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Full Research Article

Pathogenesis and Immune Responses of Labeo rohita under Experimental Co-infection with Flavobacterium columnare and Edwardsiella tarda

G
Ganesh Vijay Sundar Deva1
M
Megha K. Bedekar1,*
K
K.V. Rajendran1
R
R.P. Raman1
S
Subodh Gupta1
K
Kundan Kumar1
P
Pooja Vinde1
T
Tapas Paul2
B
B. Naveen Rajeshwar3
1ICAR-Central Institute of Fisheries Education, Mumbai-400 061, Maharashtra, India.
2College of Fisheries, Bihar Animal Sciences University, Kishanganj-855 115, Bihar, India.
3Nitte (Deemed to be University), NITTE-GOK COE | Aquamarin, Nitte University Centre for Science Education and Research (NUCSER), Deralakatte, Mangaluru-575 018, Karnataka, India.

ABSTRACT

Background: With the increase in intensification of aquaculture practices, co-infection of a variety of heterogeneous micro-organisms is frequent on commercially important fish species. Although there is rich documented literature on single pathogen infection and characterization, the mechanism of co-infection is still poorly understood in fish. In this prelude, the present study aimed to evaluate the immune response of Labeo rohita against the co-infection of Flavobacterium columnare and Edwardsiella tarda.

Methods: The experiment was conducted in triplicate as three groups viz, 1st group challenged by intraperitoneal injection of E. tarda (T1 and T2); 2nd group challenged by immersion of F. columnare (T3 and T4); 3rd group challenged by both injection and immersion of E. tarda and F. columnare (T5, T6 and T7) and control group without bacterial challenge. To compare the efficacy of different doses, three fish from each group were sacrificed on pre-challenge and 3, 6, 12, 24, 48 and 96 h post-challenge and gills, kidney, liver and spleen were collected from each fish at each time point to analyse the non-specific immune response.

Result: The results showed T6 group which received a combined infection of E. tarda and F. columnare induced the highest mortality followed by T7 and T1. The results further confirmed by immune assays and histopathological changes. The present study provides baseline information on the co-infection of F. columnare and E. tarda and their modulation of the non-specific innate immune response in L. rohita.

INTRODUCTION

Indian aquaculture has significantly flourished over the decades providing food and nutritional security to millions of people (Biswal et al., 2021a; Dheeran et al., 2025; Raghuvaran et al., 2025). Indian major carps (IMC) are the mainstay of freshwater aquaculture, accounting for more than 85% of total production. Among IMC, Labeo rohita is the most cultured fish due to its wide geographical distribution, suitability to different culture systems and preference as a food fish (Biswal et al., 2021b). However, the intensification of aquaculture practices to meet food demands results in the transmission of infectious diseases (Dhayanath et al., 2019). Pathogenic organisms like bacteria, virus, fungi and parasites cause serious infectious diseases in aquatic animals causing severe economic loss and mortality (Leung and Bates, 2013). Similarly, L. rohita culture system is subjected to different emerging pathogens having zoonotic potential causing serious concerns for aquatic and human health. The use of vaccines for aquatic diseases reduced the dependency on antibiotics. However, the lack of knowledge on fish immune systems hinders the discovery of novel vaccines against emerging diseases.
       
Among bacterial pathogens, the gram-negative bacteria Edwardsiella tarda and Flavobacterium columnare causes systemic infections in several fish species, including carp, tilapia, yellowtail, eel, flounder and turbot. Edwardsiellosis, a generalized septicaemia observed in fish infected with E. tarda show symptoms of macroscopic lesions of internal organs, distended abdomen and prolapsed rectum (Bera et al., 2020). Earlier, Hoshina (1962) reported E. tarda for the first time as Paracolobactrum anguiillimortiferum from Anguilla japonica (Japanese eel). Later, it is reported from wide range of commercially important fishes such as Ictalurus punctatus, Anguilla japonica, Pagrus major, Salvelinus fontinalis, Oncorhynchus tshawytscha, Paralichthys olivaceus, Scophthalmus maximus, Anabas testudineus, Clarias batrachus, Opsanus tau and Oreochromis niloticus (Nougayrede et al., 1994; Sahoo et al., 1998; Sahoo et al., 2000; Clavijo et al., 2002; Horenstein et al., 2004). Columnaris diseases caused by F. columnare reported of high mortality and economic loss up to 30 million dollars in commercially cultured fish species (Hawke and Thune, 1992; Wagner et al., 2002; Shoemaker and LaFrentz, 2015). Moreover, F. columnare are reported to present in freshwater fish microbiota, fish eggs and aquatic environments (Barker et al., 1991; Barnes et al., 2009).
       
In the aquatic environment, a number of heterogeneous organisms co-exist, survive and cause co-infection in fish and shrimps. Co-infections refer to the simultaneous presence of two or more genetically diverse pathogens within a host, each exerting pathogenic effects that together adversely impact the host (Bakaletz, 2004). A single pathogen has the ability to alter the host’s immune response against subsequent infections caused by additional pathogens (Lello et al., 2004; Telfer et al., 2008). This can alter host vulnerability to infection and influence host-pathogen interactions, infection biology, infection duration, disease severity and host pathology (Graham et al., 2007; Telfer et al., 2008). There were several reports of co-infection of Edwardsiella spp. and Flavobacterium columnare (Panangala et al., 2006; Dong et al., 2015; Wise et al., 2021). Although there is a rich body of literature on single-pathogen infection and characterisation, the mechanisms of co-infection are still poorly understood in fish. Hence, it is necessary to investigate interactions between two pathogens during mixed infections in farmed fish species (Johnson and Hoverman, 2012). In light of this, the present study was conducted to evaluate the co-infection model of Flavobacterium columnare and Edwardseilla tarda in Labeo rohita and to examine the host’s immunological response to the co-infection of F. columnare and E. tarda.

