with Paramphistomum cervi (KJ459936). The nucleotide blast search of 5.8S gene revealed that 40 sequences from trematodes had 98% to 99% identity with present sequence and found genetically identical to P. cervi (KJ459938) and Dicrocoelium chinensis (KF734784) from China. The ITS2 gene of investigated isolates showed no variation with Myanmar (AB743577), while blast search revealed 96-100% similarity with isolates from Myanmar, India, Bangladesh and China. This study demonstrates the utility of ITS2 and 3´ end ITS1 sequences as a valuable tool for elucidating species phylogenetic relationship in south Asia. This sequencing data will facilitate more accurate identification of G. explanatum, enabling future work to resolve many ambiguities in the literature regarding this species.

Slightly acidic electrolyzed water (SAEW) was prepared and used as wheat tempering water. This study explored the impacts of tempering with SAEW on microbial load and diversity and quality properties of wheat flour. As SAEW volume ratio increased, the residual level of total plate counts (TPC) and mould/yeast counts (MYC) decreased dramatically (p < 0.05). Based on genomics analysis, bacterial 16S rRNA gene and fungal ITS1 gene region were performed to characterize the changes in microbial communities’ composition and diversity in response to SAEW treatment. SAEW optimal volume ratio (6.5:10, v/v) of SAEW with distilled water influenced wheat microbiome composition, with a higher microbial diversity and abundance discovered on the control grains. Bacteroidetes of predominant bacterial phylum and Ascomycota of the most abundant fungal phylum were reduced after SAEW optimal volume ratio tempering. The flour yield is higher and ash content is lower than the control samples. Falling number and “b*” in terms of colour markedly increased. DSC (Differential Scanning Calorimetry) test showed that To (onset temperature), Tp (peak temperature), and Tc (conclusion temperature) were significantly decreased in thermal characteristics of flour. Gluten content, protein content, ΔH and pasting properties tests showed no significant change. It can be concluded that SAEW should be applied on wheat tempering for producing clean wheat flour. ANOVA and Tukey’s honestly significant difference (HSD) test were used for the analysis of variance and differences between the experimental and control groups, with p < 0.05.

Anthroponotic cutaneous leishmaniais (ACL) and zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania tropica and Leishmania major, respectively, are endemic vector-borne diseases in southern Saudi Arabia. In 2021, an outbreak of cutaneous leishmaniasis occurred in the province of Asir. The main objective of our investigation was to analyze the epidemiological features of CL in southern Saudi Arabia. The ministry of health recorded 194 CL patients between January and December 2021 from the Asir province. Our findings showed that the majority of CL patients (87.1%) originated from the governorates of Khamis-Mushait and Abha. Most of the patients were males (62.3%). While CL affected all age groups, those under 13 years old were the most affected (38.1%). For both genders, CL patients were mostly Saudi citizens (90.7%) compared to non-Saudi expatriates. The majority of CL patients (75.2%) suffered from a single lesion, and the majority of lesions (61.3%) were located on the face. The seasonal prevalence of CL showed two peaks, a small one in July–August and a larger one in March. Of a total of 194 Giemsa slides samples, 188 showed positive amplification of Leishmania ITS1 gene. Based on PCR-RFLP and PCR-HMR, 183 patients showed positive amplification of L. tropica and five patients showed positive amplification of L. major. Phylogenetic analysis revealed a clear distinct separation between L. major and L. tropica sequences. Our results provided strong evidence of the pre-domination of L. tropica, the main etiological agent of ACL in Asir province. We reported for the first time the presence of L. major, an etiological agent of ZCL in the study areas. The co-circulation of ACL and ZCL highlighted the complexity of the epidemiology of CL in southern Saudi Arabia, and subsequently, further studies to identify competent vectors and reservoir hosts for the establishment of control strategies are needed.

