A vaccine-matching assessment of different genetic variants of serotype O foot-and-mouth disease virus isolated in Ethiopia between 2011 and 2014.

The aim of this study was to assess the vaccine-matching and antigenic properties of foot-and-mouth disease virus (FMDV) isolates collected from Ethiopia between 2011 and 2014. Samples (n = 51) were collected from cattle and pigs with clinical signs consistent with foot-and-mouth disease (FMD) on farms in Debre-Berhan, Debre-Zeit/Bishoftu, Sidamo, Mekelle, and Addis Ababa. Infectious FMDV was isolated using BHK-21 cell cultures from 38 of the 51 field samples (74.5%). All of these FMDV-positive samples were characterized as serotype O, belonging to two East Africa topotypes (EA-3 and EA-4), and their VP1-encoding sequences demonstrated amino acid sequence variability encompassing 27 positions in comparison to the vaccine strain (O/ETH/38/2005) currently provided by the National Veterinary Institute of Ethiopia. One-dimensional virus neutralization test (1 dm VNT) results showed that O/ETH/38/2005 was antigenically matched to 10 of the 16 serotype O viruses. These findings indicate that the O/ETH/38/2005 vaccine strain can provide protection against outbreaks caused by the O/EA-3 topotype, although poorer vaccine-matching results for the O/EA-4 topotype reinforce the importance of using a good-quality vaccine with high coverage in the susceptible herds with supporting post-vaccination serosurveillance to ensure that sufficient antibody titers are generated in the vaccinated animals.

Molecular characterization of Mannheimia haemolytica isolates associated with pneumonic cases of sheep in selected areas of Central Ethiopia.

BACKGROUND: Mannheimia haemolytica has been recognized as the principal cause of pneumonic pasteurellosis in sheep and goats. It is one of the important diseases of small ruminants in Ethiopia. While annual vaccination using a monovalent vaccine (inactivated Pasteurella multocida biotype A) is common, respiratory diseases are still reported in various parts of Ethiopia. This suggests the need for further investigation into the species and strains responsible for the disease, which is vital information for development of a multivalent vaccine. The objective of the current study was to isolate M. heamolytica associated with pneumonic cases of sheep in selected areas of Central Ethiopia, determine its role and the strains/genotypes of the bacterium circulating in the study area. RESULTS: Bacteriological analysis of nasal swab samples collected from a total of 76 pneumonic cases of sheep showed that M. haemolytica was isolated from 26 of them while B.trehalosi from two cases. Further molecular analyses of the isolates using M. haemolytica species-specific and M.haemolytica serotype-1 antigen specific PCR assays revealed, 26 of the isolates were identified as M. haemolytica of which 21 of them were M. haemolytica serotype-1. Both M. haemolytica and B.trehalosi isolates were not detected in a PCR assay targeting capsular biosynthesis gene (capA) of P.multocida despite the non-specific products observed in M. haemolytica isolates. Phylogenetic analysis of M. haemolytica isolates included in this study in comparison with the reference strains with respect to PHSSA and Rpt2 genes revealed that the Ethiopian M. haemolytica isolates constituted three distinct genotypes consistent with site of origin. CONCLUSION: The study indicated that M.haemolytica is commonly associated with cases of pneumonia in sheep in the study areas of central Ethiopia although the remaining other pathogens responsible for majority of the cases are yet to be determined. Molecular characterization revealed the existence of three genotypes of M. haemolytica circulating in the study areas consistent to the site of isolation. The findings suggest further extensive work to determine all pathogens associated with sheep pneumonia and the strain distribution of M. heamolytica to understand its molecular epidemiology at national level and design cost effective prevention and control methods.

Investigation of Marek's disease virus from chickens in central Ethiopia.