MATERIALS AND METHODS

Healthy L. rohita (Rohu) advanced fingerlings were obtained from a Pen fish farm, Raigad District, Maharashtra, India. The fishes (20±6.6 g) were brought to Aquatic Animal Health Management Laboratory (AAHM), ICAR-Central Institute of Fisheries Education (CIFE), Mumbai, India and treated with potassium permanganate (KMnO4) at 5 ppm. The fishes were reared in a controlled lab conditions for a month in Fibre Reinforced Plastic (FRP) tanks (5000 L capacity) with continuous aeration. The experiment was split into three groups (only E. tarda challenge, only F. columnare challenge, co-infection with E. tarda and F. columnare) and conducted in triplicate with 24 fish per tank (n=24). The experiment was conducted in FRP tanks of 500 L capacity containing filtered freshwater under controlled laboratory conditions (pH 8.0; temperature 28°C). The experimental fishes were fed to satiation with 0.5 mm commercial carp feed pellets (CP Aquafeed, India) at 2-3% body weight throughout the experiment.
       
E. tarda ATCC® 15947 procured from Himedia, India was used in this study. The strain was confirmed using Gram staining and standard biochemical tests prior to the experiment. The bacterial culture was then revived using Brain Heart Infusion broth (BHI) and pure colonies were obtained on SS agar (Salmonella-Shigella Agar). The E. tarda (0.1 mL) was then injected into a naive L. rohita fish. After 24 h of incubation, fish were sacrificed and bacteria were reisolated from fish kidneys on BHI and SS agar plate and incubated at 28°C for 24 h. This bacterial culture was used for the median lethal dose (LD50) estimation study. Following LD50 estimation, serial dilutions (1:10) of the bacterial culture were then prepared and 0.1 mL from 10-6, 10-7 and 10-8 dilution tubes was spread on BHI agar plates and incubated at 28°C for 18-24 h to determine the viable bacterial count (CFU/mL) of the inoculum.
       
The gills of diseased L. rohita was subjected to F. columnare isolation in AAHM laboratory. The isolated strain was cultured in modified TYES broth incubated at 28°C in a shaking incubator at 100 rpm for 48 hours. Pure colonies were obtained by streaking on TYES agar plate incubated for 48 hours at 28°C. Similar to E. tarda, the viable bacterial count (CFU/mL) of the inoculum was estimated by serial dilutions (1:10) of the bacterial culture. The bacterial culture of 0.1 mL from 10-5, 10-6 and 10-7 dilution tubes were taken and spread on TYES agar plates and incubated at 28°C for 48 h.
       
100 µL of varying concentrations of E. tarda ranging from 1×104 to 1×108 CFU/mL were challenged intra-peritoneally into individual fish of 5 groups while 1 group of fish challenged with PBS (n=6 per group). Mortality was observed 12 hours post-challenge and subsequently, using Reed and Muench (1938) techniques, LD50 value was calculated. However, the cumulative Mortality is monitored for up to 96 hours. Similarly, fish were challenged with varying concentrations of F. columnare (1×102 to 1×106 CFU/mL) by immersion method (n=6 per group) to calculate LD50 based on mortality at 12 hours post-challenge.
       
The challenge experiment was conducted in three groups. The 1st group of rohu fishes were challenged with intraperitoneal injection of E. tarda (T1-7.2×107 CFU/Fish and T2- 7.2×106 CFU/Fish) while the 2nd group challenged with immersion of F. columnare (T3-5.1×105CFU/mL and T4-5.1×106 CFU/mL). The E. tarda bacterial suspension was adjusted to the required concentration (CFU/mL) using serial dilution and plate count. Each fish received 100 µL of inoculum and the final dose (CFU/fish) was calculated based on the injected volume and bacterial concentration. Further, the 3rd group challenged with both E. tarda and F. columnare (T5, T6 and T7) by injection and immersion respectively. Group T5 represents simultaneous co-infection, while T6 and T7 represent sequential infections with defined time intervals between challenges. A negative control group without bacterial challenge was also maintained with challenged groups. To compare the efficacy of different doses, three fish from each group were sacrificed on 0 (pre-challenge), 3, 6, 12, 24, 48, 96 h post-challenge and gills, kidney, liver and spleen were dissected at each time point. Mortality and morbidity symptoms were observed for 10 days after the challenge.
       
The co-infection of edwardsiellosis and columnaris disease development were tested in L. rohita fingerlings by intra peritoneal injection method and bath immersion methods. Twenty-four fish (n=24) were challenged with E. tarda at a dose of 7.2×107 CFU/Fish and 7.2×106 CFU/Fish by intraperitoneal injection (I/P). Following the E. tarda infection, the fishes were given bath immersion of F. columnare at a dose of 5.1×105 CFU/mL and 5.1× 106 CFU/mL. During bath treatment, fishes were exposed to 5 L of water for 2 h and then transferred to an experimental tank for observation of mortality for 10 days.
       
The nitrobluetetrazolium (NBT) assay was performed following the procedure established by Secombes (1990), as adapted by Stasiak and Baumann (1996). The activity of myeloperoxidase (MPO) was determined according to the standard technique established by Quade and Roth (1997), with partial modifications by Sahoo et al. (2005). Serum protein was quantified using the Biuret method (Reinhold, 1953) with the InnolineTM total protein plus kit. Immunoglobulin from plasma was separated by precipitation with polyethylene glycol following Anderson and Siwicki’s (1995) method with minor alterations.
       
