Comparative Evaluation of Molecular Diagnostic Tests for Nucleospora salmonis and Prevalence in Migrating Juvenile Salmonids from the Snake River,USA

Abstract

Nucleospora salmonis is an intranuclear microsporidian that primarily infects lymphoblast cells and contributes to chronic lymphoblastosis and a leukemia-like condition in a range of salmonid species. The primary goal of this study was to evaluate the prevalence of N. salmonis in out-migrating juvenile hatchery and wild Chinook salmon Oncorhynchus tshawytscha and steelhead O. mykiss from the Snake River in the U.S. Pacific Northwest. To achieve this goal, we first addressed the following concerns about current molecular diagnostic tests for N. salmonis: (1) nonspecific amplification patterns by the published nested polymerase chain reaction (nPCR) test, (2) incomplete validation of the published quantitative PCR (qPCR) test, and (3) whether N. salmonis can be detected reliably from nonlethal samples. Here, we present an optimized nPCR protocol that eliminates nonspecific amplification. During validation of the published qPCR test, our laboratory developed a second qPCR test that targeted a different gene sequence and used different probe chemistry for comparison purposes. We simultaneously evaluated the two different qPCR tests for N. salmonis and found that both assays were highly specific, sensitive, and repeatable. The nPCR and qPCR tests had good overall concordance when DNA samples derived from both apparently healthy and clinically diseased hatchery rainbow trout were tested. Finally, we demonstrated that gill snips were a suitable tissue for nonlethal detection of N. salmonis DNA in juvenile salmonids. Monitoring of juvenile salmonid fish in the Snake River over a 3-year period revealed low prevalence of N. salmonis in hatchery and wild Chinook salmon and wild steelhead but significantly higher prevalence in hatchery-derived steelhead. Routine monitoring of N. salmonis is not performed for all hatchery steelhead populations. At present, the possible contribution of this pathogen to delayed mortality of steelhead has not been determined.

Received September 14, 2010; accepted November 14, 2010     

草鱼细菌性烂鳃病病原菌的分离及其特异性卵黄抗体的初步研究

试验从患典型烂鳃病草鱼病变部位分离到1株柱状黄杆菌(FC-3株),采用0.4%甲醛灭活制成免疫原,接种健康120日龄桃源蛋鸡,收集鸡蛋并提纯卵黄抗体(IgY),并以此IgY为基础建立间接ELISA方法。结果:用FC-3株制备的IgY经初步提纯后电泳分离可见重链和轻链2条蛋白条带,获得了IgY多克隆抗体。建立的间接ELISA方法检测柱状黄杆菌纯培养液的检出限值为1×10^7cfu/mL,抗原包被最佳浓度和抗体最佳工作浓度分别为1∶100和1∶512,辣根过氧化物酶标记兔抗鸡IgY最佳工作浓度为1∶4000。结论:建立的间接ELISA方法对柱状黄杆菌有明显的检测特异性,与其他菌株无交叉反应,重复性好。     

Dermocystidium sp. Infection in Blue Ridge Sculpin Captured in Maryland

Raised pale cysts were observed on Blue Ridge Sculpin Cottus caeruleomentum during stream fish community surveys in Catoctin Mountain Park, Maryland. When examined histologically, preserved sculpin exhibited multiple cysts containing spherical endospores with a refractile central body characteristic of Dermocystidium spp. Cysts were not observed on the gills or internally. The portion of the watershed in which affected sculpin were observed contained lower than expected numbers of sculpin, raising concerns about the population effects of this infection. A nearby stream lacked sculpin even though they are common in this region, further suggesting the possibility of regional effects. This is the first report of a Dermocystidium infecting any fish species in the eastern United States.

