Outbreak investigations and molecular characterization of foot‐and‐mouth disease viruses circulating in south‐west Niger

In Niger, the epidemiological situation regarding foot‐and‐mouth disease is unclear as many outbreaks are unreported. This study aimed (i) to identify Foot‐and‐mouth disease virus (FMDV ) strains currently circulating in cattle herds, and (ii) to identify risk factors associated with Foot‐and‐mouth disease (FMD )‐seropositive animals in clinical outbreaks. Epithelial tissues (n  = 25) and sera (n  = 227) were collected from cattle in eight districts of the south‐western part of Niger. Testing of clinical material revealed the presence of FMDV serotype O that was characterized within the O/WEST AFRICA topotype. The antigenic relationship between one of the FMDV isolates from Niger (O/NGR /4/2015) and three reference vaccine strains was determined by the two‐dimensional virus neutralization test (2dmVNT ), revealing a close antigenic match between the field isolate from Niger and three FMDV serotype O vaccine strains. Serological analyses using a non‐structural protein (NSP ) test provided evidence for previous FMDV infection in 70% (158/227) of the sera tested. Multivariate logistic regression analysis revealed that only the herd composition (presence of both cattle and small ruminants) was significantly associated with FMDV seropositivity as defined by NSP ‐positive results (p ‐value = .006). Of these positive sera, subsequent testing by liquid‐phase blocking ELISA (LPBE ) showed that 86% (136/158) were positive for one (or more) of four FMDV serotypes (A, O, Southern African Territories (SAT ) 1 and SAT 2). This study provides epidemiological information about FMD in the south‐western part of Niger and highlights the complex transboundary nature of FMD in Africa. These findings may help to develop effective control and preventive strategies for FMD in Niger as well, as other countries in West Africa.     

Early Detection of Foot‐And‐Mouth Disease Virus from Infected Cattle Using A Dry Filter Air Sampling System

Foot‐and‐mouth disease (FMD) is a highly contagious livestock disease of high economic impact. Early detection of FMD virus (FMDV) is fundamental for rapid outbreak control. Air sampling collection has been demonstrated as a useful technique for detection of FMDV RNA in infected animals, related to the aerogenous nature of the virus. In the current study, air from rooms housing individual (n = 17) or two groups (n = 4) of cattle experimentally infected with FDMV A24 Cruzeiro of different virulence levels was sampled to assess the feasibility of applying air sampling as a non‐invasive, screening tool to identify sources of FMDV infection. Detection of FMDV RNA in air was compared with first detection of clinical signs and FMDV RNA levels in serum and oral fluid. FMDV RNA was detected in room air samples 1–3 days prior (seven animals) or on the same day (four animals) as the appearance of clinical signs in 11 of 12 individually housed cattle. Only in one case clinical signs preceded detection in air samples by one day. Overall, viral RNA in oral fluid or serum preceded detection in air samples by 1–2 days. Six individually housed animals inoculated with attenuated strains did not show clinical signs, but virus was detected in air in one of these cases 3 days prior to first detection in oral fluid. In groups of four cattle housed together, air detection always preceded appearance of clinical signs by 1–2 days and coincided more often with viral shedding in oral fluid than virus in blood. These data confirm that air sampling is an effective non‐invasive screening method for detecting FMDV infection in confined to enclosed spaces (e.g. auction barns, milking parlours). This technology could be a useful tool as part of a surveillance strategy during FMD prevention, control or eradication efforts.     

Foot‐and‐mouth disease virus transmission dynamics and persistence in a herd of vaccinated dairy cattle in India

Foot‐and‐mouth disease (FMD ) is an important transboundary disease with substantial economic impacts. Although between‐herd transmission of the disease has been well studied, studies focusing on within‐herd transmission using farm‐level outbreak data are rare. The aim of this study was to estimate parameters associated with within‐herd transmission, host physiological factors and FMD virus (FMDV ) persistence using data collected from an outbreak that occurred at a large, organized dairy farm in India. Of 1,836 regularly vaccinated, adult dairy cattle, 222 had clinical signs of FMD over a 39‐day period. Assuming homogenous mixing, a frequency‐dependent compartmental model of disease transmission was built. The transmission coefficient and basic reproductive number were estimated to be between 16.2–18.4 and 67–88, respectively. Non‐pregnant animals were more likely to manifest clinical signs of FMD as compared to pregnant cattle. Based on oropharyngeal fluid (probang) sampling and FMDV ‐specific RT ‐PCR , four of 36 longitudinally sampled animals (14%) were persistently infected carriers 10.5 months post‐outbreak. There was no statistical difference between subclinical and clinically infected animals in the duration of the carrier state. However, prevalence of NSP ‐ELISA antibodies differed significantly between subclinical and clinically infected animals 12 months after the outbreak with 83% seroprevalence amongst clinically infected cattle compared to 69% of subclinical animals. This study further elucidates within‐herd FMD transmission dynamics during the acute‐phase and characterizes duration of FMDV persistence and seroprevalence of FMD under natural conditions in an endemic setting.     

