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. 2023 Jun 30;10(2):211-221.
doi: 10.5455/javar.2023.j671. eCollection 2023 Jun.

Evaluation and development of diagnostic tools for rapid detection of Riemerella anatipestifer and Pasteurella multocida in ducks

Mohamed M M Megahed  1 , Aya M A El-Nagar  1 , Azza S El-Demerdash  2 , Mervat A Ayoub  3 , Hala M N Tolba  1
Affiliations

Evaluation and development of diagnostic tools for rapid detection of Riemerella anatipestifer and Pasteurella multocida in ducks

Mohamed M M Megahed et al. J Adv Vet Anim Res. .

Abstract

Objectives: Ducks suffer a huge economic loss as a result of infections with Pasteurella multocida and Riemerella anatipestifer, which cause high morbidity and mortality. Because these pathogens induce similar clinical symptoms when coinfections occur, it is very difficult to differentiate between them based just on clinical signs. Hence, these major pathogens must be quickly and accurately detected.

Materials and methods: A total of 104 birds ranging from 2 days to 4 weeks old were collected from Egyptian farms, and the outcomes were compared statistically. Conventional cultural identification procedures and a direct multiplex polymerase chain reaction assay were utilized to recognize both pathogens in a single tube reaction simultaneously. Then, the obtained isolates were characterized phenotypically and genotypically.

Results: Clinical signs appear at 2-4 weeks of age with respiratory distress (dyspnea), white fluid feces, and stunting. The scrutinized data demonstrated a significantly higher detection rate by PCR directly compared to classical culture procedures. Pasteurella multocida was detected only by PCR. The disc diffusion technique against ten antibiotics showed absolute susceptibilities to amikacin, doxycycline, and florfenicol. High levels of beta-lactam resistance were observed. Riemerella anatipestifer isolates were screened for pathogenicity and plasmid-borne blaTEM genes. All six isolates harbored five virulence genes: aspC, RA46, m28, pstS, and Nlp/P60. Moreover, blaTEM was identified into four isolates and deposited to GenBank with accession numbers OP347083, OP347084, OP347085, and OP347086.

Conclusion: These results suggest advanced PCR assays can be applied to the field for rapid and valuable diagnosis of two significant pathogens and focus on the worth of ducks in the propagation of transferable antibiotic resistance genes into the environment.

Keywords: Antibiotic resistance; Pasteurella multocida; Riemerella anatipestifer; ducks; multiplex RT-PCR.

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Conflict of interest statement

The authors manifested that they have no conflicts of interest.

Figures

Figure 1.
Figure 1.. Potential risk factors comprehensive breed (A), age (B), and season (C) associated with the probability of detecting Riemerella by the traditional method
Figure 2.
Figure 2.. Potential risk factors comprehensive breed (A), age (B), and season (C) associated with the probability of detecting Pasteurella by RT-PCR
Figure 3.
Figure 3.. Potential risk factors comprehensive breed (A), age (B), and season (C)associated with the probability of detecting Riemerella by RT-PCR
Figure 4.
Figure 4.. Potential risk factors comprehensive breed (A), age (B), and season (C) associated with the probability of detecting Pasteurella plus Riemerella by RT-PCR
Figure 5.
Figure 5.. Frequency of antimicrobial susceptibility of Riemerella anatipestifer isolates from ducklings. P: Pencillin G, AMC: Amoxicillin-clavulanic acid, SAM: Ampicillin/sulbactam, E: Erythromycin, NOR: Norfloxacin, N: Neomycin, SXT: Sulfamethoxazole-trimethoprim, AK: Amikacin, DO: Doxycycline, FFC: Florfenicol.
Figure 6.
Figure 6.. Agarose gel electrophoresis of PCR for amplification products of aspC gene among six Riemerella anatipestifer isolates; Lane +C: Control positive, Lane L: 100-bp ladder (marker); Lane -C: Control negative.
Figure 7.
Figure 7.. Agarose gel electrophoresis of PCR for amplification products of RA46 gene among six R. anatipestifer isolates; Lane +C: Control positive, Lane L: 100-bp ladder (marker); Lane -C: Control negative.
Figure 8.
Figure 8.. Agarose gel electrophoresis of PCR for amplification products of m28 gene among six R. anatipestifer isolates; Lane + C: Control positive, Lane L: 100-bp ladder (marker); Lane -C: Control negative.
Figure 9.
Figure 9.. Agarose gel electrophoresis of PCR for amplification products of pstS gene among six R. anatipestifer isolates; Lane +C: Control positive, Lane L: 100-bp ladder (marker); Lane -C: Control negative.
Figure 10.
Figure 10.. Agarose gel electrophoresis of PCR for amplification products of hydrolase Nlp/P60 gene among six R. anatipestifer isolates; Lane + C: Control positive, Lane L: 100-bp ladder (marker); Lane -C: Control negative.
Figure 11.
Figure 11.. Agarose gel electrophoresis of PCR for amplification products of bla TEM gene among six R. anatipestifer isolates; Lane + C: Control positive, Lane L: 100-bp ladder (marker), Lanes 1-4: Positive samples for bla TEM gene; Lane -C: Control negative.
Figure 12.
Figure 12.. Phylogenetic tree of the different blaTEM clades. The 4 MDR R. anatipestifer isolates of the sector used in this analysis were pointed with a red circle
See this image and copyright information in PMC

References

    1. Wei B, Cha S-Y, Kang M, Park I-J, Moon O-K, Park C-K, et al. Development and application of a multiplex PCR assay for rapid detection of 4 major bacterial pathogens in ducks. Poult Sci. 2013;92:1164–70. http://doi.org/10.3382/ps.2012-02823. - PubMed
    1. Cadmus KJ, Mete A, Harris M, Anderson D, Davison S, Sato Y, et al. Causes of mortality in backyard poultry in eight states in the United States. J Vet Diag Invest. 2019;31:318–26. http://doi.org/10.1177/1040638719848718. - PMC - PubMed
    1. Glisson JR, Sandhu TS, Hofacre CL. Pasteurellosis, avibacteriosis, gallibacteriosis, riemerellosis and pseudotuberculosis. A laboratory manual for the isolation, identification and characterization of avian pathogens. Georgia: Am Assoc Avian Pathol. 2008:12–8.
    1. Reuben RC, Sarkar SL, Ibnat H, Setu MAA, Roy PC, Jahid IK. Novel multi-strain probiotics reduces Pasteurella multocida induced fowl cholera mortality in broilers. Sci Rep. 2021;11:8885. http://doi.org/10.1038/s41598-021-88299-0. - PMC - PubMed
    1. Apinda N, Nambooppha B, Rittipornlertrak A, Tankaew P, Punyapornwithaya V, Nair V, et al. Protection against fowl cholera in ducks immunized with a combination vaccine containing live attenuated duck enteritis virus and recombinant outer membrane protein H of Pasteurella multocida. Avian Pathol. 2020;49:221–9. http://doi.org/10.1080/03079457.2019.1711020. - PubMed

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