Histopathology was performed using the Pirarat et al., (2006) methodology. Briefly, kidney and liver tissues of the fish from different groups that had survived bacterial challenge were dissected out and fixed in 10% neutral buffered formalin. After processing, the fixed tissues underwent sectioning (5 mm) and haematoxylin and eosin staining. After staining, the sections were observed under a microscope.
       
The software SPSS v 16.0 (IBM®, New York) was used to perform the statistical analysis. The statistical significance between treatment groups was determined at the 5% probability levels utilizing One-way ANOVA and the values were presented as mean ± standard error.

RESULTS AND DISCUSSION

Bacterial isolation and identification
 
The procured E. tarda (ATCC® 15947) challenged in L. rohita was then re-isolated from challenged fish kidney using BHI broth and SS agar media (Fig 1). E. tarda strain was confirmed by biochemical characterisation (Table 1).

Fig 1: Re-isolation of E. tarda from dead fish on SS agar.



Table 1: Biochemical confirmation of Edwardsiella tarda ATCC® 15947.


       
F. columnare isolated from diseased L. rohita gills and grown in modified TYES broth and then confirmed by using five Griffin tests, which differentiate it from other gram-negative flexing rods.
 
Confirmation of F. columnare by species-specific PCR
 
PCR amplification with F. columnare specific primers (COL-F and COL-R) consistently produced the expected 675 bp amplicon in all tested isolates (Fig 2), confirming species-specific detection.

Fig 2: Agarose gel electrophoresis (2%) showing species-specific PCR amplification of a 675 bp fragment from F. columnare isolates obtained from L. rohita.


 
Clinical signs in experimentally infected fish
 
In the present study, healthy L. rohita fingerlings were exposed to E. tarda and F. columnare by intra-peritoneal injection and immersion methods, respectively. Experimentally challenged fish exhibit marked clinical signs upon disease caused by both E. tarda and F. columnare infection. E. tarda infected fish showed abnormal swimming, dark skins and fins, redness on the ventral body, ascitic fluid in the pectoral cavity, haemorrhages and skin lesions (Fig 3 and 4). Meanwhile, F. columnare infected fish exhibit whitish-yellow pigments in the ventral part of the gills arch, eroded skins, ulcers on the surface of the body and pale discoloration on the skin. In this study, L. rohita challenged with E. tarda observed to have excess mucus secretion with gross haemorrhagic lesions on the ventral side of the body. In moribund and dead fish, the anus were protruded, haemorrhagic and swollen. These clinical signs align with earlier report on Clarias batrachus (Sahoo et al., 1998) and Anabas testudineus (Sahoo et al., 2000) infected with E. tarda. Co-infection groups showed faster and significantly higher mortality compared to fish challenged with individual bacterium in 7 days. The present results were corroborated with Dong et al., (2015), who reported significantly higher cumulative mortality in Pangasianodon hypophthalmus under co-infection of E. ictaluri and F. columnare. Further, the clinical signs of both diseases were observed in co-infected fish group. Similarly, Crumlish et al., (2010) determined that co-infection challenge of P. hypophthalmus with A. hydrophila and E. ictaluri by immersion caused higher cumulative mortalities (95%) compared to fish exposed to single infection.

Fig 3: Clinical signs in L. rohita infected with E. tarda a) skin lesions b) ascites and internal haemorrhage.



Fig 4: Clinical signs in L. rohita co-infected with E. tarda and F. columnare.


 
Pathogenicity of E. tarda and F. columnare
 
In a single infection of E. tarda, T1 and T2 groups having a bacterial dose of 7.2×107 and 7.2×105 CFU/Fish respectively were injected by an intraperitoneal method. After 7 days, T1 and T2 groups showed 60% and 50% cumulative mortality respectively. Similarly, a single infection of F. columnare in two groups T3 (5.1×106 CFU/mL) and T4 (5.1×104 CFU/mL) results in cumulative mortality of 51.4% and 47.7% respectively in L. rohita.
 
Co-infection of E. tarda and F. columnare
 
In the combinatorial infection of E. tarda and F. columnare, T5 group (7.2×105 CFU/Fish and 5.1×104 CFU/mL) and T6 group (7.2×107 CFU/Fish and 5.1×104 CFU/mL) dose were given by intraperitoneal injection and immersion methods respectively. Cumulative mortality of the T5 and T6 groups after 7 days was recorded to be 54.4% and 74.8% respectively. Further, the T7 group of bacterial dose 7.2×105 CFU/Fish and 5.1×106 CFU/mL was challenged by the bath immersion method and 60% cumulative mortality was noticed in 10 days experimental period (Fig 5).

Fig 5: Cumulative mortality (%) of L. rohita fingerlings following single and combined infections with E. tarda and F. columnare over a 7-day post-challenge period.



Non-specific immune response towards a single and combined infection
 
Total protein
 
In a single infection of E. tarda and F. columnare, there is no significant difference in total protein value within T1, T3 and T4 treatment groups at different time periods. However, total protein value was substantially (p<0.05) higher in the pre-challenged group in comparison to other time periods in T2 group. In the co-infection treatment group T5, a time-dependent decrease in total protein was observed during the experimental periods. However, T6 showed no significant difference between time periods and T7 showed a lower total protein value in 96 h (Fig 6). Total protein was found to be reduced significantly in co-infection treatment group in comparison to either single infected group and similar results were reported in E. tarda infected Nile tilapia (Benli and Yildiz, 2004). Similarly, a reduction in total protein concentration was noticed in co-infection of spring viremia and Pseudomonas fluorescens in common carp (Rehulka, 1996; Yildiz, 1998). Higher albumin and globulin level in treatment groups may be due to liver and blood cells destruction with protein synthesis dysfunction (Paul et al., 2019, 2020; Bera et al., 2020).

