The spread of Vibrio parahaemolyticus in tissues of the Pacific white shrimp Litopenaeus vannamei analyzed by PCR and histopathology.

V. parahaemolyticus are bacteria that cause the Acute Hepatopancreatic Necrosis Disease (AHPND), or Early Mortality Syndrome (EMS), in shrimp. To further understand the pathogenesis mechanisms of V. parahaemolyticus infection in shrimp, the spreading of this bacterium in various tissues was investigated. The spread of infection in shrimp that were exposed to seawater bacteria was studied by PCR and histopathology at 1 min, 1, 6, 12, 24, 48 and 72 h after exposure. The PCR results showed that V. parahaemolyticus was at its most widespread at 6 h after exposure, at which point V. parahaemolyticus was found in the gills, hepatopancreas, intestine, muscles, and hemolymph. However, examinations after 6 h of infection found only small amounts of V. parahaemolyticus in hepatopancreas and intestines. Histopathology of the hepatopancreas showed abnormalities on gross examination at 1 min-72 h after exposure. This study indicates that V. parahaemolyticus can spread quickly by using the hepatopancreas as the target tissue. After 6 h of infection, V. parahaemolyticus was eliminated by immune system while their toxins still caused damage to shrimp tissues.

Tissue specific uptake of inactivated and live Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss): visualization by immunohistochemistry and in situ hybridization.

Understanding of uptake and invasion routes of Yersinia ruckeri, causing Enteric Red Mouth Disease (ERM) in rainbow trout (Oncorhynchus mykiss), is essential for improved understanding of the pathogenicity and immune response mechanisms associated this disease. The present work shed light on areas of invasion in rainbow trout by the use of immunohistochemistry and in situ hybridization techniques. Fish were exposed to live or formalin inactivated bacteria and samples were subsequently taken for histology from various outer and inner surfaces. We applied a specific monoclonal antibody and specific oligonucleotide probes binding to Y. ruckeri (serotype O1, biotype 2) in tissue sections and were able to demonstrate a tissue specific uptake of this bacterium (both formalin inactivated and live form). Uptake and subsequent translocation dynamics at various surfaces demonstrated different site specific propensities between the formalin inactivated and live bacterial organisms. Lateral lines, dorsal fin, epidermis and gastro-intestinal tract mucosal tissue were the primary areas where bacterial uptake was demonstrated readily after exposure. The fate of internalized bacterial organisms within the host suggested that central immune organs are involved in the final antigen processing.

Activation of an immune response in Litopenaeus vannamei by oral immunization with phagocytosis activating protein (PAP) DNA.

The phagocytosis activating protein (PAP) gene has been reported to stimulate the phagocytic activity of shrimp hemocytes and to protect shrimp from several pathogens. In this study oral administration of the chitosan-PAP gene to shrimp was investigated for its ability to induce immunity. The PAP gene was cooperated into a phMGFP plasmid, named PAP-phMGFP. Chitosan-PAP-phMGFP nanoparticles were formed by mixing a low molecular weight chitosan (50 kDa) and a high molecular weight chitosan (150 kDa) with various ratios of PAP-phMGFP. The optimal ratio of chitosan PAP-phMGFP nanoparticles was first determined by transfection into Chinese Hamster Ovary (CHO) cells before being used for oral immunization in shrimp. The chitosan-PAP-phMGFP nanoparticles at a ratio of 2:1 with the low molecular weight chitosan were optimum for transfecting the CHO cells. The shrimp were then fed with 25, 50, 100 and 150 µg/shrimp/day of chitosan-PAP-phMGFP (2:1) nanoparticles then challenged by the white spot syndrome virus (WSSV). Shrimp fed with 50 µg of chitosan-PAP-phMGFP nanoparticles per day for 7 consecutive days, produced the highest relative percent survival (RPS) (94.45 ± 9.86%). The presence of PAP-phMGFP was detected in every shrimp tissue including the hemolymph, lymphoid organ, heart, hepatopancreas, intestine and muscle. The folds increase of the PAP gene expression increased significantly together with an increase of the phagocytic activity in the immunized shrimp. The stability of the PAP-phMGFP in the immunized shrimp hemolymph was detected by determination of the expression of the GFP at various days after immunization ceased. GFP expression was detected until the 15th day but not at the 30th day after immunization ceased. A quantitative analysis of the WSSV copies in shrimp heart tissue was significantly reduced in the immunized shrimp. In addition, chitosan-PAP-phMGFP nanoparticles protected shrimp against WSSV, Yellow head virus (YHV) and Vibrio harveyi with RPS values of 83.34 ± 7.86%, 55.56 ± 15.72% and 53.91 ± 5.52%, respectively. This study therefore confirms the role of the PAP gene in shrimp immunity and may lead to the development of a way to prevent microbial diseases of shrimp at an industrial level by appropriate feeding of a chitosan/DNA complex.

Stimulating the immune response of Litopenaeus vannamei using the phagocytosis activating protein (PAP) gene.

High mortality in the shrimp farming industry is caused by several pathogens such as white spot syndrome virus (WSSV), yellow head virus (YHV) and Vibrio harveyi (V. harveyi). A PAP (Phagocytosis activating protein) gene able to activate phagocytosis of shrimp hemocytes was cloned into the eukaryotic expression vector phMGFP. In vitro expression was confirmed by transfection of PAP-phMGFP into CHO (Chinese Hamster Ovary) cells and the expression of the Green Fluorescent Protein (GFP) was observed. In order to activate the phagocytic activity of shrimp, 20, 40 and 80 µg/shrimp of this PAP-phMGFP vector were injected into Litopenaeus vannamei muscle. After challenged with WSSV, 40 µg/shrimp produced the highest relative percent survival (77.78 RPS). Analysis for the expression of the GFP gene in various tissues showed the expression mostly in the hemolymph of the immunized shrimp. The expression level of PAP and proPO (Prophenoloxidase) gene were highest at 7 days after immunization. This agreed with the efficiency of protection against WSSV that also occurred 7 days after immunization with the highest RPS of 86.61%. However there was no protection 30 days after immunization. Hemocytes of shrimp injected with PAP-phMGFP had 1.9 folds and 3 folds higher percentage phagocytosis and phagocytic index than the shrimp injected with PBS. Accordingly, copies of WSSV reduced in the PAP-phMGFP injected shrimp. In addition, PAP-phMGFP also protected shrimp against several pathogens: WSSV, YHV and V. harveyi, with RPS values of 86.61%, 63.34% and 50% respectively. This finding shows that the immune cellular defense mechanisms in shrimp against pathogens can be activated by injection of PAP-phMGFP and could indicate possible useful ways to begin to control this process.