Molecular Detection and Characterization of Newcastle Disease Virus from Chickens in Mid-Rift Valley and Central Part of Ethiopia.

Background: Newcastle disease (ND) is a highly infectious poultry disease that causes major economic losses worldwide. The disease is caused by Newcastle Disease Virus (NDV) and early detection and identification of the viral strain is essential. Having knowledge of the NDV strain genotype that circulates in some regions would help in designing an effective vaccine to control the disease. In this regard, there is little information on NDV strain in chickens in mid Rift Valley and the central part of Ethiopia. Therefore, the purpose of this study was to detect and characterize NDV strain genotype from chickens in mid-Rift Valley and the central part of Ethiopia and test whether this NDV strain genotype matches the vaccine strain currently used in the study area. Methods: A total of 98 samples: 78 (tracheal and cloacal) swabs from chicken pools of five and 20 tissue samples were collected. To detect NDV strain, conserved region of the virus Matrix (M) gene was amplified by qRT-PCR. To characterize NDV strain genotypes, M-gene positive samples were specifically re-amplified by conventional PCR targeting the Fusion (F) gene region and sequenced by Sanger method. Results: 13.26% of tested samples were positive for NDV strain in the study area with statistically significant difference (P<0.05) among the study sites. Further characterization of the F genes from NDV strain isolates by phylogenetic analysis indicated that one field isolate clustered with genotype VII whereas three of the isolates clustered to genotype I, II, and III. The isolate of the current NDV strain vaccine in use in the study area clustered with genotype II. Conclusion: The current study indicates the existence of different NDV strain genotype from that of the vaccine strain currently used. Even though large-scale characterization of several isolates is required at national level, the current study laid baseline information for the existence of variations between field NDV strain genotype and vaccine strain currently used against ND in the country.

Occurrence, antimicrobial susceptibility, and resistance genes of Staphylococcus aureus in milk and milk products in the Arsi highlands of Ethiopia.

BACKGROUND: In Ethiopia, milk production and handling practices often lack proper hygiene measures, leading to the potential contamination of milk and milk products with Staphylococcus aureus (S. aureus), including methicillin-resistant strains, posing significant public health concerns. This study aimed to investigate the occurrence, antimicrobial susceptibility profiles and presence of resistance genes in S. aureus strains isolated from milk and milk products. METHODS: A cross-sectional study was conducted in the Arsi highlands, Oromia, Ethiopia from March 2022 to February 2023. A total of 503 milk and milk product samples were collected, comprising 259 raw milk, 219 cottage cheese, and 25 traditional yogurt samples. S. aureus isolation and coagulase-positive staphylococci enumeration were performed using Baird-Parker agar supplemented with tellurite and egg yolk. S. aureus was further characterized based on colony morphology, Gram stain, mannitol fermentation, catalase test, and coagulase test. Phenotypic antimicrobial resistance was assessed using the Kirby-Bauer disc diffusion method, while the polymerase chain reaction (PCR) was employed for confirming the presence of S. aureus and detecting antimicrobial resistance genes. RESULTS: S. aureus was detected in 24.9% of the milk and milk products, with the highest occurrence in raw milk (40.9%), followed by yogurt (20%), and cottage cheese (6.4%). The geometric mean for coagulase-positive staphylococci counts in raw milk, yogurt, and cottage cheese was 4.6, 3.8, and 3.2 log10 CFU/mL, respectively. Antimicrobial resistance analysis revealed high levels of resistance to ampicillin (89.7%) and penicillin G (87.2%), with 71.8% of the isolates demonstrating multidrug resistance. Of the 16 S. aureus isolates analyzed using PCR, all were found to carry the nuc gene, with the mecA and blaZ genes detected in 50% of these isolates each. CONCLUSION: This study revealed the widespread distribution of S. aureus in milk and milk products in the Arsi highlands of Ethiopia. The isolates displayed high resistance to ampicillin and penicillin, with a concerning level of multidrug resistance. The detection of the mecA and blaZ genes in selected isolates is of particular concern, highlighting a potential public health hazard and posing a challenge to effective antimicrobial treatment. These findings highlight the urgent need to enhance hygiene standards in milk and milk product handling and promote the rational use of antimicrobial drugs. Provision of adequate training for all individuals involved in the dairy sector can help minimize contamination. These measures are crucial in addressing the threats posed by S. aureus, including methicillin-resistant strains, and ensuring the safety of milk and its products for consumers.

Isolation and Determination of Antibacterial Sensitivity Characteristics of Staphylococcus aureus from Lactating Cows in West Shewa Zone, Ethiopia.

