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Estimation of the diagnostic accuracy of the glyco-C and US9 gene-based polymerase chain reaction technique for the detection of bovine Herpesvirus type 5 DNA in decomposed brain suspension from a slaughter house using Bayesian analysis, Brazil
Abstract
Brazil represents the greatest beef producer among tropical countries, and the major obstacle for meat international trade is sanitary problems especially closely related to viral encephalitis. The goal of this study was to estimate the accuracy of the glycol and US9 gene-based polymerase chain reactions (PCRs) for the detection of bovine Herpesvirus type 5 (BoHV-5) from decomposed brain samples (n = 95). For this purpose, a latent-class (bayesian) approach was used. Sensitivity (Se) was estimated to be 70% (95% probability interval, 40-80) and specificity (Sp) 100% in the statistical analysis for both PCRs used. Accordingly, a minimum of ≥40% of the calves was estimated to harbor BoHV-5 DNA even after 72 h of decomposition at room temperature. It was concluded that US9 gene-based PCR could also be considered a cost-effective alternative in sanitary programmers. However, given the importance of veterinary diagnoses, PCR-positive samples should be further confirmed through in vitro isolation and/or sequencing.
... The 5 0 nuclease assay using TaqMan probes would also be compromised by primer dimer formation , yet these products are not detected and are not expected to affect the amplification efficiency [22]. Many studies describing the conventional PCR methodology emphasize the importance of bovine b-actin reference gene [7,17]. In our study, b-actin presented the worst performance as a control gene (Fig. 1B). ...
... In fact, the US9 gene has been proven to be useful target in brain samples and also in experimental procedures using bovine semen and oocytes/em- bryos [12e14,16]. The US9 gene was generated to amplify only BoHV-5 sequences [17] . This mean an important issue in epidemiological studies since it has been documented the interspecific recombination between BoHV-1 and 5 under in vitro assay [32]. ...
... The viral DNA detection was applied to detect the US9 gene among exposed and unexposed derived-embryos, follicular liquid, oocytes and sperm, as described previously [27]. A 250-bp amplicon was generated based on the sequence of US9 region (GenBank accession number AY064172). ...
Bovine Herpesvirus type-5 (BoHV-5) is a neurovirulent α-Herpesvirus which is potentially pathogenic for cows and suspected to be associated with reproductive disorders. Interestingly, natural transmission of BoHV-5 by contaminated semen was recently described in Australia. Additionally, BoHV-5 was also isolated from the semen of a healthy bull in the same country and incriminated in a natural outbreak of reproductive disease after artificial insemination. In contrast with BoHV-1, experimental exposure of in vitro produced bovine embryos to BoHV-5 does not affect embryo viability and seems to inhibit some pathways of apoptosis. However, the mechanisms responsible for these phenomena are poorly understood. In this study, we examined mitochondrial activity, antioxidant protection, stress response and developmental rates of in vitro produced bovine embryos that were exposed and unexposed to BoHV-5.
For this purpose, bovine embryos produced in vitro were assayed for cell markers after experimental infection of oocytes (n = 30; five repetitions), in vitro fertilization and development. The indirect immunofluorescence was employed to measure the expression of superoxide dismutase 1 (SOD1), anti-oxidant like protein 1 (AOP-1), heat shock protein 70.1 (Hsp 70.1) and also viral antigens in embryos derived from BoHV-5 exposed and unexposed oocytes. The determination of gene transcripts of mitochondrial activity (SOD1), antioxidant protection (AOP-1) and stress response (Hsp70.1) were evaluated using the reverse transcriptase polymerase chain reaction (RT-PCR). MitoTracker Green FM, JC-1 and Hoechst 33342-staining were used to evaluate mitochondrial distribution, segregation patterns and embryos morphology. The intensity of labeling was graded semi-quantitatively and embryos considered intensively marked were used for statistical analysis.
The quality of the produced embryos was not affected by exposure to BoHV-5. Of the 357 collected oocytes, 313 (+/- 6.5; 87,7%) were cleaved and 195 (+/- 3.2; 54,6%) blastocysts were produced without virus exposure. After exposure, 388 oocytes were cleaved into 328 (+/- 8.9, 84,5%), and these embryos produced 193 (+/- 3.2, 49,7%) blastocysts. Viral DNA corresponding to the US9 gene was only detected in embryos at day 7 after in vitro culture, and confirmed by indirect immunofluorescence assay (IFA). These results revealed significant differences (p < 0.05) between exposed and unexposed oocytes fertilized, as MitoTracker Green FM staining Fluorescence intensity of Jc-1 staining was significantly higher (p < 0.005) among exposed embryos (143 +/- 8.2). There was no significant difference between the ratios of Hoechst 33342-stained nuclei and total cells in good-quality blastocysts (in both the exposed and unexposed groups). Using IFA and reverse transcriptase polymerase chain reaction (RT-PCR) for the set of target transcripts (SOD1, AOP-1 and Hsp 70.1), there were differences in the mRNA and respective proteins between the control and exposed embryos. Only the exposed embryos produced anti-oxidant protein-like 1 (AOP-1). However, neither the control nor the exposed embryos produced the heat shock protein Hsp 70.1. Interestingly, both the control and the exposed embryos produced superoxide dismutase (SOD1), revealing intense mitochondrial activity.
