Salmonid whirling disease: myxosporean and actinosporean stages cross-react in direct fluorescent antibody test

Abstract. Serologic relatedness of the two life stages of the salmonid whirling disease parasite Myxosoma cerebralis Hofer, 1903 — myxosporean spores from fish cartilage and actinosporean triactinomyxon spores from aquatic tubificids — were investigated. When the direct fluorescent antibody technique was used, anti-triactinomyxon and anti- M. cerebralis rabbit sera conjugated with fluorescein isothiocyanate cross-reacted with the respective heterologous life stage. Both stages showed similar locations of specific fluorescence with conjugates of either homologous or heterologous serum. Thus, serology supports the relatedness of the myxosporean M. cerebralis and the actinosporean triactinomyxon stages.     

A vital staining technique with fluorescein diacetate (FDA) and propidium iodide (PI) for the determination of viability of myxosporean and actinosporean spores

A vital staining technique with fluorescein diacetate (FDA) and propidium iodide (PI) was used for determination of viability of myxosporean stage spores of Myxobolus artus and actinosporean stage spores of M. cultus . Viable spores stained green with FDA and non-viable spores stained red with PI were clearly distinguishable in M. artus myxosporean spores released from live infected fish, while the spores collected from pseudocysts were not well stained with FDA. Immediately after release from the oligochaete, Branchiura sowerbyi , actinosporean spores of M. cultus , stained bright green in the sporoplasms and red in the spore processes. In vitro survivability tests revealed that the longevity of M. artus spores was 5 months at 25°C and 15 months at 18°C. Drying and ultraviolet irradiation (36 000 mW s cm^–2 for M. artus myxosporean spores, 600 mW s cm^–2 for M. cultus actinosporean spores) were most effective as sporicidal treatments.     

Complete developmental cycle of Myxobolus pseudodispar (Gorbunova) (Myxosporea: Myxobolidae)

Myxobolus pseudodispar (Gorbunova) is a common parasite of the muscle of roach, Rutilus rutilus L., whereas its actinosporean development occurs in two oligochaete alternate hosts. This paper reports the complete developmental cycle of this parasite in the oligochaete alternate host Tubifex tubifex and the roach. In laboratory experiments, parasite-free T. tubifex specimens were infected by myxospores of M. pseudodispar collected from roach in Lake Balaton. Parasite-free roach fingerlings were infected with floating triactinospores (TAMs) released from oligochaetes on day 69 after challenge. Young plasmodia and spores in roach were first recorded on day 80 post-exposure (p.e.). Myxospores collected from experimentally infected roach initiated a new development in T. tubifex and the resulting TAMs infected roach. No infection of roach resulted from feeding oligochaetes containing mature triactinospores.     

Characterization of glycans in the developmental stages of Myxobolus cerebralis (Myxozoa), the causative agent of whirling disease

