Susceptibility of Selected Inland Salmonids to Experimentally Induced Infections with Myxobolus cerebralis,the Causative Agent of Whirling Disease

Abstract

Laboratory exposures to the infectious stages (triactinomyxons) of Myxobolus cerebralis demonstrated a range of susceptibility to whirling disease among four species of inland salmonids. Replicate groups of each species were exposed to two concentrations of triactinomyxons, a low dose (100–200 per fish) and a high dose (1,000–2,000 per fish). Exposed fish were evaluated for clinical signs, for severity of microscopic lesions at 35 d, 2 and 5 months, and for spore concentrations in the head cartilage at 5 months. A standard strain of rainbow trout Oncorhynchus mykiss matched for age served as a susceptible species control. Rainbow trout, westslope cutthroat trout O. clarki lewisi, Yellowstone cutthroat trout O. clarki bouvieri, and bull trout Salvelinus confluentus were susceptible to M. cerebralis infections. Clinical signs, including radical swimming (“whirling”) and black tails, were observed at 7 weeks postexposure among rainbow and cutthroat trout challenged at 3 weeks of age. Clinical signs were rare among bull trout exposed at an age of 4 weeks and absent among rainbow and cutthroat trout exposed at 3 months posthatch. Most rainbow, cutthroat, and bull trout were found to be infected when examined at 5 months postexposure. The most severe microscopic lesions among infected fish at 5 months postexposure were found among rainbow trout. Cutthroat trout had less severe lesions, bull trout had mild infections, and no evidence of infection was found among Arctic grayling Thymallus arcticus. Mean spore concentrations among infected fish correlated with the severity of microscopic lesion scores. Rainbow trout had mean concentrations of spores in head cartilage reaching 10⁶, whereas more resistant species such as bull trout had 10⁴ spores; no spores were found among Arctic grayling at 5 months postexposure.     

Development of Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea) in bryozoan hosts (as examined by light microscopy) and quantitation of infective dose to rainbow trout (Oncorhynchus mykiss)

The myxozoan parasite Tetracapsuloides bryosalmonae is the causative agent of proliferative kidney disease (PKD), a highly damaging disease of cultured salmonid fish. Within this study, phylactolaemate bryozoans were collected from a river known to be endemic for PKD and subsequently cultured in the laboratory. Sequential developmental stages of T. bryosalmonae were studied by light microscopy within the living bryozoan colonies, allowing the identification of stages attached to host peritoneum, consistent with previous molecular evidence of cryptic stages. Infection resulted in the production of large numbers of spores, which were released from the bryozoans. Experimental exposure of rainbow trout (Oncorhynchus mykiss) to medium in which infected bryozoans were cultured resulted in clinical PKD. Rainbow trout were exposed to known numbers of T. bryosalmonae spores collected by micromanipulation, which had been released from mature spore sacs within colonies of the bryozoan Fredericella sultana. Exposure to one spore was sufficient to lead to development of PKD. These findings indicate that small numbers of bryozoans are capable of releasing sufficient spores to infect large numbers of fish, having implications for future control methods for PKD in salmonid farming.     

Experimental Infection of Brook Trout with Infectious Hematopoietic Necrosis Virus Types 1 and 2

Abstract

Fry of brook trout Salvelinus fontinalis became infected and diseased after immersion exposure to infectious hematopoietic necrosis virus (IHNV), but a long-lasting IHNV carrier state was not induced. Duplicate groups of 100 fish were immersed for 6 h in baths containing a type 1 (Round Butte, RB) or a type 2 (Rangen, RA) IHNV isolate at a high or low dose. Brook trout mortalities induced by immersion in a bath of the RB or RA IHNV isolate at 10² plaque-forming units (pfu) per milliliter were equivalent (1 and 0%), but fish were more susceptible to infection with RA IHNV. Only the single dead fish in the RB group was infected, but 24% of the RAexposed fish were infected 1 week after exposure. At a dose of 10⁶ pfu/mL, exposure to RB IHNV resulted in a higher mortality (35%) and prevalence of infection (89% of live fish sampled at 1 week postexposure), but no infectious virus was detectable by 5 weeks after exposure. In contrast, RA IHNV exposure at a dose of 10⁴ pfu/mL resulted in only 5% mortality, and live fish killed at 1 week postexposure had a 22% prevalence of infection, but infectious virus was not detectable by week 3. Although brook trout have been previously considered to be resistant to IHNV, this study has shown that brook trout become diseased and die after exposure to a high dose of one type I IHNV isolate and can be infected after immersion exposure to even a low dose of type 1 or type 2 IHNV.     

