In silico analysis of PirA- and PirB-like toxin genes of Vibrio spp., present in Asia and Costa Rica.

OBJECTIVE: Acute hepatopancreatic necrosis disease is an emerging infectious disease of Penaeus species. The causative agent is Vibrio species, which dispels binary toxin similar to pirA and pirB, which causes mortality in infected shrimp. The aim of this research was to investigate the evolutionary relationship of pirA and pirB homologous genes present in this Asia and Costa Rica in silico.
MATERIALS AND METHODS: The sequences for in silico analysis were all retrieved from the Basic Local Alignment Search Tool Nucleotide (BLASTN) tool of the National Center for Biotechnology Center. For pirA, a total of 25 sequences submitted from different Asian countries and Costa Rica were retrieved for analysis. Meanwhile, for pirB, a total of 11 sequences submitted from five Asian countries were retrieved. Sequences were aligned using the CLUSTAL W alignment tool under Molecular Evolutionary Genetics Analysis (MEGA) 7 software. The evolutionary history was then estimated using the Unweighted Pair Group Method with Arithmetic mean (UPGMA) method, whereas the evolutionary distances were determined using the maximum composite likelihood model with 1,000 bootstrap replications. RESULTS AND DISCUSSION: The results show that, among 27 DNA sequences analyzed for pirA gene, three groups were generated, while for pirB, 13 DNA sequences yielded only one group. The analysis revealed low genetic variation among isolates for both pirA and pirB genes.
CONCLUSION: This result suggests that the low frequency of polymorphism and geographic location cannot be attributed to the differences in V. parahaemolyticus isolates in Asian countries relative to Costa Rican isolates in pirA and pirB genes. Copyright: © Journal of Advanced Veterinary and Animal Research.

Introduction

The acute hepatopancreatic necrosis disease (AHPND, formerly known as early mortality syndrome) has reportedly been the cause of high mortalities and production in Southeast Asian countries. It induces necrosis in the hepatopancreas of the diseased shrimp, which is caused by the bacterium Vibrio species [1].

Histological sections of the hepatopancreas of Penaeus vannamei (Fig. 1A and B) show the differences between the hepatopancreas of normal and infected shrimps. Clearly, AHPND lesions are present (Fig. 1B) through the immense sloughing of hepatopancreatic tubule epithelial cells into the lumen, which only suggests severe necrosis marked by cellular detachment relating to a bacterial infection. Moreover, the distinct formation of melanized hemocytic nodules in the middle part of the hepatopancreas tubules is evident [1].

Figure 1.

Hematoxylin and H&E stained histological cross-sections of the hepatopancreas of P. vannamei sample. (A) Normal shrimp hepatopancreas tubule. (B) AHPND pathology. Actual photos were taken from the Bureau of Fisheries and Aquatic Resources, National Fisheries Laboratory Division.

Recently, the reports on the pathogenicity of strains of V. parahaemolyticus indicate that there is only a specific region in the plasmid of the bacteria that determines the pathogenicity of the bacteria [2]. Several studies illustrate that the genomes of these pathogenic isolates possess genes, which are homologous with the Photorhabdus insect-related (Pir) toxin genes pirA and pirB [3].

The mechanism of speciation in bacteria remains to be a topic of interest among researchers worldwide. Aside from its unpredictability and not to mention huge data sets to be fully explored, single-point mutations can play a tremendous role in determining the evolutionary relationship of Vibrio species across different locations. A representative of such data sets on pirA and pirB genes can give a glimpse on the genomic data of these genes. Furthermore, since in silico analyses on pirA and pirB genes from Southeast Asia and Costa Rica have not yet been thoroughly studied, this study can examine the possible diversification and/or classification of Vibrio species.

In this paper, the in silico analysis of pirA and pirB sequences from Southeast Asia and Costa Rica available in the National Center for Biotechnology Information (NCBI) was done. The phylogenetic trees of Vibrio spp. sequences of pirA and pirB toxin genes were also constructed to determine the evolutionary relationship of each isolate in different countries.

Materials and Methods

DNA sequences used in this study

The sequences for in silico analysis were all retrieved from the Basic Local Alignment Search Tool Nucleotide (BLASTN) tool of the NCBI. All of the sequences that were used are the only available sequences in NCBI as of June 25, 2019. For pirA, a total of 25 sequences submitted from different Asian countries and Costa Rica were retrieved for analysis. Two pirA sequences from Photorhabdus luminescens served as outgroup. For pirB, a total of 11 sequences submitted from five Asian countries were retrieved. Each sequence is labeled according to their corresponding accession numbers, followed by their country of origin.

Sequence and phylogenetic analyses of Vibrio spp.

Sequences were aligned using the CLUSTAL W alignment tool under Molecular Evolutionary Genetics Analysis (MEGA) 7 software. Then, the evolutionary history was inferred using Unweighted Pair Group Method with Arithmetic mean (UPGMA) method, whereas the evolutionary distances were computed using the maximum composite likelihood model with 1,000 bootstrap replications. The analysis was conducted for 27 nucleotide DNA sequences for pirA, whereas 13 nucleotide sequences were used for pirB, which include two outgroups for each target gene.

