IBR_OIE鉴定标准

NB: Version adopted by the World Assembly of Delegates of the OIE in May 2010

C H A P T E R2.4.13.

I N F E C T I O U S B O V I N E R H I N O T R A C H E I T I S/

I N F E C T I O U S P U S T U L A R V U L V O V A G I N I T I S

SUMMARY

Definition of the disease: Infectious bovine rhinotracheitis/infectious pustular vulvovaginitis (IBR/IPV), caused by bovine herpesvirus 1 (BoHV-1), is a disease of domestic and wild cattle. The virus is distributed world-wide, but has been eradicated from Austria, Denmark, Finland, Sweden, Italy (Province of Bolzano), Switzerland, Norway and parts of Germany (the ‘Oberfranken’ and ‘Oberpfalz’ districts of Bavaria). Control programmes are running in several other countries, for example in Germany and Italy.

Description of disease: The disease is characterised by clinical signs of the upper respiratory tract, such as a (muco)purulent nasal discharge, hyperaemia of the muzzle (red nose disease) and by conjunctivitis. Signs of general illness are fever, depression, inappetence, abortions and reduced milk yield. The virus can also infect the genital tract and cause pustular vulvovaginitis and balanoposthitis. Post-mortem examinations reveal rhinitis, laryngitis and tracheitis. Mortality is low, and most infections run a subclinical course. Secondary bacterial infections can lead to more severe respiratory disease, and BoHV-1 could play a role in multifactor diseases such as ‘shipping fever’.

Identification of the agent: The virus can be isolated from nasal or genital swabs from animals with respiratory signs, vulvovaginitis or balanoposthitis, taken during the acute phase of the infection, and, in severe cases, from various organs collected at post-mortem. Following infection, BoHV-1 may persist in infected animals in a latent state in sensory neurons, e.g. in the trigeminal or sacral ganglia. The virus can be reactivated and this results in virus shedding (re-excretion) without exhibition of clinical disease. Therefore, antibody-positive animals have to be classified as infected with BoHV-1 (with two exceptions: serological responses induced by vaccination with an inactivated vaccine or by colostral antibodies).

For virus isolation, various cell cultures of bovine origin are used, for example, secondary lung or kidney cells or the Madin–Darby bovine kidney cell line (MDBK). The virus produces a cytopathic effect in 2–4 days. It is identified by neutralisation or antigen detection methods using monospecific antisera or monoclonal antibodies. BoHV-1 isolates can be further subtyped by DNA restriction enzyme analysis (RFLP) into subtypes 1.1 and 1.2. BoHV-1.2 isolates can be further differentiated into 2a and 2b. Development of rhinotracheitis or vulvovaginitis/balanoposthitis depends more on the route of infection than on the subtype of the virus. The virus previously referred to as BoHV-1.3,

a neuropathogenic agent, is now classified as BoHV-5.

Viral DNA detection methods have been developed, and the polymerase chain reaction technique is increasingly used in routine diagnosis including real-time polymerase chain reaction (PCR).

Serological tests: The virus neutralisation test and various enzyme-linked immunosorbent assays (ELISA; indirect or gB-blocking) are most widely used for antibody detection. With the ELISAs, antibodies can be detected in serum or plasma, and with lower sensitivity in milk or bulk milk samples.

Requirements for vaccines and diagnostic biologicals: Inactivated and attenuated live vaccines are available. The vaccines protect cattle clinically in case of infection and markedly reduce the subsequent shedding of field virus. Although vaccination may not prevent field virus infection of individual animals, spreading of wild-type virus in infected herds is efficiently reduced. The vaccines must not induce disease, abortion, or any local or systemic reaction, and must be genetically stable.

BoHV-1 glycoprotein E deleted mutant marker vaccines are now generally available (live or

inactivated). The use of a gE-antibody-ELISA makes it possible to distinguish field virus infected

cattle from cattle vaccinated with such a marker vaccine (DIVA strategy).

A. INTRODUCTION

Infectious bovine rhinotracheitis/infectious pustular vulvovaginitis (IBR/IPV), caused by bovine herpesvirus 1 (BoHV-1), is a disease of domestic and wild cattle. BoHV-1 is a member of the genus Varicellovirus in the subfamily Alphaherpesvirinae, which belongs to the Herpesviridae family, order Herpesvirales. The viral genome consists of double-stranded DNA that encodes for about 70 proteins, of which 33 structural and more than 15 nonstructural proteins have been identified. The viral glycoproteins, which are located in the envelope on the surface of the virion, play an important role in pathogenesis and immunity. BoHV-1 can be differentiated into subtypes 1.1, 1.2a and 1.2b (Metzler et al., 1985). The BoHV-1.2 subtypes may be less virulent than subtype 1.1 (Edwards et al., 1990). The former BoHV-1.3, which may act as a neuropathogenic agent in calves, has been re-classified as BoHV-5 (Magyar et al., 1993). BoHV-1 shares antigenic and genetic close relationships with other ruminant alphaherpesviruses: BoHV-5, caprine herpesvirus 1, cervid herpesvirus 1 (red deer), cervid herpesvirus 2 (reindeer), bubaline herpesvirus 1 and elk herpesvirus 1 (Thiry et al., 2006).

After an incubation period of 2–4 days, serous nasal discharge, salivation, fever, inappetence, and depression become evident. Within a few days the nasal and ocular discharges change to mucopurulent. Where natural mating is practised, genital infection can lead to pustular vulvovaginitis or balanoposthitis. However, most infections run a very mild or subclinical course (Van Oirschot et al., 1993). Uncomplicated cases of respiratory or genital disease caused by BoHV-1 last about 5–10 days. Secondary bacterial or viral agents may contribute to a multifactor disease complex resulting in severe respiratory disease of young animals (‘shipping’ or ‘crowding fever’).

After infection via the airborne route, BoHV-1 replicates to high titres in mucous membranes of the upper respiratory tract and in the tonsils. Subsequently, the virus disseminates to conjunctivae and reaches the trigeminal ganglia by neuronal axonal transport. After genital infection, BoHV-1 replicates in the mucous membranes of the vagina or prepuce, and becomes latent in the sacral ganglia. The viral DNA remains in the neurons of the ganglia, probably for the entire life of the host (status of latency). Stress, such as transport and parturition, but also the application of corticosteroids can induce reactivation of the latent infection. Consequently, the virus may switch between latent and lytic infection and may be shed intermittently into the environment and spread to contact animals.

BoHV-1 infection elicits an antibody response and a cell-mediated immune response within 7–14 days. The immune response is presumed to persist life-long, although it may fall below the detection limit of some tests after a number of years. Maternal antibodies are transferred via colostrum to the young calf, which is consequently protected against BoHV-1-induced clinical disease (Mechor et al., 1987). Maternal antibodies have a biological half-life of about 3 weeks, but may be detected occasionally in animals up to 9 months old, and rarely in animals over this age.

The virus is distributed world-wide, with the exception of the BoHV-1-free countries, paralleling the distribution of domestic cattle. Other Artiodactyla (e.g. goats, sheep, water buffaloes, camelids) may be infected with BoHV-1. After infection, nasal viral shedding is detected for 5–14 days, with peak titres of 108–1010 TCID50 (50% tissue culture infective doses) per ml of nasal secretion. The semen of an infected bull may contain BoHV-1, and the virus can thus be transmitted by natural mating and artificial insemination (Parsonson & Snowdon, 1975). Prevention and control of BoHV-1 infections are based on thorough farm management including hygienic measures, vaccination schedules and removal of infected animals. Ideally, a 4-week quarantine period is imposed for newly introduced cattle, if the cattle are not from certified BoHV-1-free farms. Only cattle that are BoHV-1-seronegative are then admitted to a free herd. Natural mating should be avoided and only semen from BoHV-1-negative bulls should be used.

Vaccines usually prevent the development of clinical signs and markedly reduce the shedding of virus after infection, but do not completely prevent infection. Several eradication campaigns have been carried out or are currently running in different countries including test-and-removal programmes and/or vaccination campaigns (see Section C).

BoHV-1 infection may be suspected on the basis of clinical, pathological and epidemiological findings. However, to make a definite diagnosis, laboratory examinations (serology and/or virus detection) are required. A complete diagnostic procedure in the laboratory is aimed at detecting the causative virus (or viral components) and the specific antibodies they induce. Nevertheless, because of latent infection induced by BoHV-1, detection of antibodies could be sufficient for the determination of the BoHV-1 status of individual animals.

B. DIAGNOSTIC TECHNIQUES

1. Identification of the agent

a) Collection and processing of samples

Nasal swabs are collected from several (from five to ten) affected cattle in the early phase of the infection.

These cattle still have serous rather than mucopurulent nasal discharge. In cases of vulvovaginitis or balanoposthitis, swabs are taken from the genitals. The swabs should be vigorously rubbed against the mucosal surfaces. The prepuce can also be washed with saline; the washing fluid is then collected. The specimens are suspended in transport medium (cell culture medium containing antibiotics and 2–10% BoHV-1-free fetal bovine serum to protect the virus from inactivation), cooled at 4°C, and rapidly submitted to the laboratory.

During necropsy, mucous membranes of the respiratory tract, and samples of the tonsil, lung and bronchial lymph nodes are collected for virus detection. In cases of abortion, the fetal liver, lung, spleen, kidney and placental cotyledons are examined. Samples should be kept on ice and sent to the laboratory as quickly as possible.

After arrival at the laboratory, swabs are agitated at room temperature for 30 minutes in the transport medium to elute virus. Following removal of the swabs, the transport medium is clarified by centrifugation at 1500 g for 10 minutes. Tissues are homogenised to a 10–20% (w/v) suspension in cell culture medium before centrifugation at 1500 g for 10 minutes. The supernatants of these specimens are filtered through

0.45 μm filters and used for virus isolation.

The isolation of virus from semen needs some special adaptations, because the seminal fluid contains enzymes and other factors that are toxic to the cells and inhibit viral replication (see below).

b) Virus isolation

For virus isolation, bovine cells of various origins can be used. Primary or secondary bovine kidney, lung or testis cells, cell strains derived from bovine fetal lung, turbinate or trachea, and established cell lines, such as the Madin–Darby bovine kidney cell line (MDBK), are suitable for BoHV-1 propagation. Cell cultures can be grown in glass or plastic tubes, plates or dishes. When 24-well plastic plates are used, a 100–200 μl volume of the supernatants described above is inoculated into these cell cultures. After a 1-hour adsorption period, the cultures are rinsed and maintenance medium is added. The serum used as a medium supplement in the maintenance medium should be free of antibodies against BoHV-1. The cell cultures are observed daily for CPE, which usually appears within 3 days after inoculation. It is characterised by grape-like clusters of rounded cells gathered around a hole in the monolayer; sometimes giant cells with several nuclei may be observed. Experience is needed to recognise this characteristic appearance. When, after

7 days, no CPE has appeared, a blind passage must be made. The cell culture is freeze–thawed and

clarified by centrifugation, and the supernatant is used for inoculation of fresh monolayers (Brunner et al., 1988; Edwards et al., 1983).

