Veterinary Microbiology

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Isolation and Molecular Characterization of Lumpy Skin Disease Virus in Central Ethiopia
(Addis Ababa University, 2021) Mariamawit Zekarias; Dr. Shimels Tesfaye; Dr. Esayas Gelaye
Lumpy 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.
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 Getachew
Foot-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.
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 Waktole
The 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.
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 Ali
Direct 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.
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 Kumsa
Ticks 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.
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 Waktole
Salmonella 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.
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 Eguale
Fowl 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 recommended
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 Olani
Chickens 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 farms
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 Olani
Mannhemia 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.
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.
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 Getachew
Contagious 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.
Serological investigation and molecular detection of peste des petits ruminants virus in Boset and Fantale districts of East Shewa zone of Oromia region
(Addis Ababa University, 2023) Hussen Ali; Hika Waktole( Assoc. Prof.); Dr.Samson Lata; Dr Gazahegn Alemayo
A cross-sectional study was conducted for serological investigation and molecular detection of Peste des Petitis Ruminants virus from January 2023 to May 2023 in Boset and Fantale districts of East Shewa Zone of Oromia Region with the objective of revealing the sero-prevalence and associated risk factors of PPR in non-vaccinated small ruminants, estimate sero-conversion after PPR vaccination using c-ELISA and detection of PPRV circulating in the area using PCR. Of the total 331 sera samples collected, 165 samples were collected before administering vaccine in the two districts and tested for PPRV antibodies by using c-ELISA and 102 (61.82 %) were positive. Seroprevalence of 47.44 % in Boset and 74.71% in Fantale was recorded with a statistically significant difference in the prevalence between the two districts (ꭓ2=12.96, P value<0.001). Prevalence in sheep (46.88%) and (65.41%) in goat were recorded. PPRV antibody seroprevalence was higher in adult animals 65.16 % (95%CI=57.3-72.3) and lower in young animals 10 % (P< 0.001, ꭓ2=36.12) and it was statistically significant. The majority of seropositive sheep and goats were those with poor body condition (70%). With flock size, statistically significant seropositivity was observed (P< 0.001). Out of 166 vaccinated sheep and goats tested for post vaccination sero-conversion assessment starting from the second-week post-vaccination, 137 (82.53%) animals were seropositive to PPR according to c-ELISA. So, in this study, rise in herd immunity from (61.82%) prior to vaccination to (82.53%) was observed after the vaccination campaign. From 9 goat samples taken at an outbreak and tested for viral RNA detection 4 (44.4%) were positive for viral nucleic acid. Serological investigation and molecular detection results from this study show that, PPRV was circulating in Boset and Fantalle districts. Therefore, measures to control and eradicate the disease have to incorporate early reporting of the disease outbreaks, effective targeted vaccination and strict animal movement control. More studies characterizing the circulating virus are also recommended.
Assessing Farmers’ Acaricides Use and Efficacy Evaluation of Different Acaricides and Selected Herbal Extracts Against Cattle Ticks
(Addis Ababa University, 2023) Solomon Desta; Dr. Lishan Assefa
Tick-controlling activity has been carried out by using a variety of commercially available chemical acaricides. However, the extensive use of chemicals promotes resistance and resulted in toxicity to animals and the environment, and residues in food animal products. Therefore, this study was aimed at assessing farmers’ acaricides use and efficacy evaluation of different acaricides and selected herbal extracts against cattle ticks. The study was carried out using the questionnaire survey and in vitro experimental activity to evaluate the acaricidal activities of hydromethanolic leaf extracts of Calpurnia aurea, Datura stramonium, Nicotina tobaccum, and Ricinus communis against Amblyomma cohaerence by comparing with the efficacy of deltamethrin, diazinon, amitraz and five brands of ivermectin, and with DMSO (10%) as the negative control. Adult immersion test (AIT) following complete randomized design was used to test the efficacy. The result showed that farmers in the study area have poor knowledge (50%) and improper practices (91%) about acaricides use. Deltamethrin and the five brands of ivermectin were showed the highest level of efficacy (100±0.00 %) in tick mortality, while diazinon and amitraz were the least ranked (83.3±8.82% and 63.3±8.82%) within 72hrs of exposure time, respectively. There were statistical significance differences in efficacy between amitraz and other acaricides (p = 0.000). All extracts at the concentration of 100mg/ml were showed a significant difference in tick killing after 48hr post-exposure time, while below 50mg/ml showed insignificant effect (p > 0.05). At 72hr post-exposure time, N. tobaccum showed better efficacy (86.7±8.8%) followed by D. stramonium (76.7±6.7%) at 100mg/ml. Besides, R. communis showed slightly better efficacy (70±5.8%) than C. aurea with a statistical mean (63.3±8.8%). Finally, N. tobaccum and D. stramonium showed good acaricidal activity, followed by R. communis and C. aurea. While compared with commercial acaricides, all herbal extracts showed higher efficacy than amitraz 12.5%. Hence, the current study recommends, herbal extracts that showed high efficacy should be used as an alternative therapy and commercial acaricides with low efficacy should be reserved from using on animals to control tick infestation.
