Interaction of Vibrio spp. with the Inner Surface of the Digestive Tract of Penaeus monodon

Several species of Vibrio are the causative agent of gastroenteritis in humans. In aquaculture, Vibrio harveyi (Vh) and V. parahaemolyticus (Vp) have long been considered as shrimp pathogens in freshwater, brackish and marine environments. Here we show by using scanning electron microscopy (SEM) that Penaeus monodon orally inoculated with each of these two pathogens via an Artemia diet had numerous bacteria attached randomly across the stomach surface, in single and in large biofilm-like clusters 6 h post-infection. A subsequent marked proliferation in the number of V. harveyi within the biofilm-like formations resulted in the development of infections in the stomach, the upper and middle midgut, but neither in the posterior midgut nor the hindgut. SEM also revealed the induced production of peritrichous pili-like structures by the Vp attaching to the stomach lining, whilst only a single polar fibre was seen forming an apparent physical bridge between Vh and the host's epithelium. In contrast to these observations, no such adherences or linkages were seen when trials were conducted with non-pathogenic Vibrio spp. or with Micrococcus luteus, with no obvious resultant changes to the host's gut surface. In naive shrimp, the hindgut was found to be a favorable site for bacteria notably curved, short-rod shaped bacteria which probably belong to Vibrio spp. Data from the current study suggests that pathogens of P. monodon must be able to colonize the digestive tract, particularly the stomach, where chitin is present, and then they use an array of virulent factors and enzymes to infect their host resulting in disease. Oral infection is a better way of mimicking natural routes of infection; investigating the host-bacteria interactions occurring in the digestive tract may lead to new strategies for the prevention or control of bacterial infections in penaeids

Interaction of Vibrio spp. with the Inner Surface of the Digestive Tract of Penaeus monodon

Several species of Vibrio are the causative agent of gastroenteritis in humans. In aquaculture, Vibrio harveyi (Vh) and V. parahaemolyticus (Vp) have long been considered as shrimp pathogens in freshwater, brackish and marine environments. Here we show by using scanning electron microscopy (SEM) that Penaeus monodon orally inoculated with each of these two pathogens via an Artemia diet had numerous bacteria attached randomly across the stomach surface, in single and in large biofilm-like clusters 6 h post-infection. A subsequent marked proliferation in the number of V. harveyi within the biofilm-like formations resulted in the development of infections in the stomach, the upper and middle midgut, but neither in the posterior midgut nor the hindgut. SEM also revealed the induced production of peritrichous pili-like structures by the Vp attaching to the stomach lining, whilst only a single polar fibre was seen forming an apparent physical bridge between Vh and the host's epithelium. In contrast to these observations, no such adherences or linkages were seen when trials were conducted with non-pathogenic Vibrio spp. or with Micrococcus luteus, with no obvious resultant changes to the host's gut surface. In naive shrimp, the hindgut was found to be a favorable site for bacteria notably curved, short-rod shaped bacteria which probably belong to Vibrio spp. Data from the current study suggests that pathogens of P. monodon must be able to colonize the digestive tract, particularly the stomach, where chitin is present, and then they use an array of virulent factors and enzymes to infect their host resulting in disease. Oral infection is a better way of mimicking natural routes of infection; investigating the host-bacteria interactions occurring in the digestive tract may lead to new strategies for the prevention or control of bacterial infections in penaeids

Bacterial population in intestines of the black tiger shrimp (Penaeus monodon) under different growth stages.