To identify the recovered virus as BoHV-1, the supernatant of the culture should be neutralised with a monospecific BoHV-1 antiserum or neutralising monoclonal antibody (MAb). For this purpose, serial tenfold dilutions of the test supernatant are made, and to each dilution monospecific BoHV-1 antiserum or negative control serum is added. Following incubation at 37°C for 1 hour, the mixtures are inoculated into cell cultures; 3–5 days later, the neutralisation index is calculated. The neutralisation index is the virus titre (in log10) in the presence of negative control serum minus the virus titre in the presence of specific antiserum. If the neutralisation index is greater than 1.5, the isolate may be considered to be BoHV-1. To shorten the virus isolation procedure, two specimens may be inoculated into cell culture: one that has been pre-incubated with monospecific antiserum and another that has been preincubated with negative control serum.

If the CPE is inhibited by the monospecific antiserum, the isolate can be considered to be BoHV-1, although definitive confirmation would require molecular characterisation to distinguish it from related ruminant alphaherpesviruses.

An alternative method of virus identification is the direct verification of BoHV-1 antigen in cells around the CPE by an immunofluorescence or immunoperoxidase test (Kaashoek et al., 1994) with conjugated monospecific antiserum or MAb. Furthermore, the supernatant can be used as template for restriction endonuclease fragment length polymorphism (RFLP) (see Section B.1.e) and polymerase chain reaction (PCR) (see Section B.1.c) analyses.

? Virus isolation from semen (a prescribed test for international trade)

0.05 to 0.1 ml of raw semen should be tested with two passages in cell culture. Raw semen is generally

cytotoxic and should be prediluted (e.g. 1/10) before being added to cell cultures. A similar problem may

sometimes arise with extended semen. For extended semen, an approximation should be made to ensure that the equivalent of a minimum of 0.1 ml raw semen is examined (e.g. a minimum of 0.5 ml extended serum). Multiple diluted samples may need to be tested with this procedure to reach a volume equivalent to

0.1 ml raw semen (e.g. 5 × 1 ml of a 1/10 diluted sample of extended semen). A suitable test procedure is

given below. See also Brunner et al. (1988).

procedure

? Test

i) Dilute 200 μl fresh semen in 2 ml fetal bovine serum (free from antibodies against BoHV-1) with

antibiotics.

ii) Mix vigorously and leave for 30 minutes at room temperature.

iii) Inoculate 1 ml of the semen/serum mixture into a monolayer of susceptible cells (see virus isolation above) in a six-well tissue culture plate.

iv) Incubate the plates for 1 hour at 37°C.

v) Remove the mixture, wash the monolayer twice with 5 ml maintenance medium, and add 5 ml maintenance medium to each well.

vi) Include BoHV-1 negative and positive controls in the test. Special caution must be taken to avoid accidental contamination of test wells by the positive control, for example always handling the control last, and using separate plates.

vii) Observe plates under a microscope daily for the appearance of a CPE. If a CPE appears, confirmatory tests for BoHV-1 are made by specific neutralisation or immunolabelling methods (see above).

viii) If there is no CPE after 7 days, the cultures are frozen and thawed, clarified by centrifugation, and the supernatant is used to inoculate fresh monolayers.

ix) The sample is considered to be negative, if there is no evidence of a CPE after 7 days’ incubation of the passaged cultures.

c) Nucleic acid detection

During the past decade, various methods for the detection of BoHV-1 DNA in clinical samples have been described, including DNA–DNA hybridisation and the PCR. The PCR is also increasingly used in routine diagnostic submissions (Moore et al., 2000). Compared with virus isolation, the PCR has the primary advantages of being more sensitive and more rapid: it can be performed in 1–2 days. It is also possible to detect episomal DNA of non-replicating virus in sensory ganglia (Van Engelenburg et al., 1993), such as the trigeminal ganglion, in the latent phase of infection. The disadvantage is that PCR analyses are prone to contamination and therefore precautions have to be taken to prevent false-positive results. Risk of contamination is markedly reduced by new PCR techniques, such as real-time or quantitative PCR (see below) (Abril et al., 2004; Lovato et al., 2003).

So far PCR has been used mainly to detect BoHV-1 DNA in artificially (Kramps et al., 1993) or naturally (Van Engelenburg et al., 1993) infected semen samples. It is important to thoroughly optimise the PCR conditions, including the preparation of the samples, the concentration of Mg2+, primers and polymerase, and the cycle programmes. The target region for amplification must be present in all BoHV-1 strains, and its nucleotide sequence must be conserved. The TK, gB, gC, gD and gE genes have been used as targets for PCR amplification. In addition, PCRs based on detection of gE sequences can be used to differentiate between wild-type virus and gE-deleted vaccine strains (Fuchs et al., 1999; Schynts et al., 1999). Discrimination between infection with virulent IBR strains and infection with live attenuated strains is not possible with the PCR technique, and RFLP is used for this purpose. Specific PCRs have been developed that are able to discriminate between BoHV-1, BoHV-5 and other related alphaherpesviruses (Ashbaugh et al., 1997; Ros et al., 1999).

Experimentally, PCR was found to be more sensitive than virus isolation: in egg yolk-extended semen samples obtained from experimentally infected bulls, PCR detected five times as many positives as did virus isolation (Van Engelenburg et al., 1995). The detection limit of validated PCR assays amounts to only a few genome copies per PCR reaction. Nevertheless, false-negative results cannot be excluded. To identify possible false-negative results, it is recommended to spike an internal control template into the reaction tube of the semen sample to be amplified by the same primers. Such a control template may be constructed by inserting, for example, a 100 base-pair fragment into the target region. This control template also makes it possible to semi-quantify the amount of DNA that is detected (Ros et al., 1999; Van Engelenburg et al., 1993). When using an internal control, extensive testing is necessary to ensure that PCR amplification of the added internal control does not compete with the diagnostic PCR and thus lower the analytical sensitivity (see also Chapter 1.1.5 Validation and quality control of PCR methods used for the diagnosis of infectious diseases). DNA extraction and quality of the DNA preparations can also be controlled by amplification of cellular sequences (housekeeping genes) or by addition of ‘artificial’ DNA sequences prior to extraction

procedures (e.g. green fluorescent protein, non-BoHV-related viruses) as internal controls. To enhance the sensitivity and specificity of the BoHV-1 PCR, real-time PCR systems are the methods of choice.

? Real-time polymerase chain reaction (a prescribed test for international trade)

The following real-time PCR test method has been developed to detect BoHV-1 in extended bovine semen destined for trade. The method has been validated according to Chapter 1.1.5, and includes a comprehensive international inter-laboratory comparison involving six collaborating laboratories with specialist status in IBR testing (Wang et al., 2008).

A number of studies have shown that PCR assays are more sensitive than virus isolation (Smits et al., 2000;

Van Engelenburg et al., 1995; Vilcek et al., 1994; Wang et al., 2008; Wiedmann et al., 1993). Real-time PCR has been used for the detection of BoHV-1 and BoHV-5 in experimentally infected cattle and mice (Abril et al., 2004; Lovato et al., 2003) and a series of conventional PCR assays have been used for the detection of BoHV-1 DNA in artificially or naturally infected bovine semen samples (Deka et al., 2005; Grom et al., 2006;

Masri et al., 1996; Van Engelenburg et al., 1993; Weiblen et al., 1992; Wiedmann et al., 1993; Xia et al., 1995). Conventional detection of amplified PCR products relies on gel electrophoresis analysis (Rola et al., 2003). Sequence-specific primers have been selected to amplify different parts of conserved glycoprotein genes of the BoHV-1 genome, including glycoprotein B (gB) gene (Grom et al., 2006; Santurde et al., 1996), gC gene (Smits et al., 2000; Van Engelenburg et al., 1995), gD gene (Smits et al., 2000; Wiedmann et al., 1993), gE gene (Grom et al., 2006), and the thymidine kinase (tk) gene (Moore et al., 2000; Yason et al., 1995).

Real-time PCR differs from standard PCR in that the amplified PCR products are detected directly during the amplification cycle using a hybridisation probe, which enhances assay specificity. Real-time PCR assays have several advantages over the conventional PCR methods. Real-time PCR assays are able to provide sensitivity close or equal to nested PCR methods with a much lower risk of contamination. The amplification and detection of target is conducted simultaneously, and tubes have not to be opened for product analysis on agarose gels. There is no post-amplification PCR product handling, which significantly reduces the risk of contamination, and it is possible to perform quantitative analysis.

The real-time PCR described here uses a pair of sequence-specific primers for amplification of target DNA and a 5’-nuclease oligoprobe (TaqMan) for detection of amplified products. The oligoprobe is a single, sequence-specific oligonucleotide, labelled with two different fluorophores, the reporter/donor, 5-carboxyfluorescein (FAM) at the 5’ end, and the acceptor/quencher 6-carboxytetramethylrhodamine (TAMRA) at the 3’ end. This real-time PCR assay is designed to detect viral DNA of all BoHV-1 strains, including subtype 1 and 2, from extended bovine semen. The assay selectively amplifies a 97 basepair sequence of the glycoprotein B (gB) gene. Details of the primers and probes are given in the protocol outlined below.

? Sample preparation, equipment and reagents

i) The samples used for the test are, typically, extended bovine semen stored in liquid nitrogen. The

semen samples can be transported to the laboratory in liquid nitrogen, or shipped at 4°C, and stored in

liquid nitrogen or at –70°C (for long-term storage) or 4°C (for short-term storage). Storing semen at 4°C

for a short period (up to 7 days) does not affect PCR test result.

ii) Three straws from each batch of semen should be processed. Duplicate PCR amplifications should be carried out for each DNA preparation (six amplifications in total) to ensure the detection of DNA in samples containing low levels of virus.

iii) A real-time PCR detection system, and the associated data analysis software, is required to perform the assay. A number of real-time PCR detection systems are available from various manufacturers. In

the procedure described below, a RotorGene 3000, Corbett Research Ltd, Australia, was used. Other

real-time PCR detection systems can also be applied. Other equipment required for the test includes a

micro-centrifuge, a heating block, a boiling water bath, a micro-vortex, magnetic stirrer and micropipettes. Real-time PCR assays are able to detect very small amounts of target nucleic acid molecules therefore appropriate measures are required to avoid contamination1. Furthermore, a minimum of one negative sample should be processed in parallel to estimate the risk of low level contamination.

iv) The real-time PCR assay described here involves two separate procedures. Firstly, BoHV-1 DNA is extracted from semen using Chelex-100 chelating resin, along with proteinase K and DL-Dithiothreitol

(DTT). The second procedure is the PCR analysis of the extracted DNA template in a real-time PCR 1 Sources of contamination may include product carry-over from positive samples or, more commonly, from cross-

contamination by PCR products from earlier experiments. Samples and reagents should be handled in separate areas, with separate equipment for reagent and sample preparation and amplification/detection.

reaction mixture: Platinum Quantitative PCR SuperMix-UDG, Invitrogen Technologies (note that there

are a number of other commercial real-time PCR amplification kits available from various sources and

the particular kits selected need to be compatible with the real-time PCR platform selected). The

required primers and probes can be synthesised by various commercial companies. In this protocol, all

the primers and probes used were supplied by Sigma-Genosys.

