Evidence of Lumpy Skin Disease Virus Transmission from Subclinically Infected Cattle by Stomoxys calcitrans.

Lumpy skin disease virus (LSDV) is a vector-transmitted capripox virus that causes disease in cattle. Stomoxys calcitrans flies are considered to be important vectors as they are able to transmit viruses from cattle with the typical LSDV skin nodules to naive cattle. No conclusive data are, however, available concerning the role of subclinically or preclinically infected cattle in virus transmission. Therefore, an in vivo transmission study with 13 donors, experimentally inoculated with LSDV, and 13 naïve acceptor bulls was performed whereby S. calcitrans flies were fed on either subclinical- or preclinical-infected donor animals. Transmission of LSDV from subclinical donors showing proof of productive virus replication but without formation of skin nodules was demonstrated in two out of five acceptor animals, while no transmission was seen from preclinical donors that developed nodules after Stomoxys calcitrans flies had fed. Interestingly, one of the acceptor animals which became infected developed a subclinical form of the disease. Our results show that subclinical animals can contribute to virus transmission. Therefore, stamping out only clinically diseased LSDV-infected cattle could be insufficient to completely halt the spread and control of the disease.

Wildebeest-Derived Malignant Catarrhal Fever: A Bovine Peripheral T Cell Lymphoma Caused by Cross-Species Transmission of Alcelaphine Gammaherpesvirus 1.

Gammaherpesviruses (γHVs) include viruses that can induce lymphoproliferative diseases and tumors. These viruses can persist in the long term in the absence of any pathological manifestation in their natural host. Alcelaphine gammaherpesvirus 1 (AlHV-1) belongs to the genus Macavirus and asymptomatically infects its natural host, the wildebeest (Connochaetes spp.). However, when transmitted to several susceptible species belonging to the order Artiodactyla, AlHV-1 is responsible for the induction of a lethal lymphoproliferative disease, named wildebeest-derived malignant catarrhal fever (WD-MCF). Understanding the pathogenic mechanisms responsible for the induction of WD-MCF is important to better control the risks of transmission and disease development in susceptible species. The aim of this review is to synthesize the current knowledge on WD-MCF with a particular focus on the mechanisms by which AlHV-1 induces the disease. We discuss the potential mechanisms of pathogenesis from viral entry into the host to the maintenance of viral genomes in infected CD8+ T lymphocytes, and we present current hypotheses to explain how AlHV-1 infection induces a peripheral T cell lymphoma-like disease.

Duration of Immunity Induced after Vaccination of Cattle with a Live Attenuated or Inactivated Lumpy Skin Disease Virus Vaccine.

Vaccines have proven themselves as an efficient way to control and eradicate lumpy skin disease (LSD). In addition to the safety and efficacy aspects, it is important to know the duration for which the vaccines confer protective immunity, as this impacts the design of an efficient control and eradication program. We evaluated the duration of immunity induced by a live attenuated vaccine (LSDV LAV) and an inactivated vaccine (LSDV Inac), both based on LSDV. Cattle were vaccinated and challenged after 6, 12 and 18 months for LSDV LAV or after 6 and 12 months for the LSDV Inac. The LSDV LAV elicited a strong immune response and protection for up to 18 months, as no clinical signs or viremia could be observed after a viral LSDV challenge in any of the vaccinated animals. A good immune response and protection were similarly seen for the LSDV Inac after 6 months. However, two animals developed clinical signs and viremia when challenged after 12 months. In conclusion, our data support the annual booster vaccination when using the live attenuated vaccine, as recommended by the manufacturer, which could potentially even be prolonged. In contrast, a bi-annual vaccination seems necessary when using the inactivated vaccine.

Japanese Encephalitis Virus Persistence in Porcine Tonsils Is Associated With a Weak Induction of the Innate Immune Response, an Absence of IFNγ mRNA Expression, and a Decreased Frequency of CD4+CD8+ Double-Positive T Cells.