The immune response of a marine teleost, Pseudopleuronectes americanus, (winter flounder) to the protozoan parasite Glugea stephani

G. stephani is an intracellular cyst-forming microsporidan parasite that is found in the intestine of winter flounder (WF) Pseudopleuronectes americanus. No detectable humoral response was seen in parasitized fish or in fish injected with either spores or spore homogenate from this parasite. Quantification of total immunoglobulin (Ig) levels showed a decrease in Ig levels rather than enhancement, 21 days after intramuscular (IM) injections of spores (3 X 10(6)/ml). When a second injection of spores was administered on day 21 and tested 3 weeks later, a further decrease in total serum Ig's occurred. A decrease in total IgM levels also occurred in WF that were simultaneously injected with G. stephani and the antigens, horse red blood cells (HRBC) or formalin-killed Klebsiella pneumonia (KP). The total Ig levels of fish injected with an antigen plus spores was not as low as those injected with the parasite alone. The Ig levels, as well as antibody titers to HRBC and KP were however, lower when compared to fish injected only with the HRBC or bacteria. Disrupted spore homogenate injected into winter flounder, showed a less marked decrease in Ig levels when compared with whole spores. When a single IM injection of spores was given, followed by two weekly injections of indomethacin (a drug that inhibits prostaglandin activity), no decrease in Ig levels occurred and levels were comparable to control (saline injected) fish.     

Comparison of the susceptibility of brown trout (Salmo trutta) and four rainbow trout (Oncorhynchus mykiss) strains to the myxozoan Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD)

Differences in susceptibility to the myxozoan parasite Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD), between four strains of rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) were evaluated. Fish were exposed to water enzootic for the parasite in the field for 5 days and were subsequently transferred to the laboratory. Relative parasite load was determined after 2, 3 and 4 weeks post-exposure (wpe) by quantitative real-time PCR (qPCR) of kidney samples and number of parasite stages was determined in immunohistochemical stained sections of kidney, liver and spleen tissues. According to qPCR results, the highest amount of parasite DNA per equal amount of host tissue at all time points was measured in brown trout. Two of the rainbow trout strains showed lower relative parasite load than all other groups at the beginning of the experiment, but the parasite multiplied faster in these strains resulting in an equal level of relative parasite load for all rainbow trout strains at 4 wpe. A weak negative correlation of fish size and parasite load was detected. Only in samples of a few fish, single stages of T. bryosalmonae were found in sections stained by immunohistochemistry impeding quantitative evaluation of parasite numbers by this method. The results indicate a differential resistance to T. bryosalmonae between the rainbow trout strains investigated and between rainbow trout and brown trout.     

Interactions between ecto- and endoparasites in trout Salmo trutta.

Trout fry were experimentally infected with endoparasites (Anisakis sp.) and/or ectoparasites (Gyrodactylus derjavini). During the following 3 weeks the infection level of G. derjavini was significantly lowered in fish infected with live anisakids compared to fish injected with physiological saline, a corresponding amount of Anisakis protein sonicate or to untreated fish. Subsequent scanning electron microscopy (SEM) of recovered nematodes from the trout showed an extensive colonization of the worms with host macrophages. It is indicated that the activation of the fish immune system by infection with live anisakids influences the skin response of the host with a subsequent negative effect on the ectoparasites G. derjavini.     

