Phenotypic and genetic characterization of MERS coronaviruses from Africa to understand their zoonotic potential.

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.

Comparative evaluation of RBPT, I-ELISA, and CFT for the diagnosis of brucellosis and PCR detection of Brucella species from Ethiopian sheep, goats, and cattle sera.

BACKGROUND: Brucellosis is an economically devastating animal disease and has public health concern. Serological methods such as Rose Bengal Plate Test (RBPT), Complement Fixation Test (CFT), and Indirect-Enzyme-Linked Immunosorbent Assay (I-ELISA) have been used to detect brucellosis. However, there is limited comparative evaluation studies and lack of molecular confirmation of the causative agents in the study areas. The study was aimed to compare RBPT, I-ELISA, CFT, and confirmation using Polymerase Chain Reaction (PCR). A total of 2317 sera samples were collected from brucellosis-affected areas of Ethiopia with no vaccination history. All sera were subjected to comparative serological assays. Post-cross tabulation, sensitivity, and specificity were determined using Receiver Operating Characteristics (ROC) curve analysis software. PCR was performed on 54 seropositive samples using genus- and species-specific primers. RESULTS: Among the 2317 sera tested for comparative serological assays, 189 (8.16%) were positive for RBPT, 191 (8.24%) for I-ELISA, and 48 (2.07%) for CFT. Sensitivity to RBPT was 100% (95%) in shoats and 74% (95%) in cattle. Specificity on RBPT was 98.69% (95%), 99.28% (95%), 100% (95%) in sheep, goats, and cattle, respectively. CFT sensitivity was 4 (95%) in sheep, 9.65 (95%) goats, and 72 (95%) cattle. Specificity on CFT was 100% (95%) for sheep, goats, and cattle. A 223bp Brucella genus-specific and 156bp B. abortus species-specific detected. However, B. melitensis not detected. CONCLUSION: In this study, I-ELISA was the most sensitive and specific test. RBPT detected all Brucellosis-infected sheep and goats; nevertheless, it showed false positive in sheep and goats and false negative in cattle. The presence of B. abortus in small and large ruminants was confirmed by PCR. This is the first report of B. abortus detection in small ruminant in Ethiopia. B.abortus detected in non-preferred hosts. The findings suggest further study on molecular epidemiology of Brucella species.

Marek's disease in chicken farms from Northwest Ethiopia: gross pathology, virus isolation, and molecular characterization.

Marek's disease virus (MDV) is a highly contagious, immunosuppressive, and oncogenic chicken pathogen causing marek's disease (MD). In this outbreak-based study, 70 dual-purpose chickens that originated from poultry farms in Northwest Ethiopia and suspected of MD were sampled for pathological and virological study from January 2020 to June 2020. Clinically, affected chickens showed inappetence, dyspnea, depression, shrunken combs, and paralysis of legs, wings, and neck, and death. Pathologically, single or multiple greyish white to yellow tumor-like nodular lesions of various size were appreciated in visceral organs. In addition, splenomegaly, hepatomegaly, renomegaly, and sciatic nerve enlargement were observed. Twenty-seven (27) pooled clinical samples i.e. 7 pooled spleen samples and 20 pooled feathers samples were aseptically collected. Confluent monolayer of Chicken Embryo Fibroblast cells was inoculated with a suspension of pathological samples. Of this, MDV-suggestive cytopathic effects were recorded in 5 (71.42%) and 17 (85%) pooled spleen and feather samples respectively. Molecular confirmation of pathogenic MDV was conducted using conventional PCR amplifying 318 bp of ICP4 gene of MDV-1, of which, 40.9% (9/22) tested positive. In addition, 5 PCR-positive samples from various farms were sequenced further confirming the identity of MDV. The ICP4 partial gene sequences were submitted to GenBank with the following accession numbers: OP485106, OP485107, OP485108, OP485109, and OP485110. Comparative phylogenetics showed, two of the isolates from the same site, Metema, seem to be clonal complexes forming distinct cluster. The other three isolates, two from Merawi and one from Debretabor, appear to represent distinct genotypes although the isolate from Debretabor is closer to the Metema clonal complex. On the other hand, the isolates from Merawi appeared genetically far related to the rest of the 3 isolates and clustered with Indian MDV strains included in the analysis. This study presented the first molecular evidence of MDV in chicken farms from Northwest Ethiopia. Biosecurity measures should strictly be implemented to hinder the spread of the virus. Nationwide studies on molecular characteristics of MDV isolates, their pathotypes, and estimation of the economic impact associated with the disease may help justify production and use of MD vaccines within the country.

