Sex Identification in Cattle, based on Amelogenin Gene

Sex identification is considered an important step in forensic sciences, wildlife and livestock breeding management. In the current experiment we used Amelogenin gene as a biological marker for polymerase chain reaction test to identify the sex of cattle from blood remnants, collected at slaughter house. Due to the conserved region of the gene on both sex chromosomes (X and Y) a single primers pair was employed to amplify the gene in a single polymerase chain reaction. In case of band patterns, a 178 base pair fragment for AMELY and a 241 base pair fragment for AMELX genes were produced. The primer competence and exactness was initially checked on known gender cattle samples and then applied to unknown cattle samples for the validation of the experiment. PCR amplicons of unknown gender showed only one band (241-bp) for female DNA and two bands (241-bp, 178-bp) for male DNA, on the platform of agarose gel upon electrophoresis. Our findings showed that the PCR protocol based on AMELX or Y gene is a reliable technique for the identification of cattle sex.

Prevalence and Genetic Diversity of Avian Haemosporidian Parasites in Southern Iran

Avian haemosporidians are widespread and diverse and are classified in the genera Plasmodium, Haemoproteus, Leucocytozoon, and Fallisia. These species are known to cause haemosporidiosis and decreased fitness of their hosts. Despite the high diversity of habitats and animal species in Iran, only few studies have addressed avian haemosporidians in this geographic area. This study was performed in the south and southeast of Iran during the bird breeding seasons in 2017 and 2018, with the aim to partly fill in this gap. Blood samples of 237 passerine birds belonging to 41 species and 20 families were collected. Parasite infections were identified using a nested PCR protocol targeting a 479-base-pair fragment of the mitochondrial cytochrome b (cytb) gene of Haemoproteus, Plasmodium and Leucocytozoon species. The overall prevalence of haemosporidian parasites was 51.1%, and 55 different lineages were identified, of which 15 cytb lineages were new globally. The lineages of Haemoproteus predominated (63.6% of all detected lineages), followed by Leucocytozoon and Plasmodium. Nineteen new host records of haemosporidian cytb lineages were identified, and the majority of them were found in resident bird species, indicating local transmission. Thirteen co-infections (9.8% of infected individuals) of Haemoproteus and Leucocytozoon parasites in seven host species were observed. This study shows the presence of active local transmission of parasites to resident bird species in the southeast of Iran and contributes to the knowledge on haemosporidian parasite biodiversity in this poorly studied region of the world.

PROFIL GEN GROWTH HORMONE (GH) SAPI HASIL PERSILANGAN MADURA DAN LIMOUSIN DENGAN METODE PCR-RFLP

The purpose of this research was to find out the Growth Hormone (GH) gene profile of the cross breeding between Madura cattle and Limousin cattle (Madrasin). Sampl in the form of cattle blood for this research was obtained from 14 Madrasin cattles in the area of Bangkalan, Madura, East Java. DNA extraction was performed then to provide the result for PCR RFLP, which then indicated that Madrasin cattle’s GH gene profile has 432 base pair fragment length and the RFLP result indicated that Madrasin cattle’s GH genetic was cut off into 180 base pair, 250 base pair, 300 base pair, and 400 base pair. Moreover, there was no V genetic to be found in GH genetic of Madura cattle.

Sequence Analysis of the K13-Propeller Gene in Artemisinin Challenging Plasmodium falciparum Isolates from Malaria Endemic Areas of Odisha, India: A Molecular Surveillance Study

Estimation of the spread and advancement of Plasmodium falciparum artemisinin-resistant parasites can be done by probing polymorphisms in the kelch (Pfk13) domain (a validated molecular marker). This study aimed to provide baseline information for future artemisinin surveillance by analyzing the k13-propeller domain in P. falciparum field isolates collected from 24 study areas in 14 malaria hot spots of Odisha (previously Orissa) during July 2018-January 2019. A total of 178 P. falciparum mono infections were assessed. An 849-base pair fragment encoding the Pfk13 propeller was amplified by nested polymerase chain reaction and sequenced in both directions (PCR). After DNA alignment with the 3D7 reference sequence, all samples were found to be wild type. It can be anticipated that malaria public health is not under direct threat in Odisha relating to ART resistance.