Wildlife shares grazing areas with herders in the eastern Qinghai-Tibet Plateau, and humans can be infected by zoonotic nematodes through direct contact with animals or contaminated water. In this study, fecal samples (n = 296) from wild carnivores were collected to explore the infection rate and molecular genetic characteristics of nematodes by stratified random sampling in the survey areas. Host species and the nematodes they carried were then identified using 16S rRNA and 18S rRNA gene sequencing, respectively. Statistical analysis, neutrality tests, genetic diversity analysis and Bayesian inferred trees were performed to complete the study. In total, 10 species of nematodes were detected in 240 feces from six species of carnivores identified (including dominant Vulpes ferrilata and Vulpes vulpes), namely Uncinaria stenocephala, Toxascaris sp., Crenosoma vulpis, Parapharyngodon bainae, Oesophagostomum muntiacum, Aspiculuris tetraptera, Mastophorus muris, Nematodirus spathiger, Muellerius capillaris, and Molineus patens. Among these nematodes, U. stenocephala (35.83%, 86/240) and Toxascaris sp. (14.58%, 35/240) were detected at higher rates than the other nematodes (χ2 = 516.909, p < 0.05). Of 17 and 18 haplotypes were found based on the ITS1 gene for U. stenocephala and nad1 gene for Toxascaris sp., respectively. For the first time, using molecular methods, we report the infection of V. ferrilata by U. stenocephala, a potential zoonotic parasite, and suggest Toxascaris sp. may be a newly discovered nematode that lives within the fox intestine.

Background: Cystic Echinococcosis (CE) is an emergent or re-emergent zoonosis and remains a public health and economic problem all over the world. Methods: The present study was carried on the prevalence and genotypes of Echinococcus present in small ruminants in Kashmir valley. A total of 2100, sheep (2052) and goats (48), slaughtered or spontaneously dead, from various areas of Kashmir valley were screened for the presence of hydatidosis. In case of goat none of the cases were found positive for hydatidosis, whereas, all the positive cases (85) were recorded in sheep only. The overall prevalence of hydatidosis was 4.04%. The prevalence was higher in female sheep (5.46%) compared to males (2.83%). Season-wise highest prevalence was in summer (4.55%), followed by autumn (4.1%), spring (3.89%) and winter (2.5%).The liver was observed to be the most frequently infected organ with relative prevalence of 61.17% followed by lungs (38.82%).The rDNA-ITS1 fragment of positive samples was amplified with BD1 / 4S primers. Results: The length of amplified fragment for all isolated samples was 1000bps. The products obtained on PCR were digested with four restriction enzymes (Rsa 1, Alu 1, Msp 1 and Taq1). Rsa 1, Alu 1, Msp 1 yielded identical fragments, 300 and 700 bp in sheep. TaqI restriction enzyme had no effect on PCR product and after digestion; intact 1000bps fragment was seen. Conclusion: Phylogenetic analysis of ITS1 gene revealed that the common sheep strain (G1) is the predominant genotype in sheep in Kashmir valley.

Plant communities and fertilization may have an impact on soil microbiome. Most commercial olive trees are minerally fertilized, while this practice is being replaced by the use of organic amendments. Organic amendments can both fertilize and promote plant growth-promoting organisms. Our aims were (i) to describe the changes in soil bacterial and fungal communities induced by the presence of young olive trees and their interaction with organic amendments and (ii) to compare the effects of mineral and organic fertilization. We set up two parallel experiments in pots using a previously homogenized soil collected from a commercial olive orchard: in the first one, we grew olive saplings in unamended and organically amended soils with two distinct composts and compared these two soils incubated without a plant, while in the second experiment, we comparatively tested the effects of organic and mineral fertilization. OTUs and the relative abundances of bacterial and fungal genera and phyla were analyzed by 16S rRNA and ITS1 gene amplicon using high-throughput sequencing. Basal respiration and substrate-induced respiration were measured by MicroRespTM. The effects of the different treatments were analyzed in all phyla and in the 100 most abundant genera. The presence of olive saplings increased substrate-induced respiration and bacterial and fungal richness and diversity. Organic amendments greatly affected both bacterial and fungal phyla and increased bacterial richness while not affecting fungal richness. Mineral fertilization increased the relative abundance of the less metabolically active bacterial phyla (Actinobacteria and Firmicutes), while it reduced the most metabolically active phylum, Bacteroidetes. Mineral fertilization increased the relative abundance of three N2-fixing Actinobacteria genera, while organic fertilization only increased one genus of Proteobacteria. In organically and minerally fertilized soils, high basal respiration rates were associated with low fungal diversity. Basidiomycota and Chytridiomycota relative abundances positively correlated with basal respiration and substrate-induced respiration, while Ascomycota correlated negatively. Indeed, the Ascomycota phyla comprised most of the fungal genera decreased by organic amendments. The symbiotrophic phylum Glomeromycota did not correlate with any of the C sources. The relative abundance of this phylum was promoted by the presence of plants but decreased when amending soils with composts.

