Parasitism by protozoan Ichthyophthirius multifiliis enhanced invasion of Aeromonas hydrophila in tissues of channel catfish

Protozoan Ichthyophthirius multifiliis Fouquet (Ich) and bacterium Aeromonas hydrophila are two common pathogens of cultured fish, which cause high fish mortality. Currently there is no information available for the effect of parasitism by Ich on survival of channel catfish and invasion of A. hydrophila in fish tissues following exposure to A. hydrophila. A trial was conducted in this study to: (1) determine whether A. hydrophila increased fish mortality in Ich-parasitized channel catfish; and (2) compare the bacterial quantity in different tissues between non-parasitized and Ich-parasitized catfish by real-time polymerase chain reaction (qPCR). The results demonstrated that the Ich-parasitized catfish showed significantly (P<0.05) higher mortality (80%) when exposed to A. hydrophila by immersion than non-parasitized fish (22%). Low mortality was observed in catfish exposed to Ich alone (35%) or A. hydrophila alone (22%). A. hydrophila in fish tissues were quantified by qPCR using a pair of gene-specific primers and reported as genome equivalents per mg of tissue (GEs/mg). Skin, gill, kidney, liver and spleen in Ich-parasitized fish showed significantly higher load of A. hydrophila (9400-188,300 GEs/mg) than non-parasitized fish (4700-42,100 GEs/mg) after exposure to A. hydrophila. This study provides evidence that parasite infections enhance bacterial invasion and cause high fish mortality.     

Effect of water temperature on the clinical outcome of infection with Edwardsiella ictaluri in channel catfish

Channel catfish fingerlings (mean body weight, 19 +/- 3 g each) were given intraperitoneal inoculations of Edwardsiella ictaluri suspensions of 10(4), 10(5), or 10(6) bacteria. Control fish were inoculated intraperitoneally with sterile 0.85% NaCl solution. Ten-day trials were conducted at water temperatures of 17, 21, 23, 25, 28, and 32 C. Differences in mortality between E ictaluri-infected fish and fish used as controls were observed at water temperatures of 23, 25, and 28 C, but not at temperatures of 17, 21, and 32 C. Clinical signs and lesions induced by intraperitoneal inoculation of E ictaluri were comparable with those found with the intestinal form of the natural disease. The characteristic erosion of skin and muscle overlying the skull, known as the "hole-in-the-head" lesion, was not observed. A given fish may be susceptible to infection at any water temperature, but a population is at risk when water temperatures are in the 22 to 28 C range.     

In vitro evaluation of the susceptibility of Edwardsiella ictaluri, etiological agent of enteric septicemia in channel catfish, Ictalurus punctatus (Rafinesque), to florfenicol.

In vitro studies were conducted to assess the sensitivity of Edwardsiella ictaluri, the etiological agent of enteric septicemia of catfish (ESC), to the antibacterial drug florfenicol (FFC). Twelve different E. ictaluri isolates from cases submitted between 1994 and 1997 to the Thad Cochran National Warmwater Aquaculture Center fish diagnostic laboratory (Stoneville, MS) were used for testing. These isolates originated from channel catfish (Ictalurus punctatus) infected with E. ictaluri through natural outbreaks of ESC in the commercial catfish ponds in Mississippi. Seven hundred sixty-seven additional cultures of E. ictaluri were obtained from channel catfish infected experimentally with E. ictaluri. In some of these experimental infections, FFC was used for treatment. These cultures of E. ictaluri were identified by morphological and biochemical tests. Kirby-Bauer zones of inhibition (in mm) for FFC against E. ictaluri were determined using standard methods. The minimum inhibitory concentration (MIC) of FFC was determined for the natural outbreak E. ictaluri isolates and arbitrarily selected experimental cultures. The zones of inhibition for FFC tested with E. ictaluri ranged from 31 to 51 mm. The MIC for FFC tested with E. ictaluri was consistently 0.25 microg/ml. Edwardsiella ictaluri tested in these studies were highly sensitive to FFC in vitro.     

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.     