Marek's disease (MD) is a lymphoproliferative and neuropathic disease of domestic chickens and less commonly, turkeys and quails, caused by a highly contagious, cell-associated, oncogenic herpesvirus. In Ethiopia, MD is believed to be introduced with importation of exotic and crossbred to improve the poultry production and has been reported to be a potential threat to the poultry sector both in backyard and commercial farming systems. This study was aimed at isolation and molecular analysis of MD virus isolates circulating in chicken population in the central part of Ethiopia where commercial farms are populated. From September 2013 to January 2014, clinical and post-mortem examination were conducted on diseased chickens suspected of MD virus infection. Representative spleen and feather follicle samples were collected following sterile procedure, and infectious virus isolation was performed using primary chicken fibroblast cell culture. Cell culture inoculated with suspension of pathological samples developed characteristic MD virus cytopathic effect of rounding of the cells and small plaques. Further analysis of the virus was conducted by conventional PCR amplifying the ICP4 gene fragment from eleven tissue samples using MD virus specific primers. PCR products were further sequenced and analyzed. Nucleotide sequence similarity search of the local isolates resulted a high degree of sequence similarity with Gallid Herpes virus type 2 strain (Marek's disease virus type 1, JN034558). To our knowledge, the present study is the first report conducted on virus isolation and molecular characterization of MD virus isolates circulated in Ethiopia. Eleven ICP4-like gene fragment (318 bp) sequences generated in the present study were uploaded in the public database (KU842366-76). Further research on virus isolation, genetic characterization, and infection dynamics is recommended targeting chickens of all age groups reared in different agro-ecological zones under different production system.

Molecular characterization of orf virus from sheep and goats in Ethiopia, 2008-2013.

BACKGROUND: Orf is a contagious disease of sheep, goats and wild ungulates caused by orf virus (ORFV) a member of the genus Parapoxvirus, Poxviridae family. Although orf is endemic in Ethiopia, little attention has been given so far as it is not a notifiable disease by the World Organization for Animal Health. In this work, we have investigated orf outbreaks representing five different geographical locations of Ethiopia, in Amba Giorgis, Gondar zuria, Adet, Debre zeit and Adami Tulu, between 2008 and 2013. RESULTS: The viral isolation and the sequence analysis of the A32L and the B2L genes of eighteen representative isolates confirmed that sampled animals were infected by ORFVs. The phylogenetic study and the comparative analysis of the deduced amino acid profile suggests that there were two main clusters of ORFV isolates which were responsible for the investigated outbreaks. Additionally the analysis of these two genes showed limited variability to ORFVs encountered elsewhere. This is the first report on the genetic characterization of the ORFV isolates from sheep and goats in Ethiopia. CONCLUSION: The molecular characterization of Ethiopian ORFV isolates highlighted the circulation of two main clusters causing orf disease in sheep and goats. The use of laboratory based methods and a constant monitoring of Ethiopian ORFV isolates is needed to better understand the dynamic of ORFV circulating in the country and facilitate the implementation of control measures.

An international collaborative study to determine the prevalence of contagious caprine pleuropneumonia by monoclonal antibody-based cELISA.

BACKGROUND: Few serological tests are available for detecting antibodies against Mycoplasma capricolum subsp. capripneumoniae, the causal agent of contagious caprine pleuropneumonia (CCPP). The complement fixation test, the test prescribed for international trade purposes, uses a crude antigen that cross-reacts with all the other mycoplasma species of the "mycoides cluster" frequently infecting goat herds. The lack of a more specific test has been a real obstacle to the evaluation of the prevalence and economic impact of CCPP worldwide. A new competitive ELISA kit for CCPP, based on a previous blocking ELISA, was formatted at CIRAD and used to evaluate the prevalence of CCPP in some regions of Kenya, Ethiopia, Mauritius, Tajikistan and Pakistan in an international collaborative study. RESULTS: The strict specificity of the test was confirmed in CCPP-free goat herds exposed to other mycoplasma species of the "mycoides cluster". Prevalence studies were performed across the enzootic range of the disease in Africa and Asia. Seroprevalence was estimated at 14.6% in the Afar region of Ethiopia, whereas all the herds presented for CCPP vaccination in Kenya tested positive (individual seroprevalence varied from 6 to 90% within each herd). In Mauritius, where CCPP emerged in 2009, nine of 62 herds tested positive. In Central Asia, where the disease was confirmed only recently, no positive animals were detected in the Wakhan District of Afghanistan or across the border in neighboring areas of Tajikistan, whereas seroprevalence varied between 2.7% and 44.2% in the other districts investigated and in northern Pakistan. The test was also used to monitor seroconversion in vaccinated animals. CONCLUSIONS: This newly formatted CCPP cELISA kit has retained the high specificity of the original kit. It can therefore be used to evaluate the prevalence of CCPP in countries or regions without vaccination programs. It could also be used to monitor the efficacy of vaccination campaigns as high-quality vaccines induce high rates of seroconversion.

