4. DISCUSSION
Edwardsiellosis caused by E. ictaluri in Nile tilapia (O. niloticus ) was reported for the first time in the western hemisphere (Soto et al. , 2012) and later in farmed hybrid red tilapia (Oreochromis sp.) in Vietnam in 2016 (Dong et al., 2019). Comparative genomic analysis of E. ictalurifrom different fish hosts revealed that E. ictaluri isolates from tilapia are a novel genotype, which differs from the currently circulating catfish genotypes (Machimbirike et al., 2021; Reichley et al., 2017). This comprehensive follow-up investigation and findings suggest that this emerging pathogen is well established and has spread in tilapia farms in northern Vietnam. Although the introduction (source of infection) remains inconclusive, the notation of most affected farms that used imported stocks for aquaculture with improper diagnostic screening suggests a possible foreign introduction of this pathogen. Alternatively, there were a proportion of disease outbreak farms that used domestic stock sources. This implies that this pathogen may have been circulated domestically from a previously unknown introduction to Vietnam (Dong et al., 2019) and continued to spread thereafter through contaminated seeds and/or contaminated water bodies during disease outbreaks. The detection of E. ictaluri associated with disease outbreaks from two different continents (America and Asia) highlights the risk of transboundary spread and potential impact on the tilapia industry. Countries that rely on imported tilapia stocks for aquaculture, such as Vietnam, may have the same theoretical risk for the introduction of this emerging pathogen. Therefore, active surveillance, early diagnostic screening, and biosecurity measures are highly recommended for these countries.
The present study also identified some potential risk factors associated with disease outbreaks caused by E. ictaluri in tilapia, including the influence of temperature, fish size, and belated detection due to ambiguous clinical symptoms. The disease occurs in cool seasons, including autumn (ponds and cage culture in the rivers) and spring (cage culture in deep lakes/hydropower reservoirs) when the temperature range is approximately 23–29 °C due to the capacity of E. ictaluri to be motile at this temperature range (Hawke et al., 1998); thus, this temperature range is suitable for this pathogen to attach, propagate, and cause disease outbreaks. The incidence of E. ictaluriinfections associated with cool seasons in other host species, including catfish and non-catfish species, has been previously reported (Hawke et al., 1998; Pham et al., 2021; Takeuchi et al., 2016). In addition, the current survey results revealed that tilapia of a small size (< 350 g) were more susceptible to E. ictaluriinfection, which tended to cause acute death with higher mortality than at adult stages. Farming observations revealed that in cage lines raising tilapia culture on the same rivers/lakes, cages raising fish less than 350 g were found to be susceptible and have higher mortality rates than those of other cages raising marketable size fish. The fish stage influences the susceptibility to infection similar to that ofE. ictaluri infection in catfish, in which the disease occurs in all ages of catfish, but fingerlings and juveniles have been demonstrated to be more susceptible than adult fish (Dung et al., 2008; Hawke et al., 1998). Further epidemiological and experimental studies are needed to determine the optimal temperature and influence of age on the circulation and outbreaks caused by this pathogen in tilapia. Moreover, E. ictaluri- affected fish did not exhibit recognizable external symptoms, causing misleading presumptive disease diagnosis and untimely treatment efforts.
Virulence factors of pathogenic bacteria may be encoded by specific regions of the prokaryotic genome, termed pathogenicity islands (Hacker et al., 1990). Pathogenicity islands are present in the genomes of pathogenic strains, but are absent in the genomes of nonpathogenic members of the same or related species (Hacker & Kaper, 2000). The detection of identical virulence gene profiles among 26 isolates from nine provinces implied the circulation of a homologous strain. The presence of esrC , evpC , and ureA-C genes, which are well-known for enabling the bacteria to survive and replicate intracellularly (Booth et al., 2006; Chen et al., 2017; Hu et al., 2014; Moore et al., 2002; Rogge & Thune, 2011), in all the isolates identified in this study suggests their potential virulence and management difficulty using antimicrobials. The presence of these genes has also been reported in tilapia isolates from the western hemisphere by Griffin et al. (2016). However, while the virD , eseI , and its chaperone escD genes are present in US channel catfish (Ictalurus punctatus ) isolates (Rogge et al., 2013), they are absent in all tilapia isolates from this study and from the western hemisphere (Griffin et al., 2016). These findings support previous studies showing that the variability in virulence genes of E. ictaluri is related to host fish species (Griffin et al., 2016). Although E. ictaluri isolates from Asia or the western hemisphere harbor a similar pattern of six examined virulence genes, other potential virulence factors may exist in E. ictaluri isolates in Vietnam, which have contributed to and create their hypervirulence compared to those of the western hemisphere. Thus, whole genome sequencing and comparative analysis between isolates from two continents are needed to understand this evolution.
