A bread‐based lyophilized C‐strain CSF virus vaccine as an oral vaccine in pigs

A live attenuated cereal‐based classical swine fever (CSF) vaccine for oral application, a form of vaccine that farmers can use themselves, demonstrated the improvement of CSF control in backyard production systems in endemic areas. Due to the dependency on very low storage temperature (−20°C) of the cereal‐based oral CSF vaccine, a lyophilized cereal‐based oral vaccine has been developed and tested. Although some studies showed total protection against a virulent virus strain, the production procedure is still considered complex. In this study, the lyophilized oral CSF virus, which is easy to produce and could be kept in the form of a bread‐based vaccine at 4°C for an extended time, was tested for the ability to induce a humoral immune response in pigs. Among the materials tested as a base for the CSF virus vaccine, plain sliced bread was considered the most appropriate base because of its absorbing ability and virus titre maintenance. Titres of bread‐based lyophilized CSF virus vaccine were stable at around 3.67 log₁₀ TCID₅₀ per ml for 7 months at 4°C. Pigs aged 5 and 8 weeks that orally received five bread‐based lyophilized CSF virus vaccine showed seroconversion of over 90% at 14 dpv. At 28 dpv, both age groups showed 100% seroconversion. In conclusion, the bread‐based lyophilized CSF virus vaccine can be an alternative choice for oral vaccination of pigs. Due to the simple process of production and the need for less virus titre, vaccine prices could be lowered. However, vaccine thermostability has to be improved to allow the vaccine delivery to be less dependent on functioning cool chains.     

Quantification of Lumpy Skin Disease Virus Following Experimental Infection in Cattle

Lumpy skin disease along with sheep pox and goatpox are the most serious poxvirus diseases of livestock, and are caused by viruses that belong to the genus Capripoxvirus within the subfamily Chordopoxvirinae, family Poxviridae. To facilitate the study of lumpy skin disease pathogenesis, we inoculated eight 4‐ to 6‐month‐old Holstein calves intravenously with lumpy skin disease virus (LSDV) and collected samples over a period of 42 days for analysis by virus isolation, real‐time PCR and light microscopy. Following inoculation, cattle developed fever and skin nodules, with the extent of infection varying between animals. Skin nodules remained visible until the end of the experiment on day post‐inoculation (DPI) 42. Viremia measured by real‐time PCR and virus isolation was not observed in all animals but was detectable between 6 and 15 DPI. Low levels of viral shedding were observed in oral and nasal secretions between 12 and 18 DPI. Several tissues were assessed for the presence of virus at DPI 3, 6, 9, 12, 15, 18 and 42 by virus isolation and real‐time PCR. Virus was consistently detected by real‐time PCR and virus isolation at high levels in skin nodules indicating LSDV has a tropism for skin. In contrast, relatively few lesions were observed systemically. Viral DNA was detected by real‐time PCR in skin lesions collected on DPI 42. Cattle developing anti‐capripoxvirus antibodies starting at DPI 21 was detected by serum neutralization. The disease in this study varied from mild with few secondary skin nodules to generalized infection of varying severity, and was characterized by morbidity with no mortality.     

A Refined Guinea Pig Model of Foot‐and‐Mouth Disease Virus Infection for Assessing the Efficacy of Antiviral Compounds

An antiviral containment strategy for foot‐and‐mouth disease (FMD) outbreaks could support or replace current contingency plans in case of an outbreak in Europe and could spare many healthy animals from being pre‐emptively culled. Recently, substantial progress has been made towards the development of small molecule drugs that inhibit FMD virus (FMDV) replication in vitro. For the initial in vivo evaluation of antiviral lead molecules, a refined FMDV‐infection model in guinea pigs (GP) is herewith described. This GP model was validated by demonstrating the antiviral effect of T‐1105 (an influenza virus inhibitor with reported activity against FMDV). Sixteen animals were orally administered with T‐1105 twice daily (400 mg/kg/day) for five consecutive days and inoculated intraplantarly with 100 GPID₅₀ of the GP‐adapted FMDV strain O₁ Manisa 1 h after the first administration. The efficacy of T‐1105 was compared with that of prophylactic vaccination with a highly potent double‐oil emulsion‐inactivated O₁ Manisa vaccine. Ten animals received a single, full (2 ml) cattle vaccine dose and were inoculated 3 weeks later. Fourteen T‐1105‐treated and all vaccinated GP were completely protected from generalization of vesicular lesions. At 2 dpi, viral RNA was detected in serum of 9/16 T‐1105‐treated and of 6/10 vaccinated animals. At 4 dpi, viral RNA was detected in serum, organs and oral swabs of half of the T‐1105‐treated animals and only in the serum of 1/10 of the vaccinated animals. Mean viral RNA levels in serum and organs of T‐1105‐treated and vaccinated animals were reduced compared to untreated controls (P < 0.01). T‐1105 conferred a substantial clinical and virological protection against infection with O₁ Manisa, similar to the protection afforded by vaccination. These results validate the suitability of the enhanced GP model for the purpose of initial evaluation of inhibitors of FMDV replication and illustrate the potential of selective inhibitors of viral replication to control FMD outbreaks.     

