Detection of African swine fever virus in the tissues of asymptomatic pigs in smallholder farming systems along the Kenya – Uganda border : implications for transmission in endemic areas and ASF surveillance in East Africa

The persistence of African swine fever virus (ASFV) in endemic areas, with small-scale but regular outbreaks in domestic pigs, is not well understood. ASFV has not been detected using conventional diagnosis in these pigs or adjacent populations of resistant African wild pigs, that could act as potential carriers during the outbreaks. However, such data are crucial for the design of evidence-based control strategies. We conducted cross-sectional (1107 pigs) and longitudinal (100 pigs) monitoring of ASFV prevalence in local pigs in Kenya and Uganda. The horizontal survey revealed no evidence of ASFV in the serum or blood using either conventional or real-time PCR. One pig consistently tested positive using ELISA, but negative using PCR assays on blood. Interestingly, the isotype of the antibodies from this animal were strongly IgA biased relative to control domestic pigs and warthogs, suggesting a role for mucosal immunity. The tissues from this pig were positive by PCR following post-mortem. Internal organ tissues of 44 healthy pigs (28 sentinel pigs and 16 pigs from slaughter slabs) were tested with four different PCR assays; 15.9% were positive for ASFV suggesting that healthy pigs carrying ASFV exist in the swine population in the study area. P72 and p54 genotyping of ASFV revealed very limited diversity: all were classified in genotype IX at both loci, as were virtually all viruses causing recent ASF outbreaks in the region. Our study suggests that carrier pigs may play a role in ASF disease outbreaks, although the triggers for outbreaks remain unclear and require further investigation. This study significantly increases scientific knowledge of the epidemiology of ASF in the field in Africa, which will contribute to the design of effective surveillance and control strategies.


INTRODUCTION
Pigs are increasingly contributing to improved nutrition and household incomes in regions of Africa where pork consumption and pig keeping are culturally acceptable.In Uganda, for example, pork is second only to beef in terms of meat production and accounts for at least a third of the current 10 kg year À1 per capita meat consumption [1].
Household monthly income, current household monthly expenditure on meat, relative price of pork, preference for value-added pork products, prices of substitutes and response of households to improvements in pork quality have been shown to be associated with increased consumption of pork in some parts of Africa [2].However, African swine fever (ASF), an infectious and lethal disease of domestic pigs, constrains the realization of economic benefits along pig value chains and presents a major risk for farmers who invest in pig production [3,4].The disease, first reported in Kenya in 1910 [5], has spread within the African continent over the past two decades, most recently to Ethiopia [6].The disease currently has no effective chemotherapeutic treatment or vaccine available for its control.Development of innovations and interventions to effectively manage the constraint posed by ASF requires understanding the epidemiology of the disease in smallholder farming regions.Such information on constraints to pig productivity is rarely available for formulating livestock sector policy and institutional changes (http://www.africalivestockdata.org/).The ASF virus (ASFV) is known to be potentially transmitted to susceptible pigs though multiple pathways [3,4].This can be by direct contact with infected pigs, meat and slaughter waste from infected pigs, and potentially also clothing or tools transported by people who have been in contact with infected pigs.It could potentially also be transmitted or maintained in a pig population by Ornithodoros soft ticks, the presumptive ancestral arthropod reservoir and vector for transmission of the virus from warthogs, specialized African wild pigs which can carry the virus without signs of disease, to domestic pigs.
The mechanism of persistence of ASFV in endemic areas where there are sporadic outbreaks of ASF in domestic pigs, but no apparent virus reservoir in resistant African wild pigs or argasid soft ticks, is currently unclear.The potential role of carrier domestic pigs as a source of infection has been documented in Kenya [7].This study was designed to confirm the hypothesis that carrier pigs may contribute to virus transmission and thus the spread and maintenance of the disease, thereby complicating attempts at control [8].We investigated the presence of long-term infected pigs in the smallholder swine population that could be responsible for the outbreaks that are regularly reported in the border region of Kenya and Uganda, as part of an in-depth study of ASFV prevalence and ASF outbreaks in 640 households, using a randomized cluster design.The overarching goal was to increase scientific knowledge relating to the epidemiology of ASF in the region as a prerequisite for the design of effective ASF surveillance and control strategies.