This is the first demonstration of SOD1 and AOP-1 production in bovine embryos exposed to BoHV-5. Intense mitochondrial activity was also observed during infection, and this occurred without interfering with the quality or number of produced embryos. These findings further our understanding on the ability of α-Herpesviruses to prevent apoptosis by modulating mitochondrial pathways.
Bovine herpes virus 1 (BHV-1) is primarily associated with clinical syndromes such as rhinotracheitis, pustular vulvovaginitis and balanoposthitis, abortion, infertility, conjunctivitis and encephalitis in bovine species. The main sources of infection are the nasal exudates and the respiratory droplets, genital secretions, semen, fetal fluids and tissues. The BHV-1 virus can become latent following a primary infection with a field isolate or vaccination with an attenuated strain. The viral genomic DNA has been demonstrated in the sensory ganglia of the trigeminal nerve in infectious bovine rhinotracheitis (IBR) and in sacral spinal ganglia in pustular vulvovaginitis and balanoposthitis cases. BHV-1 infections can be diagnosed by detection of virus or virus components and antibody by serological tests or by detection of genomic DNA by polymerase chain reaction (PCR), nucleic acid hybridization and sequencing. Inactivated vaccines and modified live virus vaccines are used for prevention of BHV-1 infections in cattle; subunit vaccines and marker vaccines are under investigation.
The authors consider screening populations with two screening tests but where a definitive "gold standard" is not readily available. They discuss a recent article in which a Bayesian approach to this problem is developed based on data that are sampled from a single population. It was subsequently pointed out that such inferences will not necessarily be accurate in the sense that standard errors for parameters may not decrease as n increases. This problem will generally occur when the data are insufficient to estimate all of the parameters as is the case when screening a single population with two tests. If both tests are applied to units sampled from two populations, however, this particular difficulty disappears. In this article the authors further examine this issue and develop an approach based on sampling two populations that yields increasingly accurate inferences as the sample size increases.
A multiplex polymerase chain reaction (multiplex-PCR) to detect and differentiate bovine herpesvirus 1 (BoHV-1) and 5 (BoHV-5) was developed using primers for the gene sequence that encodes the glycoprotein C. The technique was assessed against the BoHV-1 and BoHV-5 cell culture adapted strains, and clinical samples collected from animals with clinical signs of BoHV-1 (n = 10) or BoHV-5 (n = 7) infection and with diagnosis confirmed by virus isolation in cell culture and semi-nested PCR. Fifteen clinical samples from asymptomatic animals were included as control group. For the evaluation of the amplifiability of the extracted nucleic acid from clinical specimens was included a bovine internal control that amplified a 626 bp fragment of the ND5 gene present in the bovine mitochondrial DNA. For DNA extraction, a combination of the phenol/chloroform/isoamyl alcohol and silica/guanidine isothiocyanate methods was used. The specificity of the BoHV-1 and BoHV-5 amplicons from standard strains were confirmed by sequence analysis. All the positive clinical samples for BoHV included in this study were characterized as BoHV-1 or BoHV-5 by the difference in length of the amplified product visualized in a agarose gel (354 bp size for BoHV-1, and 159 bp for BoHV-5). The internal control was amplified in all clinical specimens. Non-specific reactions were not observed when the multiplex-PCR was assessed with other viruses (bovine viral diarrhea virus and rabies virus) and BoHV-negative clinical samples from fetuses and adult cattle obtained from a slaughterhouse.
