Pre-registration efficacy study of a novel marker vaccine against classical swine fever on maternally derived antibody positive (MDA+) target animals.

Maternally Derived Antibodies (MDA) can have a negative effect on the efficacy of live attenuated vaccines against classical swine fever (CSF). For this reason, a marker vaccine candidate CP7_E2alf was tested for its efficacy in the presence of MDA. Pregnant sows were vaccinated four weeks before farrowing with CSF virus (CSFV) strain "Thiverval". A total of 40 piglets with MDAs were included in this study. At six weeks of age the piglets were allocated into three treatment groups using generalized randomized block design (GRBD) blocking on serological status and pen location. Of the 40 piglets with MDAs, 30 piglets were vaccinated either orally (n = 15) or intramuscularly (n = 15) with a single dose of vaccine candidate produced under Good Laboratory Practice (GLP) conditions. The ten remaining piglets were allocated into the untreated control group. All 40 piglets were oronasally challenged with 2 ml of the highly virulent CSFV strain "Koslov" 14 days after vaccination. It was revealed that presence of MDAs negatively influences the efficacy of the live marker vaccine candidate, however, the extent of this negative impact depends on the route of vaccine administration. Based on our observations, intramuscular vaccination is recommended during CSF control programs in order to develop superior immune protection.

Pre-registration efficacy study of a novel marker vaccine against classical swine fever on Maternally Derived Antibody negative (MDA-) target animals.

Classical swine fever (CSF) marker vaccine candidate CP7_E2alf produced under Good Laboratory Practice (GLP) conditions by Pfizer was tested on 40 six-week-old MDA-piglets according to the European Pharmacopoeia (Ph.Eur.) requirements. Single doses of CP7_E2alf were given to 15 piglets orally, while 15 other piglets were intramuscularly vaccinated. Ten additional animals were included as unvaccinated controls. All piglets were oronasally challenged with the highly virulent CSF virus (CSFV) strain "Koslov" 14 days after vaccination. CP7_E2alf administered i.m. provided a complete protection, while p.o. administratrion triggered only partial protection. The level of protection was determined by the development of clinical signs, viraemia and rate of mortality. The vaccine candidate met the criteria of Ph. Eur Monograph 0065, "Swine-fever vaccine (live, prepared in cell cultures), classical" 7th Edition, which claims the efficacy test is invalid if fewer than 50 per cent of the control piglets display typical signs of serious infection of CSF or die, and if fewer than 100 per cent of the control piglets show clinical signs of disease within 21 days following challenge. Fulfilling these validity criteria is a key step in the registration procedure for a vaccine candidate to become openly available.

Evaluation of classical swine fever virus antibody detection assays with an emphasis on the differentiation of infected from vaccinated animals.

The aim of this study was to evaluate the general characteristics of commercially available enzyme-linked immunosorbent assays (ELISAs) to detect antibody against classical swine fever (CSF), as well as to assess their potential use as accompanying marker tests able to differentiate infected from vaccinated animals (DIVA). The Chekit* CSF-Sero and the HerdChek* CSFV Ab, both of which detect antibodies against the E2 protein of classical swine fever virus (CSFV), had the highest sensitivity. Both tests were practicable and showed good reproducibility. Comparable sensitivity was shown by the Chekit* CSF-Marker, an Erns ELISA. However, this test does not allow differentiation between antibodies directed against ruminant pestiviruses and those against CSFV. Therefore, it is not suitable for use with the chimeric marker vaccines tested. The PrioCHECK CSFV Erns was the only ELISA suitable for use in DIVA with marker vaccines containing Erns proteins from ruminant pestiviruses. However, this test was less sensitive and selective than the E2-ELISAs and cannot be recommended.

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.

Evaluation of blowfly larvae (Diptera: Calliphoridae) as possible reservoirs and mechanical vectors of African swine fever virus.

In 2014, highly virulent African swine fever virus (ASFV) was introduced into the Baltic States and Poland, with new cases being reported almost every week from wild boar and also from domestic pigs. Contrary to initial predictions that the disease would either die out due to the high virulence of the virus strain or spread rapidly in westerly direction, the infection became endemic and spread slowly. The unexpected disease epidemiology led to the hypothesis that hitherto unconsidered factors might contribute to virus persistence and dispersal. To check whether arthropod species feeding and developing on infected carcasses might be involved, larvae of two commonly found blowfly species, Lucilia sericata and Calliphora vicina, were experimentally bred on ASFV-infected spleen tissue. After different time intervals, developing larvae and pupae were tested for infectious virus and viral DNA. By qPCR, contamination of the blowfly larvae and pupae with ASFV-DNA could be demonstrated even after several washing steps, proving the uptake of virus during feeding in the larval stage. However, infectious virus could never be isolated. By contrast, the larvae appeared to have inactivated ASFV in the offered tissue, which might be explained by the known anti-biotic effect of salivary secretions. It is concluded that immature blowfly stages do not play a relevant role as reservoirs or mechanical vectors of ASFV.

