Efficacy of Hydrogen Peroxide to Control Parasitic Infestations on Hatchery-Reared Fish

Abstract

The efficacy of hydrogen peroxide to control external parasitic infestations on juvenile (10–33-g) rainbow trout Oncorhynchus mykiss was evaluated in three clinical field trials. Fish were exposed to hydrogen peroxide concentrations ranging from 0 to 560 mg/L for 30 min once every other day for a total of three treatments. Pre- and posttreatment skin scrapes and gill wet mounts of test fish were microscopically examined to identify and enumerate external parasites. Infestation severity was classified as nonexistent (0 organisms), low (1–10 organisms), moderate (11–20 organisms), or high (?21 organisms). In trial 1, pretreatment skin examinations revealed a severe infestation of the protozoan Ambiphrya on all fish examined. Posttreatment skin examinations conducted within 24 h of the last treatment indicated that all hydrogen peroxide treatments eliminated Ambiphrya, whereas control fish remained severely infested with the protozoan. In trial 2, pretreatment examinations of skin and gill samples indicated a high infestation of the trematode Gyrodactylus (skin) and the protozoan Trichodina (gills) on all fish. Posttreatment examinations conducted within 24 h of the last treatment indicated that Gyrodactylus was eliminated from the skin of all treated fish; however, the high infestation of Trichodina remained on the gills of the test fish. All control fish had high infestation levels of both parasites. A high infestation of Ambiphrya was found on the skin of test fish before treatment (trial 3). Posttreatment examinations conducted 14 d after the last treatment revealed that 56% of the fish were parasite free, whereas the remaining test fish had low infestation levels. Control fish remained severely infested with the parasite. Based on the efficacy data, all hydrogen peroxide treatment regimens were efficacious in the control of Ambiphrya and Gyrodactylus.     

Multitiered health assessment of Atlantic menhaden in the Pamlico River, North Carolina

During the fall of 2001 and 2002, Atlantic menhaden Brevoortia tyrannus were collected from several creeks in the Pamlico River, North Carolina, to investigate recent fish kills and ulcerative skin lesions. High skin lesion prevalence (>50%) was associated with the Atlantic menhaden kills in fall 2001, whereas there were no fish kills in fall 2002 and skin lesion prevalence was lower (< or =50%). Indicators of tissue damage (histopathological analyses of gills, heart, liver, intestine, and anterior kidney), body condition (liver somatic index), and immune status (transforming growth factor-beta [TGF-beta] messenger RNA [mRNA] production, hematology, plasma chemistry, and splenosomatic index) were compared between Atlantic menhaden with and without ulcerative skin lesions in fall. Atlantic menhaden with ulcerative skin lesions had significantly higher liver somatic indices, neutrophil and monocyte percentages, and splenic mononuclear cell TGF-beta mRNA levels than did fish without lesions. Hematocrit values, plasma protein, and Ca concentrations were significantly lower in fish with ulcerative skin lesions than in those without. The indicators used in this study at multiple levels of biological organization have provided valuable baseline data for understanding the health status of lesioned and nonlesioned Atlantic menhaden in the Pamlico River.     

Multitiered Health Assessment of Atlantic Menhaden in the Pamlico River,North Carolina

Abstract

During the fall of 2001 and 2002, Atlantic menhaden Brevoortia tyrannus were collected from several creeks in the Pamlico River, North Carolina, to investigate recent fish kills and ulcerative skin lesions. High skin lesion prevalence (>50%) was associated with the Atlantic menhaden kills in fall 2001, whereas there were no fish kills in fall 2002 and skin lesion prevalence was lower (≤50%). Indicators of tissue damage (histopathological analyses of gills, heart, liver, intestine, and anterior kidney), body condition (liver somatic index), and immune status (transforming growth factor-β (TGF-β) messenger RNA (mRNA) production, hematology, plasma chemistry, and splenosomatic index) were compared between Atlantic menhaden with and without ulcerative skin lesions in fall. Atlantic menhaden with ulcerative skin lesions had significantly higher liver somatic indices, neutrophil and monocyte percentages, and splenic mononuclear cell TGF-β mRNA levels than did fish without lesions. Hematocrit values, plasma protein, and Ca concentrations were significantly lower in fish with ulcerative skin lesions than in those without. The indicators used in this study at multiple levels of biological organization have provided valuable baseline data for understanding the health status of lesioned and nonlesioned Atlantic menhaden in the Pamlico River.     