Study on Lumpy Skin Disease Vaccination Practice Based on Field Survey and Vaccine Efficacy Based on Molecular Characterization of Immunogenic Genes in and Around Bishoftu Town, Ethiopia
(Addis Ababa University, 2023) Tamirat Haile; Dr. Shimelis Tesfaye
Lumpy skin disease virus is a member of the Capripoxvirus genus of the Poxviridae family, which affects cattle and causes significant economic losses. It is controlled by vaccination with capripox live attenuated vaccines. The aim of this research was studying lumpy skin disease Vaccination practice based on field survey and vaccine efficacy based on molecular characterization of immunogenic genes in and around Bishoftu Town, Ethiopia. Questionnaires were prepared for 101 cattle owners and animal health professionals incorporating questions with a major emphasis on LSD vaccine and vaccination practices. From transport related factors, inappropriate vaccine handling and lack of transport equipment‘s and from storage related factors electric outage and refrigerator problems were taken as important factors which have negative impact on effective vaccination. Nodular skin lesions that have been collected in 2022 from the same study area by the same project were used to isolate the virus using lamb kidney cells at virology laboratory of Animal Health Institute (AHI) of Ethiopia. Polymerase chain reaction was performed targeting the immunogenic genes; LSDV 117(A27L), LSDV122 (A22R), LSD 060 (L1R) and LSD 141 (B5R). Additionally, both the field isolates and currently on use LSDV vaccine strains (KS-180) from National vaccine institute (NVI) were sequenced and analyzed. The amplicons of 7 isolates and 1 vaccine strain (KS-180) immunogenic genes (LSDV117, LSD122, LSDV060) were used for sequencing. The sequence from Neethling vaccine strain NI-2490 and NVI/CaPV vaccine strain (KS-180 have an overall similar sequence for LSD117 (A27L) but the sequences from the field sample (wild strains) have two nucleotides substituted at 325nt and 360nt positions ‗A‘ by ‗G‘ and ‗G‘ by ‗A‘, respectively. But a wide range of mutation was found on LSDV112 (EEV glycoprotein coding) gene sequence analysis. This amino acid change may have a mutative advantage for the wild strain over vaccine strains while it interacts with the immune system. Reference capripox viruses were obtained from GenBank to create the phylogenetic tree. It is recommended that genome sequence information of LSD virus circulating in a specified area (i.e. Bishoftu) should be considered in order to enhance the efficacy of the vaccine used prevent and control LSD disease in study area. Improving the transport, storage and handling of a vaccine is important for the success of disease prevention as well.
