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Abdela Bulbula,1 Bizunesh Borena,2 Biniam Tadesse,1 Abde Aliy,1 Demessa Negessu1 1Department of Bacterial Serology, Animal Health Institute (AHI), Sebeta, Oromia, Ethiopia; 2Department of Veterinary Laboratory Technology, Ambo University, Ambo, Oromia, EthiopiaCorrespondence: Abdela Bulbula, Department of Bacterial Serology, Animal Health Institute (AHI), PO Box 04, Sebeta, Oromia, Ethiopia, Tel +251 9103989, Email abdallahbulbula@gmail.comBackground: Marek’s disease virus is a devastating infection, causing high morbidity and mortality in chickens in Ethiopia.Methods: The current study was conducted from March to November, 2021 with the general objective of performing antemortem and postmortem, isolation, and molecular detection of Marek’s disease virus from outbreak cases in southwestern Ethiopia. Accordingly, based on outbreak information reported from the study sites namely, Bedelle, Yayo, and Bonga towns in southwestern Ethiopia, 50 sick chickens were sampled. The backyard and intensive farming systems of chickens were included in the sampling and priorities were given for chickens that showed clinical signs that are characteristics of Marek’s disease.Results: By clinical examinations, paralysis of legs and wings, gray eye, loss of weight, difficulty in breathing, and depression were recorded on all chickens sampled for this study and death of diseased chickens was observed. In addition, enlargement of the spleen and gross lesions of the liver and heart were recorded during postmortem examination. The death of infected chickens was observed in both vaccinated and non-vaccinated flocks. Out of 50 pooled feather follicle samples, Marek’s disease virus was isolated from 14/50 (28%) by cell culture method and out of six tissue samples, the virus was isolated from 5/6(83.30%). By Real time polymerization chain reaction technique, which was targeted to detect the Meq gene, Marek’s disease virus was detected from 18/50 feather follicles which accounts for 36% of sampled chickens.Conclusion: In general, current study showed that the circulating Marek’s disease virus in southwestern Ethiopia was caused by the oncogenic Gallid herpesvirus-2 (Serotype-1). Further research on molecular characterization of revolving virus in current and other regions is recommended for effective control of the disease through vaccination.Keywords: Ethiopia, Marek’s diseases, isolation, molecular detection, outbreak

Pacific Biosciences has developed a platform that may sequence one molecule of DNA in a period via the polymerization of that strand with one enzyme. Single-molecule real-time sequencing by Pacific BioSciences’ technology is one of the most widely utilized third-generation sequencing technologies. PacBio single-molecule real-time Sequencing uses the Zero-mode waveguide’s ingenuity to distinguish the best fluorescence signal from the stable fluorescent backgrounds generated by disorganized free-floating nucleotides. PacBio single-molecule real-time sequencing does not require PCR amplification, and the browse length is a hundred times longer than next-generation sequencing. It will only cover high-GC and high-repeat sections and is more accurate in quantifying low-frequency mutations. PacBio single-molecule real-time sequencing will have a relatively high error rate of 10%-15% (which is practically a standard flaw of existing single-molecule sequencing technology). In contrast to next-generation sequencing, however, the errors are unintentionally random. As a result, multiple sequencing will effectively rectify the bottom deviance. Unlike second-generation sequencing, PacBio sequencing may be a technique for period sequencing and doesn’t need an intermission between browse steps. These options distinguish PacBio sequencing from second-generation sequencing, therefore it’s classified because of the third-generation sequencing. PacBio sequencing produces extremely lengthy reads with a high error rate and low yield. Short reads refine alignments/assemblies/detections to single-nucleotide precision, whereas PacBio long reads provide reliable alignments, scaffolds, and approximate detections of genomic variations. Through extraordinarily long sequencing reads (average >10,000 bp) and high accord precision, the PacBio Sequencing System can provide a terribly high depth of genetic information. To measure and promote the event of modern bioinformatics tools for PacBio sequencing information analysis, a good browse machine is required.

