Communications: Experimental Production of Proliferative Gill Disease in Channel Catfish Exposed to a Myxozoan-Infected Oligochaete,Dero digitata

Abstract

Specific-pathogen-free fry of channel catfish Ictalurus punctatus raised in well water were exposed to Dero digitata (an oligochaete) collected from ponds where catfish have had proliferative gill disease (PGD) and where D. digitata is known to be infected with the triactinomyxid myxozoan Aurantiactinomyxon sp. These fry developed gill lesions and parasites characteristic of PGD. Fry exposed to suspensions of mature Aurantiactinomyxon spores obtained from squashes of infected D. digitata also developed PGD. Fry exposed to oligochaetes other than Dero spp., non-oligochaete benthic macroinvertebrates, or suspensions of squashes of D. digitata without identifiable myxozoans did not develop PGD.     

A Real-Time Polymerase Chain Reaction Assay of the Bacterium Edwardsiella ictaluri in Channel Catfish

Abstract

A rapid (4.5-h) and sensitive assay based on polymerase chain reaction (PCR) was developed to facilitate the early detection of Edwardsiella ictaluri in channel catfish Ictalurus punctatus. A 129-base-pair fragment of a sequence specific to E. ictaluri was amplified with both standard and real-time (quantitative) PCR. The sensitivity of detection was determined to be as low as the equivalent of 2.5 cells in DNA samples from both E. ictaluri cells and mixtures of blood from noninfected catfish and E. ictaluri cells. Infection levels (as determined by real-time PCR) in blood from experimentally challenged fish were compared with brain–heart-infusion-cultured bacterial colony counts to assess the accuracy of the PCR assay. The PCR-based detection level (the equivalent of 10⁵–10⁸ cells/mL) was comparable to that of traditional culturing techniques (10⁶–10⁷ cells/mL). In future applications, this assay will be applied in a comprehensive breeding program to select channel catfish that are resistant to enteric septicemia of catfish.     

Preliminary Studies of a Newly Developed Subunit Vaccine for Channel Catfish Virus Disease

Abstract

The soluble channel catfish virus (CCV) envelope was harvested and used as a vaccine for channel catfish virus disease. Three- to four-day-old eggs of channel catfish Ictalurus punctatus and 1-week-old fry were vaccinated by immersion. A booster was given to subgroups of fry 2 weeks after vaccination. Vaccinated and nonvaccinated control groups were challenged with viable CCV 8 weeks after vaccination. All challenged nonvaccinated control fry died during the first experiment, and 56% died in the second experiment. Survival offish vaccinated as eggs or fry was 31 and 82%, respectively; survival of groups given a booster dose was 81 and 89%, respectively.     

Identification of Flavobacterium and Flexibacter Species by Species-Specific Polymerase Chain Reaction Primers to the 16S Ribosomal RNA Gene

Abstract

Species-specific polymerase chain reaction (PCR) primers have been developed for the identification of the causative agents of warmwater and marine finrot in fish: Flavobacterium columnare (Flexibacter columnaris) and Flexibacter maritimus. Differences in gene sequence in the bacterial small-subunit (16S) ribosomal RNA (rRNA) were used to design the species-specific PCR primers. The previously reported species-specific PCR primers Psy1 and Psy2 for the identification of Flavobacterium psychrophilum (Flexibacter psychrophila), the causative agent of coldwater finrot, were also used to develop a speciation scheme for all three bacterial finrots. These three primer sets were successful in discriminating among yellow-pigmented bacteria as well as in speciating the three major pathogenic flexibacteria to fish. The primer sets were designed to produce uniquely sized subproducts of 16S rRNA for each species: Flavobacterium psychrophilum (1,100 base pairs, bp), F. columnare (800 bp), and Flexibacter maritimus (400 bp). These primers were shown to correctly speciate field isolates in double-blind experiments (P = 0.01).     

PCR扩增Nuc基因检测奶牛乳腺炎致病性金黄色葡萄球菌

为建立奶牛乳腺炎金黄色葡萄球菌致病菌株的检测方法,依据金黄色葡萄球菌耐热核酸酶Nuc基因序列,设计合成特异性引物,通过聚合酶链式反应扩增Nuc基因,并将扩增基因克隆入T载体,进行序列测定,对所分离的奶牛乳腺炎病原菌进行鉴定.结果显示:金黄色葡萄球菌扩增出特异性条带,大小为694bp;其他菌株未出现条带.敏感性检测的最低浓度为1.25×103 cfu/mL.本试验所建立的方法具有特异性强、敏感度高的特点.     

