Veterinary Microbiology
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Item Active Outbreak Investigation, Isolation, and Molecular Characterization of Infectious Bronchitis Virus from Poultry Farms in Mekele and Bishoftu, Ethiopia(Addis Abeba University, 2024) Nigusu Berhanu; Dr. Eyob Hirpa; Dr. Esayas GelayeAvian Infectious Bronchitis (AIBV) is a highly contagious respiratory disease that affects the poultry industry globally. In Ethiopia, AIBV has been reported in both commercial and backyard chickens. The currently used vaccine effectiveness is limited due to a lack of cross-strain protection and outbreaks continue to make mitigating the disease in Ethiopia difficult. Therefore, the objectives of this study were to isolate, genomic, and phylogenetic analysis of circulating field AIBV. A cross-sectional study was conducted from November 2023 to May 2024 in Mekele (eighteen samples) and Bishoftu (thirty-one samples) cities. Twelve tissue and thirtyseven pooled swab samples were collected, and six out of forty-nine samples (five swab samples and one tissue sample) tested positive for AIBV using real-time PCR and conventional RT-PCR. The six samples propagated into embryonated eggs and exhibited characteristic AIBV lesions and mortality over five consecutive passages. All the six isolates originating from Bishoftu (n=4) and Mekele (n=2), were amplified targeting 466 bp of the S1 gene and 433 bp of the 3'UTR using one-step RT-PCR. The purified PCR product of the five isolates targeting the 3ˊUTR region was sequenced and analyzed using bioinformatics tools. The sequence alignment of Ethiopia’s five isolates revealed a similar sequence except for one isolate (Bishoftu/03/2024) showed a single nucleotide change (A: C) resulting in amino acid change Glutamine(Q) to Proline (P). The phylogenetic analysis demonstrated the genetic distance was lower among the newly reported isolates (0.001) compared to the broader set of GenBank isolates (0.01), indicating a closer evolutionary relationship between the current local isolates and the Mexican isolates. Therefore, the findings identify genetically related local viral lineages that differ from strains used in imported vaccines. It also indicates that outbreaks were caused by infection with the IB virus which is creating a serious health risk in the poultry industry. Further research on the economic impact of AIB in poultry production, serotyping of circulating AIB viruses, and vaccine development based on the local isolates are recommended.Item Evaluation of Workers' Knowledge, attitudes, and Hygiene practices regarding food safety and the microbial load on meat and its contact surfaces in abattoirs and butcher shops in selected towns of the East Shoa Zone, Oromia, Ethiopia(Addis Abeba University, 2024) Teferi Atlaw; Dr. Biruhtesfa Asrade; Professor Bekele Megersa; Dr. Nebyou MojeA primary global goal shared by both the public and private sectors is to ensure the food safety of the meat supply chain to protect public health. Processing meat products in an unsanitary way and selling contaminated meat may harm public health. This study is aimed at the evaluation of workers’ knowledge, attitudes, and hygiene practices regarding food safety and the microbial load on meat and its contact surfaces in abattoirs and butcher shops in selected towns of the East Shoa Zone, Oromia, Ethiopia. A cross-sectional survey and sampling were conducted from November 2023 to March 2024. A total of 110 meat handlers from which 52 from slaughterhouses and 58 from butcher shops by using systematic simple random sampling methods were selected. Data was gathered via structured interviews, and 48 swab samples were randomly selected for bacterial detection. These samples aimed to demonstrate the hygienic conditions at both a slaughterhouse and a butcher shop. The statistical analysis of the data was done by STATA software version 14 for statistical analysis, with descriptive statistics and Pearson's chi-square test for evaluating sociodemographic factors' association with food safety knowledge, attitude, and practices of food handlers in slaughterhouses and butcher shops. Of the 52 slaughterhouse worker meat handlers, 75.0% had good score-level knowledge. The score level of knowledge of slaughterhouse workers was also significantly associated with socio-demographic factors of age group (ꭓ2 , 13.982; P value = 0.003), level of education (ꭓ2 , 12.515; P value = 0.002), and experiences (ꭓ2 , 7.704; P value = 0.021). The good attitude score levels of the slaughterhouse respondents were also assessed and found to be 61.5%. The study found the highest aerobic plate count (APC) in butcher shops hand and meat swabs from laughterhouses in Modjo town, with E. coli and S. aureus colonies on cutting boards. The findings emphasize the importance of knowledge and attitude in influencing food safety practices, emphasizing the need for strict protocols and trainingItem Assessment of stray dog counting and knowledge, attitudes, and practice to wards rabies in households in Addis Ababa city, Ethiopia(Addis Abeba University, 2024) Tewodros Legesse; Dr. Olana MereraRabies is a fatal viral disease of animals and people. Dogs are the primary source of infection and the majority of human rabies cases result from dog bites. Information on both domestic and stray dog populations along Knowledge Attitude Practice (KAP) assessment regarding rabies is vitally important for rabies control. However the situation of rabies is poorly known in Ethiopia, mainly in urban areas. Therefore, this study aimed to assess the demography of stray dogs, the incidence of dog bites, and the knowledge, attitude, and practice (KAP) of society concerning rabies. Questionnaire survey was collected from November, 2023 to May, 2024 using Kobo Collect toolbox in selected sub city Gulele, Yeka and Arada in order to assess KAP of the respondents towards rabies in ouseholds. In the survey, out of 384 households (96.35%) of households owned at least one dog with a total number of 463 dogs (range: 1 to 5 dogs per household); the mean number of dogs per household was 1.25 (SE 0.58). From this 96.35% of dog owning households 51.2% of respondents, owned only one dog with dog: human ratio of 1:9. 