Porcine circovirus-2 in Africa: Identification of continent-specific clusters and evidence of independent viral introductions from Europe, North America and Asia.

Porcine circovirus-2 (PCV-2) is associated with several disease syndromes in domestic pigs that have a significant impact on global pig production and health. Currently, little is known about the status of PCV-2 in Africa. In this study, a total of 408 archived DNA samples collected from pigs in Burkina Faso, Cameroon, Cape Verde, Ethiopia, the Democratic Republic of the Congo, Mozambique, Nigeria, Senegal, Tanzania and Zambia between 2000 and 2018 were screened by PCR for the presence of PCV-2. Positive amplicons of the gene encoding the viral capsid protein (ORF2) were sequenced to determine the genotypes circulating in each country. Four of the nine currently known genotypes of PCV-2 were identified (i.e. PCV-2a, PCV-2b, PCV-2d and PCV-2 g) with more than one genotype being identified in Burkina Faso, Ethiopia, Nigeria, Mozambique, Senegal and Zambia. Additionally, a phylogeographic analysis which included 38 additional ORF2 gene sequences of PCV-2s previously identified in Mozambique, Namibia and South Africa from 2014 to 2016 and 2019 to 2020 and available in public databases, demonstrated the existence of several African-specific clusters and estimated the approximate time of introduction of PCV-2s into Africa from other continents. This is the first in-depth study of PCV-2 in Africa and it has important implications for pig production at both the small-holder and commercial farm level on the continent.

Outbreak investigations and genetic characterization of foot-and-mouth disease virus in Ethiopia in 2008/2009.

The study was conducted in three regional states of Ethiopia: Amhara, Oromia, and Addis Ababa from August 2008 to April 2009 with the objectives of identifying the genetic diversity of serotypes and topotypes in Ethiopia, and determining the attack rate and associations of potential risk factors with foot-and-mouth disease (FMD) seropositivity. A total of 496 cattle were clinically and serologically examined for presence of specific lesions and nonstructural protein for FMD, respectively. Of which, 140 (28.2%) manifested clinical signs and lesions suggestive of FMD, and 219 (44.2%) were seropositive. From a total of 7,781 animals observed and recorded on a designed format in six districts, 1,409 (19.6%) were infected, and 15 (0.12%) died during outbreaks of FMD. Epidemiological investigations revealed that the morbidity rate of the disease was 21.1% in Akaki-kality sub-city, but the mortality rate was <2% in all districts. Furthermore, the mortality and case fatality rates were relatively higher, 1.6% and 8.9% in calves than the other age groups, respectively. From a total of 33 bovine epithelial tissue-cultured samples, 19 (57.6%) showed CPE for FMD virus, in which 16 samples had serotype O and EA-3 topotype, while three samples had found serotype A, Africa topotype, and G-VII strain. Various strains of FMD viruses were isolated in Ethiopia in this study, and therefore, further detailed studies on the evaluation of available vaccines and the development of a vaccine which contains cocktails of antigens of FMD virus strains in the country should be encouraged.

Immunogenicity and Efficacy Evaluation of Vero Cell-Adapted Infectious Bursal Disease Virus LC-75 Vaccine Strain.