Fig 6: Total serum protein levels in L. rohita following a) single bacterial infection challenged with E. tarda (ET; T1 and T2) and F. columnare (FC; T3 and T4) b) co-infection with E. tarda and F. columnare (EF; T5-T7).


 
Myeloperoxidase
 
Myeloperoxidase (MPO) activity in L. rohita increased in a time-dependent manner following both single infection and co-infection of pathogens. A significantly (p<0.05) different MPO activity was observed in the T1 group at different time periods, with the highest elevation in 48 h due to a single infection of E. tarda (Fig 7). MPO activity decreased significantly (p<0.05) in the co-infection treatment group compared to pre-challenged group. This might be attributed to the dysfunctioning of phagocytic enzymes and a reduction in the number of phagocytic cells due to invading pathogens. Shameena et al. (2021) reported significant alterations of MPO and RBA on Carassius auratus due to co-infection of Argulus and A. hydrophila under graded temperature corroborates with the results of the present study. Further, the results confirms that the bacterial enzymes such as superoxide dismutase, peroxidase and catalase might be responsible for free radicals detoxification by host phagocytes (Han et al., 2006; Mohanty and Sahoo, 2007).

Fig 7: Myeloperoxidase (MPO) activity (%) in L. rohita following a) Single bacterial infection challenged with E. tarda (ET; T1 and T2) and F. columnare (FC; T3 and T4) b) co-infection with E. tarda and F. columnare (EF; T5-T7).


 
Respiratory burst activity (RBA)
 
In the present study, T1 group exhibited a differential RBA pattern with higher values in 48 h and moderately decreased in 24 h and 96 h. Similarly, T2 showed the highest activity in 48 h followed by 24 h in comparison to other time periods. Interestingly, RBA was significantly (p <0.05) higher in 24 h and reduced during 48 h in the T3 and T4 treatment groups (Fig 8). In T5 group, there is a significant difference in RBA activity in early hours post-challenge. However, in T6 and T7, a significantly higher RBA was observed in 48 h followed by a decreasing trend in 96 h. Fish phagocytes after activation are able to produce superoxide anion (O2-) and its derivatives while intense oxygen consumption which is called as respiratory burst (Secombes and Fletcher, 1992). The MPO activity and respiratory burst were oxygen-dependent reactions commonly used to understand the host’s defence against pathogens (Sharp and Secombes, 1993). The increase in phagocytes killing pathogens are evidently correlated with increased respiratory burst activity. In this study, a time-dependent increased RBA activity was observed in both co-infected and individual treatment groups. Higher respiratory burst activity could be linked with increased host cellular immune response to the bacterial pathogen infection.

Fig 8: Respiratory burst activity (RBA) in L. rohita following a) Single infection with E. tarda (ET; T1-T2) and F. columnare (FC; T3-T4) at different time points (0-96 h). b) Co-infection with E. tarda and F. columnare (EF; T5-T7).


 
Histopathology
 
Histopathological examination of tissues from Rohu fish coinfected with E. tarda and F. columnare revealed pronounced pathological alterations in the liver, kidney, gills and spleen. Gills showed hyperplasia with secondary lamellae fusion, epithelial lifting, hyperaemia, haemorrhage in primary lamellae and widening of lamellae with acute inflammatory responses (Fig 9 a,b,c). Kidney sections exhibited disruption of the glomerular apparatus, necrosis and cloudy swelling of renal tubules, sloughing of tubular epithelial cells, intracellular vacuolation and glomerular atrophy with enlarged glomerular space (Fig 9 d,e,f). Liver tissue showed intracytoplasmic vacuolation, hypertrophied hepatocytes, mild mononuclear cell infiltration and alterations in sinusoidal spaces with Kupffer cell activation (Fig 9 g,h,i). In the spleen, diffuse melanomacrophage centres, disorganization of splenic parenchyma, reduced erythrocytes in the red pulp and disruption of the reticular sheath of ellipsoids were observed (Fig 9 j,k,l). Overall, these lesions indicate severe inflammatory and degenerative changes associated with bacterial coinfection in multiple organs. Histopathological alterations observed in this study aligns with earlier reports in Rohu fish infected with bacterial pathogens. Similar lesions such as lamellar hyperplasia and fusion in gills, degeneration of renal tubules, hepatocellular vacuolation and disorganization of splenic parenchyma have been previously reported during infections caused by E. tarda and F. columnare (Mohanty et al., 2010; Declercq et al., 2013). These pathological alterations are considered typical inflammatory and degenerative responses of fish tissues to bacterial invasion and systemic infection (Manoj et al., 2010). The current findings on kidney histopathological changes corroborate with Biswas et al., (2021) results of kidney tissue damage in L. rohita challenged with Aeromonas hydrophila. The present findings therefore corroborate earlier studies indicating that bacterial infections can induce severe structural damage in vital organs of rohu.

Fig 9: Histopathological showing the histological structure of gill (a,b,c); Kidney (d,e,f), Liver (g,h,i); Spleen (j,k,l) parts of Labeo rohita in co-infection of Flavobacterium columnare and Edwarsiella tarda.

CONCLUSION

In conclusion, this study enlightened that co-infection of F. columnare and E. tarda modulates the non-specific innate immune response in L. rohita, further confirmed by histology. Further, the results highlighted the importance of co-infection model in studying the pattern of infection and health status of fish by evaluating the immunological parameters. Future perspectives can be focused on evaluating the gene level expression of immune response in L. rohita against co-infection of these pathogens.

