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Getu Ayele,1 Belayneh Getachew,1 Fufa Dawo Bari,2 Berecha Bayissa,1 Ayelech Muluneh,3 Takele Abayneh,1 Esayas Gelaye,1 Bedaso Mammo Edao2 1Research and Development Directorate, National Veterinary Institute, Bishoftu, Oromia, Ethiopia; 2Department of Microbiology, Immunology and Vet Public Health, College of Veterinary Medicine, Addis Ababa University, Bishoftu, Oromia, Ethiopia; 3Department of Microbiology, Animal Health Institute, Sebeta, Oromia, EthiopiaCorrespondence: Bedaso Mammo Edao, Email edaobedaso@gmail.comIntroduction: Foot-and-mouth disease is globally one of the most economically important viral diseases of cloven-hoofed animals that can be controlled by different strategies, where vaccination plays an important role. Selection of adjuvant added to vaccine preparation is crucial in ensuring the protective effect of the vaccine. Aluminum hydroxide gel mixed with saponin (AS) is widely used adjuvant, with its suboptimal immune response in FMD vaccine. The present study was undertaken to evaluate different ingredients of adjuvants for inactivated trivalent (A, O and SAT 2) FMD vaccine and to demonstrate the effect of booster dose in cattle.Methods: Cattle were grouped into five; four experimental and one control, with six animals in each group and immunized with trivalent vaccine with various formulations of adjuvants. Immune response was measured using Solid Phase Competitive Enzyme Linked Immune Sorbent Assay (SPCE).Results: The antibody level in cattle immunised with a vaccine formulation containing a mixture of aluminum hydroxide gel and saponin (AS) were significantly lower than AS boosted group for the three serotypes (p< 0.05, t-test), which directs the need for booster dose. Whereas the antibody response in the AS + oil group was higher followed by oil alone. The AS preparation with a booster dose has shown better immune response compared to the group without.Conclusion: The findings of this study could suggest that oil based and AS with oil could replace the conventional aluminum hydroxide gel and saponin adjuvants in FMD vaccine preparations. Challenge test was not successful indicating the need for further research on the virus infectivity.Keywords: adjuvant, foot and mouth disease, immune response, serotype

Yasin Mohammed Yimer,1 Destaw Asfaw Ali,2 Belayneh Getachew Ayalew,3 Molalegne Bitew Asires,4 Esayas Gelaye3 1College of Veterinary Medicine, Samara University, Samara, Ethiopia; 2College of Veterinary Medicine and Animal Sciences, University of Gondar, Gondar, Ethiopia; 3National Veterinary Institute, Bishoftu, Ethiopia; 4Ethiopian Biotechnology Institute, Addis Ababa, EthiopiaCorrespondence: Destaw Asfaw AliCollege of Veterinary Medicine and Animal Science, University of Gondar, P.O. Box 196, Gondar, EthiopiaEmail asfawdestaw@gmail.comBackground: Marek’s disease is a chicken lymphoproliferative viral illness. As new viruses emerge, vaccination immunity is being broken and hence pathogenecity assessment and vaccine evaluation related to the pathogen is critical for developing vaccine immunity in the field.Methods: An experimental investigation was conducted to determine the pathogenicity of field isolates against Marek’s disease in antibody-free chicks and to assess the protective efficacy of the Marek’s disease vaccination. The viral isolates in question were discovered during an outbreak investigation for a previous study. The pathogenicity and effectiveness trial used a complete random design.Results: In the pathogenicity trial, chickens inoculated with Bishoftu and Mojo field isolate had lower body weight 77.7± 3.757 and 78.15± 1.95 g at 10 dpi, respectively, when compared to un-inoculated controls, 89.85± 3.838 g at 10 dpi. Incidence of early mortality syndrome (35% and 25%), lymphoma (53.8% and 40%), and overall mortality (50% and 45%) between Bishoftu and Mojo isolates, respectively, was discovered. Vaccinations with Herpes virus of turkey challenged chickens were provided complete protection against Marek’s disease.Conclusion: Based on the findings in pathogenecity assessment experimental trials, Bishoftu and Mojo isolates were designated as virulent Marek’s disease viruses. Regular vaccinations with Herpes virus of turkey vaccine and supported by biosecurity measures in poultry farms are important to prevent the disease.Keywords: chicken, Marek’s disease virus, pathogenicity, vaccine efficacy, Ethiopia