The detection and growth of bovine herpes virus type 5 (BoHV-5) was evaluated in three different media without foetal bovine serum or animal protein by infecting three different cell lines. The OPTI-PRO, VP-SFM and RPMI 1640 media were supplemented by L-glutamine, antibiotics added by 5µg of insulin-like growth factor (IGF-I) and used to adapt CER, MDBK and CRIB cells in a statistic continuous culture system. The results obtained by CRIB and MDBK cells adaptation showed steady growth after 10 th passages in OPTI-PRO medium, 20 th passages in VP-SFM medium and 30 th passages in RPMI 1640 medium, respectively. The OPTI-PRO and RPMI 1640 media were not able to support CER growth, even supplemented by IGF-1 and present apoptotic cells at 72h post-infection. The CER cells seeded after adaptation by 10 th passages in VP-SFM medium added by 5µg/20ml of IGF-1 growth factor revealed the best virus titres compared to MDBK and CRIB cell lines. It was concluded that CER, MDBK and CRIB cells infected by BoHV-5 serotype could be used for laboratory diagnosis propagated on much safer culture system of high biotechnology advances.
The detection and growth of bovine herpes virus type 5 (BoHV-5) was evaluated in three different media without foetal bovine serum or animal protein by infecting three different cell lines. The OPTI-PRO, VP-SFM and RPMI 1640 media were supplemented by L-glutamine, antibiotics added by 5µg of insulin-like growth factor (IGF-I) and used to adapt CER, MDBK and CRIB cells in a statistic continuous culture system. The results obtained by CRIB and MDBK cells adaptation showed steady growth after 10th passages in OPTI-PRO medium, 20th passages in VP-SFM medium and 30th passages in RPMI 1640 medium, respectively. The OPTI-PRO and RPMI 1640 media were not able to support CER growth, even supplemented by IGF-1 and present apoptotic cells at 72h post-infection. The CER cells seeded after adaptation by 10th passages in VP-SFM medium added by 5g/20ml of IGF-1 growth factor revealed the best virus titres compared to MDBK and CRIB cell lines. It was concluded that CER, MDBK and CRIB cells infected by BoHV-5 serotype could be used for laboratory diagnosis propagated on much safer culture system of high biotechnology advances.
It is common in population screening surveys or in the investigation of new diagnostic tests to have results from one or more tests investigating the same condition or disease, none of which can be considered a gold standard. For example, two methods often used in population-based surveys for estimating the prevalence of a parasitic or other infection are stool examinations and serologic testing. However, it is known that results from stool examinations generally underestimate the prevalence, while serology generally results in overestimation. Using a Bayesian approach, simultaneous inferences about the population prevalence and the sensitivity, specificity, and positive and negative predictive values of each diagnostic test are possible. The methods presented here can be applied to each test separately or to two or more tests combined. Marginal posterior densities of all parameters are estimated using the Gibbs sampler. The techniques are applied to the estimation of the prevalence of Strongyloides infection and to the investigation of the diagnostic test properties of stool examinations and serologic testing, using data from a survey of all Cambodian refugees who arrived in Montreal, Canada, during an 8-month period.
Factors that inhibit the amplification of nucleic acids by PCR are present with target DNAs from many sources. The inhib- itors generally act at one or more of three essential points in the reaction in the following ways: they interfere with the cell lysis necessary for extraction of DNA, they interfere by nucleic acid degradation or capture, and they inhibit polymerase ac- tivity for amplification of target DNA. Although a wide range of inhibitors is reported, the identities and modes of action of many remain unclear. These effects may have important im- plications for clinical and public health investigations, espe- cially if the investigations involve food and environmental screening. Common inhibitors include various components of body fluids and reagents encountered in clinical and forensic science (e.g., hemoglobin, urea, and heparin), food constitu- ents (e.g., organic and phenolic compounds, glycogen, fats, and Ca21), and environmental compounds (e.g., phenolic com- pounds, humic acids, and heavy metals). Other, more wide- spread inhibitors include constituents of bacterial cells, non- target DNA and contaminants, and laboratory items such as pollen, glove powder, laboratory plasticware, and cellulose. This review discusses the findings of many studies related to clinical, food, and environmental microbiology, including ap- proaches that have been used to overcome inhibition and fa- cilitate amplification for detection and typing. Few areas of biological science remain untouched by the invention of PCR (34, 81, 99). Other methods for amplifying nucleic acids (72, 123), such as Qb replicase (18), ligase chain reaction (13, 128), single-stranded sequence replication (17, 47), strand displacement amplification (126, 127), and nucleic acid sequence-based amplification (23, 122), have been de- scribed, but these methods have received less attention. Problems sometimes occur with PCR, however (124). De- spite early indications of great sensitivity, the sensitivity of PCR may be a negative aspect of the procedure, since the most commonly reported problem is false-positive results due to cross-contamination (98, 124). This problem can be overcome by UV irradiation (100), with sodium hypochlorite (92), and by photochemical or enzymic methods (25, 36, 40, 78). One problem that is less