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Avian infectious bronchitis virus is a highly contagious disease occurring in respiratory, urogenital, and reproductive tissues of chicken causing considerable losses due to death, egg drop, and reduced production. This preliminary study was conducted to investigate the prevalence of antibodies against infectious bronchitis virus (IBV) and to assess the potential risk factors in chickens of northwest Ethiopia. A cross-sectional study was conducted from November 2020 to June 2021. A total of 768 serum samples from three zones were collected. To investigate the presence of antibodies against IBV, the indirect ELISA serological test was applied. Positivity for anti-IBV antibodies was observed in 23.96% (95% CI: 20.98-27.14) of the samples. The mixed-effect logistic regression analysis of potential risk factors showed that IBV prevalence was significantly higher in young chickens than adults (p < 0.001) and higher in intensive farm type than in extensive type (p < 0.001). Based on the production purposes of the chickens, the odds of seropositivity for IB was significantly higher in layers than in broilers (p < 0.001) and dual purposes (p < 0.001). This study revealed higher seroprevalence in farms which had the "all-in-all-out" rearing method than in farms with different batches in one house with a significant difference (p < 0.001), higher seroprevalence in the poor ventilated type than in good ones (p < 0.001), and higher seroprevalence in the houses that did not remove used litter at all than houses of completely disposed and partially disposed litter (p=0.002). Moreover, disinfection of houses had significant effect on the occurrence of IB. Having personal protective equipment was significantly affecting the occurrence of IB, being higher in the farms that have no wearing clothes and shoe than in those having wearing clothes and shoe (p=0.002). In conclusion, the seroprevalence finding in the present study indicated that the organism is circulating among the population of chickens and high enough to cause significant economic losses Therefore, poultry houses should be cleaned, disinfected, and well ventilated and farm attendants should have separate farm shoe and clothes. Further studies on the virus isolation and molecular characterization of the target gene are needed in the study area.

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Anthrax is endemic in Ethiopia with sporadic outbreaks despite the regular vaccination of domestic livestock. This has raised concerns on the effectiveness of the vaccination strategy which may be associated with breaches in the vaccine cold chain maintenance. This study was aimed at demonstrating the tolerance of anthrax vaccine to cold chain breaches through evaluation of viable spore counts expressed as colony forming units per mL (CFU/mL) of freeze-dried and suspension anthrax vaccines stored at 5 °C, 20 °C and 37 °C for up to 6 months. Both vaccine formulations maintained above the recommended minimum required titre (2 × 106 culturable spores per dose for cattle, buffaloes and horses, and not <1 × 106 for sheep and goats) for up to 6 months at 5 °C storage. In storage at 20 °C, the viability of freeze-dried anthrax vaccine maintained the minimum required titre up to 6 months while up to 90 days in case of the suspension formulation. Both types of vaccine formulations maintained the minimum titre per dose for up to 30 days at 37 °C storage. Generally, both vaccine formulations showed similar trends in titre fall in all of the three storage temperatures (5 °C, 20 °C and 37 °C) as observed in the almost linearly overlapping 95% confidence intervals (CI) up to day 90 at 5 °C and 20 °C storages while up to day 30 at 37 °C storage. However, a significant (P < 0.05) drop in titre was observed after day 90 for storages at 5 °C and 20 °C, and after day 30 for 37 °C storage as observed in the non overlapping 95% CI from the average titres of previous time points. This study showed that if temperature excursion occurs above the recommended temperature range (4-8 °C) during storage or transport, the vaccine should remain effective and can still be used in vaccination programs.

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This study describes a novel multilocus variable number tandem repeat analysis (MLVA) based on six variable number of tandem repeat (VNTR) loci for genotyping of 37 Edwardsiella piscicida (previously Edwardsiella tarda) isolates from multiple sources. The number of alleles identified for each of the six VNTR loci ranged from 3 to 5 with VNTR loci 1 (DI = 0.632) and 3 (DI = 0.644), displaying the highest degrees of polymorphism. MLVA typing of the 37 E. piscicida isolates resulted in the identification of five major clusters consistent with their geographical origins, and were designated as MLVA types I, II, III, IV and V. Types III and V were resolved further into subtypes largely consistent with outbreak source. An MLVA profile comprising a string of integers representing the number of tandem repeats for each allele provided a unique identification for each MLVA type and/or strain. The MLVA protocol described in the current study is robust, relatively simple, has a higher power of resolution than multilocus sequence analysis (MLSA) and is capable of discriminating closely related isolates.

