Comparative Pathogenicity of Duck Hepatitis A Virus-1 Isolates in Experimentally Infected Pekin and Muscovy Ducklings

Abstract

Duck hepatitis virus (DHV) has always been considered one of the threats endangering duck farming in Egypt since the 1960s. In the current study, suspected DHV samples (n = 30) were obtained from commercial Pekin, Mulard (hybrid), and Muscovy duck farms and backyards in Beheira, Alexandria, Gharbia, Kafr El-Sheikh, and Giza provinces between 2012 and 2017. Diseased 3-21-day-old ducklings showed a clinical history of high mortality rates and nervous signs. Samples were screened by RT-PCR targeting the 5'UTR region and VP1 gene. The PCR-confirmed samples (n = 7) were isolated via allantoic route inoculation onto 9-day-old specific-pathogen-free embryonated chicken eggs. Embryos showed stunting, subcutaneous hemorrhages, and liver necrotic greenish-yellow foci. Duck hepatitis A virus-1 (DHAV-1) isolates were genetically analyzed in comparison to other field and vaccine strains. Phylogenetic analyses of the full-length VP1 gene sequences revealed that the obtained DHAV-1 field isolates clustered into genetic group 4 alongside other Egyptian strains isolated during the same period (95.9-99.72% similarity). Amino acid substitutions in the carboxyl-terminal of VP1 (I180T, G184E, D193N, and M213I) were identified in two strains. Also, deletion mutation at I189 was detected in three DHAV-1 strains. Additionally, the two amino acid residues E205 and N235 were common among the isolated strains and other virulent DHAV-1 strains. Two DHAV-1 isolates originated from Pekin source were selected for conducting the comparative pathogenicity testing based on detected point mutations at C-terminus of VP1. We evaluated the pathogenicity of these isolates by investigating clinical signs, mortality rates, and gross pathological and microscopic lesions. The study revealed that experimentally infected Pekin and Muscovy ducklings showed similar clinical signs including squatting down, lateral recumbency, and spasmodic kicking. Muscovy showed milder pathological changes in the liver compared to Pekin ducklings. Histopathological findings supported the gross pathological lesions detected in both breeds. In conclusion, these data provide updated information on the genetic diversity and pathotyping of Egyptian DHAV-1 strains. To the best of our knowledge, this is the first report of comparative pathogenicity of recent DHAV-1 strains in Pekin and Muscovy ducklings in Egypt and the Middle East region.

Keywords: Egypt; duck hepatitis A virus-1 (DHAV-1); duckling; muscovy; pathogenicity; pekin.

Figure 1

Geographical distribution and summary of collected samples. Map was created using Datawrapper free tool https://www.datawrapper.de/.

Figure 2

Phylogenetic relatedness of the VP1 gene of duck hepatitis A virus-1 (DHAV-1) isolates to the Egyptian vaccine and other representative Asian strains. Neighbor-joining tree generated with 1,000 bootstraps.

Figure 3

Deduced amino acid sequences of the VP1 gene of duck hepatitis A virus-1 (DHAV-1) isolates of the current study in comparison with selected Egyptian and vaccine strains. Amino acid substitutions D193N, R217K in MK510860 (Eg/HL-1/15) and MK510859 (Eg/F219/14) (arrowheads) and amino acid residues E205, N234 of DHAV-1 virulent strains (arrows) are indicated.

Figure 4

Clinical signs and gross pathological lesions of day-old Pekin and Muscovy ducklings experimentally infected with duck hepatitis A virus-1 (DHAV-1), MK510860 (Eg/HL-1/15), and MK510859 (Eg/F219/14). (A,C) Signs of opisthotonos and spasmodic kicking. (B,D) Non-infected Pekin and Muscovy controls showed normal liver appearance. (E,F) Hemorrhagic spots on the liver surface of Pekin ducklings. (G,H) Muscovy livers showed severe congestion.

Figure 5

Survival rate of day-old Pekin and Muscovy ducklings experimentally infected with duck hepatitis A virus-1 (DHAV-1), MK510860 (Eg/HL-1/15), and MK510859 (Eg/F219/14).

Figure 6

Livers of experimentally infected Pekin and Muscovy ducklings with duck hepatitis A virus-1 (DHAV-1) strains compared to non-infected negative controls. Liver of Pekin ducklings showing congested blood vessels, hepatocellular necrosis (x200) (A), and severe hemorrhage (x200) (B). Liver of Muscovy ducklings showing severely congested blood vessels (x100) (D), and vacuolar degeneration (x400) (E). Liver of Pekin ducklings showing severely congested blood vessels, hemorrhage (x100) (G), and severe necrosis of hepatocytes with few perivascular mononuclear cells infiltration (x400) (H). Liver of Muscovy ducklings showing severely congested blood vessels with proliferation of bile ductules (x100) (J), and vacuolar degeneration (x400) (K). No histological microscopic lesions were observed in the control groups (x200) (C,F) and (x400) (I,L).

Figure 7

Cerebrum and pancreas of experimentally infected Pekin and Muscovy ducklings with duck hepatitis A virus-1 (DHAV-1) strains compared to non-infected negative controls. Cerebrum of Pekin ducklings infected with Eg/HL-1/15 strain showing cerebral congestion (x200) (A). Cerebrum of Pekin ducklings infected with Eg/F219/14 strain showing neuronal degeneration with perineural edema (x400) (B). Cerebrum of Muscovy ducklings infected with Eg/HL-1/15 strain showing proliferation of glia cells (x400) (D). Cerebrum of Muscovy ducklings infected with Eg/F219/14 strain showing perineural edema (x400) (E). Pancreas of Pekin ducklings infected with Eg/HL-1/15 strain showing congested blood vessels (x200) (G). Pancreas of Pekin ducklings infected with Eg/F219/14 strain showing congested blood vessels (x200) (H). Pancreas of Muscovy ducklings infected with Eg/HL-1/15 strain showing congested blood vessels (x200) (J). Pancreas of Muscovy ducklings infected with Eg/F219/14 strain showing congested blood vessels and thickening of their walls (x200) (K). No histological microscopic lesions were observed neither in the Cerebrums of control groups (x200) (C,F) nor in Pancreas (x200) (I,L).