Unchallenged Fish Research Articles

Growth promoting and immunomodulatory effects of dietary Moringa oleifera leaf powder in rainbow trout (Oncorhynchus mykiss)

This study aimed to manifest the dietary effect of Moringa oleifera (MO) leaf powder on growth performance and immunity in rainbow trout before and after Aeromonas hydrophila infection. The fish (N = 360; 35.49 ± 0.50 g) were fed with incremental levels of MO at 0, 10, 25, and 50 g/kg for eight weeks and then challenged with A. hydrophila for 10 days in triplicates. The final weight (114.6 g) and feed conversion ratio (1.28) were significantly decreased in MO-50 compared to other MO-added groups (P < 0.05). In the unchallenged fish, the total white blood cells (WBC), lymphocyte, and neutrophil population size did not change between the experimental and the control groups (P < 0.05), while after the bacterial infection, the number of circulating lymphocytes and WBC were significantly increased in both MO-25 and MO-50 groups compared to the control group. Before the challenge test, the expression level of lysozyme gene in the head kidney was significantly (P < 0.05) increased in all MO-added groups, while the serum and skin mucus lysozyme, bactericidal, and complement activities were notably increased in MO-25 group. A similar trend was observed in the expression levels of IL-1β and TNF-1α genes in the head kidney of MO-added groups, while the total mucus immunoglobulin and protein levels were elevated in both MO-25 and MO-50 groups. The expression levels of SOD, CAT, and GPx genes were markedly down-regulated in MO-25 compared to the control group pre- and post-challenge test. The fish survival rate after challenging with A. hydrophila significantly increased from 46.7% in the control group to 76.7% in MO-25 group. In conclusion, using the supplemental diets with MO for rainbow trout at 19.3–25.0 and 27.5–30.5 g/kg is recommended based on the polynomial regression analyses to accelerate growth performance and provide immunostimulation, respectively.

Role of mucosal immune response and histopathological study in European eel (Anguilla anguilla L.) intraperitoneal challenged by Vibrio anguillarum or Tenacibaculum soleae

The external mucus layer that covers fish skin contains numerous immune substances scarcely studied that act as the first line of defence against a broad spectrum of pathogens. This study aimed to characterize and describe for the first time several humoral immune defence parameters in the skin mucus of the European eel (Anguilla anguilla) after intraperitoneal injection with Vibrio anguillarum or Tenacibaculum soleae. This study evaluated several immune-related enzymes and bactericidal activity against fish pathogenic bacteria in the skin mucus of European eels at 24, 48, and 72 h post-challenge. The results demonstrated that European eel skin mucus showed significant increments in peroxidase and lysozyme activity at 48 and 72 h after V. anguillarum challenge, compared to other experimental groups. In the case of antiprotease activity, an increase was observed at 24 h in the skin mucus of fish challenged with V. anguillarum compared to unchallenged fish, while this activity was undetected at 48 and 72 h. In contrast, protease activity had decreased at 48 and 72 h in the skin mucus of fish challenged with V. anguillarum compared to the unchallenged group. Regarding bactericidal activity, a high growth capacity of T. soleae was observed in the skin mucus of all experimental groups. Interestingly, the skin mucus from fish challenged with V. anguillarum exhibited increased bactericidal activity against this bacterium at 48 h, compared to unchallenged fish. Finally, severe histopathological alterations were observed in the gills and liver at the end of the trial (72 h), whereas the skin showed only an overspread presence of goblet cells in the challenged fish compared to unchallenged fish. The present results may give new insights into the mucosal immune system of this primitive species with potential applications in aquaculture.

Administration of dehydrated oxytetracycline effectively reduces francisellosis mortality in Nile tilapia

Francisellosis is a disease responsible for severe economic losses in aquaculture. Currently, this disease is controlled via biosecurity measures and treatment of affected batches. In this study, tilapia juveniles were challenged with Francisella orientalis via immersion and fed with oxytetracycline-medicated feed, at 100 mg kg fish−1 and 200 mg kg fish−1, under prophylactic and therapeutic strategies for 16 and 15 days respectively. All doses and treatment strategies reduced the fish mortality. The detection of the pathogen from the spleen was lower in the treated groups. Histopathology analysis showed that challenged fish that received the highest dosage presented fewer granuloma in their spleens and less inflammatory infiltrate in their livers than untreated fish. Fish that received the prophylactic treatment, regardless of the dose, presented fewer granuloma in their livers than untreated fish. Fish treated with 100 mg kg fish−1 had better scores for hepatocyte accumulations than fish treated with the higher dosage. Furthermore, 20% of the unchallenged fish that received the higher dosage presented mild inflammatory infiltrate in their livers. No differences were observed in the serum hepatocyte lesion enzyme quantifications. The results indicate that oxytetracycline-medicated feed is safe, even at the higher dose, and effective at controlling francisellosis in tilapia.