ACKNOWLEDGEMENT

The authors are thankful to The Director and Vice-chancellor, ICAR-Central Institute of Fisheries Education (CIFE), Mumbai and HoD, AEHM division for providing all possible resources to complete the research work successfully.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Pathogenesis and Immune Responses of Labeo rohita under Experimental Co-infection with Flavobacterium columnare and Edwardsiella tarda

    G
    Ganesh Vijay Sundar Deva1
    M
    Megha K. Bedekar1,*
    K
    K.V. Rajendran1
    R
    R.P. Raman1
    S
    Subodh Gupta1
    K
    Kundan Kumar1
    P
    Pooja Vinde1
    T
    Tapas Paul2
    B
    B. Naveen Rajeshwar3
    1ICAR-Central Institute of Fisheries Education, Mumbai-400 061, Maharashtra, India.
    2College of Fisheries, Bihar Animal Sciences University, Kishanganj-855 115, Bihar, India.
    3Nitte (Deemed to be University), NITTE-GOK COE | Aquamarin, Nitte University Centre for Science Education and Research (NUCSER), Deralakatte, Mangaluru-575 018, Karnataka, India.

    ABSTRACT

    Background: With the increase in intensification of aquaculture practices, co-infection of a variety of heterogeneous micro-organisms is frequent on commercially important fish species. Although there is rich documented literature on single pathogen infection and characterization, the mechanism of co-infection is still poorly understood in fish. In this prelude, the present study aimed to evaluate the immune response of Labeo rohita against the co-infection of Flavobacterium columnare and Edwardsiella tarda.

    Methods: The experiment was conducted in triplicate as three groups viz, 1st group challenged by intraperitoneal injection of E. tarda (T1 and T2); 2nd group challenged by immersion of F. columnare (T3 and T4); 3rd group challenged by both injection and immersion of E. tarda and F. columnare (T5, T6 and T7) and control group without bacterial challenge. To compare the efficacy of different doses, three fish from each group were sacrificed on pre-challenge and 3, 6, 12, 24, 48 and 96 h post-challenge and gills, kidney, liver and spleen were collected from each fish at each time point to analyse the non-specific immune response.

    Result: The results showed T6 group which received a combined infection of E. tarda and F. columnare induced the highest mortality followed by T7 and T1. The results further confirmed by immune assays and histopathological changes. The present study provides baseline information on the co-infection of F. columnare and E. tarda and their modulation of the non-specific innate immune response in L. rohita.

    INTRODUCTION

    Indian aquaculture has significantly flourished over the decades providing food and nutritional security to millions of people (Biswal et al., 2021a; Dheeran et al., 2025; Raghuvaran et al., 2025). Indian major carps (IMC) are the mainstay of freshwater aquaculture, accounting for more than 85% of total production. Among IMC, Labeo rohita is the most cultured fish due to its wide geographical distribution, suitability to different culture systems and preference as a food fish (Biswal et al., 2021b). However, the intensification of aquaculture practices to meet food demands results in the transmission of infectious diseases (Dhayanath et al., 2019). Pathogenic organisms like bacteria, virus, fungi and parasites cause serious infectious diseases in aquatic animals causing severe economic loss and mortality (Leung and Bates, 2013). Similarly, L. rohita culture system is subjected to different emerging pathogens having zoonotic potential causing serious concerns for aquatic and human health. The use of vaccines for aquatic diseases reduced the dependency on antibiotics. However, the lack of knowledge on fish immune systems hinders the discovery of novel vaccines against emerging diseases.
           
    Among bacterial pathogens, the gram-negative bacteria Edwardsiella tarda and Flavobacterium columnare causes systemic infections in several fish species, including carp, tilapia, yellowtail, eel, flounder and turbot. Edwardsiellosis, a generalized septicaemia observed in fish infected with E. tarda show symptoms of macroscopic lesions of internal organs, distended abdomen and prolapsed rectum (Bera et al., 2020). Earlier, Hoshina (1962) reported E. tarda for the first time as Paracolobactrum anguiillimortiferum from Anguilla japonica (Japanese eel). Later, it is reported from wide range of commercially important fishes such as Ictalurus punctatus, Anguilla japonica, Pagrus major, Salvelinus fontinalis, Oncorhynchus tshawytscha, Paralichthys olivaceus, Scophthalmus maximus, Anabas testudineus, Clarias batrachus, Opsanus tau and Oreochromis niloticus (Nougayrede et al., 1994; Sahoo et al., 1998; Sahoo et al., 2000; Clavijo et al., 2002; Horenstein et al., 2004). Columnaris diseases caused by F. columnare reported of high mortality and economic loss up to 30 million dollars in commercially cultured fish species (Hawke and Thune, 1992; Wagner et al., 2002; Shoemaker and LaFrentz, 2015). Moreover, F. columnare are reported to present in freshwater fish microbiota, fish eggs and aquatic environments (Barker et al., 1991; Barnes et al., 2009).
           
    In the aquatic environment, a number of heterogeneous organisms co-exist, survive and cause co-infection in fish and shrimps. Co-infections refer to the simultaneous presence of two or more genetically diverse pathogens within a host, each exerting pathogenic effects that together adversely impact the host (Bakaletz, 2004). A single pathogen has the ability to alter the host’s immune response against subsequent infections caused by additional pathogens (Lello et al., 2004; Telfer et al., 2008). This can alter host vulnerability to infection and influence host-pathogen interactions, infection biology, infection duration, disease severity and host pathology (Graham et al., 2007; Telfer et al., 2008). There were several reports of co-infection of Edwardsiella spp. and Flavobacterium columnare (Panangala et al., 2006; Dong et al., 2015; Wise et al., 2021). Although there is a rich body of literature on single-pathogen infection and characterisation, the mechanisms of co-infection are still poorly understood in fish. Hence, it is necessary to investigate interactions between two pathogens during mixed infections in farmed fish species (Johnson and Hoverman, 2012). In light of this, the present study was conducted to evaluate the co-infection model of Flavobacterium columnare and Edwardseilla tarda in Labeo rohita and to examine the host’s immunological response to the co-infection of F. columnare and E. tarda.