Vaccine matching and antigenic variability of foot-and-mouth disease virus serotypes O and A from 2018 Ethiopian isolates.

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.

Serological Evidence of Infectious Laryngotracheitis Infection and Associated Risk Factors in Chickens in Northwestern Ethiopia.

Infectious laryngotracheitis (ILT) is a disease of high economic consequence to the poultry sector. Gallid herpesvirus 1 (GaHV-1), a.k.a infectious laryngotracheitis virus (ILTV), under the genus Iltovirus, and the family Herpesviridae, is the agent responsible for the disease. Despite the clinical signs on the field suggestive of ILT, it has long been considered nonexistent and a disease of no concern in Ethiopia. A cross-sectional study was conducted from November 2020 to June 2021 in three selected zones of the Amhara region (Central Gondar, South Gondar, and West Gojjam zones), Ethiopia, with the objective of estimating the seroprevalence of ILTV in chickens and identifying and quantifying associated risk factors. A total of 768 serum samples were collected using multistage cluster sampling and assayed for anti-ILTV antibodies using indirect ELISA. A questionnaire survey was used to identify the potential risk factors. Of the 768 samples, 454 (59.1%, 95% CI: 0.56-0.63) tested positive for anti-ILTV antibodies. Mixed-effect logistic regression analysis of potential risk factors showed that local breeds of chicken were less likely to be seropositive than exotic breeds (OR: 0.38, 95% CI: 0.24-0.61). In addition, factors such as using local feed source (OR: 6.53, 95% CI: 1.77-24.04), rearing chickens extensively (OR: 1.97, 95% CI: 0.78-5.02), mixing of different batches of chicken (OR: 14.51, 95% CI: 3.35-62.77), careless disposal of litter (OR: 1.62, 95% CI: 0.49-4.37), lack of house disinfection (OR: 11.05, 95% CI: 4.09-47.95), lack of farm protective footwear and clothing (OR: 20.85, 95% CI: 5.40-80.45), and careless disposal of dead chicken bodies had all been associated with increased seropositivity to ILTV. Therefore, implementation of biosecurity measures is highly recommended to control and prevent the spread of ILTV. Furthermore, molecular confirmation and characterization of the virus from ILT suggestive cases should be considered to justify the use of ILT vaccines.

Seroprevalence and association of bovine viral diarrhea virus (BVDV) serostatus with reproductive problems in dairy cattle in central and southern Ethiopia.

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 < 0.05). Cattle from southern Ethiopia and herds of large size had 2.8 (95% CI, 1.9, 4.2) and 2.6 (95% CI, 1.5, 4.6) times higher odds of being seropositive compared to their counterparts, respectively. Serostatus to BVDV was associated with history of anestrus, repeat breeding (RB), mastitis, and extended calving interval (CI) (p < 0.05). Animals with history of extended CI and mastitis were 1.7 (95% CI, 1.0, 2.7) and 2.2 (95% CI, 1.5, 3.2) times more likely to be seropositive compared with those with normal CI and no history of mastitis, respectively. On the other hand, animals with history of anestrus and RB were less likely to be seropositive to BVDV compared to cattle with no such history. Sera from 26 selected cattle were also examined using reverse transcription (RT)-PCR for detection of BVDV RNA; however, all samples tested were negative for the presence of BVDV nucleic acid. Our study highlights the variation in BVDV status within Ethiopian dairy herds, and association with some important reproductive performance traits and potential risk factors.

