Hatch rate of channel catfish Ictalurus punctatus (Rafinesque 1818) eggs treated with 100 mg L−1 copper sulphate pentahydrate

Catfish hatcheries use copper sulphate pentahydrate (CuSO₄·5H₂O) as an economical control for saprolegniasis on eggs. This study determines hatch rate of channel catfish, Ictalurus punctatus (Rafinesque 1818), eggs in hatching troughs containing 23.8 °C flow‐through well water when treated with 100 mg L^?1 CuSO₄·5H₂O (10 times the proposed therapeutic dose). Eggs were treated daily until the embryos reached the eyed stage. Fry survival in the control and 100 mg L^?1 CuSO₄·5H₂O treatments was significantly different (15% and 71% respectively). This study demonstrates that there is a considerable margin of safety in using CuSO₄·5H₂O as a catfish egg treatment to control saprolegniasis.     

Inhibiting fungus on largemouth bass eggs with copper sulfate and its toxicity to fry and juveniles

This study determined the effectiveness of copper sulfate (CuSO₄) to inhibit fungal growth (caused by Saprolegnia spp.) on largemouth bass (LMB) eggs spawned on/in fiber mats in very high-alkalinity/-hardness waters; experiments also determined the toxicity of CuSO₄ to LMB fry and juveniles. An untreated control and three CuSO₄ concentrations (10, 20, and 40 mg/L) were tested under a flow-through scenario in the effectiveness experiment. Eggs were treated daily until hatching began. Fungal load at the time of hatch had a total area > 3.0 cm² in the untreated controls, total area 1.0 to <2.0 cm² in the 10 and 20 mg/L CuSO₄ treatments, and total area > 1.0 cm² in the 40 mg/L CuSO₄ treatments. Fungus samples were identified as Saprolegnia australis. The 24-hr median lethal concentration (LC50) values on the LMB yolk-sac and swim-up fry were 32.0 and 4.6 mg/L CuSO₄, respectively; the No Observed Effect Concentrations (NOEC) were 16.0 and 0.125 mg/L CuSO₄, respectively. Juvenile LMB were extremely tolerant to CuSO₄, and their 24-hr LC50 value was 185.5 mg/L; the NOEC was 64 mg/L. This study indicates that CuSO₄ can be an important resource for hatcheries to control egg fungus, especially in high-alkalinity/-hardness waters.     

Comparison of Copper Sulfate Concentrations to Control Ichthyophthiriasis in Fingerling Channel Catfish

ABSTRACT

Fingerling channel catfish Ictalurus punctatus were exposed to Ichthyophthirius multifiliis-infested fish until immature trophonts developed. The fish were transferred to individual static fiberglass tanks filled with 600 L of pond water (total alkalinity and total hardness was 220 mg/L and 101 mg/L, respectively) and were treated with 0, 1.1, 2.2, 3.3, or 4.4 mg/L copper sulfate (CuSO₄ · 5H₂O) every other day for four treatments to evaluate its effectiveness to control mortality associated with ichthyophthiriasis. Water temperature was maintained at 18 ± 1°C. Fish were observed for ten days post-treatment and mortalities were recorded. Results indicate that half of the recommended dose (1.1 mg/L CuSO₄) is needed to effectively control an occurrence of ichthyophthiriasis under the conditions of this study. However, fish culturists should be aware that effective CuSO₄ treatment of ichthyophthiriasis on channel catfish raised in ponds may be influenced by water chemistry characteristics and suspended materials such as pond sediments.     

Peracetic acid is effective for controlling fungus on channel catfish eggs

Peracetic acid (PAA) is a relatively new compound suggested for use to treat pathogens in aquaculture. It is approved for use in Europe, but not in the United States. This study determined the effectiveness of PAA for fungus control on channel catfish, Ictalurus punctatus (Rafinesque), eggs. The study consisted of five PAA concentrations (2.5, 5, 10, 15 and 20 mg L^?1) and an untreated control in a flow‐through system. A single spawn was used for each replication (N = 4). Eggs were treated twice daily until the embryos developed eyes. When hatching was complete for all viable eggs, fry were counted to determine the percent survival in each treatment. Fungal growth was severe in the untreated controls resulting in 11% survival. Treatments of 2.5, 5 and 10 mg L^?1 PAA were significantly different from the controls (P < 0.05). The highest percent survival of hatched fry was with 5 mg L^?1 PAA administered twice daily; the 2.5 mg L^?1 PAA treatment had slightly less survival, but gives a higher margin of safety in case of treatment error. Very little fungus was present in treatments receiving 2.5 mg L^?1 PAA or higher, and concentrations of 15 and 20 mg L^?1 PAA were toxic to the eggs. The mean survivals in the 0, 2.5, 5, 10, 15 and 20 mg L^?1 PAA treatments were 11%, 60%, 63%, 62%, 32% and 0%, respectively. Therefore, PAA may be a compound that merits further investigations regarding its use in U.S. aquaculture.     

