Haplosporidium nelsoni (MSX) in Eastern Oyster Populations of North Carolina

Abstract

Populations of the eastern oyster Crassostrea virginica from 36 locations off the coast of North Carolina were examined cytologically in the fall of 1989 for Haplosporidium nelsoni (MSX). The pathogen was found in oysters from 11 (31%) of the sites. Systemic Perkinsus marinus infections were noted in oysters from 26 (72%) of the locations.     

Lack of Hemocyte Chemiluminescence Stimulation by Perkinsus marinus in Eastern Oysters Crassostrea virginica with Dermo Disease

Abstract

Phagocytosis of zymosan A particles by hemocytes of eastern oyster Crassostrea virginica stimulates the production of reactive oxygen species (ROS), which are easily quantified by luminol-augmented chemiluminescence (CL). Antimicrobial defense mechanisms of hemocytes may involve the activity of cytotoxic ROS. The CL response to phagocytosis of zymosan by hemocytes from C. virginica with advanced Perkinsus marinus infections is more elevated than that produced by zymosan in cells from uninfected oysters. This effect is perhaps akin to macrophage activation. Phagocytosis of P. marinus cells by hemocytes withdrawn from uninfected oysters produced no detectable CL response. Hemocytes withdrawn from oysters with P. marinus infections ranging from light to heavy were evaluated for CL responses after phagocytosis of zymosan or P. marinus. Increases in CL stimulation by zymosan were seen as the intensity of infection increased. Despite avid phagocytosis of P. marinus, CL activity of the hemocytes was not seen, regardless of the level of infection of the host. Lack of hemocytic ROS stimulation by ingestion of P. marinus cells may contribute to in vivo survival of this parasite.     

Disease Susceptibility of Hatchery Snake River Spring–Summer Chinook Salmon with Different Juvenile Migration Histories in the Columbia River

Abstract

Various methods have been developed to mitigate the effects of dams on juvenile Pacific salmon Oncorhynchus spp. migrating to the Pacific Ocean through the Columbia River basin. In this study, we examined the health of hatchery Snake River spring and summer Chinook salmon relative to two mitigating strategies: dam bypass and transportation (e.g., barging). The health of out-migrants was assessed in terms of the difference in the incidence of mortality among fish, categorically grouped into no-bypass, bypass, and transportation life histories, in response to challenge with the marine pathogen Listonella anguillarum during seawater holding. These three life histories were defined as follows: (1) fish that were not detected at any of the juvenile bypass systems above Bonneville Dam were classified as having a no-bypass life history; (2) fish that were detected at one or more juvenile bypass systems above Bonneville Dam were classified as having a bypass life history; and (3) fish that were barged were classified as having the transportation life history. Barged fish were found to be less susceptible to L. anguillarum than in-river fish—whether bypassed or not—which suggests that transportation may help mitigate the adverse health effects of the hydropower system of the Columbia River basin on Snake River spring–summer Chinook salmon. The findings of this study are not necessarily transferable to other out-migrant stocks in the Columbia River basin, given that only one evolutionarily significant unit, that is, Snake River spring–summer Chinook salmon, was used in this study.     

Bacteria flora and heavy metals in cultivated oysters <Emphasis Type="Italic">Crassostrea iredalei</Emphasis> of Setiu Wetland,East Coast Peninsular Malaysia

Slipper oyster Crassostrea iredalei is a species of good demand for its sweet flavor and white coloured flesh. The filter feeding nature predisposes oysters to accumulation of pathogenic and heavy metals in waters impacted by sewage pollutions and may thus render the oysters unfit for human consumption. A study was undertaken to investigate the presence of bacteria flora and heavy metal concentrations in cultivated oysters Crassostrea iredalei at Setiu Wetland, Terengganu, the only source of cultivated oysters in East Coast of Malaysia. A total of 200 slipper oyster samples were analyzed. The bacteria were isolated using non selective agar such as TSA agar and selective agars before they were then identified using conventional methods in combination with BBL Crystal identification kit. Heavy metals such as zinc (Zn), copper (Cu), cadmium (Cd), and lead (Pb) concentrations were determined using atomic absorption spectrophotometry. Results showed that the oysters harbor predominantly Shewanella putrifaciens followed by Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio cholerae, Enterobacter cloacae, Escherichia coli and Chromobacterium violaceum. They also contain high concentration of Zn (785.68 ± 285.88 μg/g) with the lowest heavy metal was Pb (0.17 ± 0.15 μg/g), whilst the concentrations of other heavy metals were Cu (38.9 ± 13.2 μg/g) and Cd (1.60 ± 0.28 μg/g). The study is very useful to evaluate the type of bacteria and heavy metal present in oyster meat for human consumption.     

