Kenaw Birhanu’s research while affiliated with National Veterinary Laboratory and other places

Fowl cholera is a highly infectious bacterial disease in poultry production. It is caused by Pasteurella multocida ( P. multocida ) and leads to significant health risks and financial losses. Therefore, this study is aimed at isolating, molecularly detecting, and analyzing the antibiogram of P. multocida from breeder chickens in Gondar City. A cross‐sectional study design with purposive sampling was employed to collect a total of 130 tracheal swab samples from breeder chickens showing clinical signs of fowl cholera between January 2023 and December 2023, based on case availability. Bacterial isolation was performed using bacteriological and biochemical tests. The isolated P. multocida was confirmed through conventional polymerase chain reaction (PCR) using a capsular serotype‐specific primer ( capA ). The antibiogram assessment of P. multocida against 10 antimicrobial agents was conducted using the Kirby–Bauer disk diffusion method. Descriptive statistics were used to analyze the isolation rate of the bacterium. Of the 130 sampled swabs, 10 (7.69%) tested positive for P. multocida in the phenotypic assay, and 3 (30%) of those isolates were positive for the hyaD/hyaC virulence gene. The study found that all three isolates were 100% sensitive to penicillin, ampicillin, norfloxacin, and florfenicol, while showing 100% intermediate sensitivity to streptomycin and 66.7% intermediate sensitivity to gentamycin, amoxicillin, tetracycline, trimethoprim/sulphamethoxazole, and kanamycin. The study confirms that P. multocida , the causative agent of fowl cholera in breeder chickens, is circulating in the area and exhibits varying antimicrobial sensitivity profiles.

Background Avian infectious bronchitis (IB) is a highly contagious respiratory disease that affects the poultry industry globally. The disease is caused by avian infectious bronchitis virus (IBV), member of the genus Gammacoronavirus. In Ethiopia, IBV has been reported in both commercial and backyard chickens based on clinical observation. The objectives of this study were to isolate the virus, conduct molecular based identification, and phylogenetic analysis of the circulating IBV isolates. Methods and materials A cross-sectional study was conducted between November 2023 and May 2024 in Mekele and Bishoftu, Ethiopia. A total of 49 clinical samples were collected, comprising 12 tissue samples and 39 pooled swab samples. Of these, 6 samples—specifically, 5 swab samples and 1 tissue sample—tested positive for infectious bronchitis virus (IBV) through virus-specific conventional RT-PCR and real-time PCR. Nested PCR was performed using serotype-specific primers. The purified PCR products, which targeted the spike glycoprotein S1 subunit gene and the 3′ UTR of the IBV, were sequenced, followed by phylogenetic tree analysis. Results The six positive samples propagated into specific pathogen free embryonated eggs and exhibited characteristic IBV lesions and mortality observed over five consecutive passages. IBV isolates from Bishoftu (n = 4) and Mekele (n = 2) were amplified using one-step RT-PCR to target 466 bp of the S1 subunit gene and 433 bp of the 3ʹUTR. A BLAST search on the S1 partial gene and 3ʹUTR sequences, nested PCR, and phylogenetic analysis revealed that the present IBV isolates are genetically similar to the Massachusetts serotype. The S1 gene sequences of the five IBV isolates were deposited in GenBank with accession numbers PQ389500 to PQ389504. Conclusions This is the first detailed study on IB virus isolation, molecular detection, sequencing, and phylogenetic analysis in Ethiopia. The findings revealed that the outbreaks were caused by the IB virus, which created a serious health risk and economic losses in the chicken industry. To the author’s knowledge, this is the first comprehensive study on the isolation and genetic analysis of IBV in Ethiopia. Further research on the economic impact of IBV in chicken production, farm biosecurity, serotyping of circulating IB virus, and vaccine development based on the local serotypes is recommended.