Background: Aeromonas hydrophila is a zoonotic bacterial pathogen that frequently causes disease and mass mortalities among cultured and feral fishes worldwide. In Ethiopia, A. hydrophila outbreak was reported in Sebeta fish ponds and in Lake Tana fishery. However, there is no to little information on the molecular, and phenotypical characteristics of A. hydrophila in Ethiopian fisheries. Therefore, a cross-sectional study was conducted from November 2020 to May 2021 in selected Ethiopian Rift valley lakes.Results: A total of 140 samples were collected aseptically from fish (Muscle, Gill, Intestine, Spleen and Kidney) from fish landing sites, market and restaurants with purposive sampling methods. Aeromonas selective media (AMB), morphological and biochemical tests were used to isolate and identify A. hydrophila. Accordingly, the pathogen was isolated from 81 (60.45%) of samples. Among the isolates 92.59% expressed virulence trait through β hemolysis on blood agar media with 5% sheep blood. Moreover, 54 strains (66.67%) were further confirmed with Real-Time PCR (qPCR) using ahaI gene specific primers and optimized protocol. The highest (68.51%) were detected from live fish, (24.07%) were from market fish and the lowest (7.4%%) were from ready-to-eat products. Antibiogram analysis was conducted on ten representative isolates. Accordingly, A. hydrophila isolates were susceptible to ciprofloxacin (100%), chloramphenicol (100%) and ceftriaxone (100%). However, all ten isolates were resistant to Amoxicillin and Penicillin.Conclusions: The study indicates A. hydrophila strains carrying virulence ahaI gene that were ß-hemolytic and resistant to antibiotics commonly used in human and veterinary medicine are circulating in the fishery. The detection of the pathogen in 140 of the sampled fish population is alarming for potential outbreaks and zoonosis. Therefore, further molecular epidemiology of the disease should be studied to establish potential inter host transmission and antibiotic resistance traits. Therefore, raising the public awareness on risk associated with consuming undercooked or raw fish meat is pertinent.

Wastewater surveillance systems have become an important component of COVID-19 outbreak monitoring in high-income settings. However, its use in most low-income settings has not been well-studied. This study assessed the feasibility and utility of wastewater surveillance system to monitor SARS-CoV-2 RNA in Addis Ababa, Ethiopia. The study was conducted at nine Membrane Bio-reactor (MBR) wastewater processing plants. The samples were collected in two separate time series. Wastewater samples and known leftover RT-PCR tested nasopharyngeal swabs were processed using two extraction protocols with different sample conditions. SARS-CoV-2 wastewater RT-PCR testing was conducted using RIDA GENE SARS-CoV-2 RUO protocol for wastewater SARS-CoV-2 RNA testing. Wastewater SARS-CoV-2 RNA RT-PCR protocol adaptation, optimization, and detection were conducted in an Addis Ababa, Ethiopia context. Samples collected during the first time series, when the national COVID-19 case load was low, were all negative. Conversely, samples collected during the second time series were all positive, coinciding with the highest daily reported new cases of COVID-19 in Ethiopia. The wastewater-based SARS-CoV-2 surveillance approach is feasible for Addis Ababa. The COVID-19 wastewater based epidemiological approach can potentially fill the evidence gap in distribution and dynamics of COVID-19 in Ethiopia and other low-income settings.

Takele Worku,1 Morka Dandecha,2 Deraje Shegu,1 Abde Aliy,1 Demessa Negessu1 1Department of Virology and Molecular Biology, National Animal Health Diagnostic and Investigation Center, Sebeta, Oromia, Ethiopia; 2Department of Veterinary Laboratory Technology, Ambo University, Ambo, Oromia, EthiopiaCorrespondence: Morka Dandecha, Department of Veterinary Laboratory Technology, Ambo University, Ambo, Oromia, Ethiopia, Tel +251-910309600, Email morkadandecha@gmail.comBackground: Newcastle disease is a major viral disease of poultry. The virus is a major problem for chickens in Ethiopia and there is a scarcity of updated information on the virological and molecular status of confirmation of Newcastle disease outbreak cases in the country.Methods: Newcastle disease outbreaks were investigated from February 2021 to October 2021 in central Ethiopia to isolate and detect the virus by cell culture and reverse transcriptase PCR. A total of 44 pooled tissue specimens were sampled from sick and recently dead chickens showing typical clinical signs of Newcastle disease. Virus isolation were performed using DF-1 cells and detection of the virus was done by real-time PCR.Results: Out of 44 collected tissue samples, 38.63% (17/44) were positive on DF-1 cells. The result shows 17 of the clinically sick and dead chickens were positive for the virus by reverse transcriptase polymerase chain reaction. Based on the sample type, 54.54% (6/11) of the brain samples, 36.36% (4/11) of the intestines, 54.54% (6/11) of lung and trachea, 9% (1/11) of pooled liver, kidney, heart, and spleen samples were positive. Viruses were isolated in the proportions 37.5% (6/16), 25% (2/8), 50% (2/4), 25% (1/4), 50% (2/4) and 50% (4/8) from Sebeta, Bishoftu, Sululta, Nifas Silk, Kolfe and Yeka, respectively.Conclusion: This study showed that Newcastle disease is a major viral disease causing death of chickens in the study area. Therefore, any control approach should focus on the appropriate characterization of the virus strain causing the outbreak in the study area.Keywords: central Ethiopia, chickens, isolation, Newcastle diseases virus, RT-PCR

