[Show abstract][Hide abstract] ABSTRACT: Viral hemorrhagic septicemia virus (VHSV) infects a wide range of marine fish species. To study the occurrence of VHSV in wild marine fish populations in Norwegian coastal waters and fjord systems a total of 1927 fish from 39 different species were sampled through 5 research cruises conducted in 2009 to 2011. In total, VHSV was detected by rRT-PCR in twelve samples originating from Atlantic herring (Clupea harengus), haddock (Melanogrammus aeglefinus), whiting (Merlangius merlangus) and silvery pout (Gadiculus argenteus). All fish tested positive in gills while four herring and one silvery pout also tested positive in internal organs. Successful virus isolation in cell culture was only obtained from one pooled Atlantic herring sample which shows that today's PCR methodology have a much higher sensitivity than cell culture for detection of VHSV. Sequencing revealed that the positive samples belonged to VHSV genotype Ib and phylogenetic analysis shows that the isolate from Atlantic herring and silvery pout are closely related. All positive fish were sampled in the same area in the northern county of Finnmark. This is the first detection of VHSV in Atlantic herring this far north, and to our knowledge the first detection of VHSV in silvery pout. However, low prevalence of VHSV genotype Ib in Atlantic herring and other wild marine fish are well known in other parts of Europe. Earlier there have been a few reports of disease outbreaks in farmed rainbow trout with VHSV of genotype Ib, and our results show that there is a possibility of transfer of VHSV from wild to farmed fish along the Norwegian coast line. The impact of VHSV on wild fish is not well documented.
PLoS ONE 09/2014; 9(9):e108529. DOI:10.1371/journal.pone.0108529 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As adhesion and translocation through fish gut enterocytes of the pathogen Vibrio (Listonella) anguillarum are not well investigated, the effective cause of disease and mortality outbreaks in larval sea bass, Dicentrarchus labrax, suffering from vibriosis is unknown. We detected V. anguillarum within the gut of experimentally infected gnotobiotic sea bass larvae using transmission electron microscopy and immunogold labelling. Intact bacteria were observed in close contact with the apical brush border in the gut lumen. Enterocytes contained lysosomes positive for protein A‐gold particles suggesting intracellular elimination of bacterial fragments. Shed intestinal cells were regularly visualized in the gut lumen in late stages of exposure. Some of the luminal cells showed invagination and putative engulfment of bacterial structures by pseudopod‐like formations. The engulfed structures were positive for protein A‐colloidal gold indicating that these structures were V. anguillarum. Immunogold positive thread‐like structures secreted by V. anguillarum suggested the presence of outer membrane vesicles (MVs) hypothesizing that MVs are potent transporters of active virulence factors to sea bass gut cells suggestive for a substantial role in biofilm formation and pathogenesis. We put forward the hypothesis that MVs are important in the pathogenesis of V. anguillarum in sea bass larvae.
[Show abstract][Hide abstract] ABSTRACT: Viral haemorrhagic septicaemia (VHS) is a viral disease known to cause high mortality in many teleost species, both in wild and farmed populations. The effects of this virus infection on the population dynamics of Atlantic herring Clupea harengus are still unknown; however, high mortalities have been reported in infected Pacific herring C. pallasii. We investigated Atlantic herring from the Norwegian spring-spawning (NSS) stock during the spawning season and found a much higher prevalence of VHS virus (VHSV) than had been identified in previous surveillance studies. Positive results were found in 19% of the samples analysed using viral cell culture on pooled brain, spleen and kidney tissue samples from 5 fish. Real-time RT-PCR performed on the same pooled samples revealed 33% positive samples, while analysis of individual organs resulted in a prevalence of 12-13%. Gills, which were analysed only by real-time RT-PCR, displayed a prevalence of 69%. It is not clear whether the virus detected in the gills reflects an infection or a passive carrier status. Sequencing of positive samples from gills and internal organs revealed high identity levels with VHSV of genotype Ib detected previously in Norwegian herring. The high prevalence identified in our study indicates a need for further research into the significance of VHSV infection on the herring population.