Immunodeficiency Diseases in Marine Mollusks: Measurements of Some Variables

Abstract

Immunity in bivalves comprises cell-mediated and humoral mechanisms. Because bivalves (and gastropods) possess open circulatory systems, studies were conducted to ascertain whether copper and cadmium ions that enter eastern oysters Crassostrea virginica would compromise certain aspects of the animals' cell-mediated and humoral immunity. In vivo exposure to a 1-mg/L concentration of Cu²⁺ was more lethal to eastern oyster hemocytes than exposures to 1-mg/L concentrations of Cu²⁺ plus EDTA, Cd²⁺, or Cd²⁺ plus EDTA. However, in vivo exposures to 1-mg/L concentrations of both metals alone and in the chelated forms had no effect on the chemotactic attraction between hemocytes and sonicated Escherichia coli. Also, measurements of enzyme activities in eastern oysters challenged in vivo with 1-mg/L concentrations of Cu²⁺, Cu²⁺ plus EDTA, Cd²⁺, or Cd²⁺ plus EDTA revealed that Cd²⁺ inhibited the release of lysozyme into serum, Cu²⁺ inhibited the release of acid phosphatase into serum, and Cu²⁺ stimulated the intracellular synthesis of β-glucuronidase in hemocytes but inhibited the release of this enzyme into serum. In view of the above and previously published information, it is now known that in vivo exposure to certain heavy metals, as well as alterations in salinity and temperature, will compromise the internal defense mechanisms of mollusks.     

Investigation of mortality in Pacific oysters associated with Ostreid herpesvirus-1 μVar in the Republic of Ireland in 2009

High levels of mortality in Pacific oysters Crassostrea gigas in the Republic of Ireland were recorded during the summer of 2009. The new variant of Ostreid herpes 1 (OsHV-1 μVar) which first emerged in France in 2008 was identified from affected stocks. Retrospective data was collected from 70 oyster farmers through an interviewer-administered questionnaire to investigate the distribution and determinants of the mortality. Based on farmer recall, data were recorded at the batch level for cumulative mortality during 2009, start dates and duration of the mortality event and the age of animals affected. Observable mortalities were recorded in 109 out of 346 batches at 47 sites; 104 of the 109 batches were located in bays where OsHV-1 μVar had been detected. The records from bays where OsHV-1 μVar had been detected were analysed to characterize the pattern of mortality and potential risk factors. Batch mortality averaged 37% (18-65% quartiles) but showed a bimodal distribution (half the batches had mortality less than 45%). Mortalities started at the end of May and continued until early August, peaking in early July. On average oysters died over a period of 18 days. Mortality varied considerably both between and within bays. Mortality started in recently introduced batches and occurred later in the summer in established oysters, which is consistent with the introduction of an infectious agent. Mortality was significantly lower in adults compared with other age groups, which supports observations from France. Three variables were significantly (P<0.05) associated, in both bivariate screening and a logistic regression, with high batch-level mortality (>40%): oysters (i) introduced as juveniles, (ii) during or since the winter of 2008/9 and (iii) which spent less than 8h out of water (in a tidal cycle) (compared with oysters introduced as adults before the winter of 2008/9 and spending more than 8h out of water). Twenty-one percent of triploid batches experienced "high" (>40%) mortality compared with 10% for diploid batches which was significant (P<0.05) in the initial bivariate screening but not in the final logistic regression model. Future studies should develop improved methods to assess oyster mortality and follow stocks over time to better determine the influence of management and environmental factors on mortality.     

