Innate immune response of Indian major carp, Labeo rohita infected with oomycete pathogen Aphanomyces invadans

Yadav, Manoj Kumar; Pradhan, Pravata Kumar; Sood, Neeraj; Chaudhary, Dharmendra Kumar; Verma, Dev Kumar; Debnath, Chandan; Sahoo, Lakshman; Chauhan, U.K.; Punia, Peyush; Jena, J. K.

doi:10.1016/j.fsi.2014.06.005

Cited by 25 publications

(15 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Samples were then centrifuged to remove the precipitates. The absorbance (optical density; OD) of the supernatant was measured at 280 nm and anti-protease activity was calculated [ 17 , 25 ]: Reference value (RV) = Absorbance of the positive control − Absorbance of the negative control Test value (TV) = Absorbance of the test sample − Absorbance of the serum blank Percent inhibition = ((RV − TV)/RV) × 100.…”

Section: Methodsmentioning

confidence: 99%

“…Extracellular proteases produced by the oomycete A. invadans have been suggested to play a part in EUS pathogenesis. Proteases have also been reported to affect the immune response of the host and support the invasion of the hyphae in host musculature [ 17 ]. In this study, we aimed at identifying A. invadans ’ secreted proteases using SDS-PAGE and mass spectrometery and investigating their proteolytic activity against casein and fish immunoglobulin M (IgM).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

In vitro investigations on extracellular proteins secreted by Aphanomyces invadans, the causative agent of epizootic ulcerative syndrome

et al. 2017

View full text Add to dashboard Cite

BackgroundProteases produced by many microorganisms, including oomycetes, are crucial for their growth and development. They may also play a critical role in disease manifestation. Epizootic ulcerative syndrome is one of the most destructive fish diseases known. It is caused by the oomycete Aphanomyces invadans and leads to mass mortalities of cultured and wild fish in many countries. The areas of concern are Australia, China, Japan, South and Southeast Asian countries and the USA. Extracellular proteases produced by this oomycete are believed to trigger EUS pathogenesis in fish. To address this activity, we collected the extracellular products (ECP) of A. invadans and identified the secreted proteins using SDS-PAGE and mass spectrometery. A. invadans was cultivated in liquid Glucose-Peptone-Yeats media. The culture media was ultra-filtered through 10 kDa filters and analysed using SDS-PAGE. Three prominent protein bands from the SDS gel were excised and identified by mass spectrometery. Furthermore, we assessed their proteolytic effect on casein and immunoglobulin M (IgM) of rainbow trout (Oncorhynchus mykiss) and giant gourami (Osphronemus goramy). Antiprotease activity of the fish serum was also investigated.ResultsBLASTp analysis revealed that the prominent secreted proteins were proteases, mainly of the serine and cysteine types. Proteins containing fascin-like domain and bromodomain were also identified. We could demonstrate that the secreted proteases showed proteolytic activity against the casein and the IgM of both fish species. The anti-protease activity experiment showed that the percent inhibition of the common carp serum was 94.2% while that of rainbow trout and giant gourami serum was 7.7 and 12.9%, respectively.ConclusionsThe identified proteases, especially serine proteases, could be the potential virulence factors in A. invadans and, hence, are candidates for further functional and host–pathogen interaction studies. The role of identified structural proteins in A. invadans also needs to be investigated further.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

In vitro investigations on extracellular proteins secreted by Aphanomyces invadans, the causative agent of epizootic ulcerative syndrome

et al. 2017

View full text Add to dashboard Cite

show abstract

“…EUS is a seasonal epizootic condition caused by a fungal pathogen called Aphanomyces invadans listed in OIE (Office International des Epizooties) Aquatic Code as a notifiable pathogen (Yadav et al, 2014;Huchzermeyer et al, 2017). A. invadans is detected by doing histological analysis of infected fish samples to demonstrate presence of mycotic granulomas (Iberahim et al, 2018;Kar, 2016;OIE, 2018).…”

Section: Introductionmentioning

confidence: 99%

Active surveillance of infection caused by Aphanomyces invadans in Malawi

Kaphuka¹,

Njunga²,

Kamwendo³

et al. 2020

Int. J. Fish. Aquac.

