Assessment of different factors on the influence of glass wool concentration for detection of main swine viruses in water samples.

Viruses existed in wastewaters might pose a biosecurity risk to human and animal health. However, it is generally difficult to detect viruses in wastewater directly as they usually occur in low numbers in water. Therefore, processing large volumes of water to concentrate viruses in a much smaller final volume for detection is necessary. Glass wool has been recognized as an effective material to concentrate multiple in water, and in this study, we assessed the use of glass wools on concentrating pseudorabies virus (PRV), African swine fever virus (ASFV), and porcine epidemic diarrhea virus (PEDV) in water samples. The influence of pH values, water matrix, water volume, filtration rate, temperature on the effect of the method concentrating these viruses for detection was evaluated in laboratory. Our results revealed that glass wool was suitable for the concentration of above-mentioned viruses from different water samples, and demonstrated a good application effect for water with pH between 6.0-9.0. Furthermore, glass wool also showed a good recovery effect on concentrating viral nucleic acids and viral particles, as well as living viruses. In addition, combining use of glass wool with skim milk, polyethylene glycol (PEG)-NaCl, or ultracentrifuge had good effects on concentrating ASFV, PRV, and PEDV. Detection of wastewater samples (n = 70) collected from 70 pig farms in 13 regions across Hubei Province in Central China after glass-wool-concentration determined one sample positive for ASFV, eighteen samples positive for PRV, but no sample positive for PEDV. However, these positive samples were detected to be negative before glass wool enrichment was implemented. Our results suggest that glass wool-based water concentration method developed in this study represents an effective tool for detecting viruses in wastewater.

Assessment of confidence in freedom from Aujeszky's disease and classical swine fever in Danish pigs based on serological sampling--effect of reducing the number of samples.

Confirming freedom from disease is important for export of animals and animal products. In Denmark, an intensive surveillance program is in place for Aujeszky's disease (AD) and classical swine fever (CSF), including 34,974 blood samples tested for AD and 37,414 samples tested for CSF (2008 figures). In the current system, 3.5% of sows and boars for export or slaughter are tested for both diseases, as well as all boars before entering boar stations. Furthermore, nucleus herds are tested every third month for classical swine fever. We investigated, whether the sample size could be reduced without compromising the posterior probability of freedom (PostPFree) from AD and CSF by use of a scenario tree model. Conventional herds and sows or boars were defined as risk factors (compared to SPF(1) herds and finisher pigs), with a relative risk of 2 and 5, respectively. The probability of introduction was modeled as a distribution (0.0042:0.0083; 0.05), and the within-herd and between-herd design prevalence were set to 0.05 and 0.01, respectively. If 50 and 75% of the test results from exported or slaughtered sows and boars were simulated to be removed at random, while the blood samples from boar stations were kept constant (reflecting a total reduction of 28 or 43%) the PostPFree from AD was reduced from 0.989 after 1 year testing to 0.980 or 0.971, respectively. Similarly, the confidence of freedom from CSF was reduced from 0.989 to 0.982 or 0.969, when the number of serological samples from abattoirs and export sows and boars is reduced by 50 or 75%, respectively (reflecting a total reduction of 34 or 51%), and further to 0.978 or 0.963 if sampling in nucleus herds was stopped (reflecting a total reduction of 41 or 59%). The results show that a reduction in the sampling size of Danish sows will have limited effect on the PostPFree from AD and CSF, and that sampling in nucleus herds for CSF adds little to the PostPFree from CSF.

Risk assessment and cost-effectiveness analysis of Aujeszky's disease virus introduction through breeding and fattening pig movements into Spain.