? Extraction of DNA

i) In a screw top 1.5 ml tube, add:

Chelex 100 sodium (Sigma) (10% w/v in distilled deionised water) 100 μl.

Proteinase K (10 mg/ml, Sigma) 11.5 μl

DL-Dithiothreitol (1 M, Sigma) 7.5 μl

Nuclease-free water 90 μl

Semen sample 10 μl

Mix gently by pipetting2.

ii) The samples are incubated at 56°C for 30 minutes and then vortexed at high speed for 10 seconds.

iii) Subsequently, the tubes are incubated in a boiling water bath for 8 minutes and then vortexed at high speed for 10 seconds.

iv) The tubes are centrifuged at 10,000 g for 3 minutes.

v) The

supernatant3 is transferred into a new microtube and can be used directly for PCR, or stored at –20°C.

? Preparation of reagents

The real-time PCR reaction mixture (Platinum Quantitative PCR SuperMix-UDG, or other reaction mixture) is normally provided as a 2 × concentration ready for use. The manufacturer’s instructions should be followed for application and storage.

Working stock solutions for primers and probe are made with nuclease-free water at the concentration of

4.5 μM and 3 μM, respectively. The stock solutions are stored at –20°C and the probe solution should be

kept in the dark. Single-use aliquots can be prepared to limit freeze-thawing of primers and probes and extend their shelf life.

? Real-time PCR test procedure

i) Primers and probe sequences

Selection of the primers and probe are outlined in Abril et al. (2004) and described below.

Primer gB-F: 5’-TGT-GGA-CCT-AAA-CCT-CAC-GGT-3’ (position 57499–57519 GenBank?, accession

AJ004801)

Primer gB-R: 5’-GTA-GTC-GAG-CAG-ACC-CGT-GTC-3’ (position 57595–57575 GenBank?, accession

AJ004801)

TaqMan Probe: 5’-FAM-AGG-ACC-GCG-AGT-TCT-TGC-CGC-TAMRA-3’ (position 57525–57545 GenBank?, accession AJ004801)

reaction mixtures

of

ii) Preparation

The PCR reaction mixtures are prepared in a separate laboratory room. For each PCR test, appropriate

controls should be included. As a minimum, a no template control (NTC, reagents only), appropriate

negative controls, i.e. 1 per 10 test samples, and two positive controls (moderate and weak positive)

should be included. Each test sample and control is tested in duplicate. The PCR amplifications are

carried out in a volume of 25 μl.

a) PCR reagent mixtures are added in a clean room (no viral cultures, DNA extracts or post-

amplification products should be handled here)

2 × Platinum Quantitative PCR SuperMix-UDG 12.5 μl

ROX reference dye (optional) 0.5 μl

Forward primer (gB-F, 4.5 μM) 1 μl

Reverse primer (gB-R, 4.5 μM) 1 μl

Probe (3 μM) 1 μl

Nuclease free water 4 μl

2 It is important that the Chelex 100 solution is homogeneous while pipetting, as Chelex 100 sodium is not soluble. This can

be achieved by putting the vessel containing Chelex-100 solution on a magnetic stirrer while pipetting.

3 Some DNA samples can become cloudy and a thin white membrane may form occasionally after freezing and thawing.

This appears to have no influence on the PCR performance. No heating or re-centrifuging of the samples is necessary.

b) 5 μl of the DNA template are added to the PCR reagent mixture to a final volume of 25 μl. DNA

samples are prepared and added to the PCR mix in a separate room.

iii) Real-time (TaqMan) polymerase chain reaction

The PCR tubes are placed in the real-time PCR detection system in a separate, designated PCR room.

The PCR detection system is programmed for the test as follows:

PCR Reaction Parameters4

One cycle: Hold 50°C 2 minutes

One cycle: Hold 95°C 2 minutes5

45 cycles: Hold 95°C 15 seconds

Hold 60°C 45 seconds

iv) Analysis of real-time PCR data

The threshold level is usually set according to the manufacturers instructions for the selected analysis

software used. Alternatively, virus isolation negative semen samples, from sero-negative animals, can

be run exhaustively (e.g. up to 60 amplification cycles) to determine the background reaction associated with the detection system used.

? Interpretation of results

? Test

controls

Positive and negative controls, as well as reagent controls, should be included in each PCR test. Negative semen, from sero-negative bulls, can be used as a negative control. Positive semen from naturally infected bulls is preferable as a positive control. However, this might be difficult to obtain. Alternatively, positive controls can be derived from negative semen spiked with defined quantities of BoHV-1 virus.

results

? Test

Positive result: Any sample that has a cycle threshold (Ct) value equal or less than 45 is regarded as positive. The positive control should have a Ct value within an acceptable range (± 3 Ct values) as previously determined by repeatability testing. To minimise the risk of contamination by the positive control, a dilution resulting in a Ct value of about 30 to 33 should be used.

Negative result: Any sample that shows no Ct value is regarded as negative. Negative control and no template control should have no Ct values.

d) Viral antigen detection

Nasal, ocular or genital swabs can be directly smeared onto glass cover-slips, or, following centrifugation, the cell deposit (see Section B.1.a) may be spotted onto cover-slips. These cover-slips are subjected to a standard direct or indirect fluorescent antibody test. In a direct immunofluorescence test, the monospecific antiserum is conjugated to a fluorescent dye e.g. fluorescein isothiocyanate (FITC), whereas in the indirect procedure, the anti-species immunoglobulin secondary antibody is conjugated to a fluorochrome. To obtain reliable results, it is necessary to sample several animals in a herd that have fever and a slight, serous nasal discharge. Smears should be air-dried and fixed in acetone. Smears from nasal swabs from cattle with a purulent or haemorrhagic nasal discharge are often negative (Terpstra, 1979). The advantage of this antigen-detection technique is that it can lead to a same-day diagnosis. However, the sensitivity of this procedure is lower than that of virus isolation (Edwards et al., 1983) or PCR. Positive and negative controls must be included in each test.

Tissues collected at post-mortem can be examined for the presence of BoHV-1 antigen by immunofluoresence analyses of frozen sections. Immunohistochemistry may also be applied for BoHV-1 detection and determination of the antigen location in the tissues. MAbs are increasingly being used for detecting BoHV-1 antigen, leading to enhanced specificity of the test. However, such MAbs must be carefully selected, because they must be directed against conserved epitopes that are present on all isolates of BoHV-1.

Another possibility for direct rapid detection of viral antigen is the use of an enzyme-linked immunosorbent assay (ELISA). Antigen can be captured by MAbs or polyclonal antibodies coated on a solid phase, usually

4 These PCR parameters are adapted to the RotorGene 3000, Corbett Research Ltd, Australia, and may vary with different

PCR platforms.

5 PCR Taq polymerase systems from different commercial sources may require a prolonged initial denaturation (95°C) time

up to 10 minutes. Please follow the manufacturer’s instructions.

on microplates. Amounts of antigen equivalent to 104–105 TCID50 of BoHV-1 are required in order to obtain reliable positive results (Collins et al., 1988). This may not be unrealistically high, because titres of 108–109 TCID50/ml of nasal fluid can be excreted by cattle 3–5 days after infection with BoHV-1. Sensitivity can be increased by amplification systems (see Edwards & Gitao, 1987).

In contrast to virus isolation, no cell culture facilities are required for direct antigen detection techniques and

a laboratory diagnosis can be made within 1 day. The disadvantages are the lower sensitivity of direct

antigen detection and the extra requirement to perform additional virus isolation, if the isolate is required for further studies.

e) Differentiation of bovine herpesvirus 1 subtypes and of ruminant alphaherpesviruses related to

bovine herpesvirus 1

By using appropriate MAbs for immunofluorescence, radioimmunoprecipitation, immunoperoxidase or immunoblot assays, BoHV-1 subtype 1 and subtype 2b can be differentiated (Rijsewijk et al., 1999; Wyler et al., 1989). Restriction endonuclease digestion of viral DNA enables differentiation between BoHV-1 subtypes. RFLP analysis includes extraction of the DNA from virions or from infected cells, digestion of the isolated DNA by restriction endonucleases, and separation of the resulting fragments by agarose gel electrophoresis. Differentiation of the BoHV-1 subtypes 1, 2a and 2b by Hind III endonuclease digestion is based on the molecular weight of three relevant DNA fragments (I, K and L) (Metzler et al., 1985). RFLP techniques are of limited diagnostic value, but may be useful in epidemiological studies. Furthermore, RFLP pattern of virus isolates can be compared with that of live vaccine strains.

When differentiation is required between antigenically and genetically related alphaherpesviruses (BoHV-1, BoHV-5[)] caprine herpesvirus 1, cervid herpesvirus 1 and 2, elk herpesvirus 1, bubaline herpesvirus 1), improved methods are available using monoclonal antibodies (Keuser et al., 2004) or PCR amplification and sequencing (Ros et al., 1999).

results

of

f) Interpretation

The isolation of BoHV-1 from a diseased animal does not unequivocally mean that this virus is the cause of the illness. It may, for instance, be a latent virus that has been reactivated due to stressful conditions. A confirmatory laboratory diagnosis must be made from a group of animals and must be accompanied by seroconversion from negative to positive, or a four-fold or higher increase in BoHV-1-specific antibody titres.

Paired serum samples collected 3–4 weeks apart are examined in a serological test for the presence of specific antibodies (see Section B.2).

2. Serological tests

Serological tests can be used for several purposes:

i) To diagnose an acute infection: paired serum samples from the acute and convalescent stages of infection

of the same animals are examined in one test. A seroconversion from negative to positive or a four-fold or higher increase in antibody titres is considered to prove an acute infection.

ii) To demonstrate absence of infection, for instance, for international trade purposes.

iii) To determine the prevalence of infection in sero-epidemiological studies.

iv) To support eradication programmes and subsequent surveillance.

v) For research purposes, for instance, the evaluation of the antibody response after vaccination and challenge infection.

Virus neutralisation (VN) tests (Bitsch, 1978) and various ELISAs (Kramps et al., 1993) are usually used for detecting antibodies against BoHV-1 in serum. Because virus latency is a normal sequel to BoHV-1 infection, the identification of serologically positive animals provides a useful and reliable indicator of infection status. Any animal with antibodies to the virus is considered to be a carrier and potential intermittent excretor of the virus. The only exceptions are calves that have acquired passive colostral antibodies from their dam, and noninfected cattle vaccinated with inactivated vaccines. There is also a risk that calves infected under cover of maternal immunity may become serologically negative while carrying a reactivatible latent infection (Lemaire et al., 2000).

