Studies on Monoclonal Antibodies Characterization and immunohistochemical detection of Lactococcus garvieae

Abstract

Lactococcus garvieae, the aetiological agent of Lactococcosis, has recently been responsible for significant disease outbreaks in a variety of economically important freshwater and seawater fish species cultured worldwide. The development of immunological diagnostic tests to use in the control strategies against L. garvieae has been limited due to a complicated global distribution of serotypes. It appears there are serotypic differences between L. garvieae recovered from different regions, and although perhaps a common Antigen (Ag) may be expressed by all serotypes, it has not been found yet. The identification of this Ag would enable the development of specific Monoclonal Antibodies (MAbs), which could be used, in conjunction with immunological techniques, for the detection of all isolates of the pathogen in infected fish and water.

In this study, nine MAbs produced in the Aquatic Vaccine Unit of the Institute of Aquaculture (IoA), University of Stirling against L. garvieae type strain NCIMB 702928 (3 MAbs) and against a L. garvieae Japanese clinical isolate (12-99)(6 MAbs), as well as two more MAbs, kindly donated by Professor Tae Sung Jung (Laboratory of Fish and Shellfish Diseases, Gyeongsang National University, Republic of Korea) against L. garvieae Korean isolates, were screened, using Enzyme-linked immunosorbent assay (ELISA), against a collection of 12 L. garvieae isolates from Europe and Asia, and L. garvieae type strains. None of the MAbs in the study recognized all the L. garvieae isolates tested, although some of the Japanese MAbs (specifically MAbs 3 G9, 8B12, 11F8 and 11B1) recognized a higher number of isolates than the rest of MAbs (including all the type strains in the study and several Japanese and Italian isolates). Differences in the intensity of the reaction has led to the idea that the Ag can be expressed at two different levels by different isolates, or that perhaps there are two different Ags displaying similar epitopes that are recognized at two levels. European MAbs and the rest of the Japanese MAbs were very specific to certain strains, including two of the type strains and one Japanese isolate but they did not recognize any of the Italian isolates.

Korean MAbs did not recognize any isolate, and this led to think about a possible absence of MAb in the supernatant. However, none of the two L. garvieae Korean isolates were recognized by any MAb.

The MAbs were also tested for cross-reactivity using ELISA with a collection of 32 isolates from other bacterial species (including L. garvieae related and unrelated species). Low levels of cross reactivity (ranging from 0.3% to 9.6%) were detected, with the exception of a Korean MAb (U99-33) that showed a significant cross reactivity with Renibacterium salmoninarum.

An Immunohistochemistry (IHC) test was developed with the MAbs studied. A preliminary IHC test was carried out with all the MAbs, and the results obtained correlated with those from the ELISA assay. Based upon the results obtained and availability of supernatants, two MAbs, (Japan) 3G9 and (Euro) 13, were used in IHC to screen tissue samples from a L. garvieae challenge with isolate NCIMB 702928 in rainbow trout, carried out during the study. Both MAbs were able to detect specifically L. garvieae in infected tissue sections of various organs.

The pathology observed in challenged fish is described. Most prominent features on clinical examination were exophthalmos, generalized congestion and haemorrhage, hepatomegaly, splenomegaly and serositis, which sometimes extended to the myocardium. Histopathological features included several inflammatory and degenerative processes in various organs (eye, swimbladder, spleen, liver, kidney and heart).

In conclusion, the findings of the study suggest that our knowledge on serotypes and antigenic profiles of the bacteria worldwide needs to be expanded, in order to acquire a level of knowledge that will allow the development of MAbs that recognize all L.garvieae isolates worldwide. If these are developed, it will be possible to use them in IHC to detect the bacteria in infected fish tissue, and will help to differentiate lactococcosis from other streptococcal diseases.