No evidence for long-term carrier status of pigs after African swine fever virus infection.

This study targeted the assessment of a potential African swine fever virus (ASFV) carrier state of 30 pigs in total which were allowed to recover from infection with ASFV "Netherlands'86" prior exposure to six healthy sentinel pigs for more than 2 months. Throughout the whole trial, blood and swab samples were subjected to routine virological and serological investigations. At the end of the trial, necropsy of all animals was performed and viral persistence and distribution were assessed. Upon infection, a wide range of clinical and pathomorphological signs were observed. After an initial acute phase in all experimentally inoculated pigs, 66.6% recovered completely and seroconverted. However, viral genome was detectable in blood samples for up to 91 days. Lethal outcomes were observed in 33.3% of the pigs with both acute and prolonged courses. No ASFV transmission occurred over the whole in-contact phase from survivors to sentinels. Similarly, infectious ASFV was not detected in any of the tissue samples from ASFV convalescent and in-contact pigs. These findings indicate that the suggested role of ASFV survivors is overestimated and has to be reconsidered thoroughly for future risk assessments.

Simplifying sampling for African swine fever surveillance: Assessment of antibody and pathogen detection from blood swabs.

African swine fever (ASF) is a notifiable disease with serious socio-economic consequences that has been present in wild boar in the Baltic States and Poland since 2014. An introduction of ASF is usually accompanied by increased mortality, making fallen wild boar and hunted animals with signs of disease the main target for early warning and passive surveillance. It is difficult, however, to encourage hunters and foresters to report and take samples from these cases. A pragmatic and easy sampling approach with quick-drying swabs could facilitate this. In this study, we further evaluated the use of dry blood swabs for the detection of ASFV antibody and genome with samples from animal trials and diagnostic submissions (blood, bone and organs) from Estonia. Compared to serum samples, dried blood swabs yielded 93.1% (95% confidence interval: [83.3, 98.1]) sensitivity and 100% [95.9, 100.0] specificity in a commercial ASFV antibody ELISA. Similarly, the swabs gave a sensitivity of 98.9% [93.4, 100.0] and a specificity of 98.1% [90.1, 100.0] for genome detection by a standard ASFV p72 qPCR when compared to EDTA blood. The same swabs were tested in a VP72-antibody lateral flow device, with a sensitivity of 94.7% [85.4, 98.9] and specificity of 96.1% [89.0, 99.2] compared to the serum ELISA. When GenoTube samples tested in ELISA and LFD were compared, the sensitivity was 96.3% [87.3, 99.5] and the specificity was 93.8% [86.0, 97.9]. This study demonstrates reliable detection of ASFV antibody and genome from swabs. A field test of the swabs with decomposed wild boar carcasses in an endemic area in Estonia also gave promising results. Thus, this technique is a practical approach for surveillance of ASF in both free and endemic areas.

Faecal Escherichia coli as biological indicator of spatial interaction between domestic pigs and wild boar (Sus scrofa) in Corsica.

On the Mediterranean island of Corsica, cohabitation between sympatric domestic pigs and Eurasian wild boar (Sus scrofa) is common and widespread and can facilitate the maintenance and dissemination of several pathogens detrimental for the pig industry or human health. In this study, we monitored a population of free-ranging domestic pigs reared in extensive conditions within a 800-ha property located in Central Corsica which was frequently visited by a sympatric population of wild boar between 2013 and 2015. We used GPS collars to assess evidence of a spatially shared environment. Subsequently, we analysed by PFGE of XbaI-restricted DNA if those populations shared faecal Escherichia coli clones that would indicate contact and compared these results with those collected in a distant (separated by at least 50 km) population of wild boar used as control. Results showed that one of eight wild boars sampled in the study area shed E. coli XbaI clones identical to clones isolated from domestic pig sounders from the farm, while wild boar populations sampled in distant parts of the study area shared no identical clone with the domestic pigs monitored. Interestingly, within the sampled pigs, two identical clones were found in 2013 and in 2015, indicating a long-time persisting colonization type. Although the method of isolation of E. coli and PFGE typing of the isolates requires intensive laboratory work, it is applicable under field conditions to monitor potential infectious contacts. It also provides evidence of exchange of microorganisms between sympatric domestic pigs and wild boar populations.