Phagocytic activity of macrophages of rainbow trout against Vibrio anguillarum and the opsonising effect of antibody and complement

The phagocytosis of Vibrio anguillarum by peritoneal macrophages from normal rainbow trout was enhanced by antibody and complement. Treatment of either macrophages or the bacteria by antibody also enhanced opsonisation. Five weeks after immunisation with V anguillarum, the phagocytic activity of macrophages from rainbow trout was increased significantly compared with the activity of those from unvaccinated fish. Although agglutinin titres did not increase until three weeks after immunisation, seven out of 10 fish challenged one week after immunisation survived, indicating that immunised fish had developed resistance to vibrio infection before significant levels of antibody or phagocytic activity became detectable.     

Polycystic kidney disease in goldfish (Cusrassius auratus)

Abstract Thirty percent of 60 goldfish in an outside pond showed progressive development of swollen abdomens and floatation problems. The occasional fish was found dead. Necropsy of an affected fish showed an abdomen almost filled with a large bilobed ventral mass comprised of masses of cysts filled with clear fluid. On histopathological examination, this mass appeared to be composed of large cystic spaces lined by a single flattened epithelium. Occasional remnants of normal kidney tissue were seen withen the mass.     

Monitoring the spore dynamics of Aphanomyces astaci in the ambient water of latent carrier crayfish

The specialized crayfish parasite Aphanomyces astaci causes the devastating crayfish plague in European crayfish. Even though A. astaci sporulation has been thoroughly studied under pure culture conditions, little is known about the sporulation dynamic from its live host. Our purpose was to investigate the A. astaci spore dynamic in its native parasite-host relationship by monitoring the sporulation from carrier crayfish into the ambient water using agent specific qPCR. American signal crayfish (Pacifastacus leniusculus) with known positive carrier status were housed individually and communally in two experimental set-ups using multiple replicates and different temperatures. Water samples were collected weekly, and spore numbers were quantified. We demonstrate here that live latent carrier crayfish continuously released a moderate number of A. astaci spores (~2700 spores per crayfish/week) in the absence of death and moulting events. In contrast, a pronounced sporulation increase was seen already one week prior to death in moribund crayfish, suggesting a crayfish plague-like condition developing in weakened or stressed individuals. Significantly more spores were produced at 18°C compared to 4°C, while a negative correlation was detected between spore numbers and temperatures rising from 17 to 23°C. This study is the first attempt to quantify the spore release from carrier crayfish on the basis of qPCR applied on water samples, and demonstrate that the approach successfully unravel A. astaci sporulation patterns. The results emphasize that carrier crayfish pose a constant infection risk to highly susceptible crayfish species regardless of crayfish life cycle state.     

Polycystic kidney disease in goldfish (Cusrassius auratus)

Thirty percent of 60 goldfish in an outside pond showed progressive development of swollen abdomens and floatation problems. The occasional fish was found dead.

Necropsy of an affected fish showed an abdomen almost filled with a large bilobed ventral mass comprised of masses of cysts filled with clear fluid. On histopathological examination, this mass appeared to be composed of large cystic spaces lined by a single flattened epithelium. Occasional remnants of normal kidney tissue were seen withen the mass.     

Experimentally Induced Whirling Disease I. Dose Response of Fry and Adults of Rainbow Trout Exposed to the Triactinomyxon Stage of Myxobolus cerebralis

Abstract

The intensity and prevalence of whirling disease was tested by exposure of 2-monthold fry and 1-, 2-, and 3.5-year-old adults of rainbow trout Oncorhynchus mykiss to a known number of laboratory-produced Myxobolus cerebralis at the actinosporean triactinomyxon stage. Fry exposed to graded concentrations of infectivity (triactinomyxons) for 3 h were individually examined for spores of Myxobolus cerebralis 5 and 6 months later. Exposure of fish to the lowest doses, 1 and 10 triactinomyxons per fish, did not result in detectable myxosporean spores. Fish that became lightly infected by a dose of 100 triactinomyxons per fish experienced a decrease in the incidence of infection between 5 and 6 months after exposure. A linear relationship was found between the numbers of recovered myxosporean spores and doses of 100–10,000 triactinomyxons per fish, and the spore burden appeared to plateau at doses of 10,000–100,000 triactinomyxons per fish. Adult fish continuously exposed to the highest dose of triactinomyxons for 3.5 months were infected and asymptomatic, however, the severity of myxosporean infection decreased with increased age of fish. This information may help in controlling whirling disease in salmonids.     