discussed is reaction inhibition. This may be total or partial and can manifest itself as complete reaction failure or as reduced sensitivity of detection. In some cases, inhibition may be the cause of false-negative reactions, since few workers incorporate internal controls in each reac- tion tube. Early evidence of exquisite sensitivity with mamma- lian cells (53) involving detection of a single molecule of DNA from a hair was not reproduced when PCR was applied to many microbial (and some mammalian) situations, where poor sensitivity, specificity, and reproducibility have been reported (16, 82, 86, 129, 132, 134). There may also be potentially important effects in PCR typing reactions (121), and difficulties can occur in post-PCR manipulation (61). Although systematic study of inhibition has seldom been the focus of published investigations, many workers have reported these effects in the course of other studies (12, 19, 21, 124, 129, 132, 133). Con- sidering the prevalence of this problem, it is surprising that few systematic and mechanistic studies of PCR inhibition have been reported. Rossen et al. (97) contributed the most com- prehensive study of PCR inhibition, identifying inhibitory fac- tors in foods, bacterial culture media, and various chemical compounds. These inhibitory factors included organic and in- organic chemicals, detergents, antibiotics, buffers, enzymes, polysaccharides, fats, and proteins. This review lists and discusses inhibitors and methods that can overcome the attenuation of amplification in clinical, food, and environmental microbiology. It is beyond the scope of this paper to discuss in detail the various physical, enzymic, and chemical methods used in the extraction, purification, and quantitation of nucleic acids. Those methods are presented and discussed in commercial literature and elsewhere (14, 95, 96, 106, 134).
The performance of a new diagnostic test is frequently evaluated by comparison to a perfect reference test (i.e. a gold standard). In many instances, however, a reference test is less than perfect. In this paper, we review methods for estimation of the accuracy of a diagnostic test when an imperfect reference test with known classification errors is available. Furthermore, we focus our presentation on available methods of estimation of test characteristics when the sensitivity and specificity of both tests are unknown. We present some of the available statistical methods for estimation of the accuracy of diagnostic tests when a reference test does not exist (including maximum likelihood estimation and Bayesian inference). We illustrate the application of the described methods using data from an evaluation of a nested polymerase chain reaction and microscopic examination of kidney imprints for detection of Nucleospora salmonis in rainbow trout.
In this review, we critically discuss the objectives, methods and limitations of different approaches for the validation of diagnostic tests. We show (based on published data and our own experiences) that estimates for the diagnostic sensitivity and specificity may vary among populations and/or subpopulations of animals, conditional on the distribution of influential covariates. Additional variability in those parameter estimates may be attributable to the sampling strategy. The uncertainty about diagnostic parameters is of concern for the decision-maker in the context of clinical diagnosis or quantitative risk assessment as well as for the epidemiologist who uses test data for prevalence estimation or risk-factor studies. Examples for the calculation of diagnostic parameters are presented together with bias-avoidance strategies. We suggest guidelines for an epidemiologic approach to test validation of veterinary diagnostic tests.
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.
This scientific review was prompted by recent legislation to curtail the use of semen from potentially virus-infected bulls to produce embryos for import into the European Union. From studies in laboratory animals, humans and horses, it is apparent that viruses may sometimes attach to, or be integrated into, spermatozoa, although in domestic livestock, including cattle, this seems to be a rare phenomenon, and carriage of virus through the zona pellucida into the oocyte by fertilising sperm has never been described in these species. Four specific viruses; enzootic bovine leukosis (EBLV), bovine herpesvirus-1 (BoHV-1), bovine viral diarrhoea virus (BVDV) and bluetongue virus (BTV), all of which tend to cause subclinical infections in cattle, but which can occur in bovine semen, are examined with regard to the risks that use of infected semen might lead to production of infected embryos. With regard to in vivo-derived embryos, when internationally approved embryo processing protocols are used, the risks from EBLV- and BTV-infected semen are negligible, and the same is almost certainly true for semen infected with BoHV-1 if the embryos are also treated with trypsin. For BVDV, there is insufficient data on how the virus is carried in semen and how different BVDV strains can interact with sperm, oocytes and embryos. There is a potential, at least, that in vivo-derived embryos resulting from infected semen might carry BVDV, although field studies so far suggest that this is very unlikely. With regard to in vitro-produced embryos, use of semen infected with any of the four viruses, with the probable exception of EBLV, will often lead to contaminated embryos, and virus removal from these embryos is difficult even when the internationally approved embryo processing protocols are used. However, it has never been demonstrated that such embryos have resulted in transmission of infection to recipients or offspring.
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