Detection of African Swine Fever Virus and Blood Meals of Porcine Origin in Hematophagous Insects Collected Adjacent to a High-Biosecurity Pig Farm in Lithuania; A Smoking Gun?

A seasonal trend of African swine fever (ASF) outbreaks in domestic pig farms has been observed in affected regions of Eastern Europe. Most outbreaks have been observed during the warmer summer months, coinciding with the seasonal activity pattern of blood-feeding insects. These insects may offer a route for introduction of the ASF virus (ASFV) into domestic pig herds. In this study, insects (hematophagous flies) collected outside the buildings of a domestic pig farm, without ASFV-infected pigs, were analyzed for the presence of the virus. Using qPCR, ASFV DNA was detected in six insect pools; in four of these pools, DNA from suid blood was also identified. This detection coincided with ASFV being reported in the wild boar population within a 10 km radius of the pig farm. These findings show that blood from ASFV-infected suids was present within hematophagous flies on the premises of a pig farm without infected animals and support the hypothesis that blood-feeding insects can potentially transport the virus from wild boars into domestic pig farms.

Unrecognized circulation of SAT 1 foot-and-mouth disease virus in cattle herds around Queen Elizabeth National Park in Uganda.

BACKGROUND: Foot-and-mouth disease (FMD) is endemic in Uganda in spite of the control measures used. Various aspects of the maintenance and circulation of FMD viruses (FMDV) in Uganda are not well understood; these include the role of the African buffalo (Syncerus caffer) as a reservoir for FMDV. To better understand the epidemiology of FMD at the livestock-wildlife-interface, samples were collected from young, unvaccinated cattle from 24 pastoral herds that closely interact with wildlife around Queen Elizabeth National Park in Uganda, and analysed for evidence of FMDV infection. RESULTS: In total, 37 (15%) of 247 serum samples had detectable antibodies against FMDV non-structural proteins (NSPs) using a pan-serotypic assay. Within these 37 sera, antibody titres ≥ 80 against the structural proteins of serotypes O, SAT 1, SAT 2 and SAT 3 were detected by ELISA in 5, 7, 4 and 3 samples, respectively, while neutralizing antibodies were only detected against serotype O in 3 samples. Two FMDV isolates, with identical VP1 coding sequences, were obtained from probang samples from clinically healthy calves from the same herd and are serotype SAT 1 (topotype IV (EA-I)). Based on the VP1 coding sequences, these viruses are distinct from previous cattle and buffalo SAT 1 FMDV isolates obtained from the same area (19-30% nucleotide difference) and from the vaccine strain (TAN/155/71) used within Uganda (26% nucleotide difference). Eight herds had only one or a few animals with antibodies against FMDV NSPs while six herds had more substantial evidence of prior infection with FMDV. There was no evidence for exposure to FMDV in the other ten herds. CONCLUSIONS: The two identical SAT 1 FMDV VP1 sequences are distinct from former buffalo and cattle isolates from the same area, thus, transmission between buffalo and cattle was not demonstrated. These new SAT 1 FMDV isolates differed significantly from the vaccine strain used to control Ugandan FMD outbreaks, indicating a need for vaccine matching studies. Only six herds had clear serological evidence for exposure to O and SAT 1 FMDV. Scattered presence of antibodies against FMDV in other herds may be due to the occasional introduction of animals to the area or maternal antibodies from past infection and/or vaccination. The evidence for asymptomatic FMDV infection has implications for disease control strategies in the area since this obstructs early disease detection that is based on clinical signs in FMDV infected animals.

Characterization of foot-and-mouth disease viruses (FMDVs) from Ugandan cattle outbreaks during 2012-2013: evidence for circulation of multiple serotypes.

