Development of a new loop-mediated isothermal amplification test for the sensitive, rapid, and economic detection of different genotypes of Classical swine fever virus.

Background: Classical swine fever virus (CSFV) remains one of the most important pathogens in animal health. Pathogen detection relies on viral RNA extraction followed by RT-qPCR. Novel technologies are required to improve diagnosis at the point of care. Methods: A loop-mediated isothermal amplification (LAMP) PCR technique was developed, with primers designed considering all reported CSFV genotypes. The reaction was tested using both fluorometric and colorimetric detection, in comparison to the gold standard technique. Viral strains from three circulating CSFV genotypes were tested, as well as samples from infected animals. Other pathogens were also tested, to determine the LAMP specificity. Besides laboratory RNA extraction methods, a heating method for RNA release, readily available for adaptation to field conditions was evaluated. Results: Three primer sets were generated, with one of them showing better performance. This primer set proved capable of maintaining optimal performance at a wide range of amplification temperatures (60°C - 68°C). It was also able to detect CSFV RNA from the three genotypes tested. The assay was highly efficient in detection of samples from animals infected with field strains from two different genotypes, with multiple matrices being detected using both colorimetric and fluorometric methods. The LAMP assay was negative for all the unrelated pathogens tested, including Pestiviruses. The only doubtful result in both fluorometric and colorimetric LAMP was against the novel Pestivirus italiaense, ovine Italy Pestivirus (OVPV), which has proven to have cross-reaction with multiple CSFV diagnostic techniques. However, it is only possible to detect the OVPV in a doubtful result if the viral load is higher than 10000 viral particles. Conclusion: The results from the present study show that LAMP could be an important addition to the currently used molecular diagnostic techniques for CSFV. This technique could be used in remote locations, given that it can be adapted for successful use with minimal equipment and minimally invasive samples. The joined use of novel and traditional diagnostic techniques could prove to be a useful alternative to support the CSF control.

Assessing the value of PCR assays in oral fluid samples for detecting African swine fever, classical swine fever, and foot-and-mouth disease in U.S. swine.

INTRODUCTION: Oral fluid sampling and testing offers a convenient, unobtrusive mechanism for evaluating the health status of swine, especially grower and finisher swine. This assessment evaluates the potential testing of oral fluid samples with real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) to detect African swine fever, classical swine fever, or foot-and-mouth disease for surveillance during a disease outbreak and early detection in a disease-free setting. METHODS: We used a series of logical arguments, informed assumptions, and a range of parameter values from literature and industry practices to examine the cost and value of information provided by oral fluid sampling and rRT-PCR testing for the swine foreign animal disease surveillance objectives outlined above. RESULTS: Based on the evaluation, oral fluid testing demonstrated value for both settings evaluated. The greatest value was in an outbreak scenario, where using oral fluids would minimize disruption of animal and farm activities, reduce sample sizes by 23%-40%, and decrease resource requirements relative to current individual animal sampling plans. For an early detection system, sampling every 3 days met the designed prevalence detection threshold with 0.95 probability, but was quite costly. LIMITATIONS: Implementation of oral fluid testing for African swine fever, classical swine fever, or foot-and-mouth disease surveillance is not yet possible due to several limitations and information gaps. The gaps include validation of PCR diagnostic protocols and kits for African swine fever, classical swine fever, or foot-and-mouth disease on swine oral fluid samples; minimal information on test performance in a field setting; detection windows with low virulence strains of some foreign animal disease viruses; and the need for confirmatory testing protocol development.

Quantitative approach for the risk assessment of African swine fever and Classical swine fever introduction into the United States through legal imports of pigs and swine products.