Results and Discussion

Sequence and phylogenetic analyses

The pirA sequences of Vibrio spp. used in this study had an average of 306 nucleotide bases. Asian isolates range from 209 to 336 bp, whereas Costa Rican isolates range from 283 to 287 bp. The average nucleotide frequencies of Asian isolates (T = 25.10%, C = 21.20%, A = 30%, and G = 23.70%) differ from Costa Rican isolates (T = 24.60%, C = 21.46%, A = 28.78%, and G = 25.10%). The aligned sequences from the 25 pirA yielded 205 identical pairs, 32 transitional pairs, and 38 transversional pairs. On the other hand, pirB sequences had an average of 1,308 nucleotide bases (including the outgroup). The average nucleotide frequencies were as follows: T = 31.4%, C = 15.8%, A = 31.7%, and G = 21.1%. A low Guanine and Cytosine (GC) content of both pirA/B indicates recent acquisitions of these genes [4]. The high similarity between sequences was observed, having 1,010 base similarity, 103 transitional pairs, and 123 transversional pairs. This only suggests that, for pirB, low variation among isolates was observed due to high similarity.

As shown in Figure 2, phylogenetic analysis revealed the sequences of pirA genes from different Vibrio spp. from the isolates of Asian countries and Costa Rican. The pirA sequences from different Vibrio spp. include V. harveyi, Vibrio owensii, V. campbellii, and V. parahaemolyticus as shown in Figure 2.

Figure 2.

Phylogenetic tree of Vibrio spp. isolates targeting pirA gene from Malaysia, China, Japan, Thailand (Asia), and Costa Rica.

This figure showed a distinct tree topology for pirA, which attained high nodal bootstrap support. It clearly illustrates a well-known property of bootstrapping: high nodal bootstrap of 100 was generated in the main root, hence indicating that the optimal tree would be unlikely to change as sequence length increases. The constructed tree generated three distinct groups and one outgroup. Interestingly, Asian and Costa Rican isolates were grouped together for pirA gene despite differences in geographic location, thus corroborating with the previous studies that toxin genes such as pirA and pirB exhibit low polymorphism. This result only suggests that the low frequency of polymorphism and geographic location cannot be attributed to the differences in V. parahaemolyticus isolates in Asian countries relative to Costa Rican isolates in pirA gene. It answers the query of Peña-Navarro et al. [5] about the similarity of Costa Rican isolates than that of Asian genotypes. The phylogenetic relationship between these isolates has not been reported yet and thoroughly studied. The isolates of V. campbellii and V. owensii from Vietnam and China, respectively, were shown to cause AHPND in 2015 [6,7]. These isolates were confirmed to comprise a plasmid that is analogous to the one discovered in AHPND-causing V. parahaemolyticus which produces the binary toxins similar to pirA/pirB [8-10].

Consequently, it supports the theory that pirA/pirB are the main virulence factors that cause AHPND.

It clearly shows that horizontal gene transference (HGT) occurs between sister species belonging to closely related clades, as argued by Sawabe et al. [11] in their previous study. The results generated from this study will impact the shrimp industry in terms of analyzing pirA/pirB toxin genes present in isolates with AHPND.

A phylogenetic analysis of pirB showed that Asian isolates have a high similarity to each other. Clearly, based on pirB sequences, Vibrio species in Asia are grouped together with 100% bootstrap value. A sample size of pirB is smaller compared to pirA since few sequences are deposited in NCBI, whereas no sequences from Costa Rican isolates were retrieved in this study. The phylogenetic tree formed a monophyletic group and one outgroup.

As shown in Figure 3, the genetic variation is low among geographic isolates of Vibrio spp. and consistent with Figure 2 from pirA phylogenetic tree. Clearly, within the species level, Vibrio species are grouped together in one clade. This analysis could have better findings if there are more pirB sequences retrieved from NCBI.

Figure 3.

Phylogenetic tree of Vibrio spp. isolates targeting pirB gene from China, Japan, Vietnam, and Thailand.

Following the genetic variations of sequences from pirA and pirB, these will help in interpreting the evolution of this AHPND-causing Vibrio spp., which is essential for diagnostic and management strategies up to date, and subsequently, to reduce the spread of the disease and lessen its impact on commercial shrimp farms.

Conclusion

DNA sequences were analyzed in silico for pirA and pirB of Vibrio species in Asia and Costa Rica. The results showed low genetic variability among all the geographic isolates of Vibrio spp. for the genes such as pirA and pirB. This result only suggests that the low frequency of polymorphism and geographic location cannot be attributed to the differences in V. parahaemolyticus isolates in Asian countries relative to Costa Rican isolates in pirA and pirB genes. Furthermore, it can be good evidence that HGT is the mode of transmission, in which pirA/B can be spread interspecifically. An increase in sample size for pirB gene is highly recommended for further study to give comprehensive information on the phylogenetic relationship of Vibrio species across different countries.