Shazali Mohammed Awel,1,2 Getachew Mulatu Dilba,3 Bruk Abraha,2 Demeke Zewde,4 Bayeta Senbata Wakjira,4 Abde Aliy4 1Jigjiga University, College of Veterinary Medicine, Jigjiga, Ethiopia; 2Haramaya University, College of Veterinary Medicine, Dire Dawa, Ethiopia; 3Ambo University, College of Agriculture and Veterinary Science, Department of Veterinary Laboratory Technology, Ambo, Oromia, Ethiopia; 4National Animal Health Diagnostic and Investigation Center, Sebeta, EthiopiaCorrespondence: Getachew Mulatu DilbaAmbo University, College of Agriculture and Veterinary Science, Department of Veterinary Laboratory Technology, P.O. Box:19, Ambo, Oromia, EthiopiaTel +251-912717217Email getachew.mulatu@ambou.edu.etBackground: Foot and mouth disease is a debilitating and highly contagious transboundary disease of cattle that can cause a huge economical loss globally. It is a notifiable disease in Ethiopia, and it is thought to be causing a decrease in cattle productivity and production.Methods: A cross-sectional study and outbreak investigation were performed to estimate seroprevalence, identify associated factors and serotypes of FMDV in dairy cattle around Addis Ababa. A multi-stage random sampling technique was employed for the selection of sampling units for the seroprevalence study. A total of 383 blood samples were collected using plain vacutainer tubes and the obtained sera were tested by 3ABC-Ab ELISA at the NAHDIC lab. Also, from outbreak cases, 20 epithelial tissue samples were collected purposively for the molecular detection of FMDV serotypes.Results: The overall seroprevalence of FMD in dairy cattle was 72.1% (95% CI=67.27– 76.50). The seroprevalence in dairy cattle of Ada Berga, Holeta, and Sululta districts was 97.2%, 71.4%, and 57.6%, respectively. Up on Chi-square analysis, age, body condition, and management system were significantly associated with FMD seroprevalence (p< 0.05). Besides, multivariable logistic regression analysis showed that district, age, body condition, and management were significantly associated with FMD seroprevalence (p< 0.05). The odds of being seropositive to FMD were 6.9 (95% CI=1.8– 24.9; p=0.005) and 2.3 (95% CI=1.2– 4.7; p=0.01) times higher in cattle found in Ada Berga and Holeta Woreda. From outbreak cases, 18 (90.0%) were identified positive for FMDV serotype O.Conclusion: The current study revealed higher seroprevalence was recorded in the study area and associated risk factors identified statically, serotype O of FMDV was identified from outbreak cases. Therefore, it is critical to design and implement feasible control and prevention mechanisms based on the type of circulating virus serotype.Keywords: central Ethiopia, dairy cattle, FMD virus, molecular detection, seroprevalence

Bovine tuberculosis (bTB) is endemic in cattle in Ethiopia, a country that hosts the largest national cattle herd in Africa. The intensive dairy sector, most of which is peri-urban, has the highest prevalence of disease. Previous studies in Ethiopia have demonstrated that the main cause is Mycobacterium bovis , which has been investigated using conventional molecular tools including deletion typing, spoligotyping and Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR). Here we use whole-genome sequencing to examine the population structure of M. bovis in Ethiopia. A total of 134 M . bovis isolates were sequenced including 128 genomes from 85 mainly dairy cattle and six genomes isolated from humans, originating from 12 study sites across Ethiopia. These genomes provided a good representation of the previously described population structure of M. bovis , based on spoligotyping and demonstrated that the population is dominated by the clonal complexes African 2 (Af2) and European 3 (Eu3). A range of within-host diversity was observed amongst the isolates and evidence was found for both short- and long-distance transmission. Detailed analysis of available genomes from the Eu3 clonal complex combined with previously published genomes revealed two distinct introductions of this clonal complex into Ethiopia between 1950 and 1987, likely from Europe. This work is important to help better understand bTB transmission in cattle in Ethiopia and can potentially inform national strategies for bTB control in Ethiopia and beyond.

