Molecular characterization of foot-and-mouth disease viruses collected from Northern and Central Ethiopia during the 2018 outbreak

Abstract

Background and aim: Foot-and-mouth disease (FMD) is endemic in several developing countries and affects poor farmers through loss of production, death of diseased animals, and loss of animal byproducts. Forty-three samples were collected from 12 sites of five geographical located areas from suspected FMD virus (FMDV)-infected cattle during 2018. This study aimed to isolate and characterize the FMDVs using reverse transcription-polymerase chain reaction (RT-PCR) and gene sequencing.

Materials and methods: Forty-three FMDV-suspected clinical samples cultured on BHK-21 cell were examined, followed by virus serotype identification using RT-PCR and gene sequencing.

Results: Twenty-nine (67.44%) samples were cultured on BHK-21 cell, of which 14 (32.56%) were not isolated; the 43 samples were analyzed using FMDV screening primers and serotype-specific primers. The contribution of the disease-causing serotype was serotype O of 8 (18.60%) samples, serotype A of 20 (46.51%) samples, and mixed infection (O and A) of 1 (2.33%) sample. Serotypes O and A were further characterized by phylogenetic analysis, which grouped them under East Africa 3 and Africa topotypes of genotype IV, respectively. Interestingly, serotype A was isolated for the 1^st time from Keyet sub-woreda and Mulo woreda of Ethiopia, and mixed serotypes (O and A) were identified from the purchased animal.

Conclusion: Molecular test result, sequencing, and phylogenetic tree reconstruction analysis revealed that the 2018 FMD outbreak in Ethiopia was caused by FMDV serotypes O and A. FMDV serotype A was the predominant strain circulating in most study areas of the country. Infections in one sample with mixed serotypes of O and A were also reported. The authors recommend a vaccine matching study of those field isolated viruses with the vaccine strain.

Keywords: Ethiopia; foot-and-mouth disease virus serotypes; phylogenetic analysis; reverse transcription-polymerase chain reaction.

Figure-1

Map of Ethiopia display the location of sample collection sites. Where, 1: Serdo kebele, 2: Asgede-Tsimbila woreda, 3: Kilte-Awulaelo woreda, 4: Hintalo-Wajirat woreda, 5: Denkaka kebele, 6: Jilo-janjo kebele, 7: Jida woreda (Sirte town), 8: Keyet sub-woreda, 9: Tiro-brodorba kebele, 10: Mulo-kersa kebele, 11: Sendafa (kebele 01 and 02), and 12: Debre-zeit kebele 01. [The map was drawn with the help of Quantum GIS 3.10.0 with GRASS 7.8.0 software].

Figure-2

The clinical sign of foot-and-mouth disease. The representative picture was taken during sample collection. (a) Ulceration of gum; (b) Erosion of the tongue; (c) Profuse frothy mouth discharge as indicated in red arrow.

Figure-3

Characteristics of BHK-21 cell culture with and without foot-and-mouth disease virus (FMDV) infection. The representative picture was taken during FMDV isolation. (a) Uninfected (healthy) cell; (b) FMDV infected cell were rounding and cell detachment as indicated in blue arrow.

Figure-4

RT-polymerase chain reaction (PCR) amplification for the detection of foot-and-mouth disease virus (FMDV) both from the original and isolated samples. Representative gel electrophoresis picture was taken during FMDV genome screening and typing PCR product of FMDV serotypes O and A. (a) Original samples screening for FMDV shows Lane 1 and 17 of 100 bp DNA ladder, Lane 2, 7, 8, and 10-14 were FMDV negative samples during screen test, Lane 3-6 and 9 were FMDV positive, Lane 15 was negative control, and Lane 16 was positive control during screening (328 bp); (b) isolated samples screening for FMDV shows Lane 1 and 15 of 100 bp DNA ladder, Lane 12 FMDV negative samples during screen test, Lane 13 was negative control, Lane 2-11 were FMDV positive during screening (328 bp); (c) Lane 1 and 11 of 100 bp DNA ladder, Lane 9 was positive control, Lane 10 was negative control, Lane 2-8 were serotype “O” PCR product (600 bp); (d) Lane 1 and 10 were 100 bp DNA ladder, Lane 9 was positive control for serotype A, Lane 7 was negative control for serotype “A,” Lane 2-6 and 8 were serotype “A” PCR product (866 bp).

Figure-5

Phylogenetic analysis for the serotype O of foot-and-mouth disease virus. The green diamond label (ETH/37/2018) in the phylogenetic tree was mixed serotypes (O and A) isolated from the purchased calf.

Figure-6

Phylogenetic analysis for the serotype A of foot-and-mouth disease virus. The green diamond label (ETH/37/2018) in the phylogenetic tree was mixed serotypes (O and A) isolated from the purchased calf.