Background: Human hydatidosis is endemic in northeastern Iran. The present study aimed to investigate molecular diversity of Echinococcus granulosus isolates collected from human surgically. Methods: Sixty human hydatid cysts (58 lung cysts and 2 liver cysts) were collected through surgery from Ghaem and Emam Reza hospitals in Mashhad University of Medical Sciences during 2015-2016. Cysts were characterized using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer 1 (ITS1) gene and sequencing fragments of the genes coding for mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit I (nad1). Results: Overall, 55 out of 60 Echinococcus granulosus cysts (91.6%) were determined as the G1 strain, 4 cases (6.6%) were determined as the G6 strain and 1 sample was not identified. Conclusion: Although sheep strain (G1) is dominated in human patients in Great Khorasan, the prevention of camel-dog cycle should pay attention in this region.

Background: Recently, we reported that patients with mild cognitive impairment (MCI) harbor specific signature of bacteria in their gut and that a modified Mediterranean ketogenic diet (MMKD) improves Alzheimer's disease (AD) markers in cerebrospinal fluid (CSF) and the signatures of gut bacteria. However, other microbial population such as gut fungi (mycobiome) in relation to MCI/AD pathology, gut bacteria and diet remain unknown. Methods: We measure gut mycobiome by sequencing of the fungal rRNA ITS1 gene in 17 older adults (11 MCI; 6 cognitively normal [CN]) in a single-center, randomized, double-blind, crossover pilot study, before and after 6 weeks intervention of MMKD and American Heart Association Diet (AHAD), and determine its correlation with AD markers in CSF and gut bacteria. Findings: Compared to CN counterparts, patients with MCI have higher proportion of families Sclerotiniaceae, Phaffomyceteceae, Trichocomaceae, Cystofilobasidiaceae, Togniniaceae and genera Botrytis, Kazachstania, Phaeoacremonium and Cladosporium and lower abundance of Meyerozyma. Specific fungal taxa exhibit distinct correlation arrays with AD markers and gut bacteria in subjects with versus without MCI. MMKD induces broader effect on fungal diversity in subjects with MCI and increases Agaricus and Mrakia while decreasing Saccharomyces and Claviceps with differential response in subjects with or without MCI. Interpretation: The study reveals MCI-specific mycobiome signatures and demonstrates that distinct diets modulate the mycobiome in association with AD markers and fungal-bacterial co-regulation networks in patients with MCI. The findings corroborate the notion of considering gut mycobiome as a unique factor that can affect cognitive health/AD by interacting with gut bacteria and diet and facilitate better understanding of the AD and related microbiome, using unique diet or microbiome modulators.

