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

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 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 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

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