Can the protozoan parasite Bonamia ostreae infect larvae of flat oysters Ostrea edulis?

Bonamia ostreae is an intracellular protistan parasite affecting flat oysters Ostrea edulis. It can be detected in juveniles but mortalities mainly affect oysters which are more than 2 years old. The parasite is usually observed inside haemocytes and sometimes free, notably in gill epithelia suggesting a parasite release through this organ. However, the infective form and ways of entry and release remain undetermined. Flat oysters incubate their larvae in their pallial cavity for 8-10 days before releasing them into the water column. Flat oysters in Bay of Quiberon in South Brittany (France) are known to be infected with B. ostreae since 1979 and is the most important area in France for O. edulis spat collection. Flat oysters incubating larvae were sampled in this area during summertime between 2007 and 2009. Both adults and larvae were preserved and assayed by PCR and in situ hybridisation (ISH). PCR tests revealed the presence of parasite DNA in some adults and larvae. Specific labelling could be detected by ISH in gills, digestive system, gonad and mantle in adults and in the epithelium surrounding the visceral cavity of some larvae. Our results demonstrate that larvae can be infected with B. ostreae. Larvae might thus contribute to the spread of the parasite during their planktonic life. In addition, their transfer for aquaculture purpose should be controlled especially when they are exported from infected zones.     

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.     

A Marteilia-like parasite in blue mussels Mytilus edulis in China

Species of the genus Marteilia (Phylum Paramyxea) are protozoan parasites of marine mollusks. Marteilia spp. have been detected in mollusks from different parts of the world, but the presence of these parasites in China has not been previously reported. Therefore, a survey was conducted to look for the presence of Marteilia spp. in blue mussels Mytilus edulis and Asian green mussels Perna viridis collected along China's coasts. Histological and PCR analyses revealed that 5 of 180 M. edulis (prevalence = 2.8%) were positive for infection with a Marteilia-like organism, whereas the parasite was not detected in any of the 80 P. viridis individuals tested. Total genomic DNA was extracted from the infected tissue sections for PCR amplification. The PCR amplification with Marteilia primers SS1 and SAS1 yielded the expected 641-bp product. Sequencing results showed that the 18S ribosomal RNA gene fragment from the protozoans found in M. edulis from China was 88% similar to that of Marteilia refringens, a species that was reported from M. edulis and European flat oysters Ostrea edulis collected in France. This is the first report of a Marteilia-like organism infecting M. edulis in China.     

Serum cardiac troponin I concentration in dogs with ehrlichiosis

Background: Ehrlichiosis is a multisystemic disease with the potential to cause cardiomyocyte injury in naturally infected dogs.
Hypothesis: Myocardial injury occurs in dogs infected with Ehrlichia canis .
Animals: One-hundred and ninety-four dogs from Brazil with clinical and laboratory abnormalities indicative of ehrlichiosis. Sixteen healthy dogs served as controls.
Methods: Electrocardiogram, echocardiogram, noninvasive blood pressure measurement, and serum cardiac troponin I (cTnI) concentrations were evaluated. Serologic assays and PCR determined the exposure and infection status for E. canis, Anaplasma spp., Babesia canis vogeli, Bartonella spp., Borrelia burgdorferi, Dirofilaria immitis, Ehrlichia chaffeensis, Ehrlichia ewingii, Leishmania chagasi , and spotted-fever group Rickettsia . Dogs were assigned to groups according to PCR status: E. canis infected, infected with other vector-borne organisms, sick dogs lacking PCR evidence for infection, and healthy controls.
Results: E. canis -infected dogs had higher serum cTnI concentrations than controls (median: 0.04 ng/dL; range 0.04–9.12 ng/dL; control median: 0.04 ng/dL; range: 0.04–0.10 ng/dL; P = .012), and acute E. canis infection was associated with myocardial injury (odds ratio [OR]: 2.67, confidence interval [CI] 95%: 1.12–6.40, P = .027). Severity of anemia was correlated with increased risk of cardiomyocyte damage ( r = 0.84, P < .001). Dogs with clinical signs of systemic inflammatory response syndrome (SIRS) were at higher risk for myocardial injury than were other sick dogs (OR: 2.55, CI 95%: 1.31–4.95, P = .005).
Conclusions and Clinical Importance: Acute infection with E. canis is a risk factor for myocardial injury in naturally infected Brazilian dogs. Severity of anemia and SIRS might contribute to the pathophysiology of myocardial damage.     