To investigate the foot-and-mouth disease virus (FMDV) serotypes circulating in Uganda's cattle population, both serological and virological analyses of samples from outbreaks that occurred during 2012-2013 were performed. Altogether, 79 sera and 60 oropharyngeal fluid (OP)/tissue/oral swab samples were collected from herds with reported FMD outbreaks in seven different Ugandan districts. Overall, 61/79 (77%) of the cattle sera were positive for antibodies against FMDV by PrioCHECK FMDV NS ELISA and solid phase blocking ELISA detected titres ≥ 80 for serotypes O, SAT 1, SAT 2 and SAT 3 in 41, 45, 30 and 45 of these 61 seropositive samples, respectively. Virus neutralisation tests detected the highest levels of neutralising antibodies (titres ≥ 45) against serotype O in the herds from Kween and Rakai districts, against SAT 1 in the herd from Nwoya district and against SAT 2 in the herds from Kiruhura, Isingiro and Ntungamo districts. The isolation of a SAT 2 FMDV from Isingiro was consistent with the detection of high levels of neutralising antibodies against SAT 2; sequencing (for the VP1 coding region) indicated that this virus belonged to lineage I within this serotype, like the currently used vaccine strain. From the Wakiso district 11 tissue/swab samples were collected; serotype A FMDV, genotype Africa (G-I), was isolated from the epithelial samples. This study shows that within a period of less than one year, FMD outbreaks in Uganda were caused by four different serotypes namely O, A, SAT 1 and SAT 2. Therefore, to enhance the control of FMD in Uganda, there is need for efficient and timely determination of outbreak virus strains/serotypes and vaccine matching. The value of incorporating serotype A antigen into the imported vaccines along with the current serotype O, SAT 1 and SAT 2 strains should be considered.

Characterisation of recent foot-and-mouth disease viruses from African buffalo (Syncerus caffer) and cattle in Kenya is consistent with independent virus populations.

BACKGROUND: Understanding the epidemiology of foot-and-mouth disease (FMD), including roles played by different hosts, is essential for improving disease control. The African buffalo (Syncerus caffer) is a reservoir for the SAT serotypes of FMD virus (FMDV). Large buffalo populations commonly intermingle with livestock in Kenya, yet earlier studies have focused on FMD in the domestic livestock, hence the contribution of buffalo to disease in livestock is largely unknown. This study analysed 47 epithelia collected from FMD outbreaks in Kenyan cattle between 2008 and 2012, and 102 probang and serum samples collected from buffalo in three different Kenyan ecosystems; Maasai-Mara (MME) (n = 40), Tsavo (TSE) (n = 33), and Meru (ME) (n = 29). RESULTS: Antibodies against FMDV non-structural proteins were found in 65 of 102 (64%) sera from buffalo with 44/102 and 53/102 also having neutralising antibodies directed against FMDV SAT 1 and SAT 2, respectively. FMDV RNA was detected in 42% of the buffalo probang samples by RT-qPCR (Cycle Threshold (Ct) ≤32). Two buffalo probang samples were positive by VI and were identified as FMDV SAT 1 and SAT 2 by Ag-ELISA, while the latter assay detected serotypes O (1), A (20), SAT 1 (7) and SAT 2 (19) in the 47 cattle epithelia. VP1 coding sequences were generated for two buffalo and 21 cattle samples. Phylogenetic analyses revealed SAT 1 and SAT 2 virus lineages within buffalo that were distinct from those detected in cattle. CONCLUSIONS: We found that FMDV serotypes O, A, SAT 1 and SAT 2 were circulating among cattle in Kenya and cause disease, but only SAT 1 and SAT 2 viruses were successfully isolated from clinically normal buffalo. The buffalo isolates were genetically distinct from isolates obtained from cattle. Control efforts should focus primarily on reducing FMDV circulation among livestock and limiting interaction with buffalo. Comprehensive studies incorporating additional buffalo viruses are recommended.

Foot-and-mouth disease virus serotype SAT 3 in long-horned Ankole calf, Uganda.

After a 16-year interval, foot-and-mouth disease virus serotype SAT 3 was isolated in 2013 from an apparently healthy long-horned Ankole calf that grazed close to buffalo in Uganda. The emergent virus strain is ≈20% different in nucleotide sequence (encoding VP1 [viral protein 1]) from its closest relatives isolated previously from buffalo in Uganda.

A serological survey for antibodies against foot-and-mouth disease virus (FMDV) in domestic pigs during outbreaks in Kenya.