Evaluation of Enterococcus hirae LAB3 as potential probiont against Vibrio harveyi in Artemia nauplii and Asian seabass larvae (Lates calcarifer) cultures

Aim: To evaluate the ability of to inhibit the growth of V. harveyi in in-vitro co-culture assay and confer protection towards Artemia nauplii and seabass larvae against V. harveyi in in-vivo assay. Methodology: Enterococcus hirae was co-cultured with V. harveyi for 96 hrs and the samples were taken every 6 hr interval to determine the growth of the pathogen in in-vitro assay. In the in-vivo assay, E. hirae at different concentrations were introduced to the Artemia nauplii and seabass larvae cultures. After 24-hr incubation period, V. harveyi 6 -1 at concentration 10 CFU ml were added into the respective experimental tanks. Mortality was observed and recorded daily, until 50% mortality was observed in negative control group which challenged with V. harveyi only. Results: E. hirae was able to reduce the numbers of V. harveyi after 12 hr of co-culture incubation. In addition, E. hirae was administered into the Artemia nauplii and conferred protection 6 against V. harveyi with the best survival rate at concentration of 10 -1 CFUmL (70±3.1%). The untreated group seabass larvae challenged with V. harveyi presented low survival of (16.7±3.3%), 6 -1 while fish treated with probiotic E. hirae at 10 CFUml showed significantly increased survival rates (68.3±0.9%) after challenged. The survival of healthy unchallenged fish treated with probiotic was not significantly different with the control group. The pathogen loaded also reduced in groups treated with E. hirae respectively Interpretation: E. hirae strain LAB3, a potential probiont was able to reduce the number of Vibrios and conferred protection to Artemia and seabass larvae.

Molecular characterization and functional analysis of tumor necrosis factor receptor-associated factor 2/7 and tumor necrosis factor receptor 1-associated death domain protein from Larimichthys crocea

In the present study, we characterized tumor necrosis factor receptor-associated factor 2/7 (lcTRAF2/7) and TNFR1-associated death domain protein (lcTRADD) in Larimichthys crocea (L. crocea) and examined their expression profiles in tissues of Vibrio-challenged and unchallenged fish. The coding sequences of lcTRAF2, lcTRAF7, and lcTRADD were 1488, 2454, and 744 nucleotides, and they encoded proteins of 495, 344, and 248 amino acids, respectively. The results of phylogenetic analysis revealed that lcTRAF2, lcTRAF7, and lcTRADD were closest to Oplegnathus fasciatus (85%), Xiphophorus maculatus (97%), and Acanthochromis polyacanthus (65%), respectively. Multiple sequence alignment showed that lcTRAF2 and lcTRAF7 were highly conserved with other vertebrate TRAFs in their functional domains; however, lcTRADD was poorly conserved. The results of quantitative real-time polymerase chain reaction analysis indicated that lcTRAF2, lcTRAF7, and lcTRADD were constitutively expressed in the spleen, liver, kidney, heart, brain, gill, bladder, skin, fin, eye, and muscle. After challenging fish with Vibrio parahaemolyticus, the mRNA expression levels of lcTRAF2, lcTRAF7, and lcTRADD were upregulated in liver, spleen, and kidney. Immunofluorescence staining revealed that lcTRAF2 and lcTRADD were cytoplasmic in localization, whereas lcTRAF7 targeted both the cytoplasm and nucleus. In addition, the NF-κB protein level was upregulated after lipopolysaccharide stimulation in lcTRAF2, lcTRAF7, or lcTRADD overexpressing cells. Taken collectively, these results have improved our understanding of the functions of TRAF2, TRAF7, and TRADD in pathogenic infections in teleosts.

