Molecular detection and phylogenetic analysis of lumpy skin disease virus from outbreaks in Uganda 2017-2018.

BACKGROUND: Lumpy skin disease (LSD) is an infectious viral disease of cattle caused by a Capripoxvirus. LSD has substantial economic implications, with infection resulting in permanent damage to the skin of affected animals which lowers their commercial value. In Uganda, LSD is endemic and cases of the disease are frequently reported to government authorities. This study was undertaken to molecularly characterize lumpy skin disease virus (LSDV) strains that have been circulating in Uganda between 2017 and 2018. Secondly, the study aimed to determine the phylogenetic relatedness of Ugandan LSDV sequences with published sequences, available in GenBank. RESULTS: A total of 7 blood samples and 16 skin nodule biopsies were screened for LSDV using PCR to confirm presence of LSDV nucleic acids. PCR positive samples were then characterised by amplifying the GPCR gene. These amplified genes were sequenced and phylogenetic trees were constructed. Out of the 23 samples analysed, 15 were positive for LSDV by PCR (65.2%). The LSDV GPCR sequences analysed contained the unique signatures of LSDV (A11, T12, T34, S99, and P199) which further confirmed their identity. Sequence comparison with vaccine strains revealed a 12 bp deletion unique to Ugandan outbreak strains. Phylogenetic analysis indicated that the LSDV sequences from this study clustered closely with sequences from neighboring East African countries and with LSDV strains from recent outbreaks in Europe. It was noted that the sequence diversity amongst LSDV strains from Africa was higher than diversity from Eurasia. CONCLUSION: The LSDV strains circulating in Uganda were closely related with sequences from neighboring African countries and from Eurasia. Comparison of the GPCR gene showed that outbreak strains differed from vaccine strains. This information is necessary to understand LSDV molecular epidemiology and to contribute knowledge towards the development of control strategies by the Government of Uganda.

Molecular characterization and phylogenetic study of African swine fever virus isolates from recent outbreaks in Uganda (2010-2013).

BACKGROUND: African swine fever (ASF) is a highly lethal and economically significant disease of domestic pigs in Eastern Africa particularly in Uganda where outbreaks regularly occur. Sequence analysis of variable genome regions have been extensively used for molecular epidemiological studies of African swine fever virus (ASFV) isolates. By combining p72, P54 and pB602L (CVR), a high level resolution approach is achieved for viral discrimination. The major aim of this study therefore, was to investigate the genetic relatedness of ASF outbreaks that occurred between 2010 and 2013 in Uganda to contribute to the clarification of the epidemiological situation over a four year period. METHODS: Tissue samples from infected domestic pigs associated with an ASF outbreak from 15 districts in Uganda were confirmed as being infected with ASFV using a p72 gene-based polymerase chain reaction amplification (PCR) assay recommended by OIE. The analysis was conducted by genotyping based on sequence data from three single copy ASFV genes. The E183L gene encoding the structural protein P54 and part of the gene encoding the p72 protein was used to delineate genotypes. Intra-genotypic resolution of viral relationships was achieved by analysis of tetramer amino acid repeats within the hypervariable CVR of the B602L gene. RESULTS: Twenty one (21) ASF outbreaks were confirmed by the p72 ASF diagnostic PCR, however; only 17 isolates were successfully aligned after sequencing. Our entire isolates cluster with previous ASF viruses in genotype IX isolated in Uganda and Kenya using p72 and P54 genes. Analysis of the CVR gene generated three sub-groups one with 23 tetrameric amino acid repeats (TRS) with an additional CAST sequence, the second with 22 TRS while one isolate Ug13. Kampala1 had 13 TRS. CONCLUSION: We identified two new CVR subgroups different from previous studies. This study constitutes the first detailed assessment of the molecular epidemiology of ASFV in domestic pigs in the different regions of Uganda.

Prevalence of African swine fever virus in apparently healthy domestic pigs in Uganda.

BACKGROUND: African swine fever (ASF) is a contagious viral disease which can cause up to 100% mortality among domestic pigs leading to serious socio-economic impact on people's livelihoods. ASF is endemic in Uganda and there is paucity of information on the epidemiology of the disease. The major aim of this study was to determine the seroprevalence and prevalence of African swine fever virus (ASFV) in apparently healthy slaughter pigs at Wambizi slaughterhouse in Kampala city, Uganda. We also estimated the presence of ASFV antibodies and circulating viral antigens in pigs from selected districts of Uganda during targeted surveillance. We analysed 540 and 181 blood samples collected from slaughter pigs and pigs from targeted surveillance districts respectively. RESULTS: The prevalence of ASFV in slaughter pigs was 52.96% (95% CI, 48.75-57.14) and 11.5% (95% CI, 9.06-14.45) by ELISA and PCR respectively. In surveillance districts, the proportion of ASFV positive pigs was 53.59% (95% CI, 46.33-60.71) and 0.55% (95% CI, 0.1-3.06) by ELISA and PCR respectively. CONCLUSION: The study has found out a high seroprevalence of ASFV antibodies in apparently healthy slaughter pigs and also a high proportion of ASFV antibody seropositive pigs in surveyed districts in Uganda indicating exposure to ASFV. However, there was a lower prevalence of ASFV infection implying that there could be low virulent strains of ASFV circulating in domestic pigs in Uganda which requires further investigation.

Sequence Diversity of Tp1 and Tp2 Antigens and Population Genetic Analysis of Theileria parva in Unvaccinated Cattle in Zambia's Chongwe and Chisamba Districts.