The double-antigen ELISA concept for early detection of Erns -specific classical swine fever virus antibodies and application as an accompanying test for differentiation of infected from marker vaccinated animals.

Emergency vaccination with live marker vaccines represents a promising control strategy for future classical swine fever (CSF) outbreaks, and the first live marker vaccine is available in Europe. Successful implementation is dependent on a reliable accompanying diagnostic assay that allows differentiation of infected from vaccinated animals (DIVA). As induction of a protective immune response relies on virus-neutralizing antibodies against E2 protein of CSF virus (CSFV), the most promising DIVA strategy is based on detection of Erns -specific antibodies in infected swine. The aim of this study was to develop and to evaluate a novel Erns -specific prototype ELISA (pigtype CSFV Erns Ab), which may be used for CSF diagnosis including application as an accompanying discriminatory test for CSFV marker vaccines. The concept of a double-antigen ELISA was shown to be a solid strategy to detect Erns -specific antibodies against CSFV isolates of different genotypes (sensitivity: 93.5%; specificity: 99.7%). Furthermore, detection of early seroconversion is advantageous compared with a frequently used CSFV E2 antibody ELISA. Clear differences in reactivity between sera taken from infected animals and animals vaccinated with various marker vaccines were observed. In combination with the marker vaccine CP7_E2alf, the novel ELISA represents a sensitivity of 90.2% and a specificity of 93.8%. However, cross-reactivity with antibodies against ruminant pestiviruses was observed. Interestingly, the majority of samples tested false-positive in other Erns -based antibody ELISAs were identified correctly by the novel prototype Erns ELISA and vice versa. In conclusion, the pigtype CSFV Erns Ab ELISA can contribute to an improvement in routine CSFV antibody screening, particularly for analysis of sera taken at an early time point after infection and is applicable as a DIVA assay. An additional Erns antibody assay is recommended for identification of false-positive results in a pig herd immunized with the licensed CP7_E2alf marker vaccine.

Virulence of current German PEDV strains in suckling pigs and investigation of protective effects of maternally derived antibodies.

Porcine epidemic diarrhea (PED) has caused tremendous losses to the United States pig industry since 2013. From 2014, outbreaks were also reported from Central Europe. To characterize the Central European PEDV strains regarding their virulence in suckling piglets, and to assess the protective effect of maternally derived antibodies (MDA), four trial groups were randomly assigned, each consisting of two pregnant sows and their litter. To induce MDA in a subset of piglets, two sows received a cell culture-adapted PEDV strain, and another two sows were inoculated with field material from German PED outbreaks. Four sows stayed naïve. Subsequently, all piglets were inoculated with the corresponding PEDV strains at an age of 3 to 6 days, and virus shedding, clinical signs and occurrence of specific antibodies were assessed. Piglets without MDA showed a morbidity of 100% and low lethality, while almost all MDA-positive piglets stayed clinically healthy and showed considerably lower virus shedding. Taken together, the Central European PEDV strains showed rather low virulence under experimental conditions, and pre-inoculation of sows led to a solid protection of their offspring. The latter is the prerequisite for a sow vaccination concept that could help to prevent PED induced losses in the piglet sector.

Experimental Evaluation of Faecal Escherichia coli and Hepatitis E Virus as Biological Indicators of Contacts Between Domestic Pigs and Eurasian Wild Boar.

Domestic pigs and Eurasian wild boar (Sus scrofa) share several important viral and bacterial pathogens. Therefore, direct and indirect contacts between domestic pigs and wild boar present a risk of pathogen spillover and can lead to long-term perpetuation of infection. Biological indicators could be a powerful tool to understand and characterize contacts between wild boar and domestic pigs. Here, faecal Escherichia coli and Hepatitis E virus (HEV) were explored as potential biological indicators under experimental conditions. The data gained in our pilot study suggest that faecal E. coli can be used as biological indicator of contact between wild boar and domestic pig. For HEV, faecal transmission was also confirmed. However, molecular studies on full-genome basis did not reveal markers that would allow tracing of transmission direction. Based on these promising results, future field studies will especially target the practicability of E. coli microbiome molecular typing as surrogate of contacts at the wildlife-livestock interface.