In humans, Japanese encephalitis virus (JEV) causes a devastating neurotropic disease with high mortality, whereas in pigs, the virus only causes mild symptoms. Besides tropism to the central nervous system, JEV seems to harbor a particular tropism for the tonsils in pigs. This secondary lymphoid organ appears to act as a reservoir for the virus, and we show that it is found up to 21 days post infection at high viral titers. The immune response in the tonsils was studied over time upon intradermal inoculation of pigs. Entry of the virus in the tonsils was accompanied by a significant increase in anti-viral OAS1 and IFNß mRNA expression. This limited antiviral response was, however, not sufficient to stop JEV replication, and importantly, no IFNγ or innate inflammatory cytokine mRNA expression could be observed. Strikingly, the persistence of JEV in tonsils was also associated with a significant decreased frequency of CD4+CD8+ double-positive T lymphocytes. Furthermore, it is important to note that JEV persistence in tonsils occurred despite a strong induction of the adaptive immune response. JEV-specific antibodies were found after 6 days post infection in serum, and cell-mediated immune responses upon NS3 restimulation of PBMCs from experimentally infected pigs showed that CD4+CD8+ double-positive T cells were found to display the most prominent proliferation and IFNγ production among lymphocyte subtypes. Taken together, these results suggest that an inadequate induction of the innate immune response and the absence of an IFNγ antiviral response contribute to the persistence of JEV in the tonsils and is associated with a decrease in the frequency of CD4+CD8+ double-positive T cells.

Detection of Clinical and Subclinical Lumpy Skin Disease Using Ear Notch Testing and Skin Biopsies.

Lumpy skin disease (LSD) diagnosis is primarily based on clinical surveillance complemented by PCR of lesion crusts or nodule biopsies. Since LSD can be subclinical, the sensitivity of clinical surveillance could be lower than expected. Furthermore, real-time PCR for the detection of LSD viral DNA in blood samples from subclinical animals is only intermittently positive. Therefore, this study aimed to investigate an acceptable, easily applicable and more sensitive testing method for the detection of clinical and subclinical LSD. An animal experiment was conducted to investigate ear notches and biopsies from unaffected skin taken from the neck and dorsal back as alternatives to blood samples. It was concluded that for early LSD confirmation, normal skin biopsies and ear notches are less fit for purpose, as LSDV DNA is only detectable in these samples several days after it is detectable in blood samples. On the other hand, blood samples are less advisable for the detection of subclinical animals, while ear notches and biopsies were positive for LSD viral DNA in all subclinically infected animals by 16 days post infection. In conclusion, ear notches could be used for surveillance to detect subclinical animals after removing the clinical animals from a herd, to regain trade by substantiating the freedom of disease or to support research on LSDV transmission from subclinical animals.

Towards Efficient Early Warning: Pathobiology of African Swine Fever Virus "Belgium 2018/1" in Domestic Pigs of Different Age Classes.

African swine fever (ASF) is one of the most important and devastating viral diseases in wild boar and domestic pigs worldwide. In the absence of vaccines or treatment options, early clinical detection is crucial and requires a sound knowledge of disease characteristics. To provide practitioners and state veterinarians with detailed information, the objective of the present study was to characterize the ASF virus (ASFV) isolate "Belgium 2018/1" in subadult and weaning domestic pigs. To this end, two animal trials were performed. Trial A included eight subadult domestic pigs and trial B five weaner pigs. In general, clinical signs and pathological lesions were in line with previous studies utilizing highly virulent ASF genotype II viruses. However, in trial A, four subadult domestic pigs survived and recovered, pointing to an age-dependent outcome. The long-term fate of these survivors remains under discussion and would need further investigation.

The Importance of Quality Control of LSDV Live Attenuated Vaccines for Its Safe Application in the Field.

Vaccination is an effective approach to prevent, control and eradicate diseases, including lumpy skin disease (LSD). One of the measures to address farmer hesitation to vaccinate is guaranteeing the quality of vaccine batches. The purpose of this study was to demonstrate the importance of a quality procedure via the evaluation of the LSD vaccine, Lumpivax (Kevevapi). The initial PCR screening revealed the presence of wild type LSD virus (LSDV) and goatpox virus (GTPV), in addition to vaccine LSDV. New phylogenetic PCRs were developed to characterize in detail the genomic content and a vaccination/challenge trial was conducted to evaluate the impact on efficacy and diagnostics. The characterization confirmed the presence of LSDV wild-, vaccine- and GTPV-like sequences in the vaccine vial and also in samples taken from the vaccinated animals. The analysis was also suggestive for the presence of GTPV-LSDV (vaccine/wild) recombinants. In addition, the LSDV status of some of the animal samples was greatly influenced by the differentiating real-PCR used and could result in misinterpretation. Although the vaccine was clinically protective, the viral genomic content of the vaccine (being it multiple Capripox viruses and/or recombinants) and the impact on the diagnostics casts serious doubts of its use in the field.