BackgroundPhlebotomus (Larroussius) guggisbergi is among the confirmed vectors for cutaneous leishmaniasis (CL) transmission in Kenya. This scarring and stigmatizing form of leishmaniasis accounts for over one million annual cases worldwide. Most recent CL epidemics in Kenya have been reported in Gilgil, Nakuru County, where the disease has become a public health issue. However, little is known about the factors that drive its transmission. Here, we sought to determine the occurrence, distribution and host blood feeding preference of the vectors, and to identify Leishmania species and infection rates in sandflies using molecular techniques. This information could lead to a better understanding of the disease transmission and improvement of control strategies in the area.Methodology/ principal findingsAn entomological survey of sandflies using CDC light traps was conducted for one week per month in April 2016, and in June and July 2017 from five villages of Gilgil, Nakuru county; Jaica, Sogonoi, Utut, Gitare and Njeru. Sandflies were identified to species level using morphological keys and further verified by PCR analysis of cytochrome c oxidase subunit I (COI) gene. Midguts of female sandflies found to harbour Leishmania were ruptured and the isolated parasites cultured in Novy-MacNeal-Nicolle (NNN) media overlaid with Schneider's insect media to identify the species. Leishmania parasite screening and identification in 198 randomly selected Phlebotomus females and parasite cultures was done by PCR-RFLP analysis of ITS1 gene, nested kDNA-PCR and real-time PCR-HRM followed by sequencing. Bloodmeal source identification was done by real-time PCR-HRM of the vertebrate cytochrome-b gene. A total of 729 sandflies (males: n = 310; females: n = 419) were collected from Utut (36.6%), Jaica (24.3%), Sogonoi (34.4%), Njeru (4.5%), and Gitare (0.1%). These were found to consist of nine species: three Phlebotomus spp. and six Sergentomyia spp. Ph. guggisbergi was the most abundant species (75.4%, n = 550) followed by Ph. saevus sensu lato (11.3%, n = 82). Sandfly species distribution across the villages was found to be significantly different (pConclusions/ significanceThe high infection rates of L. tropica and abundance of Ph. guggisbergi in this study confirms this sandfly as a vector of L. tropica in Kenya. Furthermore, isolation of live L. tropica parasites from Ph. saevus s.l. suggest that there are at least three potential vectors of this parasite species in Gilgil; Ph. guggisbergi, Ph. aculeatus and Ph. saevus s.l. Molecular identification of L. major infections in Ph. guggisbergi suggested this sandfly species as a potential permissive vector of L. major, which needs to be investigated further. Sandfly host preference analysis revealed the possibility of zoonotic transmissions of L. tropica in Gilgil since the main vector (Ph. guggisbergi) does not feed exclusively on humans but also other vertebrate species. Further investigations are needed to determine the potential role of these vertebrate species in L. tropica and L. major transmission in the area.

We analyzed the blood from 400 one-humped camels, Camelus dromedarius (C. dromedarius), in Riyadh and Al-Qassim, Saudi Arabia to determine if they were infected with the parasite Trypanosoma spp. Polymerase chain reaction (PCR) targeting the internal transcribed spacer 1 (ITS1) gene was used to detect the prevalence of Trypanosoma spp. in the camels. Trypanosoma evansi (T. evansi) was detected in 79 of 200 camels in Riyadh, an infection rate of 39.5%, and in 92 of 200 camels in Al-Qassim, an infection rate of 46%. Sequence and phylogenetic analyses revealed that the isolated T. evansi was closely related to the T. evansi that was detected in C. dromedarius in Egypt and the T. evansi strain B15.1 18S ribosomal RNA gene identified from buffalo in Thailand. A BLAST search revealed that the sequences are also similar to those of T. evansi from beef cattle in Thailand and to T. brucei B8/18 18S ribosomal RNA from pigs in Nigeria.

Abstract Background Infectious disease is the leading cause of death worldwide, and diagnosis of polymicrobial and fungal infections is increasingly challenging in the clinical setting. Conventionally, molecular detection is still the best method of species identification in clinical samples. However, the limitations of Sanger sequencing make diagnosis of polymicrobial infections one of the biggest hurdles in treatment. The development of massively parallel sequencing or next generation sequencing (NGS) has revolutionized the field of metagenomics, with wide application of the technology in identification of microbial communities in environmental sources, human gut and others. However, to date there has been no commercial application of this technology in infectious disease diagnostic settings. Methods Credence Genomics Rapid Infection Detection™ test, is a molecular based diagnostic test that uses next generation sequencing of bacterial 16S rRNA gene and fungal ITS1 gene region to provide accurate identification of species within a clinical sample. Here we present a study comparing 16S and ITS1 metagenomic identification against conventional culture for clinical samples. Using culture results as gold standard, a comparison was conducted using patient specimens from a clinical microbiology lab. Results Metagenomics based results show a 91.8% concordance rate for culture positive specimens and 52.8% concordance rate with culture negative samples. 10.3% of specimens were also positive for fungal species which was not investigated by culture. Specificity and sensitivity for metagenomics analysis is 91.8 and 52.7% respectively. Conclusion 16S based metagenomic identification of bacterial species within a clinical specimen is on par with conventional culture based techniques and when coupled with clinical information can lead to an accurate diagnostic tool for infectious disease diagnosis.

