Trichuris Globulosa Von Linstow, 1901 from one-humped camel (Camelus dromedarius) in Egypt: prevalence, morphological and molecular study.

BACKGROUND: Trichuris spp. (whipworms) are soil-transmitted helminths distributed worldwide, parasitizing several mammalian hosts such as ruminants, primates, and rodents. Trichuris spp. is one of the most common intestinal parasites affecting both humans and animals, and it can spread directly through the fecal-oral route, resulting in severe illness and financial loss. So, this work aims to detect the frequency of Trichuris spp. in camels in Beheira Governorate, Egypt, and to identify Trichuris spp. through morphometrical studies, molecular analysis, and phylogenetic analysis. RESULTS: A total of 35 dromedaries out of 127 investigated had Trichuris spp. infection, meaning that the overall prevalence was 27.56%. The age of the camel affected the infection rate, older animals (> 5 years) having a higher prevalence of infection (24%) than animals of ages (< 3 years) (20%) than animals of ages (3-5 years) (19.14%). According to season: Trichuris spp. showed a unique pattern in camels in different seasons: summer (31.25%) > autumn (28.13%) > spring (25.8%) > winter (25%) indicating year-round infection. T. globulosa was identified morphometrically from camels in Beheira Governorate, Egypt. The BLAST analysis revealed the presence of T. globulosa isolate from camels using the Genbank database depending on nuclear small subunit ribosomal RNA (18s) and cytochrome b (Cytb) genes. CONCLUSION: A high prevalence of T. globulosa was found in camels in Beheira Governorate, Egypt. This is the first report to confirm the identification of T. globulosa from camel based on morphometrical studies and molecular and phylogenetic analysis in Egypt. More thorough studies on the incidence, molecular, and genetic analysis of Trichuris spp. in Egypt are required in addition to camel control programs.

Genetic diversity and structure of mongolian gazelle (Procapra gutturosa) populations in fragmented habitats.

BACKGROUND: The Mongolian gazelle (Procapra gutturosa) population has shown a considerable range of contractions and local extinctions over the last century, owing to habitat fragmentation and poaching. A thorough understanding of the genetic diversity and structure of Mongolian gazelle populations in fragmented habitats is critical for planning effective conservation strategies. RESULT: In this study, we used eight microsatellite loci and mitochondrial cytochrome b (Cytb) to compare the levels of genetic diversity and genetic structure of Mongolian gazelle populations in the Hulun Lake National Nature Reserve (HLH) with those in the China-Mongolia border area (BJ). The results showed that the nucleotide diversity and observed heterozygosity of the HLH population were lower than those of the BJ population. Moreover, the HLH and BJ populations showed genetic differentiation. We concluded that the HLH population had lower genetic diversity and a distinct genetic structure compared with the BJ population. CONCLUSION: The genetic diversity of fragmented Mongolian gazelle populations, can be improved by protecting these populations while reinforcing their gene exchange with other populations. For example, attempts can be made to introduce new individuals with higher genetic diversity from other populations to reduce inbreeding.

Leucocytozoon cariamae n. sp. and Haemoproteus pulcher coinfection in Cariama cristata (Aves: Cariamiformes): first mitochondrial genome analysis and morphological description of a leucocytozoid in Brazil.

The distribution of avian haemosporidians of the genus Leucocytozoon in the Neotropics remains poorly understood. Recent studies confirmed their presence in the region using molecular techniques alone, but evidence for gametocytes and data on putative competent hosts for Leucocytozoon are still lacking outside highland areas. We combined morphological and molecular data to characterize a new Leucocytozoon species infecting a non-migratory red-legged seriema (Cariama cristata), the first report of a competent host for Leucocytozoon in Brazil. Leucocytozoon cariamae n. sp. is distinguished from the Leucocytozoon fringillinarum group by its microgametocytes that are not strongly appressed to the host cell nucleus. The bird studied was coinfected with Haemoproteus pulcher, and we present a Bayesian phylogenetic analysis based on nearly complete mitochondrial genomes of these 2 parasites. Leucocytozoon cariamae n. sp. morphology is consistent with our phylogenetic analysis indicating that it does not share a recent common ancestor with the L. fringillinarum group. Haemoproteus pulcher and Haemoproteus catharti form a monophyletic group with Haemocystidium parasites of Reptilia, supporting the polyphyly of the genus Haemoproteus. We also discussed the hypothesis that H. pulcher and H. catharti may be avian Haemocystidium, highlighting the need to study non-passerine parasites to untangle the systematics of Haemosporida.