Summer Mortalities and Incidental Parasitisms of Cultured Pacific Oysters in Alaska

Abstract

During the summer of 1987, surface seawater temperatures in Alaska were unseasonably warm, periodically approaching 20°C with salinities of 29‰ in late July and early August. During this period at least one Alaskan oyster grow-out station sustained excessive mortalitygreater than 50%-within a group of 150,000 18-month-old Pacific oysters Crassostrea gigas. These oysters continued to die despite later reported declines in both seawater temperature and salinity. Histological examination of moribund oysters indicated mature or nearly mature gametes in both sexes and infiltration of tissues by opportunistic secondary invaders composed primarily of various bacterial types and the flagellate protozoan Hexamita sp. An incidental, but potentially pathogenic, rickettsia-like organism that formed cytoplasmic inclusions within vesicular connective tissues was also observed in some of the oysters. Circumstances associated with this oyster mortality were similar to those accompanying summer mortalities of raft-cultured Pacific oysters in Japan, where deaths were caused by physiological stress from rapid gonadal development or a prolonged prespawning condition.     

A Marteilia-Like Parasite in Blue Mussels Mytilus edulis in China

Abstract

Species of the genus Marteilia (Phylum Paramyxea) are protozoan parasites of marine mollusks. Marteilia spp. have been detected in mollusks from different parts of the world, but the presence of these parasites in China has not been previously reported. Therefore, a survey was conducted to look for the presence of Marteilia spp. in blue mussels Mytilus edulis and Asian green mussels Perna viridis collected along China's coasts. Histological and PCR analyses revealed that 5 of 180 M. edulis (prevalence = 2.8%) were positive for infection with a Marteilia-like organism, whereas the parasite was not detected in any of the 80 P. viridis individuals tested. Total genomic DNA was extracted from the infected tissue sections for PCR amplification. The PCR amplification with Marteilia primers SS1 and SAS1 yielded the expected 641-bp product. Sequencing results showed that the 18S ribosomal RNA gene fragment from the protozoans found in M. edulis from China was 88% similar to that of Marteilia refringens, a species that was reported from M. edulis and European flat oysters Ostrea edulis collected in France. This is the first report of a Marteilia-like organism infecting M. edulis in China.

Received May 9, 2011; accepted March 4, 2012     

牡蛎包纳米虫体外培养及鉴定

近几十年来,牡蛎包纳米虫病在欧洲等地频发,引起牡蛎大范围的死亡,对牡蛎养殖业危害极大。目前包纳米虫的研究处于初级阶段,病原的体外培养技术是深入研究包纳米虫的致病机理、与宿主的相互作用及疫病防控的基础。本研究以经PCR初步鉴定阳性的牡蛎全组织作为培养基,体外培养1个月后,采用原位杂交方法再次鉴定牡蛎包纳米虫。2次鉴定结果一致,且在包纳米虫培养1个月之后,虫体均能被原位杂交方法检测到。研究结果表明,本研究成功建立了一种简单可行的牡蛎包纳米虫培养方法。     

Localization of the initial developmental stages of Loma salmonae in rainbow trout (Oncorhynchus mykiss)

The intracellular microsporidian parasite Loma salmonae affects salmonids of the genus Oncorhynchus and is a significant cause of economic losses in pen-reared Chinook salmon (O. tshawytscha) in British Columbia. Loma salmonae infection is easily recognized by the xenomas that form in the gills, but early stages of infection are difficult to detect in histologic sections. In situ hybridization (ISH), using an L. salmonae-specific digoxigenin-labeled single-stranded DNA probe, was used to detect the parasite during the early stages of infection. Loma salmonae was detected in the gut mucosal epithelium as early as 24 hours postexposure (PE), and it localized in the lamina propria of the intestine within 24 hours of infection. After the parasite was detected in the lamina propria, dividing merogonic stages in infected cells in the heart were detected by ISH as early as 2 days PE, providing the first evidence of parasitaemia and hematogenous distribution of this parasite in infected blood cells. The parasites inside the infected cells appeared to be undergoing merogony as they passed through the heart, indicating that proliferation may start at the site of infection, before the parasite arrives to the gills for their final developmental phase. This is the first time that L. salmonae passage through the intestinal wall and migration to the heart has been visualized; however, the identity of the cells harboring the parasite has yet to be determined.     

