Proteomic analysis of liver tissue reveals Aeromonas hydrophila infection mediated modulation of host metabolic pathways in Labeo rohita

Aeromonas hydrophila (Ah) is an opportunistic Gram-negative bacterium and a serious global pathogen causing Motile Aeromonas Septicaemia (MAS) in fish and many other vertebrates. The pathogenesis of aeromonas septicaemia is complex and involves multiple perturbed pathways. Molecular analysis of host tissues could be a powerful approach to identify mechanistic and diagnostic immune signatures of disease. We performed a deep proteomic analysis of Labeo rohita liver tissue to examine changes in the host proteome during Ah infection. A total of 2525 proteins were identified of which 158 were found differentially expressed during Ah infection. Functional analysis of significant proteins identified the dysregulation of several metabolic enzymes, antioxidative proteins, cytoskeletal proteins and immune related proteins. Proteomic analysis revealed the alterations in the cellular defence mechanisms including phagolysosomal killing and apoptosis during Ah infection. Our systemic approach revealed the protein dynamics in the host cells to explore the putative biological processes underlying the metabolic reprogramming of the host cells during Ah infection. Our findings paved the way for future research into the role of Toll-like receptors (Tlr3), C-type lectins (Clec4e) and metabolic enzymes in Ah pathogenesis leading towards host directed immunotherapies to tackle the Ah infection in fish.

Comparative Pathogenicity of Aeromonas spp. in Cultured Red Hybrid Tilapia (Oreochromis niloticus × O. mossambicus)

The genus Aeromonas has been recognised as an important pathogenic species in aquaculture that causes motile Aeromonas septicaemia (MAS) or less severe, chronic infections. This study compares the pathogenicity of the different Aeromonas spp. that were previously isolated from freshwater fish with signs of MAS. A total of 124 isolates of Aeromonas spp. were initially screened for the ability to grow on M9 agar with myo-inositol as a sole carbon source, which is a discriminatory phenotype for the hypervirulent A. hydrophila (vAh) pathotype. Subsequently, LD50 of six selected Aeromonas spp. were determined by intraperitoneal injection of bacterial suspension containing 103, 105, and 107 CFU/mL of the respective Aeromonas sp. to red hybrid tilapias. The kidneys, livers and spleens of infected moribund fish were examined for histopathological changes. The screening revealed that only A. dhakensis 1P11S3 was able to grow using myo-inositol as a sole carbon source, and no vAh strains were identified. The LD50–240h of A. dhakensis 1P11S3 was 107 CFU/mL, while the non-myo-inositol utilizing A. dhakensis 4PS2 and A. hydrophila 8TK3 was lower at 105 CFU/mL. Similarly, tilapia challenged with the myo-inositol A. dhakensis 1P11S3 showed significantly (p < 0.05) less severe signs, gross and histopathological lesions, and a lower mortality rate than the non-myo-inositol A. dhakensis 4PS2 and A. hydrophila 8TK3. These findings suggested that myo-inositol utilizing A. dhakensis 1P11S3 was not a hypervirulent Aeromonas sp. under current experimental disease challenge conditions, and that diverse Aeromonas spp. are of concern in aquaculture farmed freshwater fish. Therefore, future study is warranted on genomic level to further elucidate the influence of myo-inositol utilizing ability on the pathogenesis of Aeromonas spp., since this ability correlates with hypervirulence in A. hydrophila strains.

IMMUNODIAGNOSIS INFEKSI Aeromonas hydrophila PADA IKAN

Aeromonas hydrophila causes a disease that often infects fish and is known as Motile Aeromonas Septicaemia (MAS), Hemorrhagi Septisemia, Ulcer disease or Red-Sore disease. The aims of this study were to develop polyclonal antibody of Aeromonas hydrophila in the rabbits to confirm the diagnosis of Aeromonas hydrophila in the fish by immunohistochemistry staining method. Preparation of polyclonal antibodies was performed on the rabbits used to Aeromonas hydrophila bacteria that have been tested biochemically by intravenous and intraperitoneal injection. Doses of Aeromonas hydrophila bacteria were 109 CPU/ml of 0.5 ml at first injection, 1 ml at second injection, 2 ml at thirth injection and 3 ml at fourth injection. Blood serum collection was performed at week 5 after injection from an ear and intracardial vein. The result of antibody titer was 28 = 1024 which measured by tube test. Furthermore, polyclonal antibody was used to immunohistochemistry staining with 400x dilution. The results of the staining showed that an immunopositive reaction in the liver, skin,lien, gill, kidney, and heart of fish to Aeromonas hydrophila antibody. The research conclution was polyclonal antibody from rabbit can be used to accurately confirm the diagnosis of Aeromonas hydrophila based on antigen and antibody reaction.