Horizontal transmission of foot‐and‐mouth disease virus O/JPN/2010 among different animal species by direct contact

Foot‐and‐mouth disease (FMD) is highly contagious and easily transmitted among species of cloven‐hoofed animals. To investigate the transmission of FMD virus (FMDV) among different animal species, experimental infections using the O/JPN/2010 strain were performed in cows, goats and pigs. One cow or two goats/pigs were housed with a different species of inoculated animals, and clinical observations, virus shedding and antibody responses were analysed daily. Whilst all cows and goats were infected horizontally by contact with inoculated pigs, transmission from cows to goats/pigs and from goats to cows/pigs was not observed in all in‐contact animals. In particular, no pigs were infected horizontally by contact with inoculated goats. Comparison with our previous study on experimental infections among animals of the same species indicates that horizontal transmission occurred more easily between animals of the same species than between those of the different species. These findings will be useful for establishing and performing species‐specific countermeasures in farms and regions where multiple species of animals coexist in potential future outbreaks.     

Proof of Concept for the Inhibition of Foot‐and‐Mouth Disease Virus Replication by the Anti‐Viral Drug 2′‐C‐Methylcytidine in Severe Combined Immunodeficient Mice

Recent European contingency plans envisage emergency vaccination as an animal‐friendly control strategy for foot‐and‐mouth disease (FMD). Anti‐viral drugs may be used as an alternative or complementary measure. We here demonstrate that the nucleoside analogue 2′‐C‐methylcytidine (2′CMC) protects severe combined immunodeficient (SCID) mice against lethal FMD virus infection. In brief, SCID mice were inoculated with serotype A FMD virus and treated for five consecutive days with 2′CMC. All 15 treated mice remained healthy until the end of the study at 14 days post‐infection (dpi). At that time, viral RNA was no longer detected in 13 of 15 treated mice. All eight untreated mice suffered from an acute generalized disease and were euthanized for ethical reasons on average at 4 dpi. These results illustrate the potential of small molecules to control FMD.     

Foot‐and‐Mouth Disease in Feral Swine: Susceptibility and Transmission

Experimental studies of foot‐and‐mouth disease (FMD) in feral swine are limited, and data for clinical manifestations and disease transmissibility are lacking. In this report, feral and domestic swine were experimentally infected with FMDV (A24‐Cruzeiro), and susceptibility and virus transmission were studied. Feral swine were proved to be highly susceptible to A‐24 Cruzeiro FMD virus by intradermal inoculation and by contact with infected domestic and feral swine. Typical clinical signs in feral swine included transient fever, lameness and vesicular lesions in the coronary bands, heel bulbs, tip of the tongue and snout. Domestic swine exhibited clinical signs of the disease within 24 h after contact with feral swine, whereas feral swine did not show clinical signs of FMD until 48 h after contact with infected domestic and feral swine. Clinical scores of feral and domestic swine were comparable. However, feral swine exhibited a higher tolerance for the disease, and their thicker, darker skin made vesicular lesions difficult to detect. Virus titration of oral swabs showed that both feral and domestic swine shed similar amounts of virus, with levels peaking between 2 to 4 dpi/dpc (days post‐inoculation/days post‐contact). FMDV RNA was intermittently detectable in the oral swabs by real‐time RT‐PCR of both feral and domestic swine between 1 and 8 dpi/dpc and in some instances until 14 dpi/12 dpc. Both feral and domestic swine seroconverted 6–8 dpi/dpc as measured by 3ABC antibody ELISA and VIAA assays. FMDV RNA levels in animal room air filters were similar in feral and domestic swine animal rooms, and were last detected at 22 dpi, while none were detectable at 28 or 35 dpi. The FMDV RNA persisted in domestic and feral swine tonsils up to 33–36 dpi/dpc, whereas virus isolation was negative. Results from this study will help understand the role feral swine may play in sustaining an FMD outbreak, and may be utilized in guiding surveillance, epidemiologic and economic models.     