The ND1 and CYTB genes polymorphisms associated with in vitro early embryo development of Sanjabi sheep.

This study aimed to investigate the association between polymorphisms of ND1 and CYTB genes and in vitro early embryo development of Sanjabi sheep. Blood and ovarian samples were collected from a local slaughterhouse. The cumulus-oocyte complexes with a diameter greater than 3 mm were aspirated from follicles, and in vitro maturation (IVM) and in vitro culture (IVC) rates of them were recorded. A respective 1200 bp and 980 bp fragments of ND1 and CYTB genes were genotyped using a modified single strand conformation polymorphism (SSCP) method. The results of this study revealed that four different patterns, named as A, B, C, and D were observed for both ND1 and CYTB genes. The ND1 gene polymorphisms had significant effects on the IVM and IVC rate (p < 0.05). The pattern C of the ND1 gene significantly increased the IVM rate compared to the patterns A, B and D. For the IVC, the highest and lowest means were related to the C and B patterns, respectively. The CYTB gene polymorphisms also had significant effects on IVC (p < 0.01), but the IVM did not affected (p = 0.07). Here, the pattern D had the highest and the pattern C had the lowest means for both IVM and IVC rates.

Molecular investigation of Haemoproteus and Plasmodium species of some raptors in Hatay province: new CytB lineages for raptors of Accipitriformes in Turkey.

Accipitriform raptors are significant indicators of biodiversity and environmental health. Currently, most of the studies on avian haemosporidian parasites are on passerine birds, and data on raptors is constricted, with similarities both around the world and in Turkey. This study aimed to investigate the presence of Haemoproteus and Plasmodium spp. in raptors by microscopy and nested PCR technique. The study material consisted of 47 accipitriform raptors (Buteo buteo: 14, Buteo rufinus: 7, Clanga pomarina: 8, Circaetus gallicus: 12, Milvus migrans: 6). The prevalence of haemosporidian infection was 12.8% (6/47, 1 from Buteo buteo, 4 from Clanga pomarina, 1 from Milvus migrans) microscopically and 14.9% (7/47) molecularly. One Circaetus gallicus, microscopically found to be negative, probably due to low parasitemia, was molecularly found to be positive. All PCR-positive amplicons were bidirectionally sequenced, and the identification of lineages of the isolates and phylogenetic analysis were performed using the MalAvi and GenBank databases. The study revealed H-MILANS02 lineage in Buteo buteo, H-MILANS02 and P-CIAE1 lineages in Clanga pomarina, P-GRW06 lineage in Circaetus gallicus, and P-RTSR1 lineage in Milvus migrans, respectively. While this study removes the uncertainty regarding the reporting of the H-MILANS02 lineage in Turkey, it is also the first report to reveal 3 different Plasmodium spp. CytB lineages in raptors. Moreover, the fact that the P-GRW06 lineage (Plasmodium elongatum) detected in passerine birds was detected in a raptor, Circaetus gallicus, draws attention to the need for further investigations on host-parasite interaction and gives clues about the host-shifting ability of this parasite.

Mitochondrial genome amplification of avian haemosporidian parasites from single-infected wildlife samples using a novel nested PCR approach.