PCR扩增Nuc基因检测奶牛乳腺炎致病性金黄色葡萄球菌

为建立奶牛乳腺炎金黄色葡萄球菌致病菌株的检测方法,依据金黄色葡萄球菌耐热核酸酶Nuc基因序列,设计合成特异性引物,通过聚合酶链式反应扩增Nuc基因,并将扩增基因克隆入T载体,进行序列测定,对所分离的奶牛乳腺炎病原菌进行鉴定。结果显示:金黄色葡萄球菌扩增出特异性条带,大小为694bp;其他菌株未出现条带。敏感性检测的最低浓度为1.25×103cfu/mL。本试验所建立的方法具有特异性强、敏感度高的特点。     

奶牛乳房炎常见致病菌多重PCR检测方法的建立

为建立同时快速检测奶牛奶样中无乳链球菌、停乳链球菌、乳房链球菌和金黄色葡萄球菌的方法,根据无乳链球菌sip基因、停乳链球菌isp基因、乳房链球菌pauA基因和金黄色葡萄球菌nuc基因各设计1对特异性引物,建立多重PCR检测体系。结果显示,该检测方法具有高特异性,无乳链球菌、停乳链球菌、乳房链球菌和金黄色葡萄球菌敏感性分别为105、104、105、105 CFU/mL。对临床采集的460份奶样检测结果表明,建立的多重PCR体系可以用于临床上无乳链球菌、停乳链球菌、乳房链球菌和金黄色葡萄球菌感染引起的奶牛乳房炎的检测。     

贝类折光马尔太虫半套式PCR检测方法的建立及初步应用

根据Genbank中折光马尔太虫的基因保守序列,设计了3条特异性引物,通过对半套式PCR扩增条件的优化,研究建立了检测贝类折光马尔太虫的半套式PCR方法.该方法对折光马尔太虫模板进行扩增,得到与实验设计相符的228 bp的特异性扩增带,而对单孢子虫、派琴虫、副溶血弧菌、溶藻弧菌和河弧菌等病原体的扩增,结果全为阴性.敏感性试验结果表明,该技术最低能检测到0.01 fg的折光马尔太虫质粒DNA.用该半套式PCR对福建沿海的贝类样品进行检测,结果从溢蛏中检出折光马尔太虫7份,结果提示福建沿海养殖的贝类中存在折光马尔太虫感染,建立的半套式PCR方法可以用于贝类折光马尔太虫的临床快速检测.     

A Quantitative Polymerase Chain Reaction Assay for the Detection and Quantification of Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus 3)

Epizootic epitheliotropic disease virus (EEDV; salmonid herpesvirus [SalHV3]; family Alloherpesviridae) causes a systemic disease of juvenile and yearling Lake Trout Salvelinus namaycush. No cell lines are currently available for the culture and propagation of EEDV, so primary diagnosis is limited to PCR and electron microscopy. To better understand the pervasiveness of EEDV (carrier or latent state of infection) in domesticated and wild Lake Trout populations, we developed a sensitive TaqMan quantitative PCR (qPCR) assay to detect the presence of the EEDV terminase gene in Lake Trout tissues. This assay was able to detect a linear standard curve over nine logs of plasmid dilution and was sensitive enough to detect single-digit copies of EEDV. The efficiency of the PCR assay was 99.4 ± 0.06% (mean ± SD), with a 95% confidence limit of 0.0296 (R² = 0.994). Methods were successfully applied to collect preliminary data from a number of species and water bodies in the states of Pennsylvania, New York, and Vermont, indicating that EEDV is more common in wild fish than previously known. In addition, through the development of this qPCR assay, we detected EEDV in a new salmonid species, the Cisco Coregonus artedi. The qPCR assay was unexpectedly able to detect two additional herpesviruses, the Atlantic Salmon papillomatosis virus (ASPV; SalHV4) and the Namaycush herpesvirus (NamHV; SalHV5), which both share high sequence identity with the EEDV terminase gene. With these unexpected findings, we subsequently designed three primer sets to confirm initial TaqMan qPCR assay positives and to differentiate among EEDV, ASPV, and NamHV by detecting the glycoprotein genes via SYBR Green qPCR.