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.     

A Review of OIE Country Status Recovery Using Vaccinate‐to‐Live Versus Vaccinate‐to‐Die Foot‐and‐Mouth Disease Response Policies II: Waiting Periods After Emergency Vaccination in FMD Free Countries

For countries with OIE status, FMD free country where vaccination is not practised, vaccinate‐to‐live policies have a significant economic disincentive as the trade restriction waiting period is double that of vaccinate‐to‐die policies. The disposal of healthy vaccinated animals strictly for the purpose of regaining markets with debatable scientific justification is a global concern. The feasibility of aligning the waiting periods to facilitate vaccinate‐to‐live is explored. The first article of this two‐part review (Barnett et al., 2015) explored the qualities of higher potency Foot‐and‐Mouth Disease (FMD) vaccines, performance of differentiating infected from vaccinated animals (DIVA) diagnostic assays particularly in vaccinates and carriers, as well as aspects of current limitations of post‐outbreak surveillance. Here, the history behind the OIE waiting periods for FMD free status is reviewed as well as whether the risk of vaccinated animals and their subsequent products differ appreciably at 3 versus 6 months. It is concluded that alignment is feasible for vaccinate‐to‐live using higher potency FMD vaccines within the current OIE waiting period framework of 3 and 6 months blocks of time. These waiting periods reflect precedence, historical practicalities and considered expert opinion rather than a specific scientific rationale. The future lies in updated epidemiological and diagnostic technology to establish an acceptable level of statistical certainty for surveillance or target probability of freedom of FMDV (infection or circulation) not time restricted waiting periods. The OIE Terrestrial Code limits trade from a FMD free country where vaccination is not practiced to animal products and live non‐vaccinated animals. The risk of FMDV in products derived from higher potency vaccinated animals is appreciably less than for countries with infected FMD status or even from a FMD free country where vaccination is practised for which the Code has Articles with guidelines for safe trade with time restrictions of 3 months or less. All these presume that key requirements in the implementation of emergency vaccination including appropriate vaccine match, vaccine application, susceptible population coverage, etc. are addressed.     

Genetic diversity and comparison of diagnostic tests for characterization of foot‐and‐mouth disease virus strains from Pakistan 2008–2012

We report the laboratory analysis of 125 clinical samples from suspected cases of foot‐and‐mouth disease (FMD ) in cattle and Asian buffalo collected in Pakistan between 2008 and 2012. Of these samples, 89 were found to contain viral RNA by rRT ‐PCR , of which 88 were also found to contain infectious FMD virus (FMDV ) by virus isolation (VI ), with strong correlation between these tests (κ = 0.96). Samples that were VI ‐positive were serotyped by antigen detection ELISA (Ag‐ELISA ) and VP 1 sequence acquisition and analysis. Sequence data identified FMDV serotypes A (n  = 13), O (n  = 36) and Asia‐1 (n  = 41), including three samples from which both serotypes Asia‐1 and O were detected. Serotype A viruses were classified within three different Iran‐05 sublineages: HER ‐10, FAR ‐11 and ESF ‐10. All serotype Asia‐1 were within Group VII (Sindh‐08 lineage), in a genetic clade that differs from viruses isolated prior to 2010. All serotypes O were classified as PanAsia‐2 within two different sublineages: ANT ‐10 and BAL ‐09. Using VP 1 sequencing as the gold standard for serotype determination, the overall sensitivity of Ag‐ELISA to correctly determine serotype was 74%, and serotype‐specific sensitivity was 8% for serotype A, 88% for Asia‐1 and 89% for O. Serotype‐specific specificity was 100% for serotype A, 93% for Asia‐1 and 94% for O. Interestingly, 12 of 13 serotype A viruses were not detected by Ag‐ELISA . This study confirms earlier accounts of regional genetic diversity of FMDV in Pakistan and highlights the importance of continued validation of diagnostic tests for rapidly evolving pathogens such as FMDV .     

