Detection of equine herpesvirus-1 in nasal swabs of horses by quantitative real-time PCR

Background: Early identification of inhalation-transmitted equine herpesvirus type 1 (EHV-1) infections has been facilitated by the availability of a number of real-time quantitative PCR (qPCR) tests. A direct comparison between nasal swab qPCR and traditional virus isolation (VI) requires a method for normalizing the qPCR samples and controlling for PCR inhibitors present in some clinical samples.
Objectives: To quantify EHV-1 shedding in viral swabs using an internal control and to compare fast qPCR to VI for the detection of EHV-1 in nasal swabs from horses.
Animals: Fifteen horses experimentally infected with EHV-1.
Methods: Experimental study : Nasal swab samples were collected daily after experimental infection for up to 21 days. VI was performed by conventional methods. The DNA was prepared for qPCR with the addition of a known quantity DNA of Marek's disease virus as an internal control. qPCR was performed.
Results: The qPCR method detected virus up to day 21 after challenge, whereas VI detected virus only to day 5. The median Kaplan-Meier estimates for EHV-1 detection were 12 days for qPCR and 2 days for VI ( P < .0001). When compared with VI, the sensitivity and specificity of qPCR were 97 (95% CI: 86–100) and 27% (95% CI: 20–35).
Conclusions and Clinical Importance: We conclude that fast qPCR of nasal swab samples should be chosen for diagnosis and monitoring of herpesvirus-induced disease in horses. Recommended reference ranges of C _T values are provided as well as justification of a minimum 10-day quarantine period.     

用套式PCR方法对几种猪用冻干疫苗的PCV2检测

参照Genbank已发表的猪圆环病毒2型的基因序列,取其比较保守的基因片段ORF2,利用引物设计软件Oli-go5.0设计两对PCV2型特异的引物,其中第一对引物扩增跨度为ORF2全基因片段,长度为702 bp,第二对引物扩增ORF2中间的一个小片段,跨度大小为494 bp.首先用第一对引物扩增阳性DNA,阳性DNA的浓度从10-1稀释到10-11,然后以第一次PCR产物为模板,用第二对引物进行PCR,发现这种套式PCR的方法比用普通PCR灵敏度提高102倍.同这种套式PCR方法对广东省市场上出售的几种猪用弱毒疫苗进行猪圆环病毒2型检测,结果发现这几种疫苗全部为阴性,本实验说明目前广东市场上出售的弱毒疫苗在制作过程中没有受到PCV2的污染,解除了广大猪场主的顾虑.     

Detection of respiratory herpesviruses in foals and adult horses determined by nested multiplex PCR

A nested multiplex PCR was developed as a rapid (<12h), sensitive test for the simultaneous identification of equine herpesviruses (EHV1, EHV4, EHV2 and EHV5) in clinical samples from horses. Peripheral blood and nasal swab (NS) samples from 205 weanling Thoroughbred foals on 6 different studs over 3 consecutive seasons and from 92 adult horses without clinical signs of respiratory disease were examined using direct multiplex PCR of clinical samples (direct PCR) and conventional cell culture with differentiation of EHV in cell cultures by multiplex PCR. Multiplex PCR proved a sensitive and specific technique for the detection of EHV in cell culture and clinical samples. The technique described appeared equally sensitive as one using a single set of primers for individual EHV but reduced labour and reagent costs. Cell cultures showing cytopathic effect (CPE) were always positive for EHV on PCR. EHV were also detected by multiplex PCR in 11 samples which failed to show CPE. By a combination of multiplex PCR and cell culture or direct multiplex PCR, the presence of up to three EHV in the same sample was detected. Overall, EHV5 was detected by direct multiplex PCR of peripheral blood mononuclear cells (PBMC) and/or NS samples from 78% of foals and 47% of adult horses. Repeated sampling or cell culture in combination with multiplex PCR and with the incorporation of IL-2 in culture medium increased the sensitivity for detection of EHV in PBMC and demonstrated that EHV5 DNA could be identified in PBMC from 89% of foals and 100% of adult horses. EHV2 was identified from approximately 30% of foals, but was more frequently identified in samples from 17 foals with mild respiratory disease and was isolated infrequently from adult horses. EHV1 and EHV4 were identified uncommonly in any population in the current study.     