An Investigation of the Pathology Associated with Mass Mortality Events in the Cultured Japanese Pearl Oyster Pinctada fucata martensii at Four Farms in Western Japan

Abstract

The epidemiological and histopathological characteristics of the mass mortality of cultured Japanese pearl oysters Pinctada fucata martensii were investigated. Rearing experiments with Japanese pearl oysters in farms revealed that the mass mortality occurs as a regular annual event in particular farms in western Japan. Diseased oysters had marked atrophy and red-brown discoloration of the soft parts of the body. Light microscopy revealed that the epithelia of the stomach, the ducts of the digestive diverticula (DD), and the DD themselves showed marked blebbing and necrosis to varying degrees during earlier stages of the disease. At advanced stages, muscle fibers of the adductor muscle, heart, mantle, and other parts of the body and the connective tissues of various organs involving the vascular system also exhibited considerable atrophy and necrosis. There were no remarkable changes in the branchial and pallial epithelia. No viral, bacterial, mycotic, or parasitic causative organisms were found in diseased oysters. The results of a case study of mass mortality at one farm suggested that there is some causal relationship between outbreaks of this disease and the existence of neighboring fish farms. These findings suggest that the mass mortality is not due to an infectious disease. We discuss pathological features and possible causes of this disease.     

Infectious Salmon Anemia (ISA) Virus: Infectivity in Seawater under Different Physical Conditions

Abstract

Infectious salmon anemia (ISA) virus (genus Isavirus, family Orthomyxoviridae), present in all major salmon producing countries, is the causative agent for a serious and commercially important disease affecting Atlantic Salmon Salmo salar. Nearly all ISA outbreaks occur in the marine production phase and knowledge about survival time for ISA virions in seawater is crucial for an adequate strategy to combat the disease. To acquire knowledge about this important factor, a study of ISA virus exposed to four different physical conditions was carried out. The virions’ survival was tested in sterile seawater, sterile seawater with normal ultraviolet light radiation (UVR), natural seawater, and natural seawater with UVR. During the 72-h experiment both presence of ISA virus RNA and the infectivity of ISA virions were monitored. The result of this study showed that the infectivity of ISA virions is lost within 3 h of exposure to natural seawater or sterile seawater with UVR. However, it was possible to detect ISA virus RNA throughout the experimental period. This indicates that the effect of both UVR and biological activity of natural seawater limits the survival time of ISA virions under normal conditions. The survival time of ISA virions in sterile seawater was less than 24 h. Based on the available literature and the present study it is not very likely that passive horizontal transmission in seawater over long distances can occur. This is due to the following factors: (1) the effect of UVR and biological activity on ISA virions infectivity found in the present study, (2) the speed and dilution effect in seawater currents in salmon farming areas, (3) the temperature during the major outbreak periods, and (4) the need for an infective dose of ISA virions to reach naive Atlantic Salmon.

Received August 22, 2013; accepted November 7, 2013     

Detection of Minchinia occulta in samples of pearl oysters Pinctada maxima infected by Haplosporidium hinei

Objective   To determine if juvenile pearl oysters ( Pinctada maxima ) infected with Haplosporidium hinei are also infected with another haplosporidian parasite, Minchinia occulta .
Design   Archived samples of pearl oysters infected with H. hinei were examined using polymerase chain reaction (PCR) assays and in situ hybridisation (ISH) to analyse and identify haplosporidians. A 144-bp and 220-bp region of Minchinia DNA were targeted by PCR and amplified DNA from formalin-fixed H. hinei -infected pearl oyster samples was sequenced. A 25-bp oligonucleotide probe targeting a variable section of the parasite's small subunit rRNA gene was used in ISH.
Results   The results of DNA-based diagnostic assays supported each other. The sequences obtained by PCR were found to be almost identical to M. occulta from rock oysters and the ISH assay demonstrated infection with M. occulta in affected pearl oysters. ISH indicated a prevalence of infection of 26.7% in one of the previous outbreaks.
Conclusion   Pearl oyster spat are susceptible to infection by a Minchinia parasite, most likely M. occulta , which was recently identified in rock oysters within the pearl-producing zones of Western Australia and is associated with mortalities of up to 80% in this species. The occurrence of haplosporidian co-infections in pearl oysters suggests the immunocompetence of juvenile oysters may be an important factor in preventing infection and therefore preventing mortalities such as those occurring in the recent outbreaks of pearl oyster oedema disease.     