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.

Sequence-based comparison of field and vaccine strains of infectious bursal disease virus in Ethiopia reveals an amino acid mismatch in the immunodominant VP2 protein.

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.

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.

Evaluation of spray and oral delivery of Newcastle disease I2 vaccine in chicken reared by smallholder farmers in central Ethiopia.

BACKGROUND: Newcastle disease (ND) is a highly infectious disease causing considerable economic losses to poultry farmers worldwide. Conventional vaccine delivery methods are not suitable for smallholder and rural poultry producers, and thus appropriate vaccination methods need to be sought. This study was carried out with the main objective of evaluating the efficacy of ND I2 vaccine delivered via drinking water and spray under smallholder farmers' condition in Minjar-Shenkora district, central Ethiopia. Twenty households were randomly assigned to intervention and control groups. Chickens owned by the selected households were randomly assigned to one of the three intervention groups. Blood samples were collected regularly for antibody assay from individual chicken vaccinated with ND I2 vaccine using different routes. RESULTS: At baseline, there was no difference in antibody titer among the experimental groups. After the first and booster vaccinations, the three vaccinated groups had significantly higher antibody titer (P < 0.001) than the unvaccinated control group. Interestingly, there was no statistically significant difference in antibody titer among the vaccinated groups. Out of the 40 chicken in the unvaccinated control only 14 had antibody titter≥ log23. Similarly 19/37 of chicken in the drinking water group, 19/37 of chicken in the eye drop group and 20/40 chicken in the spray group had antibody titer ≥ log23. Two weeks after the first vaccination the proportion of chicken with antibody titer ≥ log23 rose to 23/37, 30/37 and 29/40 in the group vaccinated via drinking water, eye drop and spray, respectively. The proportion remained low in unvaccinated group. Hundred percent of the vaccinated chicken survived after infection with the virulent ND virus (Alemaya strain); whereas only 40% survived from the unvaccinated control group. CONCLUSION: The results of this study showed that ND I2 vaccine administered via drinking water and spray under smallholder farmers' situation provoked protective antibody level similar to the eye drop method. The use of ND I2 vaccine could contribute to food security if used by rural poultry farmers properly.

Identification of Clade E Avipoxvirus, Mozambique, 2016.

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.

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.

Infectious bursal disease: seroprevalence and associated risk factors in major poultry rearing areas of Ethiopia.

The study was conducted in eight districts of Ethiopia with the objectives of determining the seroprevalence and associated risk factors of infectious bursal disease (IBD). From the total of 2,597 chicken serum samples examined using ELISA, 83.1 % were found positive. The highest seroprevalence was found at Mekele (90.3 %) while the lowest was recorded at Gondar district (69.8 %). These differences among the study areas were statistically significant (p < 0.05). Highest seroprevalence was found in crossbreed of chicken (91.4 %) while the lowest was recorded in indigenous breed of chicken (81.4 %). This difference was statistically significant (p < 0.05) among the three breeds of chickens, but sex was not statistically significant (p > 0.05). The seroprevalence of the disease was found high in young (≤ 8 weeks) age group (86.6 %) while the lowest prevalence was recorded in adults (>8 weeks) (72 %). This is also statistically significant (p < 0.05) between young and adult age groups. The prevalence of IBD in different production system indicated that higher seroprevalence was recorded in intensive production system (85.9 %) while the lowest was recorded in extensive production system (81.6 %). This difference is also statistically significant (p < 0.05).

A study on seroprevalence of caprine brucellosis under three livestock production systems in southern and central Ethiopia.

