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.

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.

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.

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.

Spontaneous Deletion of a 209-Kilobase-Pair Fragment from the Escherichia coli Genome Occurs with Acquisition of Resistance to an Assortment of Infectious Phages

ABSTRACT To breed resistance to an assortment of infectious phages, continuous cultures of Escherichia coli JM109 grown in a chemostat were exposed to phage mixtures prepared from sewage influent. Four sequential chemostat-grown cultures were each infected with a different phage mixture. At the end of a chemostat run, one phage-resistant colony was isolated and used to inoculate the subsequent culture. This process was repeated, and increased phage resistance of the input bacterial strain resulted from the successive challenges with different phage cocktails. Multiple mutations apparently accumulated progressively. A mutant isolated at the end of the four runs, designated D198, showed resistance to 38 of 40 phages that infect the parent strain, JM109. D198 produced less outer membrane protein C (OmpC) than JM109. However, restoration of the OmpC protein by plasmid-mediated complementation did not completely restore the susceptibility of D198 to the 38 phages. Therefore, alterations beyond the level of OmpC protein production contribute to the phage resistance of D198. PCR-based genetic analysis revealed that D198 has a genome that is 209 kbp (about 200 genes) smaller than JM109. The deletion includes the chromosomal section from ompC to wbbL that encodes the rhamnosyl transferase involved in lipopolysaccharide biosynthesis. Strains D198 and JM109 were comparable in their growth characteristics and their abilities to express a recombinant protein.

An effective PCR-based diagnostic method for the detection of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) in wood samples from lodgepole pine

A molecular diagnostic method has been designed for the detection and identification of Bursaphelenchus xylophilus. Heat shock protein 70 gene sequences from B. xylophilus and the closely related B. mucronatus were compared and used to design primers Bx701F and Bx701R which amplify a 171 base pair fragment from B. xylophilus by PCR. As a control, primers Bm701F and Bm701R were designed which specifically amplify a 168 base pair fragment from B. mucronatus. After optimisation, B. xylophilus primers were shown to be highly sensitive and could easily detect 23 target copies, or less than one nematode. Species-specific detection of B. xylophilus was carried out directly from concentrated Baermann funnel extracts using wood samples from lodgepole pine (Pinus contorta var. latifolia) trees from British Columbia, Canada, containing an unknown nematode population, thus bypassing the need for culturing or recovering the nematode before analysis.

Detection of Theileria annulata in cattle and vector ticks by PCR using the Tams1 gene sequences

A Polymerase Chain Reaction (PCR) and Southern blot hybridization for the detection of Theileria annulata are described. The PCR used primers amplifying a 785 base-pair fragment of the T. annulata gene which encodes the 30 kDa major merozoite surface antigen, Tams1. The sensitivity of the PCR in bovine blood was 1 piroplasm in 1 μl of blood. T. buffeli, T. parva, Babesia bigemina, B. bovis and B. divergens were not detected. The PCR detected down to 1 infected acinus/tick in resting and partially fed adult Hyalomma anatolicum anatolicum ticks and was negative for T. lestoquardi and T. equi, which are transmitted by this tick but are not infective to cattle. The specificity of the PCR was checked using 30 stocks of T. annulata, all of which were detected. Three stocks of T. lestoquardi, 4 of T. equi and 1 each of T. buffeli, T. parva, B. bigemina, B. bovis and B. divergens were used to ascertain there were no cross-reactions. A nested PCR using separate primers for the first reaction and the same primers for the second reaction detected T. annulata to the same sensitivity and specificity in saponin-extracted DNA samples stored for long periods at −20 °C.

Distribution of a Specific 500-Base-Pair Fragment in Mycobacterium bovis Isolates from Sardinian Cattle

Amplification of a specific, 500-bp fragment fromMycobacterium bovis isolates and use of the fragment to differentiate between Mycobacterium tuberculosis andM. bovis was previously reported (J. G. Rodriguez, G. A. Meja, P. Del Portillo, M. E. Patarroyo, and L. A. Murillo, Microbiology 141:2131–2138, 1995). In the present study, 30M. bovis isolates from Sardinian cattle were examined for the presence of this 500-bp fragment; 4 of the 30 isolates lacked the fragment. This result indicates that identification of M. bovis strains by amplification of the 500-bp sequence may lead to false-negative results.