Loma Salmonae Research Articles

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.

Intraperitoneal administration of β‐1,3/1,6‐glucan to rainbow trout, Oncorhynchus mykiss (Walbaum), protects against Loma salmonae

Intraperitoneal administration of <i>β</i>‐1,3/1,6‐glucan to rainbow trout, <i>Oncorhynchus mykiss</i> (Walbaum), protects against <i>Loma salmonae</i>

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.

Interaction of water temperature and challenge model on xenoma development rates for Loma salmonae (Microspora) in rainbow trout, Oncorhynchus mykiss (Walbaum)

This study evaluated the regulatory effects of water temperature on the development of branchial xenomas caused by Loma salmonae using a high-dose per os-challenge model compared with a low-dose cohabitation-challenge model. Approximately 275 juvenile rainbow trout (RBT), Oncorhynchus mykiss, were randomly distributed to six tanks with two tanks each maintained at 11, 15 and 19 degrees C. Fish in one tank from each temperature setting were exposed per os to macerated L. salmonae-infected gill material and fish in the other tank from each temperature setting were exposed to L. salmonae using the cohabitation-challenge model. Fish were monitored for the development of branchial xenomas beginning at day 21 post-exposure. Survival analyses were used to evaluate the effect of water temperature and challenge model on the number of days until the first visible branchial xenoma was detected. The survivor curves for the per os-challenge model revealed that there was at least one significant difference, whereas the cohabitation challenge did not reveal any significant differences amongst the temperature settings. The proportional hazards model revealed a significant interaction between the challenge model used and water temperature. This indicated that the effect of water temperature was different depending on challenge model. Additionally, from the mean xenoma intensities, on average, the per os-challenged fish showed higher xenoma intensity compared with the cohabitation-challenged fish. Overall, the impact of water temperature on disease pathogenesis was greater when the RBT were per os challenged compared with using the cohabitation model.

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.

Ultrastructural morphology suggesting a new hypothesis for development of microsporidians seen in Loma salmonae infecting the gills of rainbow and brook trout

In two variants of Loma salmonae that have specificity for rainbow trout Oncorhynchus mykiss(OA variant) and specificity to brook trout Salvelinus fontinalis(SV variant), the parasitophorous vacuole forms at the onset of sporogony. In the OA variant the merogonial stage is bound by a single plasma membrane in direct contact with host cytoplasm. The parasitophorous vacuole formation is initiated by the host cell surrounding the merogonial stages with endoplasmic reticulum (ER) as occurs in autophagy. Of the two host ER membranes surrounding the parasite, one remains in close association with the plasma membrane of the meront, while the other forms the limiting membrane of the vacuole. The sporogonial stage is bounded by two closely apposed membranes, giving the appearance of a thick electron dense plasmalemma. The observations from this study support the novel hypothesis that this microsporidian uses the intracellular process of autophagy to aid formation of a parasitophorous vacuole. The morphology of the SV‐variant is consistent with that of the OA‐variant suggesting that it uses the same mechanism for development.

Isolation of the salmonid rhamnose‐binding lectin STL2 from spores of the microsporidian fish parasite Loma salmonae

The microsporidian parasite, Loma salmonae, is the causative agent of gill disease in both wild and netpen-reared salmonids worldwide. In this paper we report the finding of a rhamnose-binding lectin from steelhead trout, Oncorhynchus mykiss, which was found bound in high concentration to the surface coat of L. salmonae spores. SDS-PAGE, immunoblot, N-terminal sequencing and mass spectrometric analyses were used to determine that the dominant 24 kDa protein lectin observed on SDS-PAGE analysis of intact spore extracts is the O. tshawytscha variant of the previously identified rhamnose-binding lectin STL2 from rainbow trout, O. mykiss. Although the physiological role of these lectins has not been clearly delineated, they have been implicated in a variety of functions, including inhibition of pathogenic bacteria by opsonization and macrophage-mediated tumour lysis.

