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.     

Genetic relationships among members of the Ichthyobodo necator complex: implications for the management of aquaculture stocks

Abstract Ichthyobodo necator (costia) is a common and important flagellate parasite that infests the skin and gills of many freshwater and marine fish. Costia infestations are often fatal and cause significant aquaculture losses worldwide. Recently it has been demonstrated that Ichthyobodo is a multispecies complex with differing host preferences. Knowing if those species have broad or narrow host specificity has important implications for the management of costia. To address the question of host specificity, genomic DNA was isolated from Ichthyobodo trophonts collected from rainbow trout, Oncorhynchus mykiss, koi, Cyprinus carpio, mirror carp, C. carpio, goldfish, Carassius auratus, channel catfish, Ictalurus punctatus, swordtail, Xiphophorus helleri, and Japanese flounder, Paralichthys olivaceus. The small subunit ribosomal RNA (SSU rRNA) gene from each isolate was analysed with previously published Ichthyobodo sequences using Bayesian phylogenetic methods. The internal transcribed spacers (ITS) from six isolates were also PCR-amplified, cloned and sequenced. Both the SSU rRNA phylogenetic analysis and the ITS rRNA sequence data support grouping the 22 Ichthyobodo isolates examined into a complex of nine different species. Many of these species were frequently isolated from multiple hosts, indicating that exchange of infested fish from one region to another has a high potential for spreading the disease. In one instance, the same species was obtained from marine and freshwater fish, further suggesting that certain Ichthyobodo species may not be limited by salinity.     

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.     

Myxosporean and microsporidian infections in cultured Pacific bluefin tuna <Emphasis Type="Italic">Thunnus orientalis</Emphasis> in Japan

During the parasitological survey of cultured juvenile Pacific bluefin tuna (PBT) Thunnus orientalis in 2007 and 2008, two myxosporeans and one microsporidian were found. Morphological and molecular analysis showed that the heart-infecting and brain-infecting myxosporeans are identified as Kudoa shiomitsui and K. yasunagai, respectively. This is a new host record for both species. High prevalence of infection (77–100%) with K. shiomitsui was observed in October to December (1–2 months post-transfer to sea cages), whereas only a few fish were infected with K. yasunagai. A microsporidian observed as white “cysts” in the trunk muscle of PBT had a resemblance to Microsporidium seriolae, the causative parasite of beko disease in yellowtail. Phylogenetic analysis showed that the microsporidian from PBT is closely related to but distinct from several other muscle-infecting species such as M. seriolae, Microsporidium sp. RSB, and Microsporidium sp. SH. Additionally, the spore dimension (2.7 × 1.5 μm on average) was remarkably smaller than the others. These results suggest that the present microsporidian is an undescribed species and designated Microsporidium sp. PBT. Prevalence and intensity of infection with Microsporidium sp. PBT reached a maximum of 100% and 20 cysts/fish, respectively. Although pathogenic effects of the two Kudoa species on fish health remain unknown, the microsporidian could be of concern to PBT aquaculture due to unsightly cysts in the musculature, reducing the market value of the fish.     

小头鲔寄生鳍缨虫的分离及鉴定

海南陵水小型金枪鱼驯养基地在小头鲔(Euthynnus affinis)养殖过程中发现有寄生虫附着现象,文章对小头鲔寄生虫进行了分离、形态观察及18S、28S rRNA分子鉴定。结果显示,经18S rRNA分子鉴定,该寄生虫与Branchiomma sp.相似性为99.13%;而28S rRNA显示,该寄生虫与黑斑鳍缨虫B.nigromaculatum相似性为100%,与Branchiomma sp.(EF116223.1)相似性为99.12%。形态观察发现,该寄生虫虫体呈墨绿色、棕黄色或红棕色,具鳃冠,全长30~90 mm,寄生于小头鲔鳃弓后缘的体表及胸鳍中后部。该寄生虫由鳃冠、胸区和腹区组成,无钙质管,其消化道包括口、口腔、咽、肠、肛门等,与黑斑鳍缨虫存在少许生活习性和形态差别。因此,该寄生虫是否为鳍缨虫属(Branchiomma)、黑斑鳍缨虫(B.nigromaculatum),仍需进一步验证。该虫附着于小头鲔体表后,小头鲔摄食量明显下降,游泳速度加快,易受惊吓。采用甲醛浸泡具有良好的去除效果,去除后小头鲔成活率为75.9%。     

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.     