Leishmaniasis is a growing health challenge in many parts of Iran, including Kerman Province. Investigating vector ecology and parasite-harboring capacity is prerequisite to the disease control measures. This study included six provincial sites namely Bam (Bm), Dehbakri (Di), Jiroft (Jt), Mohammad-Abad (Md), Rostam-Abad (Rd) and Darb-e-Behesht (Dt) where sand flies were trapped. The specimens were then identified before being exposed to DNA extraction. PCR-RFLP was used to detect leishmanial infection rates and feeding preference of vectors. Diversity indices indicated that the highest effective numbers of species was in plain sites, whereas, the highest expected numbers of species was in mountainous sites. P. papatasi and P. sergenti showed similar feeding preferences to both human and animal bloods. P. papatasi from indoor catches was found infected with Leishmania major at a 2% rate. The ITS1 gene sequences of isolated parasites were >99% similar to related GenBank haplotypes. Bam and Rostam-Abad remain active foci of both types of cutaneous leishmaniasis (CL). Md and Di are prone to visceral leishmaniasis (VL). Jt is not at risk of anthroponotic cutaneous leishmaniasis (ACL) due to absence of P. sergenti. Sand flies are absent in Dt, probably because of high elevation and cold climate. In conclusion, patterns of climate and ecosystem changes and vector-host-reservoirs interactions must be carefully scrutinized if leishmaniasis is to be controlled in the stricken sites.

BackgroundTaenia solium (T. solium) cysticercosis remains a neglected zoonotic disease in India. The current study was planned to estimate the prevalence of T. solium porcine cysticercosis in the Punjab state of India, to compare this prevalence with the disease prevalence in pigs reared outside Punjab and to assess the distribution of the parasite in pig carcasses.MethodsTwo slaughter shops were selected in each of the 22 districts of Punjab. Pigs slaughtered on the day/s of inspection were post-mortem inspected to identify the presence of T. solium cysts. Estimated true prevalence was estimated by taking into account the diagnostic sensitivity (38%) and specificity (100%) of post-mortem inspection using the Rogan-Gladen estimator. Positive carcasses were purchased and brought to the laboratory to assess the tissue distribution of T. solium cysts and to conduct PCR targeting large subunit rRNA gene, internal transcribed spacer 1 gene, ITS1 gene and Cytochrome oxidase I gene. The selected PCR products were submitted for sequencing and phylogenetic analyses were performed.FindingsWe contacted 71 shop owners to achieve a sample of 44 shops for the study. We inspected 642 pigs reared in Punjab and 450 imported from other states at these slaughter shops. In addition, we sampled 40 pigs from an abattoir located in the state capital. Of the 642 pigs reared in Punjab, 9 had T. solium cysts with an apparent prevalence of 1·40% (95% CI: 0·74%, 2·64%) and the estimated true prevalence of 3.69% (95% CI: 1·95%, 6·95%). Pigs imported from outside the state had a significantly higher prevalence (odds ratio: 2·58; 95% CI: 1·12, 5·98; p-value: 0·026) as 15 of the 450 imported pigs were positive (apparent prevalence: 3.33%; 95% CI: 2.03%, 5.43%; estimated true prevalence: 8.77%; 95% CI: 5.34%, 14.28%). None of samples was positive from the pigs sampled at the abattoir in the state capital. The PCR confirmed T. solium cysts from all the 24 positive samples. We counted a median of 897 (range 526-1964) cysts per infected pig from the 19 infected pig carcasses inspected. The phylogenetic tree based on the alignment of partial cytochrome oxidase 1 sequences indicated all positive samples to be clustered with the T. solium Asian genotype. The analysis did not indicate the presence of T. asiatica in the slaughter pigs.ConclusionsDespite the underestimation of the prevalence due to missing mildly-infected carcasses, low participation and lack of representative sampling, the presence of heavily infected carcasses containing viable cysts, particularly those imported from outside the state, indicates that T. solium cysticercosis is an important food safety concern for pork consumers in Punjab, India. Measures should be taken to reduce the disease prevalence in pigs to reduce the disease burden in the public.