Haemosporidian parasites that infect birds (Apicomplexa: Haemosporida) are blood parasites that require an invertebrate host (vector) and a vertebrate host for their lifecycle and cause malaria-like diseases. This group of parasites has provided valuable insights into host specificity, virulence, and parasite dispersal. Additionally, they have played a significant role in reshaping our understanding of the evolutionary history of apicomplexans. In order to accurately identify species and to address phylogenetic questions such as the timing of the haemosporidian radiation, the use of a sufficiently large genetic data set is crucial. However, acquiring this genetic data poses significant challenges. In this research, a sensitive nested PCR assay was developed. This assay allows for the easy amplification of complete mitochondrial genomes of haemosporidian parasites in birds, even during the chronic stage of infection. The effectiveness of this new nested PCR assay was evaluated using blood and tissue samples of birds with verified single parasite infections from previous studies. The approach involves amplifying four overlapping fragments of the mitochondrial genome and requires DNA extracts from single-infected samples. This method successfully amplified the complete mitochondrial genomes of 24 distinct haemosporidian parasite lineages found in various bird species. This data is invaluable for conducting phylogenetic analyses and accurately defining species. Furthermore, this study proposes the existence of at least 15 new haemosporidian parasite species based on the genetic information obtained. Data regarding pGRW04, previously categorized as Plasmodium relictum like pSGS1 and pGRW11, indicates that the pGRW04 lineage is actually a separate, hidden Plasmodium species.

Species Identification and Fungicide Sensitivity of Fungi Causing Alternaria Leaf Blight and Head Rot in Cole Crops in the Eastern United States.

Alternaria leaf blight and head rot is an important disease of broccoli and other cole crops. With no resistant host varieties, fungicides are utilized to manage this disease. However, anecdotal evidence suggests that, in southeastern U.S. broccoli-producing states, there is a loss of disease control through the use of quinone outside inhibitor (QoI) fungicides. To understand why there is a reduced sensitivity to QoI fungicides in these states, we isolated Alternaria spp. from symptomatic lesions on cole crops from Georgia and Virginia (two states with observations of loss of fungicide sensitivity) as well as New York (a state with no observations of loss of fungicide sensitivity). Using multilocus sequencing and phylogenetic analysis, we identified two species, Alternaria brassicicola and A. japonica. Whereas A. brassicicola was isolated in all states, A. japonica was only isolated in Georgia. Next, we wanted to determine the sensitivity of these isolates to azoxystrobin-an active ingredient in some QoI fungicides-by estimating the effective concentration at which only 50% of spores germinate (EC50). The EC50 of A. brassicicola ranged from 0.01 to 0.17 ppm, whereas that of A. japonica was 8.1 to 28.1 ppm. None of the known target-site mutations that confer resistance to QoI fungicides were identified during screening of either species. A. japonica was first reported on the east coast of the United States in 2020 in South Carolina. The substantially higher EC50 value suggests that its emergence in the southeastern United States may play at least a part in the observed loss of disease control. However, further in planta and field studies are needed to thoroughly test this hypothesis.

Metabolic selection of a homologous recombination-mediated gene loss protects Trypanosoma brucei from ROS production by glycosomal fumarate reductase.

The genome of trypanosomatids rearranges by using repeated sequences as platforms for amplification or deletion of genomic segments. These stochastic recombination events have a direct impact on gene dosage and foster the selection of adaptive traits in response to environmental pressure. We provide here such an example by showing that the phosphoenolpyruvate carboxykinase (PEPCK) gene knockout (Δpepck) leads to the selection of a deletion event between two tandemly arranged fumarate reductase (FRDg and FRDm2) genes to produce a chimeric FRDg-m2 gene in the Δpepck∗ cell line. FRDg is expressed in peroxisome-related organelles, named glycosomes, expression of FRDm2 has not been detected to date, and FRDg-m2 is nonfunctional and cytosolic. Re-expression of FRDg significantly impaired growth of the Δpepck∗ cells, but FRD enzyme activity was not required for this negative effect. Instead, glycosomal localization as well as the covalent flavinylation motif of FRD is required to confer growth retardation and intracellular accumulation of reactive oxygen species (ROS). The data suggest that FRDg, similar to Escherichia coli FRD, can generate ROS in a flavin-dependent process by transfer of electrons from NADH to molecular oxygen instead of fumarate when the latter is unavailable, as in the Δpepck background. Hence, growth retardation is interpreted as a consequence of increased production of ROS, and rearrangement of the FRD locus liberates Δpepck∗ cells from this obstacle. Interestingly, intracellular production of ROS has been shown to be required to complete the parasitic cycle in the insect vector, suggesting that FRDg may play a role in this process.