ASFV surveillance study site diagnostic results
All pigs (1107) sampled during the cross-sectional study were negative using PCR assay screening on blood, while only one was positive for ASFV using a commercial ELISA kit (Table 1).During the longitudinal study, five pigs were positive for ASFV by both conventional PCR [9] and Universal ProbeLibrary (UPL) real-time PCR [10] diagnostic assays using blood, with three of them being positive for ASFV in tissues, following post mortem (Table 1).ASFV was successfully isolated from the PCR-positive tissue samples in vitro.

Detection of the ASFV genome in tissues
Four diagnostic assays were applied in an effort to investigate the possibility of ASFV sequestration in tissues from the study area and slaughter slabs (Table 2).In vitro isolation of the virus was only successful for tissue samples that tested positive in both the conventional and UPL PCR assays.
From the longitudinal study, 28 ear-tagged sentinel pigs that tested negative for virus in blood by PCR and serological assays, with the exception of two pigs (UG64/2013 which was seropositive during the entire study period and Ken13/ busia.3 which was positive in blood by conventional and UPL PCR from the first longitudinal time point at 3 months), were euthanized and tested for the presence of ASFV in the tissues.Three out of 28 samples (10.7 %) tested PCR-positive in tissues using both conventional and UPL PCR assays; however, they were ASFV-negative by PCR using blood samples (Table 2).Using a modified Taqman qPCR assay that was initially developed by King et al. [11] implemented at the Friedrich-Loeffler-Institute (FLI; Germany), 15 out of 28 pigs were positive for ASFV in the tissues.Tissue and blood samples from 12 of the 15 pigs that had tested positive at the FLI were selected and tested with a modified PCR assay initially developed by Zsak and colleagues at the Plum Island Animal Disease Center, New York, USA [12,13].Using this assay, 9 out of 12 pigs were found to be positive for ASFV in the tissue samples, while in the blood samples 7 out of 12 pigs were positive for ASFV.Both the King et al. assay using an ABI 7500 platform and Additionally, 16 pigs sold by farmers at different times at local slaughter slabs in the Busia study district (Kenya) over a period of 1 month in July 2014 were sampled using both blood and internal tissues in order to test the extent of ASFV tissue sequestration in the pig population using an independent sampling strategy.The data revealed 25 % (4/ 16) positive pigs in the tissue, but none were ASFV-positive in blood using the UPL PCR assay.The percentage of positivity in tissues increased to 68.75 % (11/16) using the modified Taqman qPCR assay developed at the FLI (Table 2).

Samples from reported outbreaks
Several ASFV outbreaks were reported to veterinary authorities in Busia district during the study period.In two of these outbreaks, samples were obtained.Conventional PCR and UPL qPCR assays conducted on the samples collected during the outbreak (n=5) confirmed that in both cases the suspected pigs were ASFV-positive.

Virus isolation
To confirm that the tissue positives detected by PCR contained viable viral isolates, ground-up tissues were used to infect peripheral blood mononuclear cells (PBMCs) as described in Methods.This resulted in the collective isolation of nine viral isolates in culture using all four sampling strategies.Isotype analysis of anti-ASFV immune response UG64/2013 serum which was positive by diagnostic p62 competition ELISA (INgezim PPA Compac, Ingenasa, Madrid, Spain) was further characterized for isotype specific antibody responses against p72 antigen in comparison to serum from a pig vaccinated with an attenuated virus (E75CV1), two field warthog sera that were also positive by the INgezim ELISA and serum from an ASFV naïve European breed pig (Fig. 1).UG64/2013 serum had lower IgG responses compared to the E75CV1 and warthog sera, and higher IgA and IgM responses compared to all the other sera.A low IgG:IgM ratio was observed in the UG64/2013

