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Of the many arthropod species affecting hemp (Cannabis sativa L.) cultivation in the United States, one species of particular importance is the hemp russet mite (Aculops cannabicola , HRM). Hemp russet mite is a microscopic arthropod which feeds on all parts of hemp plants. Due to its minute size, HRM can proliferate undetected for a long time, complicating management efforts and causing serious economic losses. DNA sequencing and PCR assays can facilitate accurate identification and early detection of HRM in infested-plants. Therefore, a real-time SYBR Green based species-specific PCR assay (quantitative PCR, qPCR) was developed for the identification of HRM DNA by amplification of a 104 bp Internal Transcribed Spacer 1 (ITS1) sequence. The detection limit was estimated to be approximately 48 copies of the HRM marker gene sequence. The real-time-PCR assay is rapid, detects all life stages of mite under 2 hours. A 10-fold serial dilution of the plasmid DNA containing the ITS1 insert were used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay showed a strong linear relationship with HRM DNA with R 2 of 0.96. The assay was tested against several commonly found hemp pests including two-spotted spider mite and western flower thrips to determine specificity of the assay and to show that no non-target species DNA was amplified. The outcomes of this research will have important applications for agricultural biosecurity through accurate identification of HRM, early detection and timely deployment of management tactics to manage and prevent pest outbreaks. [ABSTRACT FROM AUTHOR]

Objective: Leishmaniasis is a parasitic disease caused by more than 20 Leishmania species. This disease is spread by vectors. Many researchers agree that Leishmania was spread to mammals by sandflies of the genus Phlebotomus and Lutzomyia. Leishmaniasis is still considered one of the most neglected diseases by the World Health Organization (WHO). An estimated 0.7-1 million new cases of leishmaniasis are reported annually from approximately 100 endemic countries. The types of leishmaniasis in humans are the visceral (VL), cutaneous (CL), mucocutaneous (MCL), diffuse cutaneous (DCL), and post kala-azar dermal (PKDL) forms of Leishmaniasis. The aim of this study is to perform phylogenetic analysis of Leishmania origin based on ITS1 gene region using insilico techniques. In this way, it is also aimed to take a snapshot of a meta-analysis of vertical and horizontal spread at the global level. Methods: In this study, Leishmania ITS1 region sequences presented with the GenBank data of the National Center for Biotechnology Information, USA, (NCBI) until 15.05.2019 were taken and analyzed by in-silico techniques. 914 sequences were obtained for the Leishmania ITS1 region in the NCBI database. All sequences were examined and sequences without indel problems were selected from these strains mapped according to the consensus sequence. It was decided to form a phylogenetic tree with the forms that were examined and 65 strains were obtained by removing the sub-branches. Results: The phylogenetic tree obtained in this study showed that Leishmania strains clustered in six branches according to the ITS1 region. Here, a phylogenetic tree is drawn and the molecular epidemiological and demographic data of these six generations and beyond, which are obtained as a result of the genetic relationships between the strains, are summarized. Conclusion: In conclusion, Leishmaniasis is an important public health problem that can be seen in many developing countries. In this study, the strains examined using the in-silico method were isolated from different geographies of the world between 1984 and 2018. The phylogenetic relationships between these strains show not only the vertical spread of the origins over the years, but also the horizontal spread geographically. These species were obtained from different host and tissue types. Thus, the relationships of Leishmania strains in the host-vector-reservoir chain are explained. Therefore, it is clear that there is a need for more meta-analysis studies such as this study on factors and their diffusion. [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]

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Infection with Neospora caninum parasites is a leading cause of reproduction losses in cattle worldwide. In Australia, this loss is estimated to total AU$110 million every year. However, despite this considerable economic impact, the transmission cycle and the host(s) responsible for the sylvatic transmission of the parasite remain to be defined. Dingoes (Canis familiaris) have been suggested to be a wildlife host of N. caninum in Australia, but this is yet to be proven in a nonexperimental setting. This study aimed to determine the prevalence of natural N. caninum shedding in Australian wild dogs (defined as dingoes, dingo‐domestic dog hybrids and feral dogs) by performing molecular analysis of faecal samples collected in wild dog populations in south‐east Australia. Molecular analysis allowed host species identification and dingo purity testing, while genetic analysis of Coccidia and Neospora conserved genes allowed for parasite identification. Among the 115 samples collected and determined to belong to dingoes, dingo‐domestic dog hybrids and foxes, Coccidian parasites were detected in 41 samples and N. caninum was identified in one sample of canine origin from South East Australia (Mansfield). Across all samples collected in Mansfield only 15 individuals were successfully identified by genotype. Thereby our study determined that 6.7% (1/15, 95% confidence intervals 1.2–29.9) of wild dogs were actively shedding N. caninum oocysts at this site. Further, only four individuals were identified at a second site (Swift Creek), and none were positive. This study conclusively confirms the role of wild dogs in the horizontal transmission of N. caninum parasites in Australia. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