Method: Brucella is an intracellular bacterium that affects both animals and humans. The aim of this study was to compare brucellosis diagnostic methods such as RBPT, I-ELISA, CFT and PCR detection of Brucella species. A total of (552) Sheep, (1345) Goats, and (420) Cattle sera samples were collected with no history of vaccination from brucellosis reported regions of Ethiopia. All sera samples were subjected to comparative serological assays. Post- cross tabulation, sensitivity and specificity were determined using Receiver Operating Characteristics (ROC) curve analyses software. Fifty-four sero-positive sera samples were also subjected to PCR amplification targeting Brucella Genus-specific, as well as B. abortus, and B. melitensis species specific primers. Results: Among 2317 sera samples subjected to comparative serological diagnostic tests (RBPT, I-ELISA and CFT), 189 (8.16 %) for RBPT, 191 (8.24 %) for I-ELISA and 48 (2.07 %) on CFT were found to be positive. Sensitivity on RBPT was 100 % with (95%) in Sheep, 100% (95%) Goats and 74% (95%) Cattle. Specificity on RBT was 98.69% (95%), 99.28% (95%), 100 (95%) in Sheep, Goats and Cattle, respectively. Sensitivity on CFT was 4% with (95) in Sheep 9.65 (95%) Goats and 72 (95%) Cattle. Specificity on CFT was 100 % (95%) for each Sheep, Goats and Cattle. In PCR amplified product analyses, a 223bp in Brucella genus-specific and 156bp B.abortus species specific were detected from all study animals. However, B. melitensis was not detected in all sera samples. Conclusion: In this study Indirect-ELISA was found to be the most sensitive and specific test. RBPT was also sensitive enough to detect all Brucellosis-infected Sheep and Goats. However, this test showed false positive in Sheep and Goats and false negative results in cattle. Therefore, RBT should be complemented with ELISA to solve problems associated with sensitivity and specificity of RBT. PCR assay employed in this study confirmed the presence of B. abortus in small and large ruminants consistent to the study sites. This study is the first report on the detection of B.abortus on small ruminants’ serum in Ethiopia. We could detect B. abortus in non-preferred hosts. The findings suggest further extensive works are required to determine the genetic diversity of Brucella species and understand their molecular epidemiology at national level to design cost effective prevention and control methods.

Wakjira Kebede,1 Molalegne Bitew,2 Fufa Dawo Bari,3 Bedaso Mammo Edao,3 Hawa Mohammed,1 Martha Yami,1 Belayneh Getachew,1 Takele Abayneh,1 Esayas Gelaye1 1National Veterinary Institute, Bishoftu, Ethiopia; 2Ethiopian Biotechnology Institute, Addis Ababa, Ethiopia; 3College of Veterinary Medicine and Agriculture, Addis Ababa University, Bishoftu, EthiopiaCorrespondence: Esayas GelayeNational Veterinary Institute (NVI), PO Box: 19, Bishoftu, EthiopiaTel +251 923625362Fax +251 114339300Email esayasgelaye@gmail.comFufa Dawo BariCollege of Veterinary Medicine and Agriculture, Addis Ababa University, PO Box: 34, Bishoftu, EthiopiaTel +251 929190312Fax +251 114339933Email fufa.dawo@aau.edu.etIntroduction: 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.Keywords: antibody, chicks, IBD vaccine, immunogenicity, protection, Vero cell

Background Foot-and-mouth disease is globally one of the most economically important viral diseases of cloven-hoofed animals that can be controlled by different strategies, where vaccination plays an important role. Selection of the adjuvants,that added to the vaccine is crucial in ensuring the protective effect of the vaccine. Aluminum hydroxide gel and saponin (AS) is widely used adjuvant, with its poor immune response in FMD vaccine. The present study was undertaken to evaluate different ingredients of adjuvants for inactivated trivalent (A, O and SAT 2) FMD vaccine and to demonstrate the effect of booster dose in cattle. Results Cattle were grouped into five; four experimental and one control, with six animals in each group and immunized with trivalent vaccine with various formulations of adjuvants. Immune response was measured using Solid Phase Competitive Enzyme Linked Immune Sorbent Assay (SPCE). The antibody level in cattle immunised with a vaccine formulation containing a mixture of aluminum hydroxide gel and saponin (AS) were significantly lower than AS boosted group for the three serotypes (p t. test), which directs the need for booster dose. Whereas the antibody response in the AS + oil group was higher followed by oil alone, AS boosted and AS at 95% CI. Conclusion The findings of this study could suggest that oil based and AS with oil could replace the conventional aluminum hydroxide gel and saponin adjuvants in FMD vaccine preparations. On different note, challenge test was not successful in this study indicating the need for further research on the virus infectivity.