DISCUSSION
The virus transmission dynamics and biology underpinning geographically restricted clinical foci of ASF in districts exhibiting persistent outbreaks of disease in African domestic pigs is not currently understood.ASFV prevalence in pigs has not been systematically assessed in these systems.However, such data are crucial for the design of evidencebased control strategies.We conducted both cross-sectional and longitudinal monitoring of ASFV prevalence in African genotype domestic pigs in the border region of western Kenya and eastern Uganda.Internal organ tissues of 44 healthy pigs (28 sentinel pigs and 16 pigs from slaughter slabs) were tested with four different PCR assays; 15.9 % were positive for ASFV, using all assays, suggesting that healthy pigs carrying ASFV exist in the domestic pig population in the study area.Genotyping of ASFV by sequencing the C-terminus of p72 indicated very limited diversity; all were within p72 and p54 genotype IX, which has been associated with all ASFV outbreaks in the region analysed genetically over the past decade.Our study suggests that carrier pigs may be involved in causing in ASF disease outbreaks, although the specific proximal triggers require further investigation.
Both conventional and UPL PCR analyses of 1107 pig blood and sera samples included in the horizontal survey were negative for ASFV.Similarly, whole blood and serological analysis of sentinel pigs sampled longitudinally at three time points (a baseline sampling and two subsequent longitudinal samplings at 3 month intervals) mostly exhibited negative results for ASFV according to antibody and virus detection.Only six of these pigs tested positive on whole blood and only three of these were also ASFV-positive in tissues.Interestingly, there was one exception (an animal designated UG64/2013), which was consistently seropositive, negative according to PCR applied to DNA extracted from whole blood, but positive in several tissues using a PCR assay subsequent to post-mortem.The naïve interpretation of these results was that the population of pigs sampled was minimally exposed to and free from ASFV in the blood according to the diagnostic assay methods used.The data were consistent with the hypothesis that these sentinel pigs had limited exposure to ASFV infection during the period of the study in 2012-2013.This was a surprising result given that a number of suspected ASF outbreaks had been reported to the local veterinary authorities in the immediate area and two of these were confirmed by our laboratory during the study period, as indicated in the results.This result of a low frequency of long-term ASFVinfected pigs detectable by sampling in blood, is similar to that reported from an ASFV endemic region in central Uganda [14], although key differences in this study were, first, that we were able to confirm suspected ASFV outbreaks by PCR and, second, that a percentage of animals were positive in the tissues and were confirmed by virus isolation and genotyping in vitro.However, whether these tissue positive animals represent 'carriers' in the sense that they are capable of directly transmitting the disease to uninfected naïve pigs remains to be determined.
Limitations in the sensitivity of virus detection assays could explain non-detection of positive samples in blood [15].Differences in the sensitivity of the modified Previous studies suggest that lack of success in detection of anti-ASFV antibodies in the case of genotype IX is not attributable to antigenic polymorphism in the target antigens but may, alternatively, be related to the specific characteristics of the phenotypes of African pigs which differ genetically and likely result from different domestication events [16].PCR analysis confirmed outbreaks, and the fact that virus was isolated in the two outbreak cases rules out the failure of the haemadsorption assay or a fundamental failure of PCR assays to detect the virus in the pigs that were sampled.
Recent studies in south-western Kenya [7] have demonstrated that some p72 genotype X-infected pigs were positive with ASFV from blood, but appeared asymptomatic.However, as in the case of the Busia pigs, it was unproven whether these infected pigs were 'carriers' that could transmit the virus to naïve animals on the same farms.Tissues from these animals were not tested concurrently.
To test the potential involvement of 'carrier' pigs in ASFV transmission in the Busia/Teso production system, we further investigated the presence of detectable virus DNA in tissues from apparently healthy pigs.Earlier studies have shown high ASFV prevalence through screening of blood and sera from pigs at slaughter slabs [17,18].A high prevalence as detected by antibody and virus detection assays in slaughterhouse samples is expected during the acute phase, following outbreaks, that is associated with farmers disposing of pigs by slaughter to avoid losses through mortality.

Virus characterization
Viruses from all the PCR-positive tissues sampled from the sentinel pigs, slaughter slabs and outbreak cases were genotyped.preparation), which is the major genotype associated with recent ASF outbreaks throughout both Kenya and Uganda [19,20] and different from genotype X viruses that have recently been associated with a carrier-like status in pigs in south-west and western Kenya and also observed in ticks collected from warthog burrows [7,20].An earlier Kenyan genotype X isolate from the 1950s whose complete genotype has been determined [21] induced a lethal infection in domestic pigs.However, in the south-western Kenya study, the ASFV detected in blood by PCR was not associated with clinical symptoms of ASF and the animals had no detectable ASFV antibody response using the World Organisation for Animal Health (OIE) indirect-ELISA.Sequence analysis of two genotype X isolates from a virulent pig isolate from the 1950s and a recent tick isolate plus a genotype IX isolate from a clinically sick pig demonstrate that the complete genomes of the Kenyan and Uganda isolates are very distinct from the other complete ASFV genomes sequenced to date [22].Genotypes IX and X are, however, closely related to one another and the p72 genes of genotypes IX and X comprise the most similar pairing among the 23 currently publicly available genotypes.