An interferon-induced GTP-binding protein, Mx, from the redlip mullet, Liza haematocheila: Deciphering its structural features and immune function

The interferon-induced GTP-binding protein Mx is responsible for a specific antiviral state against a broad spectrum of viral infections that are induced by type-I interferons (IFN α/β) in different vertebrates. In this study, the Mx gene was isolated from the constructed mullet cDNA database. Structural features of mullet Mx (MuMx) were analyzed using different in-silico tools. The pairwise comparison revealed that the MuMx sequence was related to Stegastes partitus Mx with an 83.7% sequence identity, whereas MuMx was clustered into the teleost category in the phylogentic analysis. Sequence alignment showed that the dynamin-type guanine nucleotide-binding domain (G_DYNAMIN_2), central interactive domain (CID), and GTPase effector domain (GED) were conserved among Mx counterparts. The transcriptional expression of MuMx was the highest in blood cells from unchallenged fish. The temporal mRNA profile showed that MuMx expression was significantly elevated in all tissues, including blood, spleen, head kidney, liver, and gills after the injection of polyinosinic-polycytidylic acid (poly I:C) at many time points. Moreover, MuMx expression increased slightly, in the blood, spleen, and head kidney at a few time points after the injection of lipopolysaccharide (LPS) and Lactococcus garvieae (L. garvieae). Results of the subcellular localization analysis confirmed that the MuMx protein was highly expressed in the cytoplasm. The analysis of the gene expression of the viral hemorrhagic septicemia virus (VHSV) under conditions of MuMx overexpression confirmed the significant inhibition of viral transcripts. The cell viability (MTT) assay and VHSV titer quantification with the presence of MuMx indicated a significant reduction in virus replication. Collectively, these findings suggest that Mx is a specific immune-related gene that elicits crucial antiviral functions against viral antigens in the mullet fish.

Dietary Aloe vera supplementation on growth performance, some haemato-biochemical parameters and disease resistance against Streptococcus iniae in tilapia (GIFT)

This study investigated effects of dietary Aloe vera on growth performance, some haemato-biochemical parameters and disease resistance against Streptococcus iniae in tilapia (GIFT). Five groups were designed including a basal diet (control) and 100% A. vera powder incorporated in fish feed at 0.5% 1%, 2%, and 4%/kg feed, which were administered for 8 weeks. Fish fed 0.5%, 1%, and 2% A. vera supplemented diet significantly improved (p < 0.05) weight gain, absolute growth rate and specific growth rate. Feed intake significantly increased in fish fed with A. vera diet at 1% and 2%/kg feed. Feed efficiency ratio, feed conversion ratio, and hepatosomatic index were significantly enhanced in 4% A. vera supplemented fish over unsupplemented ones (p < 0.05). Several haemato-biochemical indices were examined before and after fish were challenged with S. iniae pathogen containing 7.7 × 106 CFU cells mL−1. A. vera supplemented fish showed a significant increase (p < 0.05) in red blood cells, hematocrits (Hb), hemoglobin (Hb), white blood cells (WBC), neutrophils, monocytes, eosinophils, serum total protein, glucose and cortisol after challenge when compared to unsupplemented ones. Meanwhile, 4% A. vera supplemented fish showed a decrease (p < 0.05) in RBC, Hb, Ht, WBC, and mean corpuscular hemoglobin (MCH) after challenge compared to unsupplemented ones and other supplemented ones. In addition, lower mean corpuscular volume values (MCV) (p < 0.05) were observed in fish fed with A. vera diet at 2% and 4% A. vera/kg feed than those fed unsupplemented diet. Unchallenged fish fed 0.5%, 1%, and 2% A. vera showed significantly higher values (p < 0.05) of mean corpuscular hemoglobin concentration (MCHC) than those fed unsupplemented diet and 4% A. vera supplemented diet. There was a significant increase (p < 0.05) in the neutrophil to lymphocyte ratio (N/L) within experimental groups after challenge; N/L ratio in A. vera unsupplemented fish and those supplemented with A. vera diet at 1%/kg feed increased significantly (p < 0.05) throughout challenge period; while those fed 4% A. vera supplemented diet maintained higher values at all experimental stages among groups. There was a significant correlation (p < 0.05, r = 0.53) between N/L ratio and glucose concentration, 96 h after challenge. Aloe had no significant effect (p > 0.05) on the survival of the fish when compared to the control; no mortality was recorded in challenge trial. Overall, our results indicated that dietary aloe supplementation could improve growth, feed utilization, and haemato-biochemical parameters of cultured tilapia.