Staphylococcus (S.) aureus is one of the etiologies of bovine mastitis, hindering milk production and productivity in dairy farms. This study was aimed at assessing the distribution of bovine mastitis and the isolation rate of S. aureus in milked cows of West Shewa Zone. The clinical mastitis was diagnosed by physical methods including observation and palpation, whereas the subclinical mastitis was tested by the California mastitis test (CMT). All of the cows tested for mastitis were aseptically sampled (teat-milk) for bacteriology. The bacterium was primarily identified based on colony characterization, catalase, coagulase tests, and Gram stain reaction. Finally, MALDI-TOF Biotyper confirmed the species. The antibacterial sensitivity characteristics of the isolates to different antibacterial drugs were tested by the disk diffusion method. The drugs were selected based on the frequent usage in veterinary medicine in the study area. By using particular primers, the presence of the resistance (mecA and blaZ), and thermonuclease (nuc) genes were determined by polymerase chain reaction (PCR). The data were analyzed by R statistical software. The associations between the dependent variables (prevalence of mastitis and S. aureus) and the explanatory variables were analysed by chi-square (χ 2) and logistic regression tests at a 95% confidence interval (CI). Accordingly, 258 lactating cows were examined, of which 97 (37.6%) were mastitis positive. Of these mastitis positive cows, 59 (60.8%) were subclinical and 38 (39.2%) were clinical. Among the 258 milk samples, 43 (16.7%) were positive for S. aureus. According to the results of the current investigation, subclinical mastitis was significantly more prevalent than clinical mastitis (p < 0.05). The disease was found varied with the lactation stage of the animal, milking with washed hand, udder washing before milking, and tick infestation of the teat. In comparison to animals from farms with lower number of lactating cows, the prevalence of the bacteria was significantly higher in animals managed in farms with large (OR = 12.58, 95% CI = 2.33-68.54, and p < 0.05) and medium (OR = 12.58, 95% CI = 2.33-68.54, and p < 0.05) population of lactating cows per herd. The isoation rate of the bacterium was also found significantly higher in tick-infested cows (OR = 27.69, 95% CI = 9.71-93.01, and p < 0.05) than tick free cows. The antibiogram tests revealed that the isolates resisted penicillin G and tetracycline group drugs (oxytetracycline and tetracycline). Moreover, the nuc gene was confirmed to be present in all of the examined isolates. However, they were not found harboring blaZ and mecA genes. We concluded that S. aureus is sustaining as a main causative agent of bovine mastitis, and they were resistant to the frequently used antibiotics in public and veterinary medicines in the study areas. Therefore, research-based interventions need to be taken in action to combat the pathogen.

Detection of Antibodies Against Brucellosis and Associated Risk Factors in Cross Breed Dairy Cattle in Smallholder Farmers, Southern Ethiopia.

Background: A cross-sectional study was conducted in selected districts of Gamo zone, southern Ethiopia over a period of November 2019 and September 2020 to estimate seroprevalence and associated risk factors and assess knowledge and practices of smallholder farmers about bovine brucellosis. Two districts and four kebeles from each district were purposively selected, and individual animals were sampled using a simple random sampling technique. A total of 384 sera samples were collected, and concurrently, 236 dairy cattle owners were interviewed. The samples were tested for antibodies against Brucella using both Rose Bengal Plate test and Complement Fixation test following OIE standard protocol. Risk factors associated with bovine brucellosis were analyzed using univariate and multivariate logistic regressions. Results: The survey result has shown that 95% and 97% of the small holder farmers did not know the cause and symptoms of bovine brucellosis, respectively, and the majority have engaged in risky practices. Eight (2.08%) of the collected serum samples were positive with screening test (RBT) and only six (1.5%) were positive with confirmatory test (CFT). Multivariable logistic regression analysis showed a statistically significant association between herd sizes and the disease. The seropositivity of the disease is higher in small-sized herds followed by medium-sized herds. Conclusion: The seroprevalence of bovine brucellosis was found to be at a low percentage with confirmatory tests even if there was a presence of associated risk factors for the disease in the study area. Again, the results suggest that smallholder farmers have poor knowledge and risky practices, which expose them to the disease. Awareness creation about the disease is of paramount importance even if the prevalence was low in this serological study. The implementation of a test and slaughter program before the disease becomes widespread, along with the testing of new stock before introduction to the farms is recommended.

Clinicopathological and molecular studies on cattle naturally infected with lumpy skin diseases in selected districts of Wolaita Zone, Southern Ethiopia.