Comparison of Collagen Distribution and Muscle Structure Between Cultured Amberjack (Seriola dumerili) and Cultured Yellowtail (Seriola quinqueradiata)

ABSTRACT

The collagen distribution in cultured specimens of amberjack and yellowtail were assessed by histochemical observation in relation to the meat texture of the fish. Major structural alteration was observed in the level of pericellular connective tissue of yellowtail that had higher muscle lipid content; these changes are likely to be associated with the large-scale changes in muscle firmness resulting from the weakening of the cellular binding force. However, lipid deposition was limited to the myocommata in amberjack muscle; lipid deposition was not observed in the pericellular connective tissue, which is the likely reason why amberjack maintained firmer meat texture.     

Pleistophora infestation in fathead minnows, Pimephales promelas (Rafinesque)

Pleistophora infestation was observed in adult fathead minnows, Pimephales promelas, held under laboratory conditions. Fish were clinically healthy, and presented no gross findings at necropsy. Histopathology revealed parasitic stages only in the ovaries. Spores within sporophorous vesicles were mainly encountered in late vitellogenic oocytes and were ultrastructurally identified as a microsporidian parasite. Heavily parasitized oocytes underwent degeneration followed by the release of spores into the ovarian interstitium. Degenerating oocytes and interstitial spores caused ovarian inflammation. Male fish showed no parasites in the testes. Parasitic infestation was compared with body length, body weight, gonadal weight, gonadosomatic index and plasma vitellogenin levels, and revealed no statistically significant differences between non-parasitized and parasitized females. The isolated holding conditions of the fish and the presence of parasitic stages in the ovaries suggested that an infestation with Pleistophora ovariaeSummerfelt, 1964 was more probable than that with Pleistophora mirandellae (Vaney & Conte, 1901).     

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.     

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.     

Determination of primary structure of amberjack myosin heavy chain and its relationship with structural stability of various fish myosin rods

The structural stability of fish myosin depends upon species and temperatures of water in which fish live. Primary, secondary, and quaternary structures of myosin heavy chain (MyHC) from three species of fish living at different temperature ranges have been compared with those of rabbit MyHC in order to investigate the differences in stability. Primary structure of MyHC, although being accessible for warm-water and cold-water fish (carp and walleye pollack), was not available in previous for tropical-water fish literature; so in this study primary structure of MyHC of the tropical-water fish amberjack has been determined by cloning and sequencing its cDNA. The MyHC has 1938 amino acid residues (AA), which are almost as much as as those of carp and walleye pollack. The amberjack MyHC is 91–95% homologous with other fish and rabbit MyHCs. There is a discernible difference between animal species with stable myosin rod (amberjack, carp, and rabbit) and walleye pollack with unstable rod. Stable rod species have a high probability of forming coiled-coil around the COOH-terminal end of the rod, while the pollack has a low coiled-coil formation probability. In addition, the average scores of the coiled-coil for myosin rod were rabbit (1.738) > amberjack (1.691) > carp (1.680) > walleye pollack (1.674) which correlated exactly with the observed stability. The results suggest that coiled-coil forming ability, particularly around the COOH-terminal end, directs structural stability of fish myosin rod.     

Loma salmonae (Protozoa: Microspora) infections in seawater reared coho salmon Oncorhynchus kisutch

Loma salmonae (Putz et al., 1965) infections were observed in five groups of coho salmon, Oncorhynchus kisutch, reared in seawater net-pens in Washington State, U.S.A. in 1984–1986. Ultrastructural characteristics, size of spores, tissues and host infected, and geographical location identified the microsporidium as Loma salmonae. Preserved spores measured 4.4×2.3 (4–5.6×2–2.4) μm and exhibited 14–17 turns of the polar filament. Infections were evident in the gills of some fish before seawater entry, but few parasites were observed and they caused little tissue damage. Infections observed in fish after transfer to seawater were associated with significant pathological changes in the gills. A mixed inflammatory infiltrate was associated with ruptured microsporidian xenomas within the vessels and interstitium of the primary lamellae. Microsporidian spores were dispersed throughout the lesions and were often seen inside phagocytes. The parasite was also observed in the heart, spleen, kidney and pseudobranchs; however, the inflammatory lesions were common only in the heart.

Monthly examination of fish after transfer to seawater showed peak prevalences (33–65%) of gill infections during the summer. Although moribund fish were often infected with other pathogens, the high prevalence of L. salmonae infections and the severity of the lesions it caused, suggested that this parasite significantly contributed to the recurrent summer mortalities observed at this net-pen site.     

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.     