Antibody isotype analysis
Although there was only one seropositive pig observed in the current study (UG64/2013), interestingly, the isotype of the antibodies from this animal were strongly IgA biased, relative to control domestic pigs and warthogs, suggesting a role for mucosal immunity.It should be noted that this was among a sample of >1000 individual pigs that were assayed.The low IgG:IgM ratio and high IgA level in UG64/2013 may be attributable to the route of virus administration.ASFV was likely transmitted to UG64/2013 through pig-topig contact, whereas the attenuated E75CV1 was delivered via the intramuscular route.The two warthogs were almost certainly infected as neonates by Ornithodoros ticks in warthog burrows.The data on these responses is sufficiently distinct to warrant further comprehensive investigation (with even larger pig numbers >10 000) or even an entire population, from birth.
Data collected in this study confirm the presence of ASFV in the tissues of apparently asymptomatic pigs.Analysis of virus transmission during the acute phase is difficult, due to rapid death of infected pigs combined with the lack of timely reporting to veterinary authorities by farmers.This illustrates the importance of understanding both biological parameters, including transmission timescales [23], and anthropogenic factors, including the motivation underlying decisions to sell or slaughter pigs and report ASF outbreaks [24] in obtaining a more in-depth understanding of ASFV epidemiology.The analysis of the questionnaires combined with social network studies indicated that farmers probably recognized ASF symptoms at an early stage and rapidly sold animals either to local butchers or distant neighbours [24].This statement was supported by a considerably higher ASFV prevalence in apparently healthy animals sampled at slaughter slabs, than in the population at large, based on both previously published observations [12] and also the current study.
It seems possible that multiple modes of transmission may exist for ASFV: an initial one in which farmers dispose of pigs incubating disease that are not yet clinically ill, through sale or agistment [25,26], a second one during active outbreaks when farmers rapidly sell-off animals, and which is difficult to measure in the field due to the very rapid death of infected animals resulting in rapid 'burn out' of infections [24,25], and a third one involving virus sequestrated in the tissues, that we describe in this study.It seems quite conceivable that tissues from slaughter slabs, or shedding of body fluids (whose duration and frequency is unknown in a field context), may be key determinants in maintaining transmission in endemic areas.
Such a mechanism of persistence of carriers through sequestration in tissues is similar to that seen in African wild suids, particularly warthogs [27].However, there are several differences: first, unlike the African domestic pigs, adult warthogs are almost universally seropositive, and second Ornithodoros ticks infesting burrows are typically involved in the warthog 'sylvatic cycle'.
The current data demonstrate that there are potential carrier pigs, devoid of clinical signs of ASF or detectable ASFV in blood in the study region that nonetheless have ASFV sequestered in tissues.These data imply a requirement to design control approaches that limit the potential impact of these carrier pigs on the transmission of ASFV.The result indicated the importance of further sampling to test the extent of ASFV tissue sequestration in the wider population of pigs in the eastern African region.Insight into factors that could trigger latency and shedding of the virus by these carrier pigs leading to outbreaks is also required.One possibility is that co-infections may play a role; for example, burdens of gastrointestinal helminths (E.Okoth and R. Bishop, unpublished data) and enteric viruses [28,29] were high in some animals in the current study.
The data suggest that at least for genotype IX, non-invasive surveillance of key tissues using techniques such as lung lavage, or use of novel molecular markers of infection identified by techniques such as high-throughput RNA sequencing or proteomics, may be required to monitor the extent of potential 'carrier pigs' and their role in the ASF epidemiology in free-range African pig production systems.

Conclusions
Cross-sectional and longitudinal surveys of >1000 and 100 smallholder pigs, respectively, using serology, combined with real-time and conventional PCR, revealed only two putative carriers of the ASFV p72 genotype IX in the blood of African domestic pigs in a specific region on the border of Kenya and Uganda.However, sampling of other tissues from animals that died in outbreaks, animals euthanized at the end of the longitudinal survey, or samples obtained from slaughter slabs during the study period, revealed a minimum prevalence of 15.9 %, based on positivity in four different PCR assays, supported in nine cases by virus isolation.This is consistent with the possibility that carrier pigs may play a role in maintenance of ASFV in pig production systems in which persistent clinical ASF outbreaks regularly occur, although the exact role of these animals in virus transmission requires further investigation.

