Evaluation of five diagnostic methods for the detection and quantification of Myxobolus cerebralis.

Diagnostic methods were used to identify and quantify Myxobolus cerebralis, a myxozoan parasite of salmonid fish. In this study, 7-week-old, pathogen-free rainbow trout (Oncorhynchus mykiss) were experimentally infected with M. cerebralis and at 7 months postinfection were evaluated with 5 diagnostic assays: 1) pepsin-trypsin digest (PTD) to detect and enumerate spores found in cranial cartilage, 2) 2 different histopathology grading scales that provide a numerical score for severity of microscopic lesions in the head, 3) a conventional single-round polymerase chain reaction (PCR), 4) a nested PCR assay, and 5) a newly developed quantitative real-time TaqMan PCR. There were no significant differences (P > 0.05) among the 5 diagnostic assays in distinguishing between experimentally infected and uninfected control fish. The 2 histopathology grading scales were highly correlated (P < 0.001) for assessment of microscopic lesion severity. Quantification of parasite levels in cranial tissues using PTD and real-time TaqMan PCR was significantly correlated r = 0.540 (P < 0.001). Lastly, 104 copies of the 18S rDNA gene are present in the M. cerebralis genome, a feature that makes this gene an excellent target for PCR-based diagnostic assays. Also, 2 copies of the insulin growth factor-I gene are found in the rainbow trout genome, whose detection can serve both as an internal quality control for amplifiable DNA and as a basis to quantify pathogen genome equivalents present in quantitative PCR assays.     

Investigation of pseudorabies virus latency in nervous tissues of seropositive pigs exposed to field strain

The prevalence and quantity of latent pseudorabies virus (PrV) in nervous tissues of pigs exposed to field strain in Korea was investigated by nested and real-time PCR. Nervous tissues including trigeminal ganglion (TG), olfactory bulb (OB), and brain stem (BS) were collected from 94 seropositive pigs. PrV latent infection in nervous tissues was initially investigated by nested PCR targeting three glycoprotein genes (gB, gE, and gG). Based on the obtained result, latent infection was detected in 95.7% of screened animals. Furthermore, it was revealed that the examined tissues harbored different copy numbers of latent PrV genome ranging from <10(2.0) to 10(7.1) copies per microgram of genomic DNA in real-time PCR analysis. These results show that under normal conditions, levels of latent PrV in the nervous tissues of pigs can vary across a wide range. Therefore, the data presented here provides information regarding control of the endemic state of PrV in Korea.     

Clinical effects, virological and serological responses in chickens following in-ovo inoculation of reticuloendotheliosis virus

Six-day-old embryonated specific pathogen free chicken eggs were inoculated with reticuloendotheliosis virus (REV) into the yolk sac and were incubated until they hatched. The hatchability of eggs inoculated with REV was significantly less (P less than 0.025) than that of media-inoculated controls. Although there were no significant differences in the body weights of these chickens at hatching, there were differences (P less than 0.001) at 6, 25 and 51 days of age between the infected and control chickens. Six of 10 chickens hatched from eggs inoculated with REV had feathering defects at 6 days of age. All chickens hatched from infected eggs had cell-free viraemia and antigenaemia, but not precipitating antibodies. Some of these chickens had very low neutralising antibody titres (less than 45) when examined at 25 and 37 days of age, as did all 10 chickens at 51 days of age. A low rate of horizontal transmission was indicated by the detection of antibodies at 37 and 51 days of age in chickens running in contact with the chickens hatched from eggs inoculated with REV.     