Mobilization and activation of nonspecific cytotoxic cells (NCC) in the channel catfish (Ictalurus punctatus) infected with Ichthyophthirius multifiliis

Channel catfish demonstrate a shift in the tissue distribution of nonspecific cytotoxic cells (NCC) when infected with the protozoan parasite, Ichthyophthirius multifiliis. NCC, isolated from head kidney (HK) tissues (hemopoietic organ) or peripheral blood leukocytes, were assessed for cytotoxic activity against NC-37 (a transformed mammalian cell line). NCC activity from HK tissue of moribund I. multifiliis-infected fish was depressed compared to HK-NCC activity in uninfected or I. multifillis-immune fish. The activity of NCC, isolated from the peripheral blood of moribund I. multifiliis-infected fish was significantly greater than the NCC activity in peripheral blood from either immune or uninfected fish. Chromium-51 release assays were combined with effector and target conjugate assays to determine killing capacity (Vmax) and affinity (Km) for target cells of peripheral blood NCC from moribund I. multifiliis-infected and uninfected fish. These experiments indicated that the peripheral blood from the moribund infected fish contained an increased percentage of active NCC with increased killing capacity and target cell affinity compared to peripheral blood NCC activity of uninfected fish.     

Modulary effects of temperature on antibody response and specific resistance to challenge of channel catfish, Ictalurus punctatus, immunized against Edwardsiella ictaluri

The effects of various temperature treatments on the level of the humoral antibody response in channel catfish immunized with formalin killed Edwardsiella ictaluri was determined in laboratory controlled experiments. Immunized fish that were held at 25 degrees C for 30 days and 12 degrees C for an additional 30 days had higher antibody titers, and were more protected upon challenge, than immunized fish held at 25 degrees C for 60 days. Also immunized catfish held at 25 degrees C for 5 or 10 days followed by 12 degrees C water had higher antibody titers than immunized fish held at 12 degrees C or 25 degrees C for 60 days. In a field experiment carried out during winter and spring (February-May) fingerling channel catfish were vaccinated with E. ictaluri using intraperitoneal injection or immersion with either sonicated or whole cell preparations. Following challenge, the fish vaccinated by immersion in the sonicated preparation had 11.8% mortality whereas the groups immersed in whole cell bacterin, injected with the whole cell bacterin in adjuvant, or injected with sonicate showed 24.6, 57.9 and 41.7% mortality, respectively. Although the fish vaccinated by immersion with the sonicated bacteria had lower antibody titers than those vaccinated by the other methods the immersion vaccinates were more protected against challenge with the pathogen.     

Early interactions of Edwardsiella ictaluri, with Pangasianodon catfish and its invasive ability in cell lines

Commercial Pangasianodon catfish production is heavily impacted by Bacillary Necrosis of Pangasius (BNP) caused by Edwardsiella ictaluri. This study aimed to investigate the early bacterium-host interactions following immersion challenge and to compare the retrieved data with the invasion ability of the used isolates in fish cell lines. Firstly, Pangasianodon hypophthalmus fingerlings were challenged via immersion using E. ictaluri isolate HO2 or 223. At different times post inoculation, fish were sacrificed and gill and internal organ samples were taken for bacteriological, histological and immunohistochemical evaluation. The bacterial load was higher for fish inoculated with isolate HO2 compared with 223. Histological and immunohistochemical analysis revealed multifocal necrotic areas in kidney, spleen and liver of HO2 inoculated fish at 72 h post inoculation with short rod-shaped immunoperoxidase positive bacteria clustered inside cells respectively. Bacteria especially were present in the gills and intestinal tract of HO2 inoculated fish, suggesting the gastrointestinal tract and gills act as portals of entry. Following, the ability of HO2, 223 and four additional isolates to invade a Chinook salmon embryo cell line, a fat head minnow cell line and a rainbow trout liver cell line was tested. All E. ictaluri isolates were invasive in all cell lines albeit at different degrees. Isolate HO2 was highly invasive in all cell lines with a significantly higher invasion capacity than isolate 223 in the Chinook salmon embryo cell line. A correlation between in vivo virulence and in vitro invasiveness hence is suggested although further studies are needed to confirm this hypothesis.     