Bovine tuberculosis (bTB) is endemic in cattle in Ethiopia, a country that hosts the largest national cattle herd in Africa. The intensive dairy sector, most of which is peri-urban, has the highest prevalence of disease. Previous studies in Ethiopia have demonstrated that the main cause isMycobacterium bovis(M. bovis), which has been investigated using conventional molecular tools including deletion typing, spoligotyping and Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR). Here we use whole genome sequencing (WGS) to examine the population structure ofM. bovisin Ethiopia. A total of 134M. bovisisolates were sequenced including 128 genomes from 85 mainly dairy cattle and six genomes isolated from humans, originating from 12 study sites across Ethiopia. These genomes provided a good representation of the previously described population structure ofM. bovis, based on spoligotyping and demonstrated that the population is dominated by the clonal complexes African 2 (Af2) and European 3 (Eu3). A range of within-host diversity was observed amongst the isolates and evidence was found for both short- and long-distance transmission. Detailed analysis of available genomes from the Eu3 clonal complex combined with previously published genomes revealed two distinct introductions of this clonal complex into Ethiopia between 1950 and 1987, likely from Europe. This work is important to help better understand bTB transmission in cattle in Ethiopia and can potentially inform national strategies for bTB control in Ethiopia and beyond.

Bovine tuberculosis (bTB) is endemic in Ethiopia with higher prevalence in cattle, particularly in the central parts. Spread of Mycobacterium bovis (M. bovis) to wider regions is inevitable in uncontrolled conditions. This study was conducted to explore the pathology, characterize M. bovis strains, and describe genotypic diversity to demonstrate possible epidemiological links in emerging dairy areas of Ethiopia, namely, Mekelle and Gondar. Twenty-seven bTB positive cattle identified by the Single Intradermal Comparative Cervical Tuberculin (SICCT) test were subjected to post-mortem inspection to determine lesion distribution and pathological score. Samples from tissues with visible tuberculous or suspected non-visible lesions were processed and cultured following a standard protocol. Isolates identified as M. bovis by Region of Difference (RD)-based Polymerase Chain Reaction (PCR) were also spoligotyped to determine their spoligotype patterns. Post-mortem inspection of visceral organs indicated bTB suggestive lesions in 41% of the animals, with 25% being in the lungs. Lymph nodes from 77% of the animals had lesions. Fifty-five isolates identified from 24 of the slaughtered animals were confirmed as M. bovis. No other mycobacterial species were isolated. Spoligotyping classified strains from 21 of these animals into seven spoligotype patterns: SB0133, SB0134, SB1176, SB2233, SB2290, SB2467, and SB2520. More than one spoligotype were identified from five of these animals, and none of the last four spoligotypes had been reported in Ethiopia before. SB0134 was the most predominant type (47%) followed by SB0133 (25.5%). SB0133, SB2290, SB2467, and SB1176 are spoligotypes lacking spacers 3–7, characteristics of M. bovis strains of the African 2 (Af2) clonal complex, while SB0134, SB2233, and SB2520 do not belong to any of the established clonal complexes and likely to have a different evolutionary history. Despite a small sample size, the present study showed strain diversity with multiple genotypes identified in a single herd and even within a single animal, and the genotypes showed no sign of geographical localization, which could be a consequence of significant movement of bTB diseased cattle around the country, spreading the disease. Therefore, any future control programme of bTB in Ethiopia needs to address the risks of cattle movement.

Background Newcastle disease (ND) is a major infectious disease of poultry caused by a virulent strain of Avian Paramyxovirus – 1. It is a major threat to the poultry industry in many countries of the world including Ethiopia. Newcastle Disease Virus (NDV) is an enveloped, non-segmented, single-stranded negative-sense RNA virus with a helical morphology whose genome has six open reading frames (ORF) which encode for the following proteins: nucleoprotein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN) and RNA-dependent RNA polymerase (L). The aim of this study was to detect matrix gene (M-gene), for NDV by molecular tools and identify its risk factors in non-vaccinated village chicken in Central Rift Valley of Oromia, Ethiopia.Methods A total of 84 pooled in five swab samples from 420 cloacal and tracheal chickens were sampled and RNA was extracted from the 84 pooled samples to carry out real-time quantitative polymerase chain reaction (qRT-PCR). A real-time reverse transcriptase polymerase chain reaction (rRT-PCR) along with the quantification was also done for 10 positive qRT-PCR samples that were having high concentration of viral load Ct. value.Results Out of the 84 pools of five swab samples tested for M-gene using qRT-PCR, 16.7% (14/84) samples were detected which included 13 positives and 1 negative for NDV. The prevalence of ND in males was found to be 16.10% and that in females was 14.67%. Although the overall ND prevalence was 15.48% (13/84), the highest score was recorded in Adama, 42.86% (6/14), and no positive case was detected in Bote and Bishoftu (p