Microsporidial Gill Disease Research Articles

Demonstrated Efficacy of a Pilot Heterologous Whole-Spore Vaccine against Microsporidial Gill Disease in Rainbow Trout

Intraperitoneal vaccines using whole viable spores of the microsporidian Glugea anomala or Glugea hertwigi reduced the numbers of branchial xenomas by 80% and 91%, respectively, after a standard experimental infection of juvenile rainbow trout with the microsporidian Loma salmonae. Similar significant results were obtained when killed-spore preparations were used.

Ontogeny and disease responses of Langerhans-like cells in lymphoid tissues of salmonid fish

The ontogeny and disease responses of Langerhans-like cells within lymphoid tissues of Atlantic salmon, Salmo salar, and rainbow trout, Oncorhynchus mykiss, were investigated. These cells were studied in situ with the use of two markers: the ultrastructural presence of Birbeck-like granules and immunohistochemistry with an antibody against human langerin/CD207 that cross-reacts with salmonid tissues. The appearance of Birbeck-like granules was observed in rainbow trout at 2 weeks post-hatch (PH) in the thymus and anterior kidney prior to the development of the spleen. Spleen first appeared at 3 weeks PH in both Atlantic salmon and rainbow trout, and Birbeck-like granules were observed within cells of the newly developed spleens. The cross-reactivity of langerin as seen by immunohistochemistry was not clearly observed in kidney and spleen until 9 weeks PH, when a strong cytoplasmic reaction was observed. To study langerin-positive cells in spleen and kidney during disease, microsporidial gill disease (MGD) in rainbow trout was used as a known disease model inducing a strong cell-mediated adaptive immune response. Langerin-positive cells in healthy fish were seen predominantly in the spleen, and only low numbers were present in the anterior kidney. During MGD, langerin-positive cell numbers were elevated in the anterior kidney and were significantly higher during 5, 6, and 10 weeks post-exposure (PE) compared with healthy control tissue. During MGD, the distribution of langerin-positive cells in the spleen and anterior kidney shifted from having significantly higher numbers of cells in the spleen than in the kidney in controls and at 1 and 4 weeks PE to having a similar distribution of the cells in the two organs at 2, 3, 5, and 6 weeks PE. By 10 weeks PE, significantly higher numbers of langerin-positive cells occurred in the anterior kidney compared with the spleen.

Development of an Effective Whole-Spore Vaccine To Protect against Microsporidial Gill Disease in Rainbow Trout (Oncorhynchus mykiss) by Using a Low-Virulence Strain ofLoma salmonae

In determining the effective vaccine spore dose of a low-virulence strain of Loma salmonae to limit microsporidial gill disease in trout, we found that fish receiving 10(3) to 10(5) killed spores had the best protection against experimental infection, with 85% fewer xenomas in their gills than in the controls. Intraperitoneal delivery of the vaccine was effective, and the addition of adjuvant did not improve vaccine performance against this disease-causing microsporidian.

Transmission of the microsporidian gill parasite, Loma salmonae

Since it was first reported in 1987 at a hatchery in British Columbia, Loma salmonae has become increasingly important as an emerging parasite affecting the Canadian salmonid aquaculture industry. L. salmonae causes Microsporidial Gill Disease of Salmon (MGDS) in farmed Pacific salmonids, Oncorhynchus spp., resulting in respiratory distress, secondary infections and high mortality rates. In the last decade, laboratory studies have identified key transmission factors for this disease and described the pathogenesis of MGDS. L. salmonae enters the host via the gut, where it injects sporoplasm into a host cell, which then migrates to the heart for a two-week merogony-like phase, followed by a macrophage-mediated transport of the parasite to the gill, with a final development stage of a spore-laden xenoma within the endothelial and pillar cells. Xenoma rupture triggers a cascade of inflammatory events leading to severe, persistent, and extensive proliferative branchitis. The development of robust and reliable experimental challenge models using several exposure methods in marine and freshwater environments with several fish hosts, is a primary reason for the success of scientific research surrounding L. salmonae. To date, demonstrated factors affecting MGDS transmission include host species, strain and size, the length of contact time between naïve and infected fish, water temperature and flow rates.