Received October 16, 2015; accepted February 14, 2016 Published online July 25, 2016     

鲑鳟鱼对饲料中蛋白质和氨基酸的需求

鲑鳟鱼指的是鲑科鱼类,一般研究较多的是虹鳟和大马哈鱼。目前挪威以成为当今世界上鲑鳟鱼类养殖的第一大国,其产量占世界总产量的50%以上。目前世界上最主要的养殖种类是虹鳟和大西洋鲑。鲑鱼又叫大马哈鱼,是浅海中的洄游种类,每年随着生长和生育迁移生活地点。鲑鳟鱼具有极高的营养价值。其肉质鲜美,含有丰富的营养成分,对人体最有益处的是它含有大量的不饱和脂肪酸(又称Omega-3),可预防心脏病和中风,每天摄食0.5～1g可使患心脏病的机会减少40%;它对神经系统和视网膜生长极为有利,对小孩的脑部和眼睛发育有极大的帮助,而且它还能有效的防…     

Prevalence of Renibacterium salmoninarum among DownstreamMigrating Salmonids in the Columbia River

Abstract

Bacterial kidney disease (BKD) is an important contributor to mortality of salmonids in hatcheries in the Columbia River basin. However, the impact of BKD on the survival of downstream migrants is difficult to determine because there is little information on the disease-related mortality among these fish. In this study, the impact of BKD on juvenile salmonids was examined by determining the percentage of downriver migrants infected with Renibacterium salmoninarum (the causative agent of BKD) and evaluating the effects of salt water on the progress of the disease. During the 2 years of this study, approximately 20% of the three species of migrating hatchery and wild salmonids (Oncorhynchus spp.) collected were infected with R. salmoninarum. Mortality caused by BKD increased when fish were held in salt water.     

Rate of Disappearance of Gas Bubble Trauma Signs in Juvenile Salmonids

Abstract

To assess the rate of disappearance of gas bubble trauma (GBT) signs in juvenile salmonids, we exposed spring chinook salmon Oncorhynchus tshawytscha and steelhead O. mykiss to water containing high levels of dissolved gas supersaturation (DGS) for a time period sufficient to induce signs of GBT, reduced the DGS to minimal levels, and then sampled fish through time to document changes in severity of GBT. Because of the tendency of GBT signs to dissipate at different rates, we conducted trials focusing on emboli (bubbles) in the gill filaments and lateral line and separate trials that focused on bubbles in the external surfaces (fins, eyes, and opercula). Bubbles in gill filaments dissipated almost completely within 2 h after transfer of fish to water of nearly normal DGS (104%), whereas bubbles in the lateral line dissipated to negligible levels within 5 h. Bubbles on external surfaces were more persistent through time than they were in gill filaments and the lateral line. Although typically dissipating to low levels within 48 h, external bubbles sometimes remained for 4 d. Assuming a direct relation exists between easily observable signs and direct mortality, our results suggest that fish can recover quickly from the potentially lethal effects of DGS once they move from water with high DGS to water of almost normal gas saturation. These results should be of fundamental importance to fishery managers interpreting the results of monitoring for the severity and prevalence of GBT in juvenile salmonids in the Columbia River system and perhaps elsewhere.     

Gill diseases of cultured salmonids in Ontario.

Between 1977 and 1981, the Fish Pathology Laboratory of the Ontario Veterinary College received 239 cases from trout farms of southern Ontario, 51 (21.3%) of which had diseased gills. Branchial lesions in 86.3% of these 51 cases were characterized by marked lamellar epithelial hyperplasia with epithelial hypertrophy and lamellar fusion. Filamentous bacteria were seen on the surface of the branchial filaments and lamellae in 68.6% of the cases. Our observations highlight the importance of gill diseases as a production problem of farmed salmonids in southern Ontario.     