70% of dog owning households have vaccinated their dogs against rabies. Nearly all (99%) of the respondents recognized the right response regarding the route of rabies transmission and had heard of rabies. However, 64.3% of research participants had a satisfactory level of suitable rabies prevention practices score, whereas 61.8% of individuals had a moderate level of knowledge and 59.8% had an intermediate level of attitude. In this study, even though the study participants have moderate knowledge, attitude and practice towards rabies, the dog bite management and dog vaccination practice is unsatisfactory on the last three years. Age, occupation, and the source of rabies information were all significantly correlated with knowledge score (P<0.05). The counting method was by using photo capturing method and by observation. In this study, male dogs were higher than female dogs. The total dog estimation of Gulele, Arada and Yeka sub cities had estimate 1050,783, 1282 respectively. There is huge population of stray dog in the studied sites which may serve as a risk for maintaining and transmission of rabies. Along the set of study objectives, the RabiCare android app was developed to create awareness on transmission, prevention and control of rabies in three most spoken languages.Item Study of Peste des Petits Ruminants (RRP) Outbreaks: Isolation, Molecular detection, and Serological identification in Small ruminants of Borana pastoral area, Ethiopia(Addis Abeba University, 2024) Tolera Fufa; Hika Waktole; Dr. Samson LataPPR is a severe, highly transmissible transboundary virus disease that primarily affects pastoral areas by seriously compromising the health of both domestic animals and wild herbivores. An outbreak investigation was carried out from October 2023 to January 2024 in Borana and East Borana zones to figure out the PPR status in sheep and goats by isolation, molecular detection and assessing the antibodies level amongst small ruminants by purposively collecting serum(n=102) and swabs (n=48) samples from the flocks exhibiting active clinical symptoms resembling PPR.. From 48 swabs and 102 serum, 26(54.2%) and 70(68.6%) was positive for RT-PCR and b-ELISA respectively. All positive samples identified by RT-PCR underwent further processing for virus isolation by infecting Vero Dog SLAM (VDS) cells. Among the 26 samples cultured, 17 (65.4%) displayed typical cytopathic effects. To measure the agreement of the two diagnostic techniques that performed on the common animals was analyzed by Kappa statistics (κ) which revealed an agreement between the two tests was 40.7% with kappa value -0.16, it indicating that no agreement between the two tests (RT-PCR and b-ELISA). Goats were the only species suffering with clinical symptoms but no pathological evidence of PPR reported in sheep. Serologically, female animals (72.7%) were highly tested positive than male (61.1%) and the highest percentage was found in old animals followed by adults, young was the least. The sex-wise percentage of RT-PCR value for PPRV was higher in male than females. Unauthorized animal movement, flock size, rearing practices and management system, communal grazing, mixing of unknown origin, lack of quarantine practices are the main factors identified for the continuous emerging of PPRV in study area. The persistent clinical manifestations, high anti-PRV antibody levels, successful virus isolation, and precise nucleic acid detection by RT-qPCR all suggest PPRV as the primary cause of the continuous outbreak in the study area. We recommended controlling animal movement, quarantine newly purchased animals, isolating the symptomatic animals from a flock and effective mass vaccination against the disease among small ruminants.Item Risk Factors For endometritis and its Impact on fertility of postpartum dairy cows in Wolaita Sodo, Southern Ethiopia(Addis Abeba University, 2024) Tsigereda Teshome; Professor Fikadu Regassa; Tilaye DemiseDespite its significant impact on the dairy industry, there is limited research or documentation on the extent of endometritis, its specific risk factors and its impact on the reproductive performance in postpartum dairy cow in the study area. The present study was aimed to investigate incidence of postpartum endometritis, risk factors contributing to its development and reproductive consequences of endometritis in dairy farms in Wolaita Sodo, Southern Ethiopia, from October 2023 to May 2024. Longitudinal prospective study determined incidence, risk factors and reproductive outcomes prospectively. A study was carried out on 74 dairy cows with 69 clinically healthy and 5 cows diagnosed with clinical endometritis. Subclinical endometritis was diagnosed using endometrial cytology and subclinical mastitis was diagnosed using California Mastitis Test. The clinical, management and reproductive data were obtained from a weekly follow up visit of each cow and from record book of the farm. Incidence of endometritis was 44.59% (33/74) and subclinical endometritis was 40.57% (28/69). Retained fetal membranes (OR=9.23, P=0.007), assisted calving (OR= 5.06, P=0.026), dystocia (OR=7.79, P=0.014), hypocalcaemia (OR=6.49, P=0.027), mastitis (OR=5.06, P=0.026), male calf births (OR=3.06, P=0.04) and poor body condition scores (OR=2.78, P=0.003) were significantly related with subclinical endometritis. Clinical endometritis in this study were reported to be related to retain fetal membrane, vaginal prolapse, and abortion. Cows with endometritis had shown longer (P < 0.001) calving-to-conception interval, extended (P < 0.001) calving-tofirst service interval and higher (P <0.001) number of services per conception. Thus, it could be concluded that endometritis occur at higher incidence and causes a tremendous impact on the reproductive performance of dairy cows in the study area. Therefore, preventive measures should be done to reduce occurrence of endometritis through enhanced reproductive health and management practices and dairy farmers should take awareness creating trainingsItem Detection and Antimicrobial Resistance Profile of Salmonella Isolated from Cow Milk and its Products in Bishoftu Town, Central Ethiopia: Its Implication for Public Health(Addis Abeba University, 2024) Lema Temesgen; Dr. Fufa Abunna; Takele BeyeneSalmonella is a significant foodborne pathogen, with milk and milk products commonly implicated in its transmission. However, limited information is available regarding the direct link