Caprine brucellosis in Ethiopia is less commonly reported with limited information on the disease status in the country. The objective of this study was therefore to highlight the status of goat brucellosis in three distinctly different livestock production systems of southern and central Ethiopia. A total 3,315 goats of different age and sex, living with other animals in variable flock size, were sampled from 448 flocks raised in sedentary, pastoral and agro-pastoral production systems. Goats were bled aseptically and sera were collected for serial testing using Rose Bengal Plate Test as screening test and subsequently complement fixation test as confirmatory test. Questionnaire and laboratory data were analysed for descriptive, univariable and multivariable logistic regression analysis both at individual and flock level (STATA 11). The study revealed an overall animal level seroprevalence of 1.9 % (95 % CI 1.5, 2.4). In sedentary production system, the observed seroprevalence was 0.6 % (95 % CI 0.2, 0.9) while 1.9 % (95 % CI 1.1, 2.7) and 7.6 % (95 % CI 5.1, 10.1) were the proportion of seroreactors for agro-pastoral and pastoral production systems, respectively. The observed prevalence difference between the three production systems was statistically significant (P < 0.05). At the flock level analysis, 11.2 % (95 % CI 8.2, 14.1) of the flocks sampled had at least one seropositive goat among themselves. Like individual level analysis, the highest prevalence of 32.5 % (95 % CI 21.9, 43.0) was recorded for pastoral production system, followed by agro-pastoral, 13.0 % (95 % CI 7.0, 19.0) and sedentary production system, 3.6 % (95% CI 1.3, 6.0). Accordingly, the odds of Brucella seropositivity were higher (OR = 12.8) in pastoral followed by agro-pastoral (OR = 4.0) in relation to sedentary production system. Large numbers of seroreactors were observed in adult age living in larger flocks with other livestock species. However, no difference was noted between male and female goats. Finally, the need for nationwide survey and subsequent designing and implementation of appropriate control measure is suggested.

Seroprevalence and risk factors associated with bovine herpesvirus 1 (BHV-1) infection in dairy cattle in southern and central Ethiopia.

Bovine herpesvirus 1 (BHV-1) is an important pathogen of cattle with a worldwide distribution. It occurs as a subclinical, mild or severe disease. The clinical signs may vary widely with respiratory, genital, ocular and encephalomyelitis form. This cross-sectional study was carried out between May 2019 and March 2020 with the aim to estimate the seroprevalence of bovine herpesvirus 1 (BHV-1) and to identify related potential risk factors in dairy cattle in central and southern Ethiopia. A total of 954 serum samples were obtained from randomly selected dairy cattle in 98 herds. The samples were collected from animals over 6 months old and tested using a BHV-1 antibody blocking enzyme linked immunosorbent assay (b-ELISA). The study showed that the animal- and herd-level seroprevalence of BHV-1 was 30.0 % (95 % CI: 21.7, 39.9) and 75.5 % (95 % CI: 65.9, 83.1), respectively. Multiple logistic regression model demonstrated that adult animals (> 2.5 years) (OR = 2.4, 95 % CI: 1.1, 5.5) had higher seroprevalence of BHV-1 compared to their counterparts (p < 0.05). Cattle in farms using artificial insemination (AI), and both AI and bulls had a 3.9 (95 % CI: 1.2, 13.3) and 5.1 (95 % CI: 1.8, 14.8) odds of being seropositive, respectively, compared to farms using bulls only. Arrangement of animals in a tail-to-tail fashion appeared to be protective against BHV-1 infection (p < 0.05). However, source of the animal was not associated with BHV-1 serostatus (p > 0.05). The animal- and herd-level prevalence recorded in our study confirms that BHV-1 infection is widespread and remains endemic in dairy cattle of central and southern Ethiopia.

Application of structural equation modelling to inform best management strategies for Marek's disease in Amhara region, Ethiopia.