Efficient control of Japanese encephalitis virus in the central nervous system of infected pigs occurs in the absence of a pronounced inflammatory immune response.

BACKGROUND: Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia. JEV infection of mice and humans can lead to an uncontrolled inflammatory response in the central nervous system (CNS), resulting in a detrimental outcome. Pigs act as important amplification and reservoir hosts, and JEV infection of pigs is mostly subclinical. Information on virus spread in the CNS and immune responses controlling JEV infection in the CNS of pigs, however remains scarce. METHODS: Nine-week-old pigs were inoculated intranasal or intradermal with a relevant dose of 105 TCID50 of JEV genotype 3 Nakayama strain. Clinical signs were assessed daily, and viral spread was followed by RT-qPCR. mRNA expression profiles were determined to study immune responses in the CNS. RESULTS: Besides a delay of 2 days to reach the peak viremia upon intranasal compared to intradermal inoculation, the overall virus spread via both inoculation routes was highly similar. JEV appearance in lymphoid and visceral organs was in line with a blood-borne JEV dissemination. JEV showed a particular tropism to the CNS but without the induction of neurological signs. JEV entry in the CNS probably occurred via different hematogenous and neuronal pathways, but replication in the brain was mostly efficiently suppressed and associated with a type I IFN-independent activation of OAS1 expression. In the olfactory bulb and thalamus, where JEV replication was not completely controlled by this mechanism, a short but strong induction of chemokine gene expression was detected. An increased IFNy expression was simultaneously observed, probably originating from infiltrating T cells, correlating with a fast suppression of JEV replication. The chemokine response was however not associated with the induction of a strong inflammatory response, nor was an induction of the NLRP3 inflammasome observed. CONCLUSIONS: These findings indicate that an adequate antiviral response and an attenuated inflammatory response contribute to a favorable outcome of JEV infection in pigs and help to explain the limited neurological disease compared to other hosts. We show that the NLRP3 inflammasome, a key mediator of neurologic disease in mice, is not upregulated in pigs, further supporting its important role in JEV infections.

An Immunoperoxidase Monolayer Assay (IPMA) for the detection of lumpy skin disease antibodies.

During this study a new Immunoperoxidase Monolayer Assay (IPMA) was developed for the detection of antibodies against lumpy skin disease virus (LSDV) in an easy and low tech setting. Using two dilutions (1:50 and 1:300) in a duplicate format, the test was shown to be highly sensitive, specific and repeatable. In comparison to the VNT and a commercial ELISA, the LSDV-IPMA was able to detect the LSDV antibodies earlier in infected, vaccinated and vaccinated/infected animals. The assay is very flexible as it can be easily adapted for the detection of sheeppox or goatpox antibodies and it can be scaled-up to handle medium size sample sets by preparing the IPMA plates in advance. These plates are safe and can be handled in low biosafety level labs.

Assessment of cross-protection induced by a bluetongue virus (BTV) serotype 8 vaccine towards other BTV serotypes in experimental conditions.