In general, BoHV-1 serological tests can be divided into conventional and marker tests. Up to now, the only serological marker tests available are the BoHV-1 gE-antibody blocking ELISAs (Van Oirschot et al., 1997). Animals vaccinated with gE-deleted marker vaccines can be discriminated from field-virus infected animals by a negative serological reaction for gE. For conventional serology, VNT, BoHV1-antibody blocking ELISAs indirect ELISAs may be used.

ELISAs, including the gE-ELISA, are increasingly used for the detection of antibodies in (bulk) milk samples (Wellenberg et al., 1998a), but have some limitations. By testing bulk milk, a positive gB-specific test indicates that the infection has already spread in the herd (Frankena et al., 1997). With the gE-blocking ELISA, bulk milk gives a positive reaction when more than 10–15% of the herd is infected ((Wellenberg et al., 1998b). Consequently, it is not possible to declare a herd to be free from BoHV-1 infection with these tests on the basis of bulk or pooled milk samples, and a negative gE- or gB-ELISA bulk milk test should be followed up with individual blood samples from all cattle in the herd. However, indirect ELISAs optimised for use with bulk milk samples of up to 50 individual cows can indicate reliably the BoHV-1 status of these animals. For general surveillance purposes, bulk milk tank tests can give an estimate of BoHV-1 prevalence in a herd, an area or country (Nylin et al., 2000). These should be supplemented by serum testing (individual or pooled) from non-milking herds. For monitoring a BoHV-1 status in dairy herds, bulk milk samples of up to 50 animals should be tested 3–4 times per year with a suitable indirect ELISA. In herds of more then 50 cows, several bulks of milk from up to 50 animals should be tested. Positive bulk milk results have to be confirmed by testing individual blood samples from all animals included in a positive bulk milk sample.

In an extensive study, tests for the detection of antibodies as routinely used by national reference laboratories in Europe were evaluated (Kramps et al., 2004). Twelve reference laboratories from 12 European countries participated in this study. Fifty three serum samples and 13 milk samples, originating from several countries, were sent in duplicate under code to the participating laboratories. The serum samples included the three European reference sera EU1 (antibody positive), EU2 (antibody weak positive and defined as borderline sample) and EU3 (antibody negative) (Perrin et al., 1994). It was concluded that VNT and gB-specific ELISAs are the most sensitive tests for the detection of antibodies in serum. Owing to the very high sensitivity of the gB-blocking ELISAs, gB-antibody weak positive results can often not be confirmed by alternative test systems (indirect ELISA, VNT). Recently, new indirect BoHV-1 ELISAs have been developed that are highly sensitive and specific. The results of these ELISAs are comparable with those obtained using gB-blocking ELISAs (Beer et al., 2003).

gE-ELISAs are less sensitive and specific than the conventional test systems. In addition, sero-conversion against gE can be delayed, especially in vaccinated animals, and is often not detectable before day 21 to 35 post-infection. Furthermore, second generation indirect ELISAs were found to be the most sensitive tests for the detection of BoHV-1-specific antibodies in milk. Moreover, it has observed that commercial ELISAs perform better than home-made ELISAs.

a) Virus neutralisation (a prescribed test for international trade)

VN tests are performed with various modifications. Tests vary with regard to the virus strain used in the test protocol, the starting dilution of the serum, the virus/serum incubation period (1–24 hours), the type of cells used, the day of final reading and the reading of the end-point (50% versus 100%) (Perrin et al., 1993).

Among these variables, the virus/serum incubation period has the most profound effect on the sensitivity of the VNT. A 24-hour incubation period may score up to 16-fold higher antibody titres than a 1-hour incubation period (Bitsch, 1978), and is recommended where maximum sensitivity is required (e.g. for international trade purposes). Various bovine cells or cell lines are suitable for use in the VN test, including secondary bovine kidney or testis cells, cell strains of bovine lung or tracheal cells, or the established Madin–Darby bovine kidney (MDBK) cell line.

A suitable protocol for a VN test is shown below.

i) Inactivate sera, including control standard sera, for 30 minutes in a water bath at 56°C.

ii) Make doubling dilutions of test sera in cell culture medium. Start with undiluted serum and continue to 1/1024 horizontally in a 96-well flat-bottomed cell-culture grade microtitre plate, at least three wells per dilution and 50 μl volumes per well. Dilutions of a positive control serum, and of weak positive and negative internal control sera, are also included in the test. An extra well with undiluted test serum is used for toxicity control of sera.

iii) Add 50 μl per well of BoHV-1 stock at a dilution in culture medium calculated to provide 100–200 TCID50 per well. In the toxicity control wells, add 50 μl of culture medium in place of virus. Add 100 μl of culture medium to ten empty wells for cell controls.

iv) Make at least four tenfold dilutions of the residual virus stock (back titration) in culture medium, using

50 μl per well and at least four wells per dilution.

v) Incubate the plates for 24 hours at 37°C.

vi) Add 100 μl per well of the cell suspension at 3 × 104 cells per well.

vii) Incubate the plates for 3–5 days at 37°C.

viii) Read the plates microscopically for CPEs. Validate the test by checking the back titration of virus (which should give a value of 100 TCID50 with a permissible range of 30–300 TCID50), the control sera

and the cell control wells. The positive control serum should give a titre of ± 1 twofold dilution (±0.3 log10 units) from its target value. The weak positive serum should be positive. The negative serum should give no neutralisation (equivalent to a final dilution of 1/2 at the neutralisation stage). In the cell control wells, the monolayers should be intact.

ix) The test serum results are expressed as the reciprocal of the dilution of serum that neutralised the virus in 50% of the wells. If 50% of the wells with undiluted serum neutralised the virus, the (initial dilution) titre is read as 1 (1/2 using the final dilution convention). If all the undiluted and 50% of the wells with 1/2 diluted serum neutralised the virus, the (initial dilution) titre is 2 (final dilution 1/4). For qualitative results, any neutralisation at a titre of 1 or above (initial dilution convention) is considered to be positive.

If cytotoxicity is observed in the control wells, the sample is reported to be toxic (no result) unless neutralisation of the virus without cytotoxicity is observed at higher dilutions and a titre can be read without ambiguity. Where cytotoxicity of a serum interferes with the interpretation of the neutralising activity of the sample, changing the medium in the wells of the lowest two or three dilutions 16–

24 hours after the addition of cells may remove the cytotoxic effects.

b) Enzyme-linked immunosorbent assay (a prescribed test for international trade)

ELISAs for the detection of antibody against BoHV-1 appear to be gradually replacing VN tests. A standard procedure for ELISA has not been established. Several types of ELISA are commercially available, including indirect and blocking ELISAs, some of which are also suitable for detecting antibodies in milk (Kramps et al., 2004). For reasons of standardisation in a country or state, it may be desirable to compare the quality of the kits and to perform batch release tests by previously defined criteria in the national reference laboratory, before it is used by other laboratories in the country.

There are a number of variations in the ELISA procedures. The most common are: antigen preparation and coating, the dilution of the test sample, the incubation period of antigen and test sample, and the substrate/chromogen solution. Before being used routinely, an ELISA should be validated with respect to sensitivity, specificity and reproducibility (see Chapter 1.1.4 Principles of validation of diagnostic assays for infectious diseases). For this purpose, a comprehensive panel of well defined (e.g. by VN test) strong positive, weak positive and negative sera has to be tested. However, it is recommended to use commercially available ELISAs that have been shown to perform better than home-made assays (Kramps et al., 2004).

? Indirect enzyme-linked immunosorbent assay

The principle of an indirect ELISA is based on the binding of BoHV-1-specific antibodies present in the test sample to immobilised BoHV-1 antigen. The bound antibodies are detected using enzyme-labelled anti-bovine immunoglobulin antiserum. The presence of antibodies in the test sample will result in colour development after addition of the substrate/chromogen solution.

? Blocking enzyme-linked immunosorbent assay

The principle of a blocking or competitive ELISA is based on blocking the binding of an enzyme-labelled BoHV-1 antiserum or anti-BoHV-1 MAb to immobilised antigen by antibodies in the test sample. The presence of antibodies in the test sample results in reduced colour development after addition of the substrate/chromogen solution. An example of a gB blocking ELISA procedure is given below:

i) Prepare the antigen by growing BoHV-1 in cell cultures. When extensive CPE is observed, cells and

medium are frozen at –20°C. After thawing, the resulting cellular lysate is centrifuged for 4 hours at 8500 g. The virus-containing pellet is suspended in a small volume of phosphate buffered saline (PBS), cooled on ice and disrupted using an ultrasonic disintegrator. The antigen preparation is then centrifuged for 10 minutes at 800 g, and inactivated by adding detergent (final concentration of 0.5% Nonidet P 40). The antigen preparation is used at an appropriate dilution to coat the microtitre plates.

Many alternative methods of antigen production are described in the published literature.

ii) Coat the microtitre plates with antigen by adding 100 μl of diluted antigen (in 0.05 M carbonate buffer, pH 9.6) to each well. Seal the plates with tape, incubate at 37°C overnight, and store at –20°C.

iii) Before the test is performed, wash the plates with 0.05% Tween 80. Add 100 μl negative serum (fetal calf serum, FCS), 100 μl of each of the serum test samples and 100 μl of positive, weak positive and negative control sera. Usually, serum samples are tested undiluted. Shake, seal the plates and incubate overnight at 37°C. With some ELISAs, it is necessary to heat sera for 30 minutes at 56°C before testing in order to avoid nonspecific responses.

iv) Wash the plates thoroughly and add 100 μl of an anti-BoHV-1-gB-monoclonal antibody/horseradish peroxidase conjugate at a predetermined dilution, and incubate again for 1 hour at 37°C. The monoclonal antibody must be selected carefully for its specificity to gB of BoVH-1.

v) Wash the plates and add freshly prepared substrate/chromogen solution (e.g. 0.05 M citric acid buffer, pH 4.5, containing 2,2’-azino-bis-[3-ethylbenzothiazoline]-6-sulphonic acid [ABTS; 0.55 mg/ml] and a 3% solution of freshly added H2O2 [5 μl/ml]), and incubate for the appropriate time (1–2 hours at room temperature).

vi) Measure the absorbance of the plates on a microplate photometer at 405 nm.

vii) Calculate for each test sample the blocking percentage [(OD FCS – OD test sample)/OD FCS × 100%]

vii) A test sample is considered to be positive if it has a blocking percentage of e.g. 50% of higher. The test is valid if the positive and weak positive control sera are positive and the negative control serum reacts negatively. The acceptable limits for control and cut-off values must be determined for the individual assay.

c) Standardisation

In each serological test, appropriate controls of strong positive, weak positive and negative serum should be included. A scientific group in Europe, initiated by the group of artificial insemination veterinarians of the European Union (EU), has agreed on the use of a strong positive (EU1), a weak positive (EU2) and negative serum (EU3) for standardisation of BoHV-1 tests in laboratories that routinely examine samples from artificial insemination centres (Perrin et al., 1994). These sera have been adopted as OIE international standards for BoHV-1 tests and are available in limited quantities at the OIE Reference Laboratories for IBR/IPV6.