Foot-and-mouth disease (FMD) is highly infectious, limits live animal trade, and affects ranchers owing to the loss of animal yield. The present study was designed to perform vaccine matching for field FMD virus isolates from clinically diseased cattle and assess the antigenic properties of the field isolates against the current vaccine strains used for vaccine production at the National Veterinary Institute, Ethiopia. Both sequencing and reverse transcription-polymerase chain reactions were used for distinguishing between the viral strains. To evaluate the serological relationship of the vaccine strain with these field isolates (r1 value), in vitro cross-neutralization was performed using ETH/6/2000 and ETH/38/2005 antisera. Infectious field FMD viral samples represented serotypes A and O. Sequence analysis showed that serotype A VP1/1D possessed amino acid variability at positions 28 and 42 to 48, 138, 141, 142, 148, 156, 173, and 197 compared with the ETH/6/2000 vaccine strain, whereas serotype O possessed amino acid variability at positions 45, 48, 138, 139, 140, 141, and 197 compared with the ETH/38/2005 vaccine strain. Based on the one-dimensional virus neutralization test, serotypes A and O demonstrated antigenic matching of up to 13/17 (76.47%) with the vaccine strain, except for the isolates ETH/40/2018, ETH/48/2018, ETH/55/2018, and ETH/61/2018, which had r-values less than 0.3. Therefore, the currently used vaccine strains ETH/38/2005 for serotype O and ETH/6/2000 for serotype A protected against all and most field viruses characterized as serotypes O and A, respectively, and amino acid residue variation was observed in different FMD virus B-C loops, G-H loops, and C-termini of VP1 at sites 1 and 3 in both serotypes.

BackgroundPasteurellaceae families are usually considered opportunistic pathogens which inhabit normal flora on the mucosal membranes of the upper respiratory and the lower genital tracts of mammals and birds. The majority of P. multocida, M. hemolytica, and B. trehalosi isolates are opportunistic animal pathogens and cause disease only under certain conditions. Some of the target genes are shared by isolates found in the same or different genuses. The alpha / beta hydrolase kmt1 genes part of OMP and SLP are shared by all P.multocida species and FbpA is common gene for both P.multocida and M. hemolytica isolates. Other virulence related genes are used for genotyping of P.multocida, M.hemolytica and B.trehalosi isolates. The objective of the current research was focused on the detection and genotyping of different target genes that can be found in the isolates of P. multocida, M. hemolytica, and B. trehalosi using various PCR primers. The distributions of virulence-associated genes were assessed in the isolates of the three genera.Materials and methodsA total of eight isolates from P. multocida, M. hemolytica, and B. trehalosi isolated from clinical cases of cattle and sheep and stored at the Bacteriology laboratory of NVI were used as source of samples for the present study. DNeasy® Blood & Tissue Kit (Qiagen, Germany) was used for the extraction of DNA from bacterial isolates. Different primers were used for genotyping of P. multocida, P.hemolytica and B.trehalosi isolates.ResultsP.multocida was detected using species-specific kmt1 primer with an amplicon product of 460bp. Four serogroups of P.multocida (A, B, D, &E) were detected using serotype-specific primers with amplification products of 1044bp, 760bp, 657bp, and 511bp respectively. PCR was conducted to HS causing P.multocida type B and detected 620 bp amplicons. SLP genes were detected in all P.multocida serotypes with an amplification product size 1400-1553bp. In addition to that, the four P. multocida serotypes were found to be positive for BRD-PmSLP with an amplification product of 460bp but using recombinant HS-SLP primers resulted in the double band at 460 bp and 541bp for serotypes A and D. However, HS causing type B& E serotypes were detected with an amplified product of 541bp. TbpA2 has been detected with an amplicon size of approximately 750 bp for types (A and D) and 1300bp for type B using TbpA-F2/Rev primers. All of the P.multocida serotypes (A, B, D&E) and M. hemolytica (A: 1) were strongly positive for the iron acquisition FbpA gene with amplification bands at 500bp and 1000bp, respectively. A multiplex PCR assay was carried out for detection of M. haemolytica A: 1 using PHSSA and Rpt2 primers with amplification products of 327pb and 1022 bp respectively. The three isolates of B. trehalosi were identified using BtsodA primers with the result of 144bp amplicon size.ConclusionFrom this work, we understood that P.multocida and M. haemolytica shared genetic materials like iron binding proteins (FbpA) and some of the target genes (kmt1 and SLP) were commonly found in all isolates of P. multocida. However, TbpA2 was found to be in most but not in all Pasteurella isolates. We also observed that PHSSA and Rpt2 genes were exclusively found in M. hemolytica isolates whereas Bt-sodA was prominent in all B. trehalosi isolates (T3, T4&T15).