BACKGROUND: Lumpy skin disease is a contagious viral disease of cattle caused by LSDV that results in huge economic losses in the cattle industry. This study characterizes LSDV in cattle through clinicopathological and molecular techniques in selected districts of Wolaita Zone, Southern Ethiopia. METHODS: A crossectional study was conducted from November 2020 to June 2021 using Real-time polymerase chain reaction and Histopathological techniques to confirm LSDV. RESULT: This study revealed that the percentage of positivity of cattle for LSDV was 36.2%. Clinically, cattle infected with LSDV revealed fever (39-41 °C), nodular lesions on the skin and mucous membranes, and lymphadenopathy. Histopathologically, affected tissue revealed ballooning degenerations of the epidermis, infiltration of mononuclear inflammatory cells, vasculitis, and intracytoplasmic eosinophilic inclusion bodies. RT-PCR confirmed that DNA extracts from skin biopsies of virus isolates were positive for LSDV. CONCLUSION: The present study confirms that LSDV is widely circulating in cattle of selected districts of the Wolaita zone. Thus, effective control measures through regular vaccination and further confirmation of circulating strains of LSDV through detailed molecular analysis should be recommended.

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.

First molecular detection of hepatitis E virus genome in camel and pig faecal samples in Ethiopia.

BACKGROUND: Hepatitis E is an enteric and zoonotic disease caused by hepatitis E virus (HEV) that is mainly transmitted via the faecal-oral route through contaminated food or the environment. The virus is an emerging infectious agent causing acute human infection worldwide. A high seroprevalence of the disease was reported in pregnant women in Addis Ababa, Ethiopia, raising significant public health concern. The presence of HEV specific antibodies were also reported in dromedary camels in the country; however, the infectious virus and/or the viral genome have not been demonstrated to date in animal samples. METHODS: To address this gap, a total of 95 faecal samples collected from both apparently healthy pigs of uncharacterised types (50 samples) in Burayu and Addis Ababa areas and camels (Camelus dromedarius, 45 samples) in west Hararghe were screened for the presence of HEV genome using universal primers in a fully nested reverse transcription polymerase chain reaction (nRT-PCR). The protocol is capable of detecting HEV in faecal samples from both pigs and camels. RESULTS: The nRT-PCR detected HEV genes in six (12%) pig faecal samples and one camel sample (2.2%). Therefore, the results indicate that HEV is circulating in both pigs and camels in Ethiopia and these animals and their products could serve as a potential source of infection for humans. CONCLUSION: The detection of HEV in both animals could raise another concern regarding its public health importance as both animals' meat and camel milk are consumed in the country. Further studies to determine the prevalence and distribution of the virus in different animals and their products, water bodies, food chain, and vegetables are warranted, along with viral gene sequencing for detailed genetic characterisation of the isolates circulating in the country. This information is critically important to design and institute appropriate control and/or preventive measures.

Molecular Analysis of East African Lumpy Skin Disease Viruses Reveals a Mixed Isolate with Features of Both Vaccine and Field Isolates.

Lumpy skin disease (LSD), an economically significant disease in cattle caused by lumpy skin disease virus (LSDV), is endemic to nearly all of Africa. Since 2012, LSDV has emerged as a significant epizootic pathogen given its rapid spread into new geographical locations outside Africa, including the Middle East, Eastern Europe, and Asia. To assess the genetic diversity of LSDVs in East Africa, we sequenced and analyzed the RPO30 and GPCR genes of LSDV in twenty-two archive samples collected in Ethiopia, Kenya, and Sudan before the appearance of LSD in the Middle East and its incursion into Europe. We compared them to publicly available sequences of LSDVs from the same region and those collected elsewhere. The results showed that the East African field isolates in this study were remarkably similar to each other and to previously sequenced field isolates of LSDV for the RPO30 and GPCR genes. The only exception was LSDV Embu/B338/2011, a field virus collected in Kenya, which displayed mixed features between the LSDV Neethling vaccine and field isolates. LSDV Embu/B338/2011 had the same 12-nucleotide insertion found in LSDV Neethling and KS-1 vaccines. Further analysis of the partial EEV glycoprotein, B22R, RNA helicase, virion core protein, NTPase, and N1R/p28-like protein genes showed that LSDV Embu/B338/2011 differs from previously described LSDV variants carrying the 12-nucleotide insertion in the GPCR gene. These findings highlight the importance of the constant monitoring of genetic variation among LSDV isolates.

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.

Use of an Alignment-Free Method for the Geographical Discrimination of GTPVs Based on the GPCR Sequences.