Prescribed tests for international trade purposes (VN or ELISA) must be capable of scoring both the strong and weak positive standards (or secondary national standards of equivalent potency) as positive. Because of the limited availability of the international standard sera, there is a need to prepare a new extended panel of reference lyophilised serum (and milk) samples taken from infected as well as from vaccinated animals. This panel should be used to validate newly developed tests and to harmonise tests between laboratories.

Additional reference sera are available in limited quantities from the OIE Reference Laboratories (e.g. R1, R2 and R3 as positive, weak positive and very weak positive standard sera from the OIE Reference Laboratory in Germany).

d) Nonspecific reactivity in BoHV-1-serology and ‘pseudo-vaccines’

Nonspecific reactivity of sera in the BoHV-1-ELISAs should be taken into consideration, and is more often seen for the marker test than for the conventional serology. There are several reasons for nonspecific reactions:

? batch variation of the ELISA used;

? samples were tested very early after collection (freshness phenomenon);

? samples were collected within 4 weeks after vaccination (vaccination phenomenon);

? bad sample quality (e.g. haemolysed samples).

Therefore the following measures should be considered:

? validation of each test batch, and batch release tests have to be implemented;

? samples should be stored at 4°C and should not be tested before 24–48 hours after sample collection;

? samples should be subjected to a freeze–thaw cycle (–20°C) and to subsequent heat inactivation

(30 minutes/56°C);

? Cattle should not be serologically tested for BoHV-1 prior to 4 weeks after any vaccination;

? gE-ELISAs should not be used for classification of unvaccinated animals.

C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS

1. Background

a) Rationale and intended use of the product

Several attenuated and inactivated BoHV-1 vaccines are currently available. The vaccine strains have usually undergone multiple passages in cell culture. Some of the vaccine virus strains have a temperature-6 Central Veterinary Institute, Division of Virology, P.O. Box 2004, 8203 AA Lelystad, The Netherlands, and Anses Lyon,

Laboratoire de pathologie bovine, 31 avenue Tony Garnier, BP 7033, 69342 Lyon Cedex 07, France.

sensitive phenotype, i.e. they do not replicate at temperatures of 39°C or higher. Attenuated vaccines are administered intranasally or intramuscularly. Inactivated vaccines contain high levels of inactivated virus or portions of the virus particle (glycoproteins) supplemented with an adjuvant to stimulate an adequate immune response. Inactivated vaccines are given intramuscularly or subcutaneously. Vaccination against BoHV-1 is used to protect animals from the clinical outcome of infection, and as an aid in control and eradication programmes.

Marker or DIVA (differentiation of infected from vaccinated animals) vaccines are now available in various countries. These attenuated or inactivated marker vaccines are based on deletion mutants (deletion of gE) or on a subunit of the virion, for instance glycoprotein D. The use of such marker vaccines in conjunction with companion diagnostic tests allows the distinction between infected and vaccinated cattle (DIVA principle), and provides the basis for BoHV-1 eradication programmes in countries or regions with a high prevalence of field-virus infected animals. Intensive vaccination programmes can reduce the prevalence of infected animals (Bosch et al., 1998; Mars et al., 2001), which could be monitored by using an appropriate diagnostic test. In situations where it is economically justifiable, the residual infected animals could be slaughtered, resulting in a region free from BoHV-1. Control and eradication of BoHV-1 was started in some countries in the early 1980s. Different policies have been used due to differences in herd prevalence, breeding practices and disease eradication strategies. To date, in the European Union, only gE-deleted DIVA vaccines (live as well as killed) have been marketed and used for control or eradication programmes. As there is no proven advantage of conventional non-marker BoHV-1 vaccines, gE-deleted marker vaccines (live or inactivated) should be the vaccines of choice.

Guidelines for the production of veterinary vaccines are given in Chapter 1.1.8 Principles of veterinary vaccine production. The guidelines given here and in Chapter 1.1.8 are of general nature and may be supplemented by national and regional requirements.

2. Outline of production and minimum requirements for conventional vaccines

a) Characteristics of the seed

i) Biological

characteristics

The vaccine is prepared using a seed-lot system. Origin, passage history and storage conditions of the master seed virus (MSV) must be recorded. A virus identity test must be performed on the MSV. The seed lot contains BoHV-1 strains have to be attenuated to yield a live vaccine strain. The strains can be attenuated by multiple passages in cell cultures, by adapting virus to grow at low temperatures (temperature-sensitive mutants), or by genetic engineering, for example, by deleting one or more viral genes (e.g. the BoHV-1 glycoprotein E) that are nonessential for replication. There should be some means of distinguishing the live vaccine virus from field viruses (for example temperature-specific growth patterns or restriction fragment length polymorphisms). Strains used for the preparation of inactivated vaccines need not be attenuated. The seed lot must be free from contaminants.

ii) Quality criteria (sterility, purity, freedom from extraneous agents)

The seed lot is tested for absence of extraneous viruses and absence from contamination with bacteria, fungi or mycoplasma. The following extraneous viruses should be specifically excluded in BoHV-1 vaccines: adenovirus, Akabane virus, bovine coronavirus, bovine herpesviruses 2, 4 and 5, bovine parvovirus, bovine respiratory syncytial virus, bovine viral diarrhoea virus and atypical pestiviruses, bovine rotavirus, vaccinia virus, and the viruses of Aujeszky’s disease, bluetongue, bovine ephemeral fever, bovine leukaemia, bovine papilloma, bovine papular stomatitis, cowpox, foot and mouth disease, lumpy skin disease, malignant catarrhal fever, parainfluenza 3, rabies, rinderpest, and vesicular stomatitis. As bovine viral diarrhoea virus (either CPE and/or non-CPE) has regularly been found to be

a contaminant of vaccines, special attention should be paid to the absence of BVDV. In addition, new

atypical pestiviruses (HoBi or HoBi-like) have to be taken into consideration as possible contaminants.

b) Method of manufacture

i) Procedure

The cells used for vaccine production are prepared using a seed-lot system. The virus should be cultured on established cell lines that have been shown to be suitable for vaccine production, for example the Madin–Darby bovine kidney (MDBK) cell line. The history of the cell line must be known.

The cell line must be free from extraneous agents and may be tested for tumorigenicity.

ii) Requirements for substrates and media

All substances used for the manufacture of vaccines must be free from contaminants. Cells should be used that are not further than 20 passages from the master cell seed. The seed virus should not be more than five passages from the MSV. Genetically engineered vaccine virus strains are treated in the

same way as conventionally attenuated vaccine virus strains. When sufficient cells are grown, infection of the cell line with the vaccine virus takes place. The addition of antibiotics is normally restricted to cell culture fluids. The supernatant fluid is harvested at times when the virus (antigen) production peaks.

For live vaccines, the supernatant is clarified, mixed with a stabiliser, freeze-dried and bottled. For the production of classical inactivated vaccines, the supernatant is homogenised before the inactivating agent is added in order to ensure proper inactivation. After the inactivation procedure, a test for ensuring complete inactivation of the virus is carried out. The test should include at least two passages in cells. The inactivated virus suspension is then mixed with an adjuvant and bottled. The manufacture of vaccines must comply with guidelines for Good Manufacturing Practice (GMP).

iii) In-process

controls.

Working cell seed and working virus seed must have been shown to be free from contaminants. The cells must show inconspicuous morphology before being inoculated with virus. The CPE is checked during cultivation. Uninoculated control cells must have retained their morphology until the time of harvesting. A virus titration is performed on the harvested supernatant. During the production of inactivated vaccines, tests are performed to ensure inactivation. The final bulk must be tested for freedom from contaminants.

iii) Final product batch tests

The following tests must normally be performed on each batch. Example guidelines for performing batch control can be found in EU directives, the European Pharmacopoeia and the United States Department of Agriculture’s Code of Federal Regulations.

Sterility/purity

Bacteria, fungi, mycoplasma and extraneous viruses must not be present. Tests for sterility and freedom from contamination of biological material may be found in Chapter 1.1.9.

Safety

For inactivated vaccines, a twofold dose of vaccine, and for live vaccines, a tenfold dose of vaccine, must not produce adverse effects in young BoHV-1 seronegative calves.

Batch potency

It is sufficient to test one representative batch for efficacy, as described in Section C.1.c.ii. In the case of live vaccines, the virus titre of each batch must be determined and must be not higher than 1/10 of the dose at which the vaccine has been shown to be safe, and no lower than the minimum release titre.

In the case of inactivated vaccines, the potency is tested using another validated method, for instance, efficacy assessment in calves.

c) Requirements for authorisation

requirements

i) Safety

Target and non-target animal safety

A quantity of virus equivalent to ten doses of vaccine should (a) not induce significant local or systemic

reactions in young calves; (b) not cause fetal infection or abortion, and (c) not revert to virulence during five serial passages in calves. For inactivated vaccine, a double dose is usually administered. The reversion to virulence test is not applicable to inactivated vaccines.

Reversion-to-virulence for attenuated/live vaccines

The selected final vaccine strain should not revert to virulence during a minimum of five serial passages in calves.

Environmental consideration

Attenuated vaccine strains should not be able to perpetuate autonomously in a cattle population (R0 <1).

requirements

ii) Efficacy

For animal production

This must be shown in vaccination challenge experiments under laboratory conditions. Example guidelines are given in a monograph of the European Pharmacopoeia (Third Edition (1997). Briefly, the vaccine is administered to ten 2–3-month-old BoHV-1 seronegative calves. Two calves are kept as controls. All the calves are challenged intranasally 3 weeks later with a virulent strain of BoHV-1 that

gives rise to typical clinical signs of a BoHV-1 infection. The vaccinated calves should show no or only

very mild signs. The maximum (peak) virus titre in the nasal mucus of vaccinated calves should be at

least 100 times lower than that in control calves. The virus excretion period should be at least 3 days shorter in vaccinated than in control calves.

An efficacious BoHV-1 vaccine should induce protective immunity for at least 1 year, although many existing vaccines have not been tested to this standard.

For control and eradication

In addition to the above-mentioned criteria, BoHV-1-vaccines for control and eradication should be marker vaccines (e.g. gE-deleted vaccines) allowing the differentiation of infected from vaccinated animals (DIVA-strategy). Several gE-deleted vaccines (inactivated preparations as well as modified live

vaccines) are commercially available.

iii) Stability

For live vaccines, virus titrations should be carried out 3 months beyond the indicated shelf life. In addition, tests for determining moisture content, concentrations of preservatives, and pH are performed. For inactivated vaccines, the viscosity and stability of the emulsion are also tested.

The efficacy of preservatives should be demonstrated. The concentration of the preservative and its persistence throughout shelf life should be checked. The concentration must be in conformity with the limits set for the preservative.

3. Vaccines based on biotechnology

a) Vaccines available and their advantages

There is a glycoprotein E (gE)-deleted inactivated vaccine available which is based on a recombinant strain.