Foot-and-mouth disease (FMD) is endemic in Kenya and has been well studied in cattle, but not in pigs, yet the role of pigs is recognised in FMD-free areas. This study investigated the presence of antibodies against FMD virus (FMDV) in pigs sampled during a countrywide random survey for FMD in cattle coinciding with SAT 1 FMDV outbreaks in cattle. A total of 191 serum samples were collected from clinically healthy pigs in 17 districts. Forty-two of the 191 sera were from pigs vaccinated against serotypes O/A/SAT 2 FMDV. Antibodies against FMDV non-structural proteins were found in sera from 30 vaccinated and 71 non-vaccinated pigs, altogether 101/191 sera (53 %), and 91 % of these (92/101) also had antibodies measurable by serotype-specific ELISAs, predominantly directed against SAT 1 with titres of 10-320. However, only five high titres against SAT 1 in vaccinated pigs were confirmed by virus neutralisation test (VNT). Due to high degree of agreement between the two ELISAs, it was concluded that positive pigs had been infected with FMDV. Implications of these results for the role of pigs in the epidemiology of FMD in Kenya are discussed, and in-depth studies are recommended.

Laboratory capacity for diagnosis of foot-and-mouth disease in Eastern Africa: implications for the progressive control pathway.

BACKGROUND: Accurate diagnosis is pertinent to any disease control programme. If Eastern Africa is to work towards control of foot-and-mouth disease (FMD) using the Progressive Control Pathway for FMD (PCP-FMD) as a tool, then the capacity of national reference laboratories (NRLs) mandated to diagnose FMD should match this task. This study assessed the laboratory capacity of 14 NRLs of the Eastern Africa Region Laboratory Network member countries using a semi-structured questionnaire and retrospective data from the World Reference Laboratory for FMD annual reports and Genbank® through National Centre for Biotechnology Information for the period 2006-2010. RESULTS: The questionnaire response rate was 13/14 (93%). Twelve out of the 13 countries/regions had experienced at least one outbreak in the relevant five year period. Only two countries (Ethiopia and Kenya) had laboratories at biosecurity level 3 and only three (Ethiopia, Kenya and Sudan) had identified FMD virus serotypes for all reported outbreaks. Based on their own country/region assessment, 12/13 of these countries /regions were below stage 3 of the PCP-FMD. Quarantine (77%) and vaccination (54%) were the major FMD control strategies employed. The majority (12/13) of the NRLs used serological techniques to diagnose FMD, seven used antigen ELISA and three of these (25%) also used molecular techniques which were the tests most frequently requested from collaborating laboratories by the majority (69%) of the NRLs. Only 4/13 (31%) participated in proficiency testing for FMD. Four (31%) laboratories had no quality management systems (QMS) in place and where QMS existed it was still deficient, thus, none of the laboratories had achieved accreditation for FMD diagnosis. CONCLUSIONS: This study indicates that FMD diagnostic capacity in Eastern Africa is still inadequate and largely depends on antigen and antibody ELISAs techniques undertaken by the NRLs. Hence, for the region to progress on the PCP-FMD, there is need to: implement regional control measures, improve the serological diagnostic test performance and laboratory capacity of the NRLs (including training of personnel as well as upgrading of equipment and methods, especially strengthening the molecular diagnostic capacity), and to establish a regional reference laboratory to enforce QMS and characterization of FMD virus containing samples.

Application of the Ceditest® FMDV type O and FMDV-NS enzyme-linked immunosorbent assays for detection of antibodies against Foot-and-mouth disease virus in selected livestock and wildlife species in Uganda.