Mitochondrial peroxiredoxin 3 (Prx3) from rock bream (Oplegnathus fasciatus): Immune responses and role of recombinant Prx3 in protecting cells from hydrogen peroxide induced oxidative stress

Pathogenic infections and environmental factors cause a variety of stresses in fish including oxidative stress by rapid elevation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Transcriptional activation and expression of antioxidant enzymes are essential for reducing the oxidative stress. In this study, we present the molecular characterization, immune responses and ROS scavenging activity of mitochondrial peroxiredoxin 3 from Oplegnathus fasciatus (RbPrx3). Coding sequence (CDS) of RbPrx3 contains 248 amino acids polypeptide which consists of highly conserved peroxiredoxin super family domain and two cysteine residues. Pairwise sequence comparison revealed that RbPrx3 has the greatest identity (94.8%) to Sparus aurata Prx3. Transcriptional analysis of RbPrx3 indicated the ubiquitously expressed mRNA in wide array of organs showing the highest expression in the liver of rock bream. Upon immune challenge of Edwardsiella tarda, Streptococcus iniae, rock bream iridovirus (RBIV) and lipopolysaccharide (LPS), RbPrx3 mRNA level was up-regulated in immunocompetent liver tissues compared to unchallenged fish. Purified recombinant RbPrx3 treated THP-1 cells showed higher survival rate against H2O2 induced oxidative stress and significantly reduced the level of intracellular ROS. Overall results from our study suggest that RbPrx3 may be involved in broader functions such as regulating oxidative stresses by scavenging ROS and activating immune responses in rock bream.

Spleen Index and Mannose‐Binding Lectin Levels in Four Channel Catfish Families Exhibiting Different Susceptibilities to Flavobacterium columnare and Edwardsiella ictaluri

Edwardsiella ictaluri and Flavobacterium columnare are two bacterial pathogens that affect channel catfish Ictalurus punctatus aquaculture. At the Catfish Genetics Research Unit (U.S. Department of Agriculture, Agricultural Research Service), some progress has been made in selectively breeding for resistance to E. ictaluri; however, the susceptibility of these families to F. columnare is not known. Our objectives were to obtain baseline information on the susceptibility of channel catfish families (maintained as part of the selective breeding program) to E. ictaluri and F. columnare and to determine whether the spleen index and plasma levels of mannose-binding lectin (MBL) are predictive indicators of susceptibility to these pathogens. Four channel catfish families were used: family A was randomly chosen from spawns of fish that were not selectively bred for resistance; families B, C, and D were obtained after selection for resistance to E. ictaluri. All four families were immersion challenged with both bacterial pathogens; the spleen index and plasma MBL levels of unchallenged fish from each family were determined. Mean cumulative percent mortality (CPM) after E. ictaluri challenge ranged from 4% to 33% among families. Families A and B were more susceptible to F. columnare (mean CPM of three independent challenges = 95% and 93%) than families C and D (45% and 48%), demonstrating that there is genetic variation in resistance to F. columnare. Spleen index values and MBL levels were not significantly different, indicating that these metrics are not predictive indicators of F. columnare or E. ictaluri susceptibility in the four tested families. Interestingly, the two families that exhibited the highest CPM after F. columnare challenges had the lowest CPM after E. ictaluri challenge. Further research on larger numbers of families is needed to determine whether there is any genetic correlation between resistance to E. ictaluri and resistance to F. columnare.

Evaluation of the probiotics Bacillus subtilis and Lactobacillus plantarum bioencapsulated in Artemia nauplii against vibriosis in European sea bass larvae (Dicentrarchus labrax, L.)

Two potential probiotics Bacillus subtilis and Lactobacillus plantarum were evaluated for use in aquaculture as preventive measures against vibriosis. In vitro evaluation of the probiotics using co-culture assays with the pathogen Vibrio anguillarum and testing for the production of antibacterial substances showed the presence of antagonism and confirmed the production of antibacterial substances. Both potential probiotics were administered to the live fish feed Artemia franciscana nauplii, offering protection against a subsequent challenge of the nauplii with the fish pathogen V. anguillarum, with best survival rates of the nauplii and the most efficient protection offered by B. subtilis. Nauplii enriched with B. subtilis were further used to evaluate the protection of sea bass larvae against vibriosis. The untreated group of fish challenged with V. anguillarum presented low survival of 36.7 %, while the fish treated with nauplii enriched with the probiotic B. subtilis showed significantly increased survival rates of 86.7 % after challenge with the pathogen. The survival of healthy unchallenged fish treated with the probiotic was not significantly different from control unchallenged fish (90-94 %). Our results indicate that B. subtilis is a probiotic suitable to be used for the prevention of vibriosis in fish larvae and can be safely administered through their live feed Artemia nauplii.