Bluetongue disease is caused by bluetongue virus (BTV) and BTV serotype 8 (BTV8) caused great economic damage in Europe during the last decade. From 1998 to 2007, in addition to BTV8, Europe had to face the emergence of BTV1, 2, 4, 9, and 16, spreading in countries where the virus has never been detected before. These unprecedented outbreaks trigger the need to evaluate and compare the clinical, virological and serological features of the European BTV serotypes in the local epidemiological context. In this study groups of calves were infected with one of the following European BTV serotypes, namely BTV1, 2, 4, 9 and 16. For each tested serotype, two groups of three male Holstein calves were used: one group vaccinated against BTV8, the other non-vaccinated. Clinical signs were quantified, viral RNA was detected in blood and organs and serological relationship was assessed. Calves were euthanized 35 days post-infection and necropsied. Most of the infected animals showed mild clinical signs. A partial serological cross reactivity has been reported between BTV8 and BTV4, and between BTV1 and BTV8. BTV2 and BTV4 viral RNA only reached low levels in blood, when compared to other serotypes, whereas in vitro growth assays could not highlight significant differences. Altogether the results of this study support the hypothesis of higher adaptation of some BTV strains to specific hosts, in this case calves. Furthermore, cross-protection resulting from a prior vaccination with BTV8 was highlighted based on cross-neutralization. However, the development of neutralizing antibodies is probably not totally explaining the mild protection induced by the heterologous vaccination.

Evaluation of mycobacteria-specific gamma interferon and antibody responses before and after a single intradermal skin test in cattle naturally exposed to M. avium subsp. paratuberculosis and experimentally infected with M. bovis.

This study reports on the diagnostic potential of IFN-γ release assays and serology for Mycobacterium bovis in six naturally M. avium subsp. paratuberculosis (Map) exposed bulls of which four were intratracheally infected with a Belgian field strain of M. bovis. Heparinized blood, serum and fecal samples were collected at regular time intervals for mycobacteria-specific IFN-γ release assays, antibody analysis and for Map culture respectively. Single intradermal skin test (SIT) with bovine tuberculin (PPD-B) was performed on day 115 and animals were sacrificed on day 133 after M. bovis infection. Organs were collected and stored for histopathological examination, modified Ziehl-Neelsen staining and bacteriological analysis of M. bovis and Map by culture and RT-PCR. Prior to infection five animals showed positive IFN-γ responses to avian PPD (PPD-A) and four were positive in Map PCR (IS900) on faeces. Three M. bovis infected animals reacted as early as day 14 with sustained higher PPD-B than PPD-A specific IFN-γ responses, whereas the fourth animal (with the strongest PPD-A response prior to infection) showed sustained higher PPD-B specific IFN-γ levels only a day 56 after infection. Two of the infected animals had a sustained positive IFN-γ response to the ESAT-6/CFP-10/TB7.7 (QuantiFERON®-TB Gold) peptide cocktail as early as day 14, among which the animal with the initial high PPD-A response. Later during infection, positive responses were found to ESAT-6 peptides in three infected bulls and to CFP-10 peptides in all four infected bulls. One of the control animals reacted intermittently to the ESAT-6/CFP10/TB7.7 cocktail. Prior to SIT, weak but positive MPB83/MBP70 specific antibody responses were detected in two of the infected bulls. All four M. bovis infected bulls reacted with a positive skin test and showed, as reported by others, increased mycobacteria specific IFN-γ production and increased positive responses in MPB83/MBP70 specific serology after SIT. At autopsy, M. bovis lesions were detected in all four experimentally infected bulls. Our results indicate that in Map exposed cattle, M. bovis diagnosis using IFN-γ assays needs a combination of PPD-B/A and ESAT-6/CFP10 for early and optimal sensitivity and that sensitivity of MPB83/MBP70 serodiagnosis is dramatically increased by prior skin testing. Map exposure did not interfere with the development of SIT in M. bovis infected animals, but resulted in a false positive M. bovis specific IFN-γ and antibody response after SIT in one of the two control animals (which remained negative in skin-test).

Experimental bluetongue virus superinfection in calves previously immunized with bluetongue virus serotype 8.