Glycans and sugar-binding molecules (lectins) form an interactive recognition system, which may enable parasitic organisms to adhere to host cells and migrate into target tissues. The aim of the present study was to analyse surface-associated glycans in the developmental stages of Myxobolus cerebralis (Hofer), the causative agent of whirling disease. A panel of biotin-labelled plant lectins was used to detect a broad spectrum of glycan motifs with high specificity. Binding sites were detected histochemically in the tissue sections of infected rainbow trout, Oncorhynchus mykiss (Walbaum), and infected Tubifex tubifex (Müller), and were characterized by light, fluorescence and transmission electron microscopy. With mannose-specific lectins [Lens culinaris agglutinin, Pisum sativum agglutinin, Canavalia ensiformis agglutinin (LCA, PSA, CanA)] mannose-containing glycans were detected in all the developmental stages and host tissues. No binding sites for galactose-specific lectins were present in M. cerebralis spores but reactivity with host tissues occurred. Diversity in glycans was detected by N-acetyl-D-galactosamine-specific lectins in sporoplasm cells of M. cerebralis and triactinomyxon spores. In the group of lectins with monosaccharide-specificity for N-acetyl-D-glucosamine (GlcNAc), the reactivity of Datura stramonium agglutinin (DSA), Lycopersicon esculentum agglutinin (LEA) and Solanum tuberosum agglutinin (STA) was restricted to polar capsules whereas Griffonia simplicifolia agglutinin II (GSA II) also bound to sporoplasm cells of stages in the fish host but not in those present in infected T. tubifex. Moreover, Triticum vulgaris (wheat germ) agglutinin (WGA) and succinylated WGA indicated the presence of N-acetyl-D-glucosamine polymers in polar capsules. No specificity for spores was observed concerning 'bisected'N-glycans and no reactivity in parasitic stages was observed with the fucose-binding lectin Ulex europaeus agglutinin (UEA) I, Sambucus nigra agglutinin (SNA) (specific for alpha2,6-sialylated glycans) and Maackia amurensis agglutinin (MAAI) (specific for alpha2,3-sialylated glycans). Arachis hypogaea (peanut) agglutinin (PNA), Erythrina cristagalli agglutinin (ECA), GSA I, Sophora japonica agglutinin (SJA), Dolichos biflorus agglutinin (DBA) and GSA II detected reactive sites solely confined to the developmental stages of M. cerebralis and were not reactive in the fish host. These parasite-specific glycans may play a role in the adhesion process of the parasite to fish epidermis prior to infection, but may provide protection to the host by activating the complement system, or stimulating an adaptive immune response as putative antigens.     

The life cycle of Henneguya nuesslini Schuberg & Schroder, 1905 (Myxozoa) involves a triactinomyxon-type actinospore

The life cycle of the histozoic myxozoan parasite Henneguya nuesslini was investigated in two salmonid host species. Naive brown trout, Salmo trutta, and brook trout, Salvelinus fontinalis, were experimentally infected in two trials by triactinomyxon type actinospores from naturally infected Tubifex tubifex. In exposed common carp, Cyprinus carpio, no myxospore production was detected. The parasite formed cysts with mature myxospores in the connective tissue of the fish 102 days post-exposure. The morphology of both actinosporean and myxosporean stages was described by light microscopy and a 1417-bp fragment of the 18S rDNA gene was sequenced. Sequence analysis confirmed the absolute congruence of the two developmental stages and assisted in determining species identity. Host range, tissue specificity and myxospore measurements provided sufficiently distinctive features to confirm species validity and were thus crucial for identification. The triactinomyxon spores had 16 secondary germ cells, unique dimensions, a very opaque sporoplasm matrix and three conspicuously protruding, pyriform polar capsules. This is the first record of a Henneguya sp. life cycle with a triactinomyxon-type actinospore, which suggests a close relationship with the Myxobolus group and a polyphyletic origin of the genus Henneguya.     

Experimental identification of the actinosporean stage of Sphaerospora renicola Dykova & Lom 1982 (Myxosporea: Sphaerosporidae) in oligochaete alternate hosts

The extrapiscine development of Sphaerospora renicola, a myxosporean parasite of the kidney of common carp, Cyprinus carpio L., was studied in the experimentally infected oligochaetes Tubifex tubifex (Müller) and Branchiura sowerbyi (Beddard). After the infection of these tubificids with homogenized common carp kidneys containing myxospores of S. renicola, the development of actinosporean stages was first observed under light microscopy 8 days after infection in pathogen-free T. tubifex. Infection of B. sowerbyi with mature actinosporean stages was first observed 91 days after infection. At that stage of development, pansporocysts containing neoactinospores filled the intestinal epithelium of the worm. Ninety-five days after infection, pansporocysts containing actinospores and free actinospores were found in the gut lumen of B. sowerbyi. Actinospores of S. renicola emerged from B. sowerbyi after 98 days of intraoligochaete development. These were floating in the water and showed the typical form of neoactinospores. The shape of the spores was triangular in apical view and elliptical in lateral view. The prevalence of infection reached 37%. Control specimens of B. sowerbyi proved to be free of neoactinospores. Except for a single specimen of B. sowerbyi, the only early developmental stages (pansporocysts) were found in T. tubifex.     