    MATERIALS AND METHODS

    Healthy L. rohita (Rohu) advanced fingerlings were obtained from a Pen fish farm, Raigad District, Maharashtra, India. The fishes (20±6.6 g) were brought to Aquatic Animal Health Management Laboratory (AAHM), ICAR-Central Institute of Fisheries Education (CIFE), Mumbai, India and treated with potassium permanganate (KMnO4) at 5 ppm. The fishes were reared in a controlled lab conditions for a month in Fibre Reinforced Plastic (FRP) tanks (5000 L capacity) with continuous aeration. The experiment was split into three groups (only E. tarda challenge, only F. columnare challenge, co-infection with E. tarda and F. columnare) and conducted in triplicate with 24 fish per tank (n=24). The experiment was conducted in FRP tanks of 500 L capacity containing filtered freshwater under controlled laboratory conditions (pH 8.0; temperature 28°C). The experimental fishes were fed to satiation with 0.5 mm commercial carp feed pellets (CP Aquafeed, India) at 2-3% body weight throughout the experiment.
           
    E. tarda     ATCC® 15947 procured from Himedia, India was used in this study. The strain was confirmed using Gram staining and standard biochemical tests prior to the experiment. The bacterial culture was then revived using Brain Heart Infusion broth (BHI) and pure colonies were obtained on SS agar (Salmonella-Shigella Agar). The E. tarda (0.1 mL) was then injected into a naive L. rohita fish. After 24 h of incubation, fish were sacrificed and bacteria were reisolated from fish kidneys on BHI and SS agar plate and incubated at 28°C for 24 h. This bacterial culture was used for the median lethal dose (LD50) estimation study. Following LD50 estimation, serial dilutions (1:10) of the bacterial culture were then prepared and 0.1 mL from 10-6, 10-7 and 10-8 dilution tubes was spread on BHI agar plates and incubated at 28°C for 18-24 h to determine the viable bacterial count (CFU/mL) of the inoculum.
           
    The gills of diseased L. rohita was subjected to F. columnare isolation in AAHM laboratory. The isolated strain was cultured in modified TYES broth incubated at 28°C in a shaking incubator at 100 rpm for 48 hours. Pure colonies were obtained by streaking on TYES agar plate incubated for 48 hours at 28°C. Similar to E. tarda, the viable bacterial count (CFU/mL) of the inoculum was estimated by serial dilutions (1:10) of the bacterial culture. The bacterial culture of 0.1 mL from 10-5, 10-6 and 10-7 dilution tubes were taken and spread on TYES agar plates and incubated at 28°C for 48 h.
           
    100 µL of varying concentrations of E. tarda ranging from 1×104 to 1×108 CFU/mL were challenged intra-peritoneally into individual fish of 5 groups while 1 group of fish challenged with PBS (n=6 per group). Mortality was observed 12 hours post-challenge and subsequently, using Reed and Muench (1938) techniques, LD50 value was calculated. However, the cumulative Mortality is monitored for up to 96 hours. Similarly, fish were challenged with varying concentrations of F. columnare (1×102 to 1×106 CFU/mL) by immersion method (n=6 per group) to calculate LD50 based on mortality at 12 hours post-challenge.
           
    The challenge experiment was conducted in three groups. The 1st group of rohu fishes were challenged with intraperitoneal injection of E. tarda (T1-7.2×107 CFU/Fish and T2- 7.2×106 CFU/Fish) while the 2nd group challenged with immersion of F. columnare (T3-5.1×105CFU/mL and T4-5.1×106 CFU/mL). The E. tarda bacterial suspension was adjusted to the required concentration (CFU/mL) using serial dilution and plate count. Each fish received 100 µL of inoculum and the final dose (CFU/fish) was calculated based on the injected volume and bacterial concentration. Further, the 3rd group challenged with both E. tarda and F. columnare (T5, T6 and T7) by injection and immersion respectively. Group T5 represents simultaneous co-infection, while T6 and T7 represent sequential infections with defined time intervals between challenges. A negative control group without bacterial challenge was also maintained with challenged groups. To compare the efficacy of different doses, three fish from each group were sacrificed on 0 (pre-challenge), 3, 6, 12, 24, 48, 96 h post-challenge and gills, kidney, liver and spleen were dissected at each time point. Mortality and morbidity symptoms were observed for 10 days after the challenge.
           
    The co-infection of edwardsiellosis and columnaris disease development were tested in L. rohita fingerlings by intra peritoneal injection method and bath immersion methods. Twenty-four fish (n=24) were challenged with E. tarda at a dose of 7.2×107 CFU/Fish and 7.2×106 CFU/Fish by intraperitoneal injection (I/P). Following the E. tarda infection, the fishes were given bath immersion of F. columnare at a dose of 5.1×105 CFU/mL and 5.1× 106 CFU/mL. During bath treatment, fishes were exposed to 5 L of water for 2 h and then transferred to an experimental tank for observation of mortality for 10 days.
           
    The nitrobluetetrazolium (NBT) assay was performed following the procedure established by Secombes (1990), as adapted by Stasiak and Baumann (1996). The activity of myeloperoxidase (MPO) was determined according to the standard technique established by Quade and Roth (1997), with partial modifications by Sahoo et al. (2005). Serum protein was quantified using the Biuret method (Reinhold, 1953) with the InnolineTM total protein plus kit. Immunoglobulin from plasma was separated by precipitation with polyethylene glycol following Anderson and Siwicki’s (1995) method with minor alterations.
           