Intestinal bacterial communities in aquaculture have been drawn to attention due to potential benefit to their hosts. To identify core intestinal bacteria in the black tiger shrimp (Penaeus monodon), bacterial populations of disease-free shrimp were characterized from intestines of four developmental stages (15-day-old post larvae (PL15), 1- (J1), 2- (J2), and 3-month-old (J3) juveniles) using pyrosequencing, real-time PCR and denaturing gradient gel electrophoresis (DGGE) approaches. A total of 25,121 pyrosequencing reads (reading length = 442±24 bases) were obtained, which were categorized by barcode for PL15 (7,045 sequences), J1 (3,055 sequences), J2 (13,130 sequences) and J3 (1,890 sequences). Bacteria in the phyla Bacteroides, Firmicutes and Proteobacteria were found in intestines at all four growth stages. There were 88, 14, 27, and 20 bacterial genera associated with the intestinal tract of PL15, J1, J2 and J3, respectively. Pyrosequencing analysis revealed that Proteobacteria (class Gammaproteobacteria) was a dominant bacteria group with a relative abundance of 89% for PL15 and 99% for J1, J2 and J3. Real-time PCR assay also confirmed that Gammaproteobacteria had the highest relative abundance in intestines from all growth stages. Intestinal bacterial communities from the three juvenile stages were more similar to each other than that of the PL shrimp based on PCA analyses of pyrosequencing results and their DGGE profiles. This study provides descriptive bacterial communities associated to the black tiger shrimp intestines during these growth development stages in rearing facilities

Representative SEM images of the inner surface of the digestive tract of <i>P</i>. <i>monodon</i> infected with <i>Vibrio parahaemolyticus</i>.

<p>(A) At 24 post-infection, numerous straight-shaped bacteria adhering to the fibre seta, to (B) short spines and (C) to the inner surface of the stomach. Some of the spines were broken and had detached from the stomach lining (arrowheads). (D-F) Attached bacteria producing peritrichous pili-like structures. Abbreviation: fiber seta (FS), spines (Sp), cuticle (Cu), fimbria (Fi), peritrophic membrane (PM)</p

Representative SEM images of the inner surface of <i>P</i>. <i>monodon</i> infected with <i>Vibrio harveyi</i>.

<p>(A) At 1.5 h post-infection (PI), no bacteria adhering to the surface of the stomach linings were seen, but (B) numerous bacterial cells mixed with ingested food attaching the stomach surface were seen. (C) Numerous rod-shaped bacteria firmly attached to the stomach lining. (D) At 6 h PI, colonizing bacteria cover the epithelium of the anterior midgut. (E, yellow arrow) A higher magnification of the bacteria seen at 6 h show that they possess polar flagella that are linked with each other, and (F) heavy destruction of the epithelial layers by bacteria exposed of the basement membrane underneath. (G) The posterior portion of the midgut showing intact tissue with a thick peritrophic matrix or (H) with a few bacterial cells attached to the microvilli. (I) Scattered clusters of rod-shaped bacteria adhering to the lining of the hindgut. (J-L) At 24 h PI, the numbers of bacteria within the stomach of infected shrimp increased dramatically. Densely packed-bacteria were found covering the epithelium of the anterior midgut. Abbreviation: cuticle (Cu), spines (Sp), peritrophic membrance (PM), ingested food (IF), fiber seta (FS), polar flagella (PF), bacteria (Ba), basement membrane (BM), microvilli (Mv)</p

Real-time PCR presented of the ratio of the <i>gyrB</i> gene of <i>Vibrio harveyi</i> (A) and 16S rDNA bacteria, and the ratio of the <i>gyrB</i> and <i>tlh</i> gene of <i>Vibrio parahaemolyticus</i> (B) and 16S rDNA bacteria in the GI tract of <i>P</i>. <i>monodon</i>.

<p>Error bars were expressed as ±SEM. Asterisk indicates significant differences between the control and challenged group (<i>P</i><0.05).</p

Representative SEM pictures of the inner surface of the digestive tract of a suspected diseased <i>Penaeus monodon</i> from a shrimp farm.

<p>A cluster of unique rod-shaped bacteria attached to (A) fibre setae or (B) to the lining of the stomach. (C) Higher magnification image of the attached bacteria in the stomach exhibiting peritrichous pili-like structures or fimbria., where (D) a few fibres linked to the peritrophic matrix (PM) can be seen (arrowhead). (E) Many holes were created in the PM and a few granules were seen inside the holes. (F) A group of irregular-shaped bacteria were found attached to the PM. (G) A cluster of short-rod shaped bacteria with polar flagella, (h) irregular-shaped, and (I) unidentified particles were seen attached to the hindgut wall. Abbreviation: fiber seta (FS), bacteria (Ba), spines (Sp), fimbria (Fi) granule (Gr), microvilli (Mv), polar flagella (PF), unknown particles (UP), ingested food (IF)</p

Representative scanning electron microscopy (SEM) micrographs of the inner surface of the digestive of farmed <i>Penaeus monodon</i>.