Expanded geographical distribution of Myxobolus cerebralis: first detections from Alaska

The parasite responsible for salmonid whirling disease, Myxobolus cerebralis, was introduced to the USA in 1958. It has since spread across the country causing severe declines in wild trout populations, but has never been documented from Alaska. However, while assessing the risk of introduction of M. cerebralis into the state, we detected the parasite using a species-specific polymerase chain reaction (PCR) assay. Testing of 180 hatchery rainbow trout, Oncorhynchus mykiss (Walbaum), by pepsin trypsin digest (PTD) and quantitative PCR (QPCR) revealed 14 positive samples. Infection was confirmed by sequencing the parasite 18S rRNA gene and by a nested PCR assay based on the same gene. Sequence comparison of M. cerebralis from several locations demonstrated the Alaska isolates were genetically distinct and therefore not false-positives arising from contamination during processing. We were unable to visually identify myxospores, indicating that either infection was light or mature spores had not formed. A reference set of fish samples spiked with known numbers of myxospores verified the QPCR and PTD results. This paper presents DNA sequence data from the Alaska M. cerebralis isolates, provides a brief history of the fish and facility of origin, and discusses implications of different testing methods on asymptomatic fish populations.     

Development of Myxobolus bramae (Myxosporea: Myxobolidae) in an oligochaete alternate host, Tubifex tubifex

The development of Myxobolus bramae Reuss 1906, a myxosporean parasite of the gills of common bream Abramis brama L., was studied in experimentally infected oligochaetes. In five experiments, uninfected Tubifex tubifex (Müller) and Limnodrilus hoffmeisteri Claparéde were exposed to mature myxospores of M. bramae . In four experiments triactinomyxon type actinospores developed in Tubifex specimens but no infection was found in Limnodrilus . Actinospores were released from oligochaetes 70–81 days after initial exposure. At that time pansporocysts containing eight actinospores were located in the gut epithelium of experimental oligochaetes, but free actinosporean stages were also found in their gut lumen. Each actinospore had three pyriform polar capsules and a barrel-shaped sporoplasm with 32 secondary cells. The spore body joined the three caudal projections with a stout style. The total length of the actinospore was 139 μm on the average.     

荆州碘泡虫(粘体动物门,碘泡虫科)重描述及其基于18S rDNA系统发育关系分析

本实验利用现行主流分类衍征,重新对荆州碘泡虫(Myxobolus kinchowensis)进行了详细描述,其分类特征如下:包囊圆形,寄生于鲫肌肉与肾脏,肌肉包囊大小(126.7±1.8)μm,肾脏包囊直径为94.2μm;两寄生部位各形态参数无显著差异,成熟孢子正面观呈梨形,缝面观呈纺锤形,含一大一小两梨形极囊;无明显囊间突起,孢子后端无褶皱,无粘液膜。组织病理显示在两寄生部位均未引起严重的炎性反应,且感染强度不高,推测该种对宿主无显著影响。基于18S r DNA进化分析发现荆州碘泡虫与寄生在肌肉部位的碘泡虫属种类聚为一个大枝,然后该大枝又根据地理位置远近分为北美枝、欧洲枝以及亚洲枝。通过形态比较研究以及分子分析可确定荆州碘泡虫为有效种,其寄主为鲫,寄生部位为肌纤维间和肾小囊。此外,两组织差异明显的寄生部位出现说明荆州碘泡虫相应的放射孢子虫在宿主鲫体内存在不同的发育与移行途径,而肌肉可能为其常规寄生部位,肾脏为其异常寄生部位,这种寄生部位的转移与宿主转变可能是粘孢子虫物种多样性形成的机制。     

A case history of whirling disease in a drainage system: Battle Creek drainage of the upper Sacramento River basin, California, USA