Cellular immunity in salmonids infected with the microsporidial parasite Loma salmonae or exposed to non-viable spores

Following a per os challenge of naive rainbow trout with live spores of Loma salmonae, head kidney mononuclear cells (MNC) in culture were able to proliferate in response to crude soluble parasite extract or intact dead spores. A significant response was seen by week 2 post-exposure and a maximum response developed by week 6 or 8, respectively. During this initial challenge, spore filled cysts developed on the gills of challenged fish, and the cysts ruptured by week 12 as is typical for microsporidial gill disease of salmonids (MGDS). Two weeks following this, fish were re-challenged with live spores, and in these fish an enhanced in vitro proliferative response of MNC was immediately apparent, and spore filled cysts did not develop. In contrast, when naive trout were given dead spores by intraperitoneal injection, the most pronounced proliferative responses of MNC developed earlier (week 2 PE) and the response was greater when cells were incubated in vitro with dead spores rather than with crude soluble extract. When these fish were re-challenged per os with live spores, a heightened proliferation in MNC was observed 4 weeks after this exposure and the fish likewise resisted development of xenomas. In fish infected orally or injected intraperitoneally with spores, a marked increase in the response to the mitogen concanavalin A was seen for 22 weeks post-exposure when compared to controls not receiving any spores.

Ultrastructural examination of the host inflammatory response within gills of netpen reared chinook salmon ( Oncorhynchus tshawytscha) with Microsporidial Gill Disease

The sequence of host changes following the rupture of spore-laden xenomas of the microsporidian Loma salmonae during Microsporidial Gill Disease of Salmon was deduced from ultrastructural examination of the gills of naturally infected, moribund, chinook salmon from a commercial aquaculture site. The gills contained many stages of parasite development suggesting fish were chronically exposed to the parasite. Intact xenomas were generally found beneath the endothelium in arteries and arterioles and were encapsulated by a layer of collagen containing fibroblasts sometimes joined by desmosomes. Xenoma dissolution was characterized by neutrophil infiltration and loss of the xenoma plasma membrane and encapsulation. The inflammatory responses associated with ruptured xenomas ranged from acute lesions, denoted by a marked neutrophil infiltration and vascular thrombosis, to chronic lesions with a macrophage-rich infiltrate variously accompanied by neovascularization and vascular remodelling. Dendritic-like cells and plasma cells were characteristic throughout. Basement membrane damage of the primary filament epithelium and subsequent transepithelial expulsion of spores were associated with severe inflammation. An unusual previously undescribed multifocal change, in which epithelial cells invaded deeply beyond the normal boundaries of the basement membrane, affected areas of gill filament epithelium with basement membrane damage. Some neutrophils that contained L. salmonae spores, or spore polar tube, displayed morphological changes that included irregular cell shape, cytoplasmic darkening associated with an abundance of free ribosomes, lysis of neighbouring cells, and type II nuclear clefts. Fusion of apparently intact neutrophils occurred in other areas of the lesion, where close contacts between neighbouring cells were established and in some areas plasma membrane fusion occurred. Closely associated neutrophils with intact plasma membranes were observed to contain type II nuclear clefts, abundant granules and rough endoplasmic reticulum. Other neutrophils in the lesion displayed type I nuclear pockets, which is suspected to be an early stage of apoptosis.

Induction time for resistance to microsporidial gill disease caused by Loma salmonae following vaccination of rainbow trout ( Oncorhynchus mykiss) with a spore-based vaccine

Resistance to re-infection of rainbow trout to Loma salmonae, a microsporidian gill parasite has been previously documented and this study examined how rapidly this resistance develops. Naive rainbow trout were inoculated intraperitoneally (IP) with an inactivated spore-based vaccine and were then given an oral challenge with a high dose of L. salmonae spores at various weeks after being vaccinated. Non-vaccinated naive fish (exposed group) were challenged alongside of each group of vaccinated fish to ensure that the challenges were relatively standardised. In each group of fish, four weeks after the challenge, numbers of xenomas were counted on a gill arch for all fish. Vaccinated trout were completely resistant to a L. salmonae challenge six weeks after vaccination, although the onset of resistance began at approximately week 3, as observed with a reduction in the percent infected and xenoma intensity. The maximum percent infected for the vaccinated fish was 83% following a challenge two weeks following vaccination, whereas for the exposed group the maximum prevalence of 100% was reached several times. With continued research, a spore-based vaccine for L. salmonae has the potential to become the first commercially available parasite vaccine for fish.