Morphological presentation of a dendritic-like cell within the gills of chinook salmon infected with Loma salmonae

Morphological presentation of a dendritic-like cell within the gills of chinook salmon infected with Loma salmonae

Review of the sequential development of Loma salmonae (Microsporidia) based on experimental infections of rainbow trout (Oncorhynchus mykiss) and Chinook salmon (O. tshawytscha)

Loma salmonae is a common gill parasite of salmonids, and essentially all species in the genus Oncorhynchus are susceptible. Infections occur in both fresh and salt water. Loma salmonae is directly transmissible by ingestion of spores or infected tissue. The parasite infects the wall of blood vessels of various organs, but the gill is the primary site of infection. Initial infection occurs in the intestine, and xenomas are easily detected in the gills by standard histology at 4-6 wk post-exposure. A few presporogonic stages of the parasite are found in the heart endothelium prior to xenoma formation in the gills. Ultrastructure studies of early infections demonstrated that wandering blood cells transport the meronts to the gills, and that merogony occurs in pillar cells and other cells underlying the gill endothelium. Xenomas develop in these cells, resulting in hypertrophied host cells filled with spores. Xenomas ultimately rupture, and are associated with severe inflammation in which free spores are found in macrophages. The parasites are most pathogenic during this phase of the infection, resulting in severe vasculitis and clinical disease. Both rainbow trout (Oncorhynchus mykiss) and Chinook salmon (Oncorhynchus ishawytscha) recover from infections, but free spores persist in kidney and spleen phagocytes for many months after xenomas are absent in Chinook salmon. Fish that have recovered from the infection show strong immunity against the parasite, lasting up to 1 year. Fish are susceptible to infection by other routes of exposure by spores; co-habitation, anal gavage, and intramuscular, intraperitoneal and intravascular injection. Autoinfection probably occurs following release of spores in blood vessels after xenomas rupture. The optimal temperature for L. salmonae infections is 15-17 degrees C, with a permissive range of 11-20 degrees C.

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.

Differences in metabolic response to Loma salmonae infection in juvenile rainbow trout Oncorhynchus mykiss and brook trout Salvelinus fontinalis

Routine and post-exercise metabolic rates were measured for juvenile rainbow trout Oncorhynchus mykiss and brook trout Salvelinus fontinalis infected with the microsporidium gill parasite Loma salmonae under laboratory conditions. Rainbow trout increased routine and post-exercise metabolic rate in response to infection compared with controls. Brook trout, on the other hand, lowered routine metabolic rate without effecting post-exercise metabolic rate compared to controls. The result of these 2 different strategies may either reflect defense of metabolic scope or a difference in the rate of recovery of the excess post-exercise oxygen consumption between the 2 species in response to the same infection.

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.

Loma salmonae -associated xenoma onset and clearance in rainbow trout, Oncorhynchus mykiss (Walbaum): comparisons of per os and cohabitation exposure using survival analysis

Survival analysis techniques were used to compare experimental exposure methods of Loma salmonae (Microspora) in rainbow trout (RBT) by measuring xenoma onset and clearance time. Twenty-eight naive RBT were exposed per os (fed L. salmonae spores) and 28 RBT were exposed by cohabitation with 28 L. salmonae-infected RBT. Exposed fish were examined once every week (7 days) post exposure (PE). For xenoma onset, the median survival time, the time to first appearance of branchial xenomas, was 6 weeks PE for both per os and cohabitation exposure. For xenoma clearance, the median survival time, the time to total clearance of branchial xenomas, was 10 weeks for per os exposure and 12 weeks for cohabitation exposure. There was a significant difference between the survival curves of per os fish compared with cohabited fish for both xenoma onset and xenoma clearance. The incidence rate of xenoma development was greater for per os exposure (0.1745 cases per fish-week) compared with cohabitation exposure (0.1342 cases per fish-week). Similarly, the rate of xenoma clearance was greater for per os exposure (0.1028 cases per fish-week) compared with cohabitation exposure (0.0885 cases per fish-week). Differences between exposure methods were attributed to differences in the frequency and duration of exposure to spores. Survival analysis was useful for examining the onset and clearance of L. salmonae and may have applications for understanding disease dynamics in aquaculture.

Characterization of recombinant microsporidian methionine aminopeptidase type 2.