Background Avibacterium paragallinarum is a causative agent of infectious coryza (IC), a disease that affects the upper respiratory tracts and paranasal sinuses of chickens, resulting significant economic losses in the poultry industry. The objective of this study was to isolate and identify Av. paragallinarum using bacteriological and molecular methods between February 2022 and April 2024. A total of 74 swab samples were collected from chickens showing ocular and nasal discharges and swelling of the infraorbital sinuses. Method Clinical samples were collected from chickens showing symptoms of IC from six locations of Ethiopia for the isolation and identification of the causative agent. Swab samples from the nasal cavity and cheesy material from the infraorbital sinus were screened using conventional PCR and inoculated onto chocolate agar enriched with 5% sheep blood. Colonies suspected of being Av. paragallinarum were transferred to brain heart agar supplemented with horse serum. Gram staining was used to examine the morphology of bacteria in pure colonies grown on chocolate and brain heart infusion agar. Results The isolation of Av. paragallinarum on chocolate and brain heart infusion agar resulted in the observation of small, translucent, dewdrop-shaped colonies after 24 h of incubation at 37 °C in a 5% CO2 incubator. A smear prepared from a single colony of revealed Gram-negative, short rod-shaped or coccobacilli Av. paragallinarum bacteria. Biochemical tests conducted on this isolate yielded negative results for catalase, oxidase, urease, indole, methyl red, and Voges-Proskauer tests. However, the bacterium exhibited positive fermentative activity with glucose, sucrose, and maltose. Biochemical assay revealed the presence of Av. Paragallinarum. The bacterial colonies confirmed a 511 bp PCR product. The partial HPG-2 gene nucleotide sequences of eleven isolates were sequenced and deposited in GenBank with the accession number PQ565862-72. A phylogenetic tree was constructed to determine the genetic relatedness of Ethiopian isolates with isolates from other African countries and elsewhere. Conclusion The current investigation confirmed that the outbreaks were caused by Avibacterium paragallinarum and provided scientific evidence on the presence of different strains of Av. paragallinarum in Ethiopia. This is the first study in Ethiopia to detect and identify Av. paragallinarum from diseased chickens using molecular approaches. Further molecular characterization of locally circulating Av. paragallinarum isolates is recommended to be used as a vaccine strain for the prevention and control of infectious coryza.

Introduction Infectious laryngotracheitis (ILT) is a highly contagious upper respiratory tract disease of chickens caused by a Gallid herpesvirus 1 (GaHV-1). The current study was to establish molecular evidence of Infectious laryngotracheitis virus (ILTV) in the Amhara region, Ethiopia, and determine its seroprevalence in areas of high chicken population and assess the risk factors associated with the disease. Methods Serological study was conducted on 385 serum samples collected from commercial and backyard chickens in the study area, and the presence of antibodies against ILTV was determined by indirect ELISA. In addition, oropharyngeal swab samples were collected from chickens suspected of ILT infection and inoculated into embryonated chicken eggs through the Chorioallantoic membrane (CAM) route for isolation of the virus. Isolates were confirmed using polymerase chain reaction (PCR) upon amplification of ICP4 gene. Furthermore, potential factors were recorded, and their association with the virus seropositivity assessed. Results The overall seroprevalence of ILT in the study area was 19.4%. A significant difference (P < 0.05) among districts, and between commercial (14.2%) and backyard (22.9%) production systems was observed (P < 0.05). Significantly higher seroprevalence was observed in layers compared to broilers and dual-purpose chickens however, there were no significant differences in prevalence based on age and sex. Of all (n = 27) tested oropharyngeal swab samples, four were positive for ILTV by PCR targeting a 688 bp region of ICP4 gene. Three of the PCR positive cases were from backyard chickens, while one was from commercial chicken farms. Based on oropharyngeal samples tested using PCR, a quarter of the samples were positive for ILT. Discussion The result confirms the presence of ILT infection in the Amhara region of Ethiopia using serological and molecular methods. The study shows chickens shed the virus potentially spreading the infection to other birds. Vaccination strategy, strict biosecurity measures, rapid diagnosis, and detection of latent carriers are recommended to control and eradicate the disease. Further studies on clinical cases and the molecular characterization of the target gene are needed to identify circulating strains.

