Evaluation of chicken embryo extract and egg yolk extract as alternatives to basic cell culture medium supplement.

BACKGROUND: Fetal calf serum (FCS), an existing cell culture supplement, is effective but has several drawbacks, including being expensive, requiring a lengthy process of production, and requiring a hard currency. With this in mind, we planned to evaluate chick embryo extract and egg yolk extracts in cell culture as alternatives to fetal calf serum (FCS). METHODS: Specific pathogen-free eggs were purchased from the National Veterinary Institute, Bishoftu, Ethiopia, and incubated in a humidified incubator at 37 °C for 11 days. Egg yolk extract (EYE) and chick embryo extract (CEE) were collected after the egg was opened with caution not to destroy the yolk sack or the chick embryo itself. Chick fibroblasts and Vero cells were cultured in minimum essential medium (MEM) supplemented with egg yolk extract or chick embryo extract at ratios of 0:10, 1:9, 2.5:7.5, and 5:5% fetal calf serum. RESULTS: Fibroblast cell attachment was better in media supplemented with 5% CEE and 5% FCS. The confluency was also greater than 50% at this concentration. Vero cells cultured with 5% CEE and 5% FCS also exhibited very good cell attachment and a confluency of up to 70%. Viability and confluency were also observed at 5%:5% ratios of 50 and 70%, respectively. CONCLUSION: This investigation evaluated these two extracts as cell culture supplements and revealed promising results as alternatives to fetal calf serum. The limitation of this study is that it only used two cell types and additional cell lines, and different ratios should be tested. With the above findings, further research using different cell lines, ratios and conditions is warranted.

Occurrence, molecular characterization, and antimicrobial susceptibility of sorbitol non-fermenting Escherichia coli in lake water, fish and humans in central Oromia, Ethiopia.

Contaminated lake water and fish can be sources of bacterial pathogens of public health concern, including pathogenic E. coli. Within Ethiopia, specifically, Central Oromia, raw fish consumption is a common practice. Although there are few reports on occurrence of E. coli O157 in fish destined for human consumption and children under five years, information on the transmission pathways of E. coli O157 and other sorbitol non-fermenting (SN-F) E. coli from water-to-fish-to-human, and their virulence factors and antimicrobial resistant determinants along the fish supply chain is lacking. The study aimed to investigate the occurrence, molecular characteristics, and antimicrobial susceptibility of E. coli O157 and other SN-F E. coli strains in fish, lake water and humans in central Oromia, Ethiopia. A total of 750 samples (450 fish samples, 150 water samples, 150 human stool samples) were collected from five lakes and three health facilities. The samples were processed following the standard protocol recommended by European Food Safety Authority and Kirby-Bauer disc diffusion method for detection of the bacteria, and antimicrobial susceptibility tests, respectively. Molecular characterization of presumptive isolates was performed using Whole-Genome Sequencing (WGS) for serotyping, determination of virulence factors, antimicrobial resistance traits, and genetic linkage of the isolates. Overall, 3.9% (29/750) of the samples had SN-F E. coli; of which 6.7% (n = 10), 1.8% (n = 8) and 7.3% (n = 11) were retrieved from water, fish, and diarrheic human patients, respectively. The WGS confirmed that all the isolates were SN-F non-O157: H7 E. coli strains. We reported two new E. coli strains with unknown O-antigen from fish and human samples. All the strains have multiple virulence factors and one or more genes encoding for them. Genetic relatedness was observed among strains from the same sources (water, fish, and humans). Most isolates were resistant to ampicillin (100%), tetracycline (100%), cefotaxime (100%), ceftazidime (100%), meropenem (100%), nalidixic acid (93.1%) and sulfamethoxazole/trimethoprim (79.3%). Majority of the strains were resistant to chloramphenicol (58.6%) and ciprofloxacin (48.3%), while small fraction showed resistance to azithromycin (3.45%). Isolates had an overall MDR profile of 87.5%. Majority, (62.1%; n = 18) of the strains had acquired MDR traits. Genes encoding for mutational resistance and Extended-spectrum beta-lactamases (ESBL) were also detected. In conclusion, our study revealed the occurrence of virulent and MDR SN-F E. coli strains in water, fish, and humans. Although no genetic relatedness was observed among strains from various sources, the genomic clustering among strains from the same sources strongly suggests the potential risk of transmission along the supply chain at the human-fish-environment interface if strict hygienic fish production is not in place. Further robust genetic study of the new strains with unknown O-antigens, and the epidemiology of SN-F E. coli is required to elucidate the molecular profile and public health implications of the pathogens.