between antimicrobial use (AMU), dairy hygiene practices, and antimicrobial resistance (AMR) in Salmonella strains isolated from dairy products in Bishoftu town. Cross-sectional research was done from October 2023 to April 2024 to assess dairy farmers' antimicrobial usage (AMU) and hygiene practices and the occurrence of antimicrobial resistance (AMR) profiles of Salmonella isolated from raw cow milk and its products. Two hundred samples were collected from dairy farms, milk vendors, and restaurants and analyzed using standard microbiological methods. Using the OmniLog system, Salmonella enterica was successfully identified. Then, the antimicrobial susceptibility was evaluated using the Kirby-Bauer disc diffusion technique. A structured questionnaire was also used to assess the milk value chain's knowledge, attitude, and practices (KAP) regarding AMU, AMR, and hygiene practices. Data were analyzed using STATA version 14.2. Overall, 2% (n = 4) of the samples tested positive for S. enterica. of the 4 isolates 3 were identified in dairy farm samples, whereas 1 were isolated from milk vendors. However, no Salmonella was identified in cheese or yogurt samples obtained from the restaurants. Regarding the AMR profile, S. enterica isolates were resistant to amoxicillin (75%), streptomycin (75%), and tetracycline (50%). Resistant to two or more antimicrobials were identified in 75% of the isolates. Among 41 dairy farmers interviewed it was found that most of the respondents had sufficient knowledge (78%), desired attitudes (90%), and good practices (76%) regarding AMU and AMR. However, 36% of dairy farms had poor hygienic practices. In conclusion, the current investigation indicated contamination of cow milk and its products with S. enterica. Therefore, appropriate control measures, including awareness creation among personnel and improving hygienic practices at the milk value chains is recommended to mitigate cross-contaminationItem Production of monoclonal antibody for lumpy skin disease, sheep pox, and goat pox viruses to develop Enzyme-linked immunosorbent assay(Addis Abeba University, 2024) Kalkidan Asnake; Fufa DawoLumpy skin disease (LSD), sheeppox (SPP), and goatpox (GTP) are economically significant pox disease of ruminants, caused by lumpy skin disease virus (LSDV), sheeppox virus (SPPV), goatpox virus (GTPV), respectively. The new emergence of disease caused by capripoxviruses and spreading outside of their endemic regions, stressing the urgent need to develop high-throughput serological surveillance tools. This experimental study was conducted to produce Monoclonal antibodies (mAbs) against LSDV, SPPV, and GTPV to development ELISA assay. The study was conducted in African Union Pan African Veterinary Vaccine Centre (AU-PANVAC), from October 2023 to May 2024. The mAbs were produced through immunization of BALB/C mice with purified antigen of LSD, SPP, and GTP, with consecutive booster injection. A hybridoma technology was used to produce the hybridoma clones (LC 5.14 and LC 5.4) which were subsequently mass-produced and tested. These LC 5.14 and LC 5.4 mAbs were precipitated and then quantified through the Bicinchoninic Acid protein assay kit. The isotype for the two mAbs were determined through Pierce™ Rapid Antibody Isotyping Kit, and both mAbs were IgG1. The cross-reaction of the two mAbs with GTPV and SPPV Ag were studied and those two mAbs were cross-reacted with the GTPV Ag but not with the SPPV Ag LC 5.14 and LC 5.4 mAbs were then conjugated with horseradish peroxidase enzyme; hence enzyme linked mAbs were used for ELISA development. The dot blot test was conducted by using of the LSDV, SPPV, and GTPV Ags with the two conjugated mAbs on the nitrocellulose membrane. Finally, the conjugated mAbs (LC 5.14 and LC 5.4) were titrated to determine the concentration which is required for ELISA test, and the titer of 1/10 for LC 5.14 and titer of 1/5 for LC 5.4 were determined to use for ELISA assay. Further repeated tests with the positive serum from immunized cattle, sheep, and goat and the negative serum from the same species of animals are required to produce the validate kit which can be used as a diagnostic purpose for the capripoxviruses.Item Isolation and Molecular Characterization of African Horse Sickness Virus AHS Outbreak Cases In Horses in selected areas of Ethiopia(Addis Abeba University, 2021) Degu Fhetanegest; Hika Waktole; Dr. Esayas Gelaye; Dr. Hana ZewduEquines play an important role in the country‟s economy and are a lifeline for millions of people in rural and peri-urban areas of Ethiopia. However, the productivity and welfare of equids are constrained by numerous infectious diseases especially in developing nations like African Horse Sickness disease (AHS). A H S is one of the major infectious diseases that cause severe socioeconomic losses to the equine population and the national economy in general. A cross-sectional study design was undertaken in equines to isolate and detect African horse sickness virus (AHSV) from November 2019 to May 2021 in selected and epidemic areas of Ethiopia. A Total of 30 whole bloods and 2 tissue specimens were collected aseptically from recently dead and clinically sick equids that manifested prominent signs of the disease and transported under cold chain to the National Veterinary Institute, Bishoftu, Ethiopia. A total of 32 samples were subjected to conventional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) technique targeting Viral protein 7 (VP7) genes to amplify fragments of segment 7 of all serotypes using sero-group specific primers. Only 7 (21.88%) samples were detected with a band size of 102 bp fragments on a 2% agarose gel elctrophoresis. For serotyping, seven universal PCR positive samples were detected again targeting to a gene encoding viral protein 2 (VP2) using serotype-specific primers. Serotype 9 with a band size of 228 bp was identified from tissue samples. Only tissue samples were grown on Vero cells and showed cytopathic effects characterized by aggregation, rounding and detaching of cells on cell line. In conclusion, African Horse Sickness caused by serotype 9 severely affects equines results in death of horses. Strong strategic control of disease through vaccination should be done and further assessment to determine the potential of outbreaks and genotypic characterization of virus