Isolation and Molecular Detection of Newcastle Disease Virus and Vaccine Immunogenicity Evaluation in and It’s Surrounding Bishoftu, Ethiopia
(Addis Abeba University, 2023) Ayele Teshome; Hika Waktole (Associate Professor); Mirtneh Akalu
Newcastle disease (ND) is an economically important endemic viral disease of chickens that has a potential threat to village chickens and commercial poultry farms in Ethiopia. Despite intense ND vaccination, most clients were raising their doubt regarding efficacy of the current National Veterinary Institute (NVI) ND vaccines against the circulating viruses. Therefore, in this research a cross sectional study was designed to isolate and molecularly detect ND virus and to assess the protective efficacy of ND vaccines in and its surrounding Bishoftu from October 2022 to May 2023. A total of 57 samples were processed using SPF embryonated eggs, hatched chickens and DF1cell culture methods to isolate NDV. The pathogenicity of the isolates was evaluated using intracerebral athogenicity index (ICPI) scoring (60 chickens) and embryo mean death time (MDT) determination. The isolates were further identified by molecular assay, targeting primers of the M-gene (~1100 p) of avian paramyxovirus-1 (APMV-1). The result of PCR revealed that from the total 11 ND suspected samples eight of them were positive. Vaccine efficacy was evaluated in 80 chickens hatched from SPF eggs. Chickens were immunized with lentogenic vaccines of NCH1, NCL and NCTH using three vaccination schedule groups and one control group. Standard dose of 106.5 EID50 per chicken was administered at day zero and boosted at days 14 and 35. At the end of experiment period all groups were challenged with isolated NDV (1010.6 ELD50 per chicken) through occulonasal route. The result revealed that the isolated NDV was characterized as very virulent (velogenic pathotype) based on the mean death time (MDT) and ICPI score. The geometric mean (GM) haemagglutination inhibition (HI) antibody titer was peaked post-second-boost and revealed a significance difference (P < 0.05) compared to the control group. Thus, secondary boost produces higher antibody HI titer than the first boost. Besides, the challenge assay revealed that vaccinated chickens were protected against the challenge strain while unvaccinated chicken were deceased 100% within 6 days post challenge. Accordingly, it can be concluded that the current ND vaccine is efficient to protect vaccinated chickens against the circulating NDV in the study area. Thus, the study commends further investigations of ND outbreaks, training and awareness regarding ND and vaccine management.
Pharmacological Activity of Selected Medicinal Plants Extract on Pasteurella Multocida and Mannheimia Haemolytica Isolated from Small Ruminants
(Addis Abeba University, 2023-06) Beshada Asfa; Takele Beyene (Assoc. Prof.); Dr. Dereje Nigussie
Small ruminants (SR), namely sheep and goats, are playing key roles in supporting the livelihood of poor farmers particularly in the developing country due to their potential to replicate and rapid growth, and valuable commodities where they are raised for meat, milk, and wool. Despite the economic and sociocultural significance of SR in Ethiopia, the occurrence of diseases and other factors cause their productivity to be very low. Pneumonic pasteurellosis is one of the most economically important infectious diseases of SR, with a global distribution. Antibiotics may be effective in treating the disease. However, farmers residing in remote areas of Ethiopia prefer to practice herbal medicines to manage respiratory infections in SR. Hence, the aim of the current study was to evaluate the antibacterial activity of the crude extracts of selected medicinal plants. The medicinal plants, namely Nicotiana tabacum, Psidium guajava and Solanum incanum were selected based on a literature review of previous studies that showed promising effects on respiratory diseases, namely P. multocida and M. haemolytica strains. Agar well diffusion method was used to determine the antibacterial activity of methanol and chloroform extracts of the three selected medicinal plants against P. multocida and M. haemolytica strains. The phytochemical constituents of the extracts of the three medicinal plants were also investigated. The antibacterial activity evaluation results showed the methanol extracts of the three medicinal plants had good activity against the two strains at 200mg/ml concentration and was comparable to gentamicin and streptomycin. From the three selected medicinal plants S. incanum showed higher zone of inhibition (26.3mm) as compared to that of N. tabacum (19.8mm) and P. guajava (19.6mm). Similarly, the chloroform extracts showed good activity against the two strains. However, the chloroform extracts of P. guajava showed the highest activity (30.2mm) on P. multocida at 200mg/ml. The results of the phytochemical screening showed various levels of alkaloids, flavonoids, tannins, saponins and terpenoids. The results of the antibacterial activity investigation showed that the crude extracts of all tested plants inhibit the growth of the tested bacterial strains. The current findings support the traditional use of these plants against major respiratory diseases causing two bacterial strains in SR.