Assessing the population structure of trematode Metagonimus suifunensis using three mitochondrial markers.

In this work, for the first time, the genetic variability of the Metagonimus suifunensis population in the Russian southern Far East was estimated based on the full-length sequences of the nad1 gene of mitochondrial DNA. In addition, for a sample of the same size, the sequences of cox1 and cytb genes, previously used for population studies for M. suifunensis, were reanalysed. Three markers were combined to a common sequence, and the obtained data were studied. Despite the higher level of variability, nad1 and cox1 mtDNA genes did not reveal subdivisions within the population. The combined dataset made it possible to determine that the sample from the Odyr River was the centre of the species' range formation and clarified the continental migration route of the parasite from south to north. According to the data obtained, it was presumed that piscivorous birds participate in the life cycle of the parasite. The subdivision within population revealed that using all three mitochondrial markers is consistent with the features of differentiation within populations of related species, but the reasons for its formation remain unclear due to the insufficient amount of data and the use of different markers in studies of different species.

Isolation and molecular characterization of Polychromophilus spp. (Haemosporida: Plasmodiidae) from the Asian long-fingered bat (Miniopterus fuliginosus) and Japanese large-footed bat (Myotis macrodactylus) in Japan.

Bats (order, Chiroptera) account for more than one-fifth of all mammalian species in the world and are infected by various intra-erythrocytic parasites of the family Plasmodiidae (Apicomplexa: Haemosporida), including Polychromophilus Dionisi, 1899. Recent advance in the molecular characterization of haemosporidian isolates has enabled their accurate identification, particularly in the last decade. Studies are actively conducted in tropical regions, Europe, and Australia; however, data on haemosporidian infection in bats in Asian temperate areas, including Japan, remain limited. In this study, 75 bats of 4 species (Miniopterus fuliginosus, Myotis macrodactylus, Rhinolophus nippon, and Rhinolophus cornutus) were captured at three sites in western Japan (Yamaguchi Prefecture), and haemosporidian parasites were screened microscopically and molecularly via nested polymerase chain reaction (PCR) targeting the cytochrome b (cytb), cytochrome c oxidase subunit I (cox-1), apicoplast caseinolytic protease C (clpc), and nuclear elongation factor 2 (EF2) genes. The survey detected Polychromophilus melanipherus in 15 (40.5%) miniopterid bats (M. fuliginosus) and Polychromophilus murinus in 6 (46.2%) vespertilionid bats (M. macrodactylus), whereas none of the 25 rhinolophid bats (R. nippon and R. cornutus) was infected, indicating the robust host specificity for miniopterid (P. melanipherus) and vespertilionid (P. murinus) bats regardless of orthotopic nesting. The 15 Polychromophilus cytb sequences obtained from 11 miniopterid and 4 vespertilionid bats were classified into six cytb haplotypes (three for each species), showing no region-specific variation in a phylogenetic tree of Polychromophilus isolates in the Old World. Similarly, multiple haplotypes (seven for cox-1 and nine for clpc) and genotypes (three for EF2) were characterized for the Japanese isolates of Polychromophilus, and the results were consistent with those based on a haemosporidian cytb analysis. Bat flies (Nycteribia allotopa and another undetermined Nycteribia sp.) collected from the body surface of bats harbored Polychromophilus oocysts on the external surface of the midgut. This is the first study to report the isolation and molecular characterization of Polychromophilus spp. in miniopterid and vespertilionid bats in the temperate area of Asia (western Japan). Future studies should evaluate the global prevalence of haemosporidian infections in bats.