from the outbreaks and insects needs further studyItem Development of Rapid, Specific, and Field-Deployable Diagnostic Assays For Sheepox and Goatpox Viruses Using Crispr-Cas Technology(Addis Ababa University, 2024) Birtukan Zenebe; Dr. Olana Merera; Dr. Samson LetaEthiopia is home to a large population of sheep and goats, which are raised for their meat, hides, and skins. Even with this huge financial resource, animal diseases are still the main factor that limits their productivity. sheeppox and goatpox is one of the most prevalent diseases caused by a genus of Capripoxvirus that have a negative impact on sheep and goats throughout the world. One of the main obstacles to the quick diagnosis of the sheeppox and goatpox viruses is the lack of rapid, specific, and point-of-care diagnostic tests. Thus, the aim of this study is to develop a rapid, specific and field deployable diagnostic assay to detect sheeppox and goatpox viruses based on recombinase polymerase amplification (RPA) and trans-cleavage activity of clustered regularly interspaced short palindromic repeats (CRISPR/Cas12a) and to validate its diagnostic specificity. Thus, a lab-based experimental study was employed from November 2023 to April 2024 to develop this assay. RPA-CRISPR/Cas12a-lateral flow assay was developed to detect the entry fusion complex component gene of the sheeppox and goatpox viruses’ genomes. The assay was optimized to be completed within one hour, including nucleic acid extraction for 25 minutes, RPA reaction at 39°C for 15-20 minutes, CRISPR/Cas12a reaction at 37°C for 20 minutes, and visual detection through lateral flow (3 minutes). The primers for RPA, CRISPR RNA (crRNA), and ssDNA reporter for lateral flow detection (LFD) were synthesized and optimized. In this study, quick, specific, and versatile diagnostic method for goatpox and sheeppox virus was successfully developed. This innovative approach harnesses recombinase-polymerase amplification in conjunction with the CRISPR/Cas12a system, offering a cost-effective and efficient solution for identifying these viral infections. This diagnostic technique shows no cross-reactivity with Peste des petits ruminants (PPR). In conclusion, RPA-CRISPR/Cas12a is a potentially useful method for quickly and accurately diagnosing goatpox and sheeppox, especially in environments with limited resources. Therefore, Further research is needed to ensure the assay's reliability and robustness across diverse sample populations and varying environmental conditions.Item Isolation and Molecular Characterization of Lumpy Skin Disease Virus in Central Ethiopia(Addis Ababa University, 2021) Mariamawit Zekarias; Dr. Shimels Tesfaye; Dr. Esayas GelayeLumpy skin disease (LSD) is high impact viral disease affecting cattle in various parts of Ethiopia and considerable number of countries worldwide. Taxonomically, LSD virus is classified in the family Poxviridae of the genus Capripoxvirus. The disease is caused by LSD virus and is characterized by nodules on the skin that cause permanent damage to hides and skins. LSD a serious disease that has been impossible to control in Ethiopia which is made clear by the outbreak that occurs year in and year out. Even though a continuous work has been done to understand the disease, tackling it prove to be unrealizable, urging more work to be done as much as possible that can assist as a building block to bring this devastating disease to its annihilation. The current work hopes to do that by isolating and characterizing of the virus that cause this disease from the most recent outbreak incident. A purposive sampling technique was mplemented in the town of Mojo, Ejere, Ejersa and koka. Cell line originated from sheep skin (Embryonic skin of sheep/ESH-L), highly sensitive to Capripoxvirus, was used for the isolation of the infectious virus. The isolates were further processed for classical and real time PCR in order to genotype. The virus that was detected as LSDV, have been further characterized through the RPO30 gene amplification for sequencing and phylogenetic tree construction in relation to different viral isolates from a previous work done though out the country and beyond. Out of 15 samples collected 10 of them were found to be positive for LSD. Further sequencing shows there was a two nucleotide position variation when comparing the present study isolates and the vaccine (KS-1) resulting in a single nonsense amino acid mutation. Constant outbreak investigation and full gene sequencing are the major suggestion of the study.Item Evaluation of different adjuvant formulations of inactivated trivalent foot and mouth disease vaccine in cattle(Addis Ababa University, 2021) Getu Ayele; Dr. Bedaso Mammo; Dr. Belayneh GetachewFoot-and-mouth disease is globally one of the most economically important viral diseases of cloven-hoofed animals that can primarily controlled by vaccination. Selection of the effective adjuvants formulated simultaneously with the antigen in the vaccine is crucial in ensuring the quality of vaccine and the protective effect of the vaccine against FMD. Aluminum hydroxide gel and saponin (AS) is the most used adjuvant, with its shortcoming in short duration and poor immune response in FMD vaccine. Therefore, the present experimental study was undertaken to evaluate different formulation of adjuvants for inactivated trivalent FMD vaccine containing A, O and SAT 2 serotypes in cattle. Additionally, this study was also performed to demonstrate the effect of booster dose administration on immune response. The vaccines were prepared by mixing 10 % of aluminum hydroxide gel, 0.3 % of saponin with the virus suspension. Oil based was prepared with equal volume of virus suspension (50:50). Twenty-nine cattle were classified into five groups, with four experimental groups consisting of six cattle in each group (n=6) and the fifth group is a control group with five cattle (n=5). The experimental cattle were grouped as: AS, AS boosted, oil based and AS + oil. The sera sample were collected on day 0, 7, 14, 21, 28 and 42, their immune response was measured using Solid Phase Competitive Enzyme Linked Immune Sorbent Assay (SPCE). There was a significant difference in the immune response between the adjuvant groups (P<0.05, ANOVA). The results showed that, the antibody level in cattle vaccinated with AS were significantly