Poultry Producers‟ Knowledge, Attitude and Practices Regarding Antibiotics Use and Detection of Multi-Drug Resistance Profile of Escherichia Coli from Selected Farms in Bishoftu Town, Central Ethiopia
(Addis Abeba University, 2023) Dehinenet Terefe; Dr. Getachew Tadesse; Takele Beyene (Assoc. Professor); Hika Waktole (Assoc. Professor)
Antibiotics are used at sub_ therapeutic dose for growth promotion and prohylaxis in poultry are now on the rise which predisposes to the development and transfer of anti biotics resistance .A cross_ sectional study was carried out to assess the knowledge, at titudes and practice of poultry producer regarding antibiotics use and resistance using semi_ structured questionnaire to selected poultry farms in Bishoftu town, Ethiopia. Ei ghteen poultry farms were visited for sample collection to detect the resistance of E. coli from cloacal swab and litter samples using systematic random sampling, Identific ation of E. coli was carried out by MALDI_ TOF. Kirby bauer disk antimicrobial susce ptibility test method was conducted to determine antibiotics resistance of E. coli. Desc riptive tatistics, chis_ quare, and logistic regression were used to determined the assoc iation of explanatory variable with knowledge, attitude and practice, antibiotic usage history, and resistance profile of E.coli. 61%, 67% and 56% of respondents in the stud y area had sufficient knowledge, good attitude and favorable practice on antibiotics us e and resistance, respectively. Knowledge of the usage of antibiotics and resistance was associated with respondents' age (> 45, OR = 3.6, p = 0.014), and farming experi ence (> 10, OR = 14.3, p = 0.00). The respondents' attitudes were also associated with their age (> 45, OR = 16, p = 0.001), education (Graduates, OR = 6.25, p = 0.007), and farming experience (> 10; OR = 5.4, p = 0.04). Practice of responders was associated with education level (Graduate, OR = 6, p = 0.01) and farming experience (>10, OR = 4.89, p = 0.004). According to correlation analysis, attitudes and the use of antibiotics as preventive medicine have a positive linear link (r = 0.81, p = 0.00), as well as attitudes and growth promotion (r = 0.47, p = 0.00). Additionally, there were s ignificant positive correlations between the usage of prophylactic antibiotics (r = 0.81 5, p = 0.00) and the stimulation of growth (r = 0.592,p = 0.00). Tetracycline, Sulpham ethoxazole + trimethoprim and Sulfonamide were commonly used in the study farms. E. coli was confirmed in 46% of cloacal sample, andin eleven farms by MALDI_ TOF. 75% of the E. coli were showed multidrug antibiotisresistance. The study revealed co ncordance of farm antibiotic use purpose and resistance profile of the E. coli, warranti ng strict regulation to reduce antibiotics usage in poultry.
Molecular Detection and Virus Isolation of Infectious Bursal Disease Virus from Outbreak Cases in Selected Districts of Central Ethiopia
(Addis Abeba University, 2023) Endeshaw Mulu; Debebe Ashenafi ( Asst. Prof); Hika Waktole (Associate Professor)
Infectious bursal disease also called Gumboro is a highly contagious viral disease of young chickens that causes significant economic losses in the poultry industry worldwide. The disease is caused by the Infectious bursal disease virus (IBDV), which belongs to the genus Avibirnavirus of the family Birnaviridae, double strand RNA virus with a non-enveloped, icosahedral capsid with bi-segmented genome. An outbreak based cross sectional study was conducted in poultry farms located in Bishoftu, Dukem, Modjo, Gelan and Tulu-Dimtu, central Ethiopia, from December 2022 to May 2023 for molecular detection and isolation of infectious bursal disease virus (IBDV) from outbreak cases. Purposive sampling technique and a semi-structured questionnaire was employed on selected farm owner and attendants to assess management and vaccination practices of the farms. Pools of bursal samples were collected, and virus isolation and reverse transcriptase-polymerase chain reaction (RT-PCR) were performed to confirm the outbreak cases. From the total nineteen farms surveyed 47.37% vaccinate their chickens against IBDV. Only 21.05% vaccinate according to manufacturer‟s protocol and 78.95% revealed lack of knowledge about biosecurity practice, and 63.16% had poor hygienic condition, while only 26.32% practice regular cleaning and disinfectants. Out of a total of 4 bursal suspensions collected and prepared from 19 poultry farm outbreaks, all were showed characteristic cytopathic effects starting on day two post infection after second passage on differentiated fibroblasts (CEF) cell lines. Of the seven pooled samples analyzed by RT-PCR, six of them were positive results. The results indicated that there is continuous presence of IBD in poultry farms in the country suggesting that current vaccine procedures and protocols as well as management practices may not be working properly. In conclusion, IBD has become the most devastating disease of chicken in central Ethiopia, with high economic lose. Hence, sequencing of the positive samples is much needed to know the circulating virus strains in the areas.