Newcastle disease (ND) is caused by virulent strains of avian paramyxovirus type 1, also known as Newcastle disease virus (NDV). Despite vaccination, the frequency of reported outbreaks in Ethiopia has increased. From January to June 2022, an active outbreak investigation was conducted in six commercial chicken farms across areas of central Ethiopia to identify the circulating NDV strains. Thirty pooled tissue specimens were collected from chickens suspected of being infected with NDV. A questionnaire survey of farm owners and veterinarians was also carried out to collect information on the farms and the outbreak status. NDV was isolated using specific-pathogen-free (SPF)-embryonated chicken eggs and detected using haemagglutination and the reverse transcriptase–polymerase chain reaction (RT–PCR). The genotype and virulence of field NDV isolates were determined using phylogenetic analysis of fusion (F) protein gene sequences and the mean death time (MDT) test in SPF-embryonated chicken eggs. The questionnaire results revealed that ND caused morbidity (23.1%), mortality (16.3%), case fatality (70.8%), and significant economic losses. Eleven of thirty tissue specimens tested positive for NDV using haemagglutination and RT–PCR. The MDT testing and sequence analysis revealed the presence of virulent NDV classified as genotype VII of class II velogenic pathotype and distinct from locally used vaccine strains (genotype II). The amino acid sequences of the current virulent NDV fusion protein cleavage site motif revealed ¹¹²RRQKR↓F¹¹⁷, unlike the locally used avirulent vaccine strains (¹¹²GRQGR↓L¹¹⁷). The epidemiological data, MDT results, cleavage site sequence, and phylogenetic analysis all indicated that the present NDV isolates were virulent. The four NDV sequences were deposited in GenBank with accession numbers F gene (PP726912-15) and M gene (PP726916-19). The genetic difference between avirulent vaccine strains and circulating virulent NDV could explain the low level of protection provided by locally used vaccines. Further studies are needed to better understand the circulating NDV genotypes in different production systems.

Coronaviruses are enveloped, large positive-sense, and single-stranded RNA viruses. Four coronavirus genera have been identified: alpha, beta, gamma, and delta. Non-structural proteins make up two-thirds of the coronavirus genome, and some of these (ORF1ab) are essential to create the replicase-transcriptase complex. The remaining coronavirus genome encodes four major structural proteins (S, E, M, and N). Coronaviruses infect both animals and humans, and the emergences of SARS-CoV, MERS-CoV, and SARS-CoV-2 highlight the viruses' importance as emerging pathogens. Accurate viral detection is crucial for treating cases and controlling diseases. MERS was first identified in Saudi Arabia in 2012 where as SARS-CoV-2, the causal agent of coronavirus disease 2019 (COVID-19), was first identified in Wuhan, China, in December 2019 and then spread globally, resulting in a pandemic. Many countries performed fast large-scale testing as a way to halt its spread. The National Veterinary Institute (NVI) of Ethiopia was among the organizations that took part in the COVID-19 testing campaign during the pandemic. The real-time reverse transcription polymerase chain reaction (real-time RT-PCR) is the gold standard method for diagnosing suspected cases of COVID-19 and MERS-CoV. These methods, which target viral ribonucleic acid (RNA), are currently the primary approach for directly identifying the virus. In this study, three real-time RT-PCR assays were employed to detect SARS-CoV-2 in human samples: two developed by CDC China for ORF1ab and the N genes, and one ORF1ab-based assay (BGI, China), and one RT-PCR assay developed by Charité laboratory (Berlin, Germany) for E gene targets. The other two RT-PCR assays developed by Corman et al., 2012 targeting MERS-CoV's upstream E (UpE) gene and ORF1a gene were used to screen and confirm MERS-CoV in camel swab samples. The uniplex, duplex, and triplex RT-PCR assays were assessed following the recommended protocols.