The effect of a superinfection with bluetongue virus serotype 1 (BTV1) was evaluated on two groups of four calves. One group received a commercial inactivated BTV serotype 8 (BTV8) vaccine. This group and the non-vaccinated group of calves were challenged twice (4 months apart) with the European BTV8 strain isolated during the 2006-2007 epidemics. Calves were then infected with a BTV1 inoculum which was found to be unexpectedly contaminated by BTV serotype 15 (BTV15). BTV1 and BTV15 single infections were performed on two other groups of three BTV naïve calves. A severe clinical picture was obtained after superinfection with BTV1/BTV15 in both vaccinated and non-vaccinated animals and after challenge with BTV8 in non-vaccinated animals. BTV1 and BTV15 single infection caused only very slight clinical signs. After superinfection and at the viraemic peak, there were an average of above 1000 times more BTV15 genomic copies than BTV1 ones. BTV1 RNA could be detected only in the spleen of one calf whereas BTV15 RNA was found in 15 organs of seven different animals. BTV8 immunization whether it was acquired through vaccination and challenges or challenges alone did not change BTV1 or BTV15 RNA detection in superinfected animals. However in these animals a partial cross neutralization between BTV8 and BTV1 might be involved in the lower BTV1 replication versus BTV15. Infection with different serotypes can occur also in the field. Interference between virus strains, genetic reassortment and cross-protection were considered as mechanisms to explain the clinical outcomes and the other virological and immunological findings in the course of BTV1/BTV15 superinfection.

Age- and strain-dependent differences in the outcome of experimental infections of domestic pigs with wild boar pseudorabies virus isolates.

Although pseudorabies virus (PRV) has been eradicated in domestic swine in many countries, its presence in wild boars remains a threat for a reintroduction into the currently unprotected swine population. To assess the possible impact of such a reintroduction in a naive herd, an in vivo infection study using two genetically characterized wild boar PRV isolates (BEL24043 and BEL20075) representative for wild boar strains circulating in south-western and central Europe and the virulent NIA3 reference strain was performed in 2- and 15-week-old domestic pigs. Our study revealed an attenuated nature of both wild boar strains in 15-week-old pigs. In contrast, it showed the capacity of strain BEL24043 to induce severe clinical symptoms and mortality in young piglets, thereby confirming that the known age dependency of disease outcome after PRV infection also holds for wild boar isolates. Despite the absence of clinical disease in 15-week-old sows, both wild boar PRV strains were able to induce seroconversion, but to a different extent. Importantly, differences in infection and transmission capacity of both strains were observed in 15-week-old sows. Strain BEL24043 induced a more prolonged and disseminated infection than strain BEL20075 and was able to spread efficiently to contact animals, indicative of its capacity to induce a sustained infection. In conclusion, it was shown that a reintroduction of a wild boar isolate into the domestic swine population could have serious economic consequences due to the induction of clinical symptoms in piglets and by jeopardizing the PRV-negative status.

Diagnosis of the Lelystad strain of Porcine reproductive and respiratory syndrome virus infection in individually housed pigs: comparison between serum and oral fluid samples for viral nucleic acid and antibody detection.

There has been a developing interest in the use of oral fluid for the diagnosis of different pathogens such as Porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV and PRRSV-specific antibodies have been shown to be present in oral fluid samples, but the correlation between diagnostic results in oral fluid and serum samples has been insufficiently addressed. Studies investigating this correlation focused on boars older than 6 months and type 2 strains, but it is known that the outcome of a PRRSV infection is age and strain dependent. To address this gap, the current study reports on the detection of PRRSV and PRRSV-specific antibodies in serum and oral fluid samples collected over a 6-week period after an experimental infection of 8-week-old individually housed pigs with Lelystad virus, the type 1 prototype strain. Quantitative reverse transcription polymerase chain reaction analysis showed that significantly more serum samples were PRRSV RNA-positive than oral fluid until 5 days postinfection (dpi). Between 7 and 21 dpi, PRRSV RNA detection was similar in both samples but higher detection rates in oral fluid were found from 28 dpi. Compared with existing literature, this highlights that detection rates at particular time points postinfection might vary in function of strain virulence and animal age and provides useful information for the interpretation of pen-based oral fluid results. An excellent agreement between the oral fluid and serum enzyme-linked immunosorbent assay results was observed at every time point, further supporting the usefulness of oral fluid as a diagnostic sample for antibody detection.

Experimental Schmallenberg virus infection of pigs.