Detection of Influenza A Virus Nucleoprotein Antibodies in Oral Fluid Specimens From Pigs Infected Under Experimental Conditions Using a Blocking ELISA

In commercial swine populations, influenza is an important component of the porcine respiratory disease complex (PRDC) and a pathogen with major economic impact. Previously, a commercial blocking ELISA (FlockChek^™ Avian Influenza Virus MultiS‐Screen^® Antibody Test Kit, IDEXX Laboratories, Inc., Westbrook, ME, USA) designed to detect influenza A nucleoprotein (NP) antibodies in avian serum was shown to accurately detect NP antibodies in swine serum. The purpose of this study was to determine whether this assay could detect NP antibodies in swine oral fluid samples. Initially, the procedure for performing the NP‐blocking ELISA on oral fluid was modified from the serum testing protocol by changing sample dilution, sample volume, incubation time and incubation temperature. The detection of NP antibody was then evaluated using pen‐based oral fluid samples (n = 182) from pigs inoculated with either influenza A virus subtype H1N1 or H3N2 under experimental conditions and followed for 42 days post inoculation (DPI). NP antibodies in oral fluid were detected from DPI 7 to 42 in all inoculated groups, that is, the mean sample‐to‐negative (S/N) ratio of influenza‐inoculated pigs was significantly different (P < 0.0001) from uninoculated controls (unvaccinated or vaccinated‐uninoculated groups) through this period. Oral fluid versus serum S/N ratios from the same pen showed a correlation of 0.796 (Pearson's correlation coefficient, P < 0.0001). The results showed that oral fluid samples from influenza virus‐infected pigs contained detectable levels of NP antibodies for ≥42 DPI. Future research will be required to determine whether this approach could be used to monitor the circulation of influenza virus in commercial pig populations.     

Prolonged Detection of PCV2 and Anti‐PCV2 Antibody in Oral Fluids Following Experimental Inoculation

The onset, level and duration of PCV2 and anti‐PCV2 antibody in oral fluid were evaluated using samples collected from experimentally inoculated pigs for 98 days post‐inoculation (DPI). Pigs (n = 24) were obtained at 3 weeks of age and randomly allocated to 4 treatment pens of 6 pigs each: (i) negative control group; (ii) inoculated with PCV2a (strain ISU 40895) on DPI 0; (iii) inoculated with PCV2a (strain ISU‐40895) on DPI 0 and re‐challenged at DPIs 35 and 70; (iv) inoculated with PCV2a (ISU‐40895), PCV2b (PVG4072) and PCV2a (ISU‐4838) on DPIs 0, 35 and 70, respectively. Serum was collected from each animal, and one oral fluid sample was collected from each pen (group) every other day from DPI 2 through DPI 14 and weekly through 98 DPI. Oral fluid samples were assayed for the presence of PCV2 by quantitative polymerase chain reaction (qPCR) and anti‐PCV2 IgG, IgA and IgM antibody isotypes by enzyme‐linked immunosorbant assay (ELISA). Serum was assayed for anti‐PCV2 IgG by ELISA. Anti‐PCV2 antibodies (IgG, IgM and IgA) were detected in oral fluid from experimentally inoculated pigs from DPI 14 with IgG and IgA clearly present at 98 DPI. PCV2 was detected in oral fluid samples from all pens of inoculated pigs at 2 DPI. Thereafter, PCV2 was detected in oral fluid throughout 98 DPI. Overall, the data indicated that PCV2 infection in swine is prolonged, persists in the face of an active antibody response and can be efficiently monitored using oral fluid specimens.     