Survey of Pathogens in Juvenile Salmon Oncorhynchus Spp. Migrating through Pacific Northwest Estuaries

Abstract

Although the adverse impact of pathogens on salmon populations in the Pacific Northwest is often discussed and recognized, little is currently known regarding the incidence and corresponding significance of delayed disease-induced mortalities. In the study reported herein, we surveyed the presence and prevalence of selected micro- and macroparasites in out-migrant juvenile coho salmon Oncorhynchus kisutch and Chinook salmon O. tshawytscha from 12 coastal estuaries in the Pacific Northwest over a 6-year period (1996–2001). The major finding of this study was the widespread occurrence of pathogens in wild salmon from Pacific Northwest estuaries. The six most prevalent pathogens infecting both juvenile Chinook and coho salmon were Renibacterium salmoninarum, Nanophyetus salmincola, an erythrocytic cytoplasmic virus (erythrocytic inclusion body syndrome or erythrocytic necrosis virus), and three gram-negative bacteria (Listonella anguillarum, Yersinia ruckeri, and Aeromonas salmonicida). The most prevalent pathogen in both Chinook and coho salmon was N. salmincola, followed by the pathogens R. salmoninarum and the erythrocytic cytoplasmic virus. Statistically significant differences in the prevalence of R. salmoninarum and N. salmincola were observed between Chinook and coho salmon. Based on the prevalence of pathogens observed in this study, disease appears to be a potentially significant factor governing the population numbers of salmon in the Pacific Northwest. Development of a detailed understanding of the principal components influencing the ecology of infectious disease will aid in the development of management and control strategies to mitigate disease in and hence further the recovery of salmon stocks listed under the Endangered Species Act.     

Protease Inhibitory Activity in Selectively Bred Families of Eastern Oysters

Abstract

In an effort to identify potential biochemical markers for resistance to Dermo, a disease that afflicts eastern oysters Crassostrea virginica, selected stocks of these oysters were interbred to produce 10 families with high interfamily and intrafamily genetic variability; subsamples of 2-year-old oysters from each family were then assessed for protease inhibitory (PI) activity against proteases of Perkinsus marinus, the causative agent of Dermo. The prevalence and intensity of the pathogen were determined via Ray's fluid thioglycollate medium test. Family survival under field challenge, disease intensity, and PI activity were compared to evaluate family performance. In the laboratory, three families had the highest survival rates, lowest average numbers of parasite cells, and highest average PI activity. In field challenges, the same three families exhibited the lowest cumulative mortality. Among all families, disease intensity was negatively correlated with PI activity; mortality patterns also indicated an inverse relationship between disease intensity and such activity.     

Parasites of the pleasure oyster Crassostrea corteziensis cultured in Nayarit, Mexico

The pleasure oyster Crassostrea corteziensis is collected and cultured in Nayarit on the Pacific coast of Mexico, and the improvement and promotion of its culture are seen as a possible source for the economic development of coastal populations. However, information about the parasite fauna of the pleasure oyster is almost completely lacking. A histopathological survey carried out in two estuaries, Boca del Camichín and Pozo Chino, revealed the presence of hypertrophied gametes, rickettsiales-like prokaryotes (RLPs), the protozoan Perkinsus marinus, a protozoan Nematopsis sp., Ancistrocoma-like ciliates (ALCs), Sphenophrya-like ciliates, a turbellarian Urastoma sp., and encysted crustaceans. In general, prevalence and intensity of parasites were similar in both localities except that ALCs and encysted crustaceans were more prevalent in Pozo Chino than in Boca del Camichín. Perkinsus marinus and RLPs seem to represent a more significant risk for the health of pleasure oysters than do the other parasites, and surveillance and control of these parasites are needed for the development of pleasure oyster culture.     

钦州湾牡蛎线粒体16 S rRNA基因片段核苷酸序列分析

对69个钦州湾牡蛎个体的线粒体DNA16S rRNA进行PCR扩增,纯化后的PCR产物测序分析,研究16S rRNA基因在白肉牡蛎和红肉牡蛎2个群体中的遗传多样性,通过DNAman比对序列并构建系统进化树。结果得到2个群体的序列长度均为434bp,共检测到16个核苷酸突变位点,包括8个转换位点和8个颠换位点。与GenBank序列比对发现,白肉牡蛎和香港牡蛎的序列基本相同,系统进化树中显示白肉牡蛎与香港牡蛎聚为一支,红肉牡蛎与有明巨牡蛎聚为一支。证实了白肉牡蛎和红肉牡蛎是2个不同的种,它们有各自的遗传多样性,该研究为牡蛎的遗传育种提供科学依据。     