lower than AS boosted group for serotype A, O and SAT 2, indicating that the need for booster dose. Whereas the antibody response in the AS + oil group was higher followed by oil alone, AS boosted and AS. It can be concluded that oil based and AS with oil induce better antibody response relative to others and they could replace the aluminum hydroxide gel and saponin for FMD vaccine production to control the disease. On different note, challenge test was not successful in this study indicating the need for further research on the virus infectivity.Item Preparation of Freeze-Dried Clostridium Chauvoei Master Seed Bank from Local Isolate for Production of Safe, Effective and Acceptable Quality of Black Leg Vaccine(Addis Ababa University, 2021) Jaleta Shuka; Hika WaktoleThe research was accompanied from December 2020 to May 2021 amid to develop new preservation method spore form of Cl. Chauvoei from local isolate with the objective of mainly improving vaccine immunogenicity. Currently, in National Veterinary Institute (NVI) of Ethiopia viande-foie (V.F) medium is the sole medium used for preservation of the strain as a vaccine master seed bank. However, this method of preservation has its own drawbacks like low antigenicity maintenance, prone to contamination and difficulty in transportation. Intended for this purpose, samples were composed from Adea district from cattle following outbreak information of black leg disease. The samples were taken to NVI using cold chain system for isolation purpose. The blood and tissue samples were isolated as Cl. Chauvoei. The isolates were identified by cultural, morphological and biochemical features. With the aim of additional confirmation by means of pathogenicity study and acuity estimation, the isolates were inoculated on gunia pigs and up on postmortem examination it was confirmed as Cl. Chauvoei. The study of PCR also revealed the existence of Cl. Chauvoei. New method of preservation was then developed through lyophilization or freeze-drying of the isolated strain. Quality control tests like vacuum, moister content, viability, sterility, safety, lethal dose determination for potency test, and identity tests of freeze-dried spore form of Cl. Chauvoei seed bank were performed. Accordingly, the quality control tests of the freeze-dried product revealed; vacuum (1.5%), moister content (97.5%), LD50/ML (1 x 10 7.2), known to be viable, safe and free of contaminant. Regarding potency test 10 gunia pigs were challenged with LD50 dose of virulent challenge strain, and only one of them was dead, thus it is 90 % protective. It is believed that freeze-dried spore stored at -20 0 C retain viability for years, however in this specific study stability test at different storage temperatures for long period of time is essential to conclude this idea. Moreover, study reinforced by DNA sequencing is also indispensable to finalize these inferences.Item Comparative Evaluation of Direct Rapid Immuno-Histochemical Test (DRIT) with Direct Fluorescent-Antibody Test (DFAT) for laboratory diagnosis of animal Rabies in Ethiopia(Addis Ababa University, 2021) Sintayehu Abedla; Hika Waktole; Dr. Gezahegne Mamo; Dr. Abraham AliDirect fluorescent antibody test (DFAT) is used as a gold standard method for rabies virus detection. The present study aimed was to compare and evaluate DRIT with Direct Fluorescent Antibody Test (DFAT) to use equivalently as one of rabies virus diagnosing methods in areas where DFAT is not accessible. The method is based on the capture of rabies nucleoprotein (N) antigen in brain smears using a cocktail of biotinylated monoclonal antibodies specific for the N protein and color development by streptavidin peroxidase-amino ethyl carbazole and counterstaining with hematoxylin. The test was performed in parallel with the standard DFAT and mice inoculation test (MIT) using 100 brain specimens from various species of animals. The majority of them were dogs (n=88), ollowed by cats (n =8), cattle (n =3), and donkey (n =1), and also from those samples that were tested by DRIT and DFAT, we randomly selected and tested 12 brain samples by MIT. The results indicated that 63% of the tests were positive by DFAT and 64% were positive by DRIT. A slight difference was observed in such a way that one sample was negative by DFAT but positive by DRIT and MIT. The DRIT provides powerful, economical tool for rabies diagnosis to improve existing rabies surveillance, prevention and control programs in Ethiopia. Although further laboratory and field examinations are essential, our findings were providing and remark the potential value of the DRIT for countries with limited diagnostic resources.Item Isolation and Identification of Cultivable Aerobic Pathogenic Bacteria from Ticks of Cattle in Central Ethiopia(Addis Ababa University, 2021) Tekabe Gebre; Dr. Gezahegne Mamo; Prof. Bersissa KumsaTicks are well-known vectors of a variety of intracellular tick-borne pathogens associated with tick-borne diseases worldwide (TBD). There is, however, a scarcity of precise and up-to-date comprehensive information on cultivable aerobic pathogenic bacteria from ticks of cattle from Ethiopia. A cross-sectional study was conducted from November 2020 to July 2021 with the objectives to isolate and identify cultivable aerobic pathogenic bacteria from ticks infesting cattle in central Ethiopia, namely, Ada’a, Lome and, Ezha districts. All ticks used to study pathogenic bacteria were morphologically identified to species level under a stereomicroscope using standard taxonomic keys. During the study period, a total of 205 adult live ticks belonging to eight species, namely, Hyalomma truncatum (N=50; 24.4 %), Amblyomma variegatum (N=41; 20%), Amblyomma cohaerens (N=40; 19.5%), Rhipicephalus decoloratus (N=33; 16.1%), Hyalomma rufipes (N=29; 14.1%), Rhipicephalus evertsi (N=7; 3.4%), Amblyomma gemma (N=4; 1.9%), and Rhipicephalus pulchellus (N=1; 0.5%) were identified in decreasing order and collected for bacteriological examination. Bacterial identification was performed by using multiple biochemical tests and API-20E strips. Results of the study showed that out of the total of 205 ticks studied for the presence of bacteria, 107 (52.2%) ticks were positive and 98 (47.8%) ticks were found negative for bacterial isolation and a total of 107 isolates of different bacterial