[Show abstract][Hide abstract] ABSTRACT: Infections with Vibrio anguillarum and other pathogenic Vibrio spp. are a major problem for marine larviculture, and improved control of the microbiota in marine larvae cultures could ensure a more reliable and cost-effective production of juvenile fish. Phaeobacter gallaeciensis is capable of reducing V. anguillarum in live feed cultures and can, in challenge trials, protect fish larvae from vibriosis. The purpose of the present study was to estimate the probiotic potential of Phaeobacter isolates that produce different levels of the antagonistic compound tropodithietic acid (TDA). We compared the capability of three wild type Phaeobacter strains to reduce cod larvae mortalities in challenge trials with single cod (Gadus morhua) embryo/larvae cultures, and assessed the importance of the time point at which the probiotic bacteria were introduced relative to the pathogen. All three Phaeobacter strains reduced larvae mortalities, however to different degrees. The capability of the strains to prevent disease was correlated with their in vitro TDA production, as measured by in vitro inhibition of V. anguillarum. The most effective time to apply the probiotics was in advance of the pathogen, while simultaneous introduction was only effective for the two strains with the highest TDA production. This suggests that prophylactic use of Phaeobacter spp., where the probiotic bacterium is introduced early into the system, is most efficient in disease prevention.
Aquaculture 03/2013; s 384–387:82–86. DOI:10.1016/j.aquaculture.2012.12.013 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As adhesion and translocation through fish gut enterocytes of the pathogen Vibrio (Listonella) anguillarum are not well investigated, the effective cause of disease and mortality outbreaks in larval sea bass, Dicentrarchus labrax, suffering from vibriosis is unknown. We detected V. anguillarum within the gut of experimentally infected gnotobiotic sea bass larvae using transmission electron microscopy and immunogold labelling. Intact bacteria were observed in close contact with the apical brush border in the gut lumen. Enterocytes contained lysosomes positive for protein A-gold particles suggesting intracellular elimination of bacterial fragments. Shed intestinal cells were regularly visualized in the gut lumen in late stages of exposure. Some of the luminal cells showed invagination and putative engulfment of bacterial structures by pseudopod-like formations. The engulfed structures were positive for protein A-colloidal gold indicating that these structures were V. anguillarum. Immunogold positive thread-like structures secreted by V. anguillarum suggested the presence of outer membrane vesicles (MVs) hypothesizing that MVs are potent transporters of active virulence factors to sea bass gut cells suggestive for a substantial role in biofilm formation and pathogenesis. We put forward the hypothesis that MVs are important in the pathogenesis of V. anguillarum in sea bass larvae.
Journal of Fish Diseases 11/2012; 36(4). DOI:10.1111/jfd.12009 · 2.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phaeobacter gallaeciensis can antagonize fish-pathogenic bacteria in vitro, and the purpose of this study was to evaluate the organism as a probiont for marine fish larvae and their feed cultures. An in vivo mechanism of action of the antagonistic probiotic bacterium is suggested using a non-antagonistic mutant. P. gallaeciensis was readily established in axenic cultures of the two microalgae Tetraselmis suecica and Nannochloropsis oculata, and of the rotifer Brachionus plicatilis. P. gallaeciensis reached densities of 10(7) cfu/ml and did not adversely affect growth of algae or rotifers. Vibrio anguillarum was significantly reduced by wild-type P. gallaeciensis, when introduced into these cultures. A P. gallaeciensis mutant that did not produce the antibacterial compound tropodithietic acid (TDA) did not reduce V. anguillarum numbers, suggesting that production of the antibacterial compound is important for the antagonistic properties of P. gallaeciensis. The ability of P. gallaeciensis to protect fish larvae from vibriosis was determined in a bath challenge experiment using a multidish system with 1 larva per well. Unchallenged larvae reached 40% accumulated mortality which increased to 100% when infected with V. anguillarum. P. gallaeciensis reduced the mortality of challenged cod larvae (Gadus morhua) to 10%, significantly below the levels of both the challenged and the unchallenged larvae. The TDA mutant reduced mortality of the cod larvae in some of the replicates, although to a much lesser extent than the wild type. It is concluded that P. gallaeciensis is a promising probiont in marine larviculture and that TDA production likely contributes to its probiotic effect.