Effectiveness of copper sulphate,potassium permanganate and peracetic acid to reduce mortality and infestation of Ichthyobodo necator in channel catfish Ictalurus punctatus (Rafinesque 1818)

Ichthyobodo necator is a single‐celled biflagellate parasite, which in high density can cause significant mortality in young fish. Copper sulphate (CuSO₄), potassium permanganate (KMnO₄) and peracetic acid (PAA) were evaluated for effectiveness against ichthyobodosis. Treatments were: untreated control, 2.1 mg L^?1CuSO₄, 3.0 mg L^?1 KMnO₄, 1.5 mg L^?1 PAA and 3.0 mg L^?1 PAA, and were applied to flow‐through tanks on three consecutive days. The study was designed to simulate the flow‐through systems utilized in the commercial rearing of juvenile channel catfish (Ictalurus punctatus). Mortality was monitored daily to compare survival rate among treatments. Parasite intensity was assessed pre chemical exposure and 20–24 h after the third application to determine effectiveness of the treatment. An assessment was also done 7 days post application to investigate possible reoccurrence. Copper sulphate, KMnO₄ and PAA (3.0 mg L^?1) significantly reduced the infestation rate of I. necator. Copper sulphate significantly improved the survival of I. necator infested channel catfish after three flow‐through applications compared with the untreated control. The 3.0 mg L^?1 PAA resulted in significantly lower survival than the untreated control, the 1.5 mg L^?1 PAA and the KMnO₄ were not statistically different from the untreated control.     

Evaluation of a 4‐h static copper sulphate treatment against experimental infection of Flavobacterium columnare in channel catfish (Ictalurus punctatus)

The efficacy of copper sulphate (CuSO₄) against the early stages of an experimental acute infection of Flavobacterium columnare in channel catfish (Ictalurus punctatus) was evaluated. Fish were challenged, by waterborne exposure to F. columnare in an ultra‐low flow‐through water delivery system, and treated with CuSO₄ at 4.2 and 2.1 mg L^?1 at 5.5 h post challenge for 4 h. Bacterial challenged non‐treated fish acted as a positive control and non‐challenged non‐treated fish acted as a negative control. Fish challenged with F. columnare and treated with CuSO₄ at 4.2 and 2.1 mg L^?1 post challenge had mortalities of 6.4% and 18.3%, respectively, compared with the positive control, which had 90.2% mortality. The mortality of challenged fish treated with CuSO₄ at 4.2 and 2.1 mg L^?1 was significantly different from the positive control. There was no significant difference between the mortalities of the 4.2 and 2.1 mg L^?1 treatments. The results suggest that CuSO₄ has clear therapeutic value against columnaris infections.     

Saprolegnia diclina IIIA and S. parasitica employ different infection strategies when colonizing eggs of Atlantic salmon,Salmo salar L.

Here, we address the morphological changes of eyed eggs of Atlantic salmon, Salmo salar L. infected with Saprolegnia from a commercial hatchery and after experimental infection. Eyed eggs infected with Saprolegnia spp. from 10 Atlantic salmon females were obtained. Egg pathology was investigated by light and scanning electron microscopy. Eggs from six of ten females were infected with S. parasitica, and two females had infections with S. diclina clade IIIA; two Saprolegnia isolates remained unidentified. Light microscopy showed S. diclina infection resulted in the chorion in some areas being completely destroyed, whereas eggs infected with S. parasitica had an apparently intact chorion with hyphae growing within or beneath the chorion. The same contrasting pathology was found in experimentally infected eggs. Scanning electron microscopy revealed that S. parasitica grew on the egg surface and hyphae were found penetrating the chorion of the egg, and re‐emerging on the surface away from the infection site. The two Saprolegnia species employ different infection strategies when colonizing salmon eggs. Saprolegnia diclina infection results in chorion destruction, while S. parasitica penetrates intact chorion. We discuss the possibility these infection mechanisms representing a necrotrophic (S. diclina) vs. a facultative biotrophic strategy (S. parasitica).     