pathogen were identified from all the study areas. Out of the total of 107 bacterial isolates recorded, a total of 5 species of aerobic pathogenic bacteria were identified including, (N=39; 36.5%) Citrobacter freundii, (N=34; 31.8%) Escherichia coli, (N=18; 16.2%) Staphylococcus aureus, (N=8; 7.5%) Proteus mirabilis, and (N=8; 7.5%) Morganella morganii. Statistically significant differences between study districts as well as species of ticks with the isolation rates of the pathogenic bacteria were observed. In vitro efficacy evaluation of the most commonly used antibiotics demonstrated that majority of the cultivable aerobic pathogenic bacteria detected in ticks collected from cattle were susceptible to chloramphenicol, streptomycin, and gentamicin but resistance against the action of bacitracin, penicillin, and clindamycin was recorded on the disk diffusion test method. In conclusion, the high isolation rate of pathogenic bacteria in ticks collected from cattle in the current study most likely indicates that ticks play an active role in environmental contamination and increase the likelihood of pathogenic bacteria transmission to their hosts.Item Isolation, molecular detection, antibiogram profile and the associated risk factors of Salmonella from poultry farms in and around Debire Birhan(Addis Ababa University, 2021) Yonas Ayele; Hika WaktoleSalmonella infections are very common in both animals and humans that cause significant economic and public health impacts in Ethiopia. A cross-sectional study was conducted from December 2020 to June 2021 to isolate, perform molecular detection, determine antibiogram, and asses the associated risk factors of Salmonella species from poultry farms in and around Debire Birhan, Central Ethiopia. For these purposes, a total of 384 samples obtained from cloacal swab (n=136), fecal dropping (n=130), chicken feed (n=64), and drinking water (n=54) were aseptically collected and examined. Out of 26 poultry farms subjected to standard bacteriological culture method, 19 (73.07%) were found positive for Salmonella isolates at least in one of the examined sample types. The overall bacteriological prevalence of Salmonella species isolated was 14.06% out of the total 384 samples analyzed. Among the determinants, sample type and flock size were strongly associated with the isolation and identification rate of Salmonella (P<0.05). Accordingly, higher isolation and identification rates found in fecal droppings 29(22.30%) and flock size greater than 1500 chickens/farm 20(23.25%), respectively. However, the isolation rate was not affected by location, age, and breed of the chickens. The molecular detection rate of S. Typhimurium was 50% out of the 30 Salmonella isolates subjected to a conventional polymerase chain reaction. The detection rate of S. Typhimurium showed significant association with age groups (p=0.03) and flock size (p=0.04) where higher isolation rates were recorded in the age group greater than 18 weeks and flock size greater than 1500 chickens. The disk diffusion antimicrobial susceptibility finding showed that all S. Typhimurium isolates were found multidrug resistant and higher antimicrobial resistance observed to ampicillin (93.3%) followed by oxytetracycline (86.7%), sulfamethoxazole (46.7%), and tetracycline (40%). On the other hand, 100% and 73.3% of isolates were susceptible to ciprofloxacin and gentamycin, respectively. In nut shell, the present study disclosed higher isolation and detection rate of Salmonella species and also the appearance of multidrug resistant S. Typhimurium to several drugs necessitating the urgency for further detailed molecular characterization to come up with the circulating Salmonella serovars and antimicrobial resistance strains and responsible genes.Item Evaluation of protective efficacy of irradiated Salmonella Gallinarum vaccine against fowl typhoid in Sasso breed chicken(Addis Abeba University, 2022) Yitbarek Habtamu; Prof Gezahegne Mamo.; Dr.Tadesse EgualeFowl typhoid is worldwide distributed septicemic disease of chicken, turkeys, ducks, pheasants, guinea fowl, peafowl, sparrow, goose, and quail caused by Salmonella enterica subspecies enterica serovar Gallinarum. Live attenuated 9R strain of Salmonella Gallinarum (SG) is commonly used vaccine for the control of fowl typhoid. However, its persistence in vaccinated chickens causes vertical transmission through eggs and the residual virulence inducing lesions in the liver and spleen in some breeds of chicken are the drawbacks of this vaccine. In recent vaccine development efforts, alternative methods to develop a variety of vaccine types have been attempted of which radiation inactivated pathogens are some of them. Irradiation can avoid chemical contaminants from chemical inactivation and penetrate pathogens to destroy nucleic acids without damaging the pathogen surface antigens. The objective of this study was to evaluate protective efficacy of gamma irradiated Salmonella Gallinarum vaccine against fowl typhoid in poultry. After the strain identification test, inoculum of approximately 109 cfu/ml of field strain of Salmonella Gallinarum was prepared and exposed to series of radiation dose ranging from 1.1-3 kilo gray (kGy) for inactivation of which 2.6 KGy was found to be minimum lethal dose and it was used for final irradiation dose in this study. Forty two (42) days old Sasso breed of chickens were allocated randomly to six groups having 10 chickens each: G1 (vaccinated with irradiated Salmonella Gallinarum vaccine but not challenged), G2 (vaccinated with irradiated Salmonella Gallinarum vaccine and challenged), G3 (vaccinated with irradiated Salmonella Gallinarum vaccine, provided with booster dose 21 days later and challenged), G4 (vaccinated with irradiated Salmonella Gallinarum containing 20% trehalose and challenged), G5 (vaccinated with 9R commercial Salmonella Gallinarum vaccine produced at NVI, Bishoftu, Ethiopia, and challenged) and G6 (unvaccinated but challenged group as a control). Prior to the immunization process, all chickens were assessed for the presence of antibody against Salmonella Gallinarum on the 7 th weeks of age and none of them were found serologically positive using slide agglutination test (SAT). The homologous challenge infection experiment was conducted using the standard field dose of (~5.3 x 107 cfu/ml) ~ with