A Fluorescent Antibody Test for Detection of the Rickettsia Causing Disease in Chilean Salmonids

Abstract

An indirect fluorescent antibody test (IFAT) was developed for detection of the rickettsia that was causing epizootics among salmonids cultured in seawater net-pens in southern Chile. Antiserum against the rickettsial agent was produced in New Zealand white rabbits with a preparation grown in antibiotic-free chinook salmon embryo (CHSE-214) cell cultures and partially purified by a combination of filtration and centrifugation steps. The IFAT was effectively used on blood films, tissue sections, and smears. Two gram-negative and two gram-positive bacterial pathogens of salmonids did not react in this test. Detection of the rickettsial agent has previously been restricted to examination by light microscopy or isolation in salmonid cells. The IFAT provides a simple, rapid, sensitive method for detection of the agent and diagnosis of the disease. The rickettsia is thought to be a member of the tribe Ehrlichieae and was tested by IFAT with sera from animals infected with other rickettsial agents.     

Observations on the Occurrence of Bacterial Gill Disease and Amoeba Gill Infestation in Rainbow Trout Cultured in a Water Recirculation System

Abstract

Spontaneous outbreaks of bacterial gill disease (BGD) occurred in 17-44-g rainbow trout Oncorhynchus mykiss after they were placed in a water recirculation system. In each of five groups stocked from September 1991 through July 1992, BGD occurred within 6–8 d after stocking. In each instance, BGD was followed by a secondary amoeba infestation. The spontaneous BGD outbreaks did not occur among previously stocked groups that had recovered from earlier BGD disease outbreaks. Examination of gill tissue by Gram stain and indirect fluorescent antibody technique showed increased numbers of filamentous bacteria associated with BGD after the rainbow trout were stocked into the system. Bacterial numbers decreased after a 1-h treatment with chloramine-T at concentrations of 9–15 mg/L but increased within 2 d after treatment. Although the chloramine-T treatments controlled mortality related to BGD, the amoeba infestation persisted. Histological examination of gills showed some focal hyperplasia before the rainbow trout were placed into the recirculation system, but hyperplasia became more extensive and lamellar fusion and mild telangiectasis developed within a week after placement in the system. The density at which the fingerling rainbow trout were stocked and the suspended solids present in tank water may have contributed to the BGD outbreaks. Exposure of juvenile rainbow trout to tank water from the recirculation system before they were placed into the system did not afford them protection against BGD after stocking.     

Neuronal vacuolation in raccoons from Oregon.

During a 2-year period (1995-1997), vacuoles were detected in neurons of 21/50 (42% prevalence) raccoons (Procyon lotor) in Oregon. Age or sex predisposition was not apparent. Twenty of these raccoons were from within a radius of 40 km of Corvallis in western Oregon. Microscopically, the vacuoles were variable in size, were in the perikarya, and were consistently present in pontine nuclei. Brain tissues were negative for rabies virus antigen by fluorescent antibody test and for the protease-resistant protein prion by immunohistochemistry. Electron microscopic examination of the brain stem of selected animals revealed accumulation of electron-dense material within neuronal perikarya. Light and electron microscopic examination indicated that the accumulated intracellular material had a high lipid content. These lesions suggest a form of neuronal storage condition. Further research is required to identify the composition of the intracellular lipid material, to elucidate the mechanism of neuronal vacuolation in raccoons, and to understand the basis for the apparent geographic restriction of this lesion.     

麝化脓病病原研究

四川省都江堰市某养麝场自１９９９年以来，麝常发生化脓病，发病率和死亡率都较高，动物开始发病时表现精精神萎靡，食谷下降，而后出现突出于皮肤表面的硬脓包，一周左右脓包变软，并伴随体温升高，出现脓包的病麝往往用抗生素治疗无效而死亡，，剖检死亡麝发现在肺部和肝脏等实质器官有大量大小不等的化脓灶。经采取死亡麝的的肺、肝等实质器官和脓汁进行病原的分离和鉴定，分离了3 病原菌，分别化脓棒状杆菌（Ｃorynebcterium)、淋巴管隐球菌（Ｃryptococcus farciminosus)和侵肺拟杆菌（Bacteroides pneumosintes）,通过动物试验证它们均为化脓病的病原。     