Background & objectives: Aedes albopictus (Skuse) is one of the major vectors of dengue which is an emerging threat in Northeast part of India. The morphological characterisation of mosquitoes is time consuming and lacks accuracy for distinguishing closely related species. Hence, molecular methods of mosquito identification, genetic diversity and molecular phylogeny have gained increased importance. This study was aimed to identify and characterize the most abundant species of Aedes vectors collected from different breeding spots in Assam, Northeast India employing molecular as well as bioinformatics tools. Methods: Ae. albopictus species was genetically characterized with internal transcribed spacer1 (ITS1) and cytochrome c oxidase subunit I (COI) genes and sequence analysis was carried out following molecular methods like PCR amplification, DNA sequencing and multiple sequence analysis. Maximum likelihood molecular phylogeny was reconstructed to define the evolutionary relationship among studied isolates and isolates from other parts of Southeast Asia. Results: Molecular study revealed that all five subject specimens belonged to Ae. albopictus species as per both ITS1 and COI genes. Maximum likelihood tree based on ITS1 and COI genes showed that isolates were distinctly grouped into separate clusters. Almost similar pattern of amino acid frequencies in COI gene was found amongst the five studied isolates. However, amino acid frequency in ITS1 gene was found to be dissimilar, indicating polymorphisms in this gene, among the isolates. Interpretation & conclusion: This is the first report among the Northeastern states of India describing the genetic make-up of Ae. albopictus species by virtue of highly conserved mitochondrial (mt) DNA and ribosomal (r) DNA gene sequences. This study also illustrates that the sequence diversity of these two genes in this mosquito species differs geographically which differentiate a population and brings unique identity.

There is a paucity of knowledge regarding the diversity and impact(s) of disease-causing fungi in marine animals, especially shellfish. In efforts to address this knowledge gap for the shore crab Carcinus maenas, a year-long disease screen was carried out across two sites in Swansea Bay (Wales, UK) with a view to characterising putative fungal infections. Crabs were sampled between November 2017 and October 2018, and screened systematically for disease signatures using haemolymph (blood) preparations, targeted PCR and tissue histopathology. Strikingly, mycosis was confirmed in ~0.4% of total crabs tested (n = 1191) and restricted to one location only (Mumbles Pier). Clinical infections were observed in four out of four infected crabs. In these animals, the gills and hepatopancreas were congested with fungal morphotypes. In addition, some evidence indicates haemocyte (immune cell) reactivity toward the fungi. Phylogenetic placement of the partial internal transcribed spacer (ITS1) gene regions amplified from three mycotic crabs revealed the causative agent to be related to hypocrealean fungi, thereby representing a novel species.

Mesophotic coral ecosystems are an almost entirely unexplored and undocumented environment that likely contains vast reservoirs of undescribed biodiversity. Twenty-four macroalgae samples, representing four genera, were collected from a Hawaiian mesophotic reef at water depths between 65 and 86 m in the ‘Au‘au Channel, Maui, Hawai‘i. Algal tissues were surveyed for the presence and diversity of fungi by sequencing the ITS1 gene using Illumina technology. Fungi from these algae were then compared to previous fungal surveys conducted in Hawaiian terrestrial ecosystems. Twenty-seven percent of the OTUs present on the mesophotic coral ecosystem samples were shared between the marine and terrestrial environment. Subsequent analyses indicated that host species of algae significantly differentiate fungal community composition. This work demonstrates yet another understudied habitat with a moderate diversity of fungi that should be considered when estimating global fungal diversity.

