Molecular Typing, Antibiogram and PCR-RFLP Based Detection of Aeromonas hydrophila Complex Isolated from Oreochromis niloticus

Abstract

Motile Aeromonas septicemia is a common bacterial disease that affects Oreochromis niloticus and causes tremendous economic losses globally. In order to investigate the prevalence, molecular typing, antibiogram and the biodiversity of Aeromonas hydrophila complex, a total of 250 tilapia (Oreochromis niloticus) were collected randomly from 10 private tilapia farms (25 fish/farm) at El-Sharkia Governorate, Egypt. The collected fish were subjected to clinical and bacteriological examinations. The majority of infected fish displayed ulcerative necrosis, exophthalmia, and internal signs of hemorrhagic septicemia. The prevalence of A. hydrophia complex was 13.2%, where the liver was the most predominant affected organ (54.1%). Polymerase chain reaction (PCR) was used to verify the identification of A. hydrophila complex using one set of primers targeting gyrB as well as the detection of virulent genes (aerA, alt, and ahp). All isolates were positive for the gyrB-conserved gene and harbored aerA and alt virulence genes. However, none of those isolates were positive for the ahp gene. The antimicrobial sensitivity was carried out, where the recovered strains were completely sensitive to ciprofloxacin and highly resistant to amoxicillin. All retrieved strains showed the same phenotypic characteristics and were identical based on the restriction fragment length polymorphism (RFLP). Experimentally challenged fish presented a high mortality rate (76.67%) and showed typical signs as in naturally infected ones. In conclusion, the synergism of phenotypic and genotypic characterization is a valuable epidemiological tool for the diagnosis of A. hydrophila complex. RFLP is a fundamental tool for monitoring the biodiversity among all retrieved strains of A. hydrophia.

Keywords: A. hydrophila complex; O. niloticus; PCR; RFLP; antibiogram.

Figure 1

Naturally infected O. niloticus with Aeromonas hydrophila complex; (A) fish showing skin darkness (*), detached scales (white arrow), and external scattered hemorrhagic patches (black arrows); (B) fish showing ulcerative necrosis (black arrows), fin erosions (white arrows), and inflamed vent (#); (C) fish showing exophthalmia and abdominal distension (black arrows).

Figure 2

Naturally infected O. niloticus with Aeromonas hydrophila complex; (A) fish showing enlarged liver, engorged spleen, and congested kidney (white arrows); (B) fish showing sero-hemorrhagic fluids in the abdominal cavity and congested friable gills (Black arrows).

Figure 3

Electrophoretic pattern of gyrB gene of A. hydrophila complex using specific set of primer described elsewhere by Yanez et al. (2003). Lane marked L refer to 100 bp DNA ladder. CP is a control positive (A. hydrophila strain, kindly supplied by Animal Health Research Institute in Dokki, Cairo). CN is a negative control (DNA free template). Lanes 1–16, the specific DNA product amplified from the representative retrieved isolates of O. niloticus with expected amplicons size of 1100 bp.

Figure 4

Electrophoretic pattern of aerA (A), alt (B), and ahp (C) virulent genes of A. hydrophila complex using specific sets of primers described elsewhere by Li, Ni, Liu and Lu [29]. Lanes marked L refer to 100 bp DNA ladder. CP is a control positive (A. hydrophila strain, kindly supplied by Animal Health Research Institute in Dokki, Cairo). CN is a negative control (DNA free template). Lanes 1–8, the specific DNA products amplified from the representative retrieved isolates of O. niloticus with expected amplicons size of 301, 442, and 911 bp, respectively.

Figure 5

Electrophoretic patterns showing the restriction fragment length polymorphism (RFLP) analysis of A. hydrophila complex isolates following digestion of amplified gyrB gene using ECORII (A) and Eco31I (B) restriction enzymes. Lane marked L refers to 100 bp DNA ladder. CN is a negative control (DNA free template). Lanes 1–14, fragment patterns of A. hydrophila isolated from seven different tilapia farms (two representative isolates for each farm) located at El-Sharkia Governorate, Egypt.

Figure 6

Experimentally infected O. niloticus with Aeromonas hydrophila complex showing grayish irregular ulcers on the flank region surrounded by area of erythema (black arrow), scales detachment (*), and mild fin erosions (white arrows) (A), friable liver, congested kidney, and serous fluid in the peritoneal cavity (B).