Tissue reactions to microsporidian infections in fish

Abstract. Histopathological studies on natural and experimental infections of nineteen microsporidian species from fishes distinguished two types of tissue reactions.
The first type is characteristic of infections with xenoma-inducing microsporidian species and comprises three successive stages: a weakly reactive stage, a productive stage with the formation of granulomas and a stage of granuloma involution. Following the first stage, tissue reactions are directed towards the isolation of the parasite and result in its complete elimination and host tissue repair. The extent of pathological changes probably depends on the number of parasite cells which initiated the infection.
The second type is represented by Pleistophora species infecting muscles or oocytes. Host tissue reaction is surprisingly slight during the schizogony and sporogony and does not tend to isolate the invaded muscle fibres. A slight lymphocytic infiltration of myosepta indicates the first stage of tissue reaction. The tissue reaction only reaches the productive stage when mature spores completely fill the contents of the infected muscle fibre. A thick wall of fibroblasts may be formed to demarcate the parasite mass as soon as it undergoes necrotic changes. The extent of pathological changes probably depends on the ability of early developmental stages of the parasite to spread the infection within the host.
In both types of tissue reaction, the spores are destroyed by complete digestion within host phagocytic cells.     

Macrophages and giant cells associated with a microsporidian parasite causing liquefaction of the skeletal muscle of the Norway pout, Trisopterus esmarkii (Nilsson)

Abstract. A mature microsporidian infection of the skeletal muscles of Norway pout, Trisopterus esmarkii Nilsson, is described. Infected fish had noticeable liquefaction of the muscle. The white foci of infection contained packed microsporidian spores, and the surrounding infected muscle fibres were degenerate. The host response involved invasion by phagocytic cells and encapsulation by fibroblasts, forming large granulomas. Macrophages, 17–20 μm in diameter, were actively ingesting the spores and typically had a foamy cytoplasm. Some had formed Langhans-type giant cells, 35 μm in diameter, with peripherally arranged nuclei. Sporulation was complete and appeared to have occurred within sporo- phorous vacuoles in which numerous spores were formed. Individual spores measured 2·8 × 1·5 μm and were characterized by irregular electron dense areas, a short polar filament with four coils and a large posterior vacuole with three inclusions.     

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.     

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。     

Encephalomyelitis associated with microsporidian infection in farmed greater amberjack, Seriola dumerili (Risso)

An outbreak of a disease characterized by a peculiar spiral movement in farmed greater amberjack, Seriola dumerili (Risso), occurred in Kagoshima Prefecture, Japan, in May 2008, immediately after importing the fish from China. Although neither bacteria nor viruses were detected in routine diagnostic tests, histopathological observations of the affected fish revealed severe inflammation in the tegmentum of the brain including the medulla oblongata and the anterior part of the spinal cord. In addition, a microsporidian parasite was observed in the nerve cell bodies or axons in the inflamed tissues. We identified a microsporidian small subunit rRNA gene (SSU rDNA) from the lesion, and the sequence showed 96.1% identity with that of Spraguea lophii. Subsequent in situ hybridization using probes presumably specific to the SSU rRNA confirmed that the parasite observed in histopathology harboured the identified SSU rRNA. Apparently degenerated microsporidian cells or spores were also frequently observed in tissue sections. Thus, the disease was most probably caused by the infection of a hitherto unknown microsporidian parasite that has a genetic affinity to the genus Spraguea, in the central nervous system of the amberjack.     

Kudoa trifolia sp. n. - molecular phylogeny suggests a new spore morphology and unusual tissue location for a well-known genus

A new species of myxozoan, Kudoa trifolia sp. n., was found in various organs of the golden grey mullet, Liza aurata (Risso), and the thinlip mullet, L. ramada (Risso), from the western Mediterranean. Spores developed in subspherical plasmodia of 0.28-1 mm diameter within connective tissue, predominantly in the spleen, the outer wall of the gall bladder and the gut, the mesenteries and occasionally also in the gills. The spores of K. trifolia differ from the commonly known shape of Kudoa by considerable enlargement of one of the four valve cells, thus forming a 'spore body', which contains the major part of the binucleate sporoplasm. Scanning electron microscopy of the spores revealed the presence of grape-like appendages, which occur in bundles terminally on the valve cells. Phylogenetic analysis based on the 18S rDNA sequence of K. trifolia showed that this species is deeply embedded in the genus Kudoa despite its aberrant morphology and host tissue location. This suggests important amendments to the morphological diagnosis of the genus Kudoa.     