Movement of infected animals is considered the most likely route of Aujeszky's disease virus (ADV) introduction into free areas and the main obstacle to eradicating Aujeszky's disease (AD) in those areas, which have achieved a low prevalence (>0 and < or =10%) status. For this reason, the Spanish AD control and eradication program has established measures to enhance security in animal movements in an attempt to protect areas with free or low prevalence status; however, no studies have quantified the effectiveness of the current or alternative ADV introduction prevention measures. We performed a probabilistic risk assessment and cost-effectiveness analysis, using Monte Carlo simulation, to evaluate the probability of introducing ADV-infected animals into free or low prevalence areas under the Spanish AD control and eradication program. We found the mean probability of introducing ADV-infected animals, when breeding pigs were quarantined but not tested prior to shipment, is likely (up to 21%), representing 13.6 times higher risk than when breeding pigs were tested prior to shipment. The strategy of testing pigs on fattening farms 15 days prior to shipment and using a sample size sufficient to detect a prevalence of 5% with a 95% of confidence, could reduce the probability of introducing ADV-infected animals by 91% with no additional cost. Similarly, testing pigs on breeding and fattening farms using a sample size sufficient to detect a prevalence of 1% with a 95% of confidence, could reduce the probability of introducing ADV-infected animals by 99%, but with an increased cost of 81%. Results reported in this study identify factors that contribute to risk of ADV introduction and should aid the control and eradication of AD in Spain.

Assessment of viral inactivation during pH 3.3 pepsin digestion and caprylic acid treatment of antivenoms.

Antivenoms are manufactured by the fractionation of animal plasma which may possibly be contaminated by infectious agents pathogenic to humans. This study was carried out to determine whether pre-existing antivenom production steps, as carried out by EgyVac in Egypt, may reduce viral risks. Two typical manufacturing steps were studied by performing down-scaled viral inactivation experiments: (a) a pH 3.3 pepsin digestion of diluted plasma at 30 degrees C for 1h, and (b) a caprylic acid treatment of a purified F(ab')2 fragment fraction at 18 degrees C for 1h. Three lipid-enveloped (LE) viruses [bovine viral diarrhoea virus (BVDV), pseudorabies virus (PRV), and vesicular stomatitis virus (VSV)] and one non-lipid-enveloped (NLE) virus [encephalomyocarditis virus (EMC)] were used as models. Kinetics of inactivation was determined by taking samples at 3 time-points during the treatments. The pH 3.3 pepsin digestion resulted in complete clearance of PRV (>7.0 log(10)) and in almost complete reduction of VSV (>4.5 but < or =6.4 log(10)), and in a limited inactivation of BVDV (1.7 log(10)). EMC inactivation was > or =2.5 but < or =5.7 log(10). The caprylic acid treatment resulted in complete inactivation of the 3 LE viruses tested: BVDV (>6.6 log(10)), PRV (>6.6 log(10)), and VSV (>7.0 log(10)). For EMC no significant reduction was obtained (0.7 log(10)). Cumulative reduction was >13.6, >11.5, >8.3 and > or =2.5 for PRV, VSV, BVDV and EMC, respectively. Therefore the current manufacturing processes of at least some animal antisera already include production steps that can ensure robust viral inactivation of LE viruses and moderate inactivation of a NLE virus.

Detection of economically important viruses in boar semen by quantitative RealTime PCR technology.

The objective of this study was to develop quantitative real-time polymerase chain reaction (ReTi-PCR) tests for the detection of five economically important viruses in swine semen namely, pseudorabies virus (PRV), classical swine fever virus (CSFV), foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), and porcine reproductive and respiratory syndrome virus (PRRSV). Each ReTi-PCR test was validated for specificity, analytical sensitivity (detection limits), and experimental infection studies were performed to compare the conventional virus isolation methods with the newly developed ReTi-PCR tests. All five developed ReTi-PCR tests are very rapid compared to virus isolation, highly specific, and even more sensitive (lower detection limits) than conventional virus isolation methods for the detection of mentioned viruses in semen. In semen of experimentally infected boars, viruses were detected much earlier after infection and more frequently by ReTi-PCR tests than by virus isolations. The high throughput of these rapid ReTi-PCR tests makes it possible to screen large number of semen samples for the presence of viruses prior to insemination. This is a substantial advantage, in particular for boar semen the quality of which deteriorates quickly after storage. In general, the newly developed ReTi-PCR tests are valuable tools for the early, reliable and rapid detection of five economically important viruses, namely PRV, CSFV, FMDV, SVDV, and PRRSV in boar semen. These ReTi-PCR tests will improve the control of viral diseases transmitted via semen.