Development and validation of a SYBR green real-time PCR for the quantification of porcine circovirus type 2 in serum, buffy coat, feces, and multiple tissues

The emergence of multiple genotypes of PCV2, as demonstrated by phylogenetic analysis of whole genome or capsid sequences, makes it necessary to have quantitative diagnostic assays that perform equally well on all strains. The objectives of this study were to develop and validate a novel real-time polymerase chain reaction (PCR) assay targeting the highly conserved rep gene (ORF1) and investigate the effects of diagnostic specimen choice on its performance. The assay was tested in naturally infected conventional pigs, experimentally infected gnotobiotic pigs, and plasmid-spiked negative serum, lung tissue, and feces and found to have a linear detection range of 2.2x10(3) to 2.2x10(10) copies of PCV2 per mL. The assay successfully detected and quantified PCV2 DNA in serum, buffy coat, feces, and multiple lymphoid (bronchial, mesenteric, and superficial inguinal lymph nodes; thymus; tonsil; ileal Peyer's patches; and spleen), and non-lymphoid (myocardium; lung; kidney; liver; and gluteal muscle) tissues from naturally infected pigs. Across all tissues and sera of naturally infected pigs, the mean PCV2 concentration was 3.0logs higher in wasting versus non-wasting pigs. PCV2 concentration measured by tissue culture and immunohistochemical staining in homogenized liver samples of experimentally infected gnotobiotic pigs were compared to the concentrations estimated by quantitative PCR. Similar trends were noted with increasing PCV2 concentration detected in subclinically infected to severely PMWS-affected pigs across all assays. Our diagnostic assay was developed with a conserved target sequence, and performed efficiently in quantification of PCV2 in a variety of tissues from naturally and experimentally infected pigs.     

带毒蚕卵中的家蚕微孢子虫DNA提取方法研究

为促进用PCR法检测家蚕微粒子病的生产应用,对带毒蚕卵中的家蚕微孢子虫DNA提取方法进行了研究。结果表明,蚕卵经30%KOH、27℃预处理后抽提的核酸能满足PCR检测对DNA质量的要求。以该方法抽提的DNA为模板,进行蚕卵集团检测,结果10~100粒蚕卵中混有1粒“有毒”卵,即能用PCR的方法检测出。“1/100”“有毒”卵的检出概率约为80%。     

Identification of duck plague virus by polymerase chain reaction

A polymerase chain reaction (PCR) assay was developed for detecting duck plague virus. A 765-bp EcoRI fragment cloned from the genome of the duck plague vaccine (DP-VAC) virus was sequenced for PCR primer development. The fragment sequence was found by GenBank alignment searches to be similar to the 3' ends of an undefined open reading frame and the gene for DNA polymerase protein in other herpesviruses. Three of four primers sets were found to be specific for the DP-VAC virus and 100% (7/7) of field isolates but did not amplify DNA from inclusion body disease of cranes virus. The specificity of one primer set was tested with genome templates from other avian herpesviruses, including those from a golden eagle, bald eagle, great horned owl, snowy owl, peregrine falcon, prairie falcon, pigeon, psittacine, and chicken (infectious laryngotracheitis), but amplicons were not produced. Hence, this PCR test is highly specific for duck plague virus DNA. Two primer sets were able to detect 1 fg of DNA from the duck plague vaccine strain, equivalent to five genome copies. In addition, the ratio of tissue culture infectious doses to genome copies of duck plague vaccine virus from infected duck embryo cells was determined to be 1:100, making the PCR assay 20 times more sensitive than tissue culture for detecting duck plague virus. The speed, sensitivity, and specificity of this PCR provide a greatly improved diagnostic and research tool for studying the epizootiology of duck plague.     

Increased susceptibility of bobwhites (Colinus virginianus) to Histomonas meleagridis after exposure

Bobwhites given heterakid eggs but no Sevin became infected with cecal histomonads, but there was no pathological histomoniasis. Quail given 50 microgram of Sevin (10 microgram/day) behaved normally, but at necropsy they had slightly discolored livers. Quail given various doses of heterakid eggs and Sevin (Sevin increasing from 2.5 to 50 microgram) and those given various doses of heterakid eggs and 10 microgram/day of Sevin developed pathological histomoniasis and mortality rates of 36 and 63%, respectively.     

Effect of egg storage upon the survival of Campylobacter jejuni in laboratory-infected fertile poultry eggs

Fertile eggs were infected by Campylobacter jejuni in the laboratory by a temperature differential method of inoculation, which resulted in up to 10% of the hatched birds carrying C. jejuni in the intestine. When infected eggs were stored for 5 1/2 days before incubation, the infection rate of the eggs had decreased to 20% or less when set, and no infected chicks were hatched. Inoculation of eggs after 8 days in storage also failed to yield infected chicks. In all cases, the hatch ratio was no different from that of uninfected control eggs.     