In vivo pulmonary response to Aspergillus terreus spores

The fate of intratracheally instilled Aspergillus terreus spores was followed in both rabbits and rats. Phagocytosis of the spores by the pulmonary macrophage was rapid in that approx. 42% of the observed spores were associated with the macrophages immediately after instillation. Direct penetration of the lung architecture by the spores was not observed but spores were seen in the alveolar interstitium at 3 hr after instillation and in the tracheobroncheal lymph nodes at 24 hr. Granulomas formed between 48 hr and 1 week after exposure. In the absence of apparent spore or spore extract toxicity and precipitating antibodies against Aspergillus terreus, the observed reactions preclude the possibility that the lesions were the consequence of hypersensitivity. This model of pulmonary response to fungal spores may be of future value for characterizing further the pathology associated with certain occupational exposures to moldy materials.     

The Effects of Whirling Disease on Growth and Survival of Snake River Cutthroat and Colorado River Rainbow Trout Fingerlings

Abstract

Two sizes of fingerling Snake River cutthroat trout Oncorhynchus clarkii behnkei and Colorado River rainbow trout O. mykiss were raised at hatcheries testing negative for Myxobolus cerebralis and stocked into the Dolores and Cache la Poudre rivers from 1999 to 2001. Populations were resampled over a 2-year period to determine which species and size combination had the highest growth and survival rates. Fish were tested for M. cerebralis via polymerase chain reaction and pepsin?trypsin digest analyses. Growth and survival rates between the species and size groups were not significantly different in either river. In the Dolores River, annual survival for both species and sizes of fish combined ranged from 0.063 to 0.12. In the Cache la Poudre River, survival for both sizes of rainbow trout was 0.004; survival for cutthroat trout ranged from 0.182 to 0.53. Larger fish had higher growth rates than smaller fish, and cutthroat trout had higher rates than similar sizes of rainbow trout. In both rivers, a higher percentage of the rainbow trout sample was infected than in the cutthroat trout sample. Rainbow trout also had a higher mean number of spores per head than cutthroat trout, and small rainbow trout had higher spore counts than large rainbow trout. Survival rates for cutthroat trout in the Cache la Poudre River were the highest of any of the groups, suggesting a difference that is biologically significant. Raising fingerlings to sizes greater than 100 mm can improve poststocking survival. If rainbow trout are stocked into contaminated waters, raising fingerlings to a larger size does not appear to improve growth or survival rates. Stocking rainbow trout in the spring could maximize growth rates but will expose fish to greater triactinomyxon densities, resulting in higher intensities of infection.     

Activity of pulmonary intravascular macrophages in cats and dogs with and without adult Dirofilaria immitis

Pulmonary intravascular macrophages (PIMs), large (20-80 microm diameter) monocytes are present in sheep, pigs, and horses, but not in dogs, rats, rabbits, or primates. The present study evaluated the phagocytic activity of various organs in cats and dogs and determined the influence of Dirofilaria immitis infections on PIM activity. Live or dead adult heartworm (HW) was transplanted via jugular venotomy into cats and dogs. Cats (four per group) were allocated to five groups: surgical controls--no HW, dead HW for 1 week, live HW for 1 week, dead HW for 3 weeks, or live HW for 3 weeks. Radioactive technetium (Tc-99m, 1.2mCi in 0.3ml) sulfa-colloid was injected intravenously. All cats with HW were clinically asymptomatic and developed radiographic pulmonary parenchymal changes. No gross changes were visible at necropsy for cats with HW; inflammatory changes were less severe in cats with live HW. In cats with dead HW for 3 weeks, worms were present but folded, flattened, and located in distal pulmonary arteries. Uninfected control dogs and those with dead HW did not demonstrate any PIM activity. In control cats, lungs were the primary phagocytic organ after systemic IV colloid injection (72.5% of the total recovered radioactive dose). The lung and liver together represented over 95% of the recovered Tc-99m colloid in all cats. In each group of cats with HW, phagocytic activity of the lung was significantly less (p < 0.001) than the PIM activity of controls. Cats with dead HW at 1 week (50.1%) had a significant (p < 0.019) decrease in PIM activity compared with cats with dead HW at 3 weeks (59.5%). The PIM activity in cats with live HW was significantly decreased (p < 0.001) from that in groups with dead HW, but there was no significant difference between the two groups infected with live worms. There were no significant differences in recovery between any groups in pairwise analysis of the spleen, heart, skeletal muscle, kidney, bone marrow, or blood. Significant increases (p < 0.001) in liver activity for each group inversely reflected the decreased lung activity; consistent with increased hepatic uptake of Tc colloid "escaping" a relatively suppressed lung macrophage system. Transmission electron microscopy confirmed PIM glycocalyx changes and vacuolization, moderate Type 1 cell damage and Type II cell hypertrophy in cats with dead HW. There was no evidence of PIM death. The significant decrease in PIM activity in groups with dead HW and a greater decrease in groups with live HW are consistent with a down-regulation of PIM function in cats with live HW.     