The vaccine is comparable to other gE-deleted vaccines and is licensed by European Medicines Agency (EMA) for use in the European Union.

Additional recombinant vaccines like gD-subunits or genetically engineered deletion mutants of BoHV-1 (e.g.

with deletions of gE and/or gG) are described and available as prototypes.

Advantages of BoHV-1 vaccines based on biotechnology could be the possibility of additional marker features for the differentiation of infected from vaccinated animals (DIVA; e.g. gB-antibody-ELISAs for gD-subunit vaccines or gG-antibody-ELISAs for the respective deletion mutants).

b) Special requirements for biotechnological vaccines, if any

Recombinant vaccines, which are destined for use in the European Union have to be licensed by EMA.

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M ETZLER A.E.,M ATILE H.,G ASSMANN U.,E NGELS M.&W YLER R. (1985). European isolates of bovine herpesvirus 1: a comparison of restriction endonuclease sites, polypeptides, and reactivity with monoclonal antibodies. Arch. Virol., 85, 57–69,

M OORE S.,G UNN M.&W ALLS D. (2000). A rapid and sensitive PCR-based diagnostic assay to detect bovine herpesvirus 1 in routine diagnostic submissions. Vet. Microbiol., 75, 145–153.

N YLIN B.,S TROGER U.&R ONSHOLT L.(2000). A retrospective evaluation of a bovine herpesvirus-1 (BHV-1) antibody ELISA on bulk-tank milk samples for classification of the BHV-1 status of Danish dairy herds. Prev. Vet. Med., 47, 91–105.

P ARSONSON I.M.&S NOWDON W.A. (1975). The effect of natural and artificial breeding using bulls infected with, or semen contaminated with, infectious bovine rhinotracheitis virus. Aust. Vet. J., 51, 365–369.

P ERRIN B.,B ITSCH V.,C ORDIOLI P.,E DWARDS S.,E LOIT M.,G UERIN B.,L ENIHAN P.,P ERRIN M.,R ONSHOLT L.,V AN O IRSCHOT J.T.,V ANOPDENBOSCH E.,W ELLEMANS G.,W IZIGMANN G.&T HIBIER M. (1993). A European comparative study of serological methods for the diagnosis of infectious bovine rhinotracheitis. Rev. sci. tech. Off. int. Epiz., 12, 969–984.

P ERRIN B.,C ALVO T.,C ORDIOLI P.,C OUDERT M.,E DWARDS S.,E LOIT M.,G UERIN B.,K RAMPS J.A.,L ENIHAN P., P ASCHALERI E.,P ERRIN M.,S CHON J.,V AN O IRSCHOT J.T.,V ANOPDENBOSCH E.,W ELLEMANS G.,W IZIGMANN G.& T HIBIER M. (1994). Selection of European Union standard reference sera for use in the serological diagnosis of infectious bovine rhinotracheitis. Rev. sci. tech. Off. int. Epiz., 13, 947–960.

R IJSEWIJK F.A.,K AASHOEK M.J.,L ANGEVELD J.P.,M ELOEN R.,J UDEK J.,B IENKOWSKA-S ZEWCZYK K.,M ARIS-V ELDHUIS M.A.& VAN O IRSCHOT J.T. (1999). Epitopes on glycoportein C of bovine herpesvirus-1 (BHV-1) that allow differentiation between BHV-1.1 and BHV-1.2 strains. J. Gen. Virol., 80, 1477–1483.

R OLA J.,P OLAK M.&Z MUDZINSKI J. (2003). Amplification of DNA of BHV 1 isolated from semen of naturally infected bulls. Bull. Vet. Inst. Pulaway, 47, 71–75.

R OS C.,R IQUELME M.E.,O HMAN F ORSLUND K.&B ELAK S. (1999). Improved detection of five closely related ruminant alphaherpesviruses by specific amplification of viral genome sequences. J. Virol. Methods, 83, 55–65.

S ANTURDE G.,S ILVA N.D.,V ILLARES R.,T ABARES E.,S OLANA A.,B AUTISTA J.M.,C ASTRO J.M.&D A S ILVA N. (1996). Rapid and high sensitivity test for direct detection of bovine herpesvirus-1 genome in clinical samples. Vet. Microbiol.,49, 81–92.

S CHYNTS F.,B ARANOWSKI E.,L EMAIRE M.&T HIRY E. (1999). A specific PCR to differentiate between gE negative vaccine and wildtype bovine herpesvirus type 1 strains. Vet. Microbiol., 66, 187–195.

S MITS C.B.,V AN M AANEN C.,G LAS R.D,D E G EE A.L.,D IJKSTRAB T,V AN O IRSCHOT J.T.&R IJISEWIJK F.A. (2000). Comparison of three polymerase chain reaction methods for routine detection of bovine herpesvirus 1 DNA in fresh bull semen. J. Virol. Methods,85, 65–73.

T ERPSTRA C. (1979). Diagnosis of infectious bovine rhinotracheitis by direct immunofluorescence. Vet. Q., 1, 138–144.

T HIRY J.,K EUSER V.,M UYLKENS B.,M EURENS F.,G OGEV S.,V ANDERPLASSCHEN A.&T HIRY E. (2006). Ruminant alphaherpesviruses related to bovine herpesvirus 1. Vet. Res., 37, 169–190.

V AN E NGELENBURG F.A.,M AES R.K.,V AN O IRSCHOT J.T.&R IJSEWIJK F.A. (1993). Development of a rapid and sensitive polymerase chain reaction assay for detection of bovine herpesvirus type 1 in bovine semen. J. Clin. Microbiol., 31, 3129–3135.

V AN E NGELENBURG F.A.C.,V AN S CHIE F.W.,R IJSEWIJK F.A.M.&V AN O IRSCHOT J.T. (1995). Excretion of bovine herpesvirus 1 in semen is detected much longer by PCR than by virus isolation. J. Clin. Microbiol., 33, 308–312.

V AN O IRSCHOT J.T.,K AASHOEK M.J.,M ARIS-V ELDHUIS M.A.,W EERDMEESTER K.&R IJSEWIJK F.A.M. (1997). An enzyme-linked immunosorbent assay to detect antibodies against glycoprotein gE of bovine herpesvirus 1 allows differentiation between infected and vaccinated cattle. J. Virol. Methods, 67, 23–34.

V AN O IRSCHOT J.T.,S TRAVER P.J.,V AN L IESHOUT J.A.H.,Q UAK J.,W ESTENBRINK F.&V AN E XSEL A.C.A. (1993). A subclinical infection of bulls with bovine herpesvirus type 1 at an artificial insemination centre. Vet. Rec., 132, 32–35.

V ILCEK S.,N ETTLETON P.F.,H ERRING J.A.&H ERRING A.J. (1994). Rapid detection of bovine herpesvirus 1 (BHV 1) using the polymerase chain reaction. Vet. Microbiol., 42, 53–64.

W ANG J.,O’K EEFE J.,O RR D.,L OTH L.,B ANKS M.,W AKELEY P.,W EST D.,C ARD R.,I BATA G.,V AN M AANEN K.,T HOREN P.,I SAKSSON M.&K ERKHOFS P. (2008). An international inter-laboratory ring trial to evaluate a real-time PCR assay for the detection of bovine herpesvirus 1 in extended bovine semen. Vet. Microbiol., 126, 11–19.

W EIBLEN R.,K REUTZ L.,C ANABOROO T.F.,S CHUCH L.C.&R EBELATTO M.C. (1992). Isolation of bovine herpesvirus 1 from preputial swabs and semen of bulls with balanoposthitis. J. Vet. Diag. Invest., 4, 341–343.

W ELLENBERG G.J.,V ERSTRATEN E.R.A.M.,M ARS M.H.&V AN O IRSCHOT J.T. (1998a). Detection of bovine herpesvirus 1 glycoprotein E antibodies in individual milk samples by enzyme-linked immunosorbent assays. J. Clin. Microbiol., 36, 409–413.

W ELLENBERG G.J.,V ERSTRATEN E.R.A.M.,M ARS M.H.&V AN O IRSCHOT J.T.(1998B). ELISA detection of antibodies to glycoprotein E of bovine herpesvirus 1 in bulk milk samples. Vet Rec.,142, 219–220.

W IEDMANN M.,B RANDON R.,W AGNER P.,D UBOVI E.J.&B ATT C.A. (1993). Detection of bovine herpesvirus-1 in bovine semen by a nested PCR assay. J. Virol. Methods,44, 129–140.

W YLER R.,E NGELS M.&S CHWYZER M. (1989). Infectious bovine rhinotracheitis/vulvovaginitis (BHV1). In: Herpesvirus Diseases of Cattle, Horses and Pigs, Wittmann G., ed. Kluwer Academic Publishers, Boston, USA, 1–72.

X IA J.Q.,Y ASON C.V.&K IBENGE F.S.(1995). Comparison of dot blot hybridization, polymerase chain reaction, and virus isolation for detection of bovine herpesvirus-1 (BHV-1) in artificially infected bovine semen. Can. J. Vet. Res., 59, 102–109.

Y ASON C.V.,H ARRIS L.M.,M C K ENNA P.K.,W ADOWSKA,D.&K IBENAGE F.S.B. (1995). Establishment of conditions for the detection of bovine herpesvirus-1 by polymerase chain reaction using primers in the thymidine kinase region. Can. J. Vet. Res.,59, 94–101.

*

* *

NB: There are OIE Reference Laboratories for Infectious bovine rhinotracheitis/infectious pustular vulvovaginitis (see Table in Part 3 of this Terrestrial Manual or consult the OIE Web site for the most up-to-date list:

www.oie.int).