兽用疫苗中霉形体污染的套式PCR检测技术建立及初步应用

根据GenBank公布的鸡毒霉形体、猪肺炎霉形体、猪滑液霉形体和絮状霉形体的16s rRNA基因序列,利用引物设计软件DNAStar和Primer5.0自行设计3对特异性引物,以市场上常见的兽用疫苗为检测对象进行试验,优化反应体系,建立了兽用疫苗霉形体污染检测的套式PCR方法。同时,本试验还对市场上的兽用疫苗进行随机抽查检测,其检出率分别为24.2%(23/95)、21.1%(20/95)和14.7%(14/95)。     

应用多重PCR检测和区分3个型的马疱疹病毒

针对马疱疹病毒（EHV）的EHV-1、EHV-2和EHV-4糖蛋白B基因序列，设计、合成了3对特异性引物进行多重PCR，不仅可以在数小时内分别检测这3个型的EHV，而且在同一反应系统内可以清晰地区分EHV-1、EHV-2和EHV-4，其PCR产物大小分别为226、333、570bp，符合预期的片段大小，序列分析证实与已发表的序列一致；该检测方法的灵敏度达到10^3 TCID50；分别从血清学阳性但病毒分离为阴性的1匹进口马组织样品和一些出口前检疫马的鼻咽样品检测到EHV-1和EHV-4特异性核酸。     

High frequency of Mycobacterium bovis DNA in colostra from tuberculous cattle detected by nested PCR

We evaluated by nested PCR reaction, different cow secretions from a herd with 48% of prevalence of bovine tuberculosis (BTB), seeking to determine niches where Mycobacterium bovis could be found. Postmortem examination of 18 (75%) tuberculin reacting cows allowed demonstrates BTB-compatible lesions in six, all of them PCR positives in milk and four in colostra samples. Our results showed that up to 62% of the colostra analysed contained M. bovis DNA, whereas only 18% of milk gave a positive reaction. Moreover, in bronchoalveolar lavages from cattle with compatible lesions in lungs or lymph nodes, where macrophages account up to 90% of cells, we did not find evidences of M. bovis. Altogether, these results suggest that differences in the anti-bacterial capacity of bovine macrophages, dependent upon microenvironment and organ-specific factors, exist. Alternatively, we hypothesize that hypoxic conditions that are encountered in mammary glands macrophages could induce M. bovis entrance into a 'dormancy-like' state, and that the high number of colostra samples were M. bovis was detected, could be an indicator of reactivation during 'peripartum'.     

Development of a differential multiplex PCR assay for equine herpesvirus 1 and 4 as a diagnostic tool

In this study, a multiplex polymerase chain reaction (PCR) procedure was developed for differentiation of strains and field isolates of equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4). Specific oli-gonucleotide primers were combined to amplify the thymidine kinase (TK) gene region of EHV-1 and EHV-4, which would yield fragments of different lengths for each virus in the same amplification reaction. The specificity of the largest PCR amplicon for EHV-4 was confirmed by restriction digestion with HindIII. The multiplex PCR proved to be a fast and sensitive method for typing EHV-1 and EHV-4 isolates and for detection and differentiation of both viruses in field samples in which infectious virus is no longer available. The sensitivity was improved by combining cycling optimization and visualization of PCR products in ethidium bromide and silver-stained acrylamide gels.     

Survival of equine herpesvirus-4, feline herpesvirus-1, and feline calicivirus in multidose ophthalmic solutions

Objective To determine survival over time of infectious equine herpesvirus‐4, feline herpesvirus‐1, and feline calicivirus in three commercially available and commonly used ophthalmic solutions (eyewash, fluorescein, and proparacaine HCl). Sample population Viruses used in this study were originally isolated from eyes of animals referred to the University of Illinois. Equine herpesvirus‐4 was propagated in MDBK cells and feline herpesvirus‐1 and feline calicivirus in CRFK cells. Procedure After separately inoculating a designated solution with a specific titer of an individual virus, solutions were incubated per manufacturer's recommendations, either at 4 °C or 25 °C. Virus titers within solutions were subsequently measured at 1, 8, and 24 h and 3, 5 and 7 days post inoculation using either plaque or TCID₅₀ assays. Results Equine herpesvirus‐4, feline herpesvirus‐1, and feline calicivirus were present in eyewash for 7 days, 5 days, and 7 days, respectively. Eyewash did not decrease survival time of any virus when compared to controls. Equine herpesvirus‐4 and feline herpesvirus‐1, both enveloped viruses, were not recovered at any time ≥ 1 h post inoculation in fluorescein. Feline calicivirus, a nonenveloped virus, was present in fluorescein for 7 days. Equine herpesvirus‐4 and feline herpesvirus‐1 did not remain infectious in proparacaine at any time ≥ 1 h post inoculation, but feline calicivirus was recovered at up to 24 h post inoculation. Conclusions Equine herpesvirus‐4, feline herpesvirus‐1, and feline calicivirus may be readily transmissible via the eyewash solution used in this study. Risk of iatrogenic transmission of the three viruses used in this study was significantly reduced in both fluorescein and proparacaine solutions. Feline calicivirus, the only nonenveloped virus evaluated, remained viable longer in both fluorescein and proparacaine solutions.     