The US livestock safety strongly depends on its capacity to prevent the introduction of Transboundary Animal Diseases (TADs). Therefore, accurate and updated information on the location and origin of those potential TADs risks is essential, so preventive measures as market restrictions can be put on place. The objective of the present study was to evaluate the current risk of African swine fever (ASF) and Classical swine fever (CSF) introduction into the US through the legal importations of live pigs and swine products using a quantitative approach that could be later applied to other risks. Four quantitative stochastic risk assessment models were developed to estimate the monthly probabilities of ASF and CSF release into the US, and the exposure of susceptible populations (domestic and feral swine) to these introductions at state level. The results suggest a low annual probability of either ASF or CSF introduction into the US, by any of the analyzed pathways (5.5*10-3). Being the probability of introduction through legal imports of live pigs (1.8*10-3 for ASF, and 2.5*10-3 for CSF) higher than the risk of legally imported swine products (8.90*10-4 for ASF, and 1.56*10-3 for CSF). This could be caused due to the low probability of exposure associated with this type of commodity (products). The risk of feral pigs accessing to swine products discarded in landfills was slightly higher than the potential exposure of domestic pigs through swill feeding. The identification of the months at highest risk, the origin of the higher risk imports, and the location of the US states most vulnerable to those introductions (Iowa, Minnesota and Wisconsin for live swine and California, Florida and Texas for swine products), is valuable information that would help to design prevention, risk-mitigation and early-detection strategies that would help to minimize the catastrophic consequences of potential ASF/CSF introductions into the US.

Assessment of international inter-laboratory comparison tests for the diagnosis of classical swine fever from 1998 until 2007.

The inter-laboratory comparison tests for classical swine fever (CSF) laboratory diagnosis organised by the European Community Reference Laboratory for CSF are regularly performed within European Union Member States. The objective of this study was to evaluate the results of the inter-laboratory comparison tests carried out over the last decade, from 1998 until 2007, by using a statistical approach. A set of five or six lyophilised sera was sent to participants. These included sera containing CSF antibodies, sera containing antibodies against ruminant pestiviruses, sera containing CSF virus and negative sera. This study focused on the results of the diagnostic reference methods for CSF: the neutralisation test for the detection of CSF antibodies (including its interpretation) and virus isolation for the detection of CSF virus. For the detection of CSF antibodies, results were closest to what was expected by the Community Reference Laboratory when only neutralisation tests were performed. The percentage of correct results decreased as soon as the results of CSF antibody enzyme-linked immunosorbent assay were included or when sera with antibodies to ruminant pestiviruses were added to the panel. The results for the detection of CSF antibodies are still valid today, as no additional method has been introduced recently. Regarding CSF virus detection, CSF virus isolation is well established but on the way to being superseded as the reference test by reverse-transcription polymerase chain reaction.

[Nonspecific clinical signs in pigs and use of exclusion diagnosis for classical swine fever: a survey among pig farmers and veterinary practitioners]. / Toepassing van uitsluitdiagnostiek voor klassieke varkenspest bij aspecifieke klinische problemen op varkensbedrijven: een enquête onder varkenshouders en dierenartsen.

Outbreaks of Classical Swine Fever (CSF) occurred in spring 2006 in Germany close to the Dutch border. On 6th April Dutch pig farmers were given the possibility to submit blood samples directly via their veterinary practitioner to the National Reference Laboratory for CSF if their pigs had non-specific clinical symptoms or if pigs were being treated with antibiotics. The pig farm was not quarantined and was not visited by the veterinary authorities. Over a period of 9 weeks 156 pig farmers submitted whole blood samples via 50 different veterinary practices. All samples tested negative in the PCR test. These pig farmers and veterinary practitioners were asked to respond to a postal questionnaire with questions regarding their experience with this new diagnostic possibility, the distribution of the costs involved, a comparison with other instruments, such as official notification or use of a leukocyte count test, and their knowledge of clinical signs of CSF. 65 pig farmers (42%) and 33 veterinary practices (66%) returned the questionnaire. The main results indicated that pig farmers (72%) would use this type of exclusion diagnostics sooner than that they would approach the veterinary authorities (practitioners: 86%). Moreover the respondents considered the fact that the farm was not quarantined immediately to be an advantage (pig farmers, 79%; practitioners, 88%). 32 percent of the pig farmers were not aware that they were required to submit blood samples if pigs were being treated with antibiotics (practitioners: 11%). The majority of pig farmers and practitioners were not satisfied with the current distribution of the costs involved: in their opinion the costs of the PCR test, the costs of the veterinary practitioner and the costs for shipping the samples to the reference laboratory should be paid out of the Animal Health Fund (50% government and 50% industry) or by the government. If the current distribution of the costs is not changed, a large proportion of the pig farmers indicated that they would not use this form of exclusion diagnostics for CSF in the future. Pig farmers appeared to have a rather limited knowledge of the clinical signs of CSF: 33% of the pig farmers could mention maximally three clinical signs of CSF, and 7% could not mention a single clinical sign of CSF and said they were entirely dependent on the practitioners' ability to judge a CSF-suspect situation.