Challenges for Serology‐Based Characterization of Foot‐and‐Mouth Disease Outbreaks in Endemic Areas; Identification of Two Separate Lineages of Serotype O FMDV in Uganda in 2011

Control of foot‐and‐mouth disease (FMD) in Uganda by ring vaccination largely depends on costly trivalent vaccines, and use of monovalent vaccines could improve the cost effectiveness. This, however, requires application of highly specific diagnostic tests. This study investigated outbreaks of FMD in seven Ugandan districts, during 2011, using the PrioCHECK^® FMDV NS ELISA, solid‐phase blocking ELISAs (SPBEs) and virus neutralization tests (VNTs), together with virological analyses for characterization of the responsible viruses. Two hundred and eighteen (218) cattle and 23 goat sera as well as 82 oropharyngeal fluid/epithelial tissue samples were collected. Some 50% of the cattle and 17% of the goat sera were positive by the PrioCHECK^® FMDV NS ELISA, while SPBEs identified titres ≥80 for antibodies against serotype O FMD virus (FMDV) in 51% of the anti‐NSP positive cattle sera. However, 35% of the anti‐NSP positive cattle sera had SPBE titres ≥80 against multiple serotypes, primarily against serotypes O, SAT 1 and SAT 3. Comparison of SPBEs and VNTs for the detection of antibodies against serotypes O, SAT 1 and SAT 3 in 72 NSP positive cattle sera showed comparable results against serotype O (P = 0.181), while VNTs detected significantly fewer samples positive for antibodies against SAT 1 and SAT 3 than the SPBEs (P < 0.001). Detection of antibodies against serotype O was consistent with the isolation of serotype O FMDVs from 13 samples. Four of these viruses were sequenced and belonged to two distinct lineages within the East Africa‐2 (EA‐2) topotype, each differing from the currently used vaccine strain (EA‐1 topotype). The relationships of these lineages to other serotype O viruses in the Eastern Africa region are discussed. To enhance the control of FMD in Uganda, there is need to improve the specificity of the SAT‐SPBEs, perform vaccine matching and implement improved regional FMD control.     

Detection and genetic characterization of foot‐and‐mouth disease viruses in samples from clinically healthy animals in endemic settings

A total of 1501 oral swab samples from Pakistan, Afghanistan and Tajikistan were collected from clinically healthy animals between July 2008 and August 2009 and assayed for the presence of foot‐and‐mouth disease virus (FMDV) RNA. The oral swab samples from two (of four) live animal markets in Pakistan (n = 245), one (of three) live animal market in Afghanistan (n = 61) and both the live animal markets in Tajikistan (n = 120) all tested negative. However, 2 of 129 (∼2%) samples from Gondal and 11 of 123 (9%) from Chichawatni markets in Pakistan were positive for FMDV RNA. Similarly, 12 of 81 (15%) samples from Kabul and 10 of 20 (50%) from Badakhshan in Afghanistan were found to be positive. Serotypes A and O of FMDV were identified within these samples. Oral swab samples were also collected from dairy colonies in Harbanspura, Lahore (n = 232) and Nagori, Karachi (n = 136), but all tested negative for FMDV. In the Landhi dairy colony, Pakistan, a cohort of 179 apparently healthy animals was studied. On their arrival within the colony, thirty‐nine (22%) of these animals were found positive for FMDV RNA (serotype A was identified), while 130 (72.6%) had antibodies to FMDV non‐structural proteins. Thus, newly introduced animals may be a significant source of the disease in the colony. Only two animals from the cohort were detected as becoming positive for FMDV RNA during a follow‐up period of 4 months; however, only 10 animals remained negative for anti‐NSP antibodies during this period.     