    Histopathology was performed using the Pirarat et al., (2006) methodology. Briefly, kidney and liver tissues of the fish from different groups that had survived bacterial challenge were dissected out and fixed in 10% neutral buffered formalin. After processing, the fixed tissues underwent sectioning (5 mm) and haematoxylin and eosin staining. After staining, the sections were observed under a microscope.
           
    The software SPSS v 16.0 (IBM®, New York) was used to perform the statistical analysis. The statistical significance between treatment groups was determined at the 5% probability levels utilizing One-way ANOVA and the values were presented as mean ± standard error.

    RESULTS AND DISCUSSION

    Bacterial isolation and identification
     
    The procured E. tarda (ATCC® 15947) challenged in L. rohita was then re-isolated from challenged fish kidney using BHI broth and SS agar media (Fig 1). E. tarda strain was confirmed by biochemical characterisation (Table 1).

    Fig 1: Re-isolation of E. tarda from dead fish on SS agar.



    Table 1: Biochemical confirmation of Edwardsiella tarda ATCC® 15947.


           
    F. columnare     isolated from diseased L. rohita gills and grown in modified TYES broth and then confirmed by using five Griffin tests, which differentiate it from other gram-negative flexing rods.
     
    Confirmation of F. columnare by species-specific PCR
     
    PCR amplification with F. columnare specific primers (COL-F and COL-R) consistently produced the expected 675 bp amplicon in all tested isolates (Fig 2), confirming species-specific detection.

    Fig 2: Agarose gel electrophoresis (2%) showing species-specific PCR amplification of a 675 bp fragment from F. columnare isolates obtained from L. rohita.


     
    Clinical signs in experimentally infected fish
     
    In the present study, healthy L. rohita fingerlings were exposed to E. tarda and F. columnare by intra-peritoneal injection and immersion methods, respectively. Experimentally challenged fish exhibit marked clinical signs upon disease caused by both E. tarda and F. columnare infection. E. tarda infected fish showed abnormal swimming, dark skins and fins, redness on the ventral body, ascitic fluid in the pectoral cavity, haemorrhages and skin lesions (Fig 3 and 4). Meanwhile, F. columnare infected fish exhibit whitish-yellow pigments in the ventral part of the gills arch, eroded skins, ulcers on the surface of the body and pale discoloration on the skin. In this study, L. rohita challenged with E. tarda observed to have excess mucus secretion with gross haemorrhagic lesions on the ventral side of the body. In moribund and dead fish, the anus were protruded, haemorrhagic and swollen. These clinical signs align with earlier report on Clarias batrachus (Sahoo et al., 1998) and Anabas testudineus (Sahoo et al., 2000) infected with E. tarda. Co-infection groups showed faster and significantly higher mortality compared to fish challenged with individual bacterium in 7 days. The present results were corroborated with Dong et al., (2015), who reported significantly higher cumulative mortality in Pangasianodon hypophthalmus under co-infection of E. ictaluri and F. columnare. Further, the clinical signs of both diseases were observed in co-infected fish group. Similarly, Crumlish et al., (2010) determined that co-infection challenge of P. hypophthalmus with A. hydrophila and E. ictaluri by immersion caused higher cumulative mortalities (95%) compared to fish exposed to single infection.

    Fig 3: Clinical signs in L. rohita infected with E. tarda a) skin lesions b) ascites and internal haemorrhage.



    Fig 4: Clinical signs in L. rohita co-infected with E. tarda and F. columnare.


     
    Pathogenicity of E. tarda and F. columnare
     
    In a single infection of E. tarda, T1 and T2 groups having a bacterial dose of 7.2×107 and 7.2×105 CFU/Fish respectively were injected by an intraperitoneal method. After 7 days, T1 and T2 groups showed 60% and 50% cumulative mortality respectively. Similarly, a single infection of F. columnare in two groups T3 (5.1×106 CFU/mL) and T4 (5.1×104 CFU/mL) results in cumulative mortality of 51.4% and 47.7% respectively in L. rohita.
     
    Co-infection of E. tarda and F. columnare
     
    In the combinatorial infection of E. tarda and F. columnare, T5 group (7.2×105 CFU/Fish and 5.1×104 CFU/mL) and T6 group (7.2×107 CFU/Fish and 5.1×104 CFU/mL) dose were given by intraperitoneal injection and immersion methods respectively. Cumulative mortality of the T5 and T6 groups after 7 days was recorded to be 54.4% and 74.8% respectively. Further, the T7 group of bacterial dose 7.2×105 CFU/Fish and 5.1×106 CFU/mL was challenged by the bath immersion method and 60% cumulative mortality was noticed in 10 days experimental period (Fig 5).

    Fig 5: Cumulative mortality (%) of L. rohita fingerlings following single and combined infections with E. tarda and F. columnare over a 7-day post-challenge period.



    Non-specific immune response towards a single and combined infection
     
    Total protein
     
    In a single infection of E. tarda and F. columnare, there is no significant difference in total protein value within T1, T3 and T4 treatment groups at different time periods. However, total protein value was substantially (p<0.05) higher in the pre-challenged group in comparison to other time periods in T2 group. In the co-infection treatment group T5, a time-dependent decrease in total protein was observed during the experimental periods. However, T6 showed no significant difference between time periods and T7 showed a lower total protein value in 96 h (Fig 6). Total protein was found to be reduced significantly in co-infection treatment group in comparison to either single infected group and similar results were reported in E. tarda infected Nile tilapia (Benli and Yildiz, 2004). Similarly, a reduction in total protein concentration was noticed in co-infection of spring viremia and Pseudomonas fluorescens in common carp (Rehulka, 1996; Yildiz, 1998). Higher albumin and globulin level in treatment groups may be due to liver and blood cells destruction with protein synthesis dysfunction (Paul et al., 2019, 2020; Bera et al., 2020).