PLoS ONE 08/2012; 7(8):e43996. DOI:10.1371/journal.pone.0043996 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Immunomodulatory feed additives are expected to exert their primary influence at the intestinal level through the expression of cytokines, which in turn affect the immune responses in fish. In two separate experiments a yeast-derived mannan oligosaccharide product (YM) or a purified β-glucan (BG) product were fed to Atlantic cod (Gadus morhua L.) for 5 weeks, after which they were bath-challenged with a bacterial pathogen--Vibrio anguillarum. The transcription of selected cytokines (proinflammatory--il1b, il8, ifng; anti-inflammatory--il10) in different intestinal segments was analysed using qPCR. In the case of YM study, the effect of the compound was observed in both the posterior intestine and rectum of Atlantic cod, upon challenge with the pathogen. iIl1b expression in the posterior intestine and rectum of post-challenge fish was significantly higher than that of pre-challenge fish. In the case of il8 the difference was confined to rectum. The expression of ifng was altered only in the anterior intestine upon YM feeding. In the BG trial, the additive had a differential effect on the expression of the cytokine genes. In anterior intestine and rectum, the purified β-glucan additive significantly elevated the expression of il1b when challenged with V. anguillarum. An effect of BG on the anti-inflammatory cytokine il10 was visible in the rectum after the pathogen challenge. The differential responses of cytokines in the intestine of fish upon exposure to V. anguillarum suggest that both mannan oligosaccharides and β-glucans impact the ability of Atlantic cod to respond to the pathogen.
[Show abstract][Hide abstract] ABSTRACT: For most marine aquaculture species, one of the main bottlenecks is the stable production of high quality juveniles. The high and unpredictable mortality in the first weeks after hatching of marine fish larvae remains a challenging problem that needs to be solved. The severity of the problem differs between species, but cannot be considered adequately solved for any species. Both scientific evidence and experience in hatcheries for a variety of fish, shrimp and shellfish species are accumulating as support for the hypothesis that detrimental fish–microbe interactions are the cause of these problems. Host–microbe interactions in reared fish are still poorly understood, except for a few pathogens, and empirical data of the quality required to test this hypothesis, are lacking. This article provides an overview on the current knowledge of the microbial environment of fish larvae, including methodological aspects to characterize the microbial community (both using culture-dependent and culture-independent methods). Further, the current knowledge of the immunology of fish larvae is reviewed, including recent advances in the understanding of toll-like receptors, inflammatory cytokines, mast cells and piscidins, and the ontogeny of the adaptive immune system. Finally, we provide an overview of the state of the art with respect to steering of microbial communities associated with fish larvae – both steering of community composition and of its activity (e.g. by quorum sensing interference).
Reviews in Aquaculture 06/2012; 5(4):1-25. DOI:10.1111/j.1753-5131.2012.01082.x · 3.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vibrio splendidus is a pathogen that can cause major losses during the early stages of larval turbot rearing when live feed (rotifers or Artemia) is used. As haemolytic bacteria have often been associated with larval rearing losses, we studied the role of the V. splendidus haemolysin in infection of larvae. From a bank of over 10,000 transposon mutants of V. splendidus, two different types of haemolysin-negative mutants were obtained. Both had lost virulence for larval fish, and immunohistochemistry showed that the transposon mutant studied colonized the turbot larval intestinal tract at a similar level to the wild-type organism but did not cause damage or signs of enteritis found with the wild-type organism. One transposon insertion site was located within a gene with high homology to aerolysin, the cytolytic toxin produced by several Aeromonas spp. The haemolysin, which we have termed vibrioaerolysin, had properties similar to aerolysin and osmotic protection studies showed that it formed pores in the membranes of erythrocytes of similar diameter to those of aerolysin. The Tn10 insertion site of the second transposon mutant was in an adjacent ToxR-like gene, suggesting that this might control expression of the vibrioaerolysin. The gastroenteritis caused by Aeromonas spp. in humans is considered to be due to production of aerolysin causing cyclic AMP-dependent chloride secretion in cells of the gastrointestinal tract. Damage to the intestinal tract of marine fish larvae could occur in a similar way, and it is possible that several Vibrio spp. found in the developing bacterial flora of the larval fish gut can secrete aerolysin-like toxins leading to death of larvae in the early rearing stages. Routine bacteriological screening on blood agar plates of live feed is recommended with measures to reduce the concentrations of haemolytic bacteria in rearing systems.