<p>Inner surface of (A) the dorsal (B) ventral, (C) peritrophic membrane, and (D) fiber seta of the stomach were devoid of bacteria. (E) bacterial cells (arrowed), however, were seen in association with food inside the stomach. (F, G, H, I, J) Healthy midgut have intact microvilli, and a large number of bacteria were observed attached to the peritrophic membrane and food particle in the midgut. (F, I) Massive granules among the epithelial cells can be seen projecting into the midgut lumen between the microvilli. (K, L) only a few bacteria were seen attached to the cuticle lining of the hindgut. Abbreviation: cuticle (cu), spines (Sp), peritrophic membrane (PM), fiber seta (FS), ingested food (IF), bacteria (Ba), microvilli (Mv), granule (Gr), pit (Pi)</p

Representative SEM images of the inner surface of the digestive tract from <i>P</i>. <i>monodon</i> receiving non-pathogenic bacteria (<i>Micrococcus luteus</i> and non-pathogenic <i>Vibrio</i> B4-24).

<p>No observed attachment of bacteria to the stomach (A—<i>M</i>. <i>luteus</i>, B- <i>Vibrio</i> B4-24) or to the midgut (C- <i>Vibrio</i> B4-24). The bacteria found in the hindgut were variable in number where most of the hindgut tissues were intact (D—<i>M</i>. <i>luteus</i>, E- <i>Vibrio</i> B4-24). A high number of pits were found across the surface of the midgut (G, H- <i>Vibrio</i> B4-24), where a large number of cocci- and spindle-shaped granules which resided in the epithelial cells were seen (I- <i>Vibrio</i> B4-24). Abbreviation: spines (Sp), fiber seta (FS), cuticle (Cu), microvilli (Mv), bacteria (Ba), pit (Pi), granule (Gr)</p

Characterization of Intestinal Bacteria in Wild and Domesticated Adult Black Tiger Shrimp (<i>Penaeus monodon</i>)

<div><p>The black tiger shrimp (<i>Penaeus monodon</i>) is a marine crustacean of economic importance in the world market. To ensure sustainability of the shrimp industry, production capacity and disease outbreak prevention must be improved. Understanding healthy microbial balance inside the shrimp intestine can provide an initial step toward better farming practice and probiotic applications. In this study, we employed a barcode pyrosequencing analysis of V3-4 regions of 16S rRNA genes to examine intestinal bacteria communities in wild-caught and domesticated <i>P. monodon</i> broodstock. Shrimp faeces were removed from intestines prior to further analysis in attempt to identify mucosal bacterial population. Five phyla, <i>Actinobacteria</i>, <i>Fusobacteria</i>, <i>Proteobacteria</i>, <i>Firmicutes</i> and <i>Bacteroidetes,</i> were found in all shrimp from both wild and domesticated environments. The operational taxonomic unit (OTU) was assigned at 97% sequence identity, and our pyrosequencing results identified 18 OTUs commonly found in both groups. Sequences of the shared OTUs were similar to bacteria in three phyla, namely <i>i) Proteobacteria</i> (<i>Vibrio</i>, <i>Photobacterium</i>, <i>Novosphingobium</i>, <i>Pseudomonas</i>, <i>Sphingomonas</i> and <i>Undibacterium</i>), <i>ii) Firmicutes</i> (<i>Fusibacter</i>), and <i>iii) Bacteroidetes</i> (<i>Cloacibacterium</i>). The shared bacterial members in <i>P. monodon</i> from two different habitats provide evidence that the internal environments within the host shrimp also exerts selective pressure on bacterial members. Intestinal bacterial profiles were compared using denaturing gradient gel electrophoresis (DGGE). The sequences from DGGE bands were similar to those of <i>Vibrio</i> and <i>Photobacterium</i> in all shrimp, consistent with pyrosequencing results. This work provides the first comprehensive report on bacterial populations in the intestine of adult black tiger shrimp and reveals some similar bacterial members between the intestine of wild-caught and domesticated shrimp.</p></div