Molecular detection and prevalence of Rotavirus with acute gastroenteritis among the children of rural and urban areas

Rotavirus is the main infective agent of acute gastroenteritis (AGE) in children under the age of five years and causing significant morbidity as well as mortality throughout the world. The study was carried out to detect the prevalence rate, genotypes strain and risk factors of Rotavirus among the children of rural and urban areas of district Bannu Khyber Pakhtunkhwa Pakistan. A total of 180 stool samples were collected from children under the age of 5 years from two major hospitals of Bannu from January to December (2015). The samples were analyzed by Reverse-transcriptase Polymerase Chain Reaction (RT-PCR) for the detection of Rotavirus, positive samples were further processed for genotyping (G and P type) through specific PCR. Of the total, 41 (23%) samples were positive for Rotavirus. The most prevalent G genotypes found were: G3, G8, G9 (each 29%), followed by G10 (15%), and G11 (10%). Whereas the prevalent P genotypes were: P-8 (25%), P-4 and P-10 (each 20%), P-9 (15%), followed by P-6 and P-11 (each 10%). Moreover, Rotavirus infection was more prevalent in summer (23.73%) and winter (22.7%) than spring (20%) and autumn (21.4%). Rotavirus infection exhibited high frequency in June (14%), October (8%) and November (6%). It is concluded that Rotavirus is more prevalent in children and various genotypes (G and P) of Rotavirus are present in the study area. Lack of studies, awareness and rarer testing of Rotavirus are the principal reasons of virus prevalence in district Bannu, Pakistan.

Circulating Phylotypes of White Spot Syndrome Virus in Bangladesh and Their Virulence

White Spot Syndrome Virus (WSSV) has emerged as one of the most prevalent and lethal viruses globally and infects both shrimps and crabs in the aquatic environment. This study aimed to investigate the occurrence of WSSV in different ghers of Bangladesh and the virulence of the circulating phylotypes. We collected 360 shrimp (Penaeus monodon) and 120 crab (Scylla sp.) samples from the south-east (Cox’s Bazar) and south-west (Satkhira) coastal regions of Bangladesh. The VP28 gene-specific PCR assays and sequencing revealed statistically significant (p < 0.05, Kruskal–Wallis test) differences in the prevalence of WSSV in shrimps and crabs between the study areas (Cox’s Bazar and Satkhira) and over the study periods (2017–2019). The mean Log load of WSSV varied from 8.40 (Cox’s Bazar) to 10.48 (Satkhira) per gram of tissue. The mean values for salinity, dissolved oxygen, temperature and pH were 14.71 ± 0.76 ppt, 3.7 ± 0.1 ppm, 34.11 ± 0.38 °C and 8.23 ± 0.38, respectively, in the WSSV-positive ghers. The VP28 gene-based phylogenetic analysis showed an amino-acid substitution (E→G) at the 167th position in the isolates from Cox’s Bazar (referred to as phylotype BD2) compared to the globally circulating one (BD1). Shrimp PL artificially challenged with BD1 and BD2 phylotypes with filtrates of tissue containing 0.423 × 109 copies of WSSV per mL resulted in a median LT50 value of 73 h and 75 h, respectively. The in vivo trial showed higher mean Log WSSV copies (6.47 ± 2.07 per mg tissue) in BD1-challenged shrimp PL compared to BD2 (4.75 ± 0.35 per mg tissue). Crabs infected with BD1 and BD2 showed 100% mortality within 48 h and 62 h of challenge, respectively, with mean Log WSSV copies of 12.06 ± 0.48 and 9.95 ± 0.37 per gram tissue, respectively. Moreover, shrimp antimicrobial peptides (AMPs), penaeidin and lysozyme expression were lower in the BD1-challenged group compared to BD2 challenged shrimps. These results collectively demonstrated that relative virulence properties of WSSV based on mortality rate, viral load and expression of host immune genes in artificially infected shrimp PL could be affected by single aa substitution in VP28.

Distribution of the Beta-Casein Gene Variants in Three Cattle Breeds Reared in Benin

The beta-casein gene is one of the most functional genetic candidate that affect milk quality and composition traits. Among its variants, the A1/A2 are the most common. Therefore, the aim of this study was to identify the distribution of the Beta-casein gene variants (A1/A2) in three different cattle breeds in order to determine which of the breed produce a better milk for consumers&rsquo; health. 152 blood samples which comprises 72 (Muturu), 40 (Azawak) and 40 Girolando were used to carry out this study. Genomic DNA was extracted from the blood samples and each variant was subsequently amplified from the extracted DNA samples using an Allele-Specific PCR technique and then confirmed by running the PCR products on 1% agarose gel. The result showed that there were three genotypes (A1A1, A2A1 and A2A2) in the three breeds. The average percentage genotypic frequencies obtained from this study were 42.76%, 31.58% and 25.66% respectively for A1A1, A1A2 and A2A2 genotypes while the percentage allelic frequencies were 58% and 42% respectively for A1 and A2 allele. The genetic parameters of Azawak breed were higher than that of the other breeds, what implies that there was a higher polymorphism and genetic diversity in the Azawak breed in the beta-casein gene compare to the other breeds. The A2 beta-casein variant in milk has been found to be desirable for milk consumer&rsquo;s health and nutrition. This study therefore showed that the Azawak breed provides a good potential for increasing this favorable allele through appropriate breeding techniques of cattle.