应用双重实时荧光定量PCR方法检测鸡马立克氏病血清1型病毒

本研究建立了鸡马立克氏病血清1型病毒（MDV1）绝对定量检测方法。研究中选择MDV1特有的Meq基因的一段保守序列作为检测对象，将其克隆到质粒载体中，作为阳性标准品；同时将管家基因．鸡卵铁转蛋白（Ovo）特异性基因片段克隆到质粒载体上作为内参照的标准品。经荧光定量PCR（FQ-PCR）法扩增获得MDV1的FQ-PCR两条标准曲线，建立了MDV1双重FQ-PCR检测方法。应用该方法绝对定量检测了实验攻毒鸡及吉林省某地发病鸡只的羽髓、淋巴细胞等组织样本中单位细胞病毒拷贝数，并与琼脂扩散（AGP）、常规PCR等检测方法进行比较。结果表明，不论实验攻毒鸡还是自然发病鸡，羽髓中病毒富含量均高于其它组织，每百万宿主细胞内病毒含量为10^7～10^8拷贝；FQ-PCR检测MD发病鸡只的阳性率高于AGP，达100％；该方法的灵敏度比常规PCR检测高10～100倍，在单位细胞内可灵敏地检测到2．78个拷贝的病毒。该方法可以在不同的样品中有效的绝对定量检测MDVl。     

Differentiation of pathogenic and non-pathogenic serotype 1 Marek's disease viruses (MDVs) by the polymerase chain reaction amplification of the tandem direct repeats within the MDV genome.

There are no simple, direct methods to reliably distinguish oncogenic serotype 1 Marek's disease viruses (MDVs) from their attenuated variants. The present study was an attempt to apply polymerase chain reaction (PCR) to develop a rapid and sensitive assay for the presence of the MDV genome. PCR oligos were chosen to flank the 132-base-pair tandem direct repeats in the serotype 1 MDV genome. The PCR reaction was specific for serotype 1 MDVs, amplifying fragments corresponding to one to three copies of the tandem repeats present in Md11/8, JM/102W, and GA viruses. A high-molecular-weight DNA smear was observed when the DNA from an attenuated Md11/100 was PCR-amplified. Use of the PCR technique allowed the detection of two copies of the 132-base-pair repeat in the DNA extracted from MDV-induced lymphomas removed from two chickens. No DNA was amplified from the DNA extracted from lymphomas induced by either an avian leukosis virus (RAV-1) or reticuloendotheliosis virus (chick syncytial virus).     

Rapidly quantitative detection of Nosema ceranae in honeybees using ultra-rapid real-time quantitative PCR

BackgroundThe microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen.ObjectivesThe present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees.MethodsA procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration.ResultsUR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 10⁴ spores/bee, and the stable detection level was ≥ 2.40 × 10⁵ spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 10⁴ copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min.ConclusionsUR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.     

Immunologic effects of low levels of ochratoxin A in ovo: utilization of a chicken embryo model

Ochratoxin A (OA) was administered to 13-day-old chicken embryos via the chorioallantoic membrane. The 7-day LD50 value (day 20 incubation) of OA was calculated at 7.9 micrograms of OA. Ochratoxin-treated embryos (2.5 micrograms) had slight but significant changes in numbers of immunoglobulin-bearing cells in the bursa but not in the spleen. Chicks hatched from in ovo-treated eggs were challenged with 9 X 10(4) colony-forming units (CFU) of beta-hemolytic Escherichia coli (O1:K1) at 7 days of age via the thoracic air sac. Lesion scores of OA-treated chicks were equal to or less severe than those of controls. Hatchmates of the above chicks were vaccinated with a homologous killed E. coli bacterin (O1:K1) at both 2 and 4 weeks of age and challenged with 10(4) CFU of E. coli at 7 weeks. Post-challenge lesions were present in three vaccinated untreated controls and no OA-treated chicks. We conclude that although in ovo exposure to OA may marginally suppress immunoglobulin-bearing cells of bursa, chicks hatched from OA-treated eggs respond as well as controls to an antigen and resist infection by a virulent organism.     