Comparative analysis of the mitochondrial proteins reveals complex structural and functional relationships in Fasciola species.

Mitochondria is a cellular source of energy, appears to play an essential role in dealing with cellular stress induced by environmental stimuli. The genetic diversity of mitochondrial genes involved in oxidative phosphorylation affecting the production of cellular energy and regional adaptation to various ecological (climatic) pressures affecting amino acid sequences (variants of protein). However, little is known about the combined effect of protein changes on cell-level metabolic alterations in simultaneous exposure to various environmental conditions, including mitochondrial dysfunction and oxidative stress induction. The present study was designed to address this issue by analyzing the mitochondrial proteins in Fasciola species including Cytochrome oxidase (COX1, COX2, COX3, and CYTB) and NADH dehydrogenase (ND1, ND2, ND3, ND4, ND5, and ND6). Mitochondrial proteins were used for detailed computational investigation, using available standard bioinformatics tools to exploit structural and functional relationships. These proteins in Fasciola hepatica, Fasciola gigentica, and Fasciola jacksoni were functionally annotated using public databases. The results showed that the protein of COX1 of F. hepatica, F. gigantica, and F. jacksoni consist of 510, 513, and 517 amino acids, respectively. The alignment of proteins showed that these proteins are conserved in the same regions at ten positions in COX and CYTB proteins while at twelve locations in NADH. Three-dimensional structure of COX, CYTB, and NADH proteins were compared and showed differences in additional conserved and binding sites in COX and CYTB proteins as compared to NADH in three species of Fasciola. These results based on the amino acid diversity pattern were used to identify sites in the enzyme and the variations in mitochondrial proteins among Fasciola species. Our study provides valuable information for future experimental studies, including identification of therapeutic, diagnostic, and immunoprophylactic interests with novel mitochondrial proteins.

KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum.

BACKGROUND: The emergence and spread of anti-malarial resistance continues to hinder malaria control. Plasmodium falciparum, the species that causes most human malaria cases and most deaths, has shown resistance to almost all known anti-malarials. This anti-malarial resistance arises from the development and subsequent expansion of Single Nucleotide Polymorphisms (SNPs) in specific parasite genes. A quick and cheap tool for the detection of drug resistance can be crucial and very useful for use in hospitals and in malaria control programmes. It has been demonstrated in different contexts that genotyping by Kompetitive Allele Specific PCR (KASP), is a simple, fast and economical method that allows a high-precision biallelic characterization of SNPs, hence its possible utility in the study of resistance in P. falciparum. METHODS: Three SNPs involved in most cases of resistance to the most widespread anti-malarial treatments have been analysed by PCR plus sequencing and by KASP (C580Y of the Kelch13 gene, Y86N of the Pfmdr1 gene and M133I of the Pfcytb gene). A total of 113 P. falciparum positive samples and 24 negative samples, previously analysed by PCR and sequencing, were selected for this assay. Likewise, the samples were genotyped for the MSP-1 and MSP-2 genes, and the Multiplicity of Infection (MOI) and parasitaemia were measured to observe their possible influence on the KASP method. RESULTS: The KASP results showed the same expected mutations and wild type genotypes as the reference method, with few exceptions that correlated with very low parasitaemia samples. In addition, two cases of heterozygotes that had not been detected by sequencing were found. No correlation was found between the MOI or parasitaemia and the KASP values of the sample. The reproducibility of the technique shows no oscillations between repetitions in any of the three SNPs analysed. CONCLUSIONS: The KASP assays developed in this study were efficient and versatile for the determination of the Plasmodium genotypes related to resistance. The method is simple, fast, reproducible with low cost in personnel, material and equipment and scalable, being able to core KASP arrays, including numerous SNPs, to complete the main pattern of mutations associated to P. falciparum resistance.

Plasmodium ouropretensis, n. sp., a new case of non-erythrocytic species within lizard malaria parasites.

