A cross-sectional study was conducted to assess the prevalence, molecular detection, and antimicrobial resistance of Salmonella isolates within 162 poultry farms in selected urban and peri-urban areas of central Ethiopia. A total of 1515 samples, including cloacal swabs (n = 763), fresh fecal droppings (n = 188), litter (n = 188), feed (n = 188), and water (n = 188), were bacteriologically tested. The molecular detection of some culture-positive isolates was performed via polymerase chain reaction (PCR) by targeting spy and sdfl genes for Salmonella Typhimurium and Salmonella Enteritidis, respectively. Risk factors for the occurrence of the bacterial isolates were assessed. Antimicrobial susceptibility testing of PCR-confirmed Salmonella isolates was conducted using 12 antibiotics. In this study, it was observed that 50.6% of the farms were positive for Salmonella. The overall sample-level prevalence of Salmonella was 14.4%. Among the analyzed risk factors, the type of production, breed, and sample type demonstrated a statistically significant association (p < 0.05) with the bacteriological prevalence of Salmonella. The PCR test disclosed that 45.5% (15/33) and 23.3% (10/43) of the isolates were positive for genes of Salmonella Typhimurium and Salmonella Enteritidis, respectively. The antimicrobial susceptibility test disclosed multi-drug resistance to ten of the tested antibiotics that belong to different classes. Substantial isolation of Salmonella Typhimurium and Salmonella Enteritidis in poultry and on poultry farms, along with the existence of multi-drug resistant isolates, poses an alarming risk of zoonotic and food safety issues. Hence, routine flock testing, farm surveillance, biosecurity intervention, stringent antimicrobial use regulations, and policy support for the sector are highly needed.

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.

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. 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. 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. 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.

Lumpy skin disease (LSD) is an economically significant viral disease because of its high morbidity and high production loss. Vaccination of cattle using LSD vaccines is a more effective disease preventive and control strategy in endemic countries such as Ethiopia. Despite high vaccination coverage, there is an increasing number of field reports of the disease outbreaks. Thus, an observational study was designed to investigate disease, characterize the disease‐causing agent, and isolate the virus from a local isolate for future vaccine development. Wera Jarso and Amuru districts in North West Oromia were chosen based on outbreak occurrence. For this study skin, 13 pooled biopsy samples were collected from affected cattle. In this outbreak investigation, the morbidity rate was 6.50%, the mortality rate was 0.50%, and the case fatality rate was 7.77%. The virus was isolated from all skin samples on both lamb testis and lamb kidney primary cells and confirmed to be LSDV using conventional and real‐time PCR genotyping. Therefore, after each suspected LSD outbreak, a molecular test should be carried out to confirm the cause of the disease, targeting the previously suggested RPO30 or GPCR genes. Further studies targeting more regions and outbreaks, including full genome sequencing to check for genetic differences between the field viruses and vaccine strains, are recommended.

BackgroundPasteurellaceae 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 genuses. 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.hemolytica 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 methodsA 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.ResultsP.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 serotypes 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.ConclusionFrom 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).