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AIMS: This study describes a novel species within the genus Edwardsiella based on phenotypic and genetic characterization of fish pathogenic Edwardsiella isolates previously identified as E. tarda. METHODS AND RESULTS: Phenotypic characterization, DNA-DNA hybridization and phylogenetic analysis of representative Edwardsiella isolates from fish previously identified as E. tarda were conducted and compared with E. tarda type strain (ATCC 15947(T)). Phenotypically, strains from fish grow with pin-point colonies producing slight ß-haemolysis under the colony. In contrast to the E. tarda type strain, fish strains did not [corrected] degrade ß-methyl-D-glucoside [corrected] (with the exception of NCIMB 2034), citric acid and L-proline. [corrected]. With the exception of strain ETK01, all fish strains were highly pathogenic to zebra fish, while ATCC 15947(T) and NCIMB 2034 were nonpathogenic. DNA-DNA hybridization (DDH) levels between representative fish isolates and the E. tarda type strain ranged from 15 to 43·6%, while NCIMB 2034 hybridised with the type strain at the level of 63·2%. DDH values between the various fish isolates ranged from 68·2 to 93·9% defining a new and separate DNA hybridization group differing from the E. tarda type strain consistent with the findings of phylogenetic analysis, in which the fish isolates comprised a separate clade. CONCLUSIONS: Phenotypical and genetic characterizations demonstrated that Edwardsiella isolates from fish described in this study do not belong to the species E. tarda or any of the previously established taxa within the genus Edwardsiella. The fish related strains studied here (excluding NCIMB 2034) represent, therefore, a novel species within the genus Edwardsiella for which we propose the name Edwardsiella piscicida sp. nov, with strain ET883(T) (NCIMB 14824(T) = CCUG 62929) as the type strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The current finding will improve the diagnosis, understanding of the epidemiology and in establishment of effective control measures against this serious fish pathogen.

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Edwardsiella tarda is an enteric fish pathogen that has caused significant economic losses in a range of fish species residing in diverse ecological conditions. Several molecular methods relying on DNA fingerprinting (RAPD, RFLP and ERIC-PCR) and the gyrB gene marker have been used to characterize E. tarda isolates. However, all had drawbacks in resolving power and reproducibility. The present study was aimed at developing a novel Multi-locus Sequence Analysis (MLSA) scheme for genetic characterization of E. tarda isolates originating from multiple sources. MLSA has been described as an effective molecular tool with superior discriminatory power and reproducibility for exploring intra-species genetic diversity of several bacterial species. Nucleotide sequence fragments of eight protein coding housekeeping genes (gyrB, mdh, adk, dnaK, phoR, metG, pyrG and aroE2) were obtained from 23 fish pathogenic E. tarda isolates of different geographical origins, one human isolate and 3 reference strains. The phylogenetic relationships between isolates in individual gene analyses were not consistent, although some common patterns were apparent. Phylogenetic analysis based on concatenated sequences of seven gene loci, however, buffered the conflicting phylogenetic signals and resolved isolates according to their geographical origin and/or fish host. The MLSA revealed two major genetically diverging clusters in E. tarda isolates examined, one cluster representing isolates from fish and the other representing (in the main) human isolates, with E. ictaluri cluster situated in between. The results suggest, therefore, that the fish pathogenic E. tarda isolates may have been previously misclassified and probably represent one or more as yet unrecognized taxa within the genus Edwardsiella. The MLSA described here was robust enough in discriminating E. tarda isolates not only with respect to their geographical origins but also within different hosts from the same geographical location, high-lighting its potential application in tracing the source of infection and understand the epidemiological relationships among isolates of environmental, fish, other domestic animals or human origins.

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