Species Sensitivity to Copper

Abstract

Channel catfish, Ictalurus punctatus (Rafinesque 1818), and sunshine bass, female white bass Morone chrysops (Rafinesque 1820) × male striped bass M. saxatilis (Walbaum 1792), juveniles (10 and 12 g, respectively) were exposed to copper sulfate (CuSO₄) in a series of static toxicity tests to observe species sensitivity. The water used in this study was 18.9°C filtered well water with initial pH of 8.71, and total alkalinity and total hardness of 224 and 110 mg/L, respectively. Estimates of mean 96-hour median lethal concentration (LC50) values were 1.75 mg/L Cu (6.89 mg/L CuSO₄) for channel catfish and 0.85 mg/ L Cu (3.35 mg/L CuSO₄) for sunshine bass; LC50 values were calculated using nominal CuSO₄ concentrations. These values differed significantly. This study demonstrates that sunshine bass juveniles are less tolerant of CuSO₄ than channel catfish fingerlings when exposed concurrently in waters from the same source.     

Chemical Prevention and Treatment of Winter Saprolegniosis (“Winter Kill”) in Channel Catfish Ictalurus puncfatus1

The efficacies of formalin, potassium permanganate, sodium chloride, and copper sulfate as prophylactic treatments for saprolegniosis (“winter kill”) in channel caffish Icralums puncratus were evaluated. Formalin and copper sulfate were also evaluated as postinfective treatments for the disease. Each of the five experiments was conducted with 5–to 7-g channel catfish placed in static water aquaria maintained in refrigerated tanks. Water temperature was reduced from 25 to 10 C within 36 h and maintained at 10 ± 0.5 C for 28 d. Fish were exposed to cultured Saprolegnia sp. at 20 C, and morbidity and mortality data were recorded for 28 d. Saprolegnia sp.-associated mortalities occurred 7–10 d after exposure. Formalin (25 mg/L) was effective as both a prophylactic and postinfective treatment for saprolegniosis. Copper sulfate was effective in preventing saprolegniosis at a concentration of 0.1 mg/L, but was ineffective as a postinfective treatment. Sodium chloride at 5,000 mg/L was effective in preventing saprolegniosis, but was not practical for use in the commercial catfish industry. Potassium permanganate (up to 0.5 mg/L) was not effective in preventing or treating saprolegniosis in channel caffish. Based on the results of this study, it may be possible to prevent saprolegniosis in channel catfish using formalin or copper sulfate during winter when fish are immunosuppressed by rapid decreases in water temperature or to treat infected fish with formalin. However, the routine use of chemicals to control this disease should not be recommended until studies on their efficacy under typical caffish culture conditions and the effect of long-term use of chemicals on fish and on the environment are completed.     

Acute Toxicity of Copper to Juvenile Freshwater Prawns,Macrobrachium rosenbergii

Abstract

Copper sulfate is an algicide that is commonly used for phytoplankton and filamentous algae control and has been used as a therapeutant in aquaculture. The objectives of this study were to determine the acute toxicity of copper sulfate and the safe level for use in freshwater prawn, Macrobrachium rosenbergii, production ponds in a high calcium and alkalinity environment. Six concentrations of copper sulfate (0, 0.2, 0.4, 0.6, 0.8, 1.0 mg/L) were tested in 8-L glass aquaria for 48 hours with three replicate aquaria per treatment. Concentrations of calcium hardness and alkalinity were set at 100 mg/L using calcium chloride and sodium bicarbonate, respectively. After 48 hours, survival of the control treatment (0% CuSO₄) averaged 97%, which was significantly higher (P< 0.05) than that of all other treatments. The survival in the 0.2 mg/L and 0.4 mg/L (70% and 73%, respectively) concentrations of CuSO₄ were significantly greater (P< 0.05) than higher dose treatments; but were not significantly different from each other (P> 0.05). Treatments containing 0.6, 0.8, and 1.0-mg/L copper sulfate demonstrated a dramatic decrease in prawn survival, which averaged 30, 7, and 0%, respectively. Regression analysis of the data predicted 48-hour LC₅₀ for copper sulfate tobe0.46 mg/L. Since recommended application rates for use of copper sulfate as an algicide are 1.0 mg/L or more for water with alkalinities of 100 mg/L, copper sulfate treatments are not recommended for prawn production ponds.     