Delimiting and describing Plasmodium species in reptiles remains a pressing problem in Haemosporida taxonomy. The few morphological characters used can overlap, and the significance of some life-history traits is not fully understood. Morphologically identical lizard Plasmodium forms have been reported infecting different cell types (red and white blood cells) in the same host and have been considered the same species. An example is Plasmodium tropiduri tropiduri, a species known to infect erythrocytes, thrombocytes and lymphocyte-like cells. Here, both forms of P. t. tropiduri were analysed using light microscope-based morphological characteristics and phylogenetic inferences based on almost complete mitochondrial genomes of parasites naturally infecting lizards in southeastern Brazil. Although morphologically similar, two distinct phylogenetic lineages infecting erythrocytes and non-erythrocytic cells were found. The lineage found in the erythrocytes forms a monophyletic group with species from Colombia. However, the non-erythrocytic lineage shares a recent common ancestor with Plasmodium leucocytica, which infects leucocytes in lizards from the Caribbean islands. Here, Plasmodium ouropretensis n. sp. is described as a species that infects thrombocytes and lymphocyte-like cells.

Identification of Leishmania infantum in blood donors from endemic regions for visceral leishmaniasis.

Visceral leishmaniasis is an endemic protozoonosis observed in over 60 countries, with over 500 000 new cases recorded annually. Although the diagnostic procedure of its symptomatic forms is well established, for asymptomatic patients, who represent about 85% of those infected, there is no consensus on the best method for its identification. Recent studies have presented molecular techniques as viable identification methods, with good sensitivity and specificity indices in asymptomatic individuals. Therefore, we aimed to use molecular methods to assess their effectiveness in identifying the presence of asymptomatic infection by Leishmania infantum (L. infantum) individuals from endemic regions of Brazil. Screening was performed by real-time polymerase chain reaction (qPCR) and confirmed by sequencing the cytochrome B gene. Of the 127 samples [from 608 blood donors who had participated in a previous study, of which 34 were positive by the enzyme-linked immunosorbent assay (ELISA) rK39] tested by qPCR, 31 (24.4%) were positive. In the sequencing of 10 qPCR-positive samples, five were identified as L. infantum. Complimentary samples of the ELISA rK39 and conventional PCR showed only reasonable and low agreement with qPCR, respectively. The qPCR confirmed the presence of infection in five of the 10 sequenced samples, ELISA confirmed three, and the conventional PCR confirmed none.

Haplotype comparisons of Echinococcus granulosus sensu lato via mitochondrial gene sequences (co1, cytb, nadh1) among Pakistan and its neighbouring countries.

Echinococcus granulosus sensu lato (s.l.) is a zoonotic parasite that causes cystic echinococcosis (CE) in humans. However, E. granulosus sensu stricto (s.s.) is considered the predominant species in CE infections worldwide. According to the population genetic diversity and structure of E. granulosus s.l., gene flow can explain the parasite drift among the neighbouring countries of Pakistan. The mitochondrial (mt) co1 (n = 47), nadh1 (n = 37) and cytb (n = 35) nucleotide sequences of E. granulosus s.l. isolates from Pakistan, Iran, China and India were retrieved from the National Centre for Biotechnology Information database to determine the genealogical relationships. The sequences were grouped as the mt-co1 (genotypes G1 and G3, G6-G7), mt-cytb (genotypes G1 and G3), and mt-nadh1(genotypes G1 and G3). The data were analysed using bioinformatic tools. A total of 19 polymorphic sites for the mt-co1 sequence (374 bp) were observed of which 31.6% (6/19) were parsimony-informative sites. Unique singleton haplotypes within the E. granulosus s.s. haplotype network based on the mt-co1 gene were highly prevalent (68.4%; 13/19) in Pakistani isolates followed by Chinese, Indian and Iranian isolates; four polymorphic sites were detected in the E. canadensis (G6/G7). In E. canadensis mt-co1 haplotype network, 75% (3/4) unique singleton haplotypes were from the Iranian isolates. Twelve polymorphic sites were found using the mt-cytb sequence (547 bp); 25% (3/12) were parsimony-informative and there were 66.7% (8/12) unique singleton haplotypes within the mt-cytb haplotype network in E. granulosus s.s. with the most reported from Pakistan followed by Iran and China. 20 polymorphic sites were detected in E. granulosus s.s. mt-nadh1 sequences (743 bp); 20% (4/20) were parsimony-informative. There were 66.7% (8/12) main single haplotypes within the mt-nadh1 haplotype network, with the most reported from Pakistan followed by that from India, Iran and China. The sequence analyses show low nucleotide diversity and high haplotype diversity in general.