Translation elongation factor 1-alpha gene as a marker for diagnosing of Candida onychomycosis

Background and Purpose: Culture-based identification methods have been the gold standard for the diagnosis of candidal onychomycosis. Molecular technologies, such as polymerase chain reaction (PCR) assays, can provide an alternative for the rapid detection of Candida species. The present study was conducted to investigate a pan-Candida PCR assay based on the translation elongation factor 1-alpha (TEF-1α) gene for the detection of the most prevalent pathogenic Candida species. Materials and Methods: For the purpose of the study, an optimized pan-Candida PCR primer pair was designed, and the target was amplified and sequenced. The analytical and clinical diagnostic performance of the designed primers was tested using 17 reference strains, 137 nail scrapings suspected of onychomycosis, and 10 healthy nail specimens. Results: The use of the universal pan-Candida primers designed on TEF-1α gene resulted in the successful amplification of a 270-base pair fragment in all Candida species tested, except for C. glabrata, and reacted neither with other fungi nor with E. coli. The sequence difference count matrix showed poor insertion/deletion differences (0-2 nt) among Candida species. Among 137 nail specimens, 35% (n=48), 30.7% (n=42), and 40.1% (n=55) of the samples were found to be positive by direct microscopy, culture, and pan-Candida PCR, respectively. Conclusion: Based on the findings, the PCR-based detection targeting the DNA TEF-1α gene is a rapid and simple procedure for the diagnosis of candidal onychomycosis directly from nail sample.

Taxonomic status of the nominal forms assigned to Necromys lactens (Rodentia, Cricetidae) as revealed by molecular and morphometric evidence

Necromys is a genus of sigmodontine rodent that inhabits grasslands and scrublands in South America. Eight extant species are recognized in the genus; one of these is Necromys lactens, which inhabits high-elevation grasslands in the Yungas from south-central Bolivia to northwestern Argentina. Morphological variation in N. lactens has been recognized by the description of three nominal forms. Geographically structured genetic diversity also has been observed, but a thorough revision of these nominal forms within an integrative framework has yet to be performed. We conducted a phylogeographic assessment based on an 801 base-pair fragment of the cytochrome-b gene that guided morphometric analyses (univariate and multivariate comparisons) of patterns of geographic variation in the species, and the distinction of its nominal forms. Haplotypes of N. lactens form a well-supported and geographically structured clade. Within it, there are two main clades; haplotypes from the northern range form a well-supported clade, sister and allopatric to a weakly supported southern clade, which includes variants collected at or near the type localities of three nominal forms. In turn, both main clades are composed by two allopatric subclades. Morphometric analyses indicated no differences in shape of the skull among the three nominal forms or between the recovered clades and subclades. Taking together all the available evidence, we consider N. lactens to be a monotypic species. Necromys es un género de roedor sigmodontino que habita los pastizales y arbustales de América del Sur. Se reconocen ocho especies actualmente en existencia en el género; una de ellas, Necromys lactens, habita pastizales de altura en las Yungas, desde el centro-sur de Bolivia hasta el noroeste de Argentina. Se ha reconocido variación morfológica en N. lactens con base en descripciones de tres formas nominales; también se ha observado diversidad genética geográficamente estructurada, pero una revisión exhaustiva de esas formas nominales dentro de un esquema integrativo aún no se ha llevado a cabo. Realizamos una evaluación filogeográfica basada en un fragmento de 801 pares de bases del gen citocromo b que orientó análisis morfométricos (comparaciones univariadas y multivariadas) respecto al patrón geográfico de variación de la especie y la distinción de sus formas nominales. Los haplotipos de N. lactens forman un clado bien apoyado y geográficamente estructurado. Dentro de este clado, los haplotipos del norte de su área de distribución forman un clado bien apoyado que es hermano y alopátrico con respecto de un clado austral débilmente apoyado, el cual incluye variantes colectadas en las localidades tipo de las tres formas nominales o sus cercanías. A su vez, ambos clados principales están compuestos por dos sub-clados alopátricos. Los análisis morfométricos no revelaron diferencias en la forma del cráneo entre las tres formas nominales ni entre los clados y sub-clados recuperados. Teniendo en cuenta toda la evidencia disponible, consideramos que N. lactens es una especie monotípica.