Comparative efficacy of Pyceze® (bronopol) in controlling mortality of brown trout Salmo trutta eggs

The efficacy of Pyceze^® (Novartis Animal Vaccines) and Proxitane^® 0510 (Solvay Interox) in controlling the mortality of eggs was studied in brown trout Salmo trutta eggs under the usual incubation conditions in a hatchery affected by saprolegniosis. Eggs from eight spawnings and from two lines of brown trout were used. The cumulative mortality of eggs at the start of the eyed stage (M₁) and at hatching (M₂) was measured, as was the percentage of eggs with fungal infection at weekly intervals during the green stage. Mortality at M₂ with Pyceze^® ranged between 2.38% and 12.61% depending on the batch, with a mean of 6.10%. Mortality at M₂ with Proxitane^® varied between 5.83% and 43.86%, with a mean of 22.36%. Fungal colonization at the start of the eyed stage ranged between 0% and 0.15% when Pyceze^® was used and between 0.82% and 12.50% with Proxitane^®. Mortality rates were higher among those eggs left untreated. The results indicate that Pyceze^® (bronopol) is efficacious in controlling mortality caused by Saprolegnia spp. and other biological factors in fertilized brown trout eggs, as has been demonstrated previously in other salmonid species.     

Cryopreservation of sperm of common carp, Cyprinus carpio L.

Milt of common carp, Cyprinus carpio L. was cryopreserved in pellet form with the use of 12 extenders. Most efficient were: BE2 original extender (containing 85 mM NaCl, 50 mM KCl, 3 mm CaCl₂, 1 mm MgCl₂ with 10% dimethyl-acetamide (DMA) and 10% addition of hen's egg yolk) and Kurokura et al.'s extender with 15% DMA and 10% yolk (about 73% and 69% of eyed eggs, about 61% and 52% of swim-up larvae, respectively). Within the most effective treatments, survival from the eyed-egg stage to the swim-up stage was similar to that observed in the control group. Survival from the eyed-egg stage to the swim-up stage (percentage of eyed eggs was considered as 100%) was highly significantly and positively correlated with the actual rate of swim-up larvae.     

Dietary copper effects survival of channel catfish challenged with Flavobacterium columnare

Columnaris disease is an important bacterial disease of commercially grown channel catfish, Ictalurus punctatus. Copper sulphate (CuSO₄) has been shown to be therapeutic and prophylactic as a water treatment for columnaris disease. Copper is an essential dietary component in animal feeds and CuSO₄ is typically included in base diets; a study was conducted to evaluate whether fish feed supplemented with additional CuSO₄ at 0, 40 and 80 mg kg^?1 of diet and fed at a daily rate of 3% body weight would affect survival to columnaris disease. Results indicate fish fed the copper‐supplemented diet for 2 weeks significantly increased survival following F. columnare challenge. This increase appeared to be dose‐dependent. The mean per cent survival (±SEM) for fish fed the base diet (unsupplemented) for 2 weeks and then challenged was 2.0% ± 1.1. Fish fed the base plus 40 mg CuSO₄ kg^?1 had a mean survival of 22.0% ± 11.0. Fish fed the base plus 80 mg CuSO₄ kg^?1 had a mean survival of 29.3% ± 13.4. The mean per cent survival for fish fed the base diet for 4 weeks and then challenged was 28.3% ± 9.0. Fish fed the base plus 40 mg CuSO₄ kg^?1 for 4 weeks had a mean survival of 12.5% ± 6.3. Fish fed the base plus 80 mg CuSO₄ kg^?1 for 4 weeks had a mean survival of 40.5% ± 8.1. There was a significant effect after 4 weeks with fish fed the base plus 80 mg CuSO₄ kg^?1 mg not with 40 mg kg^?1.     