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。     

Detection of circovirus infection in pigeons by in situ hybridization using cloned DNA probes.

Degenerate primers were designed based on known sequence information for the circoviruses psittacine beak and feather disease virus and porcine circovirus and applied by polymerase chain reaction (PCR) to known virus-infected bursa of Fabricius (BF) from a pigeon. A 548-bp DNA fragment was amplified and shown to be specific to a novel circovirus, named pigeon circovirus (PiCV), and was used to produce sensitive and specific probes for detection of circovirus DNA by in situ hybridization (ISH). Using ISH on BF from 107 pigeons submitted for necropsy, infection was detected in 89%, compared with a histologic detection rate of 66%. Using the ISH technique, infected cells were also found in liver, kidney, trachea, lung, brain, crop, intestine, spleen, bone marrow, and heart of some birds. Large quantities of DNA were present in some of these tissues, and in the absence of BF, liver in particular is identified as a potentially useful organ to examine for presence of PiCV. This high prevalence of infection in diseased birds is noteworthy, emphasizing the need for studies to determine the precise role of this virus as a disease-producing agent.     

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.     

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.     

Detection of Myxobolus cerebralis in Rainbow Trout and Oligochaete Tissues by Using a Nonradioactive in Situ Hybridization (ISH) Protocol

Abstract

A nonradioactive in situ hybridization (ISH) protocol was developed to detect Myxobolus cerebralis, the causative organism of whirling disease, in its primary host, rainbow trout Oncorhynchus mykiss, and in its alternate oligochaete host, Tubifex tubifex. A cocktail of three oligonucleotide primers (derived from the small subunit ribosomal DNA sequence) directed at target sequences of the parasite DNA was tailed at the 3′ end with digoxigenin-labeled deoxyuridine triphosphate (DIG-dUTP). Labeled probes were hybridized to parasite DNA present in deparaffinized tissue sections from infected trout and oligochaetes. The bound probes were visualized after modifications of existing ISH protocols. By using the new ISH procedure, the parasite was found in target tissues of subclinically and clinically infected fish and tubificid oligochaetes after exposures of these hosts to triactinomyxons and mature spores, respectively. The probe did not bind with salmonid tissues infected with two other myxosporean parasites, Ceratomyxa shasta or the PKX organism, or to a Myxobolus sp. infecting the cartilage of plain sculpin Myoxocephalus jaok. These initial results indicate that ISH is an effective and specific test for detecting Myxobolus cerebralis in its fish and oligochaete hosts.     

Effect of Nanophyetus salmincola and Bacterial Co-Infection on Mortality of Juvenile Chinook Salmon

The freshwater trematode Nanophyetus salmincola has been demonstrated to impair salmonid immune function and resistance to the marine pathogen Vibrio anguillarum, potentially resulting in ocean mortality. We examined whether infection by the parasite N. salmincola similarly increases mortality of juvenile Chinook Salmon Oncorhynchus tshawytscha when they are exposed to the freshwater pathogens Flavobacterium columnare or Aeromonas salmonicida, two bacteria that juvenile salmonids might encounter during their migration to the marine environment. We used a two-part experimental design where juvenile Chinook Salmon were first infected with N. salmincola through cohabitation with infected freshwater snails, Juga spp., and then challenged with either F. columnare or A. salmonicida. Cumulative percent mortality from F. columnare infection was higher in N. salmincola-parasitized fish than in nonparasitized fish. In contrast, cumulative percent mortality from A. salmonicida infection did not differ between N. salmincola-parasitized and nonparasitized groups. No mortalities were observed in the N. salmincola-parasitized-only and control groups from either challenge. Our study demonstrates that a relatively high mean intensity (>200 metacercariae per posterior kidney) of encysted N. salmincola metacercariae can alter the outcomes of bacterial infection in juvenile Chinook Salmon, which might have implications for disease in wild fish populations.