Nested Polymerase Chain Reaction Assay for Detection of Mycobacterium shottsii and M. pseudoshottsii in Striped Bass

Abstract

Wild striped bass Morone saxatilis in Chesapeake Bay are experiencing a high prevalence of mycobacteriosis, which produces granulomatous lesions of the skin and visceral organs. Culture-based studies have indicated that the newly described species Mycobacterium shottsii and M. pseudoshottsii are the dominant isolates from diseased fish. The classical fish pathogen M. marinum is also found, albeit at much lower frequencies. Both M. shottsii and M. pseudoshottsii are extremely slow-growing on standard selective media, and up to 12 months may be required for isolation and characterization. Epidemiological studies of mycobacteriosis in Chesapeake Bay would therefore benefit from rapid molecular assays with which to detect these species in fish. In this paper, we describe the development of polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) assays capable of detecting M. shottsii, M. pseudoshottsii, and, in most instances, coinfections thereof in striped bass tissues. In addition, PCR–RFLP assays were designed to detect M. marinum and other as-yet-undescribed Mycobacterium spp. present in Chesapeake Bay striped bass. Comparison of these molecular assays with culture-based techniques using splenic tissue from wild striped bass yielded generally concordant results and demonstrated the applicability of these techniques to the study of wild fish.     

Surgical removal of cataracts due to Diplostomum species in Gulf sturgeon (Acipenser oxyrinchus desotoi).

Twenty 6-yr-old (1995-yr-class) Gulf of Mexico sturgeon (Acipenser oxyrinchus desotoi) were diagnosed as having bilateral cataracts. Histopathologic assessment of the lenses of two of the fish revealed the presence of a diplostomid trematode. Pharmacological treatment of the trematodes may be effective for killing the parasites, but the damage to the lenses and resulting cataracts are nonreversible. Because these animals were to be used in a subsequent study as sentinels in the natural environment, it was necessary to return the animals' vision to as close to normal as possible. Electroretinograms were performed on each fish's eyes to ensure that retinal function was present. Cataracts then were surgically removed by phacoemulsification and aspiration. The animals tolerated the surgical procedures well. This report is the first known report of surgical correction of cataracts in sturgeon species. It also is the first known attempt to correct vision problems in fish being returned to the wild.     

Quantitation of immune response gene expression and cellular localisation of interleukin-1beta mRNA in Atlantic salmon, Salmo salar L., affected by amoebic gill disease (AGD)

The characterisation of selected immune response genes during amoebic gill disease (AGD) in Atlantic salmon, Salmo salar L., was performed using semi-quantitative RT-PCR, quantitative real-time RT-PCR (qRT-PCR), and in situ hybridisation (ISH). The immune response genes of interest were interleukin-1beta (IL-1beta), inducible nitric oxide synthase (iNOS), serum amyloid A (SAA), and serum amyloid P-like pentraxin (SAP). Atlantic salmon were inoculated with the ectoparasite Neoparamoeba sp., the causative agent of AGD, and gill, liver and anterior kidney tissue sampled at 0, 7 and 14 d post-inoculation (p.i.). Semi-quantitative RT-PCR was performed on the tissue samples to identify up/down-regulated mRNA expression relative to uninfected control fish and normalised to the housekeeping gene, beta-actin. Interleukin-1beta (IL-1beta) was the only immune response gene of those investigated whose mRNA was differentially regulated in any of the tissues and was found to be up-regulated in the gills by semi-quantitative RT-PCR. Increased gill IL-1beta mRNA expression was then accurately quantitated and confirmed using probe-based qRT-PCR. The cellular localisation of the IL-1beta mRNA expression in the gills of uninfected and infected fish was then determined by ISH using an IL-1beta-specific biotinylated cRNA probe. Expression of IL-1beta mRNA was localised to filament and lamellar epithelium pavement cells in gills of uninfected and infected Atlantic salmon. These data implicate the involvement of IL-1beta at the site of infection, the gills, of Atlantic salmon during AGD. This work supports previous studies that suggest IL-1beta is important in the regulation of the fish immune response to parasitic infection but additionally shows the cellular localisation of fish IL-1beta mRNA expression during infection.     