optical density value of 0.6. On day 21, G4 and G5 showed strong antibody production than other groups, 80% and 90%, respectively. Fifty (50%) of G1 showed strong antibody production and 50% of them moderate reaction. However, only 20% and 30% of G2 and G3 respectively produce strong reaction on day 21 post vaccination. As G6 was unvaccinated group, there was no reaction throughout the experiment. But G3 on booster dose after 2 weeks of vaccination showed 60% strong agglutination on day 35 of first vaccination. Up on the challenge, chickens in G3 and G4 showed significant difference in survival rate (70%) over G2 and G6 which only 20% of them survived. Survived and sacrificed chickens at the end of experiment showed significantly lower lesions and bacterial re-isolation from the liver, spleen and gizzard as compared to those birds died during challenge experiment. There was significantly higher number of survivors among vaccinated G3 & G4 as compared to non-vaccinated group (G6) (p<0.0001). There was significant difference (p<0.05) in level of protection between G2 and G4 as well as between G2 and G5. Survivors in G5 were 100% that showed commercial 9R vaccine conferred strong protection as compared to G2, G3 and G4 and their protection was 20%, 70% and 70% respectively. There was no significant level of protection in chickens in G2 compared to unvaccinated control group (p>0.05). Addition of 20% trehalose and booster dose improved protection of irradiated vaccine by50%. In conclusion, subcutaneously administered irradiated SG candidate vaccine with 20% trehalose and booster dose of irradiated vaccine without 20% trehalose showed promising safety, immunogenicity and protective efficacy. Further studies on safety, shelf life, radiation dose optimization for trehalose added irradiated Salmonella Gallinarum vaccine and quantification of antibody response using ELISA and other immunological methods are recommendedItem Identification, Antimicrobial Susceptibility Profiles and Molecular Detection of Salmonella from Chicken Farms in Holeta, Sululta and Sebeta Towns, Central Ethiopia(Addis Abeba University, 2022) Ebisa Mezgebu; Hika Waktole (Associate Professor); Dr.Abebe OlaniChickens Salmonellosis is one of the leading causes of heavy losses in chicken industry and has a significant public health impact. In Ethiopian chicken farms, determining the antimicrobial susceptibility test (AST) status of Salmonella with respect to its reported serovars was not very prevalent. This study was conducted with the objectives of identification, molecular detection and determination of AST profiles of Salmonella species from chickens’ farms in central Ethiopia. A cross-sectional study was conducted in selected potential chicken raising areas including Holeta, Sululta and Sebeta from November 2021 to May 2022. A 425 cloacal swabs were sampled by simple random sampling technique, and 18 feed and 18 water samples were collected from 18 farms before providing it for chickens. Out of 461 samples (176, 130 and 155) samples from Holeta, Sululta and Sebeta respectively; 3 (0.65%) Salmonella were identified. From these three isolates 2 (1.14%) and 1 (0.65%) were identified from Holeta and Sebeta respectively. However, no sample was found positive from Sululta. Out of the three isolates, 2 (0.47%) and 1(5.56%) Salmonella were identified from a total of 425 cloacal and 18 feed samples respectively. Biochemically isolated and Omnilog identified as Salmonella enterica Paratyphi B, and Salmonella Enterica and Salmonella Gallinarum (identified from feed, and the later were from cloacae swabs) samples. The invA gene was detected in all of them. Then AST was assessed by 9 antimicrobials of all Oxoid disks; So, Salmonella Gallinarum was resistant to streptomycin and tetracycline. Whereas, Salmonella enterica Paratyphi B and Salmonella Enterica were intermediate to eropenem and streptomycin disks. Only sample type variable was statistically significant (p < 0.05). The findings showed that Salmonella can be present in chickens and their environments. Even though isolates numbers were low, all of them were resistant and intermediate to some of the antimicrobials; and if it transmitted to other animals and humans with their resistant genes, it can pose a serious risk of transmission of resistant developed genes. This warrants the implementation of strong biosecurity policy, and proper use of antimicrobials by excluding resistance developed antimicrobials from the market. Moreover, awareness should be created to the chicken farm owners on measures to avoid biosecurity and management risk factors of Salmonellosis and the occurrence of antimicrobials resistance in chicken farmsItem MALDI TOF MS and Molecular Detection of Mannheimia haemolytica from Sheep and Goats in Holeta and Sebeta town, Oromia Special Zone, Ethiopia(Addis Abeba University, 2022) Abdi Ahmed; Dr. Debebe Ashenaf; Dr.Abebe OlaniMannhemia haemolytica is one of the most important bacteria among causative agent of pneumonic pasteurellosis in small ruminants throughout worldwide. It is one of the most economically devastating pathogen in sheep and goats in Ethiopia. A cross sectional study was carried out from November 2021 to May 2022 with the aim to identify Mannheimia haemolytica from sheep and goat in Sebeta and Holeta town, Oromia special zone, Ethiopia. A total of 235 samples (213 nasal swabs and 22 whole blood) were collected from sheep and goats for Mannheimia haemolytica identification. Sheep and goat with clinical signs suggestive of pneumonic pasteurollosis were purposively sampling. Bacterial identification was conducted using biochemical, Biolog, Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI TOF MS) and Real time PCR detection. Moreover, antimicrobials susceptibility test was also conducted on the identified bacterial isolates using disc diffusion method. The result showed that from a total of 235 samples, only two nasal swab samples were positive for M. haemolytica (0.85%). The two isolates were confirmed by all the tests and similar result was obtained by; biochemical, Biolog, MALDI TOF MS and real time PCR. Up on antimicrobial susceptibility testing, the two isolates were resistant to Streptomycin, Erythromycin and Clindamycin whereas they were susceptible to Tetracycline, Chloramphenicol, Trimethoprim/sulfonamides and