Received February 24, 2015; accepted September 7, 2015     

Development of in situ nest PCR and comparison of five molecular biological diagnostic methods for the detection of intracellular viral DNAs in paraffin sections

Nest polymerase chain reaction (PCR), in situ hybridization (ISH), in situ PCR, in situ PCR/hybridization (PCR-ISH) and in situ nest PCR were compared for the detection and localization of intracellular viral DNAs in paraffin sections. MDBK cells were infected with alcelapine herpesvirus 1 ranging from 10(1) to 10(5) 50% tissue culture infected doses (TCID(50)), incubated 18 hr, then fixed and processed into paraffin blocks. Sections of the cell preparation were subjected to nest PCR, ISH, in situ PCR, PCR-ISH and in situ nest PCR using specific oligonucleotide primers or probes directed against the viral open reading frame 50. In situ nest PCR and nest PCR were found to be capable of detecting the viral DNA in the cells infected with the lowest virus titer. As compared with other molecular biological methods for the detection of the virus, in situ nest PCR was found to be more sensitive than ISH, in situ PCR and PCR-ISH. In situ nest PCR has wide applications for sensitive localization of low copy viral sequences within cells to investigate the role of viruses in a variety of clinical conditions.     

Detection of White Spot Syndrome virus and Yellowhead virus in prawns imported into Australia

Objective To determine whether viable White Spot Syndrome virus (WSSV) or Yellowhead virus (YHV) were present in prawn products imported into Australia.
Procedure A sample of fourteen uncooked prawns was obtained from a consignment imported from southeast Asia. Each of the prawns was examined for WSSV by polymerase chain reaction (PCR), and then a bioassay was conducted in which a 10% homogenate of cuticular epithelium from each of the prawns was inoculated intramuscularly into healthy challenge prawns ( Penaeus monodon ) from Australia. The latter were then monitored for clinical signs of disease, and tissue samples were processed for electron microscopy, histological examination and for detection of WSSV by in situ hybridization (ISH) using a commercial kit. Limited numbers of haemolymph samples from inoculated challenge prawns were also examined by PCR for the presence of WSSV and YHV. All work was carried out under microbiologically secure conditions.
Results Results of the initial PCR examination for WSSV on the imported prawns were not definitive. However, in the bioassay, several of the challenge prawns inoculated with homogenates from the imported prawns showed clinical signs of disease (inappetence and lethargy) within 24 h post inoculation (pi) and died at 1 to 4 days pi. Tissue samples from a number of moribund prawns demonstrated lesions typical of White Spot Disease (WSD), and the presence of the virus was confirmed by electron microscopy, ISH and PCR. YHV was also demonstrated by PCR in two challenge prawns inoculated with homogenates.
Conclusion Viable WSSV and YHV were present in frozen prawn products imported into Australia for human consumption from southeast Asia. Importation of frozen infected products may present a risk of transferring virus to wild and farmed populations of crustaceans in this country. To date, WSD and Yellowhead Disease remain exotic to Australia.     

Salmonella Dublin infection in Queensland dairy cattle

Objective: To investigate the presence of Salmonella Dublin in Queensland cattle.
Design: An epidemiological study using diagnostic laboratory information and farm records.
Procedure: Outbreaks of gastroenteritis or pneumonia in calves, and abortions and enteritis in cows were routinely investigated for the presence of salmonellae. Where S Dublin was isolated, attempts were made to gather further epidemiological information.
Results: Prior to 1983 only two outbreaks of S Dublin have been recorded in Queensland dairy cattle. In 1983 S Dublin abortions were diagnosed in dairy heifers introduced from southern Australia to south-east Queensland. Sampling indicated that at least 10% of the 500 introduced heifers were faecal excretors of S Dublin. On 3 of the 7 farms from which S Dublin was recorded, infection spread to other cattle that were in contact. From February 1985 to February 1996, 29 outbreaks of S Dublin in cattle occurred on 29 farms (28 in south east Queensland and 1 in north Queensland). Calves were primarily affected. Continuing outbreaks were confirmed on only 4 of these 29 farms. On 15 farms S Dublin infections were associated with the purchase of infected calves or cows, while another farm adjoined 2 previously infected farms. No source of S Dublin was evident for the other 13 farms, where histories were often inadequate.
Conclusion: There has been a marked increase in S Dublin outbreaks in Queensland dairy cattle since 1983. Introduction of S Dublin carrier and aborting dairy heifers from southern Australia, where S Dublin is not uncommon, was associated with the initial outbreaks.