Molecular characterization, immune responses and DNA protection activity of rock bream (Oplegnathus fasciatus), peroxiredoxin 6 (Prx6)

In this study, we describe the molecular characterization, immune responses of rock bream, Oplegnathus fasciatus peroxiredoxin 6 cDNA (RbPrx6) and DNA protection activity of its recombinant protein. The full-length cDNA sequence of RbPrx6 was identified after pyrosequencing of rock bream cDNA library. RbPrx6 consists of 663 bp open reading frame (ORF) that codes for a putative protein of 221 amino acids with predicted molecular mass of 27 kDa. It showed characteristic peroxiredoxin super-family domain similar to vertebrate Prx counterparts. In the pair-wise comparison, RbPrx6 showed the highest amino acid identity (92.8%) to Scophthalmus maximus Prx6.Real-time RT-PCR analysis revealed that constitutive expression of RbPrx6 transcripts in eleven tissues selected from un-challenged fish showing the highest level in liver. Synthetic polyinosinic:polycytidylic acid (poly I:C) and iridovirus containing supernatant, up-regulated the RbPrx6 mRNA in liver. Purified recombinant RbPrx6 protein was able to protect supercoiled plasmid DNA from damages that is induced by metal-catalyzed generation of reactive oxygen species. Our results suggest that RbPrx6 may play an important role in regulating oxidative stress by scavenging of ROS, involving immune reactions and minimizing the DNA damage in rock bream.

Dietary Korean mistletoe enhances cellular non-specific immune responses and survival of Japanese eel ( Anguilla japonica)

The present study was performed to investigate the immunostimulatory effects of Korean mistletoe extract (KM-110; Viscum album Coloratum) on the non-specific immune response and protection against Aeromonas hydrophila infection in Japanese eel ( Anguilla japonica). Eels were fed under 4 regimes, 0%, 0.1%, 0.5% and 1.0% KM-110 mixed diet. On day 14 after feeding, 15 fish from each group were injected i.p. with live A. hydrophila (3 × 10 6 CFU) and the remaining unchallenged fish from each group were used to study the innate immune response. On 14 days post-infection, the total survival rates were 26.6% in control, and 33.3%, 66.6% and 80% in 0.1%, 0.5% and 1% KM-110-treated groups, respectively. The maximum lysozyme activity was observed in the 1% KM-110-treated group. There was no significant difference of lysozyme activity between 0.1% and 0.5% KM-110 group. Superoxide anion (O 2 −) production was significantly ( p < 0.05) augmented in the 0.5% and 1% KM-110 groups compared to the control and 0.1% KM-110 group. No significant difference of (O 2 − production was found between 0.5% and 1% KM-110 group. Likewise, there was a significant increase in phagocytic activity in the 0.5% KM-110 group compared with the 0.1% group ( p < 0.05), but no significant difference between the 0.5% and the 1% KM-110 group indicating that 0.5% KM-110 concentration is suitable for stimulating maximum phagocytic activity resulting in a high amount of ROI production. Considering the present results, KM-110 could be utilized as a promising immunostimulating substance for a diet in aquaculture.

Passive immunization of channel catfish Ictalurus punctatus with anti-Flavobacterium columnare sera

Passive immunization of channel catfish Ictalurus punctatus (Rafinesque) was conducted to determine if anti-Flavobacterium columnare serum was protective when injected intraperitoneally (i.p.) into channel catfish. The anti-F. columnare serum was produced by actively immunizing (i.p. injection) channel catfish with sonicated whole cells or purified lipopolysaccharide (LPS) of F. columnare in Freund's adjuvant. Serum anti-F. columnare activity was verified by Western blotting and ELISA of serum. Normal serum and sterile culture broth were used as controls. Complement was inactivated in all sera by heating. After 48 h, passively immunized fish were challenged with virulent F. columnare by i.p. injection. A group of unchallenged fish served as controls. The immune response of catfish to the antigenic fractions was different when examined by Western blotting. Antibody produced with whole-cell antigen responded to a broad range of molecular weight components, while LPS antigens were restricted to a pair of bands near 20 kDa. Control fish injected with culture medium experienced 100% mortality 14 d post-challenge. Relative percent survival was 77 and 73 for catfish passively immunized with anti-LPS and anti-whole-cell serum, respectively. Results suggest that antibodies in the serum are involved in the protective immune response against columnaris disease in channel catfish.