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. ispartof: TRANSBOUNDARY AND EMERGING DISEASES vol:67 issue:6 pages:2983-2992 ispartof: location:Germany status: published

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-2g) 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. This article is protected by copyright. All rights reserved.

Background Bovine Respiratory Disease (BRD) is a multifactorial and economically important illness of cattle. The current study was designed to characterize the major bacterial pathogens associated with BRD and determine the antibiotic susceptibility patterns of isolates. Samples were collected from 400 pneumonic cases of cattle. Results Laboratory assay revealed isolation of 376 (94.0%) bacterial pathogens. The most prevalent bacterial pathogens recovered were Mannheimia haemolytica (M. haemolytica) followed by Pasteurella multocida (P. multocida), Histophilus somni (H. somni), and Bibersteinia trehalosi (B. trehalosi) from 191 (50.80%), 81 (21.54%), 56 (14.89%), and 48 (12.77%) samples, respectively. M. haemolytica strains were confirmed using multiplex PCR assay through the amplification of PHSSA (~ 325 bp) and Rpt2 (~ 1022 bp) genes. Capsular typing of P. multocida revealed amplification of serogroup A (hyaD-hyaC) gene (~ 1044 bp) and serogroup D (dcbF) gene (~ 657 bp). B. trehalosi isolates displayed amplification of the sodA gene (~ 144 bp). Besides, serotyping of M. haemolytica showed the distribution of serotype A:1 (82.20%), A:2 (10.47%), and A:6 (7.33%). Whereas, biotyping of P. multocida revealed a higher prevalence of biotype A:3 (83.95%), then A:1 (8.64%), A:2 (4.94%), and A:12 (2.47%). The majority of the retrieved isolates showed remarkable susceptibility to enrofloxacin, ciprofloxacin, sulfamethoxazole-trimethoprim, florfenicol, and ceftiofur (100%). Besides, varying degree of antimicrobial resistance was observed against streptomycin, gentamicin, penicillin-G, and ampicillin. Conclusions The current findings confirmed that M. haemolytica (A:1) strain is the most common bacterial pathogen identified from BRD cases in the study areas of Ethiopia. Hence, continuous outbreak monitoring and evaluation of antibiotics susceptibility patterns of bacterial pathogens associated with BRD are indispensable to reduce the impact of BRD in the study areas. Further investigation of bacterial pathogens and genotypic analysis of pathogens from a wider area of the country is essential to design a cost-efficient control strategy.

Marek's disease is a chicken lymphoproliferative viral illness. As new viruses emerge, vaccination immunity is being broken and hence pathogenecity assessment and vaccine evaluation related to the pathogen is critical for developing vaccine immunity in the field.An experimental investigation was conducted to determine the pathogenicity of field isolates against Marek's disease in antibody-free chicks and to assess the protective efficacy of the Marek's disease vaccination. The viral isolates in question were discovered during an outbreak investigation for a previous study. The pathogenicity and effectiveness trial used a complete random design.In the pathogenicity trial, chickens inoculated with Bishoftu and Mojo field isolate had lower body weight 77.7±3.757 and 78.15±1.95 g at 10 dpi, respectively, when compared to un-inoculated controls, 89.85±3.838 g at 10 dpi. Incidence of early mortality syndrome (35% and 25%), lymphoma (53.8% and 40%), and overall mortality (50% and 45%) between Bishoftu and Mojo isolates, respectively, was discovered. Vaccinations with Herpes virus of turkey challenged chickens were provided complete protection against Marek's disease.Based on the findings in pathogenecity assessment experimental trials, Bishoftu and Mojo isolates were designated as virulent Marek's disease viruses. Regular vaccinations with Herpes virus of turkey vaccine and supported by biosecurity measures in poultry farms are important to prevent the disease.