Diagnosis and control of Foot-and-mouth disease virus (FMDV) requires rapid and sensitive diagnostic tests. Two antibody enzyme-linked immunosorbent assay (ELISA) kits, Ceditest® FMDV-NS for the detection of antibodies against the nonstructural proteins of all FMDV serotypes and Ceditest® FMDV type O for the detection of antibodies against serotype O, were evaluated under African endemic conditions where the presence of multiple serotypes and the use of nonpurified vaccines complicate serological diagnosis. Serum samples from 218 African buffalo, 758 cattle, 304 goats, and 88 sheep were tested using both kits, and selected samples were tested not only in serotype-specific ELISAs for antibodies against primarily FMDV serotype O, but also against other serotypes. The FMDV-NS assay detected far more positive samples (93%) than the FMDV type O assay (30%) in buffalo (P < 0.05), with predominant antibodies against the South African Territories (SAT) serotypes, while the seroprevalence was generally comparable in cattle with antibodies against serotype O elicited by infection and/or vaccination. However, some districts had higher seroprevalence using the FMDV type O assay indicating vaccination without infection, while 1 cattle herd with antibodies against the SAT serotypes had far more positive samples (85%) using the FMDV-NS versus the FMDV type O (10%), consistent with the latter test's lower sensitivity for antibodies against SAT serotypes. Based on the current investigation, the FMDV type O ELISA may be limited by the presence of SAT serotypes. The FMD NS assay worked well as a screening test for antibodies against all FMDV serotypes present in Uganda; however, as long as nonpurified vaccines are applied in the region, this test cannot be used to differentiate between vaccinated and infected animals.

Analysis of the acute phase responses of serum amyloid a, haptoglobin and type 1 interferon in cattle experimentally infected with foot-and-mouth disease virus serotype O.

A series of challenge experiments were performed in order to investigate the acute phase responses to foot-and-mouth disease virus (FMDV) infection in cattle and possible implications for the development of persistently infected "carriers". The host response to infection was investigated through measurements of the concentrations of the acute phase proteins (APPs) serum amyloid A (SAA) and haptoglobin (HP), as well as the bioactivity of type 1 interferon (IFN) in serum of infected animals. Results were based on measurements from a total of 36 infected animals of which 24 were kept for observational periods exceeding 28 days in order to determine the carrier-status of individual animals. The systemic host response to FMDV in infected animals was evaluated in comparison to similar measurements in sera from 6 mock-inoculated control animals.There was a significant increase in serum concentrations of both APPs and type 1 IFN in infected animals coinciding with the onset of viremia and clinical disease. The measured parameters declined to baseline levels within 21 days after inoculation, indicating that there was no systemically measurable inflammatory reaction related to the carrier state of FMD. There was a statistically significant difference in the HP response between carriers and non-carriers with a lower response in the animals that subsequently developed into FMDV carriers. It was concluded that the induction of SAA, HP and type 1 IFN in serum can be used as markers of acute infection by FMDV in cattle.

Rescue of foot-and-mouth disease viruses that are pathogenic for cattle from preserved viral RNA samples.

BACKGROUND: Foot and mouth disease is an economically important disease of cloven-hoofed animals including cattle, sheep and pigs. It is caused by a picornavirus, foot-and-mouth disease virus (FMDV), which has a positive sense RNA genome which, when introduced into cells, can initiate virus replication. PRINCIPAL FINDINGS: A system has been developed to rescue infectious FMDV from RNA preparations generated from clinical samples obtained under experimental conditions and then applied to samples collected in the "field". Clinical samples from suspect cases of foot-and-mouth disease (FMD) were obtained from within Pakistan and Afghanistan. The samples were treated to preserve the RNA and then transported to National Veterinary Institute, Lindholm, Denmark. Following RNA extraction, FMDV RNA was quantified by real-time RT-PCR and samples containing significant levels of FMDV RNA were introduced into susceptible cells using electroporation. Progeny viruses were amplified in primary bovine thyroid cells and characterized using antigen ELISA and also by RT-PCR plus sequencing. FMD viruses of three different serotypes and multiple lineages have been successfully rescued from the RNA samples. Two of the rescued viruses (of serotype O and Asia 1) were inoculated into bull calves under high containment conditions. Acute clinical disease was observed in each case which spread rapidly from the inoculated calves to in-contact animals. Thus the rescued viruses were highly pathogenic. The availability of the rescued viruses enabled serotyping by antigen ELISA and facilitated genome sequencing. CONCLUSIONS: The procedure described here should improve the characterization of FMDVs circulating in countries where the disease is endemic and thus enhance disease control globally.