Cysticercusbovis and Taenia Saginata: Prevalence, Public Health Significance and Community Perception about Meat Borne Zoonosis in Three Selected Districts of West Shoa Zone of Oromia Region, Ethiopia
(AAU, 2014-06) Worku, Abate; Hailu, Yacob(Dr.
A cross-sectional study was conducted from November 2013 to March 2014 on bovine cysticercosis in cattle slaughtered at Holata, Addis Alem and Ginchi municipal abattoirs in west Shoa zone of Oromia Regional State with the objective of estimates the prevalence of Taenia saginala/cystiercosis, organ distribution, viability of the cysts, associated risk factors and community knowledge about meat-borne zoonosis. Routine meat inspection method and questionnaire survey on conveniently selected respondents in the study areas were used. Out of 600 carcasses examined during the study period in three municipality abattoirs 2.5 % (15/600) were infected with C. hovis. A prevalence of 2.5% (95% CI: 0.3%-4.7%) (5/200), 1.6% (95% CI: 0.2%-3.5%) (3/180), and 3.2% (95% Cl: 0.9%-5.5%) (7/220) in Holeta, Addis Alem and Ginchi were observed, respectively. Cysts wcre found in heart (46.6%), tongue (33.33%) and shoulder muscle (20%). Out of the cysts 46.6 % (7/15) were viable, while 53.3% (8/15) were non-viable. The questionnaire survey revealed that T. saginata/taeniosis is a wide spread problem in these three towns and surrounding rural areas. Out of 110 respondents 63.6% (70/110) had contracted T. saginata. Age, sexe, religion, occupation, education status, raw meat consumption, knowledge about the disease and presence or absence of the latrine was found as potential risk factors of taeniosis. The present study indicate that Taenia saginata/cysticercosis is highly distributed in the study areas warranting professional intervention and community based control programs should be introduced.
Cysticercusbovis and Taenia Saginata: Prevalence, Public Health Significance and Community Perception about Meat Borne Zoonosis in Three Selected Districts of West Shoa Zone of Oromia Region, Ehiopia
(AAU, 2014-06) worku, Abate; Mamo, Bedaso(Dr
A cross-sectional study was conducted from November 2013 to March 2014 on bovine cysticercosis in cattle slaughtered at Holata, Addis Alem and Ginchi municipal abattoirs in west Shoa zone of Oromia Regional State with the objective of estimates the prevalence of Taenia saginala/cystiercosis, organ distribution, viability of the cysts, associated risk factors and community knowledge about meat-borne zoonosis. Routine meat inspection method and questionnaire survey on conveniently selected respondents in the study areas were used. Out of 600 carcasses examined during the study period in three municipality abattoirs 2.5 % (15/600) were infected with C. hovis. A prevalence of 2.5% (95% CI: 0.3%-4.7%) (5/200), 1.6% (95% CI: 0.2%-3.5%) (3/180), and 3.2% (95% Cl: 0.9%-5.5%) (7/220) in Holeta, Addis Alem and Ginchi were observed, respectively. Cysts wcre found in heart (46.6%), tongue (33.33%) and shoulder muscle (20%). Out of the cysts 46.6 % (7/15) were viable, while 53.3% (8/15) were non-viable. The questionnaire survey revealed that T. saginata/taeniosis is a wide spread problem in these three towns and surrounding rural areas. Out of 110 respondents 63.6% (70/110) had contracted T. saginata. Age, sexe, religion, occupation, education status, raw meat consumption, knowledge about the disease and presence or absence of the latrine was found as potential risk factors of taeniosis. The present study indicate that Taenia saginata/cysticercosis is highly distributed in the study areas warranting professional intervention and community based control programs should be introduced

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