Communications: Specificity of the Channel Catfish Antibody to Edwardsiella ictaluri

Abstract

The specificity of channel catfish Ictalurus punctatus serum antibody to Edwardsiella ictaluri was characterized by microtiter agglutination assay. There was no correlation between antibody titer to Aeromonas hydrophila and antibody titer to E. ictaluri in wild or feral channel catfish. Anti-E. ictaluri antibodies in naturally infected channel catfish were not removed by adsorption by nine other species of bacteria found in the channel catfish intestine and fish ponds. Channel catfish immunized with nine other species of bacteria did not develop substantial antibody titer to E. ictaluri. The antibody response of channel catfish to E. ictaluri is highly specific, and the microtiter agglutination test is a specific indicator of previous exposure to E. ictaluri     

Role of Ichthyophthirius multifiliis in the Infection of Aeromonas hydrophila

Aeromonas hydrophila and Ichthyophthirius multifiliis are important fish pathogens. To clarify their relationship, two host models, goldfish with trauma and those infected with I. multifiliis, were used to study infection kinetics of A. hydrophila 4332 tagged with plasmid pGFPuv (Ah4332(GFP)). The results showed that in fish suffering from ichthyophthiriosis, the bacterial infection process was accelerated and more persistent than in the other groups. It is suggested that I. multifiliis plays an important role in the infection of A. hydrophila. Mechanical trauma caused by I. multifiliis might act as a portal of entry for A. hydrophila present in the water, thereby facilitating the invasion of bacteria. The ectoparasite might also serve as a vector and reservoir of pathogenic bacteria.     

Specificity of Humoral Responses of Convalescent Channel Catfish to Edwardsiella ictaluri

Abstract

Serum samples from 15 yearling channel catfish Ictalurus punctatus convalescing after experiencing enteric septicemia of catfish were distributed into three representative serum pools, each containing equal volumes of serum from five fish. Serologic recognition of each pooled serum sample against Edwardsiella ictaluri and Escherichia coli whole cells and against secretory antigen (exoantigen) derived from E. ictaluri was measured by enzyme-linked immunosorbent assays (ELISA). Serum samples were purified by affinity chromatography with a heterologous Ra,-mutant lipopolysaccharide that was derived from rough Salmonella typhimurium TV 119 and was covalently bound to an agarose matrix. Removal of antibodies recognizing the lipopolysaccharide by cross-reactive affinity purification caused significant decreases in serologic recognition of E. ictaluri (P < 0.10) and E. coli (P < 0.01) whole cells; however, serologic recognition of the E. ictaluri-specific exoantigen was not significantly decreased. These results indicate that serologic recognition of the exoantigen is highly specific and that cross-reactive immune responses recognizing homologous gram-negative core antigens will not cause false-positive test results when the specific capture ELISA is used to detect exposure to E. ictaluri     

Prevention of Ichthyophthirius multifiliis Infestation in Channel Catfish Fingerlings by Copper Sulfate Treatment

Abstract

The ability of copper sulfate to control Ichthyophthirius multifiliis tomites was determined in pond water and in settled, decanted pond water at 23°C for 7 d. Results indicate that copper concentrations of 0.15 and 0.02 mg/L prevented tomites from infesting channel catfish Ictalurus punctatus in pond water (total alkalinity = 366 mg CaCO₃ /L) and in settled, decanted pond water (total alkalinity = 224 mg CaCO₃ /L), respectively.     

Transmission of Edwardsiella ictaluri from Infected,Dead to Noninfected Channel Catfish

Abstract

Enteric septicemia of catfish (ESC) was transmitted horizontally from channel catfish Icialurus punctatus that had died from Edwardsiella ictaluri infection to contact channel catfish during 2 d of habitation in a tank. The contact channel catfish became positive for E. ictaluri antibody, became infected with this bacterium, and had signs of ESC and died within 12 d postexposure. Edwardsiella ictaluri was recovered from 24 of the 30 contact channel catfish that died from ESC, as well as from 9 of the 25 tested contact survivors. The cannibalizing of E. ictaluri-infected fish, or the shedding of E. ictaluri from dead fish, or both, were shown to be mechanisms of horizontal transmission of ESC among channel catfish.     