The role of African buffalos (Syncerus caffer) in the maintenance of foot-and-mouth disease in Uganda.

BACKGROUND: To study the role of African buffalos (Syncerus caffer) in the maintenance of foot-and-mouth disease in Uganda, serum samples were collected from 207 African buffalos, 21 impalas (Aepyceros melampus), 1 giraffe (Giraffa camelopardalis), 1 common eland (Taurotragus oryx), 7 hartebeests (Alcelaphus buselaphus) and 5 waterbucks (Kobus ellipsiprymnus) from four major National Parks in Uganda between 2005 and 2008. Serum samples were screened to detect antibodies against foot-and-mouth disease virus (FMDV) non-structural proteins (NSP) using the Ceditest® FMDV NS ELISA. Solid Phase Blocking ELISAs (SPBE) were used to determine the serotype-specificity of antibodies against the seven serotypes of FMDV among the positive samples. Virus isolation and sequencing were undertaken to identify circulating viruses and determine relatedness between them. RESULTS: Among the buffalo samples tested, 85% (95% CI = 80-90%) were positive for antibodies against FMDV non-structural proteins while one hartebeest sample out of seven (14.3%; 95% CI = -11.6-40.2%) was the only positive from 35 other wildlife samples from a variety of different species. In the buffalo, high serotype-specific antibody titres (≥ 80) were found against serotypes O (7/27 samples), SAT 1 (23/29 samples), SAT 2 (18/32 samples) and SAT 3 (16/30 samples). Among the samples titrated for antibodies against the four serotypes O, SAT 1, SAT 2 and SAT 3, 17/22 (77%; CI = 59.4-94.6%) had high titres against at least two serotypes.FMDV isolates of serotypes SAT 1 (1 sample) and SAT 2 (2 samples) were obtained from buffalo probang samples collected in Queen Elizabeth National Park (QENP) in 2007. Sequence analysis and comparison of VP1 coding sequences showed that the SAT 1 isolate belonged to topotype IV while the SAT 2 isolates belonged to different lineages within the East African topotype X. CONCLUSIONS: Consistent detection of high antibody titres in buffalos supports the view that African buffalos play an important role in the maintenance of FMDV infection within National Parks in Uganda. Both SAT 1 and SAT 2 viruses were isolated, and serological data indicate that it is also likely that FMDV serotypes O and SAT 3 may be present in the buffalo population. Detailed studies should be undertaken to define further the role of wildlife in the epidemiology of FMDV in East Africa.

Patterns, risk factors and characteristics of reported and perceived foot-and-mouth disease (FMD) in Uganda.

Patterns of outbreaks of foot-and-mouth disease (FMD) in Uganda were elucidated from spatial and temporal retrospective data retrieved from monthly reports from District Veterinary Officers (DVOs) to the central administration for the years spanning 2001-2008. An assessment of perceived FMD occurrence, risk factors and the associated characteristics was made based on semi-structured questionnaires administered to the DVOs. During this period, a total of 311 FMD outbreaks were reported in 56 (70%) out of Uganda's 80 districts. The number of reported FMD outbreaks changed over time and by geographical regions. Occurrence of FMD was significantly associated with the dry season months (p = 0.0346), the time when animals movements are more frequent. The average number of FMD outbreaks was higher for some sub-counties adjacent to national parks than for other sub-counties, whilst proximity to international border only seemed to play a role at the southern border. DVOs believed that the major risk factor for FMD outbreaks was animal movements (odds ratio OR 50.8, confidence interval CI 17.8-144.6) and that most outbreaks were caused by introduction of sick animals.

Phylogeography, population dynamics, and molecular evolution of European bat lyssaviruses.