Schmallenberg virus (SBV) is a newly emerged virus responsible for an acute non-specific syndrome in adult cattle including high fever, decrease in milk production and severe diarrhea. It also causes reproductive problems in cattle, sheep and goat including abortions, stillbirths and malformations. The role of pigs in the epidemiology of SBV has not yet been evaluated while this could be interesting seen their suggested role in the epidemiology of the closely related Akabane virus. To address this issue, four 12 week old seronegative piglets were subcutaneously infected with 1 ml of SBV infectious serum (FLI) and kept into contact with four non-infected piglets to examine direct virus transmission. Throughout the experiment blood, swabs and feces samples were collected and upon euthanasia at 28 dpi different organs (cerebrum, cerebellum, brain stem, lung, liver, iliac lymph nodes, kidney and spleen) were sampled. No clinical impact was observed and all collected samples tested negative for SBV in rRT-PCR. Despite the absence of viremia and virus transmission, low and short lasting amounts of neutralizing antibodies were found in 2 out of 4 infected piglets. The limited impact of SBV infection in pigs was further supported by the absence of neutralizing anti-SBV antibodies in field collected sera from indoor housed domestic pigs (n=106). In conclusion, SBV infection of pigs can induce seroconversion but is ineffective in terms of virus replication and transmission indicating that pigs have no obvious role in the SBV epidemiology.

An essential role for γ-herpesvirus latency-associated nuclear antigen homolog in an acute lymphoproliferative disease of cattle.

Wildebeests carry asymptomatically alcelaphine herpesvirus 1 (AlHV-1), a γ-herpesvirus inducing malignant catarrhal fever (MCF) to several ruminant species (including cattle). This acute and lethal lymphoproliferative disease occurs after a prolonged asymptomatic incubation period after transmission. Our recent findings with the rabbit model indicated that AlHV-1 infection is not productive during MCF. Here, we investigated whether latency establishment could explain this apparent absence of productive infection and sought to determine its role in MCF pathogenesis. First, whole-genome cellular and viral gene expression analyses were performed in lymph nodes of MCF-developing calves. Whereas a severe disruption in cellular genes was observed, only 10% of the entire AlHV-1 genome was expressed, contrasting with the 45% observed during productive infection in vitro. In vivo, the expressed viral genes included the latency-associated nuclear antigen homolog ORF73 but none of the regions known to be essential for productive infection. Next, genomic conformation analyses revealed that AlHV-1 was essentially episomal, further suggesting that MCF might be the consequence of a latent infection rather than abortive lytic infection. This hypothesis was further supported by the high frequencies of infected CD8(+) T cells during MCF using immunodetection of ORF73 protein and single-cell RT-PCR approaches. Finally, the role of latency-associated ORF73 was addressed. A lack of ORF73 did not impair initial virus replication in vivo, but it rendered AlHV-1 unable to induce MCF and persist in vivo and conferred protection against a lethal challenge with a WT virus. Together, these findings suggest that a latent infection is essential for MCF induction.

Experimental co-infections of calves with bluetongue virus serotypes 1 and 8.

The contemporary circulation of multiple bluetongue virus (BTV) serotypes or strains within the same territory can imply the co-infection of the ruminant and/or the vector populations. As a consequence, the clinical and pathological outcomes of co-infections as well as the biological properties of the viral progeny could be influenced and exhibit relevant variation. In this study, two independent co-infection experiments were carried out in calves using European strains of BTV serotypes 1 and 8 (BTV-1 and BTV-8, respectively), with the objective of studying the clinical and virological outcomes in comparison with BTV-1 and BTV-8 single infections. Synchronous co-infections using the same titre for the two viral strains were performed and the clinical signs were quantified using a standardized clinical form. Serotype-specific real-time RT-PCRs and viral isolation were used to monitor the course of viraemia. Neutralizing antibody titres were measured during the experiments, and necropsy with viral detection in the affected organs was performed. In the co-infected calves, a high BTV-8 viraemia was detected, while BTV-1 viraemia was irregular and sporadic. During BTV-1 single infection the development of viraemia and high titres of anti-BTV-1 neutralizing antibodies proved that the inoculum was infectious and the detection protocols were efficient. Several hypotheses could explain the predominant detection of BTV-8 in the co-infected calves, such as the occurrence of a privileged BTV-8 segment 2 reassortment, as recently described during in vitro BTV-1/BTV-8 co-infections; interference between the two viral strains; or a higher BTV-8 tropism for the bovine species.