Cellular Inflammatory Response of Rainbow Trout to the Protozoan Parasite that Causes Proliferative Kidney Disease

Abstract

The cellular inflammatory response of rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri) to the myxozoan parasite PKX that causes proliferative kidney disease was investigated. The response was studied from 3 to 20 weeks after the fish were injected with infected kidney homogenate. Kidney samples were examined by light and electron microscopy. In contrast to most myxosporeans, PKX provoked a severe host response. Parasites were found in peritubular capillaries and sinusoids 3 weeks postinjection. The initial response to PKX was hemopoietic hyperplasia followed by a marked granulomatous nephritis that was resolved by termination of the study at 20 weeks postinjection. The macrophage was the predominant cell type involved in the inflammatory response to PKX. We presume that the macrophage effectively interrupts the development of PKX and eliminates the parasite from the host.     

Contribution of food deprivation to the immune response in rainbow trout (Oncorhynchus mykiss) vaccinated against Cryptobia salmositica and Aeromonas salmonicida

The aims of the present study were to determine (a) the effectiveness of an attenuated live Cryptobia salmositica vaccine; (b) the effects of food deprivation on the immune response and its duration in rainbow trout (Oncorhynchus mykiss) immunised with a live C. salmositica vaccine or with a killed Aeromonas salmonicida vaccine. The fish were divided into three groups (I, II and III; 14 fish per group), those in Groups I and II were under food deprivation (0.40% of body weight), while Group III fish were fed to satiety. The study showed that the attenuated strain of C. salmositica did not cause anaemia and disease, and the fish were protected from clinical disease when they were challenged with virulent parasites. Parasitaemia in all fish vaccinated and challenged with virulent C. salmositica fluctuated and was relatively low; however, fish in Group III had higher parasitaemia than those in Groups I and II between weeks 8 and 14. The numbers of activated neutrophils increased [nitroblue tetrazolium (NBT) assay] after immunisation with both Cryptobia and Aeromonas vaccines and they remained high throughout the experiment. Antibody production (ELISA values) increased after vaccination and were slightly higher in Group III. ELISA titres against A. salmonicida increased after vaccination and decreased after 5 weeks. The titres increased again after the vaccinated fish were given booster, and they were higher than those in the first vaccinated fish.     

Protection against murine potomac horse fever by an inactivated Ehrlichia risticii vaccine

