RESUMO
Background and Aim: Various methods can detect foot-and-mouth disease (FMD) in cows, but they necessitate resources, time, costs, laboratory facilities, and specific clinical specimen submission, often leading to FMD virus (FMDV) diagnosis delays. The 2022 FMD outbreak in East Java, Indonesia, highlighted the need for an easy, inexpensive, rapid, and accurate detection approach. This study aims to devise a one-step reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) technique and phylogenetic analysis to detect the serotype O FMDV outbreak in East Java. Materials and Methods: Swab samples were collected from the foot vesicles, nasal secretions, and saliva of five suspected FMDV-infected cows in East Java between June and July 2022. The RT-LAMP design used hydroxy naphthol blue dye or SYBR Green I dye, with confirmatory analysis through reverse transcriptase polymerase chain reaction (RT-PCR) targeting 249 base pairs. PCR products underwent purification, sequencing, and nucleotide alignment, followed by phylogenetic analysis. Results: The RT-LAMP method using hydroxy naphthol blue dye displayed a positive reaction through a color shift from purple to blue in the tube. Naked-eye observation in standard light or ultraviolet (UV) light at 365 nm, with SYBR Green I stain, also revealed color change. Specifically, using SYBR Green I dye, UV light at 365 nm revealed a color shift from yellow to green, signifying a positive reaction. Nucleotide alignment revealed mutations and deletion at the 15th sequence in the JT-INDO-K3 isolate from the East Java FMDV outbreak. Despite differing branches, the phylogenetic tree placed it in the same cluster as serotype O FMDV from Malaysia and Mongolia. Conclusion: JT-INDO-K3 exhibited distinctions from Indonesian serotype O FMDV isolates and those documented in GenBank. Then, the RT-LAMP method used in this study has a detection limit 10 times higher latter than the conventional RT-PCR limit, without any cross-reactivity among strains.
RESUMO
Background and Aim: Questions about the origin of coronavirus and its introduction to human beings have persisted. The detection of a variety of coronavirus related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in bats and pangolins led to the widespread belief that SARS-CoV-2 originated from wild ani-mals and was introduced to humans through an inter-mediate animal. Thus, coronaviruses from animals, especially those in close contact with humans, have attracted particular attention. This study aimed to phylogenetically analyze feline enteric coronavirus (FECV), feline infectious peritonitis virus (FIPV), and SARS-CoV-2 found in cats in Surabaya amid the COVID-19 pandemic. The results will provide a basis for developing basic preventive and pet healthcare strategies. Materials and Methods: Samples were collected on physical examinations of domestic and Persian cats (males and females) from March 2020 to March 2022. Samples were collected if there were clinical signs of FECV and FIP based on a veterinarian's diagnosis in several clinics in Surabaya. Laboratory examinations in this study were performed by reverse-transcription-polymerase chain reaction (RT-PCR) with primers for conserved regions of FIP and FECV, DNA sequencing was performed with Applied Biosystem Genetic Analyzer protocol, homology analysis was performed using Basic Local Alignment Search Tool NCBI, phylogenetic analysis was carried out with BioEdit 7.2 software, and sequences were compared with references from GenBank. Results: Samples were collected from ten cats showing clinical signs of FECV and FIP, based on a veterinarian's diagnosis. On RT-PCR examinations performed with specifically designed primers for detecting FIPV in blood, peritoneal fluid, and feces, only one sample showed positivity for FIPV (1/10), namely, a peritoneal sample from a domestic cat in Surabaya. Homology analysis of the FIPV Surabaya isolate showed 98% similarity with FECV and FIPV reported in GenBank (MT444152 and DQ010921, respectively). In phylogenetic analysis, the FIPV Surabaya isolate was clustered together with SARS-CoV-2 of Clade A (MT198653) from Spain, SARS-CoV-2 Clade A (MT192765) from the USA, SARS-CoV-2 Clade D (039888) from the USA, and SARS-CoV-2 Clade F (MT020781) from Finland. Conclusion: This study revealed a relationship between the SARS-CoV-2 viruses that infect humans and cats (FECV), which is an important finding for those keeping cats at home. However, this finding requires further comprehensive support from laboratory studies.