Immunohistochemical and polymerase chain reaction studies in Neospora caninum experimentally infected broiler chicken embryonated eggs

The diagnostic characteristics of immunohistochemistry (IHC) and polymerase chain reaction (PCR) methods were studied in the tissues of broiler chicken embryos experimentally infected by Neospora caninum. An infection with N. caninum NC-1 isolate was conducted in 70 broiler chicken embryonated eggs randomly divided into seven equal groups. After 8 days of incubation, six groups were inoculated with 10, 10(2), 10(3), 10(4), 10(5), and 10(6) doses of tachyzoites/embryonated egg. The 7th group was considered as control. The mortality rate and pathological changes of the dead embryos and hatched chickens up to 60 days old were noticed. Consecutive sections to those used for histopathological examination including the liver, heart, brain, and chorioalantoic (CA) membrane were subjected to IHC. The intensity and distribution of the immunostaining was graded as highly to mildly positive. For PCR procedure, DNA was extracted from 50mg of the tissues and primer pair Np21/Np6 was used for amplification of the Nc-5 gene. The results of the immunosignaling ranged from variable degrees of mild to moderate staining as dark-brown to brown and coarsely to finely granular, mostly within the cytoplasm of infected cells such as the endothelial cells of blood vessels. The parasite aggregation was more predominant in the heart than other tissues. Immunoreactivity for N. caninum antigen was multifocally moderate positive in the heart, liver and CA of the 10(3) dose, and also heart, liver, brain and CA of the 10(4) dose. IHC showed mildly positive in the liver and heart of the chicken embryos infected with 10 and 10(2) tachyzoites, as well. The results of the PCR confirmed the existence of the parasite in all of the examined tissues from the 10(3) and 10(4) doses. In conclusion, the results indicate a good agreement between IHC and PCR in diagnosis of neospora antigen in the infected tissues.     

TaqMan荧光定量PCR检测鸡产蛋下降综合征病毒方法的建立及应用

根据GenBank公布的鸡产蛋下降综合征病毒六邻体蛋白基因的高度保守序列,设计了2对特异性引物和1条TaqMan探针.以构建的阳性重组质粒为标准品,绘制标准曲线,建立了一种快速检测鸡产蛋下降综合征病毒的TaqMan荧光实时定量PCR方法.该方法最小检出量达10 copies·μL-1,在1.0×102～1.0×108copies· μL-1检测范围间有良好的线性关系,特异性、稳定性和重复性也较好.用建立的本方法检测感染鸡产蛋下降综合征病毒鸡群的产蛋分离物,与普通PCR相比,该荧光实时定量PCR方法具有更高的敏感性,可更好地用于鸡产蛋下降综合征病毒的临床检测.     

A real-time PCR assay for rapid detection and quantitation of canine parvovirus type 2 in the feces of dogs

We describe a rapid, sensitive and reproducible real-time PCR assay for detecting and quantifying canine parvovirus type 2 (CPV-2) DNA in the feces of dogs with diarrhea. An exogenous internal control was added to control the assay performance from extraction to amplification. The method was demonstrated to be highly specific and sensitive, allowing a precise CPV-2 DNA quantitation over a range of eight orders of magnitude (from 10(2) to 10(9) copies of standard DNA). The reproducibility of the CPV-2 real-time PCR assay was assessed by calculating the coefficients of variation (CV) intra-assay and inter-assay for samples containing amounts of CPV-2 DNA spanning the whole range of the real-time PCR standard curve. Then, fecal specimens from diarrheic dogs were analyzed by hemagglutination (HA), conventional PCR and real-time amplification. Comparison between these different techniques revealed that real-time PCR is more sensitive than HA and conventional gel-based PCR, allowing to detect low viral titers of CPV-2 in infected dogs.     