Pleistophora mirandellae Vaney & Conte, 1901, a microsporidian parasite in cyprinid fish of rivers in Herault: taxonomy and histopathology

Abstract. Cyprinid fish from the Lergue and Lez rivers in Herault, southern France, were found to be parasitized by the microsporidian Pleistophora mirandellae Vaney & Conte, 1901. The parasite was found only in the gonads, forming cysts in the connective tissue between the seminiferous tubules of testes or occurring in the oocytes of the ovaries. Two types of uninuclear spores are produced. The synonomy of microsporidia parasitizing the gonads of cyprinid fish is discussed.     

Spraguea (Microsporida: Spraguidae) infections in the nervous system of the Japanese anglerfish, Lophius litulon (Jordan), with comments on transmission routes and host pathology

Anglerfish from the genus Lophius are a globally important commercial fishery. The microsporidian Spraguea infects the nervous system of these fish resulting in the formation of large, visible parasitic xenomas. Lophius litulon from Japan were investigated to evaluate the intensity and distribution of Spraguea xenomas throughout the nervous system and to assess pathogenicity to the host and possible transmission routes of the parasite. Spraguea infections in L. litulon had a high prevalence; all fish over 403 mm in standard length being infected, with larger fish usually more heavily infected than smaller fish. Seventy percent of all fish examined had some gross visible sign of infection. The initial site of development is the supramedullary cells on the dorsal surface of the medulla oblongata, where all infected fish have parasitic xenomas. As the disease progresses, a number of secondary sites typically become infected such as the spinal, trigeminal and vagus nerves. Fish with infection in the vagus nerve bundles often have simultaneous sites of infection, in particular the spinal nerves and along the ventral nerve towards the urinary bladder. Advanced vagus nerve infections sometimes form xenomas adjacent to kidney tissue. Spraguea DNA was amplified from the contents of the urinary bladders of two fish, suggesting that microsporidian spores may be excreted in the urine. We conclude that supramedullary cells on the hindbrain are the primary site of infection, which is probably initiated at the cutaneous mucous glands where supramedullary cells are known to extend their peripheral axons. The prevalence of Spraguea infections in L. litulon was very high, and infections often extremely heavy; however, no associated pathogenicity was observed, and heavily infected fish were otherwise normal.     

A hyperparasitic microsporidian infecting the salmon louse, Lepeophtheirus salmonis: an rDNA-based molecular phylogenetic study

The sea louse, Lepeophtheirus salmonis, is an obligate ectoparasitic copepod that lives on the external surface of salmonid fish. It is the most common ectoparasite of marine cage-reared salmonids, causing major economic loss to the aquaculture industry. During a sea louse monitoring programme, samples of L. salmonis were found to harbour an unreported microsporidian parasite. The microsporidian was observed in pre-adult and adult stages of both male and female copepods, with a prevalence of up to 5%. Unfixed spores were slightly pyriform in shape measuring 2.34 microm by 1.83 microm (+/- 0.01 microm) and were not observed to be enclosed by a sporophorous vesicle. The microsporidian infection was observed in all areas of the copepods' body, xenoma-like cysts forming directly under the cuticle in the epidermal tissue layer. In the present study, rDNA (530f-580r) sequence data gathered from the unidentified microsporidian parasite isolated from infected sea lice were compared with equivalents available in the databases in an attempt to identify its systematic position. The microsporidian was found to group within the phylogenetic clade containing the family Enterocytozoonidae, being most similar to members of the intranuclear genus Nucleospora. This is the first report of a hyperparasitic microsporidian infecting a caligid copepod.     