Humoral Immune Response in Calves Vaccinated with Monovalent Vaccines or a Trivalent Combination Thereof and Matching of These Vaccines to the Selected Circulating Foot-and-Mouth Disease Viruses in Ethiopia.

Foot-and-mouth disease (FMD) is an endemic, highly contagious, and devastating disease of livestock production in Ethiopia. Control of this disease relies mainly on prophylactic vaccination by willing farmers without a countrywide vaccination program. The objectives of this study were to quantify the humoral immune response and evaluation of the serological relationship of the vaccine strain used with representative field strain isolates. This was performed by primo vaccination of 6-9-month-old Holstein Friesian calves (35 treatment and 4 control calves) on day one and booster vaccination on day 28. Calves were vaccinated using the locally available National Veterinary Institute (NVI), Bishoftu, Ethiopia, inactivated aluminum hydroxide adjuvant monovalent (either O, A, SAT-2 alone) or trivalent (combination of A, O, SAT-2) vaccine (A/ETH/6/2000 (G-VII, O/ETH/38/2005(EA-3) and SAT-2/ETH/64/2009(XIII)). A 2 mL or 4 mL dose was used to vaccinate all calves except the animals that served as a control. In the case of the trivalent vaccine, a 4 mL dose was used to vaccinate calves. The serum was collected at 7, 14, 21, 28, and 56 days post-vaccination (d.p.v.). The humoral immune response was quantified by the solid-phase competitive enzyme-linked immunosorbent assay (SPC ELISA) and the virus-neutralization test (VNT). The serological relationship of heterologous and homologous viruses was also evaluated by adjuvant vaccine matching tests. The r1-value was determined using serum collected 21 d.p.v. An increase in immune response was observed from 7 d.p.v. to 28 d.p.v. in calves who received a 4 mL dose containing a 107.24 antigen load of 100 tissue culture infective dose (100TCID50) virus titer in the formulation. Upon receiving a booster dose on day 28, the humoral immune response was checked on the 56th day post-initial vaccination. Amounts of 54%, 72%, 79%, and 72% of inhibition for A, O, SAT-2, and trivalent vaccine in the three serotypes SPCE, respectively, was measured. Here, it was found that the immune response of calves increased from day 7 to 56, as evidenced by SPCE analysis. Likewise, an increase in antibody titer measured by a one-dimensional virus neutralization assay was also in line with SPCE analysis. This indicates that the vaccine is capable of inducing a neutralizing antibody that confers a protective immune response in 70%, 62%, and 100% heterologous field strains of A, O, and SAT-2 isolates, respectively, and has an average antigenic relationship of >0.3 with a standard deviation of +0.05 (N = 3) to the vaccine strains A/ETH/6/2000, O/ETH/38/2005 and SAT-2/ETH/64/2009, respectively, when using the one-dimensional virus neutralization test. The contribution and importance of this study is a confirmation of the vaccine and the field strain serological relationship for serotype SAT-2 strain and further research/change of vaccination strategy/ improvement in the currently used vaccine to cover a wide range of prevailing genotypes/lineages and induction of sound immune response after vaccination for serotype A and O strain. This study suggests that the trivalent vaccine produced by the National Veterinary Institute containing viral isolates from serotype O, A, and SAT-2 has a good serological relationship with the majority of circulating field strains in Ethiopia.

Viral Protein 1 (VP1) Sequence-Based Genetic Diversity of SAT 2 FMDV Circulating in Ethiopia from 1990 to 2015.

Introduction: Pathogen molecular epidemiology determines the origin of specific outbreaks locality of foot-and-mouth disease virus serotype South African Territories-2 sequence-based analysis of highly variable Viral Protein 1 (VP1), which helps to identify the evolution of this virus through time and space. The objective of this study was to compare the differences between SAT-2 VP1 sequences of FMDV circulated in Ethiopia from 1990 to 2015 at the genetic level. Methods: The nucleotide and amino acid sequences were analyzed using Basic Local Alignment Search Tools (BLAST), Multiple sequence alignment and sequence editing and Phylogenetic tree reconstruction. The nucleotide and amino acid sequences alignment, distance matrix, and phylogenetic tree constructions were done using the MEGA 6.0 software package. Result and Discussion: In this analysis, we found 76% nucleotide identities and amino acid similarities among the sequences. The overall group mean distance at nucleotide level was 19% with a mean intra-population diversity of 2%. The lowest sequence variation was observed among sequences obtained from the years 2007/09/10, 2014/15, and 1990/91 which was less than 5% among them. This analysis revealed that in the last 25 years, four different topotypes of the FMDV SAT-2 were circulating in Ethiopia. The Arg-Gly-Asp (RGD) amino acid (AA) motif at AA position 144-146 within the G-H loop of the VP1 protein of FMDV is conserved, but up- and downstream hyper-variable AA sequences are identified. In this study, it was observed that four topotypes (IV, XIV, XIII, and VII) were circulating in Ethiopia for 25 years. Further, compared with sequences from neighboring countries (Sudan, Kenya) confirmed the presence of these topotypes. Conclusion: Pertinent to this genetic diversity control strategies in Ethiopia should be based on having regular antigenic and genetic vaccine matching tests with the circulating strain within a defined period, space, transboundary nature of the disease and applying biosecurity measures.