Evidence of pyrethroid resistance in Anopheles amharicus and Anopheles arabiensis from Arjo-Didessa irrigation scheme, Ethiopia

Background Indoor residual spraying and insecticide-treated nets are among the key malaria control intervention tools. However, their efficacy is declining due to the development and spread of insecticide resistant vectors. In Ethiopia, several studies reported resistance of An. arabiensis to multiple insecticide classes. However, such data is scarce in irrigated areas of the country where insecticides, pesticides and herbicides are intensively used. Susceptibility of An. gambiae s.l. to existing and new insecticides and resistance mechanisms were assessed in Arjo-Didessa sugarcane plantation area, southwestern Ethiopia. Methods Adult An. gambiae s.l. reared from larval/pupal collections of Arjo-Didessa sugarcane irrigation area and its surrounding were tested for their susceptibility to selected insecticides. Randomly selected An. gambiae s.l. (dead and survived) samples were identified to species using species-specific polymerase chain reaction (PCR) and were further analyzed for the presence of knockdown resistance (kdr) alleles using allele-specific PCR. Results Among the 214 An. gambiae s.l. samples analyzed by PCR, 89% (n = 190) were An. amharicus and 9% (n = 20) were An. arabiensis. Mortality rates of the An. gambiae s.l. exposed to deltamethrin and alphacypermethrin were 85% and 86.8%, respectively. On the other hand, mortalities against pirmiphos-methyl, bendiocarb, propoxur and clothianidin were 100%, 99%, 100% and 100%, respectively. Of those sub-samples (An. amharicus and An. arabiensis) examined for presence of kdr gene, none of them were found to carry the L1014F (West African) allelic mutation. Conclusion Anopheles amharicus and An. arabiensis from Arjo-Didessa sugarcane irrigation area were resistant to pyrethroids which might be synergized by extensive use of agricultural chemicals. Occurrence of pyrethroid resistant malaria vectors could challenge the ongoing malaria control and elimination program in the area unless resistance management strategies are implemented. Given the resistance of An. amharicus to pyrethroids, its behavior and vectorial capacity should be further investigated.

Lesion Material From Treponema-Associated Hoof Disease of Wild Elk Induces Disease Pathology in the Sheep Digital Dermatitis Model

A hoof disease among wild elk (Cervus elaphus) in the western United States has been reported since 2008. Now present in Washington, Oregon, Idaho, and California, this hoof disease continues to spread among elk herds suggesting an infectious etiology. Causing severe lesions at the hoof-skin junction, lesions can penetrate the hoof-horn structure causing severe lameness, misshapen hooves, and in some cases, sloughed hooves leaving the elk prone to infection, malnutrition, and premature death. Isolated to the feet, this disease has been termed treponeme-associated hoof disease due to the numerous Treponema spp. found within lesions. In addition to the Treponema spp., treponeme-associated hoof disease shares many similarities with digital dermatitis of cattle and livestock including association with several groups of anaerobic bacteria such as Bacteroides, Clostridia, and Fusobacterium, neutrophilic inflammatory infiltrate, and restriction of the disease to the foot and hoof tissues. To determine if there was a transmissible infectious component to this disease syndrome, elk lesion homogenate was used in a sheep model of digital dermatitis. Ten animals were inoculated with lesion material and lesion development was followed over 7 weeks. Most inoculated feet developed moderate to severe lesions at 2- or 4-weeks post-inoculation timepoints, with 16 of 18 feet at 4 weeks also had spirochetes associated within the lesions. Histopathology demonstrated spirochetes at the invading edge of the lesions along with other hallmarks of elk hoof disease, neutrophilic inflammatory infiltrates, and keratinocyte erosion. Treponema-specific PCR demonstrated three phylotypes associated with elk hoof disease and digital dermatitis were present. Serum of infected sheep had increased anti-Treponema IgG when compared to negative control sheep and pre-exposure samples. Analysis of the bacterial microbiome by sequencing of the bacterial 16S rRNA gene showed a community structure in sheep lesions that was highly similar to the elk lesion homogenate used as inoculum. Bacteroidies, Fusobacterium, and Clostridia were among the bacterial taxa overrepresented in infected samples as compared to negative control samples. In conclusion, there is a highly transmissible, infectious bacterial component to elk treponeme-associated hoof disease which includes several species of Treponema as well as other bacteria previously associated with digital dermatitis.