《劳动标准》最新劳动能力鉴定标准

劳动能力鉴定—职工工伤与职业病致残等级分级 GB／T 16180—2006 前言 本标准的全部内容为推荐性的。 本标准参考了世界卫生组织有关“损害、功能障碍与残疾”的国际分类，以及美国、英国、日本等国家残疾分级原则和基准。 根据《工伤保险条例》（中华人民共和国国务院第375号令）制定本标准。本标准代替GB／T 16180 —1996《职工工伤与职业病致残程度鉴定》。 本标准参考与协调的国家文件、医学技术标准与相关评残标准有：残疾人标准，革命伤残军人评定标准等。 为使劳动能力鉴定适应我国当前社会经济发展的要求，保障因工作遭受事故伤害或者患职业病的劳动者获得医疗救治和经济补偿，对工伤或患职业病劳动者的伤残程度做出更加客观、科学的技术鉴定，在总结分析10余年工伤评残实践经验基础上，对GB／T 16180 —1996进行了修订与完善，并与我国劳动能力鉴定法规制度相配套，将原标准更名为《劳动能力鉴定职工工伤与职业病致残等级》，并对以下技术原则作了调整： ——增加了总则中4. 1. 3医疗依赖的分级判定； ——取消了总则中关于工伤、职业病证明的规定； ——取消了总则中关于重新鉴定的规定； ——伤残类别增加了十二指肠的损伤，同时取消了单列的耳廓缺损； ——智能减退改为智能损伤，增加记忆商（MQ）判定指标； ——取消了利手与非利手的表述； ——增加了低氧血症的判断标准； ——增加了活动性肺结核诊断要点的判定； ——增加了大血管的界定； ——增加了瘢痕诊断的界定； ——增加了贫血诊断标准与分级； ——修订了6. 4. 1肝功能损害的判定与分级； ——修订了6. 5. 4中毒性肾病和6. 5. 5肾功能不全的判定指标； ——取消了辅助器具如安装假肢的表述； ——修订了人格改变的判定基准指标； ——全身瘢痕的最低下限由≤30％修改为<5％，但≥1％； ——对附录A判定基准补充的A. 1智能损伤表述内容作了调整； ——取消了判定基准补充的A. 3人格障碍与人格改变的表述，同时增加了“与工伤、职业病相关的精神障碍的认定”的表述； ——伤残条目由470条调整为572条； ——根据国家工伤保险法规及有关文件精神，对“于国家社会保险法规所规定的医疗期满后……”的表述改为“于国家工伤保险法规所规定的停工留薪期满……”，达到与相关法规相衔接，以便于判断与执行。 本标准的附录A、附录B是规范性附录。 本标准的附录C是资料性附录。 本标准由中华人民共和国劳动和社会保障部、卫生部共同提出。 本标准由劳动和社会保障部工伤保险司归口。

施工质量评定标准

5 施工质量评定 5.1 合格标准 5.1.1合格标准是工程验收标准。不合格工程必须按要求处理合格后，才能进行后续工程施工或验收。水利水电工程施工质量等级评定的主要依据有：1国家及相关行业技术标准； 2《单元工程评定标准》； 3经批准的设计文件、施工图纸、金属结构设计图样与技术条件、设计修改通知书、厂家提供的设备安装说明书及有关技术文件； 4工程承发包合同中采用的技术标准； 5工程施工期及试运行期的试验和观测分析成果。 5.1.2单元（工序）工程施工质量合格标准应按照《单元工程评定标准》或合同约定的合格标准执行。当达不到合格标准时，应及时处理。处理后的质量等级按下列规定确定： 1全部返工重做的，可重新评定质量等级。 2经加固补强并经设计和监理单位鉴定能达到设计要求时，其质量评为合格。 3处理后的工程部分质量指标仍达不到设计要求时，经设计复核，项目法人及监理单位确认能满足安全和使用功能要求，可不再进行处理；或经加固补强后，改变外形尺寸或造成永久性缺陷的，经项目法人、监理及设计确认能基本满足设计要求，其质量可定为合格，但应按规定进行质量缺陷备案。 条文中“处理后部分质量指标达不到设计要求”指单元工程中不影响工程结构安全和使用功能的一般项目质量未达到设计要求。“可不再进行处理”者，应按4.4.3条的及4.4.4条的规定进行质量缺陷备案。技术标准、设计文件、图纸、质检资料、合同文件等是工程施工质量评定的依据。试运行期的观测资料可综合反映工程建设质量，是评定工程施工质量的重要依据。 5.1.3分部工程施工质量同时满足下列标准时，其质量评为合格： 1所含单元工程的质量全部合格。质量事故及质量缺陷已按要求处理，并经检验合格； 2原材料、中间产品及混凝土（砂浆）试件质量全部合格，金属结构及启闭机制造质量合格，机电产品质量合格。 分部工程施工质量合格标准，内容与SL176—1996相同。 5.1.4单位工程施工质量同时满足下列标准时，其质量评为合格： 1所含分部工程质量全部合格； 2质量事故已按要求进行处理；

司法鉴定档案标准

司法鉴定案件归档格式文本（试行）

卷内目录

司法鉴定风险告知书 根据我国诉讼法的有关规定，司法鉴定是司法机关为了解决案件中涉及的专门性问题，依照《全国人大常委会关于司法鉴定管理问题的决定》的规定，委托列入司法鉴定人名册的司法鉴定人所从事的鉴别和判断并提供鉴定意见的活动。由于鉴定涉及专业问题，因此有必要将其中的一些情况予以告知、提示： 一、鉴定意见属于证据 鉴定意见是鉴定人根据相关学科的专业知识，依据法律规定，客观、公正、科学地提出的专业性意见，属于法律规定的证据之一。是否成为定案的根据，取决于法官的审查和判断，鉴定人并无决定权和影响法官采信鉴定意见的能力。如果法官不予采信，根据诉讼法的规定，法官有权启动新的鉴定程序。 二、法医临床鉴定一般需要检查被鉴定人的身体 法医学鉴定除了需要审查送检的鉴定资料之外，一般需要对被鉴定人的身体进行必要的检查或者做必要的辅助检查，特殊情况下才实施书面鉴定。有关费用由被鉴定人先行垫付，请被鉴定人予以配合。 三、鉴定可能得出不明确的鉴定意见 鉴定意见的提出依赖于委托单位及当事人提交的鉴定材料，有时由于委托方不能按要求补充鉴定材料或者受客观条件的制约，可能得出不明确的鉴定意见或依照规定中止鉴定。所开支费用由被鉴定人承担，请被鉴定人予以配合。 四、鉴定意见具有科学、公正性 鉴定需要解决的问题是办案机关处理案件过程中遇到的疑难专业问题，鉴定人遵循科学、公正的宗旨，鉴定活动也是围绕委托单位提出的鉴定目的，依据现有送检材料来进行的。因此，鉴定意见可能对被告不利，也可能对原告不利，与鉴定申请提出方没有必然的关系。 五、鉴定费的承担 国家有关法律明确规定鉴定需要交纳有关费用，当事人支付鉴定费，鉴定实际支出费，都属于垫付，最终由哪一方承担，由法官在裁判案件时一并决定，鉴定机构不解决有关法律问题。 六、鉴定活动具有严肃性 鉴定人可以就鉴定委托单位和被鉴定人提出的有关鉴定文书中的专业性问题进行解释，如果被鉴定人仍然有意见或者异议，只能通过庭审质证或者申请其他鉴定机构重新鉴定来解决。 委托方对以上内容已知，无异议。 委托方签名（或盖章）： 年月日

员工技能鉴定管理办法

后英集团海城钢铁有限公司大屯分公司 机电修岗位等级鉴定规定 一、技术等级设定 1 维修工、电工、操作工，等级分为初、中、高共三级； 2 技术员等级分为初、中共两级； 3专业技术工程师等级分为助理工程师、工程师、主任工程师共三级。 二、技能鉴定申报条件 对于不同技术等级的员工，在初次鉴定时只要符合条件之一即可申报。 1 初级工：新入岗最基本评级为初级工。 2 中级工 2.1 连续从事本职业工作2年以上。 2.2 在本公司连续从事本职业工作12个月以上。 2.3 取得以中级技能为培养目标的中等以上职业学校本职业毕业证书。 2.4 取得本职业中级资格证书。 3 高级工 3.1 连续从事本职业工作5年以上。 3.2在本公司连续从事本职业工作24个月以上。 3.3取得以高级技能为培养目标的大专及以上毕业证书。 3.4取得本职业高级资格证书。 4 初级技术员 4.1初步掌握本专业的基础理论知识或专业技术知识。 4.2取得本职业相关专业中专以上学历，连续从事本职业工作3年以上（新入职时大专1年以上，本科及以上不做工作时间限制）。 5 中级技术员 5.1能够运用本专业的基础理论知识和专业技术知识。具有完成一般性技术工作的实际能力。 5.2取得本职业相关专业中专以上学历，连续从事本职业工作5年以上（大专，2年以上；本科，1年以上）。 5.3在本公司连续从事本职业工作24个月以上。 5.4在本专业领域能独立工作，凭借已有的工作经验，发现生产中潜在的问题，分析原因、采取措施。

6 助理工程师 6.1 能够运用本专业的基础理论知识和专业技术知识。具有完成较为复杂性技术工作的实际能力。 6.2 取得本职业相关专业中专以上学历，连续从事本职业工作8年以上（大专，5年以上；本科，3年以上）。 6.3 在本公司连续从事本职业工作36个月以上。 6.4 在本专业领域能独立工作，凭借已有的工作经验，发现生产中潜在的问题，分析原因、采取措施。 6.5 取得本职业助理工程师职称。 7 工程师/主任工程师 7.1系统、深厚的理论功底和扎实专业技术知识，对所从事的专业领域有较深入的研究和独到的见解，在本部门有较高的权威性。 7.2全面掌握与本专业有关的技术标准、技术规范和技术规程，具有对工程设计、科研项目进行鉴定和评估的能力。 7.3能进行技术可行性、经济性和市场前景分析，所取得的成果能满足市场需求，具有较强的技术创新能力。 7.4取得本职业相关专业中专以上学历，连续从事本职业工作10年以上（大专，8年以上；本科，6年以上）。 7.5取得本职业中级/高级工程师职称。 三、技能鉴定项目及方法 1 主要包括理论知识考试、实际操作技能考核、个人素质评价三个方面。理论考试主要为所属工种的相关理论知识、操作规程、安全常识、常见问题分析等。由各专业相关专业人员拟定考试题库，设备部组织考试。 2操作技能考核的内容，设备部门根据评定对象所从事的工种确定相应的考核项目，在指定场地通过现场实操的形式进行考核。 3 素质测评由部门、车间垂直管理层级确定。 4三项考核满分均为100分，三项均达到60分以上（含60分）者为合格，其中有一项达不到60分者为不合格。 5 对于文化水平较低者，可采用口头提问的方式进行理论考试。

质量检验评定的等级标准

4工程质量检验评定的等级标准 4.0.1本标准的分项、分部、单位工程质量划分为“合格,,与“优良"两个等级。 4.0.2分项工程的检验项目分为保证项目、基本项目和允许偏差项目,其质量等级应符合下列规定: 4.0.2.1合格: (l)保证项目必须符合本标准的规定: (2)基本项目抽检的处(件)应符合本标准合格的规定: (3)允许偏差项目抽检的点数中,有80%及以上的实测值应在本标准规定的允许偏差范围内,其余实测值也应基本接近本标准的规定。 4.0.2.2优良: (l)保证项目必须符合本标准的规定: (2)基本项目每项抽检处〈件〉的质量应符合本标准合格的规定:每项抽检的处(件)中有60%及以上符合优良规定,该项应为优良项,优良项数应占检验项目数50%及以上,该基本项目应评为优良. (3)允许偏差项目抽检的点数中,有90%及以上的实测值应在本标准的允许偏差范围内,其余测值也应基本接近本标准的规定。 4.0.3分部工程的质量等级应符合下列规定: 4.0.3.1合格: 所含分项工程的质量等级应全部合格。 4.0.3.2优良: 所含分项工程的质量等级必须全部合格其中达到优良等级的分项工程数量不应少于50%,且主要分项工程质量等级应为优良。 4.0.4单位工程的质量等级应符合下列规定: 4.0.4.1合格: 〈1〉所含分部工程的质量等级应全部合格; 〈2〉质量保证资料(P2)应基本齐全完整: 〈3〉单位工程总体检验压力试验、气密试验、仪表单校、联校试运转等)结果必须合格。 4.0.4.2优良: (1) 所含分部工程的质量评定等级全部合格其中有50%及以上为优良: (2) 质量保证资料(P2)应齐全完整; (3) 单位工程总体检验(高低压实验、变电所受、送电及电动机联动试运转等)结果必须合格。 4.0.5分项工程质量不符合本标准的合格规定时,必须及时处理,并按以下规定评定质量等级: 4.0. 5.1返工重做的可重新评定等级。 4.0. 5.2经加固补强返修达到合格的分项工程,其质量等级仅能评为合格,不能评为优良. 4.0. 5.3让步接收的分项工程仅能评为合格。 （3）