Altered tissue distribution of Loma salmonae: effects of natural and acquired resistance

This study compared and contrasted the fate of the microsporidian Loma salmonae , a branchial pathogen of salmonids of the genus Oncorhynchus , upon exposure of (1) naive susceptible rainbow trout (RT) O. mykiss , (2) naive RT passively immunized with sera from RT previously exposed to L. salmonae , (3) previously exposed and resistant RT and (4) two species believed to be innately resistant to the parasite, Atlantic salmon (AS) Salmo salar and brook trout (BT) Salvelinus fontinalis . The fish were infected per os with viable L. salmonae spores. The infection was followed in the fish by detection of parasite DNA by polymerase chain reaction (PCR) at several times post-infection. Spore germination and intestinal invasion by the parasite occurred in all groups of fish. In the susceptible RT, parasite DNA was detected in the heart by day 3 post-exposure (PE), followed by the gill at 2 weeks PE, whereas visible xenoparasitic complexes (xenomas) were detected by week 4 PE. In the passively immunized RT, the parasite's fate was similar to that of controls, however, its arrival in the heart was delayed by 1 week. A delay was also detected in RT which had been previously exposed to L. salmonae and then recovered from disease. In these resistant fish, the parasite was able to reach the heart by week 3 PE, however, it failed to reach the gill and form xenomas. In AS and BT, the parasite reached the heart and gills quickly, where it remained for 2 weeks before being cleared; xenomas never formed. We speculate that failure to complete the life cycle in AS, BT and resistant fish might be because of interference by the immune system in the development of the parasite, resulting in the absence of disease in these fish.     

Detection of the intranuclear microsporidian Enterospora nucleophila in gilthead sea bream by in situ hybridization

Enterospora nucleophila is an intranuclear microsporidian responsible for emaciative microsporidiosis of gilthead sea bream (GSB). Its minute size and cryptic nature make it easily misdiagnosed. An in situ hybridization (ISH) technique based on antisense oligonucleotide probes specific for the parasite was developed and used in clinically infected GSB in combination with calcofluor white stain (CW) and other histopathological techniques. The ISH method was found to label very conspicuously the cells containing parasite stages, with the signal concentrating in merogonial and sporogonial plasmodia within the infected cell nuclei. Comparison with CW demonstrated limited ISH signal in cells containing mature spores, which was attributed mostly to the scarcity of probe targets present in these stages. Although spores were detected in other organs of the digestive system as well as in the peripheral blood, proliferative stages or parasite reservoirs were not found in this work outside the intestines. The study demonstrated a frequent disassociation between the presence of abundant spores and the intensity of the infections as determined by the parasite activity. The ISH allows confirmatory diagnosis of GSB microsporidiosis and estimation of infection intensity and will be a valuable tool for a more precise determination of parasite dissemination pathways and pathogeny mechanisms.     

Effect of water temperature manipulation on a thermal unit predictive model for Loma salmonae

Loma salmonae is an important microsporidian pathogen affecting the mariculture of Pacific salmon, Oncorhynchus tshawytscha (Walbaum). Clinical signs associated with infection arise when the parasite enters sporogony and forms xenoparasitic complexes (xenomas) within the gill. The present study tested a thermal unit (TU) model, which was devised to predict the timing of xenoma formation, under conditions in which water temperature changed during the course of infection. In vivo trials with juvenile trout showed that the model TU for xenoma onset= (days post exposure) (C above 7C) accurately predicted the onset time for xenomas when fish were moved from either 11 or 5C to 15C at various intervals after exposure. These findings suggest that the TU model may allow aquaculturists to predict disease onset. However, the model failed when fish were moved from 15 to 5C at intervals after exposure. This finding suggests that the temperature-constrained phase of the life-cycle of L. salmonae occurs early on after the spore is ingested by the fish.     

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。     

舟山地区大棚凡纳滨对虾生长缓慢病因的调查分析

2013年以来,浙江省舟山地区大棚养殖的凡纳滨对虾(Penaeus vannamei)普遍出现生长缓慢、养殖成功率低的现象。为了查明该原因,本研究采用分子生物学和组织病理学等方法对引起对虾生长缓慢的病因开展了调查分析。结果显示,采集的270份病虾样本中,对虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)PCR阳性检出率高达85.19%,传染性皮下及造血器官坏死病毒(infectious hypodermal and hematopoietic necrosis virus,IHHNV)检出率为0;所有采集的病虾样本中也未分离到常见的致病菌;54份正常的对虾样本中EHP和IHHNV均未检出。将病虾PCR扩增产物进行序列测定和比对分析,结果获得的序列片段与Gen Bank中已有EHP相关序列相似性高达99.55%;病虾的肝胰腺组织病理切片观察显示,在虾肝胰腺组织中可观察到处于各个生长发育阶段的EHP。通过上述研究,初步认为EHP是引起舟山地区大棚养殖对虾生长缓慢的一个重要病原。     