Pengendalian Infeksi Bakteri Aeromonas hydrophila pada Ikan Nila (Oreochromis niloticus) dengan Ekstrak Rimpang Lengkuas Merah (Alpinia purpurata)

MAS diseases (Motile Aeromonas Septicaemia) is caused by the bacterium Aeromonas hydrophila. One of the natural ingredients that can be used as an alternative to inhibit bacterial activity is the extract of red galangal rhizome (Alpinia purpurata). Active substances possessed of this material have potential as antibacterial. The purpose of this study is to determine the effectiveness of red galangal rhizome extract in the control of MAS disease in tilapia. The type of this research is experimental research in Completely Random Design (RAL) with 6 treatments, which are positive control, negative control, 250, 500, 750, and 1000 ppm. The result of the research on the activity of the galangal extract bacteria on A. hydrophila obtained the widest clear zone at 1000 ppm constipation, which is 9.7625 mm. The treatment has been conducted by immersion of Tilapia fish into the extracted red galangan rgizomes for 5 minutes, then it was put into aquarium for maintenance for 7 days. 0.1 ml A. hydrophila (108 cfu/ml) was injected to Tilapia. The result of the research on antibacterial shoes the wides clear zone at 1000 ppm with constipation, which is 9.7625 mm and the effectiveness test of red galangal extract on tilapia infected by A. hydrophila bacteria experience (ulcer) healing after 7 days with the concentration of rhizome extract of galangal 1000 ppm. Key words: red galangal rhizome extract, A. hydrophila, A. purpurata.

The effect of vaccination in controling bacterial disease caused by Aeromonas hydrophila in nile tilapia (Oreochromis niloticus)

This study aims to apply vaccination against bacterial disease. The purpose of vaccination is to trigger the immune respone both non-specific and specific of fish against bacteria Motile Aeromonas Septicaemia (MAS) caused by Aeromonas hydrophila. The vaccination for fish with different ages: 2-3 weeks and 5 weeks were done using immersion method. The fish were re-vaccinated (booster) after two weeks of the first vaccination. The survival rate was < 50 % for juveniles 2-3 weeks and > 50% for juveniles 5 weeks. Survival rate for juveniles 5 weeks was higher than juveniles of 2-3 weeks. This shows that organs of juveniles of 5 weeks were more complete than the 2-3 weeks juveniles. The age of fish is one of the important factors for successfully vaccination. Penelitian ini bertujuan untuk melakukan penerapan vaksinasi terhadap penyakit bakterial. Vaksinasi ditujukan untuk merangsang respon kekebalan non- spesifik dan spesifik pada tubuh ikan terhadap penyakit Motile Aeromonas Septicaemia (MAS) yang disebabkan oleh bakteri Aeromonas hydrophila. Vaksin diberikan pada benih ikan nila yang berbeda umur yaitu 2-3 minggu dan 5 minggu dengan menggunakan metode perendaman. Ikan divaksinasi ulang (booster) setelah 2 (dua) minggu dari vaksinasi yang pertama. Prosentase kelangsungan hidup < 50 % untuk benih umur 2-3 minggu dan > 50% untuk benih umur 5 minggu. Jumlah kematian benih umur 2-3 minggu lebih tinggi dari 5 minggu. Hal ini menunjukkan bahwa fungsi organ benih umur 5 minggu telah lebih lengkap dari pada benih umur 2-3 minggu. Umur ikan merupakan salah satu faktor penting penentu keberhasilan suatu kegiatan vaksinasi.

DETECTION OF AEROLYSIN (aerA) GENE IN AEROMONAS HYDROPHILA STRAINS ISOLATED FROM DISEASED CARP

Bacterial septicemia caused by motile aeromonads is common infection in the intensive fish production. Aeromonas (A.) hydrophila is often present in fish populations. Ubiquitous distribution of these bacteria in the aquatic environment, and the stress caused by intensive breeding are predisposing factors for the occurence of the disease. A. hydrophila is considered a major cause of septicaemia caused by motile aeromonads. Several A. hydrophila extracellular products (ECP) are considered as important factors in pathogenesis, primarily aerolysin (aerA), the extracellular lipase, cytolytic enterotoxin, hemolytic toxin and extracellular proteases. PCR detection of aerolysin (aerA) is considered a reliable method of identifying potentially pathogenic Aeromonas strains. In spring 2012, after a sudden increase in water temperature, disease occured in common carp population in one fish farm in Serbia. Five specimens of the one-year-old carp with clinical symptoms of motile aeromonas septicaemia were used for isolation of the bacteria. Identification of A. hydrophila was done on the basis of morphological, physiological, cultural and biochemical characteristics. PCR amplification of DNA from A. hydrophila isolates revealed presence of aerolysin (aerA) gene in all examined A. hydrophila isolates from carp with motile aeromonas septicaemia.