Histopathology of Five Species of Didymocystis spp. (Digenea: Didymozoidae) in Cage-reared Atlantic Bluefin Tuna (Thunnus thynnus thynnus)

The first parasitological examinations of Atlantic bluefin tuna Thunnus thynnus thynnus reared in the Adriatic Sea revealed high numbers of different digenean trematodes, belonging to family Didymozoidae. Five species were the most frequently isolated: Coeliodidymocystis, abdominalis, Didymocystis wedli, Koellikerioides internogastricus, K. intestinalis and K. apicalis. Parasites were isolated from gills, gill rakers, intestine, serosa of pyloric caeca, and inner layers of the fundic region of stomach, encysted in pairs in connective tissue capsules originating from host tissue. Gross pathology revealed no pathological changes. Histological alterations comprised tissue reaction resulting in lymphocyte infiltration and formation of a collagenous envelope around the parasite cysts; the intensity of observed reaction depended on the size of the cyst and on the tissue infected. The most notable changes were observed in the intestine under provocation by K. intestinalis, while the most potentially pathogenic didymozoid under fish rearing conditions is D. wedli, which if present in high abundance can induce secondary bacterial infections.     

Abundance of the Parasitic Copepod Caligus elongatus on Wild Pollock near Commercial Salmonid Net-Pens

Abstract

Between July and December 1993, wild pollock Pollachius virens collected near salmonid net-pens on the Atlantic coast of Maine in the United States were examined for parasitic copepods of the family Caligidae to determine the role of pollock as host reservoirs. Only Caligus elongatus was detected on 1,456 pollock sampled. Most pollock (97.39%) had two or fewer sea lice per fish. A maximum prevalence (percent of fish infested) of 50%, an abundance (average number per fish) of 0.9, and an intensity (average number per infested fish) of L79 were recorded. Only one larval C. elongatus was found. No seasonal or geographic trends in infestation statistics were observed.     

Clinical and Pathological Effects of the Polyopisthocotylean Monogenean,Gamacallum macroura in White Bass

Abstract

An aquaculture research facility experienced high mortality rates in white bass Morone chrysops associated with a monogenean infestation of the gills, but not in striped bass Morone saxatilis in the same facility. All mortalities had pale gills. Monogeneans, identified as Gamacallum macroura (MacCallum and MacCallum 1913) Unnithan 1971, were found on the gills. Pale-gilled and healthy white bass were selected with no particular attention to condition for venipuncture and euthanasia for postmortem examination, including parasite counts from gills. The median packed cell volume (PCV) of fish with gill pallor was 12.5% (range 9–37%) while PVC of fish with more normal color was 30% (27–33%). Association between the PCV and gill pallor score was statistically significant, as was the association between PCV and the number of monogeneans found on the gills of each fish. Median estimated white blood cell count of fish with gill pallor, at 12.05 × 10^3/μL (range 3.8–24.7), was significantly lower than of apparently healthy fish: 24.7 × 10³/μL (17.3–31.5). Histopathology of the gill arches of pale-gilled fish revealed multifocal moderate to severe branchitis, focal areas of dilated hyperplastic lamellae occluded by fibrin, and monogeneans attached to the lamellae. Fish that were apparently healthy had grossly similar histologic lesions, but at lower frequency and severity.

Received May 27, 2011; accepted July 12, 2012     

Proliferative Gill Disease of Fish from the Tennessee-Tombigbee Waterway,Mississippi

Abstract

Proliferative gill disease (PGD), a condition not previously reported in wild fish, was found in two channel catfish Ictalurus punctatus sampled from the Tennessee-Tombigbee Waterway in Mississippi during June and July 1989. The parasite thought to cause PGD was observed in only one of the fish, but the distinctive lesions associated with this disease were prominent in both of the channel catfish. Organisms resembling the PGD parasite were also found in the gills of 4 of 18 largemouth bass Micropterus salmoides and 6 of 20 bluegills Lepomis macrochirus, but there was little or no host response to these parasites.     