Preventive efficacy of sodium hypochlorite against water mold infection on eggs of chum salmon Oncorhynchus keta

ABSTRACT:   To protect chum salmon eggs from water mold infection during incubation, the eggs were treated daily with sodium hypochlorite solution (NaOCl) at 10 mg/L residual chlorine concentration for 15 min during their developmental period from fertilization to eyed stages. The number of infected eggs and number of eyed eggs were observed on day 23 of incubation. The percentage of infected eggs to total eggs was significantly lower with NaOCl treatment (1.8 : 33.4%) than in the control (11.3 : 59.3%, P  < 0.01). The percentage of eyed eggs to total eggs was significantly higher with NaOCl (85.9 : 98.6%) than in the control (66.1 : 97.5%, P  < 0.01). The antifungal activity of NaOCl resulted in improving egg survival. Accordingly, NaOCl is a useful antifungal agent against water mold infection on chum salmon eggs.     

A thicker chorion gives ova of Atlantic salmon (Salmo salar L.) the upper hand against Saprolegnia infections

Since the ban of malachite green in the fish farming industry, finding alternative ways of controlling Saprolegnia infections has become of utmost importance. Much effort has been made to elucidate the mechanisms by which Saprolegnia invades fish eggs. Little is known about the defence mechanisms of the hosts, making some eggs more prone to infection than others. One clue might lie in the composition of the eggs. As the immune system in the embryos is not developed yet, the difference in infection levels could be explained by factors influenced by the mother herself, by either transferring passive immunity, influencing the physical aspects of the eggs or both. One of the physical aspects that could be influenced by the female is the chorion, the extracellular coat surrounding the fish egg, which is in fact the first major barrier to be overcome by Saprolegnia spp. Our results suggest that a thicker chorion in eggs from Atlantic salmon gives a better protection against Saprolegnia spp. In addition to the identification of differences in sensitivity of eggs in a fish farm set‐up, we were able to confirm these results in a laboratory‐controlled challenge experiment.     

Efficacy of clove oil and ethanol against Saprolegnia sp. and usability as antifungal agents during incubation of rainbow trout Oncorhynchus mykiss (Walbaum) eggs

Inhibitory concentrations of clove oil and ethanol against growth of Saprolegnia sp. hyphae were screened by a modification of the hemp (Cannabis sativa L.) seed MicroPlate (HeMP) method and their usability as antifungal agents during incubation of rainbow trout Oncorhynchus mykiss eggs was tested. In vitro experiment showed that in continuous static exposure, clove oil at 100 mg L^?1 significantly inhibited the growth of Saprolegnia, whereas in bath exposures, clove oil at 500 mg L^?1 had no significant effect at any exposure time tested (15, 60 and 240 min), but clove oil at 10 000 mg L^?1 significantly inhibited growth at all exposure times. Clove oil and ethanol treatments had no visible effects on the onset or spread of the fungus during incubation of rainbow trout eggs. Clove oil at 1000 mg L^?1 resulted in 95–100% mortality before the eyed stage was reached. Sublethal concentrations of clove oil and ethanol had no effects on the development rate of the embryo or growth and yolk utilization efficiency after hatching. This study suggests that clove oil and ethanol may not be options in controlling aquatic fungi infestations during incubation of rainbow trout eggs.     

双氧水预防白点鲑卵水霉病发生的效果研究

在白点鲑(Salvelinus leucomaenis Pallas)受精卵至发眼期间,为了替代甲醛,采用浓度分别为0mg/L(对照组)、600mg/L、900mg/L、1200mg/L、1500mg/L、1800mg/L的双氧水预防水霉菌的发生。消毒时间为隔天一次上午7:00-8:00,持续时间15min后换至正常流水孵化。在水温(4.5±0.4)℃,水体溶解氧(6.75±0.35)mg/L,pH(6.6±0.45)的情况下,经过40d的孵化,对各处理组的数据统计和分析,认为在浓度为1200mg/L可以有效预防水霉菌的发生,发眼率、上浮率和畸形率分别为89%、80.6%、0.7%、,而对照组相应为72%、70.2%、0.45%。当双氧水浓度低于1200mg/L时,受精卵孵化率和双氧水浓度呈正相关;高于1200mg/L时,受精卵孵化率和双氧水浓度呈负相关。另外,在孵化后期,发眼卵死亡率、仔鱼死亡率和畸形率则随着双氧水浓度的增加呈现明显上升的趋势。     