Epidemiological Dynamics of Foot-and-Mouth Disease in the Horn of Africa: The Role of Virus Diversity and Animal Movement.

The Horn of Africa is a large area of arid and semi-arid land, holding about 10% of the global and 40% of the entire African livestock population. The region's livestock production system is mainly extensive and pastoralist. It faces countless problems, such as a shortage of pastures and watering points, poor access to veterinary services, and multiple endemic diseases like foot-and-mouth disease (FMD). Foot-and-mouth disease is one of the most economically important livestock diseases worldwide and is endemic in most developing countries. Within Africa, five of the seven serotypes of the FMD virus (FMDV) are described, but serotype C is not circulating anymore, a burden unseen anywhere in the world. The enormous genetic diversity of FMDV is favored by an error-prone RNA-dependent RNA polymerase, intra-typic and inter-typic recombination, as well as the quasi-species nature of the virus. This paper describes the epidemiological dynamics of foot-and-mouth disease in the Horn of Africa with regard to the serotypes and topotypes distribution of FMDV, the livestock production systems practiced, animal movement, the role of wildlife, and the epidemiological complexity of FMD. Within this review, outbreak investigation data and serological studies confirm the endemicity of the disease in the Horn of Africa. Multiple topotypes of FMDV are described in the literature as circulating in the region, with further evolution of virus diversity predicted. A large susceptible livestock population and the presence of wild ungulates are described as complicating the epidemiology of the disease. Further, the husbandry practices and legal and illegal trading of livestock and their products, coupled with poor biosecurity practices, are also reported to impact the spread of FMDV within and between countries in the region. The porosity of borders for pastoralist herders fuels the unregulated transboundary livestock trade. There are no systematic control strategies in the region except for sporadic vaccination with locally produced vaccines, while literature indicates that effective control measures should also consider virus diversity, livestock movements/biosecurity, transboundary trade, and the reduction of contact with wild, susceptible ungulates.

Occurrence of Aflatoxins in Poultry Feed in Selected Chicken Rearing Villages of Bishoftu Ethiopia.

Background: Aflatoxins (AFs) are major contaminants of feed used in the poultry industry that negatively affect animal and human health. In Ethiopia, previous studies on AFs mainly considered cattle feed and milk but scarce information exists for poultry feeds. Methods: The aim of this study was to determine the occurrence of AFs in poultry feed in selected chicken rearing villages of Bishoftu. The study was conducted from December 2018 to May 2019. Thirty-three compound poultry feed samples were collected and analyzed for aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and total AFs (AFT) using high performance liquid chromatography (HPLC). The moisture content of the samples was also determined. Results: The result indicated that 31 (94%) from a total of 33 samples were contaminated with AFs. The mean levels of AFB1, AFB2, AFG1, AFG2 and AFT were 70.11 µg/kg, 13.50 µg/kg, 88.55 µg/kg, 18.00 µg/kg and 190.18 µg/kg, respectively. This study found AFs at a level above the limit of FDA regulatory levels of 20 µg/kg in 25 (72.75%) samples for AFT and 22 (66.67%) samples for AFB1. The analysis of moisture content of the samples, ranges from 7.33% to 11.17%, indicating all were at optimal value (<12%). Conclusion: The study showed the high contamination of AFs in poultry feeds with optimal moisture content and hence further investigations are needed to address the cause. The study also supports the need for preventive strategies of AFs contamination in poultry feeds in Bishoftu.

Temporal and Spatial Patterns and a Space-Time Cluster Analysis of Foot-and-Mouth Disease Outbreaks in Ethiopia from 2010 to 2019.