Abstract. Whirling disease was diagnosed in steelhead trout, Salmo gairdneri Richardson, during 1985 at Coleman National Fish Hatchery in the Battle Creek drainage of the upper Sacramento River basin, California, USA. Early confirmation of the aetiologic agent, Myxobolus cerebralis Hofer, 1903, was difficult. However, later investigation confirmed the identity of the agent and led to a management decision to destroy the 1985 brood year of steelhead trout at the hatchery. Sentinel fish and collections of feral trout were used to survey Battle Creek watershed to determine the source of infectivity. Feral rainbow trout, Salmo gairdneri Richardson, from South Fork Battle Creek were found to be infected with M. cerebralis and other infected trout were later found at two commercial rainbow trout hatcheries. No reinfection with M. cerebralis has been detected at the Coleman hatchery since ozonation was begun in early 1986.     

Ultrastructure and development of Pleistophora senegalensis sp. nov. (Protozoa, Microspora) from the gilt-head sea bream, Sparus aurata L. (Teleost, Sparidae) from the coast of Senegal

Abstract. The microsporidian Pleistophora senegalensis sp. nov. parasitizes the gilt-head sea bream, Sparus aurata L, The parasite is found in the intestinal wall where it forms small xenomas in the muscularis. The development cycle of this species is described by light and electron microscopy. Meronts are rounded plasmodia dividing by plasmotomy and bounded by an amorphous and very regular wall. At the onset of sporogony, sporophorous vesicles are formed by separation of the plasma membrane from the external wall which then becomes a characteristic mesh. Mature spores (4.45 × 2.37μm) are ovoid and slightly pyriform with a large posterior vacuole.     

Dynamics of experimental production of Thelohanellus hovorkai (Myxozoa: Myxosporea) in fish and oligochaete alternate hosts

The dynamics of development and production of Thelohanellus hovorkai (Myxozoa) were examined to investigate factors inducing haemorrhagic thelohanellosis in carp, Cyprinus carpio L. Fresh actinospores of T. hovorkai were harvested from the oligochaete alternate host, Branchiura sowerbyi, and used for infection experiments with myxosporean-free carp. Visualization of actinospores by fluorescent labelling revealed that sporoplasms penetrated the gill filaments of carp immersed in an actinospore suspension as early as 30 min post-exposure (PE). Plasmodia of T. hovorkai developed in the connective tissues of various organs and matured 3-5 weeks PE; dispersion of myxospores from degenerate plasmodia occurred 5-7 weeks PE. Challenges with a high dose of actinospores (4.5 x 10(6) spores per fish) resulted in the onset of disease, which was more easily achieved by the oral intubation of actinospores than by immersion in an actinospore suspension. Actinosporean-free B. sowerbyi were exposed to different densities of myxospores (10(4)-10(6) spores per oligochaete) and subsequently reared at different temperatures (15, 20, 25 degrees C). At 20 and 25 degrees C, actinospore releases were first detected 40-43 days PE, with multiple peaks of release (max. 7 x 10(5) actinospores day(-1)) during the next 60 days. We concluded that the developmental cycle of T. hovorkai was completed within 3-5 months at 20-25 degrees C, and that the ingestion of large numbers of actinospores orally, possibly by feeding on infected oligochaetes, resulted in a disease condition in carp.     

Infection of the epaulette shark, Hemiscyllium ocellatum (Bonnaterre), by the nematode parasite Proleptus australis Bayliss (Spirurida: Physalopteridae)

Seventy-two epaulette sharks, Hemiscyllium ocellatum (Bonnaterre), were infected with the nematode parasite Proleptus australis Bayliss, 1933. The parasite population was overdispersed. Infection intensity ranged from 3 to 1002 worms per fish stomach, and there was a positive correlation between shark length and number of parasites present. The majority of worms were attached to the stomach wall, and scanning electron microscopy and histological examination showed that worms penetrated the stomach lining. Worms were observed within the lamina propria of the stomach and occasionally penetrated the muscularis mucosa. Little to no inflammatory or cellular immune reaction to the presence of the parasites was observed, except in one case where a worm was being degraded by a host tissue response. There was a large amount of connective tissue proliferation as a result of nematode attachment, but no obvious effects on the overall health of the sharks were seen. Three sharks were also found to be infected by the cestode Callitetrarhynchus sp.     