Effect of Salinity on Embryonic Development,Hatchability, and Growth of African Catfish,Clarias gariepinus,Eggs and Larvae

Abstract

Effects of salinity on embryonic development and growth of African catfish, Clarias gariepinus, eggs and larvae were studied. Eggs were incubated at 27-29°C in 2,4,6,8, and 10 ppt sodium chloride. Rate of embryonic development was delayed in all salt solutions by 15, 15,28 and 30 minutes, in 2,4,6, and 8 ppt sodium chloride, respectively, when compared with the control group (0% salt); total mortality occurred at 12 hours after gastrula stage in the 10 ppt concentration. Percentage hatching was 45.1,47.7, 59.5,49.2, and 26.6% while percentage deformity was 10.4, 16.1, 52.0, 28.6, and 71.6% in 0, 2, 4, 6, 8, and 10 ppt salt treatments, respectively. There were significant differences (P <0.05) in the hatching percentage and in deformity percentage between 4, 6, and 8 ppt. Rate of yolk absorption was significantly faster in the control and 2 ppt salt treatments, but slower in 4, 6, and 8 ppt. Rate of increase in length was slower with increasing salinity. The optimum salinity for African catfish eggs and was between 0-2 ppt and acceptable up to 6 ppt. The results suggest that increasing salinity delayed hatching and development of African catfish eggs and larvae, respectively, as well as increased the deformity of the larvae.     

In Vitro Screening of Fungicidal Chemicals for Antifungal Activity against Saprolegnia

Saprolegnia is an important fish fungal pathogen that often results in significant economic losses to freshwater aquaculture. To find effective drugs to control saprolegniasis, 30 fungicidal chemicals used in agriculture were screened, in which kresoxim‐methyl and azoxystrobin, with minimum inhibitory concentration (MIC) values of 1.0 and 0.5 mg/L, respectively, showed good in vitro antifungal activities against Saprolegnia. Azoxystrobin has the most promising anti‐Saprolegnia activity with 50% effective concentration (EC₅₀) value of 0.212 mg/L against mycelial growth and minimum fungicidal concentration (MFC) value of 0.13 mg/L against spores, while EC₅₀ and MFC values to kresoxim‐methyl are 0.240 and 0.25 mg/L, respectively. Through the acute toxicity assay using goldfish, Carassius auratus, azoxystrobin exhibited wider margin of safety with a safe concentration (SC) value of 0.553 mg/L than kresoxim‐methyl with an SC value of 0.131 mg/L. These findings demonstrated that azoxystrobin has the potential for the development of therapy for the control of Saprolegnia in aquaculture. Both kresoxim‐methyl and azoxystrobin were tested with a post‐antifungal effects (PAFE) assay and the results revealed that the two chemicals had no significant effect on fungal growth inhibition after a 1‐hour exposure, indicating that the treatment needs to be carried out over an extended period.     

Antifungal Activity of Rhein and Aloe‐Emodin from Rheum palmatum on Fish Pathogenic Saprolegnia sp.

Saprolegnia infections cause severe economic losses among freshwater fish farming. In this study, two known compounds, rhein and aloe‐emodin, were isolated from Rheum palmatum, and the in vitro inhibitory activity of both compounds against mycelial growth and spore germination of Saprolegnia was tested. Both rhein and aloe‐emodin were able to decrease Saprolegnia mycelial growth and spore activity in all tested concentrations after exposure for 48 h. Complete inhibition of mycelial growth was observed at 20 mg/L for rhein and at 50 mg/L for aloe‐emodin, while spore germination was 100% prevented at 16 and 40 mg/L for rhein and aloe‐emodin, respectively. Because rhein showed stronger in vitro anti‐Saprolegnia activity, it was further tested in vivo to measure the prevention and treatment efficacy on Saprolegnia infection of grass carp. Its acute activity to grass carp was also evaluated. The results revealed that exposure to rhein at 20 mg/L for 7 d could prevent 93.3% of infections by Saprolegnia in abraded grass carp, while 67.7% of infected fish could be recovered by treatment with rhein. The 48‐h median lethal concentration (48 h‐LC₅₀) to grass carp was 148.5 mg/L, which is about 7.4 times the effective dose indicating the safety for the use of rhein. This study suggests that rhein has promising anti‐Saprolegnia activity and may be an option in preventing and controlling Saprolegnia infection.