特异PCR方法用于鸡的3种艾美耳球虫混合感染鉴别的研究

用单卵囊分离法获得的鸡的3种艾美耳球虫（每种各2株）卵囊：柔嫩艾美耳球虫（Eimeria tenella）、巨型艾美耳球虫（E．maxima）、堆型艾美耳球虫（E．acervulina）。经纯化、提取基因组DNA后，用报道的种特异引物做PCR扩增分析，以确定是否为纯种。结果发现这3种球虫均存在混合感染的情况。该结果为进一步研究这3种球虫奠定了基础，并说明特异PCR方法能够有效地、快速地鉴别球虫虫种。     

Prevalence of Salmonella in beef feeder steers as determined by bacterial culture and ELISA serology

Results are presented for monitoring Salmonella infection by bacteriological culture and immune response (enzyme linked immunosorbent assay (ELISA) and haptoglobin) testing of samples collected from beef cattle at a single feedyard sampled over time. A total of 120 beef steers were examined on entry to the feedyard and at days 30, 60, and at time of slaughter (120-150 days). Isolations of Salmonella decreased over time from 40% of the steers sampled at day 0 to 0% at slaughter, whereas serological results varied by serogroup. Seropositivity increased for Salmonella group B up to day 60, and subsequently decreased to about half of the 60-day positivity rate at the time of slaughter. No significant changes in seropositivity were detected during the course of the study for the four other Salmonella serogroups (C1, C3, D1, and E1). Haptoglobin measurements were not a good indicator of Salmonella infection status. Sequential Salmonella testing either by culture, ELISA, or both could be used to monitor pathogen control practices.     

Detection and genotypic differentiation of Campylobacter jejuni and Campylobacter coli strains from laying hens by multiplex PCR and fla-typing

In total, 26 Campylobacter (C.) strains, isolated from liver, spleen, caecal or jejunal content of laying hens from different flocks were examined. In these flocks a drop in egg production, an increasing mortality and livers with whitish-grey lesions as post-mortem finding were observed. Suspected Campylobacter colonies were differentiated using a modified m-PCR in 13 Campylobacter jejuni and 13 Campylobacter coli strains. All isolates were characterised by typing of the flaA and flaB gene each with two restriction enzymes. To compare the four different profiles for all strains an artificial “fla-type” was generated. Different and identical fla-types of C. jejuni and C. coli were recovered from both intestinal and extra-intestinal organs of the laying hens and even from individual birds. One significant observation is that some fla-types of C. jejuni or C. coli were detected in intestinal and systemic sites but not all fla-types of both species appeared to be equally able to invade internal organs.     

Prevalence of canid herpesvirus-1 infection in stillborn and dead neonatal puppies in Denmark

Background

Canid herpesvirus-1 (CaHV-1) infection in puppies less than three weeks of age is often reported to be associated with a lethal generalized necrotizing inflammation and since the discovery of the virus in 1965 several reports of neonatal infections have been published. However, the significance of CaHV-1 for peri- and neonatal mortality in puppies remains unclear. Therefore, we examined stillborn and dead neonatal puppies in Denmark to determine the prevalence of infection and further to correlate infection levels with necropsy findings to assess the possible significance of the infection.

Results

From a cross-sectional study of 57 dead puppies, 22.8% (n = 13) were confirmed positive for CaHV-1 by real-time polymerase chain reaction (PCR) of tissue pools of lung/liver and/or spleen/kidney. Specimens from PCR positive cases were further investigated by histology and in situ hybridization (ISH). High levels of CaHV-1 DNA were present in only one case in which lesions and ISH staining consistent with CaHV-1 infection were found as well. CaHV-1 concentrations in the other cases were low and a range of lesions not consistent with CaHV-1 were found. Similar, ISH staining was mostly negative in these except for one case with a few positive cells.

Conclusion

CaHV-1 infection in stillborn and dead neonatal puppies in Denmark seems to be common, but the direct significance for puppy mortality remains unclear as only one of 13 PCR positive puppies (7.7%) had pathognomonic lesions.

Electronic supplementary material

The online version of this article (doi:10.1186/s13028-014-0092-9) contains supplementary material, which is available to authorized users.     

