First detection of African swine fever (ASF) virus genotype X and serogroup 7 in symptomatic pigs in the Democratic Republic of Congo.

BACKGROUND: African swine fever (ASF) is a highly contagious and severe hemorrhagic viral disease of domestic pigs. The analysis of variable regions of African swine fever virus (ASFV) genome led to more genotypic and serotypic information about circulating strains. The present study aimed at investigating the genetic diversity of ASFV strains in symptomatic pigs in South Kivu province of the Democratic Republic of Congo (DRC). MATERIALS AND METHODS: Blood samples collected from 391 ASF symptomatic domestic pigs in 6 of 8 districts in South Kivu were screened for the presence of ASFV, using a VP73 gene-specific polymerase chain reaction (PCR) with the universal primer set PPA1-PPA2. To genotype the strains, we sequenced and compared the nucleotide sequences of PPA-positive samples at three loci: the C-terminus of B646L gene encoding the p72 protein, the E183L gene encoding the p54 protein, and the central hypervariable region (CVR) of the B602L gene encoding the J9L protein. In addition, to serotype and discriminate between closely related strains, the EP402L (CD2v) gene and the intergenic region between the I73R and I329L genes were analyzed. RESULTS: ASFV was confirmed in 26 of 391 pigs tested. However, only 19 and 15 PPA-positive samples, respectively, were successfully sequenced and phylogenetically analyzed for p72 (B646L) and p54 (E183L). All the ASFV studied were of genotype X. The CVR tetrameric repeat clustered the ASFV strains in two subgroups: the Uvira subgroup (10 TRS repeats, AAAABNAABA) and another subgroup from all other strains (8 TRS repeats, AABNAABA). The phylogenetic analysis of the EP402L gene clustered all the strains into CD2v serogroup 7. Analyzing the intergenic region between I73R and I329L genes revealed that the strains were identical but contained a deletion of a 33-nucleotide internal repeat sequence compared to ASFV strain Kenya 1950. CONCLUSION: ASFV genotype X and serogroup 7 was identified in the ASF disease outbreaks in South Kivu province of DRC in 2018-2019. This represents the first report of ASFV genotype X in DRC. CVR tetrameric repeat sequences clustered the ASFV strains studied in two subgroups. Our finding emphasizes the need for improved coordination of the control of ASF.

Direct in vitro propagation of avian germ cells from an embryonic gonad biorepository.

Direct introduction of cryopreserved embryonic gonadal germ cells (GGC) into a sterile chicken surrogate host to reconstitute a chicken breed has been demonstrated as a feasible approach for preserving and utilizing chicken genetic resources. This method is highly efficient using male gonads; however, a large number of frozen female embryonic gonads is needed to provide sufficient purified GGC for the generation of fertile surrogate female hosts. Applying this method to indigenous chicken breeds and other bird species is difficult due to small flock numbers and poor egg production in each egg laying cycle. Propagating germ cells from the frozen gonadal tissues may be a solution for the biobanking of these birds. Here, we describe a simplified method for culture of GGC from frozen embryonic 9.5 d gonads. At this developmental stage, the germ cells are autonomously shed into medium, yielding hundreds to thousands of mitosis-competent germ cells. The resulting cultures of GGC have over 90% purity, uniformly express SSEA-1 and DAZL antigens and can re-colonize recipient's gonads. The GGC recovery rate from frozen gonads are 42% to 100%, depending on length of cryopreservation and the breed or line of chickens. Entire chicken embryos can also be directly cryopreserved for later gonadal isolation and culture. This storage method is a supplementary approach to safeguard local indigenous chicken breeds bearing valuable genetic traits and should be applicable to the biobanking of many bird species.

Identification of Bacillus anthracis, Brucella spp., and Coxiella burnetii DNA signatures from bushmeat.

Meat from wildlife species (bushmeat) represents a major source of dietary protein in low- and middle-income countries where humans and wildlife live in close proximity. Despite the occurrence of zoonotic pathogens in wildlife, their prevalence in bushmeat remains unknown. To assess the risk of exposure to major pathogens in bushmeat, a total of 3784 samples, both fresh and processed, were collected from three major regions in Tanzania during both rainy and dry seasons, and were screened by real-time PCR for the presence of DNA signatures of Bacillus anthracis (B. anthracis), Brucella spp. (Brucella) and Coxiella burnetii (Coxiella). The analysis identified DNA signatures of B. anthracis (0.48%), Brucella (0.9%), and Coxiella (0.66%) in a total of 77 samples. Highest prevalence rates of B. anthracis, Brucella, and Coxiella were observed in wildebeest (56%), dik-dik (50%), and impala (24%), respectively. Fresh samples, those collected during the rainy season, and samples from Selous or Serengeti had a greater relative risk of being positive. Microbiome characterization identified Firmicutes and Proteobacteria as the most abundant phyla. The results highlight and define potential risks of exposure to endemic wildlife diseases from bushmeat and the need for future investigations to address the public health and emerging infectious disease risks associated with bushmeat harvesting, trade, and consumption.