Although disease caused by E. ictaluri was first detected only a few years ago and affected farmed tilapia in Vietnam, the high levels of antibiotic resistance pose potential risks; thus, emergent action is needed to mitigate the disease and spread of this pathogen. The high resistance frequencies of this bacterium to various antimicrobials belonging to several classes and subclasses may be the consequence of inappropriate usage of these drugs for disease control in tilapia farms, leading to 12 multi-drug resistance phenotypes (Dang et al., 2021). Of note, these MAR isolates may have also been introduced to Vietnam elsewhere and continue to spread and acquire more resistance. Some resistance was likely intrinsic. Specifically, approximately 85% of theE. ictaluri isolates from tilapia were resistant to erythromycin, which is consistent with a previous report on the intrinsic resistance of Edwardsiella species to macrolides (Stock & Wiedemann, 2001). The intrinsic resistance of gram-negative bacteria and specifically ofEdwardsiella species to glycopeptides has also been widely shown (Breijyeh et al., 2020; Stock & Wiedemann, 2001) and is reaffirmed by the extremely high frequency of vancomycin resistance in E. ictaluri . However, some resistance likely resulted from the misuse of antimicrobials. For example, florfenicol is highly effective in E. ictaluri infection control in catfish (Gaunt et al., 2015), but allE. ictaluri isolates from tilapia in this study are resistant to this drug. Similarly, high resistance frequencies with two other approved antibiotics used in aquaculture in Vietnam, namely amoxicillin (80.8%) and sulfamethoxazole/trimethoprim (100%), were also recorded. Nevertheless, alternatives to antibiotics, such as vaccines, probiotics, and bioactive metabolites, should be further explored to tackle this emerging, pathogenic, multi-drug resistant bacterium.
The multidrug-resistant E. ictaluri isolates in this study were extremely highly virulent. In comparison to previous studies where the LD50 was 3.2×104 CFU/mL in yellow catfish (Kim & Park, 2015) and 5.1 ×104 CFU/mL in Nile tilapia (Soto et al., 2013), E. ictaluri isolates in this study required only 42–61 CFU/fish to kill 50% of the tilapia population. At higher doses, the results are similar to those reported by Dong et al. (2019) when fish challenged with 105–107 CFU/fish result in 95%–100% mortality within 9 days post infection. Gross signs and histopathological manifestations are similar to those of previous studies that show severe tissue destruction, especially in the spleen and head kidney, two major lymphoid organs that play important roles in defense against infection (Soto et al., 2012; Dong et al., 2019). The failure of these organs may explain the high mortality in experimentally challenged fish (Dong et al., 2019; Soto et al., 2013). Since the current investigation suggested homologous strains of the collectedE. ictaluri isolates, an autogenous vaccine might be the best option to combat this emerging disease in the present time before a better vaccine candidate for a wider region is discovered.
In conclusion, this follow-up investigation from a previous case report highlights the establishment and widespread use of extremely virulent, multidrug-resistant E. ictaluri isolates as an emerging threat to the tilapia farming industry in Vietnam. The arrival of this pathogen likely involved both imported and domestic stocks. Therefore, increased awareness, early diagnostic testing, and biosecurity measures at both national and international levels are needed to prevent its transboundary spread and negative impact on the tilapia industry.