Thermal burns of the spectacle associated with supplementary heating in native New Zealand geckos

ABSTRACT

Case history: Gradual onset of ocular opacity was observed in three gold-striped geckos (Woodworthia chrysosiretica), and five Pacific geckos (Dactylocnemis pacificus) held in two adjacent terrariums in a zoological institution located in the North Island of New Zealand. Ultraviolet light and heat had been provided for the previous 3–4 years by a fluorescent bulb, but in the last 4 weeks of winter a ceramic heat bulb had been added, situated 10?cm above the upper mesh of the cage

Clinical findings: All eight geckos presented with mostly bilateral lesions of varying severity confined to the central or upper quadrant of the spectacles. These lesions ranged from variable areas of opacity within the stroma of the spectacle to similarly distributed ulcers of the surface epithelium of both spectacles. The spectacle lesions in the Pacific geckos responded well to treatment with topical combined antimicrobial therapy, within 18–29 days. The gold-striped geckos suffered complications including dysecdysis, severe spectacle ulceration and perforation, mycotic spectaculitis, and widespread mycotic dermatitis resulting in death or leading to euthanasia.

Pathological findings: In the three gold-striped geckos, there were extensive areas of deep ulceration and replacement of the spectacle with a thick serocellular crust containing large numbers of fungal elements. The affected areas of the stroma were expanded by large deposits of proteinaceous and mucinous material, pyknotic cellular debris and moderate numbers of heterophils and macrophages as well as infiltrating fungal hyphae.

Diagnosis: Mycotic spectaculitis with ulceration and perforation, and disseminated mycotic dermatitis likely secondary to thermal burns.

Clinical relevance: This is the first report of thermal burns of the spectacle in any reptile. There was species variation in the burn severity with gold-striped geckos showing more severe lesions, possibly due to a mix of behavioural and anatomical factors. The thermal burns to the spectacles in three cases were complicated by delayed healing, perforation, dysecdysis and severe mycotic infection.     

Detection of Myxobolus (Myxosoma) cerebralis in Salmonid Fishes in Oregon

Abstract

Myxobolus (Myxosoma) cerebralis, the etiological agent of whirling disease, was detected in salmonid fish populations in northeastern Oregon. This is the first record of M. cerebralis in the Pacific Northwest of the USA. During an epizootiological survey for the parasite, two methods for spore detection were compared, and an efficient procedure for determining M. cerebralis infection in adult fish was developed. The enzyme digest method was more efficient than the plankton centrifuge procedure for examination of numerous individual lots of fish processed during the survey. Sampling only the area around the otoliths was at least as effective as sampling entire heads for detection of spores in infected fish.     

Infecting Pacific Herring with Ichthyophonus sp. in the Laboratory

The protistan parasite Ichthyophonus sp. occurs in coastal populations of Pacific Herring Clupea pallasii throughout the northeast Pacific region, but the route(s) by which these planktivorous fish become infected is unknown. Several methods for establishing Ichthyophonus infections in laboratory challenges were examined. Infections were most effectively established after intraperitoneal (IP) injections with suspended parasite isolates from culture or after repeated feedings with infected fish tissues. Among groups that were offered the infected tissues, infection prevalence was greater after multiple feedings (65%) than after a single feeding (5%). Additionally, among groups that were exposed to parasite suspensions prepared from culture isolates, infection prevalence was greater after exposure by IP injection (74%) than after exposure via gastric intubation (12%); the flushing of parasite suspensions over the gills did not lead to infections in any of the experimental fish. Although the consumption of infected fish tissues is unlikely to be the primary route of Ichthyophonus sp. transmission in wild populations of Pacific Herring, this route may contribute to abnormally high infection prevalence in areas where juveniles have access to infected offal.