    Fig 6: Total serum protein levels in L. rohita following a) single bacterial infection challenged with E. tarda (ET; T1 and T2) and F. columnare (FC; T3 and T4) b) co-infection with E. tarda and F. columnare (EF; T5-T7).


     
    Myeloperoxidase
     
    Myeloperoxidase (MPO) activity in L. rohita increased in a time-dependent manner following both single infection and co-infection of pathogens. A significantly (p<0.05) different MPO activity was observed in the T1 group at different time periods, with the highest elevation in 48 h due to a single infection of E. tarda (Fig 7). MPO activity decreased significantly (p<0.05) in the co-infection treatment group compared to pre-challenged group. This might be attributed to the dysfunctioning of phagocytic enzymes and a reduction in the number of phagocytic cells due to invading pathogens. Shameena et al. (2021) reported significant alterations of MPO and RBA on Carassius auratus due to co-infection of Argulus and A. hydrophila under graded temperature corroborates with the results of the present study. Further, the results confirms that the bacterial enzymes such as superoxide dismutase, peroxidase and catalase might be responsible for free radicals detoxification by host phagocytes (Han et al., 2006; Mohanty and Sahoo, 2007).

    Fig 7: Myeloperoxidase (MPO) activity (%) in L. rohita following a) Single bacterial infection challenged with E. tarda (ET; T1 and T2) and F. columnare (FC; T3 and T4) b) co-infection with E. tarda and F. columnare (EF; T5-T7).


     
    Respiratory burst activity (RBA)
     
    In the present study, T1 group exhibited a differential RBA pattern with higher values in 48 h and moderately decreased in 24 h and 96 h. Similarly, T2 showed the highest activity in 48 h followed by 24 h in comparison to other time periods. Interestingly, RBA was significantly (p <0.05) higher in 24 h and reduced during 48 h in the T3 and T4 treatment groups (Fig 8). In T5 group, there is a significant difference in RBA activity in early hours post-challenge. However, in T6 and T7, a significantly higher RBA was observed in 48 h followed by a decreasing trend in 96 h. Fish phagocytes after activation are able to produce superoxide anion (O2-) and its derivatives while intense oxygen consumption which is called as respiratory burst (Secombes and Fletcher, 1992). The MPO activity and respiratory burst were oxygen-dependent reactions commonly used to understand the host’s defence against pathogens (Sharp and Secombes, 1993). The increase in phagocytes killing pathogens are evidently correlated with increased respiratory burst activity. In this study, a time-dependent increased RBA activity was observed in both co-infected and individual treatment groups. Higher respiratory burst activity could be linked with increased host cellular immune response to the bacterial pathogen infection.

    Fig 8: Respiratory burst activity (RBA) in L. rohita following a) Single infection with E. tarda (ET; T1-T2) and F. columnare (FC; T3-T4) at different time points (0-96 h). b) Co-infection with E. tarda and F. columnare (EF; T5-T7).


     
    Histopathology
     
    Histopathological examination of tissues from Rohu fish coinfected with E. tarda and F. columnare revealed pronounced pathological alterations in the liver, kidney, gills and spleen. Gills showed hyperplasia with secondary lamellae fusion, epithelial lifting, hyperaemia, haemorrhage in primary lamellae and widening of lamellae with acute inflammatory responses (Fig 9 a,b,c). Kidney sections exhibited disruption of the glomerular apparatus, necrosis and cloudy swelling of renal tubules, sloughing of tubular epithelial cells, intracellular vacuolation and glomerular atrophy with enlarged glomerular space (Fig 9 d,e,f). Liver tissue showed intracytoplasmic vacuolation, hypertrophied hepatocytes, mild mononuclear cell infiltration and alterations in sinusoidal spaces with Kupffer cell activation (Fig 9 g,h,i). In the spleen, diffuse melanomacrophage centres, disorganization of splenic parenchyma, reduced erythrocytes in the red pulp and disruption of the reticular sheath of ellipsoids were observed (Fig 9 j,k,l). Overall, these lesions indicate severe inflammatory and degenerative changes associated with bacterial coinfection in multiple organs. Histopathological alterations observed in this study aligns with earlier reports in Rohu fish infected with bacterial pathogens. Similar lesions such as lamellar hyperplasia and fusion in gills, degeneration of renal tubules, hepatocellular vacuolation and disorganization of splenic parenchyma have been previously reported during infections caused by E. tarda and F. columnare (Mohanty et al., 2010; Declercq et al., 2013). These pathological alterations are considered typical inflammatory and degenerative responses of fish tissues to bacterial invasion and systemic infection (Manoj et al., 2010). The current findings on kidney histopathological changes corroborate with Biswas et al., (2021) results of kidney tissue damage in L. rohita challenged with Aeromonas hydrophila. The present findings therefore corroborate earlier studies indicating that bacterial infections can induce severe structural damage in vital organs of rohu.

    Fig 9: Histopathological showing the histological structure of gill (a,b,c); Kidney (d,e,f), Liver (g,h,i); Spleen (j,k,l) parts of Labeo rohita in co-infection of Flavobacterium columnare and Edwarsiella tarda.

    CONCLUSION

    In conclusion, this study enlightened that co-infection of F. columnare and E. tarda modulates the non-specific innate immune response in L. rohita, further confirmed by histology. Further, the results highlighted the importance of co-infection model in studying the pattern of infection and health status of fish by evaluating the immunological parameters. Future perspectives can be focused on evaluating the gene level expression of immune response in L. rohita against co-infection of these pathogens.

    ACKNOWLEDGEMENT

    The authors are thankful to The Director and Vice-chancellor, ICAR-Central Institute of Fisheries Education (CIFE), Mumbai and HoD, AEHM division for providing all possible resources to complete the research work successfully.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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