猪圆环病毒2型遗传标记毒株的构建及生物学特性鉴定

以临床分离的猪圆环病毒2型（PCV2）为材料，利用PCR法扩增病毒基因组，将2个基因组顺式连接插入到质粒载体中构建感染性分子克隆。通过引物设计替换碱基，在病毒基因组内插入Sal Ⅰ限制性内切酶位点作为遗传标记，经重组质粒转染细胞获得带有遗传标记的新毒株。采用免疫过氧化物酶单层细胞试验、免疫电镜、核酸序列分析表明，在病毒感染细胞中检出病毒特异抗原，其抗原性、病毒形态学及基因序列与亲本毒株一致。鉴于新病毒基因组内插入一个SalⅠ酶切住点，用PCR与限制性片段长度多态性分析法可与野生型病毒相鉴别。新毒株经细胞连续传60代，体外培养增殖性能稳定，毒价可达10^6.6CID50/mL。取病毒培养物经静脉和滴鼻途径接种35日龄抗体阴性仔猪4头，接种后表现出体温升高、进行性消瘦、被毛粗糙及体表淋巴结肿胀症状，迫杀后多种脏器中均能检测到病毒抗原与核酸。研究表明，利用感染性分子克隆手段构建带有遗传标记的PCV2新毒株，为进一步开展该病毒的致病性、疫苗免疫、分子诊断等研究奠定了基础。     

Alterations in the rainbow trout (Oncorhynchus mykiss) eggs exposed to ionizing radiation during induced androgenesis

Ionizing radiation (IR) is applied to inactivate nuclear genome in the salmonid eggs to induce androgenetic development. However, it has been considered that doses of IR used to damage maternal chromosomes may also affect morphology of the eggs and decrease their developmental potential. Thus, the main goal of the present research was to assess alterations in the rainbow trout (Oncorhynchus mykiss) eggs caused by the high dose of IR administered during androgenesis. In the present research, rainbow trout eggs were irradiated with 350 Gy of X‐rays, inseminated and exposed to the high hydrostatic pressure (HHP) shock to develop as androgenetic doubled haploids (DHs). The distribution of lipid droplets in the irradiated and non‐irradiated rainbow trout eggs, survival rates and morphology of larvae from androgenetic and control groups were compared. It has been observed that non‐irradiated and irradiated eggs exhibited altered distribution of lipid droplets. Most of the eggs before IR treatment displayed rather equal distribution of the oil droplets. In turn, majority of eggs studied after irradiation had coalesced lipid droplets, a pattern found in eggs with reduced quality. Incidences of abnormally developed larvae were more frequently observed among fish that hatched from the irradiated eggs. Observed changes suggest X‐rays applied for the genetic inactivation of rainbow trout eggs may lead to decrease of their developmental competence.     

Development of a diagnostic PCR assay based on novel DNA sequences for the detection of Mycoplasma suis (Eperythrozoon suis) in porcine blood

An efficient method of control of porcine eperythrozoonosis (PE) caused by Mycoplasma suis is eradication of infection by detection and removal of infected carrier animals. At present, only a few tests are available for the diagnosis of these latent M. suis infections in pigs. The objective of this study was to develop a PCR assay based on novel DNA sequences for the identification of M. suis-infected pigs. A 1.8 kb EcoRI DNA fragment of the M. suis genome was isolated from the blood of pigs experimentally infected with M. suis. Specificity of the DNA fragment was confirmed by DNA sequence analysis and PCR using primers directed against sequences contained in the 1.8 kb fragment. PCR products of 782 bp in size were amplified only from M. suis particles prepared from the blood of experimentally infected pigs but not from any controls, comprising blood from gnotobiotic piglets and a panel of bacteria including other porcine mycoplasmas. PCR results were confirmed by dot blot hybridisation. The applicability of the PCR assay to diagnose M. suis infections in pigs was evaluated by investigating blood samples from 10 symptomatic pigs with clinical signs typical of porcine eperythrozoonosis and blood samples from 10 healthy pigs. The M. suis-specific PCR product was amplified from all samples taken at episodes of acute disease as well as from samples taken during the latent stage of infection, thus demonstrating the suitability of the PCR assay for detecting latent infected carrier animals.