Cytochrome b as a more promising marker for analysing the distribution vector for Metagonimus suifunensis (Trematoda: Heterophyidae).

In this study of Metagonimus suifunensis (M. suifunensis) in the Russian Southern Far East, the variability of the full-length sequences of the cytochrome b (cytb) mtDNA gene was assessed for the first time. In addition, the cox1 mtDNA gene sequences were also obtained for this species from new localities. In total, 87 and 81 sequences of the cytb and cox1 genes, respectively, were used in the current study. The cytb gene proved more promising and revealed two haplogroups that are associated with the spatial distribution of the species: geographical isolation caused the fixation of differences between northern and southern populations. In addition, the results obtained for the cytb gene opened up new perspectives in the analysis of sequences of the cox1 gene, which was not sufficiently effective as a sole marker. Based on data for both mitochondrial genes, molecular processes influencing the formation of the modern population were analysed for M. suifunensis. The new data confirmed the previously expressed opinion that this species colonized the study territory from north to south and will form the basis for determining possible ways of its further expansion, which is important for predicting the emergence of new foci of metagonimosis.

Molecular Phylogeny of the Subgenus Karstomys Reveals Genetic Signature of Post-Glacial Colonization of Apodemus mystacinus (Rodentia: Muridae) in the Zagros Mountains from Different Refugia.

Eastern broad-toothed field mouse, Apodemus mystacinus, is a rocky habitat dwelling rodent distributed in Asia Minor, the Levant, the Caucasus, and the Zagros Mountains. In this study, we investigated the phylogenetic relationship between different populations of A. mystacinus throughout its range, based on the mitochondrial cytb marker. Phylogenetic analyses revealed the existence of five separately evolving lineages within A. mystacinus, of which two previously unrecognized lineages were identified in the Zagros Mountains and the Levant. Divergence between two major clades of the subgenus Karstomys, corresponding to A. mystacinus and Apodemus epimelas, is inferred to coincide with the Messinian Salinity Crisis (Late Miocene), whereas the splits between major lineages of A. mystacinus are inferred to have occurred during the Pleistocene. Colonization of the Zagros may have occurred from different refugia via eastward migration of the Turkish population and then again by a more recent colonization from the Caucasus, after reopening of the land corridor between the Caucasus and the Zagros Mountains during the Holocene drought.

Apparent density, trypanosome infection rates and host preference of tsetse flies in the sleeping sickness endemic focus of northwestern Uganda.