Similar frequency of Porcine circovirus 3 (PCV‐3) detection in serum samples of pigs affected by digestive or respiratory disorders and age‐matched clinically healthy pigs

Porcine circovirus 3 (PCV‐3) has been identified in pigs affected by different disease conditions, although its pathogenicity remains unclear. The objective of the present study was to assess the frequency of PCV‐3 infection in serum samples from animals suffering from post‐weaning respiratory or digestive disorders as well as in healthy animals. A total of 315 swine serum samples were analysed for PCV‐3 DNA detection by conventional PCR; positive samples were further assayed with a quantitative PCR and partially sequenced. Sera were obtained from 4 week‐ to 4 month‐old pigs clinically diagnosed with respiratory (n = 129) or digestive (n = 126) disorders. Serum samples of age‐matched healthy animals (n = 60) served as negative control. Pigs with clinical respiratory signs had a wide variety of pulmonary lesions including suppurative bronchopneumonia, interstitial pneumonia, fibrinous‐necrotizing pneumonia and/or pleuritis. Animals with enteric signs displayed histopathological findings like villus atrophy and fusion, catarrhal enteritis and/or catarrhal colitis. Overall, PCV‐3 DNA was detected in 19 out of 315 analysed samples (6.0%). Among the diseased animals, PCV‐3 was found in 6.2% (8 out of 129) and 5.6% (7 out of 126) of pigs with respiratory and digestive disorders, respectively. The frequency of PCV‐3 PCR positive samples among healthy pigs was 6.7% (4 out of 60). No apparent association was observed between PCR positive cases and any type of histopathological lesion. The phylogenetic analysis of the partial genome sequences obtained showed high identity among viruses from the three groups of animals studied. In conclusion, PCV‐3 was present in the serum of diseased and healthy pigs to similar percentages, suggesting that this virus does not seem to be causally associated with respiratory or enteric disorders.     

A Review of OIE Country Status Recovery Using Vaccinate‐to‐Live Versus Vaccinate‐to‐Die Foot‐and‐Mouth Disease Response Policies I: Benefits of Higher Potency Vaccines and Associated NSP DIVA Test Systems in Post‐Outbreak Surveillance

To rapidly return to trade, countries with OIE status, FMD‐free country where vaccination is not practised, have destroyed emergency vaccinated animals, raising ethical concerns with respect to social values, the environment, animal welfare and global food security. This two‐part review explores whether science could support eligibility to return to previous OIE status in 3 months irrespective of vaccinate‐to‐live or vaccinate‐to‐die policies. Here, we examine the benefits of higher potency (≥ 6 PD₅₀), high‐purity vaccines formulated from antigen banks for emergency use, their efficacy and performance in differentiating infected from vaccinated animals (DIVA) assays for post‐outbreak surveillance. From an intensive programme of research, we conclude that high‐quality, higher potency vaccines are proven to reduce FMD virus (FMDV) subclinical circulation and the risk of carriers. Broader coverage than predicted by serology suggests the potential to hold a few ‘key’ vaccine strains improving logistics and reducing the financial burden of antigen banks. The OIE should adopt formal definitions for emergency vaccination and emergency vaccines. In terms of supportive tools, we consider that the lack of OIE recognition of DIVA tests other than those of PANAFTOSA in cattle is a shortcoming. There is need for research on maternal antibody interference with DIVA tests and on the use of such tests to establish whether greater purification of vaccines improves performance. We consider that alignment of waiting periods for vaccinate‐to‐live and vaccinate‐to‐die in OIE Code Article 8.5.9 1 b. and c. is feasible until an acceptable level of statistical certainty for surveillance or target probability of freedom is established to substantiate the absence of FMDV infection or circulation. It is surveillance intensity rather than waiting periods that establishes the risk of residual FMDV. EU Directive 2003/85/EC implicitly recognizes this, permitting derogation of the OIE waiting periods.     

Development of a Microsphere‐based Immunoassay for Serological Detection of African Horse Sickness Virus and Comparison with Other Diagnostic Techniques