INTRODUCTION: Infectious bursal disease virus (IBDV) is an avian viral pathogen that causes infectious bursal disease (IBD) of chickens. The disease has been endemic in Ethiopia since 2002, and vaccination has been practiced as the major means of disease prevention and control. An IBD vaccine is produced in Ethiopia using primary chicken embryo fibroblast (CEF) cell, which is time-consuming, laborious, and uneconomical. The present study was carried out to develop cell-based IBDV LC-75 vaccine using Vero cells and to evaluate the safety, immunogenicity and protection level. METHODS: Identity of the vaccine seed was confirmed with gene-specific primers using reverse transcription polymerase chain reaction (RT-PCR). Confluent monolayer of Vero cells was infected with vaccine virus and serial passage continued till passage 10. A characteristic virus-induced cytopathic effect (CPE) was observed starting from passage 2 on the third day post-infection. The infectious titer of adapted virus showed a linear increment along the passage level. The virus-induced specific antibody was determined using indirect ELISA after vaccination of chicks through ocular route. RESULTS: The antibody titer measured from Vero cells vaccinated chicks revealed similar level with the currently available CEF cell-based vaccine, hence no significant difference. Chicks vaccinated with Vero cell adapted virus showed complete protection against very virulent IBDV, while unvaccinated group had 60% morbidity and 25% mortality. CONCLUSION: It is concluded that the IBDV vaccine strain well adapted on Vero cells and found to be immunogenic induces antibody development and successfully protects chicks against challenge with the circulating field IBDV isolate. Hence, it is recommended to produce IBD vaccine using Vero cell culture at the industrial scale to conquer the limitations caused by using CEF cells and thus to vaccinate chicks population to protect against the circulating IBDV infection.

Establishing a Robust Manufacturing Platform for Recombinant Veterinary Vaccines: An Adenovirus-Vector Vaccine to Control Newcastle Disease Virus Infections of Poultry in Sub-Saharan Africa.

Developing vaccine technology platforms to respond to pandemic threats or zoonotic diseases is a worldwide high priority. The risk of infectious diseases transmitted from wildlife and domestic animals to humans makes veterinary vaccination and animal health monitoring highly relevant for the deployment of public health global policies in the context of "one world, one health" principles. Sub-Saharan Africa is frequently impacted by outbreaks of poultry diseases such as avian influenza and Newcastle Disease (ND). Here, an adenovirus-vectored vaccine technology platform is proposed for rapid adaptation to ND or other avian viral threats in the region. Ethiopian isolates of the Newcastle Disease virus (NDV) were subjected to sequence and phylogenetic analyses, enabling the construction of antigenically matched vaccine candidates expressing the fusion (F) and hemagglutinin-neuraminidase (HN) proteins. A cost-effective vaccine production process was developed using HEK293 cells in suspension and serum-free medium. Productive infection in bioreactors (1-3L) at 2 × 106 cells/mL resulted in consistent infectious adenoviral vector titers of approximately 5-6 × 108 TCID50/mL (approximately 1011VP/mL) in the harvest lysates. Groups of chickens were twice immunized with 1 × 1010 TCID50 of the vectors, and full protection against a lethal NDV challenge was provided by the vector expressing the F antigen. These results consolidate the basis for a streamlined and scalable-vectored vaccine manufacturing process for deployment in low- and medium-income countries.

Molecular characterization of foot-and-mouth disease viruses circulating in Ethiopia between 2008 and 2019.

One of the constraints to controlling foot-and-mouth disease (FMD) in East Africa is the incomplete knowledge of the specific FMD virus (FMDV) strains circulating and the way in which these viruses move across countries in the region. This retrospective study focuses on Ethiopia, which has one of the largest FMD-susceptible livestock populations in Africa. Analyses of FMDV positive samples collected between 2008 and 2019 demonstrate that serotypes O (n = 175), A (n = 51) and SAT 2 (n = 33) were present in the country. Phylogenetic analysis of the VP1 sequences for these viruses showed that there were at least seven different FMD viral clades circulating during this period: O/EA-3, O/EA-4, A/AFRICA/G-I, A/AFRICA/G-IV, A/AFRICA/G-VII, SAT2/VII and SAT2/XIII. Although these results only represent a snapshot and might not reflect all FMDV lineages that were present, they highlight the importance of serotype O, as well as the complexity and co-existence of FMDV serotypes in Ethiopia and surrounding countries. These sequence data also support the idea that there are two FMDV ecosystems existing in East Africa. Data from retrospective studies, such as these presented here, will be beneficial for vaccine selection and vaccination campaigns to control FMDV within Ethiopia.

Genetic characterization of poxviruses in Camelus dromedarius in Ethiopia, 2011-2014.