中国肾型IBV毒株的基因型及其S1基因的巢式PCR

用本研究建立的ＩＢＶＳ１基因的巢式ＰＣＲ方法鉴定了国内５个肾型传染性支气管炎病毒（ＩＢＶ）分离株。以此５个分离株及３个标准毒株的ＲＴ－ＰＣＲ产物（１７４９ｂｐ,含Ｓ１基因）为模板,１对内部引物经巢式ＰＣＲ都获得１７２９ｂｐ的产物,另１对内部引物的巢式ＰＣＲ有大小不同的产物（１６７８ｂｐ或０．５、０．６ｋｂ）。参照以前的ＲＦＬＰ分析结果,探讨了中国肾型ＩＢＶ毒株的基因型状况。     

Prevalence and genotyping of bovine Cryptosporidium species in the Mediterranean and Central Anatolia Region of Turkey

The prevalence of Cryptosporidium species in calves and heifers with relation to diarrhea from several herds was investigated in this study. Fecal samples were collected from 135 and 120 pre-weaned calves and 79 and 130 heifers raised in the Central Anatolia (CAR) and Mediterranean Regions (MR) of Turkey, respectively. A total of 86 post-weaned calves in CAR were also included in the study. For diagnostic comparison, all samples were examined by microscopic examination, SSU rRNA nested PCR and TaqMan real-time PCR for the presence of oocyst and Cryptosporidium DNA. In total, 102 (34.0 %) and 93 (37.2 %) of the examined samples from CAR and MR were found positive for Cryptosporidium DNA with both nested PCR and real-time PCR analyses, respectively with an overall prevalence of 35.5 %. The diagnostic sensitivity and specificity of microscopic examination were determined as 68.7 % and 100.0 % compared to molecular tools, respectively. RFLP and sequence analyses of the SSU rRNA from the PCR products revealed that 138 (70.8 %) out of 195 positive isolates were C. parvum further confirming the species-specific real-time PCR results. Among the remaining 57 (29.2 %) positive isolates, 30 (15.4 %) and 27 (13.8 %) were characterized as C. ryanae and C. bovis, respectively. C. parvum was the dominant species in pre-weaned calves especially with diarrhea while C. bovis and C. ryanae were mostly found in post-weaned calves and heifers. The sequence analyses of the gp60 gene of C. parvum isolates revealed two subtypes (IIaA13G2R1, IIaA14G1R1) belonging to zoonotic family IIa, with IIaA13G2R1 being the most common in diarrheic calves.     

Seroprevalence of equine granulocytic anaplasmosis and lyme borreliosis in Canada as determined by a point-of-care enzyme-linked immunosorbent assay (ELISA)

Equine granulocytic anaplasmosis (EGA) and Lyme borreliosis (LB) are an emerging concern in Canada. We estimated the seroprevalence of EGA and equine LB by testing 376 convenience serum samples from 3 provinces using a point-of-care SNAP^® 4Dx^® ELISA (IDEXX Laboratories, Westbrook, Maine, USA), and investigated the agreement between the point-of-care ELISA and laboratory-based serologic tests. The estimated seroprevalence for EGA was 0.53% overall (0.49% in Saskatchewan, 0.71% in Manitoba), while the estimated seroprevalence for LB was 1.6% overall (0.49% in Saskatchewan, 2.86% in Manitoba). There was limited agreement between the point-of-care ELISA and an indirect fluorescent antibody test for EGA (kappa 0.1, PABAK 0.47) and an ELISA/Western blot combination for LB (kappa 0.23, PABAK 0.71). While the SNAP^® 4Dx^® ELISA yielded expected seroprevalence estimates, further evaluation of serologic tests for the purposes of disease exposure recognition may be needed.     

Allele frequency of hereditary equine regional dermal asthenia in American Quarter horses in Brazil determined by quantitative real-time PCR with high resolution melting analysis

Hereditary equine regional dermal asthenia (HERDA) is a genetic disorder that occurs in the American Quarter horse (AQH) and is caused by a c.115G>A missense mutation in the peptidylprolyl isomerase B (PPIB) gene. Using a quantitative real-time PCR high resolution melting analysis genotyping assay for the PPIB mutation, the estimated HERDA allele and carrier frequencies in a sample of Brazilian AQHs were 2.9% and 5.8%, respectively.     

Detection of challenge virus in fetal tissues by nested PCR as a test of the potency of a porcine parvovirus vaccine

To estimate the potency of a porcine parvovirus (PPV) vaccine, three vaccinated and three non-vaccinated pregnant gilts were infected with PPV and the distribution of the virus was studied in the tissues of their 51 fetuses. Virus detection was attempted using haemagglutination (HA) and immunofluorescence (IF) assays, as well as by standard (single) and nested polymerase chain reactions (PCR). None of the detection methods yielded positive results when used to test for the presence of virus in suspensions of organs from the fetuses from the vaccinated gilts. However, the virus was detected in the fetuses from non-vaccinated gilts as follows: HA was positive in 14 cases out of 23 (60.8%), IF in 16/23 (69.5%), standard PCR in 12/20 (60%), and the nested PCR in 19/23 (82.6%). Although the correlation among the results of various methods of virus detection was rather close (r<0.83), the sensitivity of the nested PCR was the highest, both when testing dilutions of PPV and when analysing the fetal organs. The nested PCR therefore provides a reliable approach for studies of virus distribution in fetal organs, with special reference to potency tests on vaccines.