Effect of Vitamin E on the Immune Response of Channel Catfish to Edwardsiella ictaluri

Abstract

Three-month-old fingerling channel catfish Ictalurus punctatus were fed purified diets supplemented with ∝-tocopherol acetate to provide 0, 60, and 2,500 mg vitamin E/kg for 180 d. A 30-s immersion bath and an oral booster were used to deliver a bacterin of formalin-killed Edwardsiella ictaluri to half of the fish from each dietary treatment. Resistance of red blood cells to peroxidation was used as an index of antioxidant status. The susceptibility of red blood cells to oxidative hemolysis decreased with increasing levels of dietary vitamin E. Vaccinated and nonvaccinated fish were evaluated for agglutinating antibody titers and macrophage activity. Humoral antibody titers in response to E. ictaluri were significantly (P ≤ 0.05) higher in vaccinated fish than in nonvaccinated fish; however, no such differences in agglutinating antibody titers were detected among any of the dietary treatment groups. Both vaccination and vitamin E significantly enhanced the ability of macrophages to phagocytize virulent E. ictaluri. Results of this study indicate that elevated levels of dietary vitamin E may affect the ability of channel catfish to respond immunologically to bacterial pathogens.     

南方鲇鮰爱德华氏菌感染的病理损伤特征及病原分布研究

为研究鮰爱德华氏菌(E.ictalur)对南方鲇的病理损伤特征及病原在组织中的分布,本研究采用腹腔注射E.ictaluri (4× 105 cfu/尾)的方式感染体质量约20 g的南方鲇,并分别于接种后不同时间随机迫杀5尾实验鱼,采集各组织样品进行组织病理、细胞病理学以及病原定位检测.结果表明,感染初期实验鱼主要表现为尾柄及上颌须褪色,感染后期褪色区溃烂,断须、断尾,部分感染鱼呈头朝上尾朝下的“悬挂”姿态.组织病理学和细胞病理学结果表明,E.ictaluri最早引起肾、肝、脾出现损伤,并且这3个脏器的损伤也最为严重,表现为广泛的变质性炎症,逐渐波及到脑、心脏、鳃及肠道.免疫组化结果表明,细菌最先可在肾组织中检测到,之后出现在脾脏,然后出现在肝脏并进入肝血窦.随着病程的延长,可在脑和心肌组织中检测到病原,最后出现在肠道等组织器官.以上结果表明南方鲇感染E.ictalud主要病理变化为体表皮肤褪色、溃烂及肝脾肾变质性炎症;细菌可分布在肾、脾、肝、脑、心、肠道.     

Cool Weather Feeding Influences Responses of Channel Catfish to Edwardsiella ictaluri Challenge

Abstract

A pond study was conducted from November 1 through April 30 with young (age-0; average size, 43 g) and market-size (age-2; average size, 660 g) channel catfish Ictalurus punctatus to compare three management regimens: No feeding, partial feeding (no feeding in December, January, and February), and continuous feeding according to fish size and water temperature. Weight change, feed conversion ratio, and responses to experimental challenge with Edwardsiella ictaluri were evaluated. No significant difference in weight gain occurred between partially fed and continuously fed fish in either age-group; average weight increase for fed age-0 fish was 99% and for fed age-2 fish was 38%. The nonfed age-0 fish lost 12.3% and the nonfed age-2 fish lost 7% of their initial weight. Feed conversion ratios were significantly lower in partially fed fish than in continuously fed fish in both age-groups. Mortality from E. ictaluri challenge among age-0 fish was significantly higher in the nonfed fish, but among the age-2 fish, mortality was significantly lower in the nonfed fish. No difference in mortality rate occurred between partially fed and continuously fed fish in either age-group. Starvation induced lower antibody production against E. ictaluri antigen in the age-0 fish but higher antibody production in the age-2 fish. Phagocytic index was lower in nonfed fish than in fed fish from both age-groups. This study indicates no benefit from feeding age-0 and age-2 channel catfish during December, January, or February if feeding is reintroduced in March and continued through April. Although starvation was immunosuppressive in small channel catfish, it enhanced resistance to bacterial infection in larger fish. However, more research information is needed before reduced feeding can be recommended to enhance resistance of channel catfish to E. ictaluri.     