Moritella viscosa bypasses Atlantic salmon epidermal keratocyte clearing activity and might use skin surfaces as a port of infection

Moritella viscosa is considered the main causative agent of winter ulcer disease in salmonid fish. In order to obtain more details on route of infection, we challenged Atlantic salmon (Salmo salar) epidermal keratocytes with M. viscosa and performed an Atlantic salmon immersion challenge. Although keratocytes were able to remove M. viscosa from surfaces, their engulfment capability appeared inefficient with reduced ability to reepithelialise superficial wounds (scale less skin surfaces) challenged with the bacterium. The immersion challenge revealed a significant connection between exposure area and mortality. Enhanced invasion ability and mortality was observed by M. viscosa exposure of the head and gill region compared to exposure of: the right side of the body; the left side of the body; or the body from pectoral to caudal fin (p=0.04). Ulcer development corresponded to area exposed (p=0.002), suggesting skin ulcer formation to result primarily from direct skin surface colonization. Ulceration of surfaces exposed to M. viscosa in parallel with occurrence of septicaemia suggests that both skin and gills may act as possible initiation sites for M. viscosa infections.     

Induction and Evaluation of Proliferative Gill Disease in Channel Catfish Fingerlings

Abstract

Proliferative gill disease (PGD) was first reported in channel catfish Ictalurus punctatus at commercial farms in 1981 and is caused by the myxozoan parasite Henneguya ictaluri. The disease affects the gills and is characterized by severe branchial inflammation, epithelial hyperplasia, lamellar fusion, and lysis of chondrocytes. Presumptive diagnosis is based on the presence of lytic areas in the cartilage of the primary lamellae on microscopic examination and is confirmed histologically by the presence of the organism. In these trials, PGD was induced by exposing channel catfish fingerlings to fresh or aged infectious water collected from a pond containing fish diagnosed with severe PGD. The severity of disease was graded by histological scoring and microscopic examination of wet mounts to determine the percentage of gill filaments containing chondrolytic lesions. Exposure of fish to infectious pond water was shown to produce pathological lesions consistent with PGD, and the percentage of gill filaments containing chondrolytic lesions was positively correlated with histological scoring of gill pathology. The number of trophozoite stages in the gills was shown to increase with the severity of the disease. In most cases, however, parasitic cells were not observed in tissue samples with chondrolytic lesions during the early stages of infection. These observations indicate that pathology and lysis of chondrocytes can occur prior to detection of the organism by histopathology. Exposing fish to infectious pond water that was aged for 1 d produced negligible gill pathology and implies that the infectivity of the H. ictaluri actinospore stage is short lived. Removing fish from the source of infection promoted repair of damaged gill tissue; within 14 d of fish transfer to clean water, gill pathology associated with the acute infection was negligible.     

Iridovirus infections among Missouri River sturgeon: initial characterization, transmission, and evidence for establishment of a carrier state

Iridovirus infections of the integument were associated with disease and mortality among hatchery-reared populations of juvenile pallid sturgeon Scaphirhynchus albus and shovelnose sturgeon S. platorynchus from the Missouri River. Virus-infected cells in the integument of fins and body were greatly enlarged, possessed pleomorphic and eccentric nuclei, and exhibited an amphophilic to eosinophilic staining of the cytoplasm in hematoxylin-and-eosin-stained sections. Virus particles found in the host cell cytoplasm were composed of an outer hexagonal capsid measuring 254 nm in diameter and surrounding a dense nucleoid. Despite numerous attempts, the virus could not be propagated on routine cell lines used in fish viral diagnostics or from established cell lines from white sturgeon Acipenser transmontanus, pallid sturgeon, or shovelnose sturgeon. Bath exposures of healthy juvenile pallid sturgeon to a crude extract or a 0.45-microm-filtered extract from the fins of infected fish resulted in transmission of the virus and mortality. At water temperatures of 15 degrees C, the first deaths occurred at approximately 1 month; mortality peaked between 50 and 60 d postexposure, after which surviving fish recovered. Presence of the virus was confirmed among dead and moribund pallid sturgeon by both histology and detection of viral DNA by polymerase chain reaction methods. Feeding of infected tissues and cohabitation with virus-infected shovelnose sturgeon also resulted in successful virus transmission to juvenile pallid sturgeon. Virus infections among experimentally exposed pallid sturgeon that recovered from clinical episodes persisted for at least 8.5 months, and these apparently healthy fish transmitted the virus and disease to juvenile pallid sturgeon by cohabitation. The newly described Missouri River sturgeon iridovirus (MRSIV) as found in pallid sturgeon and shovelnose sturgeon shares many properties with a group of iridoviruses associated with serious skin and gill infections in several species of sturgeon.     