Seeding nets with neutral spores of the red alga Porphyra umbilicalis (L.) Kützing for use in integrated multi-trophic aquaculture (IMTA)

Nets in traditional Porphyra mariculture are seeded with conchospores derived from the conchocelis phase, and spend a nursery period in culture tanks or calm coastal waters until they reach several centimeters in length. Some species of Porphyra can regenerate the foliose phase directly through asexual reproduction, which suggests that the time, infrastructure, and costs associated with conchocelis culture might be avoided by seeding nets with asexual spores. Here, we present work from a short-term mariculture study using nets seeded with asexual spores (neutral spores) of a native Maine species of Porphyra. Porphyra umbilicalis (L.) Kützing was selected for this proof of concept research because of its reproductive biology, abundance across seasons in Maine, and evidence of its promise as a mariculture crop. We studied the maturation, release, and germination of the neutral spores to develop an appropriate seeding protocol for nets, followed by development of a nursery raceway to provide an easily manipulated environment for the seeded nets. Neutral spores were produced throughout the year on the central Maine coast; however, there was a temporal variability in the number and survival of released neutral spores, depending upon thallus position in the intertidal zone. Small thalli were strictly vegetative, but most thalli reproduced by neutral spores; sexual reproduction was absent. Neutral spores germinated quickly at 10 and 15 °C, but germination was delayed at 5 °C. Unlike some algal zygotes and spores, neutral spores of P. umbilicalis required light to germinate; however, irradiances of 25 and 100 μmol photons m^− 2 s^− 1 were equally sufficient for germination. Rafts of seeded nets were deployed in Cobscook Bay, Maine, at two distances from salmon aquaculture pens and at a control site on a nearby, fallow aquaculture site (no salmon). There was no difference in nitrogen content of harvested thalli; however, both the density and the surface area of harvested thalli were different among the sites. The possible causes of these differences are discussed in the context of potential use of P. umbilicalis in IMTA.     

异育银鲫武汉单极虫病发生、发展、消退和消失的病理变化与PCR分析

为探究武汉单极虫病疾病过程和病理变化等特点,根据患病异育银鲫体表孢囊内有无成熟孢子和成熟孢子的崩溶解程度,将该病划分为发生、发展、消退和消失4个疾病时期,并分别进行病理变化观察和巢式PCR分析。结果发现,发生期的病鱼体表刚形成的孢囊使鳞片和皮肤微微隆起,孢囊乳白色,孢囊内分布着正在繁育逐渐增多的营养体,尚未出现成熟孢子;发展期的病鱼孢囊内已出现成熟孢子,孢囊逐渐增多增大,其表面黑色素细胞增加而呈灰黑色,感染强度高的病鱼出现死亡或畸形;消退期的成熟孢子通过破裂孢囊流入水体或不同步地崩溶解而减少直至全部溶解,孢囊随之逐渐缩小,使得黑色素细胞更为密集,此时期病鱼病情减轻不再出现死亡现象;消失期的病鱼孢囊平坦,内已无成熟孢子,只残留逐渐减少的成熟孢子崩溶解物质,黑色素细胞逐渐减少,最后原孢囊部位被结缔组织取代。巢式PCR分析结果表明,巢式第一轮和第二轮PCR在4个疾病时期都分别能扩增出1 584和853 bp的武汉单极虫目的条带,但在消失期的后期只有巢式第二轮PCR扩增出853 bp目的条带,说明残留的核酸物质含量逐渐减少,10月下旬后原孢囊部位巢式PCR扩增已无条带出现。本研究不仅揭示了武汉单极虫寄生部位在4个疾病时期的病理变化特点,而且明确了疾病消退和消失的2种方式,孢囊内成熟孢子通过破裂孢囊进入水体的方式和首次发现的孢囊内成熟孢子通过崩溶解的方式。     