African horse sickness (AHS) is a viral disease that causes high morbidity and mortality rates in susceptible Equidae and therefore significant economic losses. More rapid, sensitive and specific assays are required by diagnostic laboratories to support effective surveillance programmes. A novel microsphere‐based immunoassay (Luminex assay) in which beads are coated with recombinant AHS virus (AHSV) structural protein 7 (VP7) has been developed for serological detection of antibodies against VP7 of any AHSV serotype. The performance of this assay was compared with that of a commercial enzyme‐linked immunosorbent assay (ELISA) and commercial lateral flow assay (LFA) on a large panel of serum samples from uninfected horses (n = 92), from a reference library of all AHSV serotypes (n = 9), on samples from horses experimentally infected with AHSV (n = 114), and on samples from West African horses suspected of having AHS (n = 85). The Luminex assay gave the same negative results as ELISA when used to test the samples from uninfected horses. Both assays detected antibodies to all nine AHSV serotypes. In contrast, the Luminex assay detected a higher rate of anti‐VP7 positivity in the West African field samples than did ELISA or LFA. The Luminex assay detected anti‐VP7 positivity in experimentally infected horses at 7 days post‐infection, compared to 13 days for ELISA. This novel immunoassay provides a platform for developing multiplex assays, in which the presence of antibodies against multiple ASHV antigens can be detected simultaneously. This would be useful for serotyping or for differentiating infected from vaccinated animals.     

Control of Bovine Brucellosis from Persistently Infected Holdings Using RB51 Vaccination with Test‐and‐Slaughter: A Comparative Case Report from a High Incidence Area in Portugal

Bovine brucellosis due to Brucella abortus infection causes significant reproductive and production losses in cattle and is a major zoonosis. Eradication of this disease has proved difficult to achieve in Portugal where it still occurs in some regions despite an ongoing national eradication programme. In 2004, the Alentejo region, a major cattle producing area, reported one of the highest levels of bovine brucellosis in the country, especially in one divisional area. In that area, bovine brucellosis was particularly problematic in a holding of ten herds, the largest extensive cattle unit in the country, which remained infected despite an extensive test‐and‐slaughter programme and depopulation of five herds. A 5‐year programme of RB51 vaccination with biannual test‐and‐slaughter was thus implemented in 2004. The apparent animal seroprevalence decreased from 19% (646/3,400) to 3% (88/2930) on the third herd‐level test and remained below 0.8% (27/3324) after the fourth test. After the tenth test, the holding had a prevalence of 0.1% (2/2332) and only one herd remained positive with a within‐herd prevalence of 1.1% (2/177). The results were compared to all other herds (n = 10) in the divisional area that were also persistently infected but were subject only to test‐and‐slaughter before being depopulated. In these herds, the strategy of test‐and‐slaughter did not reduce the prevalence, which remained significantly higher than the vaccinated group (median = 0.48% and 8.5% in vaccinated versus non‐vaccinated herds; Wilcoxon rank sum test; P < 0.01). The success of this pilot programme in continental Portugal provided a valuable case study to the official veterinary services by illustrating the value of RB51 vaccination with parallel testing and improved biosecurity as a comprehensive and sustainable strategy for bovine brucellosis control in persistently infected herds.     

Antibodies Against Foot‐and‐mouth Disease (FMD) Virus in African Buffalos (Syncerus caffer) in Selected National Parks in Uganda (2001–2003)

In East Africa, the foot‐and‐mouth disease (FMD) virus (FMDV) isolates have over time included serotypes O, A, C, Southern African Territories (SAT) 1 and SAT 2, mainly from livestock. SAT 3 has only been isolated in a few cases and only in African buffalos (Syncerus caffer). To investigate the presence of antibodies against FMDV serotypes in wildlife in Uganda, serological studies were performed on buffalo serum samples collected between 2001 and 2003. Thirty‐eight samples from African buffalos collected from Lake Mburo, Kidepo Valley, Murchison Falls and Queen Elizabeth National Parks were screened using Ceditest^® FMDV NS to detect antibodies against FMDV non‐structural proteins (NSP). The seroprevalence of antibodies against non‐structural proteins was 74%. To characterize FMDV antibodies, samples were selected and titrated using serotype‐specific solid phase blocking enzyme linked immunosorbent assay (ELISAs). High titres of antibodies (≥1 : 160) against FMDV serotypes SAT 1, SAT 2 and SAT 3 were identified. This study suggests that African buffalos in the different national parks in Uganda may play an important role in the epidemiology of SAT serotypes of FMDV.     