Assessment of replication and virulence of attenuated pseudorabies virus in swine.

A nonclinical study was conducted to characterize the replication behavior of a modified live gE-deleted pseudorabies virus (PRV MS+1) in swine and potential for reversion to virulence after animal passages. Two to 3 week-old weaned pigs, negative for PRV, were maintained in isolation and challenged by intranasal instillation. For the first passage, 6 pigs were given 1 mL of PRV MS+1 (10(7.3)TCID(50)/mL) and 2 were necropsied at 3, 4 and 5 days post-inoculation (PI). Brain and secondary lymphoid tissues were collected, homogenized and the supernatants individually pooled for virus isolation, and PRV was recovered from each sample. No clinical signs of PRV infection were observed, but each pig had a nasal swab suspect or positive for PRV. For the second passage, 5 pigs were given 1 mL of the homogenate of mixed tissues from 1 animal in the previous passage (PRV at 10(1.9) TCID(50)/mL). At 5 days PI, all pigs were necropsied, and PRV was not recovered from their tissue homogenates or nasal swabs, and no clinical signs were observed. During a second attempt at a second passage, tissue homogenates from all pigs in the first passage (PRV at approximately 10(1.7)TCID50(50)/mL) were pooled and used to inoculate 15 pigs with 2 mL for 3 consecutive days. Ten pigs were monitored for clinical signs and seroconversion through 21 days PI, and 5 pigs were necropsied at 5 days PI. No clinical signs or PRV antibodies were detected in the 10 monitored pigs, and no PRV was recovered from the homogenates or nasal swabs of the 5 necropsied pigs. Thus, no evidence of reversion to virulence was demonstrated in pigs given the attenuated PRV.

An analysis of a presumed major outbreak of pseudorabies virus in a vaccinated sow herd.

We describe a major outbreak of pseudorabies virus (PRV) in a sow herd in which the sows were vaccinated simultaneously three times a year with a vaccine containing Bartha strain. Also in the associated rearing herd in which the gilts were vaccinated twice an outbreak of PRV occurred. The outbreak was analysed with mathematical models, statistical methods and Monte-Carlo simulation. Under the assumption that the outbreak started with one introduction of virus the reproduction ratio R(ind)--as a measure of transmission of PRV between individuals--in the sow herd was estimated with a Generalized Linear Model to be 1.6. Also under the assumption of one introduction of virus R(ind) in the rearing herd was estimated with a martingale estimator to be 1.7. Both estimates were significantly larger than 1. Mathematical analysis showed that heterogeneity in the sow herd, because of the presence of not-optimally immunized replacement sows could not be the only cause of the observed outbreak in the sow herd. With Monte-Carlo simulations, the duration of an outbreak after a single introduction of virus and R(ind) = 1.6 did not mimic the data and thus the hypothesis of a single introduction with R(ind) = 1.6 could also be rejected and R(ind) is thus, not necessarily above 1. Moreover, with statistical analysis, endemicity in the combination of herds as a cause for the observed outbreak could be rejected. Endemicity in the rearing herd alone could not be excluded. Therefore, multiple introductions from outside and most probably from the rearing herd were possibly the cause of the observed outbreak(s). The implications for eradication of pseudorabies virus were discussed.

Challenges of the final stages of the ADV eradication program.

The authors were chairpersons in the session on epidemiology and control of Aujeszky's disease (AD). In this document, they focus on several issues, such as vaccination, compliance and surveillance, which influence the eradication programs. Also, some research topics which may need attention in the future are indicated. The main conclusion is that eradication programs for AD virus have made good progress in different parts of the world and that we have the knowledge and tools to do the job. It must be realized, however, that setbacks can occur. As prevalence declines, susceptibility increases and producers may let their guard down so that the virus may spread again in susceptible areas.