Microsporidia are obligate intracellular protists related to fungi that are pathogens in both vertebrate and invertebrate hosts [21,23,25]. In humans microsporidiosis most often presents as a diarrheal illness, but keratoconjunctivitis, disseminated disease, hepatitis, myositis, sinusitis, renal infection, prostatitis, ascites, and cholangitis have also been described [21]. Of the over 144 genera of microsporidian the following species are human pathogens: Nosema, Vittaforma, Pleistophora, Encephalitozoon (Septata intestinalis is now Encephalitozoon intestinalis), Enterocytozoon, Brachiola, Trachipleistophora and Microsporidium [21]. Benzimidazoles, such as albendazole, are therapeutic agents of choice for infections due to Encephalitozoon sp., however, they are not very efficacious for infections due to Enterocytozoon bieneusi [2,24]. Fumagillin and its derivatives, have been effective for the treatment of microsporidiosis in fish (Pleistophora sp., Loma salmonae, Nucleospora fsyn Enterocytozoong salmonis) and in insects (Nosema kingi, Nosema apis, Octosporea muscaedomesticae) [2,7] Fumagillin has also been demonstrated to have in vitro and in vivo activity against Encephalitozoon sp. and Vit. corneae [2,3,14,17]. Importantly, fumagillin also had efficacy in the treatment of AIDS patients that had Ent. bieneusi infection [13]. We have demonstrated that TNP470 and other fumagillin derivatives are also active both in vitro and in vivo against microsporidia [3,7,22]. Fumagillin and TNP470 bind irreversibly to a common bifunctional protein identified by mass spectrometry as methionine aminopeptidase type 2 (MetAP2) [5,18]. These drugs selectively block the aminopeptidase, but not the elF-2 phosphorylation activity, of human MetAP2 and do not bind or inhibit methionine aminopeptidase type 1 (MetAP1). We have previously demonstrated that microsporidia have MetAP2 activity using the substrates methionine-para-nitroanilide and L-methionine-AFC. In addition, we have previously used homology PCR to obtain partial MetAP2 genes from Enc. cuniculi, Enc. hellem, Enc. intestinalis, Glugea americanus and Brachiola algerae [22] (Weiss, L.M. & Zhang, H. 2002. Microsporidian Methionine Aminopeptidases as Therapeutic Targets, Woods Hole Molecular Parasitology Meeting XIII, 284A, abstract). Phylogenetic analysis of these genes suggested that the microsporidian MetAP2 genes, while related to fungal MetAP2, formed a distinct clade (Weiss, L.M. & Zhang, H., 2002, Woods Hole Molecular Parasitology Meeting XIII, 284A, abstract). We have used 59 and 39 RACE techniques to obtain the fulllength MetAP2 sequences from Enc. cuniculi (GeneBank AF440270), Enc. intestinalis (GeneBank AY224693), and Enc. hellem (GeneBank AY224694). By homology PCR we could not demonstrate MetAP1 in Enc. cuniculi [22] and the subsequently published Enc. cuniculi genome confirms that this organism lacks MetAP1 [8]. We have now cloned Enc. cuniculi MetAP2 into a baculovirus expression system and have been able to purify enzymatically active microsporidian MetAP2. We also used data on the crystal structure of the human MetAP2 protein to model the Enc. cuniculi MetAP2 sequence.

Ultrastructural study of the late stages of Loma salmonae development in the gills of experimentally infected rainbow trout.

The main objective of this investigation was to examine the ultrastructural features of gills from rainbow trout experimentally infected with Loma salmonae to determine the morphological events that occur during the late stages of development of this parasite. Peripheral distribution of the mature parasites inside round xenomas was observed at weeks 5 and 6 postexposure (PE), but eventually the parasite occupied the entire xenoma. Degenerative changes were observed only in immature parasites at week 7 PE, and eventually an inflammatory reaction with a cellular infiltration was directed against mature spores. Round, flattened, and irregular shaped xenomas were observed at week 8 PE. The round xenomas showed a severe inflammatory response with disintegration of the xenoma membrane. This event was accompanied by eversion of polar tubes within the attacked xenoma and by the simultaneous presence of 2 tubular appendages, the type I and II tubules. Flattened xenomas were observed below the endothelium of gill lamella arteries. The irregular xenomas were located in the connective tissue of the gill filament and showed multiple projections occupied by spores. Both flattened and irregular xenomas showed no evidence of inflammatory reaction. An earlier proposed hypothesis is expanded to explain how L. salmonae is implanted beneath lamellar endothelium and within filament connective tissue.

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.

Failure of Vaccination in Brook Trout Salvelinus fontinalis against Loma salmonae (Microspora).

The effectiveness of vaccines made of viable spores of two strains of Loma salmonae (Microspora) delivered orally to rainbow trout Oncorhynchus mykiss and brook trout Salvelinus fontinalis was evaluated under experimental conditions of re-challenge. Whereas prior exposure to either of two strains of L. salmonae led to marked resistance of rainbow trout to a second challenge, brook trout failed to mount an effective protective response.

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.

Ultrastructural Study of the Early Development and Localization of Loma salmonae in the Gills of Experimentally Infected Rainbow Trout

Ultrastructural Study of the Early Development and Localization of Loma salmonae in the Gills of Experimentally Infected Rainbow Trout