Pasteurellaceae families are usually considered opportunistic pathogens which inhabit normal flora on the mucosal membranes of the upper respiratory and the lower genital tracts of mammals and birds. The majority of P. multocida, M. hemolytica, and B. trehalosi isolates are opportunistic animal pathogens and cause disease only under certain conditions. Some of the target genes are shared by isolates found in the same or different genera.. The alpha / beta hydrolase kmt1 genes part of OMP and SLP are shared by all P.multocida species and FbpA is common gene for both P.multocida and M. hemolytica isolates. Other virulence related genes are used for genotyping of P.multocida, M.he-molytica and B.trehalosi isolates. The objective of the current research was focused on the detection and genotyping of different target genes that can be found in the isolates of P. multocida, M. hemolytica, and B. trehalosi using various PCR primers. The distributions of virulence-associated genes were assessed in the isolates of the three genera. Materials and methods A total of eight isolates from P. multocida, M. hemolytica, and B. trehalosi isolated from clinical cases of cattle and sheep and stored at the Bacteriology laboratory of NVI were used as source of samples for the present study. DNeasy® Blood & Tissue Kit (Qiagen, Germany) was used for the extraction of DNA from bacterial isolates. Different primers were used for genotyping of P. multocida, P.hemolytica and B.trehalosi isolates. Results P.multocida was detected using species-specific kmt1 primer with an amplicon product of 460bp. Four serogroups of P.multocida (A, B, D, &E) were detected using serotype-specific primers with amplification products of 1044bp, 760bp, 657bp, and 511bp respectively. PCR was conducted to HS causing P.multocida type B and detected 620 bp amplicons. SLP genes were detected in all P.multocida serotypes with an amplification product size 1400-1553bp. In addition to that, the four P. multocida serotypes were found to be positive for BRD-PmSLP with an amplification product of 460bp but using recombinant HS-SLP primers resulted in the double band at 460 bp and 541bp for sero-types A and D. However, HS causing type B& E serotypes were detected with an amplified product of 541bp. TbpA2 has been detected with an amplicon size of approximately 750 bp for types (A and D) and 1300bp for type B using TbpA-F2/Rev primers. All of the P.multocida serotypes (A, B, D&E) and M. hemolytica (A: 1) were strongly positive for the iron acquisition FbpA gene with amplification bands at 500bp and 1000bp, respectively. A multiplex PCR assay was carried out for detection of M. haemolytica A: 1 using PHSSA and Rpt2 primers with amplification products of 327pb and 1022 bp respectively. The three isolates of B. trehalosi were identified using BtsodA primers with the result of 144bp amplicon size. Conclusion From this work, we understood that P.multocida and M. haemolytica shared genetic materials like iron binding proteins (FbpA) and some of the target genes (kmt1 and SLP) were commonly found in all isolates of P. multocida. However, TbpA2 was found to be in most but not in all Pasteurella isolates. We also observed that PHSSA and Rpt2 genes were exclusively found in M. hemolytica isolates whereas Bt-sodA was prominent in all B. trehalosi isolates (T3, T4&T15).

BACKGROUND ANDMETHODS: Brucellosis is a dreadful zoonotic disease affecting humans and all domestic animals including camels worldwide. Serological evidence for Brucella infection in camels has been reported from all pastoralist and agro-pastoralist regions of Ethiopia. Investigations have shown that antibody concentrations are lower in camels than in cattle. However, serological diagnostic kits have been developed for cattle brucellosis is directly transposed for camels without adequate assay validation and genotyping of Brucella species is still absent in camel population in Ethiopia. This study aimed to evaluate gaps associated with Rose Bengal Plate Test (RBPT) and molecular detection of Brucella species from apparently healthy camels slaughtered at the Akaki abattoir. The study applied Brucella genus speci c, Brucella abortus (B.abortus) and Brucella melitensis (B.melitensis) species-speci c primers on RBPT-positive and retropharyngeal lymph node samples collected from 100 camels' heads. RESULTS: RBPT revealed the presence of anti-Brucella antibodies in 5 of 100 (5%) slaughtered camels. All RBPT-positive were also positive for PCR. Among the100 lymph node samples examined, 35 (35%) were Brucella positive by PCR. All were found to be B. abortus;however, B. melitessis was not detected in either the serum or lymph node samples. CONCLUSION: To the authors' knowledge, this investigation is the rst report on the molecular detection of B.abortus from camel in Ethiopia. Sequence data con rmed the presence of B. abortus from apparently healthy camels slaughtered at Akaki abattoir, Ethiopia. B. abortus molecular detection rate on lymph nodes samples was seven times greater than that of RBPT. We recommend that advanced research be conducted on camel milk and meat and those camel herders emphasize Ethiopian pastoral areas in particular to understand Brucella epidemiology and its public health signi cance.