Journal of Fish Diseases 02/2012; 35(2):153-67. DOI:10.1111/j.1365-2761.2011.01331.x · 2.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effect of water treatment and flow rate on young Atlantic cod juveniles was investigated in a 36-days experiment. Four different flow rates (10, 20, 40, and 70 times the effective tank volume per day) were set up in triplicate tanks within each of three rigs with recirculated, UV-radiated, and untreated water, respectively. Each of the 36 tanks was stocked with 200 weaned cod juveniles at a mean weight of 0.048 g. Fish mortality was recorded daily in all tanks, and growth (wet weight) was determined at the end of the experiment. The microflora in the rearing water was investigated by means of PCR-DGGE and flow cytometry. Observed mortality was significantly higher at low flow rates while otherwise unexplained mortality (presumed to be due to cannibalism) was lowest in the recirculation system. No correlation was found between survival and growth. Growth was significantly affected by both water exchange rate and treatment, as the juveniles from high flow rates and the UV-treatment showed elevated growth rates. Both growth and survival scaled in accordance with metabolic factors like oxygen saturation and unionized ammonia. Bacterial concentrations increased in all tanks and treatment from the beginning of the experiment to the end. The UV-treated and untreated water started at typical seawater concentrations (0.5–1 × 106 mL−1) and increased five to tenfold during the experiment. The recycled water tanks started with bacterial concentrations 2–5 times higher than the UV- and untreated experiments at the time of fish transfer, and ended up with 10 times higher concentrations in the end. Cluster analysis of the DGGE profiles separated the recirculation tanks, including the respective inlet water, from the flow-through systems, with one exception (the highest flow rate). Eighty-five% of the sequences clustered within the Gammaproteobacteria, further divided into four distinct clusters. One of the clusters was only detected in the recirculation system, and showed highest affiliation to bacteria belonging to the Alteromonas/Pseudoalteromonas genera. In contrast, bacteria belonging to the family Vibrionaceae were detected in the flow-through systems.
[Show abstract][Hide abstract] ABSTRACT: To evaluate whether two commercial nitrifying bacterial consortia can function as biocontrol agents in ornamental fish transporting systems.
The consortia were applied in a simulated set-up using zebrafish as the model organism in three trials. The efficacy of the bacterial consortia in controlling the ammonia level was validated by measuring water quality parameters such as total ammonia, nitrate and pH of the transport water. The bacterial community structure in the transport unit was studied using denaturing gradient gel electrophoresis. The consortia tested improved the nitrifying activity that in turn facilitated the reduction of ammonia that had accumulated during the transport. Bacterial profiles revealed the presence of both ammonia-oxidizing and nitrite-oxidizing bacteria in the transport bags.
The application of the consortia during the transportation of zebrafish could profoundly improve the water quality by curbing ammonia accumulation.
The potential of applying nitrifying bacteria as a bioremediation practice during the transport of ornamental fish has been demonstrated and this innovative approach contributes to the amelioration of current fish welfare in ornamental fish trade.
[Show abstract][Hide abstract] ABSTRACT: Eggs of Atlantic halibut (Hippoglossus hippoglossus) and turbot (Scophthalmus maximus) were exposed to Flexibacter ovolyticus and pathogenic Vibrio sp. strains prior to, and during hatching. Activity, buoyancy and mortality of the yolk sac larvae were monitored from hatching until time of first feeding. The halibut larvae showed reduced activity and increased mortality in response to the challenge of bacteria, compared to uninfected control groups. In addition, the infected halibut larvae showed increased specific density compared to the uninfected larvae. These responses were not found for turbot. However, turbot larvae infected with Vibrio anguillarum had lower activity than larvae infected with F. ovolyticus. The reduced activity of halibut larvae occurred 1–2 weeks prior to the increased mortality, allowing infections to be detected at an early stage. The results suggest that the behaviour of fish larvae is influenced by bacterial infection.