Goatpox virus (GTPV) belongs to the genus Capripoxvirus, together with sheeppox virus (SPPV) and lumpy skin disease virus (LSDV). GTPV primarily affects sheep, goats and some wild ruminants. Although GTPV is only present in Africa and Asia, the recent spread of LSDV in Europe and Asia shows capripoxviruses could escape their traditional geographical regions to cause severe outbreaks in new areas. Therefore, it is crucial to develop effective source tracing of capripoxvirus infections. Earlier, conventional phylogenetic methods, based on limited samples, identified three different nucleotide sequence profiles in the G-protein-coupled chemokine receptor (GPCR) gene of GTPVs. However, this method did not differentiate GTPV strains by their geographical origins. We have sequenced the GPCR gene of additional GTPVs and analyzed them with publicly available sequences, using conventional alignment-based methods and an alignment-free approach exploiting k-mer frequencies. Using the alignment-free method, we can now classify GTPVs based on their geographical origin: African GTPVs and Asian GTPVs, which further split into Western and Central Asian (WCA) GTPVs and Eastern and Southern Asian (ESA) GTPVs. This approach will help determine the source of introduction in GTPV emergence in disease-free regions and detect the importation of additional strains in disease-endemic areas.

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.

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

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

Characterization of Newcastle disease virus isolates obtained from outbreak cases in commercial chickens and wild pigeons in Ethiopia.

Newcastle disease (ND), caused by virulent avian paramyxovirus type 1, is one of the most important diseases responsible for devastating outbreaks in poultry flocks in Ethiopia. However, the information about genetic characteristics of the Newcastle disease viruses (NDVs) circulating in commercial chickens and wild birds is scarce. In this study, we characterized isolates obtained from ND suspected outbreaks during 2012-2014 from poultry farms (n = 8) and wild pigeons (n = 4). The NDVs isolated from pathological specimens, through inoculation in embryonated chicken eggs, were characterized biologically by conventional intracerebral pathogenicity indices (ICPI), and genetically on the basis of Phylogenic analysis of partial F-gene sequences (260 bp) encompassing the cleavage site. The ICPI values of isolates from chickens ranged from 0.9 to 1.8; whereas, the ICPI of pigeon isolates was 1.4. All isolates contained multiple basic amino acids at the deduced cleavage site of fusion protein, which is a typical feature of virulent viruses. Phylogenic analysis of the partial cleavage site of F-gene (260 bp) indicated that all the sequences of viruses obtained from pigeons were identical and clustered within the genotype VIh while the sequences of viruses obtained from chickens were clustered together within the genotype VIf. The similarity between the viruses obtained from chickens and those obtained from pigeons ranged from 82.5 to 85.6 %. This suggests that different sub genotypes of genotype VI are circulating in chicken and wild pigeon population in Ethiopia. This warrants further study to understand the role of wild birds in the epidemiology of NDV in Ethiopia and as well highlights the importance of continuous surveillances both in wild birds and domestic poultry.

High prevalence of bovine tuberculosis in dairy cattle in central ethiopia: implications for the dairy industry and public health.

BACKGROUND: Ethiopia has the largest cattle population in Africa. The vast majority of the national herd is of indigenous zebu cattle maintained in rural areas under extensive husbandry systems. However, in response to the increasing demand for milk products and the Ethiopian government's efforts to improve productivity in the livestock sector, recent years have seen increased intensive husbandry settings holding exotic and cross breeds. This drive for increased productivity is however threatened by animal diseases that thrive under intensive settings, such as bovine tuberculosis (BTB), a disease that is already endemic in Ethiopia. METHODOLOGY/PRINCIPAL FINDINGS: An extensive study was conducted to: estimate the prevalence of BTB in intensive dairy farms in central Ethiopia; identify associated risk factors; and characterize circulating strains of the causative agent, Mycobacterium bovis. The comparative intradermal tuberculin test (CIDT), questionnaire survey, post-mortem examination, bacteriology, and molecular typing were used to get a better understanding of the BTB prevalence among dairy farms in the study area. Based on the CIDT, our findings showed that around 30% of 2956 tested dairy cattle from 88 herds were positive for BTB while the herd prevalence was over 50%. Post-mortem examination revealed gross tuberculous lesions in 34/36 CIDT positive cattle and acid-fast bacilli were recovered from 31 animals. Molecular typing identified all isolates as M. bovis and further characterization by spoligotyping and MIRU-VNTR typing indicated low strain diversity within the study area. CONCLUSIONS/SIGNIFICANCE: This study showed an overall BTB herd prevalence of 50% in intensive dairy farms in Addis Ababa and surroundings, signalling an urgent need for intervention to control the disease and prevent zoonotic transmission of M. bovis to human populations consuming dairy products coming from these farms. It is suggested that government and policy makers should work together with stakeholders to design methods for the control of BTB in intensive farms in Ethiopia.