Received April 20, 2015; accepted November 10, 2015     

Development of a Multiplex Polymerase Chain Reaction for the Differentiation of Bovine Herpesvirus‐1 and ‐5

Bovine herpesvirus‐1 (BHV‐1) and bovine herpesvirus‐5 (BHV‐5) are closely related viruses which exhibit some important differences at the genetic and immunogenic levels which may explain the differences in their pathogenicity and epidemiological characteristics. A multiplex polymerase chain reaction (M‐PCR) was developed to detect and differentiate between BHV‐1 and BHV‐5. In this M‐PCR two pairs of primers (TK1, TK2 and GD1, GD2) were used in the same reaction mix to amplify a thymidine kinase genomic region (183 bp) of BHV‐1 and one genomic region of the glycoprotein D (564 bp) of BHV‐5. The specificity of the M‐PCR was demonstrated when using both primers pairs simultaneously with BHV‐1 and BHV‐5 templates. The two expected bands were amplified without the apparition of non‐specific products. However, when other herpesvirus strains were used, there was no amplification. To evaluate the sensitivity of the assay, dilutions of purified viral DNA were made for M‐PCR amplification. The detection limit was 7 pg for BHV‐1 and 22 pg for BHV‐5. It was also determined by comparing the M‐PCR with viral isolation. M‐PCR was able to detect one log₁₀ more than viral isolation for BHV‐1 and for BHV‐5 was two logarithms lower. The applicability of M‐PCR was demonstrated on different specimens. Twenty isolates from field samples (11 BHV‐1 and nine BHV‐5) were positive by M‐PCR, and the results were completely coincident with previous characterization using the immunoperoxidase assay. M‐PCR could detect viral DNA in organ samples from natural infections, such as semen and brain. In addition, M‐PCR detected more positive samples than observation of the citophatic effect in cell culture of nasal swabs from experimentally infected animals in two different assays. Owing to the difference in size of the M‐PCR products which allows easy identification in an electrophoretic run, it is not necessary to use extra blotting and hybridization steps or a second round of amplification to differentiate clearly between BHV‐1 and BHV‐5.     

Field Evaluation of Sensitivity and Specificity of a Polymerase Chain Reaction (PCR) for Detection of Nucleospora salmonis in Rainbow Trout

Abstract

We determined the sensitivity and specificity of a nested polymerase chain reaction (PCR) for detection of the microsporidian parasite Nucleospora salmonis in kidney tissue of rainbow trout Oncorhynchus mykiss. Kidney tissues were sampled on three dates from 162 juvenile rainbow trout obtained from a California State fish hatchery where the organism was endemic. Kidney tissues were used to prepare imprints stained with May–Grünwald Giemsa and for extraction of genomic DNA for a nested PCR test for N. salmonis. Positive PCR results for N. salmonis were obtained from 1 of 100, 2 of 32, and 27 of 30 kidneys collected on the first, second, and third sample dates, respectively. Kidney tissues from 3 of 27 trout in the third sample that tested positively by PCR also had microscopic evidence of parasites in stained kidney imprints. No parasites were detected in the remaining 159 kidney samples examined microscopically. Sensitivity and specificity of the PCR assay were estimated by using maximum likelihood estimation based on cross-classified test results. This method yielded estimates of sensitivity of 99.99% and specificity of 99.87%. This field evaluation supports experimental evidence that the nested PCR test will be a valuable diagnostic tool for prevention and control of N. salmonis as well as for risk assessment associated with fish movements.     

上海市某奶牛场牛型结核菌素皮内变态反应疑似结果影响因素分析

为评估奶牛年龄分布、棚舍位置、胎次、泌乳等因素,对牛型结核菌素(PPD)皮内变态反应试验特异性的影响,对上海市某奶牛场2 031头奶牛的PPD皮内变态反应试验初筛结果进行分析和回顾性问卷调查,并利用Epi info~(TM) 7软件,对初筛结果和问卷调查结果进行单因素分析。初筛和问卷调查结果显示:该奶牛场PPD皮内变态反应试验初筛疑似反应率为15.75%,疑似反应主要集中在2岁以下、0～2胎的青年奶牛,其中1.5～2岁青牛奶牛疑似反应率最高;随着年龄、泌乳量和胎次的增加,疑似反应率呈下降趋势。单因素分析结果显示:未配种奶牛(OR=2.02,P0.01),未怀孕奶牛(OR=1.37,P0.01),未泌乳奶牛或干奶牛(OR=3.72,P0.01)是导致奶牛场出现PPD皮内变态反应试验疑似反应的危害性风险因素。结果表明:2岁以下青年奶牛易出现PPD皮内变态反应试验疑似反应,呈现出显著的年龄相关性;是否配种、怀孕以及泌乳量多少均能够影响PPD皮内变态反应试验的特异性。建议奶牛场将犊牛结核病首次检测日龄提前至90日龄以内,并对初筛出现疑似反应的奶牛立即隔离,采用γ-干扰素试验和PPD比较皮内变态反应试验的平行检测策略进行确诊。本研究全面了解了奶牛场初筛疑似反应奶牛的个体特征和风险因素,有助于提高检测方法的敏感性,减少假阴性。