Detection of Foot‐and‐mouth Disease Virus RNA and Capsid Protein in Lymphoid Tissues of Convalescent Pigs Does Not Indicate Existence of a Carrier State

A systematic study was performed to investigate the potential of pigs to establish and maintain persistent foot‐and‐mouth disease virus (FMDV) infection. Infectious virus could not be recovered from sera, oral, nasal or oropharyngeal fluids obtained after resolution of clinical infection with any of five FMDV strains within serotypes A, O and Asia‐1. Furthermore, there was no isolation of live virus from tissue samples harvested at 28–100 days post‐infection from convalescent pigs recovered from clinical or subclinical FMD. Despite lack of detection of infectious FMDV, there was a high prevalence of FMDV RNA detection in lymph nodes draining lesion sites harvested at 35 days post‐infection, with the most frequent detection recorded in popliteal lymph nodes (positive detection in 88% of samples obtained from non‐vaccinated pigs). Likewise, at 35 dpi, FMDV capsid antigen was localized within follicles of draining lymph nodes, but without concurrent detection of FMDV non‐structural protein. There was a marked decline in the detection of FMDV RNA and antigen in tissue samples by 60 dpi, and no antigen or viral RNA could be detected in samples obtained at 100 dpi. The data presented herein provide the most extensive investigation of FMDV persistence in pigs. The overall conclusion is that domestic pigs are unlikely to be competent long‐term carriers of infectious FMDV; however, transient persistence of FMDV protein and RNA in lymphoid tissues is common following clinical or subclinical infection.     

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.     

Circulation of bluetongue virus 8 in French cattle,before and after the re‐emergence in 2015

Bluetongue virus serotype 8 (BTV ‐8) re‐emerged in Central France in August 2015. The viral strain identified is nearly identical to the one that circulated during the 2006/2009 massive outbreak throughout Europe. To address the question of an undetected BTV ‐8 circulation on the French territory, a serological study was conducted on young cattle along a transect of seven departments, three of them located in areas where the virus presence had been confirmed by RT ‐PCR by winter 2015/2016. Sera from 2,565 animals were collected during the winters preceding and following the re‐emergence, with 414 animals being sampled in each of the two consecutive years. All samples were tested by competitive ELISA (IDV et) and, when enough serum was available, ELISA ‐positive samples were confirmed by seroneutralization tests. In areas with infected holdings, seropositive animals were found before the re‐emergence (N  = 14 of 511), significantly more on the following year (N  = 17 of 257), and eight animals (N  = 158) seroconverted over 2015. Seropositive animals were also detected as early as winter 2014/2015 in one department without known infected holdings (N  = 12 of 150), and in winter 2015/2016 in three of them (N  = 21 of 555), where seven animals (N  = 154) seroconverted over 2015. These results suggest that BTV ‐8 may have spread at low levels before the re‐emergence, even in areas considered virus‐free. Unfortunately, whole blood from the seropositive animals was not available to definitely confirm the virus presence by RT ‐PCR .     

Model of persistent foot‐and‐mouth disease virus infection in multilayered cells derived from bovine dorsal soft palate

Foot‐and‐mouth disease virus (FMDV) causes a highly contagious vesicular disease in livestock, with serious consequences for international trade. The virus persists in the nasopharynx of cattle and this slows down the process to obtain an FMDV‐free status after an outbreak. To study biological mechanisms, or to identify molecules that can be targeted to diagnose or interfere with persistence, we developed a model of persistent FMDV infection in bovine dorsal soft palate (DSP). Primary DSP cells were isolated after commercial slaughter and were cultured in multilayers at the air‐liquid interface. After 5 weeks of culture without further passage, the cells were infected with FMDV strain O/FRA/1/2001. Approximately, 20% of cells still had a polygonal morphology and displayed tight junctions as in stratified squamous epithelia. Subsets of cells expressed cytokeratin and most or all cells expressed vimentin. In contrast to monolayers in medium, multilayers in air demonstrated only a limited cytopathic effect. Integrin α_Vβ₆ expression was observed in mono‐ but not in multilayers. FMDV antigen, FMDV RNA and live virus were detected from day 1 to 28, with peaks at day 1 and 2. The proportion of infected cells was highest at 24 hr (3% and 36% of cells at an MOI of 0.01 and 1, respectively). At day 28 after infection, at a time when animals that still harbour FMDV are considered carriers, FMDV antigen was detected in 0.2%–2.1% of cells, in all layers, and live virus was isolated from supernatants of 6/8 cultures. On the consensus level, the viral genome did not change within the first 24 hr after infection. Only a few minor single nucleotide variants were detected, giving no indication of the presence of a viral quasispecies. The air‐liquid interface model of DSP brings new possibilities to investigate FMDV persistence in a controlled manner.