Foot-and-mouth disease (FMD) is an endemic disease in Ethiopia, although space-time cluster and monthly variation studies have never been assessed at national level. The current study aimed to identify the spatial and temporal distribution of FMD outbreaks in Ethiopia from national outbreak reports over a period of ten years from 1 January 2010 to 31 December 2019. To this end, a total of 376,762 cases and 1302 outbreaks from 704 districts were obtained from the Minister of Agriculture for analyses. In general, the dry periods, i.e., October to March, of the year were recorded as the peak outbreak periods, with the highest prevalence in March 2012. The monthly average and the outbreak trends over ten years show a decrease of outbreaks from 2010 to 2019. Decomposing the FMD outbreak data time series showed that once an outbreak erupted, it continued for up to five years. Only 12% of the reported outbreaks were assigned to a specific serotype. Within these outbreaks, the serotypes O, A, SAT-2, and SAT-1 were identified in decreasing order of prevalence, respectively. When a window of 50% for the maximum temporal/space cluster size was set, a total of seven FMD clusters were identified in space and time. The primary cluster with a radius of 380.95 km was identified in the southern part of Ethiopia, with a likelihood ratio of 7.67 (observed/expected cases). The third cluster, with a radius of 144.14 km, was identified in the northeastern part of the country, and had a likelihood ratio of 5.66. Clusters 1 and 3 occurred from January 2017 to December 2019. The second cluster that occurred had a radius of 294.82 km, a likelihood ratio of 6.20, and was located in the central and western parts of Ethiopia. The sixth cluster, with a radius of 36.04 km and a likelihood ratio of 20.60, was set in southern Tigray, bordering Afar. Clusters 2 and 6 occurred in the same period, from January 2014 to December 2016. The fourth cluster in northern Tigray had a calculated radius of 95.50 km and a likelihood ratio of 1.17. The seventh cluster occurred in the north-central Amhara region, with a radius of 97 km and a likelihood ratio of 1.16. Clusters 4 and 7 occurred between January 2010 and December 2013. The spatiotemporal and cluster analysis of the FMD outbreaks identified in the context of the current study are crucial in implementing control, prevention, and a prophylactic vaccination schedule. This study pointed out October to March as well as the main time of the year during which FMD outbreaks occur. The area that extends from the south to north, following the central highlands, is the main FMD outbreak area in Ethiopia.

Evaluation of Postmortem Inspection Procedures to Diagnose Bovine Tuberculosis at Debre Birhan Municipal Abattoir.

Routine meat inspection in the abattoir was used to examine carcass for subsequent approval for consumption. However, the chance of missing lesions results in approval of carcass and/or the offal with lesions of tuberculosis. A cross-sectional study was conducted at Debre Birhan Municipal abattoir from October 2016 to May 2017. Lesion prevalence estimation and two meat inspection procedures' efficacy evaluation was attempted. The breeds of the animals inspected were zebu breeds. Routine abattoir meat inspection involves visual inspection, palpation and incision of intact organs such as the liver and kidneys, as well as inspection, palpation and incision of tracheobronchial, mediastinal and prescapular lymph nodes. The detailed meat inspection involves inspection of each of the carcass. In this case, the seven lobes of the two lungs, lymph nodes and organs were also thoroughly examined. The cut surfaces were examined under bright light sources for the presence of an abscess, cheesy mass, and tubercles in detail. The study involved and compared both routine and detailed meat inspections at the abattoir. Chi-square test of independence and odds ratio were used to see the association of lesion and different risk factors. Based on detailed meat inspection, the overall lesion prevalence of bovine tuberculosis in the carcass of cattle slaughtered at Debre Birhan municipality abattoir was found to be 4.7% but only 0.5% of the carcass examined had detectable bovine tuberculosis lesions when routine abattoir meat inspection alone was used. The majority of the lesions were distributed to the lungs and associated lymph nodes. There was a significant association (p < 0.05) in TB infection rate and body condition score. In conclusion, this study has clearly indicated the prevalence of bovine tuberculosis lesions in the abattoir that are missed by routine abattoir meat inspection. In addition, it showed low sensitivity of the routine meat inspection procedure used. Hence, our study warrants immediate attention to strengthen the current meat inspection practices at Debre Birhan public abattoir.

Applicability of commercial clinical chemistry test kits for horse serum.

