Hygienic status of beef butcher shop facilities and antibiotic resistance profile of Salmonella enterica in Ethiopia.

The microbiological quality of meat is influenced by the conditions of hygiene prevailing during production and handling. Thus, this study aimed to assess the prevalence of Salmonella enterica and its antimicrobial resistance, load of hygiene indicator bacteria including E. coli (ECC), coliforms (CC), total coliform (TCC), Enterobacteriaceae (EB) and aerobic plate count (APC), and meat handler's food safety knowledge and hygiene practices in butcher shops in two cities, Addis Ababa and Hawassa in Ethiopia, during 2020 and 2021. A total of 360 samples of beef carcasses (n = 120), knives (n = 60), chopping boards (n = 60), weighing balance (n = 60), and personnel's hands (n = 60) were randomly collected for microbial analysis. Besides, 120 participants were selected to participate in a food safety knowledge and hygiene practices assessment. The S. enterica isolates were identified by agglutination test followed by qPCR targeting invA gene. Phenotypic antimicrobial resistance profiles of S. enterica were determined using disk diffusion assays as described in CLSI. The ECC, CC, TCC, EB, and APC populations were quantified by plating onto petrifilm plates. A structured questionnaire was used to determine food safety knowledge and hygiene practices of participants. Overall prevalence of S. enterica was 16.7% (95% CI, 8.3-26.7) and location seems to have no effect (p = 0.806). Only 20% of the S. enterica were resistant to ampicillin and tetracycline. However, the majority (80%) of S. enterica isolates were susceptible to the panel of 11 antimicrobials tested. The overall mean ± SD (log CFU/cm2) of ECC, CC, TCC, EB, and APC were 4.31 ± 1.15; 4.61 ± 1.33; 4.77 ± 1.32; 4.59 ± 1.38 and 5.87 ± 1.52, respectively. No significant difference (p = 0.123) in E. coli contamination was observed between samples of beef carcasses and chopping boards. The EB contamination showed no significant difference (p > 0.05) among sample sources. The APC contamination levels on beef carcass were significantly higher (p > 0.05) than other sample sources. A total of 56% (95% CI: 46.7 - 65.0) of the participants had poor knowledge and 65% (95% CI: 56.7 - 73.3) had poor hygiene practices towards food safety. This study highlighted the poor hygiene status of butcher facilities with a potential risk of beef safety. Thus, appropriate food safety control strategies and inspection is needed at retail establishments.

Isolation and Molecular Detection of Marek's Disease Virus from Outbreak Cases in Chicken in South Western Ethiopia.

Background: 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.

Isolation and Molecular Detection of Newcastle Disease Virus from Field Outbreaks in Chickens in Central Ethiopia.

Background: 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.

Community Wastewater-Based Surveillance Can Be a Cost-Effective Approach to Track COVID-19 Outbreak in Low-Resource Settings: Feasibility Assessment for Ethiopia Context.

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.

Spoligotype analysis of Mycobacterium bovis isolates from cattle and assessment of zoonotic TB transmission among individuals working in bovine TB-infected dairy farms in Ethiopia.

Bovine tuberculosis (bTB) is a disease with impact on dairy productivity, as well as having the potential for zoonotic transmission. Understanding the genetic diversity of the disease agent Mycobacterium bovis is important for identifying its routes of transmission. Here we investigated the level of genetic diversity of M. bovis isolates and assessed the zoonotic potential in risk groups of people working in bTB-infected dairy farms in central Ethiopia. M. bovis was isolated and spoligotyped from tissue lesions collected from slaughtered cattle as well as from raw milk collected from bTB positive cows in dairy farms from six urban areas of central Ethiopia. From consented dairy farm workers, knowledge and practices related to zoonotic TB transmission, together with demographic and clinical information, was collected through interviews. Sputum or Fine Needle Aspirate (FNA) samples were collected from suspected TB cases. Spoligotyping of 55 M. bovis isolates that originated either from cattle tissues with tuberculous lesion or from raw milk revealed seven spoligotype patterns where SB1176 was the most prevalent type (47.3%). Most isolates (89.1%) were of the M. bovis African 2 clonal complex. All sputum and FNA samples from 41 dairy farm workers with symptoms of TB were culture negative for any mycobacteria. Among the 41 TB suspected farm workers, 61% did not know about bTB in cattle and its zoonotic potential, and over two-third of these workers practiced raw milk consumption. Our spoligotype analysis suggests a wider transmission of a single spoligotype in the study area. The results reported here may be useful in guiding future work to identify the source and direction of bTB transmission and hence design of a control strategy. Isolation of M. bovis from milk, knowledge gap on zoonotic TB and practice of consumption of raw milk in the study population showed potential risk for zoonotic transmission.

Research Note: Molecular surveillance of Avian Paramyxovirus type-1 in nonvaccinated village chickens in Central Rift Valley of Oromia, Ethiopia.

Newcastle disease (ND) is a major infectious disease of poultry caused by a virulent strain of Avian Paramyxovirus type-1 (APMV-1). It is a major threat to the poultry industry in many countries of the world including Ethiopia. The aim of this study was to conduct molecular surveillance of ND Virus and identify potential risk factors for nonvaccinated village chicken in Central Rift Valley of Oromia, Ethiopia. A total of 84 pooled swab samples, each made from pools of 5 swabs for analysis, from cloacal and tracheal sites of chickens in the Central Rift Valley were collected, and RNA was extracted to carry out real-time quantitative polymerase chain reaction. Out of the 84 pooled swab samples tested for M-gene, 13 (15.48%) samples were found positive for APMV-1. 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 prevalence was recorded in Adama, 42.86% (6/14), and no positive case was observed in Bote and Bishoftu (P < 0.05), while intermediate prevalence was obtained from Batu, Arsi-negele, and Shashemene (P > 0.05). In general, the present study provides important information on the epidemiology of ND based on M-gene assay in Central Rift Valley of Oromia, Ethiopia, and highlights the importance of implementing molecular surveillances practice in live poultry markets and village chickens.

Seroprevalence and Molecular Detection of Foot and Mouth Disease Virus in Dairy Cattle Around Addis Ababa, Central Ethiopia.

BACKGROUND: 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.

Population structure and transmission of Mycobacterium bovis in Ethiopia.

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.

Genotype Diversity of Mycobacterium bovis and Pathology of Bovine Tuberculosis in Selected Emerging Dairy Regions of Ethiopia.

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.