Cutaneous leishmaniasis (CL) is caused by Leishmania spp, this disease is a problem in Iraq. The purpose aims of the study is using a Qualitative - Real-time PCR (Q-R.T) to diagnosis compared with traditional methods to investigation of Leishmania species in Baghdad. Total 75 suspected patient with CL from both sex was enrolled in the present work study during the period between March / 2016 to March / 2017 who attended, AL-Yarmook, AL- Karamaa Al-Kadhimiya, Al-Kindi, Teaching Hospitals in Baghdad, all the demographic feature of the diseases, were recorded. The direct Microscopic examination was carried out by direct smears using Giemsa stain, aspiration fluid from lesions were cultured on Novy-MacNeal-Nicolle (NNN) medium and used R T-PCR for detection of internal transcribed spacer1 (ITS1) and Kinetopast (KDNA) genes. The results revealed that 43% of cases were positive by Microscopic examination while 16% by culture and 91% by R.T- PCR positive for kDNA gene , 60% for (ITS1) gene , it was found , 62% were typed as L. tropica and 38% of isolates were typed as L.major . Highly Significant (P<0.01) correlation was observed between diagnosis methods. [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]

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]

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.

Epidemiology and molecular characterization of Eimeria was carried in goats reared under semi-arid region of west Uttar Pradesh, India. A total of 1285 faecal samples from different goat breeds (Jamunapari, Jakhrana and Barbari) were examined for presence of Eimerian oocysts over a period of eight months along with faecal oocysts count. All raw data of faecal oocyst counts (FOC) were transformed by log e (OPG+ 100) before analysis. All fixed effects like breed, age, months of sample collections along with their interaction were considered in analysis. The overall prevalence of Eimeria infection in goats was 73.85%. Breed wise prevalence in Barbari, Jamunapari and Jakhrana breed was 68.62, 79.70 and 72% respectively. Prevalence observed in 2-6M, 6-12M and >12M was as 70.83, 79.88 and 71.74% respectively. Gender wise prevalence as observed in male and female goats was 71.95 and 74.43% respectively. In oocyst per gram (OPG) data analysis the fixed effects like breed, age, months of sample collection and age versus gender interaction had significant effect on log transformed faecal oocysts counts (LFOC). The overall least square means of OPG was 4.673±0.007 (1403OPG). Of the three goat breeds, Jamunapari had highest OPG (2886OPG) compared to Jakhrana (875OPG) and Barbari (523OPG). Mean OPG in 2-6month age goats was significantly higher than the corresponding values in 6-12 and >12months, significant variation was found among monthly OPG means and wet months showed higher faecal oocysts discharge. Nine Eimeria species were identified infecting goats and E. arloingi and E. ninakohlyakimovae were most frequent and predominant species. Molecular characterization for coccidial infection was conducted using two genes i.e. 18S rDNA and ITS-1 genes which amplified 637bp and <500bp (E. ninakohlyakimovae) and >500bp (E. christenseni and E. alijevi) respectively. The ITS1 gene was analysed by sequencing, E. christenseni was found showing nucleotide similarity with E. bovis and E. ellipsoidalis whereas 3' end of the sequence were highly conserved. The ITS1 gene of E. ninakohlyakimovae was found more homologous to E. bovis, E. ellipsoidalis and E. zuernii but for 33rd nucleotide thymidine residue deletion and 5th position G→A mutation. The 18S rDNA sequences of E. ninakohlyakimovae and E. christenseni were studied for evolutionary divergence analysis and maximum divergence was noticed between E. ninakohlyakimovae and E. christenseni (0.0605). The phylogenetic tree showed E. ninakohlyakimovae was placed in same clade with other Eimeria spp. compared, but E. christenseni being placed in a different clade as an out-group., (Copyright © 2017 Elsevier B.V. All rights reserved.)