蓝莓拟盘多毛孢叶斑病病原菌鉴定

在广西蓝莓产区发现一种叶斑病,为明确该病害致病菌种类,通过组织分离和纯化、致病性测定,并结合其产孢细胞,分生孢子的颜色、大小、附属丝等形态学特征和分子生物学特性对该病原菌进行鉴定。结果表明,该病原菌可致蓝莓叶片产生褐色病斑,边缘有深褐色晕圈,后期产生黑色分生孢子盘。该菌菌落呈白色,背面乳白色至淡黄色,产孢细胞无色透明,圆棒状。分生孢子21.5-25.8 μm×5.5-7.5 μm,中间3个色胞同为橄榄色,具2-3根顶端附属丝,长11.4-21.6 μm。结合ITS序列系统发育分析确定该病原菌为 Pestalotiopsis tranchicarpicola。这是Pestalotiopsis tranchicarpicola引起蓝莓叶斑病在国内的首次报道。     

The epizootiology of gastrointestinal nematodes of cattle in selected areas of Oregon.

A study was initiated to (1) determine species of gastrointestinal nematodes present in cattle in selected areas of Oregon, (2) examine seasonal abundance of these nematodes as an indicator of periods of transmission, and (3) determine when developmental inhibition occurs in the major genera of nematodes encountered. Four study sites were chosen: Corvallis, Langlois, near Fort Rock, and southeast of Klamath Falls on the Oregon-California border. Eight sets of three to four tracer calves each were introduced onto pasture at each site over a 2 year period with time of turn out onto pasture corresponding to late spring, mid-summer, late fall and late winter. At Corvallis, a distinct seasonality in parasite transmission was evident, peaking during the fall and winter. At Langlois, transmission was fairly constant throughout the year. No discernible patterns were evident at either Klamath Falls or Fort Rock. Eight nematode genera were found during the study. Ostertagia, Cooperia, Nematodirus and Trichostrongylus were present at all study sites and were the most common genera at each. Trichuris was found at all sites except Klamath Falls. Oesophagostomum was present in tracers only from Langlois and Corvallis while Haemonchus was found only at Klamath Falls and Fort Rock. Capillaria was only present at Klamath Falls. Where possible, specific transmission patterns for Nematodirus, Cooperia, and Ostertagia were determined for each site. Nematodirus was transmitted fairly steadily at both Langlois and Corvallis but was quite variable at Fort Rock. Developmental arrest was detected in this genus at all study sites during the fall and/or winter. Cooperia exhibited the most seasonally defined pattern of transmission, peaking during the fall and winter at Langlois, Corvallis and Klamath Falls. Hypobiotic larvae of Cooperia were present during the fall and/or winter only at Langlois and Corvallis. Peak transmission of Ostertagia at Langlois and Corvallis occurred during the fall and winter. At Fort Rock, transmission was lowest in the fall and increased in the winter. Hypobiotic larvae were evident in the fall and winter at Corvallis, Fort Rock and Klamath Falls. These data suggest Type II ostertagiasis may occur in late winter through spring in these areas. Hypobiotic larvae of Ostertagia were not detected at Langlois. The lack of appropriate environmental stimuli is one possible explanation for the apparent lack of hypobiosis at that site.     