METHODS
Cross-sectional and longitudinal studies were conducted to determine prevalence and incidence of ASF, respectively.Sampling of pigs taken to slaughter was also performed.Blood, sera and, during the longitudinal phase of the study, internal tissues were sampled from apparently healthy pigs and where suspected outbreaks were reported by farmers or veterinary authorities.

Study areas
The study was conducted in western Kenya and eastern Uganda along the border (Fig. 2), a region characterized by mixed rain-fed farm production systems in a humid/subhumid environment.Pig farmers in the area are classified into three categories: farrow to weaner, farrow to finish, and weaner to finish or sale to other farmers (mixed), with some farmers using a combination of the three systems [30].The study area along the Kenya-Uganda border was selected in order to help understand the management of ASF as a trans-boundary animal disease.Indications that outbreaks probably originating in this region had spread to central and eastern Kenya in the recent past [8,19], and that the number of outbreaks was markedly under-reported were also factors in its selection.Data collection and analysis were designed to inform identification of key intervention points for disease surveillance and control.

Cross-sectional survey
The target population in this study was pigs in the border region of Kenya and Uganda.Using an estimated incidence of ASF-positive (+ve) pigs (classified in ASF p72 genotype X) as 0.28 based on [7], 95 % CL and an error margin of 0.05, a sample size of 640 households was indicated after the design effect of cluster sampling was accounted for in the sample size calculation.A stratified multi-stage sampling approach was then applied to select the number of pigs.Briefly, in the first sampling stage eight administrative locations/sub-counties per country were randomly selected by spatial random sampling executed using GIS and the 2008 Kenyan and 2010 Ugandan administrative boundaries, the most recent datasets that were available to the project.Administrative locations were selected from Busia county: four each from Teso and Busia districts in Kenya.On the Ugandan side, eight administrative sub-counties were selected: four each from Busia and Tororo districts.The next stage randomly selected two sub-locations/parishes from each location or sub-county in Kenya and Uganda, respectively, using computer-generated random numbers, making a total of 32 sub-locations/parishes selected along the border.The third stage was a random selection of two villages from each sub-location/parish making a total of 64 villages.A current list of villages in each selected sublocation/parish was obtained from veterinary officials and village leaders.The fourth stage was random selection of ten pig-keeping households from each selected village totalling 640 pig keeping households selected for a baseline crosssectional study.Households sampled in village clusters are shown in Fig. 2. Finally, a maximum of four pigs (1-4 pigs) were selected for sampling from each household.A total of 1107 pigs were selected for the initial cross-sectional sampling.

Longitudinal survey
A random selection was used to sub-sample pigs within a target age range from the 1107 pigs selected for the initial cross-sectional sampling for inclusion in the longitudinal study.Of the 640 households from the cross-sectional survey, 114 households with pigs aged 3-4 months were randomly selected for longitudinal sampling (as 'sentinel pigs').The pigs were purchased by the project and left in the farmer's care.Three repeat samples were obtained from the sentinel pigs, the first taken during the cross-sectional survey, the second sampling taken 3.95 (+/-0.78)months after the initial cross-sectional survey sample and the third sampling taken 3.8 (+/-0.31)months after the first longitudinal survey.The sentinel pigs were either right censored due to mortalities, agistments or sales.A sub-set of the sentinel pigs that survived throughout the longitudinal survey period were humanely sacrificed at the end of the study and blood and tissue samples collected.All other pigs in the sentinel households were also sampled.

Investigation of ASFV in tissue
Twenty-eight sentinel pigs that survived till the end of longitudinal study were selected for sacrifice from the study area after the second longitudinal survey.They were humanely euthanized and tissue, blood and serum samples were collected for tissue sequestration investigation.All slaughter slabs in the study area were also identified within the same period and an additional 16 domestic pigs were sampled on specific days when the animals were taken for slaughter, usually coinciding with village market days.Blood and serum sampling of pigs at slaughter slabs were performed ante mortem and tissues were further sampled post mortem.Additionally, during the longitudinal study period, domestic pigs were also sampled from outbreaks reported by veterinary authorities around the study site.
Animal sampling: live pigs were physically restrained prior to sampling.Blood was collected from the jugular veins using BD Vacutainer needles (gauge Â length: 21Â1-1/2 inch) into 10 ml BD Vacutainer glass serum tube and 4.5 ml 15 % EDTA tubes (Becton, Dickinson and Company, UK).Non-EDTA blood was allowed to clot, and the serum separated.Both serum and EDTA blood aliquots were dispensed into 2 ml cryo-vials (Greiner bio-one, Germany) and stored at À20 C. The tissues sampled post mortem included tonsils, lymph nodes, heart, lungs, spleen, kidney and liver.