View full text Add to dashboard Cite

In Malawi, fish is regarded as a cheapest source of animal proteins and other macronutrients. Recent epizootic ulcerative syndrome (EUS) outbreaks reported in countries sharing Chobe-Zambezi river system like Zambia, Democratic Republic of Congo, Botswana, Namibia, Zimbabwe and Republic of South Africa, have posed a major threat to fish production. Malawi's biggest and important Shire River connects to Zambezi River and there is sharing of waters with Zambia during floods in some areas in north western Malawi. Active surveillance in Malawi was, therefore, conducted in four high risk areas to establish the presence or absence of EUS. Fish were inspected for EUS-like lesions by a trained surveillance team. No fish was found with EUS-like lesions. However, one Barbus paludinosus from Vwaza Marsh had a reddish and swollen caudal peduncle which after doing laboratory tissue squash did not show any evidence of fungal hyphae to suspect EUS but numerous inflammatory cells were seen.

show abstract

“…Although a large amount of data on the gross and microscopic clinical lesions of EUS and the involvement of different pathogens during the course of the disease already exist [ 9 , 16 – 18 ], there still is a paucity of information regarding pathogenicity, host-parasite interactions, molecular mechanisms of the disease and the functional genomics of A. invadans . Similar to many pathogenic microorganisms, proteases secreted by A. invadans are key in the pathogenesis of EUS [ 19 ]. Different extracellular proteins, most of which are proteases, secreted by A. invadans , have recently been identified and at least one of these proteases (serine protease belonging to peptidase_S8 domain) is believed to be involved in the degradation of the fish immunoglobulin M [ 20 ].…”

Section: Introductionmentioning

confidence: 99%

Editing the genome of Aphanomyces invadans using CRISPR/Cas9

et al. 2018

View full text Add to dashboard Cite

BackgroundThe clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is increasingly being used for genome editing experiments. It is a system to add, delete and/or replace parts of a gene in situ in a time- and cost-efficient manner. The genome of many organisms has been edited using this system. We tested the CRISPR/Cas9 system in Aphanomyces invadans, an oomycete, which is the causative agent of epizootic ulcerative syndrome (EUS) in many fish species. Extracellular proteases produced by this oomycete are believed to play a role in EUS virulence.MethodsWe designed three single guide-RNAs (gRNA) to target A. invadans serine protease gene. These gRNAs were individually combined with the Cas9 to form ribo-nucleo-protein (RNP) complex. A. invadans protoplasts were then transfected with RNP complexes. After the transfection, the target gene was amplified and subjected to sequencing. Zoospores of A. invadans were also transfected with the RNP complex. Three groups of dwarf gourami (Trichogaster lalius) were then experimentally inoculated with (i) non-treated A. invadans zoospores; (ii) RNP-treated A. invadans zoospores; and (iii) autoclaved pond water as negative control, to investigate the effect of edited serine protease gene on the virulence of A. invadans in vivo.ResultsFluorescence microscopy showed sub-cellular localization of RNP complex in A. invadans protoplasts and zoospores. Sequencing results from the protoplast DNA revealed a point mutation in the target gene. A matching mutation was also detected in zoospores after similar treatment with the same RNP complex. In vivo results showed that the CRISPR/Cas9-treated A. invadans zoospores did not produce EUS clinical signs in the fish. These results were then confirmed by histopathological staining of the muscle sections using Gomori’s methenamine silver nitrate and hematoxylin and eosin stains.ConclusionsResults obtained in this study indicate that the RNP complex caused effective mutation in the target gene. This hindered the production of serine protease, which ultimately impeded the manifestation of EUS in the fish. Our methods thus establish a promising approach for functional genomics studies in A. invadans and provide novel avenues to develop effective strategies to control this pathogen.

show abstract

Innate immune response of Indian major carp, Labeo rohita infected with oomycete pathogen Aphanomyces invadans

Cited by 25 publications

References 36 publications

In vitro investigations on extracellular proteins secreted by Aphanomyces invadans, the causative agent of epizootic ulcerative syndrome

In vitro investigations on extracellular proteins secreted by Aphanomyces invadans, the causative agent of epizootic ulcerative syndrome

Active surveillance of infection caused by Aphanomyces invadans in Malawi

Editing the genome of Aphanomyces invadans using CRISPR/Cas9

Contact Info

Product

Resources

About