OBJECTIVE: Validation of a test method is critical for confirming that the test can generate accurate and precise data. Although commercial biochemical test kits exist there are no specific and validated commercial clinical chemistry test kits designed for horses. The aim of this study was to validate commercial clinical chemistry test kits designed for a human serum for use in horses. RESULTS: Blood samples were collected from 29 apparently healthy adult male horses and pooled serum was prepared. Validation comprises replication and recovery experiments. Total observable error (TEo), sigma (σ) metrics, and quality goal index (QGI) were used to support the validation studies. Intra- and inter-assay variability was 2.05% and 2.08%, 2.26% and 1.89%, 2.4% and 1.63%, for total cholesterol, urea and total protein, respectively; recovery was 99.46%, 97.32%, and 100.1% for total cholesterol, urea and total protein, respectively. TEo% for the specified analytes was within the total allowable error (TEa). All three analytes satisfied the recommended requirement (> 3σ). The QGI for urea, as it had below 6σ was 0.95 indicating imprecision and inaccuracy. The results endorse the suitability of the studied commercial test kits and illustrated the acceptance criteria for horse's serum.

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.

Veterinary medicinal product usage among food animal producers and its health implications in Central Ethiopia.

BACKGROUND: Antimicrobials and anthelmintics are the most commonly used veterinary drugs to control animal diseases. However, widespread use of these drugs could contribute to the emergence of drug resistance. Information on the practice of antimicrobial usage among food animal raising communities in Central Ethiopia is scarce. We used a standardised questionnaire survey to assess knowledge, awareness, and practices related to drug use and resistance in food animals among the farmers in and around Bishoftu town. RESULTS: Of the total of 220 livestock owners interviewed, around 80% of the respondents were not able to define what antimicrobials are and for what purposes they are used. Only 14.1% (n = 31) of the respondents had awareness about antimicrobial resistance (AMR) and its consequences; and 35.5% (n = 11/31) and 9.7% (n = 3/31) of them agreed that the irrational use of antimicrobials in animals could lead to AMR in animals and humans. Oxytetracycline was the most commonly available antibiotic in veterinary drug shops/pharmacies and the most widely used drug in the area. However, 43.3% of the respondents did not see clinical improvements after using antibiotics. Similarly, the respondents explained that no response was observed in 73.3, 70.8 and 52.5% of the cases after medication with anthelmintics, antiprotozoal and acaricides, respectively. About 56.7% of the respondents considered traditional medicines equally important to modern medicines. It was also noted that there were illegal drug vendors, dispensing medicines under unfavourable conditions which include a direct exposure to sunlight, which practice violates the drug handling and storage recommendations given by WHO. CONCLUSION: The study revealed that there is a general lack of awareness among food animal owners about the correct use of antibiotics and anthelmintics. The widespread misuse and improper drug dispensing and handling practices observed in this study can affect the drug quality and can also contribute to the development of drug resistance in central Ethiopia.

Risk Perceptions and Protective Behaviors Toward Bovine Tuberculosis Among Abattoir and Butcher Workers in Ethiopia.

Bovine Tuberculosis (BTB) is a serious cause of economic losses and public health threat, especially in developing countries. Humans acquire BTB through consumption of raw or undercooked meat, inhalation of aerosol and occupational exposure. A cross-disciplinary approach to study diseases connecting society and biology helps to understand the ways in which social, cultural, behavioral, and economic circumstances influence a healthy life. The objective of this study was to assess the risk perceptions and protective behaviors toward BTB among abattoir and butcher workers in central Ethiopia. A health belief model was used to generate the desired data following health belief model constructs. A total of 300 meat handlers working in local abattoirs, export abattoirs and butcher houses in Bishoftu, Modjo, Dukem, and Akaki towns of central Ethiopia were selected using a systematic random sampling method. Univariate and multivariable logistic regression analysis were used to assess factors associated with risk of exposure to BTB through the consumption of raw meat. The results showed that among the study participants, 95% heard about BTB and 93% knew that eating raw meat could be a source of BTB for humans. More than 62.7% of the respondents in the high risk group strongly agreed that contracting BTB would prevent them from coming to work, keep them in bed for an extended period of time and cause death. The majority of the respondents believed that free provision of personal protective clothing, compensation with test and slaughter campaigns, television and radio advertisements, educational programs and government-imposed penalties would help in prevention of BTB. Despite the high perceived severity and risk perception, the multivarable logistic regression model showed low-risk protective behavior among male (OR: 2.3, 95% CI: 1.2-4.3) and older age (>30) individuals (OR: 14.4 95% CI: 2.1-125.8). The study also noted the importance of media for health education as means for prevention of BTB. The authors strongly recommended the need of promotion of behavioral change toward the consumption of raw meat wich would have potential implications for the public health impacts of zoonotic tuberculosis and ultimately help national and global efforts toward prevention and control of tuberculosis.