Observations of Dermocystidium sp. infections in bullheads, Cottus gobio L., from a river in southern England

Bullheads, Cottus gobio, with macroscopic external cysts on the skin and fins measuring up to 3 mm in diameter were detected in the River Allen and its tributaries in southern England between 1992 and 1998. The prevalence of these cysts was up to 50% at some sites. Examination of cyst contents revealed the presence of numerous spores, typical of the genus Dermocystidium, measuring 8 microm in diameter. The parasite developed within well-defined cysts, which were located in the hypodermal connective tissues of the host. No cysts were present on the fins of any of the fish examined. Histological examination revealed a cyst wall consisting of an inner layer of dense eosinophilic material similar to that reported for Dermocystidium spp. forming coenocytic hyphae. No evidence was found of systemic infection or hyphal formation. Spores contained a prominent refractile body, which gave a weakly positive reaction for polysaccharides with the periodic-acid Schiff reaction and was positively stained with acidic dyes. Several examples of ruptured cysts were seen in histological sections and in some of these cases the host epithelial layer was breached, allowing release of the spores to the environment. Morphological features of, and host response towards, the Dermocystidium sp. in bullheads are compared with similar infections in salmonids and other freshwater fish species.     

Hepatic microsporidiosis of juvenile grey mullet, Chelon labrosus (Risso), due to Microgemma hepaticus gen.nov. sp.nov.

Abstract. Microgemma hepaticus gen.nov. sp.nov. is described from the liver of juvenile grey mullet, Chelon labrosus (Risso). Development occurs within xenomas (diameter 500μm) which have microvillar surfaces, encircling bands of mitochondria and a reticulate hypertrophic nucleus. Vegetative developmental stages, meronts, are plasmodial and divided by plasmotomy. These stages are enclosed by host membranes. Sporogonic stages are free in the cytoplasm and divide by multiple exogenous budding. Uninucleate spores (2·4μm × 4·2μm) possess 7–9 coils of the polar filament and a lamellar polaroplast. Xenomas are associated with liver connective tissue, and cause necrosis of adjacent liver cells in certain circumstances. Host response to infection involves leucocyte infiltration and granuloma formation, with spores being destroyed by repeated macrophage phagocytosis and necrosis and resulting in gradual resolution of the lesion. Although juveniles apparently tolerate large parasite burdens there is some evidence of a contribution by the parasite to stress-related mortality. The transmission of the disease and its potentially high pathogenicity to larval fishes is discussed.     

Development of In situ hybridization and real‐time PCR assays for the detection of Hepatospora eriocheir,a microsporidian pathogen in the Chinese mitten crab Eriocheir sinensis

A microsporidian parasite, Hepatospora eriocheir, is an emerging pathogen for the Chinese mitten crab Eriocheir sinensis. Currently, there is scant information about the way it transmits infection in the crustacean of commercial importance, including its pathogenesis, propagation and infection route in vivo. In this study, chromogenic in situ hybridization (ISH) and quantitative real‐time PCR (qPCR) assays were developed to address this pressing need, and we provided an advance in the detection methods available. Pathogens can be seen in situ with associated lesions using ISH. Positive hybridization signals were noted inside the epithelial cells of the hepatopancreas, and putative free parasite spores were observed within the tubule lumen, which were associated with lesions detected by electron microscopy and haematoxylin and eosin (H&E) analysis. qPCR allows the determination of parasite loads in infected tissues, which is important for understanding disease progression and transmission. The hepatopancreas displayed the biggest statistical copy numbers among different tissues of infected crabs, confirming a tissue‐specific pathogen infection characteristic. The qPCR assay also proved to be suitable for the diagnosis of asymptomatic carrier crabs. Combination of the two methods could facilitate the study of H. eriocheir infection mechanism in E. sinensis, enhance the early diagnosis of the pathogen and improve the management of microsporidian diseases in commercial crustaceans.