Significance The absence of zoonotic MERS-CoV in Africa in spite of an abundance of MERS-CoV–infected dromedaries has remained an enigma. We demonstrate that geographically and genetically distinct viruses from Africa have low replication competence in the human lung, providing a possible explanation for the absence of severe MERS disease in Africa. The findings suggest that MERS-CoV now entrenched in the Arabian Peninsula has acquired increased pathogenic potential for humans. We demonstrate that the spike protein contributes to this phenotypic difference. If pathogenic clade B viruses from the Arabian Peninsula are introduced into Africa, they are likely to become dominant, as they have in the Arabian Peninsula, and to be associated with adverse health impacts in Africa and increased pandemic threat., Coronaviruses are pathogens of pandemic potential. Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. More than 70% of MERS-CoV–infected dromedaries are found in East, North, and West Africa, but zoonotic MERS disease is only reported from the Arabian Peninsula. We compared viral replication competence of clade A and B viruses from the Arabian Peninsula with genetically diverse clade C viruses found in East (Egypt, Kenya, and Ethiopia), North (Morocco), and West (Nigeria and Burkina Faso) Africa. Viruses from Africa had lower replication competence in ex vivo cultures of the human lung and in lungs of experimentally infected human-DPP4 (hDPP4) knockin mice. We used lentivirus pseudotypes expressing MERS-CoV spike from Saudi Arabian clade A prototype strain (EMC) or African clade C1.1 viruses and demonstrated that clade C1.1 spike was associated with reduced virus entry into the respiratory epithelial cell line Calu-3. Isogenic EMC viruses with spike protein from EMC or clade C1.1 generated by reverse genetics showed that the clade C1.1 spike was associated with reduced virus replication competence in Calu-3 cells in vitro, in ex vivo human bronchus, and in lungs of hDPP4 knockin mice in vivo. These findings may explain why zoonotic MERS disease has not been reported from Africa so far, despite exposure to and infection with MERS-CoV.

Fufa D Bari,1,2 Esayas Gelaye,3 Berhe Gebreegziabher Tekola,3 Timm Harder,1 Martin Beer,1 Christian Grund1 1Institute of Infectology, Friedrich-Loeffler-Institut, Greifswald, Germany; 2Department of Microbiology, Immunology and Veterinary Public Health, College of Veterinary Medicine and Agriculture, Addis Ababa University, Bishoftu, Ethiopia; 3National Veterinary Institute, Bishoftu, EthiopiaCorrespondence: Fufa D BariDepartment of Microbiology, Immunology and Veterinary Public Health, College of Veterinary Medicine and Agriculture, Addis Ababa University, P O Box 34, Bishoftu, EthiopiaTel +251929190312Fax +251114339933Email fufa.dawo@aau.edu.etIntroduction: Newcastle disease virus (NDV) cultures held in the isolate collections in Ethiopia between 1976 and 2008 were not characterized using biological and molecular techniques. The already characterized NDV isolates belonged to genotype VI but the genetic nature of previously collected isolates, which could shade light on the history of introduction into the country and their evolutionary relationships, were not established.Methods: A total of 14 NDVs (11 obtained from outbreak cases in chickens and three commercial vaccinal strains used in the country) were inoculated into specific pathogen free (SPF) embryonated chicken eggs (ECE). Allantoic fluids harvested from grown SPF ECE were tested by heamagglutination (HA) and heamagglutination inhibition (HI) tests. Partial F gene sequences were generated for all samples and molecular evolutionary relationships were reconstructed together with reference sequences freely available online. The pathogenicities of the isolates were assessed in vivo by determining their intracerebral pathogenicity index (ICPI) in day-old chicks and molecularly by determination of F gene cleavage sites.Results: Of these, 12 viruses (two vaccines and 10 outbreaks) were successfully propagated as evidenced by a positive heamagglutination (HA) test. These 12 propagated viruses were further characterized by heamagglutination inhibition (HI) test, of which only three viruses reacted with monoclonal antibody (MAb 617/616) specific for pigeon paramyxovirus-1. In addition, all 14 viruses were characterized by partial fusion (F) gene sequencing and phylogenetic tree reconstruction. The Ethiopian NDV isolates clustered with genotype VI viruses, forming two clades (groups 1 and 2) that have ancestral relationships with Egypt-1990 and Sudan-1975 like viruses.Discussion: The characterized genotype VI NDVs were genetically similar to currently circulating NDVs in Ethiopia. The isolates had cleavage sites consistent with mesogenic/velogenic NDV with a mean ICPI value of 1.76, indicating that the isolates were velogenic. Two and four highly virulent viruses were thermostable at 56°C for 2 hours and 1 hour, respectively. To reduce chicken mortality and production losses, proper control of the disease should be instituted using high quality and protective vaccines together with strong biosecurity measures.Keywords: antigenic, NDV, fusion gene sequence, molecular characterization, pathogenicity, velogenic