Penicillin. Generally, this study revealed that M. haemolytica is among the causative agent of pneumonic pasteurellosis in sheep and goat in the study area. Although, the other remaining bacteria responsible for the disease. The research suggests that a combination of diagnostic methods such as MALDI TOF MS, Biolog, and realtime PCR should be used, as well as for a more in-depth investigation to identify the strain or serotype of M. haemolytica using advanced molecular sequencing and also analysis of the remaining causal agent from various species and locations in countries that are significant for disease control and prevention and also to address the present vaccination and antibiotic resistance issues.Item Isolation, Antimicrobial Sensitivity Test, MALDI-TOF Confirmation and Molecular Characterization of Salmonella and Escherichia coli from Commercial Poultry Farms in Bishoftu, Ethiopia(Addis Ababa University, 2023) Bilisuma Abebe; Hika Waktole( Assoc. Prof.)In Ethiopia, the poultry sector, which is becoming the main source of economic activity, is being challenged by infectious diseases and the frequent use of antibiotics which may results in antibiotic resistance that leads to economic crises and public health issues. The current research work was conducted from November 2022 to June 2023 with the objectives of identifying, antimicrobial sensitivity testing, MALDI-TOF confirmation and molecular characterization of pathogens from layer and broiler farms in Bishoftu, Ethiopia. A cross-sectional study design and rapid questionnaire were employed using random sampling method to collect a total of 284 samples (cloacal swabs, litter droppings, water, and feed) from diverse farm size. The rapid questionnaire survey was conducted to assess the commonly available antibiotics with regard to their usage practices and management in the selected commercial poultry farms. The samples were transported to laboratory and the isolates were identified using primary and secondary isolation and confirmed by MALDI-TOF and conventional Polymerase Chain Reaction (PCR). Out of 284 samples processed for both Escherichia coli and Salmonella, 40 (25 from layer and 15 from broiler) and 38 (12 from layer and 14 from broiler) samples were confirmed by primary isolation and biochemical tests respectively. From a total of 38 Confirmed isolates of Salmonella only 26 isolates were furtherly subjected to MALDI-TOF and PCR for confirmation and remained negative. Out of the 40 samples subjected to MALDI-TOF confirmation for Escherichia coli, 17 isolates were found to be positive. All confirmed isolates of Escherichia coli were further subjected to antimicrobial sensitivity test using nine types of antibiotics. The antimicrobial sensitivity test revealed highest resistance against tetracycline and ciprofloxacin in layers and amoxicillin in broilers. The findings of the present study disclosed that Escherichia coli to be widespread and prevalent in the study area with alarmingly high level of resistance for tetracycline where most of the farms were using it commonly. The rapid questionnaire on the use of antibiotics indicated indiscriminate utilization for prevention-treatment, for treatment and for treatment-prevention-growth purposes in a farm which might attribute to higher level of antimicrobial resistance. As a general, improvement of poultry biosecurity is recommended along with rational usage of antimicrobials. More specifically, the test agreement /discrepancy among the diagnostic tests should be further evaluated.Item Evaluation of the Safety and Immunogenicity of Saponin-, Heat-, and Minor Formaldehyde-Inactivated Contagious Caprine Pleuropneumonia Whole Culture Vaccine(Addis Abeba University, 2022) Abiyot Abebe; Dr. Bedaso Mammo; Dr. Belayneh GetachewContagious caprine pleuropneumonia (CCPP) is a fatal disease of goats imposing significant economic losses through the goat production system. Addressing effective vaccine is the most cost-effective technique in the control of the disease. In National Veterinary Institute (NVI), inactivated protein-based Mycoplasma capricolum subspecies capripneumoniae (Mccp) F-38 strain whole culture vaccine is in use since a few years ago. The vaccine is inactivated by formaldehyde (in a 0.5% proportion) and adjuvated with saponin. Despite the efficacy of the vaccine in Ethiopia, using saponin and heat inactivation has not yet been well considered. While saponin could be used both as inactivant and adjuvant simultaneously, extra formaldehyde is applied for inactivation. This could affect the immunogenicity of the vaccine, and it is not economical to invest on extra formaldehyde. Formaldehyde also has a residual effect. In addition, it is toxic to the laboratory workers, while heat treatment is relatively safer and cheaper. On the other hand, applying high amount of formaldehyde affects the immunogenicity of the vaccine. The aim of this study, therefore, was to evaluate the safety and immunogenicity of the vaccine inactivated alternatively by saponin and heat to replace formaldehyde inactivation, and by minor amount of formaldehyde to make use of such amount in the inactivation process of the vaccine. The vaccine was prepared using World Organization for Animal Health (OIE) guideline for CCPP vaccine production and the standard operating procedure of the manufacturer, NVI. The prepared Mccp culture aliquots were inactivated separately by saponin, heat treatment, and minor formaldehyde proportion. Thirty Mccp antibody-free goats were arranged into 5 groups, and a single dose of every vaccine formulation of a different inactivant was applied to each respective group. The goats were observed for 1 month for the safety and immunogenicity evaluation. All of the inactivation protocols were effective in inactivating the vaccine, and the respective vaccine preparations were safe. While the preparations inactivated by heat and 0.1% formaldehyde showed seroconversion values of equal significance with that of the conventional vaccine (p > 0.05), the saponin-inactivated vaccine brought unsatisfactory results. Accordingly, it was concluded that after a field trial and/or challenge study, heat and 0.1% formaldehyde, but not saponin, can be applied as alternative inactivating agents for the CCPP whole culture vaccine, which may improve the vaccine quality and occupational safety.