Influence of feeding ratio and size on susceptibility to microsporidial gill disease caused by Loma salmonae in rainbow trout, Oncorhynchus mykiss (Walbaum)

Two trials were designed to quantify the effect of feeding ratio and fish size on the cohabitation transmission of Loma salmonae, the causative agent of microsporidial gill disease (MGD) in salmonids, Oncorhynchus spp. To evaluate the effect of feeding ratio on disease onset, groups of 45 rainbow trout, O. mykiss (Walbaum) (RBT), were fed daily at 1% (low), 2% (medium) or 4% (high) of the fish biomass in the tank. There were three tanks at each feeding level: two tanks were exposed to the pathogen and one was a control. For the second objective, 300 RBT were separated into seven tanks so that the weight classifications were small (17-23 g), medium (32-38 g) and large (57-63 g). Each size class was done in duplicate with one control tank containing medium-sized fish. Separately for each trial, on day 0 post-exposure (PE) five highly infectious RBT were added to each tank (not including the control tanks) to begin the cohabitation exposure period. Beginning on day 21 PE and continuing biweekly until days 70 and 77 PE for the feeding and fish size trials, respectively, each fish was evaluated for visible branchial xenomas to determine disease onset time. Using survival analysis, the survival curves for the low, medium and high feedings were not significantly different from each other. However, there were significant differences amongst the small, medium and large weight classes in the size trial. The median numbers of days to the development of branchial xenomas was 31, 38 and 42 for small, medium and large size fish, respectively. On any given day, a medium or large sized fish had a hazard ratio for developing branchial xenomas of 0.66 and 0.63, respectively, compared with a small fish. In addition to host species and host strain differences, fish size is now considered a host risk factor for the development of MGD.

Effect of the number of infected fish and acute exposure period on the horizontal transmission of Loma salmonae (Microsporidia) in rainbow trout, Oncorhynchus mykiss

Loma salmonae is an intracellular obligate parasite of farmed chinook salmon ( Oncorhynchus tshawtscha) and is the causative agent for microsporidial gill disease (MGD). The infection occurs mainly in secondary gill lamellae, with the formation of large white spore-laden xenomas within the endothelial and pillar cells. The overall objective of the study was to investigate the minimum exposure time required for disease transmission between naive rainbow trout ( Oncorhynchus mykiss) (RBT) and L. salmonae-infected fish using the cohabitation challenge model. In trial I, low (1), medium (5) or high (10) numbers of infected RBT were added to tanks containing 45 naive fish for a cohabitation period of 21 days. Trial II limited the cohabitation period between five L. salmonae-infected fish in a tank of 45 naive RBT to 1, 12, 24 or 96 h. For both trials, each treatment level was completed in duplicate and an additional tank of 45 naive RBT served as the control and did not receive infected fish. Beginning on day 21 post exposure (PE) and continuing biweekly until day 83 PE and 105 PE for each trial, respectively, fish were evaluated for branchial xenomas to determine xenoma onset time and intensity. The effect of adding one infected fish produced similar median xenoma onset time and mean xenoma intensity compared with adding 5 or 10 infected fish (trial I). Significant differences were observed amongst the survivor curves for the fish exposed to infected RBT for 1, 12, 24 and 96 h, with the median onset time equal to 63 days for the lowest exposure period. This study demonstrated that L. salmonae transmission was possible with as little as 1-h exposure time between 5 cohabiting infected and 45 naive RBT. Adding five L. salmonae-infected fish (at week 7 PE) to a group of naive RBT for a period of 1 h resulted in over 94.5% disease prevalence in cohabiting fish.