The occurrence of Contracaecum sp. larvae (Nematoda: Anisakidae) in the catfish Clarias gariepinus (Burchell) from Lake Chivero, Zimbabwe

Clarias gariepinus were collected from Lake Chivero, Zimbabwe, and examined for nematode parasites from November 2000 to May 2002. Of the 202 specimens collected, 42.6% were infected with third-stage larvae of Contracaecum sp. in the body cavity. The intensity of the infection was 1-7 worms per fish (mean intensity = 2.2). Seasonal variation in the prevalence of the parasite was not obvious and there was no significant difference in the prevalence of infection between males and females (chi 2 = 2.228; P > 0.05). No significant relationship between host size and prevalence was established. There was also no significant relationship between intensity and the body condition factor (r = 0.11; P > 0.05). The low parasite prevalence may have been caused by the disruption of the infection cycle since piscivorous birds, which are the final hosts of the parasite, do not feed on C. garieplnus in Lake Chivero.     

Cryptobia iubilans infection in juvenile discus

Four commercial producers of discus (Symphysodon aequifasciatus) were found to have fish infested with the flagellate Cryptobia iubilans. Affected fish had granulomatous gastritis, and many also had granulomatous disease of other organs. The parasite had to be differentiated from the related flagellates Spironucleus spp, which induce different lesions. Transmission electron microscopy was found to be useful in detecting and identifying the parasite. Morbidity and mortality rates in the various fish populations appeared to be linked to a number of variables, including water quality, presence of other parasites and bacteria, diet, species, size, and age of the fish, and optimization of husbandry appeared to be important in alleviating the severity of disease. Metronidazole was not effective for treatment of C iubilans, but bath treatments with dimetridazole (80 mg/L for 24 hours, repeated daily for 3 days) or 2-amino-5-nitrothiazol (10 mg/L for 24 hours, repeated daily for 3 days) may be useful in decreasing the prevalence of infestation.     

Aspects of the pathology of Lamproglena clariae (Copepoda: Lernaeidae) on gills of Clarias gariepinus from the Vaal River system,South Africa

Lamproglena clariae attaches to gill filaments of Clarias gariepinus, penetrates the gill tissue and consumes blood. Hence, the purpose of this study was to investigate the mechanical damage caused by this parasite on gill filaments of the host. Infestation intensity and haematocrit values of fish blood were determined and correlated. Fish were killed, gills dissected out and observed with a dissection microscope. Gill filaments with adult females in situ were fixed in a solution of alcohol, formaldehyde and acetic acid and preserved in 70% ethanol. Fixed specimens were studied by histological examination following staining with Heidenhein’s azan solution. Gross morphology revealed varying degrees of swelling of gill filaments near the attached specimens of L. clariae. Epithelial hyperplasia, resulting in fusion of gill lamellae and necrosis of the host tissue in the vicinity of the parasite were revealed through histological examination. No correlation was evident between the infestation intensity and fish haematocrit values. Although results showed that L. clariae causes localized infestation, high intensity of infestation may be detrimental to the fish. Hyperplasia of epithelial tissue resulting in fusion of lamellae would result in restriction of air passages and thus hinder the process of respiration of the host.     