Camelpox and camel contagious ecthyma are infectious viral diseases of camelids caused by camelpox virus (CMLV) and camel contagious ecthyma virus (CCEV), respectively. Even though, in Ethiopia, pox disease has been creating significant economic losses in camel production, little is known on the responsible pathogens and their genetic diversity. Thus, the present study aimed at isolation, identification and genetic characterization of the causative viruses. Accordingly, clinical case observations, infectious virus isolation, and molecular and phylogenetic analysis of poxviruses infecting camels in three regions and six districts in the country, Afar (Chifra), Oromia (Arero, Miyu and Yabello) and Somali (Gursum and Jijiga) between 2011 and 2014 were undertaken. The full hemagglutinin (HA) and partial A-type inclusion protein (ATIP) genes of CMLV and full major envelope protein (B2L) gene of CCEV of Ethiopian isolates were sequenced, analyzed and compared among each other and to foreign isolates. The viral isolation confirmed the presence of infectious poxviruses. The preliminary screening by PCR showed 27 CMLVs and 20 CCEVs. The sequence analyses showed that the HA and ATIP gene sequences are highly conserved within the local isolates of CMLVs, and formed a single cluster together with isolates from Somalia and Syria. Unlike CMLVs, the B2L gene analysis of Ethiopian CCEV showed few genetic variations. The phylogenetic analysis revealed three clusters of CCEV in Ethiopia with the isolates clustering according to their geographical origins. To our knowledge, this is the first report indicating the existence of CCEV in Ethiopia where camel contagious ecthyma was misdiagnosed as camelpox. Additionally, this study has also disclosed the existence of co-infections with CMLV and CCEV. A comprehensive characterization of poxviruses affecting camels in Ethiopia and the full genome sequencing of representative isolates are recommended to better understand the dynamics of pox diseases of camels and to assist in the implementation of more efficient control measures.

Capripox disease in Ethiopia: Genetic differences between field isolates and vaccine strain, and implications for vaccination failure.

Sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV) of the genus Capripoxvirus (CaPV) cause capripox disease in sheep, goats and cattle, respectively. These viruses are not strictly host-specific and their geographical distribution is complex. In Ethiopia, where sheep, goats and cattle are all affected, a live attenuated vaccine strain (KS1-O180) is used for immunization of both small ruminants and cattle. Although occurrences of the disease in vaccinated cattle are frequently reported, information on the circulating isolates and their relation to the vaccine strain in use are still missing. The present study addressed the parameters associated with vaccination failure in Ethiopia. Retrospective outbreak data were compiled and isolates collected from thirteen outbreaks in small ruminants and cattle at various geographical locations and years were analyzed and compared to the vaccine strain. Isolates of GTPV and LSDV genotypes were responsible for the capripox outbreaks in small ruminants and cattle, respectively, while SPPV was absent. Pathogenic isolates collected from vaccinated cattle were identical to those from the non-vaccinated ones. The vaccine strain, genetically distinct from the outbreak isolates, was not responsible for these outbreaks. This study shows capripox to be highly significant in Ethiopia due to low performance of the local vaccine and insufficient vaccination coverage. The development of new, more efficient vaccine strains, a GTPV strain for small ruminants and a LSDV for cattle, is needed to promote the acceptance by farmers, thus contribute to better control of CaPVs in Ethiopia.

Testing a new formulation for Peste des Petits Ruminants vaccine in Ethiopia.

In this paper extended tests on a new candidate formulation for Peste des Petits Ruminants (PPR) vaccine carried out at National Veterinary Institute (NVI) in Ethiopia are presented. This work was performed in the frame of the VACNADA project from GALVmed which aimed at procuring vaccines against neglected veterinary diseases to African vaccine producing laboratories, in particular PPR. After the eradication of Rinderpest, Peste des Petits Ruminants became the next veterinary disease on target for elimination, requiring an effective and thermostable vaccine. In this work a Tris/Trehalose formulation was evaluated in thermal stability studies in comparison to the current used formulation of the live attenuated PPR vaccine, the Weybridge medium. The extended results presented herein show an increased thermal stability of the vaccine, especially at 37 and 45 °C, as expected from previously published results (Silva A.C. et al., 2011). Furthermore, during the course of this project, the NVI teams have clearly demonstrated ability to produce higher quality PPR vaccines after a successful transfer of the technology. These results should significantly enhance the utility of the vaccine in the eradication of PPR.