High Prevalence of Highly Variable Atypical Porcine Pestiviruses Found in Germany.

Recently, a novel atypical porcine pestivirus (APPV) with significant distribution was described in the USA. Subsequent screening of the German pig sector showed a high prevalence of APPV with high variability among strains. First indication of a cell culture isolate is provided which will allow further investigations like pathogenesis studies.

Biological characterization of African swine fever virus genotype II strains from north-eastern Estonia in European wild boar.

Due to its impact on animal health and pig industry, African swine fever (ASF) is regarded as one of the most important viral diseases of pigs. Following the ongoing epidemic in the Transcaucasian countries and the Russian Federation, African swine fever virus was introduced into the Estonian wild boar population in 2014. Epidemiological investigations suggested two different introductions into the southern and the north-eastern part of Estonia. Interestingly, outbreak characteristics varied considerably between the affected regions. While high mortality and mainly virus-positive animals were observed in the southern region, mortality was low in the north-eastern area. In the latter, clinically healthy, antibody-positive animals were found in the hunting bag and detection of virus was rare. Two hypotheses could explain the different behaviour in the north-east: (i) the frequency of antibody detections combined with the low mortality is the tail of an older, so far undetected epidemic wave coming from the east, or (ii) the virus in this region is attenuated and leads to a less severe clinical outcome. To explore the possibility of virus attenuation, a re-isolated ASFV strain from the north-eastern Ida-Viru region was biologically characterized in European wild boar. Oronasal inoculation led to an acute and severe disease course in all animals with typical pathomorphological lesions. However, one animal recovered completely and was subsequently commingled with three sentinels of the same age class to assess disease transmission. By the end of the trial at 96 days post-initial inoculation, all animals were completely healthy and neither virus nor viral genomes were detected in the sentinels or the survivor. The survivor, however, showed high antibody levels. In conclusion, the ASFV strain from north-eastern Estonia was still highly virulent but nevertheless, one animal recovered completely. Under the experimental conditions, no transmission occurred from the survivor to susceptible sentinel pigs.

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.

Replication of classical swine fever virus strains and isolates in different porcine cell lines.

Classical swine fever virus (CSFV) is an economically important pathogen of domestic pigs and wild boar. Due to the highly variable clinical picture of CSF, laboratory methods are essential for an unambiguous diagnosis. Virus isolation using cell culture is still considered the gold standard. It is based on the incubation of permissive cells with organ or leukocyte preparations followed by antigen detection. In the "EU Diagnostic Manual for CSF Diagnosis", the permanent cell line PK(15) (porcine kidney) is recommended. In the European Reference Laboratory (EURL) a clone of this cell line, PK(15)A, and the STE (swine testicular epitheloid) cell line are in use for propagation of CSFV. The aim of this work was to assess the relative ability of eleven permanent cell lines derived from various organs of wild boar and domestic pig, respectively, to support the replication of different strains and isolates in comparison to these cell lines. An avirulent and a highly virulent laboratory CSFV strain, and several recent field isolates from domestic pigs and wild boars were used. Titers were determined after one, two and three virus passages, and after 48 and 120 h of incubation. Of the eleven cell lines analyzed, two were found that replicated all the tested CSFV strains and field isolates. Those may be useful for improving diagnosis of CSFV and for preparing low-passaged virus stocks of new isolates.

Attitudes and Beliefs of Pig Farmers and Wild Boar Hunters Towards Reporting of African Swine Fever in Bulgaria, Germany and the Western Part of the Russian Federation.

This study investigated the attitudes and beliefs of pig farmers and hunters in Germany, Bulgaria and the western part of the Russian Federation towards reporting suspected cases of African swine fever (ASF). Data were collected using a web-based questionnaire survey targeting pig farmers and hunters in these three study areas. Separate multivariable logistic regression models identified key variables associated with each of the three binary outcome variables whether or not farmers would immediately report suspected cases of ASF, whether or not hunters would submit samples from hunted wild boar for diagnostic testing and whether or not hunters would report wild boar carcasses. The results showed that farmers who would not immediately report suspected cases of ASF are more likely to believe that their reputation in the local community would be adversely affected if they were to report it, that they can control the outbreak themselves without the involvement of veterinary services and that laboratory confirmation would take too long. The modelling also indicated that hunters who did not usually submit samples of their harvested wild boar for ASF diagnosis, and hunters who did not report wild boar carcasses are more likely to justify their behaviour through a lack of awareness of the possibility of reporting. These findings emphasize the need to develop more effective communication strategies targeted at pig farmers and hunters about the disease, its epidemiology, consequences and control methods, to increase the likelihood of early reporting, especially in the Russian Federation where the virus circulates.