Intratumor morphologic and molecular genetic heterogeneity in astrocytomas of different grade of malignancy in the material from the first operation

Introduction. Intratumor heterogeneity is one of the key reasons for unfavourable prognosis in malignant tumors. Astrocytic tumors are known to develop therapy resistance inevitably during the course of disease. One of possible reason is tumor heterogeneity. Purpose. The aim of this work was to assess the intratumor morphologic and molecular heterogeneity in diffuse astrocytoma, anaplastic astrocytomas and primary glioblastomas. Material and methods. We conducted morphologic (n=22) and molecular-genetic (n=8) analysis of surgical specimens obtained from primarily operated glioblastoma giv (gb), anaplastic astrocytomas giii (aa) and diffuse astrocytoma gii (da) patients aged 18 years and older in whom total or subtotal tumor resection was performed. Tissue sampling for the analysis was performed from 5 equidistant areas of each tumor. Morphologic diagnosis was established according to who classification of central nervous system tumors (2007/2016). Mgmt, c-kit, top2a, pdgfr-α, ercc1, vegf genes mrnaexpression was assessed by rt-pcr. Idh1 and idh2 mutational status was evaluated by allele-specific pcr. Results. Morphologic heterogeneity was evident in 72,7 % tumors (16/22) overall. Heterogeneity was observed in 68,8 % (11/16) of gb, 80 % (4/5) of aa and in the only case of da. In 50 % of cases at least 3 different morphologic variants were seen in different areas of the tumor. This morphologic heterogeneity presented as the combination of different grades of anaplasia (gii – giv) in one tumor. Molecular profile was assessed in 48 expression analysis of genes: mgmt, c-kit, top2a, pdgfr-α, ercc1, vegf from 8 patients. Intratumoral molecular heterogeneity was revealed in 41,7 % of cases (20/48). Conclusion. The presence of intratumoral heterogeneity should be taken into account during surgery for adequate tumor sampling for histologic and molecular analysis which is critical for proper assessment of prognosis and following treatment planning.

Identification and Fine-Mapping of Clubroot (Plasmodiophora brassicae) Resistant QTL in Brassica rapa

European fodder turnips (Brassica rapa ssp. rapifera) were identified as sources of clubroot resistance (CR) and have been widely used in Brassica resistance breeding. An F2 population derived from a cross between a resistant turnip and a susceptible Chinese cabbage was used to determine the inheritance and locating the resistance Quantitative Trait Loci (QTLs). The parents showed to be very resistant/susceptible to the field isolates (pathotype 4) of clubroot from Henan in China. After inoculation, 27 very resistant or susceptible individuals were selected to construct bulks, respectively. Next-generation-sequencing-based Bulk Segregant Analysis Sequencing (BSA-Seq) was used and located resistance QTL on chromosome A03 (3.3–7.5 Mb) and A08 (0.01–6.5 Mb), named Bcr1 and Bcr2, respectively. Furthermore, an F3 population including 180 families derived from F2 individuals was phenotyped and used to verify and narrow candidate regions. Ten and seven Kompetitive Allele-Specific PCR (KASP) markers narrowed the target regions to 4.3–4.78 Mb (A03) and 0.02–0.79 Mb (A08), respectively. The phenotypic variation explained (PVE) of the two QTLs were 33.3% and 13.3% respectively. The two candidate regions contained 99 and 109 genes. In the A03 candidate region, there were three candidate R genes, namely Bra006630, Bra006631 and Bra006632. In the A08 candidate region, there were two candidate R genes, namely Bra030815 and Bra030846.

Preparing Gene of interest for GateWay cloning (2 step PCR process) v2

GateWay recombination cloning is achieved by flanking your gene of interest with GateWay attachment sites. In our case attB1 and attB2. Those sites are added to the PCR product via primers with 5' extensions. Since those primes create 31 bp and 30 bp 5' primer extensions respectively, plus about 20 bp of actual binding primer sequence it becomes expensive fast if you need 2 x ~50 bp primers for every GOI. We therefore use a 2 step PCR process to attach GateWay attB1 and attB2 sites. We first run a gene specific PCR with primers carrying short 5' extesions, and then a second PCR utilizing universal GateWay primers which bind to the short extension of the first PCR product to create the full attB1 and attB2 sites. This protocol has been adapted from: 2-STEP GATEWAY PCR EXPERIMENTS