Role of apoptosis in pathogenesis of infectious pancreatic necrosis in rainbow trout(Oncorhynchus mykiss Walbaum, 1792)

Abstract

In this study, total of 12 rainbow trout (average weight 5 g) from a fish farm were examined. Clinically, the fish showed inappetite and irregular swimming. In histopathological examination, focal to diffuse acinar cell degeneration and necrosis were observed in pancreas. Immunohistochemically, IPN viral antigens appeared especially in the pancreas, liver, cardiac and skeletal muscles, and occasionally in the kidneys, spleen, stomach, intestines and gills. In Reverse transcriptase-polymerase chain reaction (RT- PCR) examination, appropriately sized product (206 bp) were amplified from tissue samples and specific signals for IPN virus were observed. Apoptosis signals TUNEL (Terminal Transferase dUTP Nick End Labeling), caspases-3, -8 and -9 positivities) indicated that apoptosis was induced both by intrinsic and extrinsic pathways in the skeleton and cardiac muscles, whereas by only intrinsic pathway in the liver, spleen, kidneys, gills, stomach and intestines. However, signals of apoptotic cell death were not clearly detected in pathogenesis of IPN in the pancreas as the main target organ. In conclusion, our results suggest that apoptosis, a cell death mechanism, could be appeared in favor of IPN virus in pathogenesis of the disease in field conditions

Keywords

• Apoptosis
• IPN
• immunohistochemistry
• rainbow trout
• RT-PCR
• TUNEL

Gökkuşağı alabalıklarında (Oncorhynchus mykiss Walbaum, 1792) enfeksiyöz pankreatik nekrozun patogenezisinde apoptozisin rolü

Abstract

Çalışmada, bir balık işletmesinde iştahsızlık ve yüzme bozuklukları gösteren, ortalama 5 g ağırlığında, 12 adet gökkuşağı alabalığı incelendi. Histopatolojik incelemede, pankreasta ekzokrin bezlerin ve Langerhans adacıklarının fokal ya da diffuz nekrozları görüldü. İmmunohistokimyasal incelemede; infeksiyöz pankreatik nekroz (IPN) viral antijenleri, yoğun olarak pankreas, karaciğer, kalp ve iskelet kasları daha az olarak da, böbrek, dalak, mide, bağırsaklar ve solungaçlarda ortaya konuldu. Ters transkriptaz polimeraz zincir reaksiyonu (RT- PCR) sonucunda; IPN virüsünü işaret eden yaklaşık 206 bp aralığında görüntü elde edildi. Apoptozis göstergelerinden TUNEL (Terminal Transferase dUTP Nick End Labeling), kaspaz-3, -8 ve -9 pozitiflikleri; apoptozisin iskelet ve kalp kasında içsel ve dışsal uyarımlarla, karaciğer, dalak, böbrekler, sekonder lameller, mide, pilorik keseler ve bağırsaklarda içsel yol ile indüklendiğini göstermektedir. Buna karşın, IPN virüsü için primer hedef organ olan pankreasta apoptotik hücre ölümü, hastalığın patogenezisinde çok etkin değildi. Sonuçta; çalışma bulguları IPN’un patogenezisinde apoptotik hücre ölüm mekanizmasının virüsün lehine olduğunu göstermektedir

Keywords

• Apoptozis
• gökkuşağı alabalığı (Oncorhynchus mykiss)
• IPN
• immunohistokimya
• RT-PCR