Anthrax is endemic in Ethiopia with sporadic outbreaks despite the regular vaccination of domestic livestock. This has raised concerns on the effectiveness of the vaccination strategy which may be associated with breaches in the vaccine cold chain maintenance. This study was aimed at demonstrating the tolerance of anthrax vaccine to cold chain breaches through evaluation of viable spore counts expressed as colony forming units per mL (CFU/mL) of freeze-dried and suspension anthrax vaccines stored at 5 °C, 20 °C and 37 °C for up to 6 months. Both vaccine formulations maintained above the recommended minimum required titre (2 × 106 culturable spores per dose for cattle, buffaloes and horses, and not

Bovine viral diarrhea (BVD) is an economically important cattle disease with worldwide distribution and characterized mainly by suboptimal fertility in the affected herds. The objectives of this study were to estimate the seroprevalence of BVDV within dairy cattle, to identify potential risk factors, and to assess the association with occurrence of reproductive problems. Sera (n = 954) collected from dairy cattle from 98 herds in southern and central Ethiopia were tested for BVDV antibodies using a commercial ELISA. Among screened sera samples, 20.9% (95% CI, 18.4, 23.6) tested positive to BVDV antibodies. The herd prevalence was 50% (95% CI, 40.1, 59.9) and the intra-herd prevalence ranged between 2.6 and 100% (mean = 31.4%) in positive herds. Geographic region, herd size, and animal arrangement in the farm had significant association with serostatus (p

Background: Infectious bursal disease virus (IBDV) is an avian viral pathogen that causes infectious bursal disease (IBD) of chickens. The disease is endemic in Ethiopia since 2002 and vaccination is the major means of disease prevention and control. 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 immunogenicity and protection level.Results: Identity of the vaccine seed was confirmed using gene-specific primers using reverse transcription polymerase chain reaction. Confluent monolayer of Vero cells was infected with vaccine virus and serial passage continued till passage ten. Characteristic virus induced cytopathic effect 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. Virus induced specific antibody was determined using indirect ELISA after vaccination of 14 days old chicks through ocular route. Accordingly, the antibody titer measured from Vero cells vaccinated chicks revealed similar level with the currently available CEF cell-based vaccine. Chicks vaccinated with Vero cell adapted virus showed complete protection against very virulent IBDV, while unvaccinated group had 60% morbidity and 25% mortality.Conclusions: The IBDV vaccine strain well adapted on Vero cells and found to be immunogenic induces antibodies development and successfully protects chicks against challenging with the circulating field IBDV isolate. Hence, it is recommended to produce IBD vaccine using Vero cell culture with enough quantity to conquer the limitations using CEF cells and thus to vaccinate chicks to protect against IBDV infection.