Risk indicators for the seroprevalence of Mycoplasma hyopneumoniae, porcine influenza viruses and Aujeszky's disease virus in slaughter pigs from fattening pig herds.

Epidemiological aspects of Mycoplasma hyopneumoniae (Mh), influenza H1N1 and H3N2 viruses, and Aujeszky's disease virus (ADV) were investigated in slaughter pigs from 50 fattening pig herds. Herd factors as potential risk indicators for respiratory disease were obtained by means of a questionnaire. At slaughter, blood samples were collected from each herd, and the proportion of seropositive pigs per herd was assessed for each of these pathogens. The median herd-level seroprevalence of the agents were: Mh 88%, H1N1 100%, H3N2 60% and ADV 90%. The percentage of herds in which all investigated fattening pigs were seronegative for these agents was: Mh 0%, H1N1 0%, H3N2 12% and ADV 18%. The percentage of herds in which all investigated fattening pigs were seropositive for these agents was: Mh 8%, H1N1 71%, H3N2 22% and ADV 40%. A positive association was found between influenza H1N1 and H3N2 viruses, and a negative association between influenza H3N2 virus and ADV. There were no risk indicators for the seroprevalence of Mh. Three risk indicators were associated with the seroprevalence of influenza H1N1 virus: a fully slatted floor, an increasing number of pigs in the municipality and dry feeding. Three risk indicators were found for the seroprevalence of influenza H3N2 virus: purchase of pigs from > or = two herds, an increasing number of pigs in the municipality and natural ventilation. The seroprevalence of ADV was influenced by two risk indicators: an increasing number of pig herds in the municipality and an increasing number of pigs per pen.

Implications derived from a mathematical model for eradication of pseudorabies virus.

Simple mathematical models based on experimental and observational data were applied to evaluate the feasibility of eradicating pseudorabies virus (PRV) regionally by vaccination and to determine which factors can jeopardise eradication. As much as possible, the models were uncomplicated and our conclusions were based on mathematical analysis. For complicated situations, Monte-Carlo simulation was used to support the conclusions. For eradication, it is sufficient that the reproduction ratio R (the number of units infected by one infectious unit) is < 1. However, R can be determined at different scales: at one end the region with the herds as units and at the other end compartments with the pigs as units. Results from modelling within herds showed that contacts between groups within a herd is important whenever R between individuals (R(ind)) is > 1 in one or more groups. This is the case within finishing herds. In addition, if the R(ind) is more than 1 within a herd, the size of the herd determines whether PRV can persist in the herd and determines the duration of persistence. Moreover, when reactivation of PRV in well-vaccinated sows is taken into account, R(ind) in sow herds is still less than 1. In sow herds with group-housing systems, it is possible that in those groups R(ind) is > 1. Results from modelling between herds showed that whether or not Rherd is < 1 in a particular region is determined by two factors: (1) the transmission of infection between nucleus herds and rearing herds through transfer of animals and (2) contacts among finishing herds and among rearing herds. The transmission between herds can be reduced by reduction of the contact rate between herds. reduction of the herd size, and reduction of the transmission within herds.

Quantitative assessment of genomic similarity from restriction fragment patterns.

A quantitative algorithm for comparing restriction fragment patterns (RFPs) was developed and used to estimate the genomic similarity of 18 isolates of pseudorabies virus of known origin. Variants of this algorithm using either untransformed or square-root-transformed differences between fragment sizes were investigated with regard to their ability to classify RFPs. Multidimensional scaling was used to represent spatially the genomic relatedness among samples, with 3 dimensions producing the most meaningful results. The square-root transformation provided more interpretable dimensions. The first dimension differentiated samples geographically, separating North American from European isolates. The second and third dimensions differentiated isolates with specific gene deletions (gE and gG, respectively) from those not having these deletions. Clusters of isolates were identified that were related either by collection from the same geographic area during a specific time period, or by laboratory intervention to create vaccines. These methods offer increased precision in the determination of genetic relatedness based on RFPs, and thus offer increased diagnostic accuracy for the determination of sources of infection.