MHC class II+ cells in the gills of Atlantic salmon (Salmo salar L.) affected by amoebic gill disease

Amoebic gill disease (AGD) is characterised by the association of Neoparamoeba sp. with hyperplastic gill tissue of affected fishes, however, the identity and role of host cells associated with AGD lesions are not known. Here, we investigated cells with an immunological role that were associated with AGD lesions by locating cellular MHC class II β chain. A tank housing Atlantic salmon (Salmo salar) was inoculated with Neoparamoeba sp., and MHC class II β chain expression in the gills was qualitatively assessed by immunohistochemistry. In AGD-naïve control fish, MHC class II⁺ cells were detected basolateral to the interlamellar epithelium as well as upon the interlamellar and secondary epithelium. In the gills of AGD affected fish MHC class II⁺ cells were observed in both affected and unaffected tissue. Within AGD lesions, numerous MHC class II⁺ cells were present and these cells exhibited variable levels of expression suggesting that like mammals, MHC class II expression is highly regulated. The presence of MHC class II⁺ cells within gill lesions is indicative of immune cell trafficking and these cells could contribute in an antigen presentation capacity to the development of an antibody response in fish chronically affected by AGD.     

Microhabitat use, not temperature, regulates intensity of Gyrodactylus cichlidarum long-term infection on farmed tilapia--are parasites evading competition or immunity?

Gyrodactylids (Monogenea) are ectoparasites of fish, some of which negatively affect commercially valuable fishes. Temperature strongly regulates population dynamics of these viviparous flatworms in farmed and wild fish populations, with most gyrodactylid species showing positive temperature-abundance associations. In agreement with epidemiological theory, numerous laboratory studies demonstrate that these parasites cannot persist in confined fish populations without periodic introduction of susceptible hosts. Extinction of gyrodactylid populations is due to host immunity, which develops in several fish species. In this one-year study, we followed populations of the recognized pathogen Gyrodactylus cichlidarum infecting four genetic groups of confined tilapia (wild type Nile tilapia Oreochromis niloticus niloticus, red O. n. niloticus, Mozambique tilapia O. mossambicus and a red synthetic population called Pargo-UNAM) kept under farming conditions and subject to natural environmental fluctuations. Based on the antecedents given, we postulated the following three hypotheses: (1) parasite abundance will be regulated by water temperature; (2) parasites will induce host mortality, particularly during periods of rapid infrapopulation growth; and (3) gyrodactylid populations will eventually become extinct on confined fish hosts. We disproved the three hypotheses: (1) parasite numbers fluctuated independently of temperature but were associated to changes in microhabitat use; (2) although gyrodactylid populations exhibited considerable growth, no evidence was found of negative effects on the hosts; and (3) infections persisted for one year on confined fish. Microhabitat use changed over time, with most worms apparently migrating anteriorly from the caudal fin and ending on the pectoral fins. Gyrodactylid populations followed similar trajectories in all fish, aggregating and dispersing repeatedly. Several instances were found where increased parasite dispersion coincided with increased intensity of infection; as well as the opposite, where increased aggregation coincided with parasite population declines. Three alternative explanations could account for these observations: that parasites (1) experience differential mortality on different anatomical regions of the fish; (2) migrate to avoid intraspecific competition; and (3) migrate to escape localized immune responses induced by infection. Our data do not allow us to demonstrate which of these alternatives is correct, so we discuss the merits of each. We provide circumstantial evidence in support of the third explanation, because as shown in other fish host-gyrodactylid interactions where immune responses have been characterized, in this study worms progressively moved away from fins with high mucus cell density to those with low density - what would be anticipated if immune defenses occur and reach the fish surface through mucus.     

Experimental handling stress as infection-facilitating factor for the goldfish ulcerative disease

Experimental handling stress (EHS) was applied to clinically asymptomatic farmed goldfish (Carassius auratus L.). EHS affected the gills and skin integrity of the fish and was accompanied by increased levels of plasma glucose, cortisol and interleukin-10 (IL-10). EHS application was followed by highly significant enhancement of the rate of infection with a virulent Aeromonas salmonicida isolate. Cumulative ulceration at the initial phase of the ensuing goldfish ulcerative disease (GUD) evidenced a facilitating role of EHS in the onset of GUD. Host susceptibility to the pathogen increased from 40% in unstressed fish to 90% in the stressed fish. A. salmonicida could be reisolated from the early-stage skin lesions only, whereas opportunistic strains, other than A. salmonicida (A. sobria and A. hydrophila), were recovered from progressive-stage ulcers. The implication of these findings in fish aquaculture is discussed.