Chemotactic and Chemokinetic Responses of Channel Catfish Macrophages to Exoantigen from Edwardsiella ictaluri

Abstract

The ability of Edwardsiella ictaluri to attract macrophages of channel catfish Ictalurus punctatus was investigated. Exoantigen from E. ictaluri was tested for macrophage chemotactic activities both in vitro and in vivo. The exoantigen was chemotactic and chemokinetic for macrophages in vitro. Peritoneal injection of 750 μg of exoantigen protein into normal (E. ictaluri-free) channel catfish induced a marked increase in macrophage accumulations at 24 and 48 h. Neutrophil accumulation did not occur at the injection sites. Edwardsiella ictaluri exoantigen attracts macrophages, and this attraction may play an important role in macrophage responses during E. ictaluri infections.     

Use of a Mini-Transposon to Study Chondroitinase Activity Associated with Edwardsiella ictaluri

Abstract

Edwardsiella ictaluri, the etiological agent of enteric septicemia of catfish (ESC), is the leading cause of bacterial disease in commercially raised channel catfish Ictalurus punctatus. Little work has been conducted at a genotypic level to determine potential virulence characteristics, but the production of chondroitin sulfatase is a suspected virulence factor. Using transpositional mutagenesis, we created stable E. ictaluri mutants that are deficient in chondroitinase activity. Channel catfish were challenged by injection with E. ictaluri transposon mutant MI15. None of the catfish challenged with the mutant died or showed signs of ESC. These fish were held for 2 weeks and then challenged by injection with the known virulent parent strain of E. ictaluri. The challenged naive control fish showed clinical signs of and a mortality rate consistent with ESC, whereas catfish that had been injected with MI15 prior to challenge with the parent strain were resistant to disease. This work represents a preliminary study to suggest a possible role of chondroitin sulfatase activity in the virulence of E. ictaluri.     

Uptake of channel catfish virus from water by channel catfish and bluegills

Radiolabeled channel catfish virus entered the gills of juvenile channel catfish (Ictalurus punctatus) and was then concentrated in the gut and the liver over 48 hours. Diminution of radioactivity was not detected in these tissues over the course of the experiment. Bluegills (Lepomis macrochirus) were capable of clearing the virus during a period of 48 hours. [3H]Thymidine alone had a different distribution in the channel catfish than did labeled virus.     

Oxidative stress parameters in silver catfish (Rhamdia quelen) juveniles infected with Ichthyophthirius multifiliis and maintained at different levels of water pH

The aim of this study was to determine oxidative stress parameters in the liver, gill and muscle of silver catfish juveniles infected with Ichthyophthirius multifiliis and maintained at pH 5.0 or 7.0 for three days. Juveniles were infected by adding one I. multifiliis-infected juvenile and water containing theronts to tanks. After the appearance of white spots on the skin, infected juveniles exposed to pH 5.0 and 7.0 showed significantly higher thiobarbituric acid reactive substances (TBARS) levels in the liver and gills compared to uninfected juveniles. Liver of infected juveniles exposed to pH 7.0 showed higher catalase (CAT) and lower glutathione-S-transferase (GST) activities, but those maintained at pH 5.0 showed significantly higher GST activity than uninfected juveniles. The gills of infected juveniles showed significantly higher CAT (day two) and GST activity at both pH 5.0 and 7.0 compared to uninfected juveniles. Muscle of infected juveniles showed significantly lower CAT and GST activity and TBARS levels (at day three) when maintained at both pH 5.0 and 7.0 compared to uninfected juveniles. In conclusion, I. multifiliis infection induces liver and gill damage via lipid peroxidation products in silver catfish, but higher antioxidant enzyme activity could indicate a greater degree of protection against this parasite.