Canadian Journal of Fisheries and Aquatic Sciences 04/2011; 50(12):2552-2557. DOI:10.1139/f93-278 · 2.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Isolates of aerobic intestinal bacterial flora from unfed Atlantic halibut (Hippoglossus hippoglossus) larvae revealed a population dominated by nonfermentative rods of the Cytophaga/Flexibacter/Flavobacterium group. Following the onset of feeding, the flora gradually changed towards a fermentative flora dominated by the Vibrio/Aeromonas group. This transition occurred faster in a group which was reared in water containing cultivated phytoplankton, in contrast with groups where the larvae were kept in filtered water. No major differences in the bacterial flora of the tank water were found among the groups.
Canadian Journal of Fisheries and Aquatic Sciences 04/2011; 51(8):1899-1903. DOI:10.1139/f94-190 · 2.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The commensal microbiota plays an important role in the well-being of the host organism, and it would be worthwhile to know the tenacious communities among them. Therefore, a study was undertaken to examine the changes in constitution of the intestinal microbiota of wild fish consequential to captivity. At first, the composition of intestinal microorganisms of Atlantic cod caught from the coastal area off Bodø, Norway, was examined. Thereafter, the changes in the bacterial community of the captive fish after offering them artificial feed or subjecting them to starvation were studied. The microbiota from the intestinal contents and wall segments were analyzed quantitatively by spread plate technique and DAPI staining and qualitatively by denaturing gradient gel electrophoresis. The study revealed that the counts of intestinal microbes in wild-caught Atlantic cod were not affected by captive rearing for 6 weeks, either when fed or when starved. However, the diversity of intestinal bacterial community was reduced in response to artificial feeding, whereas the change was restricted upon starvation.
[Show abstract][Hide abstract] ABSTRACT: I 2009 startet prosjektet "Viral Haemorrhagic Septica-emia Virus (VHSV) in wild and farmed fish in Norway" hvor Havforskningsinstituttet og Veterinaerinstituttet samarbeider om å få frem økt kunnskap om smittefaren av VHS-virus mellom villfisk og oppdrettsfisk. Prosjektet skal gå over tre år, ut 2011. En viktig del av dette prosjektet er screening av ulike ville fiskebestander for VHS-virus i nor-ske kystnaere farvann. Flere av Havforskningsinstituttets tokt vil bli brukt for å samle inn materiale. Viral hemoragisk septikemi (VHS), tidligere kalt Eg-tvedsyke da den første gang ble påvist i Egtved, Danmark, i slutten av 1940-årene på regnbueørret (Oncorhynchus mykiss) i ferskvann. Selve viruset ble første gang påvist ved dyrking i cellekultur av Mogens Jensen i 1963. VHS-viruset er et negativt enkelttrådet RNA (–ssRNA) virus i familien Rhabdoviridae, genus Novirhabdovirus. Dette vi-ruset er kjent for å vaere tilpasningsdyktig med mange po-tensielle verter. Hittil er viruset isolert fra mer enn 50 arter, og både ferskvanns-og saltvannsfisk er rapportert å vaere baerere av ulike varianter av viruset. Et verdenskart med alle VHS-virus påvisninger kan studeres på følgende link: www.fishpathogens.eu/vhsv/geo_distribution.php. Viruset kan forårsake systemisk infeksjon i fisk, og dø-deligheten varierer med fiskeart, livsstadium og genotypen av viruset. Både i oppdrett av regnbueørret og piggvar (Sco-phthalmus maximus) har VHS vaert et problem. Det er ikke uvanlig at dødelighet hos regnbueørretyngel ligger opp mot 80-100 %, mens dødeligheten hos eldre og større regnbue-ørret ofte er noe lavere (Review av Skall et al. 2005). Det samme gjelder for piggvar. VHS-virus har ikke blitt påvist ved temperaturer over 18 °C og utbrudd kommer gjerne om våren ved varierende eller hurtig stigende temperaturer. Viruset er påvist på laks i Nord-Amerika, men hittil har ikke viruset vaert påvist på laks i Europa. I Norge har både laks og regnbueørret i oppdrett vaert overvåket for VHS-virus gjennom et overvåkningsprogram som startet i 1994. I direktiv 91/67 EEC, som dette overvåkningsprogrammet baserer seg på, heter det seg at i løpet av en toårsperiode skal alle oppdrettsanlegg med arter mottakelige for VHS-virus, undersøkes. Prøver av milt, hodenyre og enten hjerte eller hjerne fra tretti fisk fra hver lokalitet undersøkes vha dyrking i cellekultur.