Molecular versus morphological approaches to diet analysis of the caracal (Caracal caracal)

Diet analysis is an essential part in understanding the biology of a species and functioning of ecosystems. Traditional morphological identification of undigested remains in the scats and molecular analyses of prey species’ DNA have previously been used to assess diet. In the present study, caracal diet in the Abbasabad Wildlife Refuge, Central Iran, was investigated using both molecular and morphological methods. We collected 22 scat samples from caracal dens in the region. Feces were washed on sieves and their remaining components were morphologically identified. We also targeted a 307-base pair fragment of the cytochrome b gene to amplify and sequence the species’ DNA. Morphological analyses revealed that 76% of the diet comprised rodent species. We identified a total of nine prey taxa using the molecular method, including six rodents, one hare, one hedgehog and one wild goat. There was a general agreement between the molecular and morphological results; however, molecular methods tended to allow a better identification of the prey species. Therefore, the DNA-based approach acts as a valuable complement to current morphological methods in the study of a rare felid’s diet when no hair reference library exists.

THE PREVALENCE OF OCCULT HEPATITIS B AMONG HBSAG-NEGATIVE PERSONS WITH HIV IN VELIKY NOVGOROD

Aim: to estimate the prevalence of the occult hepatitis B virus among HIV-infected patients with the virological ineffectiveness of antiretroviral therapy in Veliky Novgorod. Materials and methods. Blood plasma samples from 76 HBsAg-negative HIVinfected patients with virological inefficiency of antiretroviral therapy from Veliky Novgorod were used in the work. For the detection of the hepatitis B virus, nucleic acids were isolated using the commercial kit AmplePrime Ribo-prep. For amplification and sequencing, overlapping pairs of specific primers were used, jointly flanking a 1475 base pair fragment including the 1169 base pair Pre-S1/Pre-S2/S region recommended for genotyping the hepatitis B virus. Results. Among 76 samples of hepatitis B virus DNA, 44 samples were found, which was 57,89%. None of the patients had HBsAg, and 6 patients (13,63%) had HBcor IgG and HBe IgG antibodies. On the basis of phylogenetic analysis, it was shown that only genotype D, which is the most common genotype of the hepatitis B virus in the Russian Federation, was detected in the examined group. The subgenotype D2 (47,72%) prevailed in comparison with the subgenotype D1 (34,09%) and the subgenotype D3 (18,18%). The distribution possible ways of the subgenotype D1 hepatitis B virus, which is uncharacteristic for the region, are discussed. Only one isolate of hepatitis B virus with mutations of drug resistance to nucleotide / nucleoside analogue therapy has been identified — amino acid substitution in the polymerase gene of the virus (L180M, M204V) associated with the development of resistance to lamivudine, entecavir, telbivudine and tenofovir. Conclusion. The high prevalence of occult hepatitis B among HIVinfected patients indicates a lack current tests for the diagnosis of chronic HBV infections. The identification of occult hepatitis B in HIV-infected individuals seems appropriate for timely treatment of patients and requires the use more sensitive methods.

Molecular evidence for multiple introductions of the banded grove snail (Cepaea nemoralis) in North America

Global identification and monitoring programs for invasive species aim to reduce imminent impacts to biodiversity, ecosystem services, agriculture, and human health. This study employs a 658 base pair fragment of the cytochrome c oxidase subunit I (COI) gene to identify and categorize clades of the banded grove snail (Cepaea nemoralis (Linnaeus, 1758)) from native (European) and introduced (North American) ranges using a maximum-likelihood phylogeny and haplotype networks. This work corroborates the existence of eight clades within C. nemoralis and further identified three clades that were common to both Europe and North America (A, D, O). Clades A and D were found in eastern Canada, Ontario (Canada), and British Columbia (Canada), whereas clade O was restricted to Ontario, possibly introduced from Poland or central Europe. Haplotype networks suggest clade A was introduced from northern Europe, whereas clade D was introduced from western and central Europe. Networks contained many private haplotypes and a lack of haplotype sharing, suggesting strong genetic structure in this system, possibly resulting from reduced dispersal in this species. This study describes the contemporary distribution of C. nemoralis in Canada and demonstrates the efficacy of DNA barcoding for monitoring the spread of invasive species, warranting its widespread adoption in management policies.

First record of the millipede genus Nesorthomorpha Jeekel, 1980 in Vietnam with description of a new species (Diplopoda, Polydesmida, Paradoxosomatidae)

The genus Nesorthomorpha Jeekel, 1980 is recorded in Vietnam for the first time based on the discovery of the new species Nesorthomorpha montana n. sp. from the Highlands of Vietnam. An identification key to the new and 8 previously known Nesorthomorpha species is also provided. The relationship between the new species and 9 other species of 5 genera of the tribe Orthomorphini was analyzed using a 500 base pair fragment of the mitochondrial 16S rRNA gene. The result indicates that the new species appears to be closely related to the genus Orthomorpha.