Avian infectious bronchitis virus is a highly contagious disease occurring in respiratory, urogenital, and reproductive tissues of chicken causing considerable losses due to death, egg drop, and reduced production. This preliminary study was conducted to investigate the prevalence of antibodies against infectious bronchitis virus (IBV) and to assess the potential risk factors in chickens of northwest Ethiopia. A cross-sectional study was conducted from November 2020 to June 2021. A total of 768 serum samples from three zones were collected. To investigate the presence of antibodies against IBV, the indirect ELISA serological test was applied. Positivity for anti-IBV antibodies was observed in 23.96% (95% CI: 20.98–27.14) of the samples. The mixed-effect logistic regression analysis of potential risk factors showed that IBV prevalence was significantly higher in young chickens than adults ( p < 0.001 ) and higher in intensive farm type than in extensive type ( p < 0.001 ). Based on the production purposes of the chickens, the odds of seropositivity for IB was significantly higher in layers than in broilers ( p < 0.001 ) and dual purposes ( p < 0.001 ). This study revealed higher seroprevalence in farms which had the “all-in-all-out” rearing method than in farms with different batches in one house with a significant difference ( p < 0.001 ), higher seroprevalence in the poor ventilated type than in good ones ( p < 0.001 ), and higher seroprevalence in the houses that did not remove used litter at all than houses of completely disposed and partially disposed litter ( p = 0.002 ). Moreover, disinfection of houses had significant effect on the occurrence of IB. Having personal protective equipment was significantly affecting the occurrence of IB, being higher in the farms that have no wearing clothes and shoe than in those having wearing clothes and shoe ( p = 0.002 ). In conclusion, the seroprevalence finding in the present study indicated that the organism is circulating among the population of chickens and high enough to cause significant economic losses Therefore, poultry houses should be cleaned, disinfected, and well ventilated and farm attendants should have separate farm shoe and clothes. Further studies on the virus isolation and molecular characterization of the target gene are needed in the study area.

Background: Pasteurella multocida is a heterogeneous species and opportunistic pathogen that causes bovine respiratory disease. This disease is one of an economically important disease in Ethiopia. Losses due to mortality and associated expenses are estimated to be higher in the country. Studies revealed that limited information is available regarding the capsular types, genotypes, and antimicrobial sensitivity of P. multocida isolates circulating in the country. This suggests, further molecular advances to understand the etiological diversity of the pathogens representing severe threats to the cattle population. Results: Bacteriological analysis of nasopharyngeal swab and pneumonic lung tissue samples collected from a total of 400 samples revealed isolation of 61 (15.25%) P. multocida subspecies multocida. 35 (20.59%) were isolated from calves and 26 (11.30%) from adult cattle. Molecular analysis using PCR assay targeting KMT1 gene (~460 bp) amplification was shown in all presumptive isolates. Capsular typing also confirmed the presence of serogroup A (hyaD-hyaC) gene (~1044 bp) and serogroup D (dcbF) gene (~657 bp) from 56 (91.80%) and 5 (8.20%) isolates, respectively. The biovar typing identified 48 (78.69%) isolates of biovar 3. Phylogenetic analysis based on the hyaD-hyaC gene nucleotide sequences showed that P. multocida serogroup A are genetically closely related to the different P. multocida isolates retrieved from the GenBank. Antibiogram of isolates revealed high sensitivity towards chloramphenicol (100%), amoxicillin (95.08%), and amikacin (90.16%). Resistance was observed in cloxacillin (88.52%) and Penicillin-G (77.05%). Conclusions: In the present study, P. multocida capsular type A biovar 3 was predominantly isolated from pneumonic cases in cattle in the study areas. The finding provides useful information for further research and choice of antimicrobials for treatment. Hence, further comprehensive molecular epidemiological investigation is proposed covering wider areas of the country to identify pathogens associated with BRD for an effective control strategy.

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 &ldquo, one world, one health&rdquo, 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&ndash, 3L) at 2 &times, 106 cells/mL resulted in consistent infectious adenoviral vector titers of approximately 5&ndash, 6 &times, 108 TCID50/mL (approximately 1011VP/mL) in the harvest lysates. Groups of chickens were twice immunized with 1 &times, 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.