Visceral leishmaniasis (VL) is a debilitating, often fatal disease caused by Leishmania donovani complex; however, it is a neglected tropical disease. L. donovani complex comprises two closely related species, L. donovani that is mostly anthroponotic and L. infantum that is zoonotic. Differentiation between these two species is critical due to the differences in their epidemiology and pathology. However, they cannot be differentiated morphologically, and their speciation using isoenzyme-based methods poses a difficult task and may be unreliable. Molecular characterization is now the most reliable method to differentiate between them and to determine their phylogenetic relationships. The present study aims to characterize Leishmania species isolated from bone marrows of Yemeni pediatric patients using sequence analysis of the ribosomal internal transcribed spacer-1 (ITS1) gene. Out of 41 isolates from Giemsa-stained bone marrow smears, 25 isolates were successfully amplified by nested polymerase chain reaction and sequenced in both directions. Phylogenetic analysis using neighbor joining method placed all study isolates in one cluster with L. donovani complex (99% bootstrap). The analysis of ITS1 for microsatellite repeat numbers identified L. infantum in 11 isolates and L. donovani in 14 isolates. These data suggest the possibility of both anthroponotic and zoonotic transmission of VL-causing Leishmania species in Yemen. Exploring the possible animal reservoir hosts is therefore needed for effective control to be achieved.

Deoxynivalenol (DON) is commonly detected in cereals, and is a threat to human and animal health. The effects of microbiological detoxification are now being widely studied. A total of 24 pigs (over four months) were randomly divided into three treatments. Treatment A was fed with a basal diet as the control group. Treatment B was fed with naturally DON-contaminated wheat as a negative control group. Treatment C was fed with a contaminated diet that also had Clostridium sp. WJ06, which was used as a detoxicant. Growth performance, relative organ weight, intestinal morphology, and the intestinal flora of bacteria and fungi were examined. The results showed that after consuming a DON-contaminated diet, the growth performance of the pigs decreased significantly (p < 0.05), the relative organ weight of the liver and kidney increased significantly (p < 0.05), and the integrity of the intestinal barrier was also impaired, though the toxic effects of the contaminated diets on growing pigs were relieved after adding Clostridium sp. WJ06. The data from MiSeq sequencing of the 16S ribosomal ribonucleic acid (rRNA) gene and internal transcribed spacer 1 (ITS1) gene suggested that the abundance of intestinal flora was significantly different across the three treatments. In conclusion, the application of Clostridium sp. WJ06 can reduce the toxic effects of DON and adjust the intestinal microecosystem of growing pigs.

A number of recent studies suggest that interspecific competition plays a key role in determining the structure of ectomycorrhizal (ECM) fungal communities. Despite this growing consensus, there has been limited study of ECM fungal community dynamics in abiotically stressful environments, which are often dominated by positive rather than antagonistic interactions. In this study, we examined the ECM fungal communities associated with the host genus Alnus, which live in soils high in both nitrate and acidity. The nature of ECM fungal species interactions (i.e., antagonistic, neutral, or positive) was assessed using taxon co-occurrence and DNA sequence abundance correlational analyses. ECM fungal communities were sampled from root tips or mesh in-growth bags in three monodominant A. rubra plots at a site in Oregon, USA and identified using Illumina-based amplification of the ITS1 gene region. We found a total of 175 ECM fungal taxa; 16 of which were closely related to known Alnus-associated ECM fungi. Contrary to previous studies of ECM fungal communities, taxon co-occurrence analyses on both the total and Alnus-associated ECM datasets indicated that the ECM fungal communities in this system were not structured by interspecific competition. Instead, the co-occurrence patterns were consistent with either random assembly or significant positive interactions. Pair-wise correlational analyses were also more consistent with neutral or positive interactions. Taken together, our results suggest that interspecific competition does not appear to determine the structure of all ECM fungal communities and that abiotic conditions may be important in determining the specific type of interaction occurring among ECM fungi.