甘肃苜蓿斑点病的初步研究

近年来甘肃省苜蓿(Medicago stativa L.)发生一种叶部病害—-斑点病,严重地降低了苜蓿的产量和饲用价值。为了有效地控制该病害对苜蓿的危害,在室内对其病原菌进行了形态学鉴定及生物学特性研究。结果表明:苜蓿斑点病的病原菌为链格孢菌(Alternaria alternata (Fr.:Fr) Keissler),该病原菌能很好地利用麦芽糖、蔗糖、甘氨酸;其生长最适温度为25℃,最适pH为6.0-7.0,最适宜的RH为98%以上,最适培养基为5%AL和PSA。在上述环境条件下,菌丝生长快,菌丝茂盛,产孢量大,孢子萌发率高。     

Loma sp. in Salmonids from the Eastern United States: Associated Lesions in Rainbow Trout

Abstract

A microsporidian of the genus Loma was noted in the gills of rainbow trout Oncorhynchus mykiss from a state hatchery (Buford Trout Hatchery) in Georgia. Mortalities of varying severity occur at this hatchery every fall, and the microsporidian was noted during an experiment from August 1991 to January 1992 to determine the effects of water source on disease. Infections first appeared to be systemic in the October sample; xenomas were observed in heart, spleen, and peripheral vessel walls. The presence of unidentified intracellular material preceded the appearance of xenomas in all tissues, but whether this material was associated with inflammation or represented immature stages of the parasite has yet to be determined. These structures were also noted in the intestine and liver, although xenomas were not noted in these organs. Mature xenomas did not elicit an inflammatory response but appeared to be short-lived. When the xenoma wall ruptured and released spores, an inflammatory response was again observed. The prevalence and severity of the infection were determined in fish maintained in troughs with well water, Chattahoochee River water, or hatchery (treated river) water. The infection tended to be more prevalent and more severe in fish maintained in the hatchery or river water than in those maintained in the well water. Stress induced by poor water quality may increase mortality from this parasite. This report extends the range of Loma sp. into the eastern United States.     

Experimental Induction of Proliferative Gill Disease in Specific-Pathogen-Free Channel Catfish

Abstract

Specific-pathogen-free channel catfish Ictalurus punctatus were exposed to sediment and mud from a pond containing channel catfish with proliferative gill disease. In one experiment, fish were to exposed to mud and sediment for 2 months in water maintained at 19°C. Fish were necropsied weekly, and certain tissues were examined histologically and ultrastructurally. Four trials were conducted with sediment samples from different epizootics of proliferative gill disease. In a second experiment, fish were exposed to sediment for 7 d in water maintained at 16, 19, or 26°C; the fish were then moved to clean water held at 16, 19, or 26°C. Fish were necropsied before transfer to clean water and weekly thereafter for 2 months. Channel catfish held at 19°C developed proliferative gill disease within 2 d of exposure to sediment. Primary cells of a uninucleate myxosporean parasite were present in the gills at the base of lamellae. These developed into plasmodia with numerous secondary cells, and some primary cells disintegrated, releasing their internal secondary cells. Similar development was observed in internal organs 1 week after appearance of the parasite in gills. Complete sporogony did not occur over the 2 months of this study. Plasmodia became necrotic and were not detected after 60 d. In fish exposed to sediment for 7 d at 16, 19, and 21°C, similar organisms were detected, but clinical disease occurred only at 19 and 26°C. Proliferative gill disease may be attributed to extrasporogonic stages of a myxosporean resembling Sphaerospora spp.     

Seed Shatter Dates of Antelope Bitterbrush in Oregon

Seed shatter dates for antelope bitterbrush (Purshia tridentata [Pursh] DC) were estimated from collections at 192 sites in Oregon and surrounding states. Shatter date was strongly correlated to elevation (r = 0.74) and an equation that included elevation, latitude, longitude, and longitude squared explained 79% of the variation in seed shatter dates. In general, earlier shatter dates were associated with more southerly latitudes, easterly longitudes, and lower elevations. Examination of climatic data confirmed the expectation that earlier shatter dates were associated with warmer sites. This information can assist those needing to schedule seed collection activities at multiple locations.     

动物废弃物中病原体的污染及其控制

本文概述了畜牧业废弃物中的病原体种类、数量及其污染与控制措施.     

动物废弃物中病原体的污染及其控制

本文概述了畜牧业废弃物中的病原体种类、数量及其污染与控制措施。