轻伤分级鉴定标准

人体轻伤分级鉴定标准 第一章总则 第一条本标准以《中华人民共和国刑法》和最高人民法院、最高人民检察院、公安部和司法部颁布的《人体轻伤鉴定标准(试行)》为根据，以医学、法医学的理论和技术为基础，结合我省检案实践经验，为人体轻伤分级提供科学依据和统一标准，特拟定本分级鉴定标准，供我省法院系统参照执行。 第二条轻伤是指物理、化学及生物等各种外界因素作用于人体，造成组织、器官结构一定程度的损害或者部分功能障碍，尚未构成重伤又不属轻微伤害的损伤。 第三条鉴定损伤程度，应该以外界因素对人体直接造成的原发性损害及后果为依据，包括损伤当时的伤情、损伤后的并发症和后遗症等，全面分析，综合评定。 第四条本标准将人体损伤所致的轻伤由重至轻分为轻伤甲级、轻伤乙级和轻伤丙级三个级别。 损伤分级 甲级 乙级 丙级 第五条帽状腱膜下血肿面积达100平方厘米(儿童达50平方厘

米)；头皮撕脱伤面积达50平方厘米(儿童达25平方厘米)；头皮外伤性缺失面积达25平方厘米(儿童达15平方厘米) 。帽状腱膜下血肿面积达50平方厘米(儿童达30平方厘米)头皮撕脱伤面积达35平方厘米 (儿童达15平方厘米)；头皮外伤性缺失达15平方厘米 (儿童达10平方厘米) 。帽状腱膜下血肿；头皮撕脱伤面积达20平方厘米 (儿童达10平方厘米)；头皮外伤性缺失达10平方厘米 (儿童达5平方厘米) 第六条头皮锐器创口累计长度达20厘米(儿童达15厘米)；钝器创口累计长度达15厘米(儿童达10厘米) 。头皮锐器创口单个长度达10厘米，累计长度达15厘米(儿童单个达6厘米，累计达8厘米)；钝器创口单个长度达8厘米，累计长度达12厘米(儿童单个达4厘米，累计长达6厘米) 。头皮锐器创口累计长度达8厘米(儿童达6厘米)；钝器创口累计长度达6厘米(儿童达4厘米) 。 第七条颅骨凹陷性骨折、粉碎性骨折；颅脑损伤，CT扫描提示脑挫伤或者蛛网膜下腔出血颅骨线形骨折 第八条头部损伤当时出现昏迷达30分钟；头部损伤确证出现短暂意识障碍和近事遗忘，伴有一定程度的颅脑损伤临床症状、体征；头部损伤确证出现短暂意识障碍和近事遗忘 第九条 1、眼睑损伤影响面容或者功能； 2、眶部粉碎性骨折或眶骨骨折有移位； 3、眼球部分结构损伤，致两眼矫正视力0.2以下,单眼矫正视力0.1以下；

质量检验员岗位级别评定标准

质量检验员岗位级别评定标准 一、目的: 根据公司管理的需要和公司领导对我公司管理的新要求,现我公司应对质量检验员进行岗位级别评定,以满足新形势下职工对未来自我职业价值提升的期望和需求。最终目的是积极培养和留住优秀员工为企业的质量管理和产品品质提升多做贡献。 二、适用范围 底盘公司从事一线质量检验和质量管理工作的员工。 三、评定要求： 1.评定级别要求及奖励:中级职称每年评选1-2名，公司在原工资基础上每月额 外有200-300元津贴补助；初级职称3-4名公司在原工资基础上每月额外有100-200元津贴补助。 中级职称工作年限按上限执行，初级职称工作年限按下限执行。以上为基本要求，符合上述条件才能进行提报。

3.职业技能要求（见下表）：

4，注意初级质检员只考核《质量员职称申报评分卡》表中1-5项，中级质检员除考核1-5项外，还应追加6-7项考核。 5，评定流程及办法：申请初级质量员和中级质量员的职工应填写《岗位职称评审申报表》（见附表一），完整填写后，由质量部部长组织3-5人评审小组对参与评选人员按《质量员职称申报评分卡》进行分项认真考评打分，每位评委应对每位申报人进行分项考评打分，取其平均总分为个人考评最终得分。然后按从高分到低分的原则从初级质量员申请人中选出3-4名为初级质量员，由质量部批准并报综合部批准备案；按从高分到低分的原则，从中级质量员申请人中选出1名人员为中级质量员，由质量部批准并报综合部批准备案。 6，以下人员不得参与评聘初级和中级质量员。 6.1我公司非质量专业从业人员。 6.2虽从事质量管理但具有行政职称和职务的人员。 6.3重大安全事故的直接责任人，根据公司规定2年内不得参与职称评聘。 6.4 在年度内请假时间累计超过20天者（不包括公休假期）； 6.5 年度内累计旷工超过7天者； 6.6两年内犯重大过失，给公司造成较大损失的直接责任人。 批准：审核：编制：

司法鉴定档案标准

. 司法鉴定案件归档格式文本（试行）卷内目录

. 司法鉴定风险告知书 根据我国诉讼法的有关规定，司法鉴定是司法机关为了解决案件中涉及的专门性问题，依照《全国人大常委会关于司法鉴定管理问题的决定》的规定，委托列入司法鉴定人名册的司法鉴定人所从事的鉴别和判断并提供鉴定意见的活动。由于鉴定涉及专业问题，因此有必要将其中的一些情况予以告知、提示： 一、鉴定意见属于证据 鉴定意见是鉴定人根据相关学科的专业知识，依据法律规定，客观、公正、科学地提出的专业性意见，属于法律规定的证据之一。是否成为定案的根据，取决于法官的审查和判断，鉴定人并无决定权和影响法官采信鉴定意见的能力。如果法官不予采信，根据诉讼法的规定，法官有权启动新的鉴定程序。 二、法医临床鉴定一般需要检查被鉴定人的身体 法医学鉴定除了需要审查送检的鉴定资料之外，一般需要对被鉴定人

的身体进行必要的检查或者做必要的辅助检查，特殊情况下才实施书面鉴定。有关费用由被鉴定人先行垫付，请被鉴定人予以配合。 三、鉴定可能得出不明确的鉴定意见 鉴定意见的提出依赖于委托单位及当事人提交的鉴定材料，有时由于委托方不能按要求补充鉴定材料或者受客观条件的制约，可能得出不明确的鉴定意见或依照规定中止鉴定。所开支费用由被鉴定人承担，请被鉴定人予以配合。 四、鉴定意见具有科学、公正性 鉴定需要解决的问题是办案机关处理案件过程中遇到的疑难专业问题，鉴定人遵循科学、公正的宗旨，鉴定活动也是围绕委托单位提出的鉴定目的，依据现有送检材料来进行的。因此，鉴定意见可能对被告不利，也可能对原告不利，与鉴定申请提出方没有必然的关系。五、鉴定费的承担 国家有关法律明确规定鉴定需要交纳有关费用，当事人支付鉴定费，鉴定实际支出费，都属于垫付，最终由哪一方承担，由法官在裁判案件时一并决定，鉴定机构不解决有关法律问题。 六、鉴定活动具有严肃性 鉴定人可以就鉴定委托单位和被鉴定人提出的有关鉴定文书中的专 业性问题进行解释，如果被鉴定人仍然有意见或者异议，只能通过庭审质证或者申请其他鉴定机构重新鉴定来解决。 委托方对以上内容已知，无异议。 委托方签名（或盖章）：

司法鉴定技术规范

视觉功能障碍法医鉴定指南 司 法 鉴 定 技 术 规 范 中华人民共和国司法部 司 法 鉴 定 管 理 局 发布

目 录 前言.............................................................................II 1 范围 (1) 2 定义 (1) 3 鉴定原则 (2) 附录A （规范性附录）视觉功能障碍检查 (4) 附录B （规范性附录）视觉功能实验室及鉴定人员的规范要求 (15) 附录C （资料性附录）视觉功能障碍程度分级标准 (16) 附录D （参考性附录）眼外伤法医鉴定检验结果记录单(范本) (18)

前言 制定本技术规范的依据包括以下国家或行业标准：由司法部、最高人民法院、最高人民检察院和公安部于1990年9月29日颁布实施的司发[1990]070号《人体重伤鉴定标准》；由最高人民法院、最高人民检察院、公安部、司法部于1990年4月2日颁布实施的法（司）发[1990]6号《人体轻伤鉴定标准（试行）》；由公安部颁布实施的GA/T 146-1996《中华人民共和国公共安全行业标准·人体轻微伤的鉴定》；由国家质量监督检验检疫总局发布，于2002年11月1日开始实施的GB18667-2002《中华人民共和国国家标准·道路交通事故受伤人员伤残程度评定》；由国家质量监督检验检疫总局和国家标准化管理委员会发布，于2007年5月1日开始实施的GB/T 16180-2006《中华人民共和国国家标准·劳动能力鉴定 职工工伤与职业病致残等级》；由公安部发布的于2005年3月1日开始实施的GA/T 521-2004《中华人民共和国公共安全行业标准·人身伤害受伤人员误工损失日评定准则》。 本技术规范参考了American medical Association（美国医学会）编著的《Guides to the Evaluation of Permanent Impairment》（Fifth Edition）（《永久性残损评定指南》第5版）。本指南还参考了视觉电生理国际标准化委员会发布的视觉电生理国际标准化文件（包括：《视网膜电图国际标准》、《图像视网膜电图国际标准》、《临床眼电图法国际标准》、《视诱发电位法国际标准》）。 本技术规范运用医学、法医学理论和技术，结合法医学检验、鉴定的实践而制定，为眼外伤后视觉功能障碍的法医鉴定提供科学、规范、统一的方法和标准。 本技术规范的附录A、B为规范性附录，附录C为资料性附录，附录D为参考性附录。 本技术规范由司法部司法鉴定科学技术研究所提出。 本技术规范由司法部司法鉴定科学技术研究所负责起草。 本技术规范主要起草人：夏文涛，刘瑞珏，朱广友，范利华，翁春红，陈捷敏，刘夷嫦。

国家职业技能鉴定标准(终审稿)

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