饲喂枯草芽孢对草鱼粪便、养殖水体中芽孢数量和水质的影响

分别在饲料中添加不同含量的枯草(Bacillus subtilis)芽孢(A组实测芽孢数量为2.6×107 cfu/g,B组为4.4×106 cfu/g,空白组为0),饲喂静水水族箱中100 g左右的草鱼(Ctenopharyngodon idellus)10 d,测定饲喂枯草芽孢后草鱼粪便、养殖水体中芽孢数量和水质的变化。结果表明:(1)9 d内,水体中芽孢数量A组和B组在前6 d时不断增加,在6 d时达到稳定,其中A组和B组稳定时的含量分别为2.22×106 cfu/g和3.98×105 cfu/g。(2)10 d内,草鱼粪便干物质中的芽孢数量与饲料中的枯草芽孢含量相比,A组和B组粪便中的枯草芽孢损失率分别为89.76%～90.71%和83.83%～86.84%。(3)草鱼饲喂枯草芽孢后,A组和B组粪便中排出的枯草芽孢对水体中亚硝酸盐和氨氮有降低的趋势,但各组之间差异不显著(P>0.05),对化学需氧量(COD)没有影响。     

The life cycle of Chloromyxum auratum (Myxozoa) from goldfish, Carassius auratus (L.), involves an antonactinomyxon actinospore

The myxozoan parasite Chloromyxum auratum Hallett, Atkinson, Holt, Banner & Bartholomew, 2006 , was shown experimentally to have a two‐host life cycle which involved a previously undescribed antonactinomyxon actinospore stage. Myxospores obtained from gall bladders of naturally infected feral goldfish, Carassius auratus (L.), were used to infect samples of mixed species of oligochaete worms obtained from the same locality as the fish: Fern Ridge Dam, Oregon, USA. After some 110 days post‐exposure, actinospores were detected from the water above the oligochaetes. The 18S rDNA sequence of these actinospores was identical to the original myxospores. Spore release was sporadic, of low intensity and short duration, which confounded efforts to identify the host oligochaete species and infect naïve fish. This is the first life cycle that incorporates an actinospore of the collective group Antonactinomyxon, and the first life cycle demonstrated in the laboratory for a species of Chloromyxum.     

Infection of Cherax quadricarinatus (Decapoda: Parastacidae) by the microsporidium Thelohania sp. (Microsporida: Nosematidae)

Abstract The microsporidian parasite Thelohania sp. was present in a population of Cherax quadriearinatus in the Mitchell River, north Queensland. An infection rate of 7--8% was recorded for the population sampled. Cohabitation, passage of spores through fish, ingestion of spores and injection of blood from an infected individual failed to elicit infection in laboratory held animals. Injection of spores elicited a host responseconsisting of encapsulation and melanization of spores. Further studies are necessary to clarify the taxonomic position and mode of transmission of this parasite.     

The food habits of four species of triakid sharks, Triakis scyllium, Hemitriakis japanica, Mustelus griseus and Mustelus manazo, in the central Seto Inland Sea, Japan

ABSTRACT:   The food habits of 595 houndsharks of four species, Triakis scyllium ( n  = 179, 42–148 cm total in length), Hemitriakis japanica ( n  = 57, 42–102 cm), Mustelus griseus ( n  = 193, 39–100 cm), and Mustelus manazo ( n  = 166, 43–120 cm), found in the central Seto Inland Sea, Japan, from March 1997 to October 1999 and May 2000 to July 2002, were studied. T. scyllium changed their main food items from shrimps to echiuran worms then to cephalopods with their growth. Comparing food habits by the value of similarity (maximum = 1), the small-sized T. scyllium had a low value (0.17) compared to larger sharks. T. scyllium gradually increased the diversity of food until it reached 700 mm long in total length, however, after that it decreased. H. japanica appeared mainly in summer and autumn and ate cephalopods and fishes. M. griseus preyed on various crustaceans and decreased the diversity of food with growth. M. manazo preferred crustaceans and polychaetes. There was no certain tendency in the diversity of the food habit for M. manazo .