Received June 25, 2015; accepted September 12, 2015     

Survey of Salmonid Pathogens in Ocean-Caught Fishes in British Columbia,Canada

Abstract

A survey of wild fishes captured around marine net-pen salmon farms and from open waters for certain salmonid pathogens was conducted in the coastal waters of British Columbia. Viral hemorrhagic septicemia virus was detected in Pacific herring Clupea pallasi, shiner perch Cymatogaster aggregata, and threespine sticklebacks Gasterosteus aculeatus. Infectious hematopoietic necrosis (IHN) virus was detected in one Pacific herring (collected well away from the farms) and in tube-snouts Aulorhynchus flavidus and shiner perch collected from a farm experiencing an IHN outbreak. Renibacterium salmoninarum was observed in moribund Pacific hakes Merluccius productus collected from within a net-pen and was also detected in several ocean-caught salmon. Aeromonas salmonicida subsp. salmonicida (typical strain) was isolated from a juvenile chinook salmon Oncorhynchus tshawytscha, whereas the atypical strain of this organism was isolated from a lingcod Ophiodon elongatus. Loma salmonae (Microsporea) was observed in chinook salmon, chum salmon Oncorhynchus keta, coho salmon O. kisutch, sockeye salmon O. nerka, and pink salmon O. gorbuscha, all of which were captured well away from net-pens. Loma spp. (Microsporea) were observed in the gills of shiner perch, lingcod, Pacific tomcod Microgadus proximus, Pacific cod Gadus macrocephalus, walleye pollock Theragra chalcogramma, and sablefish Anoplopoma fimbria; all but the first species represent new hosts for Loma. Epitheliocystis, caused by a chlamydia-like organism, was detected in the gills of chinook salmon, chum salmon, coho salmon, pink salmon, lingcod, Pacific cod, Pacific hakes, Pacific tomcod, walleye pollock, sablefish, shiner perch, Dover soles Microstomus pacificus, Pacific sanddabs Citharichthys sordidus, and various species of rockfish Sebastes spp., most of which represent new host records for this infection.     

Epidermal Tumors on Rainbow Smelt and Atlantic Salmon from Nova Scotia

Abstract

Epidermal tumors on rainbow smelt Osmerus mordax are described in detail for the first time. These tumors apparently progress from small preneoplastic lesions to papillomas, and to carcinomas on some males. These tumors and similar tumors on Atlantic salmon Salmo salar have not been previously reported from Canada. Tumors were found on rainbow smelt mainly in freshwater during the spring spawning run and on Atlantic salmon grilse in freshwater in October, These tumors varied in size and number on each fish. The smaller growths were usually translucent and whitish, with a smooth surface. Some larger tumors were papillomatous and appeared reddened because of increased vascularization. Preneoplastic lesions and papillomas had a thickened epidermis with an intact basement membrane, and most of the cells were enlarged. fn papillomas of rainbow smelt, tumor cells that were not close to papillae were vacuolated, and necrotic regions were present. Carcinomas on the heads of male rainbow smelt had regions of pleomorphic cells which invaded the subepithelial tissues and penetrated the cartilage of the mandible of two specimens found in brackish and seawater. Intranuclear basophilic inclusions suggestive of a viral agent were present in the tumors of both rainbow smelt and Atlantic salmon.     

Experimental infection of Pacific oyster Crassostrea gigas spat by ostreid herpesvirus 1: demonstration of oyster spat susceptibility

ABSTRACT: In 2008 and 2009, acute mortalities occurred in France among Pacific cupped oyster, Crassostrea gigas, spat. Different hypothesis including the implication of environmental factors, toxic algae and/or pathogens have been explored. Diagnostic tests indicated that OsHV-1 including a particular genotype, termed OsHV-1 μVar, was detected in most of samples and especially in moribund oysters with the highlighting of virus particles looking like herpes viruses by TEM examination. In this study, an experimental protocol to reproduce OsHV-1 infection in laboratory conditions was developed. This protocol was based on the intramuscular injection of filtered (0.22 μm) tissue homogenates prepared from naturally OsHV-1 infected spat collected on French coasts during mortality outbreaks in 2008. Results of the experimental trials showed that mortalities were induced after injection. Moreover, filtered tissue homogenates induced mortalities whereas the same tissue homogenates exposed to an ultraviolet (UV) treatment did not induce any mortality suggesting that oyster spat mortalities require the presence of a UV sensitive agent. Furthermore, analysis of injected oyster spat revealed the detection of high amounts of OsHV-1 DNA by real-time quantitative PCR. Finally, TEM analysis demonstrated the presence of herpes virus particles. The developed protocol allowed to maintain sources of infective virus which can be useful for the development of further studies concerning the transmission and the development of OsHV-1 infection.