Evaluating protection against Loma salmonae generated from primary exposure of rainbow trout, Oncorhynchus mykiss (Walbaum), outside of the xenoma-expression temperature boundaries

A series of challenge and re-challenge studies was conducted in which juvenile rainbow trout were exposed to the pathogen Loma salmonae , a microsporidian which typically causes xenoma formation during sporogony and inflammation in the gills as the xenomas undergo dissolution. The specific goal was to determine if a primary exposure, conducted at a water temperature outside of the range which permits the parasite to undergo sporogony and form branchial xenomas, would stimulate a protective response in the fish to a later challenge conducted under temperature conditions optimal for the parasite. Primary challenge of fish to L. salmonae at 7 °C or 21 °C blocked or limited xenoma formation, as discussed in a previous study. However, these fish had a relative percentage protection (RPP) against a second optimized exposure which matched, or was not significantly less than, the degree of protection (100%) that developed in other groups of fish that received a primary exposure throughout the range of water temperatures which permits xenoma formation. When the primary exposure was conducted at 5 °C, the RPP against the second exposure was adversely affected and declined to 61%. These findings have application to the control of L. salmonae within aquaculture, in that it may be possible to expose hatchery stocks of susceptible salmonid species to spores of L. salmonae when hatchery water temperature is at 7 °C. At this temperature, the risks of disease stemming from this primary exposure appear minimal, since xenomas fail to form. However, the degree of protection appears promising, and may be sufficient to protect fish from spore exposure occurring at netpen marine sites where the parasite may be endemic.     

Comparative Sensitivity of Yellowtail Seriolu quinqueradiata and Goldstriped Amberjack S. aureovittata to Photobacterium damsela subsp. piscicida

The pathogenicity of Photobacterium damsela subsp. piscicida to yellowtail Seriola quinqueradiata and goldstriped amberjack S. aureovittata was investigated. The 10-d cumulative mortality of yellowtail and goldstriped amberjack challenged by 1.2 × 10² CFU/fish of P. damsela piscicida was 70% and 10%. respectively. Serum bactericidal activity of goldstriped amberjack was higher than that of yellowtail. Although the phagocytic activity against P. damsela piscicida was not observed in kidney leukocytes of yellowtail and goldstriped amberjack, the production of superoxide anion in goldstriped amberjack kidney leukocytes against P. damsela piscicida was higher than that of yellowtail.     

Amberjack <Emphasis Type="Italic">Seriola dumerili</Emphasis> interleukin-10 negatively suppresses host cell-mediated immunity

Interleukin-10 (IL-10) is an anti-inflammatory cytokine and negatively regulates cell-mediated immunity (CMI) induction by inhibiting cytokine production in type 1 T helper cells. IL-10 genes have been isolated from several fish, and inflammatory cytokine inhibition by IL-10 has been well examined. However, a CMI regulator of IL-10 in fish has not yet been identified. In this study, we cloned the IL-10 gene in amberjack Seriola dumerili and analyzed its function using its recombinant protein (rIL-10). In an in vitro culture experiment, gene expression of inflammatory cytokines was suppressed in leukocytes incubated with rIL-10 compared with cells that only received Nocardia seriolae stimulation. This result suggests amberjack IL-10 has conserved function as an inflammatory cytokine inhibitor. Bactericidal activity of amberjack cells against intracellular pathogen stimulation was decreased in a rIL-10 dose-dependent manner. Furthermore, a significant reduction in the T-bet/GATA-3 ratio was observed in N. seriolae living cell (LC)?+?rIL-10-injected fish. Taken together, these results suggest amberjack rIL-10 suppresses CMI induction both in vitro and in vivo. In addition, the number of IgM⁺ cells among spleen leukocytes in N. seriolae?+?rIL-10-injected fish was higher than in only N. seriolae LC, suggesting that Th2-dominant immunity was induced by adding rIL-10.     

First record of Tetramicra brevifilum in lumpfish (Cyclopterus lumpus,L.)

A microsporidian species with 98.3–98.4% nucleotide identity to Tetramicra brevifilum (Journal of Fish Diseases, 3 , 1980, 495) was diagnosed in lumpfish (Cyclopterus lumpus, L.) broodstock held at a breeding and rearing facility in western Ireland. The fish were wild‐caught from the west coast of Ireland, and the first case was diagnosed one year after capture. Clinical signs included severe bloating, lethargy, exophthalmos, anorexia, white patches on the cornea and externally visible parasitic cysts on skin and fins. Necropsy revealed severe ascites, white nodules and vacuoles in all the internal organs and partial liquefaction of the skeletal muscle. On histological examination, microsporidian xenomas were observed in all internal organs, the skin, skeletal muscle, gills and the eyes. The microsporidian species was identified by molecular analysis and transmission electron microscopy. This is the first record of T. brevifilum infecting lumpfish, and the disease is considered to be of potential significance to the rising aquaculture industry of this species.     

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。