BACKGROUND: African trypanosomiasis, caused by protozoa of the genus Trypanosoma and transmitted by the tsetse fly, is a serious parasitic disease of humans and animals. Reliable data on the vector distribution, feeding preference and the trypanosome species they carry is pertinent to planning sustainable control strategies. METHODOLOGY: We deployed 109 biconical traps in 10 villages in two districts of northwestern Uganda to obtain information on the apparent density, trypanosome infection status and blood meal sources of tsetse flies. A subset (272) of the collected samples was analyzed for detection of trypanosomes species and sub-species using a nested PCR protocol based on primers amplifying the Internal Transcribed Spacer (ITS) region of ribosomal DNA. 34 blood-engorged adult tsetse midguts were analyzed for blood meal sources by sequencing of the mitochondrial cytochrome c oxidase 1 (COI) and cytochrome b (cytb) genes. RESULTS: We captured a total of 622 Glossina fuscipes fuscipes tsetse flies (269 males and 353 females) in the two districts with apparent density (AD) ranging from 0.6 to 3.7 flies/trap/day (FTD). 10.7% (29/272) of the flies were infected with one or more trypanosome species. Infection rate was not significantly associated with district of origin (Generalized linear model (GLM), χ2 = 0.018, P = 0.895, df = 1, n = 272) and sex of the fly (χ2 = 1.723, P = 0.189, df = 1, n = 272). However, trypanosome infection was highly significantly associated with the fly's age based on wing fray category (χ2 = 22.374, P < 0.001, df = 1, n = 272), being higher among the very old than the young tsetse. Nested PCR revealed several species of trypanosomes: T. vivax (6.62%), T. congolense (2.57%), T. brucei and T. simiae each at 0.73%. Blood meal analyses revealed five principal vertebrate hosts, namely, cattle (Bos taurus), humans (Homo sapiens), Nile monitor lizard (Varanus niloticus), African mud turtle (Pelusios chapini) and the African Savanna elephant (Loxodonta africana). CONCLUSION: We found an infection rate of 10.8% in the tsetse sampled, with all infections attributed to trypanosome species that are causative agents for AAT. However, more verification of this finding using large-scale passive and active screening of human and tsetse samples should be done. Cattle and humans appear to be the most important tsetse hosts in the region and should be considered in the design of control interventions.

[Identification of Chinese medicine sea snake based on cytochrome B (Cytb)].

The identification of species primordium has been one of the hot issues in the identification of traditional Chinese medicine. Sea snake is one of the most valuable Chinese medicinal materials in China. In order to understand the origin and varieties of sea snake in the market, we studied the molecular identification of 46 sea snakes by cytochrome B(Cytb). After comparison and manual correction, the sequence length was 582 bp, and the content of A+T(58.9%) was higher than that of G+C(41.1%). There exist 197 variable sites and 179 parsimony-informative sites of the sequence. There are 44 kinds of sequence alignment with consistency equal to 100%, and 2 kinds equal to 96%. A total of 408 Cytb effective sequences were downloaded from GenBank database, with a total of 68 species. Phylogenetic tree of a total of 454 sea snake sequences with the samples in this study were constructed by neighbor-joining trees and Bayesian inference method, respectively, which can identify 42 samples of medicinal materials, while 4 samples can not be identified because of their low node support. The results showed that the species of the sea snake medicine were at least from 2 genera and 5 species, namely, Aipysurus eydouxii, Hydrophis curtus, H. caerulescen, H. curtus, H. ornatus and H. spiralis. This study suggested that the original species of commercial sea snake are very complex and can provide insight into the identification of sea snakes.

Authentication of camel meat using species-specific PCR and PCR-RFLP.

In India and some of the African countries, one of the unconventional meats receiving the latest attention in meat adulteration is camel meat. So, the objective of this study was to develop a species-specific PCR based on mitochondrial cytochrome b (CYTB) gene and a PCR-RFLP assay of mitochondrial 12S rRNA to identify camel meat in suspected samples. Known sample of camel meat, samples suspected to be from illegally slaughtered camel and known samples of cattle, buffalo, sheep, goat, pork and chicken were used in the study. DNA were extracted from all samples following spin column method and PCR amplification were carried out using both CYTB and 12S rRNA gene primers. The CYTB gene amplification produced amplicon with a size of 435 bp without any non-specific spurious amplification towards other species studied. Further, the 12S rRNA PCR products were analysed both by sequencing and by RFLP using enzyme AluI. On BLAST analysis the 448 bp sequence obtained from suspected samples showed > 99% sequence homology to previously reported Camelus dromedaries (accession no: AM 9369251.1). On AluI digestion of the 448 bp product from both known and suspected camel samples, a specific RFLP pattern with three distinct products of 90, 148 and 210 bp size were evident, which were significantly different from the pattern of cattle, sheep, goat, chicken and buffalo. Further, after in-house validation, this cost effective and rapid method of camel meat identification is placed into practice for regular screening of vetero-legal samples in the lab.