Economic analysis of alternative AD control programmes.

The threat imposed by its virulence brings a presumption that Aujeszky's disease (AD) must be controlled because potential losses are high. Viewed as an economic problem, the decision on whether and how to control AD hinges on comparing the costs of doing so with the benefits (in terms of reduced production losses) to be gained. Four strategies are considered: (a) doing nothing, (b) suppressing and maintaining the disease at low prevalence levels by vaccination, (c) suppressing to low levels and then eradicating by culling remaining positive animals and (d) eradicating in one step by means of a test-and-slaughter policy. The net economic merits of each strategy are examined using data derived from specific vaccination studies established in Germany and the Netherlands. A computer model is developed to estimate disease costs under different technical, epidemiological and economic assumptions, allowing the economically optimal strategies to be explored. In general no single strategy can be recommended as the 'best' for dealing with AD, since it depends on a host of factors relating to pig density, prevalence levels, production system, trade relations, etc. As usual, economic realities complicate the quest for operational simplicity in disease control. However, for the regions of high pig density studied the most economic AD control strategy is to lower herd prevalence by intensive vaccination before completing eradication by test-and-removal of remaining positive animals.

Detection of virus or virus specific nucleic acid in foodstuff or bioproducts--hazards and risk assessment.

There are two possibilities for virus contamination of foodstuff and bioproducts of animal origin: i) the presence of endogenous virus as a result of an acute or subclinical infection of animal raw material used for food processing or ii) contamination of food in the course of processing or thereafter. The latter must be considered as the highest risk for human consumers since the viral contamination mostly is caused by virus shedding people and the transmitted viruses are obligate human pathogens. Food from animals consumed as raw material (e.g. oysters) is listed in a high risk category concerning viral contamination (e.g. hepatovirus). Virus contamination of bioproducts such as vaccines, blood products or biological material used in surgery and for transplantations also is more hazardous because the application of contaminating virus usually occurs by circumvention of the natural barrier systems of the body. Moreover, in many cases immunosuppressed people are treated with bioproducts. Due to an enclosing shield of high protein and lipid content in food and bioproducts viruses are well protected against physical and chemical influences, however most preparation procedures for food are destructive for viruses. The detection of pseudorabies virus and pestivirus in biological fluids was tested using polymerase chain reaction (PCR), reverse transcriptase (RT)-PCR and cell culture propagation. PCR is a powerful method to detect viral nucleic acid whereas the detection of infectious virus in cell cultures is more limited, e.g. due to protein and lipid destroying conditions. Virus contamination of bioproducts should be considered as a hazard no matter which method has been used for its detection. Examples are given about the contamination of cell lines and vaccines.

Variation in seropositivity for some respiratory disease agents in finishing pigs: epidemiological studies on some health parameters and farm and management conditions in the herds.

The relationship between the extent of seropositivity for Aujeszky's disease virus (ADV), Actinobacillus pleuropneumoniae (App.) serotype 2 and porcine influenza (PI) viruses serotype H1N1 and H3N2 in pigs on the one hand and the health status of the pigs and some farm and management conditions in the herds on the other hand was studied in 45 pig finishing herds, all members of one integration group. The health status was assessed by the extent of clinical signs, the use of veterinary drugs and the prevalence of pathological lesions in pigs at slaughter. There was no relationship between the extent of seropositivity on the one hand and clinical signs and use of veterinary drugs on the other hand. However, there was a positive relationship between the extent of seropositivity and the percentage of pigs with lesions of the respiratory tract at slaughter. Furthermore, the study indicates that the variation in seropositivity between pigs herds is associated with management related factors that particularly influence the possibility of the spreading of viruses. A sero-epidemiological investigation in 14 pig herds with recurrent pneumonia problems revealed a high percentage of seropositive pigs per herd. Furthermore, in a large number of herds, pigs were simultaneously seropositive for ADV and App. serotype 2, for ADV and PI serotype H1N1 or for ADV and PI serotype H3N2.