[Show abstract][Hide abstract] ABSTRACT: In intensive aquaculture systems, high mortalities are frequently observed during the early life stages of marine fish. The aim of this study was to investigate differences in the susceptibility of turbot Scophthalmus maximus, halibut Hippoglossus hippoglossus and cod Gadus morhua to various strains of Vibrio anguillarum (serotypes O1, O2alpha and O2beta), V salmonicida and V splendidus. The bath challenge experiments were performed using a multidish system, with 1 egg well-1. Unchallenged eggs and larvae were used as controls. Larvae in challenged groups that suffered high mortality rates were examined by immunohistochemistry. The overall results with respect to mortality showed that the O2alpha serotype was pathogenic to all 3 species, while the O1 serotype was pathogenic to halibut and cod. The immunohistochemical examinations revealed differences in histopathology. The O1 serotype produced more severe and highly developed infections than the O2alpha serotype. In larvae exposed to the O1 serotype, necrosis and bacterial cells were seen in the dermis, gastrointestinal tract, brain and eye area, while in larvae exposed to the O2alpha serotype, bacteria were usually limited to the gastrointestinal tract. These results suggest either that there are undetermined species differences in host immunity or that these pathogens are host-specific even in the early life stages of fish. The O2beta strain did not cause an increased mortality to halibut and turbot.
[Show abstract][Hide abstract] ABSTRACT: In teleost fish, the head kidney, thymus, and spleen are generally regarded as important immune organs. In this study, the ontogeny of these organs was studied in Atlantic halibut (Hippoglossus hippoglossus), larvae at various stages of development. We observed that the kidney was present at hatching, the thymus at 33days post hatch (dph), while the spleen was the last organ to be detected at 49dph. All three lymphoid organs were morphologically well developed during late metamorphic stages. Real time RT-PCR analysis showed that IgM mRNA expression could be observed at 66dph and later, which correlates well with in situ hybridisation data showing that a few IgM positive cells could be detected in the anterior kidney and spleen from 66dph. Our data also showed that the highest levels of IgM mRNA could be detected in halibut spleen. Immunostaining using a monoclonal antibody against halibut IgM detected IgM positive cells at 94dph in both the head kidney and the spleen, which is much later than the IgM mRNA. Numerous cells expressing both IgM mRNA and protein could be detected in the spleen and anterior kidney and also to some extent in thymus specimens from adult halibut.
[Show abstract][Hide abstract] ABSTRACT: In intensive aquaculture systems, high concentrations of nutrients and high densities of fish larvae provide favorable conditions for opportunistic pathogenic bacteria to flourish. We screened potentially pathogenic bacterial strains isolated from moribund Atlantic cod Gadus morhua larvae, pollack Pollachius pollachius, coalfish Pollachius virens, Atlantic halibut Hippoglossus hippoglossus, rotifers, algae and water samples from different hatcheries. Three identical challenge experiments tested a total of 53 strains. A multidish system was used: cod eggs were placed in single wells, together with 2 ml of sterile seawater, and exposed to the bacterial cultures. Final bacterial concentrations in the wells were 10(6) and 10(4) CFU ml(-1). Eggs and larvae not exposed to bacteria were used as unchallenged controls. Challenged controls were exposed to Vibrio anguillarum strain 610. Eggs were challenged approximately 48 h prior to hatching and mortality was recorded daily throughout the yolk-sac period. In spite of the high challenge dose of 106 CFU ml(-1), only 5 bacterial strains tested caused higher mortality than the unchallenged controls. Four of these strains were identified by 16S rDNA and gyrase B gene (GyrB) sequencing as resembling V. anguillarum and 1 strain resembled Carnobacterium sp. Most of the larvae exposed to these strains died within 10 d of challenge. Serotyping of the strains resembling V. anguillarum gave inconclusive results. This indicates differences in serology compared to the serotypes O1, O2 and O3, associated with disease. Three bacterial strains seemed to have a slower infection rate, indicating a longer incubation period. The remaining 45 strains did not seem to have a negative effect on larval survival, suggesting that these are not primary pathogens.