BackgroundLeishmania major and an uncharacterized species have been reported from human patients in a cutaneous leishmaniasis (CL) outbreak area in Ghana. Reports from the area indicate the presence of anthropophilic Sergentomyia species that were found with Leishmania DNA.Methodology/principal findingsIn this study, we analyzed the Leishmania DNA positive sand fly pools by PCR-RFLP and ITS1 gene sequencing. The trypanosome was determined using the SSU rRNA gene sequence. We observed DNA of L. major, L. tropica and Trypanosoma species to be associated with the sand fly infections. This study provides the first detection of L. tropica DNA and Trypanosoma species as well as the confirmation of L. major DNA within Sergentomyia sand flies in Ghana and suggests that S. ingrami and S. hamoni are possible vectors of CL in the study area.Conclusions/significanceThe detection of L. tropica DNA in this CL focus is a novel finding in Ghana as well as West Africa. In addition, the unexpected infection of Trypanosoma DNA within S. africana africana indicates that more attention is necessary when identifying parasitic organisms by PCR within sand fly vectors in Ghana and other areas where leishmaniasis is endemic.

This study was conducted to (i) determine the prevalence of African Animal Trypanosomosis (AAT) in tsetse challenged areas, (ii) compare conventional with qPCR detection systems and (iii) evaluate the host genetic background and biology as risk factors. AAT prevalence studies are often confronted with low levels of parasitaemia. Hence, we designed a novel qPCR assay using primers and species specific probes amplifying the Internal Transcribed Spacer 1 (ITS1) gene. Thereby all three AAT species could be detected simultaneously. 368 individuals from three cattle types (Baoulé, Zebu and hybrids) originating from 72 farms in Burkina Faso were analysed. Farmers were interviewed and morphometric measurements of the cattle taken. A chi-squared test and a logistic regression model were calculated to detect associations with infection. In our study, the overall rate of prevalence detected with the novel qPCR assay was 11.14%. Compared to conventional PCR we identified a concordance of 91.30%. We tested 41 animals positive for trypanosome DNA, five animals showed multiple infections. Zebus were twice as often infected (21.74%) compared to Baoulé (9.70%) and hybrids (9.57%). Trypanosoma vivax is the dominant species (9.24%), as compared to T. congolense (2.44%) and T. brucei (0.82%). The chi-squared tests linking the infection events to the breeds (Zebu vs. Baoulé and Zebu vs. hybrids) were on the border of significance. No significant association with other tested parameters could be detected. We introduce a novel qPCR technique for the fast, sensitive and simultaneous detection of the three AAT species. Our results suggest that associations with breed and infection exist since Zebu cattle are more likely to be infected compared to Baoulé and hybrids. Indigenous taurine cattle breeds, like the Baoulé, therefore provide a unique and valuable genetic resource.

BackgroundThree major forms of human disease, cutaneous leishmaniasis, visceral leishmaniasis and mucocutaneous leishmaniasis, are caused by several leishmanial species whose geographic distribution frequently overlaps. These Leishmania species have diverse reservoir hosts, sand fly vectors and transmission patterns. In the Old World, the main parasite species responsible for leishmaniasis are Leishmania infantum, L. donovani, L. tropica, L. aethiopica and L. major. Accurate, rapid and sensitive diagnostic and identification procedures are crucial for the detection of infection and characterization of the causative leishmanial species, in order to provide accurate treatment, precise prognosis and appropriate public health control measures.Methods/principal findingsHigh resolution melt analysis of a real time PCR product from the Internal Transcribed Spacer-1 rRNA region was used to identify and quantify Old World Leishmania in 300 samples from human patients, reservoir hosts and sand flies. Different characteristic high resolution melt analysis patterns were exhibited by L. major, L. tropica, L. aethiopica, and L. infantum. Genotyping by high resolution melt analysis was verified by DNA sequencing or restriction fragment length polymorphism. This new assay was able to detect as little as 2-4 ITS1 gene copies in a 5 microl DNA sample, i.e., less than a single parasite per reaction.Conclusions/significanceThis new technique is useful for rapid diagnosis of leishmaniasis and simultaneous identification and quantification of the infecting Leishmania species. It can be used for diagnostic purposes directly from clinical samples, as well as epidemiological studies, reservoir host investigations and vector surveys.