Economic analysis of an epizootic of pseudorabies and subsequent production following the institution of a vaccination program in a Pennsylvania swine herd.

The economic impact of pseudorabies was examined in a commercial swine herd. At the onset of clinical signs, a modified-live virus vaccine was administered to the sow herd and repeated at 3-month intervals. According to production data from the 320-sow farrow-to-feeder unit, preweaning mortality increased twofold, and subsequently, the number of pigs weaned per litter decreased by 19% (P less than 0.005) during the 5-week epizootic. Also, the number of pigs born alive decreased by 6% during the epizootic (P less than 0.05). No significant differences in production were observed between the 6-month periods before and after the epizootic. Actual cash flow analysis for the farm under isomarket conditions revealed a decreased net return of $2.40/inventoried sow/week, which was attributed to the disease during the epizootic, and a $0.46 decrease in net return/inventoried sow/week in the postepizootic period. Most seropositive herds have clinical signs less severe than those described in this herd, and the cost of eradication of the virus from a swine herd can be in excess of $200/inventoried sow. Thus, we believe that sufficient financial incentives are not available to all swine producers to ensure their enthusiastic cooperation in the effort to eradicate pseudorabies from the US swine population.

In vitro assessment of viral antigen content in inactivated aluminum hydroxide adjuvanted vaccines.

Antigen capture enzyme immunoassays (ELISA) were developed to assess the antigenic content of inactivated aluminum hydroxide (AH) adjuvanted porcine parvovirus, pseudorabies, and infectious bovine rhinotracheitis vaccines. Reference preparations of these viruses were constructed as a basis for comparison. Because AH-associated ELISA interference was largely circumvented, the need for isotopic or complex antigen-adjuvant desorption methods was eliminated. A 4-parameter logistic model related optical density to vaccine dilution. High correlation coefficients (r) were routinely achieved with commercial monovalent and polyvalent vaccines, and reference preparations. The procedure quantified antigen in both aqueous and AH-associated phases. The method may be generally applicable as a partial substitute for in vivo vaccine potency testing by allowing in vitro estimation of inactivated viral antigenic content in AH adjuvanted vaccines.

[Stich-plaque test--an economic method for quantitative determination of viruses]. / Stich -- Plaque -- Test, ein ökonomisches Verfahren zur quantitativen Virusbestimmung

An economic method for quantitative assay of viruses is presented. In this "canule stick-plaque test" (German abbreviation SPT) samples of viruses, geometrically diluted and taken up by a canule, are inoculated by sticking into monolayer cell cultures overlayed with agar medium. A plaquelike CPE detectable by neutral red staining develops in the area of the inoculation. The frequency of this CPE formation depends on the concentration of viruses in the inoculated dilution. This dose-response allows calculation of the ID 50. In this way it is possible to carry out titration involving 6 dilutions and 10 inoculations per dilution using 3 common Petri dishes (6 cm in diameter), only. The sensitivity , accuracy, and reproductibility of this method are described and discussed.

[Stick-plaque test--an economic method of quantitative determination of viruses]. / Stich-plaque--test, ein ökonomisches verfahren zur quantitativen virusbestimmung

An economic method for quantitative assay of viruses is presented. In this "canule stick-plaque test" (German abbreviation SPT) samples of viruses, geometrically diluted and taken up by a canule, are inoculated by sticking into monolayer cell cultures overlayed with agar medium. A plaquelike CPE detectable by neutral red staining develops in the area of the inoculation. The frequency of this CPE formation depends on the concentration of viruses in the inoculated dilution. This dose-response allows calculation of the ID50. In this way it is possible to carry out titration involving 6 dilutions and 10 inoculations per dilution using 3 common Petri dishes (6 cm in diameter), only. The sensitivity, accuracy, and reproductibility of this method are described and discussed.