Ehrlichia risticii propagated in a murine macrophage cell line were freed from the host cell by hypotonic lysis of the infected cells. The cell-free ehrlichiae were inactivated with beta-propiolactone and combined or not combined with polymyxin-B. The vaccines were administered to mice with Quil-A (saponin) as an adjuvant twice at 2 to 3 week intervals and the mice were challenged with live E. risticii 2 to 3 weeks after the last vaccination. With or without the addition of polymyxin-B, the vaccine preparations protected mice from developing clinical signs and gross pathologic changes such as thymic atrophy, splenomegaly, and increase in whole intestinal weight. Mice vaccinated with or without polymyxin-B developed high titer IgG antibody against E. risticii before and after the challenge with live E. risticii. Spleen lymphocyte proliferative response assay at 11 days post challenge revealed that with polymyxin-B a higher lymphocyte proliferation occurred as compared with that of the mice which received polymyxin-B-free vaccine. Spleen lymphocytes of the placebo (polymyxin-B and Quil-A) pretreated/challenged mice showed no proliferative activity. Western blot analysis revealed that vaccinated mice reacted mainly with 110, 57 and 33 kDa antigen bands before and after challenge. The placebo (polymyxin-B and Quil-A)/challenged mice showed a very weak response to ehrlichial antigens at day 10 to 11 post challenge. Comparison with inactivated Renografin-purified E. risticii or 0.25% SDS-insoluble fraction of E. risticii with the inactivated host cell-free vaccine revealed no increased protection. These results indicate that inactivated host cell-free E. risticii can protect mice from murine Potomac horse fever. The presence of polymyxin-B appeared to be not harmful but rather beneficial for lymphocyte proliferation response upon challenge with live E. risticii.     

Observations on Cysticercus bovis in slaughter cattle in the Matabeleland province of Zimbabwe

Cysticercus bovis was detected at meat inspection in 2.16 per cent of 102,087 carcases of cattle from Matabeleland Province, slaughtered in Bulawayo during a period of 11 months. Lightly infested (detained) carcases accounted for 95 per cent of those with cysticercosis. The head was the only site affected in 58.4 per cent of the detained carcases, the shoulder in 20.1 per cent and the heart in 7.9 per cent. In more heavily infested (condemned) carcases 81.1 per cent had at least three sites affected. The incidence of cysts was highest in older male animals but in younger animals it was highest in cows. In the detained carcases an average of 1.5 cysts were found, of which 76 per cent were live and 24 per cent dead. Condemned carcases had more than 58 cysts on average, of which 98 per cent were live and 2 per cent dead. Live cysts were more common in older cattle and dead cysts were more common in younger animals. The incidence of C bovis in carcases of cattle originating from communal areas was 3.2 per cent compared with 1.6 per cent in those originating from commercial farms.     

Immune Response of Rainbow Trout to Extracellular Products of Mycobacterium spp.

Abstract

A primary intraperitoneal (IP) vaccination of extracellular products (ECP) from Mycobacterium spp. (strains TB40, TB267, or Mycobacterium marinum) mixed with Freund's incomplete adjuvant and followed by a secondary IP injection at 8 weeks resulted in the elevation of both the nonspecific and the specific immune responses of rainbow trout Oncorhynchus mykiss. Increased nitroblue tetrazolium and phagocytosis activity were observed in these fish; peaks in activity occurred at weeks 2 and 6 after primary immunization with a third peak at week 10. Lysozyme activity, on the other hand, peaked at weeks 2 and 8 after primary immunization except in the TB40-immunized group. A third peak of lysozyme activity was observed at week 10 after primary immunization. The activity of the specific immune response was monitored by an enzyme-linked immunosorbent assay and Western blot. The results indicate that antibodies to the ECP of Mycobacterium spp. were present in rainbow trout serum and that they reacted with major ECP antigens at 65 and 16 kDa (kilodaltons) as well as with some minor antigens at 48, 46, and 40 kDa.     

Blood Parameters and Immune Status of Rainbow Trout with Proliferative Kidney Disease

Abstract

Blood parameters, disease resistance, and the immune response were sequentially evaluated in rainbow trout Oncorhynchus mykiss with proliferative kidney disease (PKD). The fish were maintained under laboratory conditions, and the study group went through a full cycle of the disease. Hematological and serological changes occurred primarily in those fish with severe kidney lesions. Fish infected with the parasite that causes PKD demonstrated a greater resistance to bacterial challenge, and their immune responses were heightened when compared with those of uninfected fish. These data suggest that PKD alone is not a predisposing factor for secondary infections if the fish does not incur severe renal lesions.