Herd Immunity Against Foot‐and‐Mouth Disease Under Different Vaccination Practices in India

A systematic vaccination programme is ongoing in India to control the three prevailing serotypes (A, O, Asia1) of foot‐and‐mouth disease (FMD) virus. Under the programme, more than 120 million bovine (term bovine applicable to both cattle and buffalo in this study) population of 221 of the 666 districts in the country are being bi‐annually vaccinated with trivalent vaccine since 2010. Although clinical disease has reduced in these districts because of the systematic vaccinations, an abrupt increase in the number of FMD cases was recorded in 2013. Hence, a longitudinal field study was conducted in the year 2014 to estimate the serological herd immunity level in bovines, the impact of systematic vaccinations and field efficacy of the vaccines used. Serum samples (n = 115 963) collected from 295 districts of the 18 states of the country were analysed to estimate antibody titres against structural proteins of the three serotypes. The efficacy of the vaccine was demonstrated in the control group (group‐D) where animals of the group were identified by ear tags for the purpose of repeated sampling after vaccination. Progressive building of the herd immunity in the field after systematic vaccination was demonstrated. The mean antibody titre against the serotypes O, A and Asia1 was estimated as log₁₀ 1.93 (95% CI 1.92–1.93), 2.02 (2.02–2.02) and 2.02 (2.02–2.02), respectively, in the states covered under the control programme. However, in other states herd immunity was significantly low [mean titre log₁₀ 1.68 (95% CI 1.67–1.69), 1.77 (1.76–1.78) and 1.85 (1.84–1.86) against the three serotypes]. Inverse relationship between the herd immunity and FMD incidences was observed the states following different vaccination practices. The study helped in demarcation of FMD risk zones in the country with low herd immunity. Estimation of herd immunity kinetics in the field helped in refining the vaccination schedule under the control programme.     

Collection of Oral Fluids Using Cotton Ropes as a Sampling Method to Detect Foot‐and‐Mouth Disease Virus Infection in Pigs

In high‐density farming practices, it is important to constantly monitor for infectious diseases, especially diseases that have the potential to spread rapidly between holdings. Pigs are known to amplify foot‐and‐mouth disease (FMD) by excreting large amounts of virus, and it is therefore important to detect the virus quickly and accurately to minimize the spread of disease. Ropes were used to collect oral fluid samples from pigs, and each sample was compared to saliva samples collected from individual animals by detecting FMD virus RNA using real‐time PCR. Two different experiments are described where groups of pigs were infected with different serotypes of FMD virus, either with or without vaccination, and unvaccinated pigs were kept in aerosol contact. The sensitivity of the rope sampling varied between 0.67 and 0.92, and the statistical agreement between this method and individual sampling ranged from substantial to moderate for the two different serotypes. The ease of collecting oral fluids using ropes together with the high sensitivity of subsequent FMD detection through PCR indicates that this could be a useful method to monitor pig populations for FMD virus infection. With further validation of the sensitivity of detection of FMD virus RNA, this can be a cost‐effective, non‐invasive diagnostic tool.     

Evidence of reduced viremia,pathogenicity and vector competence in a re‐emerging European strain of bluetongue virus serotype 8 in sheep

The outbreak of bluetongue virus (BTV) serotype 8 (BTV‐8) during 2006–2009 in Europe was the most costly epidemic of the virus in recorded history. In 2015, a BTV‐8 strain re‐emerged in France which has continued to circulate since then. To examine anecdotal reports of reduced pathogenicity and transmission efficiency, we investigated the infection kinetics of a 2007 UK BTV‐8 strain alongside the re‐emerging BTV‐8 strain isolated from France in 2017. Two groups of eight BTV‐naïve British mule sheep were inoculated with 5.75 log₁₀TCID₅₀/ml of either BTV‐8 strain. BTV RNA was detected by 2 dpi in both groups with peak viraemia occurring between 5–9 dpi. A significantly greater amount of BTV RNA was detected in sheep infected with the 2007 strain (6.0–8.8 log₁₀ genome copies/ml) than the re‐emerging BTV‐8 strain (2.9–7.9 log₁₀ genome copies/ml). All infected sheep developed BTV‐specific antibodies by 9 dpi. BTV was isolated from 2 dpi to 12 dpi for 2007 BTV‐8‐inoculated sheep and from 5 to 10 dpi for sheep inoculated with the remerging BTV‐8. In Culicoides sonorensis feeding on the sheep over the period 7–12 dpi, vector competence was significantly higher for the 2007 strain than the re‐emerging strain. Both the proportion of animals showing moderate (as opposed to mild or no) clinical disease (6/8 vs. 1/8) and the overall clinical scores (median 5.25 vs. 3) were significantly higher in sheep infected with the 2007 strain, compared to those infected with the re‐emerging strain. However, one sheep infected with the re‐emerging strain was euthanized at 16 dpi having developed severe lameness. This highlights the potential of the re‐emerging BTV‐8 to still cause illness in naïve ruminants with concurrent costs to the livestock industry.     