The contribution of work-up or additional views to the accuracy of diagnostic mammography.

This study examines the influence of work-up on the accuracy of diagnostic mammography in symptomatic women. Subjects were sampled from all women consecutively attending a symptomatic breast clinic and aged 25-55 years (240 women shown to have breast cancer and 240 age-matched women shown not to have cancer). Mammography films were prospectively reported by two radiologists independently of each other and in a blinded manner using two phases of film reading: initially only baseline films were read, then mammography was re-read with work-up films. The accuracy of reporting mammography with and without work-up was compared using sensitivity and specificity, likelihood ratios, and receiver operating characteristic (ROC) curves. Reporting the mammogram with work-up (compared to without any work-up films) improved sensitivity (75.3% vs 69.0%, P=0.059) for one radiologist, with a non-significant gain in specificity (84.5% vs 79.4%, P=0.38). For the other radiologist, it resulted in a non-significant decrease in sensitivity (79.5% vs 83.7%, P=0.14) with a significant increase in specificity (85.6% vs 61.9%, P=0.00001). ROC curves for both radiologists showed that reporting mammography with work-up resulted in significant improvement (4.5% for R1, 6.8% for R2) in overall test accuracy. Our findings support the use of work-up mammography in the diagnostic setting.

A case of mistaken identity. A rare presentation of gonadal dysgenesis.

BACKGROUND: The incidence of gonadal dysgenesis (hermaphroditism) is recognised to be low. Rarer still is an initial late presentation in the general practice setting. OBJECTIVE: To present a case study of a 35 year old man diagnosed as a hermaphrodite after routine investigations in general practice for lower abdominal pain. He has normal male external genitalia, a fully formed uterus and vagina, with no identifiable gonads. DISCUSSION: This incidental finding in general practice is supported by a 46,X,i(Yp)/45,X karyotype and mosaicism for an isochromosome of the short arm of the Y. It is not unusual that with normal male genitalia, such patients are likely to survive undiagnosed or incorrectly diagnosed into adulthood.

Analysis of spatio-temporal patterns of African swine fever cases in Russian wild boar does not reveal an endemic situation.

African swine fever (ASF) is a highly lethal viral disease of domestic pigs and wild boar. ASF was introduced into the southern Russian Federation in 2007 and is now reported to be spreading in populations of wild and domestic suids. An endemic situation in the local wild boar population would significantly complicate management of the disease in the livestock population. To date no sound method exists for identifying the characteristic pattern of an endemic situation, which describes infection persisting from generation to generation in the same population. To support urgent management decisions at the wildlife-livestock interface, a new algorithm was constructed to test the hypothesis of an endemic disease situation in wildlife on the basis of case reports. The approach described here uses spatial and temporal associations between observed diagnostic data to discriminate between endemic and non-endemic patterns of case occurrence. The algorithm was validated with data from an epidemiological simulation model and applied to ASF case data from southern Russia. Based on the algorithm and the diagnostic data available, the null hypothesis of an endemic situation of ASF in wild boar of the region was rejected.

Cytokine and immunoglobulin isotype profiles during CP7_E2alf vaccination against a challenge with the highly virulent Koslov strain of classical swine fever virus.

CP7_E2alf is a promising marker vaccine candidate against classical swine fever (CSF). To better understand the mechanisms of protection, cytokine and isotype-specific antibody profiles were investigated in CP7_E2alf vaccinated pigs before and after challenge with the highly virulent CSFV strain "Koslov" at 14 days or 6 months post-vaccination. The interference of vaccination with CSFV pathogeny-related cytokine responses, previously described following a moderately virulent challenge, was confirmed. However, the levels of additional cytokines, TNF-α and IL-6, were significantly attenuated by vaccination following highly virulent challenge. This vaccine interference with cytokine response was not dependent on the immunization route or the consequence of competition between vaccine and challenge strain. Interestingly, IFN-γ enhancement and persistent high IgG2 levels suggested an important role of cell-mediated immunity in long-term protection against CSFV induced by CP7_E2alf vaccination. IgA production also revealed a stimulation of mucosal immunity, especially after oral administration of the vaccine.