Predictive Modelling of Post-onset Xenoma Growth During Microsporidial Gill Disease ( Loma salmonae) of Salmonids

Loma salmonae, an obligate intracellular microsporidian parasite, is the causal agent of microsporidial gill disease of salmon (MGDS), characterized by the production, growth and eventual rupture of spore-filled xenomas. MGDS in farmed chinook salmon remains occult until xenoma rupture, at which time the infected fish respond with intense branchitis and high rates of mortality. The present study showed that in experimentally infected fish the rate of change of xenoma diameter could be modelled through regression analysis, particularly through the period of 4–9 weeks post-infection, yielding the predictive equation: xenoma diameter=−42.9 microns +15.3 microns x (number of weeks post-infection). This provides a tool for diagnosticians to predict the time to xenoma rupture and hence to the initiation of the clinical phase of MGDS.

Effect of Water Temperature and Flow Rate on the Transmission of Microsporidial Gill Disease Caused by Loma salmonae in Rainbow Trout Oncorhynchus mykiss

Two studies were designed to quantify the effect of water temperature and flow rate on the transmission potential of the important salmonid gill pathogen, Loma salmonae. Using survival analysis, increased water temperature and low flow rates were determined as risk factors for the transmission of microsporidial gill disease caused by L. salmonae in rainbow trout Oncorhynchus mykiss. Fish were experimentally infected with L. salmonae via a cohabitation exposure model and monitored for the development of branchial xenomas. On any given day, fish held at 11°C and 15°C had a hazard ratio equal to 0.80 and 0.68, respectively, for the development of branchial xenomas compared with fish held at 19°C. From the flow rate trial, fish housed in a low flow tank (0.83 L/min) had an increased chance of developing branchial xenomas when compared to fish in tanks at normal (1.67 L/min) and high (2.5 L/min) flow rates with hazard ratios reported as 0.69.

Effects of monensin dose and treatment time on xenoma reduction in microsporidial gill disease in rainbow trout, Oncorhynchus mykiss (Walbaum)

AbstractThe objectives of the study were (1) to determine an acceptable dose of dietary monensin against microsporidial gill disease (MGD) of salmon caused by Loma salmonae and (2) to determine the life stage of L. salmonae at which the action of monensin was most effective at reducing clinical disease. Rainbow trout (RBT) were fed monensin diets at concentrations of 0, 10, 100, 1000 and 5000 ppm starting 1 week before per os exposure to L. salmonae infected gill material. Lethal sampling occurred at weeks 6, 7, 8 and 9 post‐exposure (PE). The acceptable dose of dietary monensin was determined to be 1000 ppm because it was the lowest concentration that produced a significant and constant reduction in xenoma formation. RBT were fed monensin at 1000 ppm beginning at 0, 1, 2 and 3 weeks before exposure and at 1, 2 and 3 weeks after exposure and continuing until week 9 PE. Fish in which treatment was initiated at the time of exposure or 1 week before, showed the greatest reduction in xenoma production when compared with the untreated RBT.

Xenoma formation during microsporidial gill disease of salmonids caused by Loma salmonae is affected by host species (Oncorhynchus tshawytscha, O. kisutch, O. mykiss) but not by salinity.

Host species and salinity often affect the development of disease in aquatic species. Eighty chinook salmon Oncorhynchus tshawytscha, 80 coho salmon O. kisutch and 80 rainbow trout O. mykiss were infected with Loma salmonae. Forty of each species were reared in seawater and 40 in freshwater. The mean number of xenomas per gill filament was 8 to 33 times greater in chinook salmon than in rainbow trout (RBT). Coho salmon had a mean xenoma intensity intermediate to that of chinook salmon and RBT. In contrast to the differences between species, salinity had no significant effect on xenoma intensity in any of these host species. The onset of xenoma formation occurred at Week 5 postexposure (PE) for chinook salmon and RBT, and at Week 6 PE for coho salmon. RBT had cleared all visible branchial xenomas by Week 9 PE, whereas xenomas persisted in coho and chinook salmon at Week 9 PE. Histologically, xenomas were visible in the filament arteries of the branchial arch in chinook and coho salmon gills but were absent from RBT gills. Fewer xenomas were seen in the central venous sinusoids of RBT than in chinook and coho salmon. The lower xenoma intensity, shorter duration of infection and pathological characteristics, common to microsporidial gill disease in RBT, suggest a degree of resistance to clinical disease that is not seen in coho and chinook salmon.