Assessment of classical swine fever diagnostics and vaccine performance.

Rapid and accurate diagnosis is of the utmost importance in the control of epizootic diseases such as classical swine fever (CSF), and efficacious vaccination can be used as a supporting tool. While most of the recently developed CSF vaccines and diagnostic kits are mostly validated according to World Organisation for Animal Health (OIE) standards, not all of the well-established traditional vaccines and diagnostic tests were subject to these validation procedures and requirements. In this report, data were compiled on performance and validation of CSF diagnostic tests and vaccines. In addition, current strategies for differentiating infected from vaccinated animals are reviewed, as is information on the control of CSF in wildlife. Evaluation data on diagnostic tests were kindly provided by National Reference Laboratories for CSF in various European countries.

Cost-effectiveness of measures to prevent classical swine fever introduction into The Netherlands.

Recent history has demonstrated that classical swine fever (CSF) epidemics can incur high economic losses, especially for exporting countries that have densely populated pig areas and apply a strategy of non-vaccination, such as The Netherlands. Introduction of CSF virus (CSFV) remains a continuing threat to the pig production sector in The Netherlands. Reducing the annual probability of CSFV introduction (P(CSFV)) by preventive measures is therefore of utmost importance. The choice of preventive measures depends not only on the achieved reduction of the annual P(CSFV), but also on the expenditures required for implementing these measures. The objective of this study was to explore the cost-effectiveness of tactical measures aimed at the prevention of CSFV introduction into The Netherlands. For this purpose for each measure (i) model calculations were performed with a scenario tree model for CSFV introduction and (ii) its annual cost was estimated. The cost-effectiveness was then determined as the reduction of the annual P(CSFV) achieved by each preventive measure (DeltaP) divided by the annual cost of implementing that measure (DeltaC). The measures analysed reduce the P(CSFV) caused by import or export of pigs. Results showed that separation of national and international transport of pigs is the most cost-effective measure, especially when risk aversion is assumed. Although testing piglets and breeding pigs by a quick and reliable PCR also had a high cost-effectiveness ratio, this measure is not attractive due to the high cost per pig imported. Besides, implementing such a measure is not allowed under current EU law, as it is trade restrictive.

The effectiveness of classical swine fever surveillance programmes in The Netherlands.

Consequences of classical swine fever (CSF) epidemics depend on the control measures, but also on the number of infected herds at the end of the high-risk period (HRP). Surveillance programmes aim to keep this number as low as possible, so the effectiveness of surveillance programmes can be measured by the number of infected herds at the end of the HRP. In this paper, an evaluation of the effectiveness of the following five Dutch CSF surveillance programmes is presented: (1) routine gross pathology of severely diseased pigs; (2) routine virological tests of tonsils of all pigs, submitted under 1; (3) daily clinical observation by the farmer; (4) periodic clinical inspection by a veterinarian; (5) leucocyte counts in blood samples from diseased animals on a herd where antimicrobial 'group therapy' is started. The evaluation was done by a modelling study, in which virus transmission, disease development, and actions and diagnostic tests in surveillance programmes were simulated. Also, the yearly costs of the programmes were calculated, and direct costs of CSF epidemics were related to the number of infected herds at the end of the HRP. It appeared that the current Dutch surveillance programmes, without the leucocyte counts, keep the number of infected herds at the end of the HRP below 20 with 95% probability. Leaving out the most-expensive programme of periodic inspection (12.5M per year) does not change this result - indicating that (for CSF surveillance) the programme could well be stopped. If the leucocyte programme, which is currently not effective due to the low sample submission rate, optimally were applied, the 95th percentile could be reduced to 10 infected herds. However, whether application is beneficial is unclear, because of uncertainty of the economic benefits due to the many expected false-positive herds each year.