Efficacy of Orally Administered Florfenicol in the Treatment of Furunculosis in Atlantic Salmon

This study was performed to determine the efficacy of orally administered florfenicol to treat experimentally induced furunculosis in Atlantic salmon Salmo salar held in seawater. A strain of the causative bacterium, Aeromonas salmonicida subsp. salmonicida, sensitive to florfenicol was used. In two trials, cohabitation challenges were performed by introducing six (trial 1) or eight (trial 2) fish—challenged in advance by an intraperitoneal injection of 2.2 × 104 colony-forming units—to eight aquaria, each containing 40 healthy fish. The treatment groups in both trials consisted of three groups receiving medication at 10, 13, and 16 d, respectively, postchallenge and one control group. An unchallenged, unmedicated group was used to determine the natural mortality in the population. The recommended therapeutic dose of 10 mg florfenicol/kg fish daily for 10 d was used. A higher mortality rate was observed in both trials for challenged unmedicated control fish than for unchallenged fish. In both trials, the cumulative mortality of fish treated with florfenicol was strikingly lower than the mortality of fish in the control groups. The mortality for the two untreated control groups was quite different; the lower mortality rate occurred in the group with the lower number of cohabitants. The mortality rates of the treated groups in trial 1 differed only slightly from each other, whereas in trial 2, the group starting medication at 10 d had a substantially lower mortality rate than the group starting medication at 16 d postchallenge.

Evaluation by Enzyme-Linked Immunosorbent Assay (ELISA) ofRenibacterium salmoninarumBacteras Affected by Persistence of Bacterial Antigens

Rainbow trout Oncorhynchus mykiss were injected intraperitoneally with a bacterin containing killed Renibacterium salmoninarum cells delivered alone or in an oil-based adjuvant. We evaluated the relative abilities of the bacterins to prevent the initiation or progression of infection in fish challenged by waterborne exposure to R. salmoninarum. Sixty-one days after vaccination, fish were held for 24 h in water containing either no bacteria or approximately 1.7 × 103, 1.7 × 105, or 5.3 × 106 live R. salmoninarum cells/mL. An enzyme-linked immunosorbent assay (ELISA) was used to monitor changes in the levels of R. salmoninarum antigen in live fish before and after the immersion challenges. High levels of R. salmoninarum antigens were detected by ELISA in kidney–spleen tissue homogenates from vaccinated fish immediately before the challenges. Levels of those antigens remained high in the tissues of unchallenged fish throughout the study. We found that the ELISA used in this study may be unsuitable for evaluating the efficacy of bacterins because it did not distinguish antigens produced by the challenge bacteria during an infection from those of the bacterins. Groups of control and vaccinated fish also were injected with either L7 × 104 or l.7 × 106 R. salmoninarum cells and served as R. salmoninarum virulence controls. Relative survival among the various subgroups in the injection challenge suggests that adverse effects might have been associated with the adjuvant used in this study. The lowest survival at both injection challenge levels was among fish vaccinated with bacteria in adjuvant.

Changes in selected haematological parameters in juvenile chinook salmon subjected to a bacterial challenge and a toxicant

An infection and a toxicant were presented singly and in combination to juvenile chinook salmon. The fish were challenged with Renibacterium salmoniarum and exposed to two levels (0.05 and 0.50 × 96 h LC50) of the toxicant sodium pentachlorophenate. The experiment comprised measurement of seven blood parameters and observations on mortality and behaviour at 4‐day intervals over a period of 40 days.All fish receiving the combined treatment of bacterial challenge and higher level of toxicant died 12 days post‐infection on the 8th day of toxicant exposure. Mortalities also occurred in the groups of diseased fish and fish receiving the toxicant alone. There were no mortalities in the appropriate control groups of unchallenged fish in clean water.Most of the haematological changes pointed to a general haemodilution probably arising from progressive damage to the kidney and haematopoietic tissue by bacteria but which did not appear to be greatly affected by combined exposure to the toxicant. The blood parameters of the fish in clean water as well as those exposed to just the toxicant remained relatively constant. Higher mortality and sustained lower values of haematocrit, haemoglobin and glucose concentrations as well as red blood cell counts were present in both the diseased group of fish held in clean water and the disease plus low level toxicant group compared to the control fish; white blood cell counts were low initially but increased markedly as the infection progressed. This latter treatment combination also produced lower total protein and blood urea nitrogen concentrations.