Infectious bronchitis (IB) is a highly contagious disease of the respiratory and urogenital tract of chickens, caused by infectious bronchitis virus (IBV), a member of the family Coronaviridae. Due to the serious infectious and transmission features of the disease mostly in the reproductive and respiratory systems it causes potential economic loss. Hence a cross-sectional study was conducted from February 2022 to June 2022 on chicken serum and tracheal swab samples from backyard and commercial farms in central Gondar zone, Ethiopia, to determine the seroprevalence of IB, associated risk factors and for isolation and molecular detection of the virus. A total of 384 blood samples were collected and tested by an indirect ELISA and Anti-IBV antibody positivity was noted in 92.19% (95% confidence interval (89 %-94.6 %) of the samples. Logistic regression analysis was performed to determine the impact of possible risk factors on seropositivity. Higher prevalence was noted in young chickens than in adults (p< 0.05) and in exotic breeds than in local breeds (p<0.05). Though the difference was not statistically significant (p>0.05), higher prevalence was obtained in dual purpose chickens (93.75%) than in layers (92.17%) and broilers (90.98%). Higher prevalence was also noted in females (92.7%) than in males (90.98%) and in intensively managed chickens (93.39%) than in extensively managed chickens (90.69%) with p>0.05. Conventional RT-PCR test was also performed for the molecular detection of virus. The test was done on 52 tracheal swab samples collected from intensive and backyard unvaccinated chickens that were pooled in to 26 samples. Accordingly, 3 (11.54%) of the 26 pooled samples were IBV positive. The result showed that this was the first molecular evidence found in the study area. The seroprevalence of the disease in this study was very high for all age groups, breed types, and farm types. The risk factors mentioned and the management methods may have raised the likelihood of infection and the prevalence of the disease. Vaccination and biosecurity measures are advised to manage the disease. Identification and characterization of persistent IBV serotypes that are present in the field is also recommended to manage the disease.

Background Brucellosis is an economically devastating animal disease and has public health concern. Serological methods such as Rose Bengal Plate Test (RBPT), Complement Fixation Test (CFT), and Indirect-Enzyme-Linked Immunosorbent Assay (I-ELISA) have been used to detect brucellosis. However, there is limited comparative evaluation studies and lack of molecular confirmation of the causative agents in the study areas. The study was aimed to compare RBPT, I-ELISA, CFT, and confirmation using Polymerase Chain Reaction (PCR). A total of 2317 sera samples were collected from brucellosis-affected areas of Ethiopia with no vaccination history. All sera were subjected to comparative serological assays. Post-cross tabulation, sensitivity, and specificity were determined using Receiver Operating Characteristics (ROC) curve analysis software. PCR was performed on 54 seropositive samples using genus- and species-specific primers. Results Among the 2317 sera tested for comparative serological assays, 189 (8.16%) were positive for RBPT, 191 (8.24%) for I-ELISA, and 48 (2.07%) for CFT. Sensitivity to RBPT was 100% (95%) in shoats and 74% (95%) in cattle. Specificity on RBPT was 98.69% (95%), 99.28% (95%), 100% (95%) in sheep, goats, and cattle, respectively. CFT sensitivity was 4 (95%) in sheep, 9.65 (95%) goats, and 72 (95%) cattle. Specificity on CFT was 100% (95%) for sheep, goats, and cattle. A 223bp Brucella genus-specific and 156bp B. abortus species-specific detected. However, B. melitensis not detected. Conclusion In this study, I-ELISA was the most sensitive and specific test. RBPT detected all Brucellosis-infected sheep and goats; nevertheless, it showed false positive in sheep and goats and false negative in cattle. The presence of B. abortus in small and large ruminants was confirmed by PCR. This is the first report of B. abortus detection in small ruminant in Ethiopia. B.abortus detected in non-preferred hosts. The findings suggest further study on molecular epidemiology of Brucella species.