Quantitative assessment of clinical signs for the detection of classical swine fever outbreaks during an epidemic.

The performance of clinical signs as a diagnostic test for the detection of classical swine fever (CSF) outbreaks during the 1997-1998 CSF epidemic in The Netherlands was evaluated by constructing and analysing a receiver operating characteristic (ROC) curve. This curve assesses the discriminating ability of a diagnostic test over a range of test signals. The cut-off values for a defined diagnostic test to detect CSF outbreaks were set by different combinations of clinical signs observed. The area under the ROC curve, which is a quantitative measure of test performance, was significantly (P<0.001) larger than the area under the random ROC curve. This indicates that clinical signs have a significantly higher performance as a diagnostic test for the detection of CSF than for flipping a coin. However, the gain in diagnostic performance compared to a random process is not as much as we would wish it to be. The optimal efficient diagnostic test combined a sensitivity of 72.7% with a specificity of 52.7%, with a combination of the following clinical signs: unsteady gait/ataxia, not eating, not reacting to antibiotic treatment, conjunctivitis, hard faecal pellets.

[The leukocyte count is a valuable parameter for detecting classical swine fever]. / Leukocytentelling is een waardevol hulpmiddel voor het opsporen van klassieke varkenspest.

In this paper we describe a study of the use of the white blood cell count (wbcc) as a parameter for detecting outbreaks of Classical Swine Fever (CSF). Meta-analysis of the results of challenge experiments revealed that oronasal infection of SPF-pigs with the virulent CSF virus (CSFV) strains Brescia or NL9201 resulted in a significant decrease in the average white blood cell count during the first week after inoculation of the virus. Challenge of conventional finishing pigs and sows with the moderately virulent strain Paderborn also resulted in a significant decrease in the average wbcc. However, this decrease was not observed after inoculation of SPF pigs with the mildly virulent CSFV strains Henken, Zoelen, or Bergen. The usefulness of clinical inspection in combination with wbcc to detect CSF outbreaks in the field was examined using the results of 214 EDTA blood specimens collected from 22 infected herds and 7250 EDTA blood specimens collected from 1450 non-infected herds. Half of the infected herds had been infected with the moderately virulent CSFV strain Venhorst (closely related to strain Paderborn) during the 1997-98 epidemic in the Netherlands. The other half had been infected with the moderately virulent CSFV strain Loraine. Using these data as a starting point, 1000 samples of one to ten specimens were generated by Monte Carlo simulation. These simulated samples and the samples of the non-infected herds were analysed by use of Receiver Operating Characteristic curves. On the basis of that analysis, the optimal number of animals whose wbcc needed to be determined to detect a CSF outbreak was five. With this number of animals, in conjunction with the threshold of 8000 white blood cells per mm3 (meaning that a herd is designated as CSF suspect if one or more of the five specimens has a white blood cell count of 8000 leukocytes/mm3 or less), the test procedure had a herd sensitivity (HSE) of 94.5% and a herd specificity (HSP) of 97.2%). The HSE is defined as the percentage of samples of infected herds with a positive result of the test procedure; HSP is defined as the percentage of uninfected herds with a negative result of the test procedure. We conclude that the wbcc can help the veterinary practitioner to detect outbreaks of CSF caused by (moderately) virulent CSFV strains. However, for the detection of outbreaks caused by mildly virulent CSFV strains, the contribution of the wbcc is doubtful. Development of additional tools that can improve the clinical diagnosis of the veterinary practitioner remains desirable.