Sequencing of the VP2 region was carried out to identify amino acid mismatches between vaccine strains and field isolates of infectious bursal disease virus (IBDV). Viruses were isolated in chicken embryo fibroblast (DF-1) cells using pooled samples of bursa collected from nine outbreaks, which affected 30,250 chickens in five localities, with an overall mortality of 47.87%. Virus strains were identified by comparing the deduced amino acid sequence between positions 232 and 446 of the immunodominant VP2 epitope. All of the pooled samples were positive for IBDV. RT-PCR yielded a 645-bp DNA fragment of the VP2 gene. Phylogenetic analysis of this fragment revealed clustering of these isolates with very virulent IBDV strains. The amino acid sequences of these isolates were identical to those of the European very virulent strains UK 661 and DV 86, except at position 222, but differed from the vaccine strains used in Ethiopia, suggesting the possible introduction of virulent virus strains to Ethiopia from Europe. Our study demonstrates the widespread presence of very virulent strains of IBDV on poultry farms in Ethiopia and demonstrates the need to evaluate the protective level of existing vaccines against circulating field viruses.

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.

Sheep pox (SPP) and goat pox (GTP) are viral diseases of sheep and goats caused by sheep pox virus (SPPV) and goat pox virus (GTPV), respectively. SPPV and GTPV belong to the genus Capripoxvirus of the family Poxviridae, together with lumpy skin disease virus (LSDV) of cattle. They have double-stranded DNA genomes of approximately 134–147 kbp. SPPV and GTPV are closely related to LSDV though they possess specific nucleotide differences suggesting distinct phylogeny. SPP and GTP are notifiable diseases to the World Organisation for Animal Health (OIE). They are highly contagious diseases: the viruses spread through direct contact with lesions or contaminated objects, feed, and wool. SPP and GTP are endemic in Africa (except southern Africa), central Asia, the Indian subcontinent, the Middle East, Turkey, Greece, and some eastern European countries. Clinically, the presence of nodular skin lesions, mostly around the mouth and perineum regions, is a typical sign of the disease. Capripoxviruses classification and their nomenclature have been mainly based on the affected host species, creating a challenge for isolates naming. For a more accurate naming, it is better to use molecular methods as support to identify and classify capripoxvirus isolates. Conventional and real-time PCR methods are available that could help with the simultaneous detection and genotyping of the viruses. SPPV and GTPV as well as LSDV cross-react serologically, making it difficult to differentiate them using serological methods. To prevent and control SPP and GTP, illegal animal movement restrictions and vaccination campaigns with adequate vaccines and sufficient vaccination coverage are two very effective measures. The development of a high-throughput serological assay (ELISA) with better sensitivity and specificity and the development of a safe and effective vaccine, which can support the differentiation of infected from vaccinated animals (DIVA), are highly required.

Lumpy skin disease (LSD) is a viral disease of cattle caused by lumpy skin disease virus (LSDV). LSDV shares high degree of sequence homology with goatpox virus (GTPV) and sheeppox virus (SPPV), the two other members of the genus Capripoxvirus of the family Poxviridae. Genetically LSDV is a double-stranded DNA genome of approximately 151 kbp. LSD is an economically important and notifiable animal disease by the World Organisation for Animal Health (OIE). Clinically LSD is characterized by fever and the appearance of nodules on the skin and mucous membranes. In severe and chronic cases, nodular skin lesions cover the entire body and become deep scab and eroded. Transmission of the disease occurs predominantly by insects possibly through mechanical vectors, contaminated feed and water, infected saliva, and rarely natural contact. LSD is endemic in many African countries and mostly coexists with sheeppox and goatpox. Recently, LSD has been rapidly spreading to the Middle East, Turkey, and Russia, the Balkan and European Union countries. Diagnosis is mainly based on observation of clinical signs and the detection of virus genome using conventional and real-time PCR methods. In Africa, prevention and control of LSD relies on vaccination using live attenuated vaccines derived from Kenyan or South African LSDV strains. Vaccine that can allow the differentiation of infected from vaccinated animals (DIVA) and high-throughput serological method for the detection of specific antibody need to be developed. Alternatively, a 12 nucleotide deletion that exists only on the G-protein-coupled chemokine receptor gene of LSDV field isolates can be used to differentiate wild-type from vaccine strains by sequencing and real-time PCR.

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.

Analysis of scab samples collected from poultry during outbreaks of fowlpox in Mozambique in 2016 revealed the presence of clade E avipoxviruses. Infected poultry were from flocks that had been vaccinated against fowlpox virus. These findings require urgent reevaluation of the vaccine formula and control strategies in this country.