Direct detection and characterization of foot‐and‐mouth disease virus in East Africa using a field‐ready real‐time PCR platform

Effective control and monitoring of foot‐and‐mouth disease (FMD ) relies upon rapid and accurate disease confirmation. Currently, clinical samples are usually tested in reference laboratories using standardized assays recommended by The World Organisation for Animal Health (OIE ). However, the requirements for prompt and serotype‐specific diagnosis during FMD outbreaks, and the need to establish robust laboratory testing capacity in FMD ‐endemic countries have motivated the development of simple diagnostic platforms to support local decision‐making. Using a portable thermocycler, the T‐COR ™ 8, this study describes the laboratory and field evaluation of a commercially available, lyophilized pan‐serotype‐specific real‐time RT ‐PCR (rRT ‐PCR ) assay and a newly available FMD virus (FMDV) typing assay (East Africa‐specific for serotypes: O, A, Southern African Territories [SAT ] 1 and 2). Analytical sensitivity, diagnostic sensitivity and specificity of the pan‐serotype‐specific lyophilized assay were comparable to that of an OIE ‐recommended laboratory‐based rRT ‐PCR (determined using a panel of 57 FMDV ‐positive samples and six non‐FMDV vesicular disease samples for differential diagnosis). The FMDV ‐typing assay was able to correctly identify the serotype of 33/36 FMDV ‐positive samples (no cross‐reactivity between serotypes was evident). Furthermore, the assays were able to accurately detect and type FMDV RNA in multiple sample types, including epithelial tissue suspensions, serum, oesophageal–pharyngeal (OP ) fluid and oral swabs, both with and without the use of nucleic acid extraction. When deployed in laboratory and field settings in Tanzania, Kenya and Ethiopia, both assays reliably detected and serotyped FMDV RNA in samples (n  = 144) collected from pre‐clinical, clinical and clinically recovered cattle. These data support the use of field‐ready rRT ‐PCR platforms in endemic settings for simple, highly sensitive and rapid detection and/or characterization of FMDV.     

Investigations into the Cause of Foot‐and‐Mouth Disease Virus Seropositive Small Ruminants in Cyprus During 2007

In 2007, serological evidence for foot‐and‐mouth disease (FMD) infection was found as a result of differential diagnostic testing of Cypriot sheep suspected to be infected with bluetongue or contagious ecthyma. Seropositive sheep and goats were subsequently uncovered on ten geographically clustered flocks, while cattle and pigs in neighbouring herds were all seronegative. These antibodies were specific for serotype‐O FMD virus, reacting with both structural and non‐structural (NS) FMD viral proteins. However, no FMD virus could be recovered from the seropositive flocks. FMD had not been recorded in Cyprus since 1964 and there has been no vaccination programme since 1984. Since all the seropositive animals were at least 3 years old and home‐bred, it was concluded that infection had occurred approximately 3 years previously had passed un‐noticed and died out spontaneously. It therefore appears that antibodies to FMD virus NS proteins can still be detected around 3 years after infection of small ruminants, but that virus carriers cannot be detected at this time. This unusual situation of finding evidence of historical infection in a FMD‐free country caused considerable disruption and alarm and posed questions about the definition of what constitutes a FMD outbreak.     

Isolation,identification and retrospective study of foot‐and‐mouth disease virus from affected Mithun (Bos frontalis) in north‐eastern India

Foot‐and‐mouth disease (FMD ) is a contagious disease of cloven‐hoofed animals that causes substantial and perpetual economic loss. Apart from the contagious nature of the disease, the FMD virus can establish in a “carrier state” among all cloven‐hoofed animals. The Mithun (Bos frontalis ), popularly called the “Cattle of Mountain,” is found in the geographically isolated, hilly region of north‐east India: Arunachal Pradesh, Nagaland, Manipur and Mizoram. Despite the geographical inaccessibility, infection by FMD virus has emerged as the single most devastating disease among Mithun after the eradication of rinderpest from this region. Samples from outbreaks of FMD in Mithun were analysed by sandwich ELISA , multiplex RT ‐PCR (MRT ‐PCR ) and liquid‐phase blocking enzyme‐linked immunosorbent assay and isolated in the BHK ‐21 cell line. The results indicate the presence of FMDV serotype “O.” The sequencing and molecular phylogenies have revealed close relationships in the lineage of type “O” isolates from Bangladesh. The findings will provide useful information for further research and development of a sustainable programme for the progressive control of FMD in the Mithun population.