European bat lyssaviruses types 1 and 2 (EBLV-1 and EBLV-2) are widespread in Europe, although little is known of their evolutionary history. We undertook a comprehensive sequence analysis to infer the selection pressures, rates of nucleotide substitution, age of genetic diversity, geographical origin, and population growth rates of EBLV-1. Our study encompassed data from 12 countries collected over a time span of 35 years and focused on the glycoprotein (G) and nucleoprotein (N) genes. We show that although the two subtypes of EBLV-1--EBLV-1a and EBLV-1b--have both grown at a low exponential rate since their introduction into Europe, they have differing population structures and dispersal patterns. Furthermore, there were strong constraints against amino acid change in both EBLV-1 and EBLV-2, as reflected in a low ratio of nonsynonymous to synonymous substitutions per site, particularly in EBLV-1b. Our inferred rate of nucleotide substitution in EBLV-1, approximately 5 x 10(-5) substitutions per site per year, was also one of the lowest recorded for RNA viruses and implied that the current genetic diversity in the virus arose 500 to 750 years ago. We propose that the slow evolution of EBLVs reflects their distinctive epidemiology in bats, where they occupy a relatively stable fitness peak.

Age-dependent differences in cytokine and antibody responses after experimental RSV infection in a bovine model.

Respiratory syncytial virus (RSV) causes severe respiratory disease in both infants and calves. As in humans, bovine RSV (BRSV) infections are most severe in the first 6 months of life. In this study, experimental infection with BRSV was performed in calves aged 1-5, 9-16 or 32-37 weeks. Compared to younger animals, older calves showed significantly less fever and lower TNFalpha levels and less virus-specific IFNgamma release. In addition, blood from older animals had more mononuclear cells, more B cells and stronger BRSV-specific IgA and neutralising antibody responses to infection. A strong "inflammatory" but weak humoral antiviral response in very young animals suggests that enhanced inflammation contributes to disease during RSV infection during the early postnatal period.

Marked induction of IL-6, haptoglobin and IFNgamma following experimental BRSV infection in young calves.

Bovine respiratory syncytial virus (BRSV) has been identified worldwide as an important pathogen associated with acute respiratory disease in calves. An infection model has been developed reflecting accurately the clinical course and the development of pathological signs during a natural BRSV-infection. In the experiments described in the present study, calves were infected at 13-21 weeks of age and reinfected 14 weeks later. Blood samples from the entire infection period were analysed for acute phase protein (haptoglobin) by ELISA and for expression (mRNA level in peripheral blood mononuclear cells) of the cytokines interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6) and interferon-gamma (IFNgamma) by quantitative real-time reverse transcribed polymerase chain reaction (RT-PCR). IFNgamma, interleukin-6 and haptoglobin were markedly induced together with development of clinical signs in response to the first infection with BRSV. The IFNgamma response was biphasic, with an early peak at day 1-3 post infection (p.i.) and a later increase between day 5 and 8 p.i. Reinfection also resulted in an induction of IFNgamma, but without induction of clinical signs, IL-6 and haptoglobin. These results indicate that early mediators connected with the innate responses are induced on a first encounter with the pathogen, but not on a second encounter (reinfection) where the adaptive immune system may act as the first line defence.

Replication and clearance of respiratory syncytial virus: apoptosis is an important pathway of virus clearance after experimental infection with bovine respiratory syncytial virus.

Human respiratory syncytial virus is an important cause of severe respiratory disease in young children, the elderly, and in immunocompromised adults. Similarly, bovine respiratory syncytial virus (BRSV) is causing severe, sometimes fatal, respiratory disease in calves. Both viruses are pneumovirus and the infections with human respiratory syncytial virus and BRSV have similar clinical, pathological, and epidemiological characteristics. In this study we used experimental BRSV infection in calves as a model of respiratory syncytial virus infection to demonstrate important aspects of viral replication and clearance in a natural target animal. Replication of BRSV was demonstrated in the luminal part of the respiratory epithelial cells and replication in the upper respiratory tract preceded the replication in the lower respiratory tract. Virus excreted to the lumen of the respiratory tract was cleared by neutrophils whereas apoptosis was an important way of clearance of BRSV-infected epithelial cells. Neighboring cells, which probably were epithelial cells, phagocytized the BRSV-infected apoptotic cells. The number of both CD4(+) and CD8+ T cells increased during the course of infection, but the T cells were not found between the epithelial cells of the bronchi up until apoptosis was no longer detected, thus in the bronchi there was no indication of direct contact-dependent T-cell-mediated cytotoxicity in the primary infection.