East Coast Fever (ECF), caused by Theileria parva, is a major constraint to improved livestock keeping in east and central Africa, including Zambia. To understand the dynamics and determine the candidates for immunization in Zambia's Chongwe and Chisamba districts, a combination of Tp1 and Tp2 gene sequencing and microsatellite analysis using nine markers was conducted from which an abundance of Muguga, Kiambu, Serengeti and Katete epitopes in the field samples was obtained. Phylogenetic analysis showed six (Tp1) and three (Tp2) clusters with an absence of geographical origin clustering. The majority of haplotypes were related to Muguga, Kiambu, Serengeti and Katete, and only a few were related to Chitongo. Both antigens showed purifying selection with an absence of positive selection sites. Furthermore, low to moderate genetic differentiation was observed among and within the populations, and when vaccine stocks were compared with field samples, Chongwe samples showed more similarity to Katete and less to Chitongo, while Chisamba samples showed similarity to both Katete and Chitongo and not to Muguga, Kiambu or Serengeti. We conclude that the use of Katete stock for immunization trials in both Chongwe and Chisamba districts might produce desirable protection against ECF.

Molecular characterization and population genetics of Theileria parva in Burundi's unvaccinated cattle: Towards the introduction of East Coast fever vaccine.

Theileria parva (T. parva) is a protozoan parasite that causes East Coast fever (ECF). The disease is endemic in Burundi and is a major constraint to livestock development. In this study, the parasite prevalence in cattle in six regions namely; Northern, Southern, Eastern, Western, Central and North Eastern was estimated. Furthermore, the sequence diversity of p67, Tp1 and Tp2 genes was assessed coupled with the population genetic structure of T. parva using five satellite markers. The prevalence of ECF was 30% (332/1109) on microscopy, 60% (860/1431) on ELISA and 79% (158/200) on p104 gene PCR. Phylogenetic analysis of p67 gene revealed that only allele 1 was present in the field samples. Furthermore, phylogenetic analysis of Tp1 and Tp2 showed that the majority of samples clustered with Muguga, Kiambu and Serengeti and shared similar epitopes. On the other hand, genetic analysis revealed that field samples shared only two alleles with Muguga Cocktail. The populations from the different regions indicated low genetic differentiation (FST = 0.047) coupled with linkage disequilibrium and non-panmixia. A low to moderate genetic differentiation (FST = 0.065) was also observed between samples and Muguga cocktail. In conclusion, the data presented revealed the presence of a parasite population that shared similar epitopes with Muguga Cocktail and was moderately genetically differentiated from it. Thus, use of Muguga Cocktail vaccine in Burundi is likely to confer protection against T. parva in field challenge trials.

An alternative cold chain for storing and transporting East Coast fever vaccine.

East Coast fever (ECF) is an often fatal, economically important cattle disease that predominantly affects eastern, central, and southern Africa. ECF is controlled through vaccination by means of simultaneous injection of oxytetracycline and cryogenically preserved stabilate containing live, disease-causing parasites. Storage and transportation of the stabilate requires liquid nitrogen, a commodity that is commonly unreliable in low-resource settings. Here we show that storage of conventionally prepared stabilate at -80 °C for up to 30 days does not significantly affect its ability to infect cultured peripheral blood mononucleated cells or live cattle, suggesting an alternative cold chain that maintains these temperatures could be used to effectively manage ECF.

Sequence diversity of cytotoxic T cell antigens and satellite marker analysis of Theileria parva informs the immunization against East Coast fever in Rwanda.

BACKGROUND: East Coast fever (ECF) caused by Theileria parva is endemic in Rwanda. In this study, the antigenic and genetic diversity of T. parva coupled with immunization and field challenge were undertaken to provide evidence for the introduction of ECF immunization in Rwanda. METHODS: Blood collected from cattle in the field was screened for T. parva using ELISA and PCR targeting the p104 gene. Tp1 and Tp2 gene sequences were generated from field samples and from Gikongoro and Nyakizu isolates. Furthermore, multilocus genotype data was generated using 5 satellite markers and an immunization challenge trial under field conditions using Muguga cocktail vaccine undertaken. RESULTS: Out of 120 samples, 44 and 20 were positive on ELISA and PCR, respectively. Antigenic diversity of the Tp1 and Tp2 gene sequences revealed an abundance of Muguga, Kiambu and Serengeti epitopes in the samples. A further three clusters were observed on both Tp1 and Tp2 phylogenetic trees; two clusters comprising of field samples and vaccine isolates and the third cluster comprising exclusively of Rwanda samples. Both antigens exhibited purifying selection with no positive selection sites. In addition, satellite marker analysis revealed that field samples possessed both shared alleles with Muguga cocktail on all loci and also a higher proportion of unique alleles. The Muguga cocktail (Muguga, Kiambu and Serengeti) genotype compared to other vaccine isolates, was the most represented in the field samples. Further low genetic sub-structuring (FST = 0.037) coupled with linkage disequilibrium between Muguga cocktail and the field samples was observed. Using the above data to guide a field immunization challenge trial comprising 41 immunized and 40 control animals resulted in 85% seroconversion in the immunized animals and an efficacy of vaccination of 81.7%, implying high protection against ECF. CONCLUSIONS: Antigenic and genetic diversity analysis of T. parva facilitated the use of Muguga cocktail vaccine in field conditions. A protection level of 81.7% was achieved, demonstrating the importance of combining molecular tools with field trials to establish the suitability of implementation of immunization campaigns. Based on the information in this study, Muguga cocktail immunization in Rwanda has a potential to produce desirable results.