Marek's disease, a highly contagious and an economically significant oncogenic and paralytic viral diseases of poultry, is becoming a serious problem in Ethiopia's poultry sector. The aim of the study was to examine the relationship between risk factors and their contribution to develop risk with the intentions to implement MD control measures in the different chicken production systems of Ethiopia using the SEM framework. A questionnaire was designed based on the framework and each model constructed was measured using a set of rating scale items. Thus, a sample size of 200 farmers from different production systems were chosen for the data collection. From the analysis, Cornbrash's Alpha (coefficient of reliability) based on the average inter-item correlations were evaluated for each parameter. The result showed that when litter management goes up by 1, the number of sick goes down by 37.575, the number of staff goes up by 1, the number of sick goes down by 7.63, litter management goes up by 1, the number of deaths goes down by 2.505, flock size goes up by 1, the number of deaths goes down by 0.007 than the rest of the activities. The result of this structural equation modeling finding indicates that the data fit the model well (χ2 = 0.201, RMSEA = 0.000, CFI = 1.00, TLI = 1.496, Degrees of freedom = 2) and the model was appropriated. In conclusion, flock size, litter management and number of staff activities have more impact on the numbers of sick, drops in egg production and the number of deaths. Therefore, practicing regular awareness creation for producers regarding management techniques is recommended.

Combined Adjuvant Formulations Enhanced an Immune Response of Trivalent Foot and Mouth Disease Vaccine in Cattle.

Introduction: 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.

Intranasally Delivered Adenoviral Vector Protects Chickens against Newcastle Disease Virus: Vaccine Manufacturing and Stability Assessments for Liquid and Lyophilized Formulations.

Newcastle disease (ND) remains a critical disease affecting poultry in sub-Saharan Africa. In some countries, repeated outbreaks have a major impact on local economies and food security. Recently, we developed an adenovirus-vectored vaccine encoding the Fusion protein from an Ethiopian isolate of Newcastle disease virus (NDV). The adenoviral vector was designed, and a manufacturing process was developed in the context of the Livestock Vaccine Innovation Fund initiative funded by the International Development Research Centre (IDRC) of Canada. The industrially relevant recombinant vaccine technology platform is being transferred to the National Veterinary Institute (Ethiopia) for veterinary applications. Here, a manufacturing process using HEK293SF suspension cells cultured in stirred-tank bioreactors for the vaccine production is proposed. Taking into consideration supply chain limitations, options for serum-free media selection were evaluated. A streamlined downstream process including a filtration, an ultrafiltration, and a concentration step was developed. With high volumetric yields (infectious titers up to 5 × 109 TCID50/mL) in the culture supernatant, the final formulations were prepared at 1010 TCID50/mL, either in liquid or lyophilized forms. The liquid formulation was suitable and safe for mucosal vaccination and was stable for 1 week at 37 °C. Both the liquid and lyophilized formulations were stable after 6 months of storage at 4 °C. We demonstrate that the instillation of the adenoviral vector through the nasal cavity can confer protection to chickens against a lethal challenge with NDV. Overall, a manufacturing process for the adenovirus-vectored vaccine was developed, and protective doses were determined using a convenient route of delivery. Formulation and storage conditions were established, and quality control protocols were implemented.

Molecular evidence of very virulent infectious bursal disease viruses in chickens in Ethiopia.

Infectious bursal disease virus (IBDV) is an important immunosuppressive pathogen of chickens worldwide. The introduction and evolution of IBDV in most African countries, especially in Ethiopia, remains unclear. We have investigated IBDV isolates obtained from commercial broilers, indigenous chickens, and pullets. The hypervariable region of the virus protein (VP) 2 and the 5' two-thirds of VP1 of 11 IBDV isolates were characterized by RT-PCR and further sequencing. All isolates were identified as very virulent (vv) IBDV based on the predicted amino acid (aa) sequences of the VP2 protein. Interestingly, the sequence analysis of the 5' two-thirds of VP1 indicated that the Ethiopian IBDV strains have aa residues typical for vvIBDV and for attenuated IBDV strains. Among all IBDV strains included in this study for phylogenetic comparison of VP2 nucleotide sequences, Ethiopian strains form a cluster within the vvIBDV lineage. We have also shown that Ethiopian IBDV strains have mutations in the VP1 region. Their roles in IBDV virulence may require further in vivo studies. As depicted in this study, the nucleotide and aa sequence analysis of VP1 in addition to VP2 is necessary to obtain a clear picture of the molecular evolution of IBDV.