References

1. Alnemri ES (1997): Mammalian cell death proteases: a family of highly conserved aspartate specific cysteine proteases. J Cell Biochem, 64, 33–42.
2. Ashkenazi A, Dixit VM (1998): Death receptors: signaling and modulation. Science, 281, 1305–1308.
3. Benedict CA, Norris PS, Ware CF (2002): To kill or be killed: viral evasion of apoptosis. Nat Immunol, 3, 1013– 1018.
4. Candan A (2002): First report on the diagnosis of infectious pancreatic necrosis (IPN) based on reverse transcription polymerase chain reaction (RT-PCR) in Turkey. Bull Eur Assoc Fish Pathol, 22, 45-47.
5. Chomyznki, P., Sacchi, N. (1987). Single–step method of RNA isolation by acid guanidinium thiocyanate-phenol- chloroform extraction. Anal Biochem, 162, 156-159.
6. Cohen GM (1997): Caspases: the executioners of apoptosis. Biochem J, 326, 1–16.
7. Dockrell DH (2001): Apoptotic cell death in the pathogenesis of infectious diseases. J Infect, 42, 227–234.
8. Eleouet JF, Druesne N, Chilmonczyk S, Monge D, Dorson M, Delmas B (2001): Comparative study of in-situ cell death induced by the viruses of viral haemorrhagic septicaemia (VHS) and infectious pancreatic necrosis (IPN) in rainbow trout. J Comp Path, 124, 300–307.

9. Erer H (2002): Balık Hastalıkları.Selçuk Üniversitesi Basımevi, Konya.
10. Everett H, McFadden G (1999): Apoptosis: an innate immune response to virus infection. Trends Microbiol, 7, 160–165.
11. Hay S, Kannourakis G (2002): A time to kill: viral manipulation of the cell death program. J Gen Virol, 83, 1547–1564.
12. Hong JR, Lin TL, Hsu YL, Wu JL (1998): Apoptosis precedes necrosis of fish cell line with infectious pancreatic necrosis virus infection. Virology, 250, 76–84.
13. Hong JR, Hsu YL, Wu JL (1999): Infectious pancreatic necrosis virus induces apoptosis due to down-regulation of survival factor MCL-1 protein expression in a fish cell line. Virus Res, 63, 75–83.
14. Hong JR, Lin TL, Yang JY, Hsu YL, Wu JL (1999): Dynamics of nontypical apoptotic morphological changes visualized by green fluorescent protein in living cells with infectious pancreatic necrosis virus infection. J Virol, 73, 5056–5063.
15. Huppertz B, Frank HG, Kaufmann P (1999): The apoptosis cascademorphological and immunohistochemical methods for its visualization. Anat Embryol (Berl), 200, 1– 18.
16. O’Brien V (1998): Viruses and apoptosis. Gen Virol, 79, 1833–1845.
17. Reed, J. C. (2000). Mechanisms of apoptosis. Am J
18. Pathol, 157, 1415-1430.
19. Roberts RJ (2001): Fish Pathology. 3 rd ed., W. B. Saunders, Toronto.
20. Roulston A, Marcellus RC, Branton PE (1999): Viruses and apoptosis. Annu Rev of Microbiol, 53, 577–628.
21. Santi N, Song H, Vakharia VN, Evensen O (2005): Infectious pancreatic necrosis virus VP5 is dispensable for virulence and persistence. J Virol, 79, 14, 9206-9216.
22. Smail DA, Bain N, Bruno DW, King JA, Thompson F, Pendrey DJ, Morrice S, Cunningham CO (2006): Infectious pancreatic necrosis virus in Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland: virus identification, histopathology, immunohistochemistry and genetic comparison with Scottish mainland isolates. J Fish Dis, 29, 31-41.
23. Sweeney A, Blake S, Singer JT, Nicholson BL (1997): Detection and identification of infectious pancreatic necrosis virus (IPNV) and infectious hematopoietic necrosis virus (IHNV) by polymerase chain reaction (PCR), p. 42–47. In Inui Y, Winton J (ed.), New Approaches to Viral Disease of Aquatic Animals. National Research Institute of Aquaculture, Nansei, Japan.
24. Williams K, Blake S, Sweeney A, Singer JT, Niholson BL (1999): Multiplex reverse transcriptase PCR Assay for simultaneous detection of three fish viruses. J Clin Microbiol, 4139–4141.