ASF diagnosis
Antibody detection: the blocking enzymatic immunoassay (INgezim PPA Compac -R.1.1.PPA.K3) developed by Ingenasa (Spain) was used in the detection of ASF antibodies following the manufacturer's instructions.

Virus detection
DNA was extracted directly from serum, blood or 10 % suspensions of ground tissues using the Qiagen DNAeasy blood and tissue kit (Qiagen, CA) following the manufacturer's instructions.Positive and negative extraction controls that consisted of a known ASFV positive blood sample and sterile PBS (pH 7.0), respectively, were included in the DNA extraction process to ensure that the protocol worked effectively and also to check for possible contamination.A hot-start gel-based conventional PCR assay using the ASF diagnosis primers PPA1/PPA2 that target the ASFV VP73 (p72) coding region [31] was used for primary detection of the presence of ASFV in the extracted DNA.ASFV-positive samples were determined by the amplification of a 257 bp product that was visible on agarose gel using a UV transilluminator.A UPL real-time PCR assay, which is more specific and sensitive than the conventional PCR, was conducted as described by Fernandez-Pinero et al. [10] to confirm the gel-based PCR results.Suspected ASFV samples that generated C t values less than 40 were considered positive while those that yielded C t values greater than 40 were considered doubtful/negative.Positive and negative amplification controls that consisted of DNA obtained from known ASFVpositive tissue samples and nuclease-free sterile water, respectively, were run alongside the test samples as positive and negative controls.
Additionally, two qPCR assays were applied to tissue samples obtained from the study area and slaughter slabs to confirm our results.The first PCR assay was a modification of the Taqman qPCR assay developed by King et al. at the Pirbright Laboratory, Institute for Animal Health, UK [11] that was conducted at the OIE reference laboratory at the FLI.Briefly The second qPCR assay was performed on the smartcycler (Cepheid) platform in our laboratory at the BecA-ILRI hub using Taqman qPCR assay protocol developed by Zsak et al. in Plum Island Animal Disease Center, New York, USA [13] with minor modifications, as previously described by Thomas et al. [12].

Virus isolation
Virus isolation was performed on tissues derived from domestic pigs that had tested positive for ASFV upon diagnosis with conventional PCR.PBMC (macrophage) cultures used for the isolation of the virus were derived from naïve domestic pigs as previously described [32].Briefly, cells were seeded into 96-well tissue culture grade microtitre plates (volume 200 µl; 300 000 cells per well) in autologous swine serum, and incubated in a humidified atmosphere containing 5 % CO 2 at 37 C. Three-day cultures were infected at a multiplicity of infection (m.o.i.) of 1 : 10 with 10 % suspensions of ground tissues supplemented with 5 µg ml À1 gentamycin sulfate (Bio Whittaker) and incubated for 24 h at 37 C.After inoculation, a preparation of 1 % autologous red blood cells in buffered saline (pH 7.0) was added to each well.The plates were examined for haemadsorption over a 6-day period.The samples were blind-passaged three times.
ASFV isotype-specific antibody responses ASFV isotype-specific antibody responses were assessed using an in-house direct p62 ELISA.Briefly, ELISA plates were coated overnight at 4 C with 100 ng recombinant p62 antigen in 100 µl per well carbonate/bicarbonate coating buffer, after which they were washed four times with 200 µl per well PBS with 0.05 % Tween-20 (PBS-T) and blocked with PBS-T with 5 % skimmed milk (PBS-M) for 1 h at 37 C.After a second wash step, 100 µl per well serum diluted 1 : 100 in PBS-M was added to the plate in triplicate and incubated for 1 h at 37 C. Following a third wash step, horseradish peroxidase (HRP) conjugated anti-pig isotype specific antibodies [anti-Pig IgA, M and G (Thermo Scientific, Rockford, IL, USA)], were added in 100 µl per well PBS-M (1 : 5000) and incubated for 1 h at 37 C.An irrelevant species HRP conjugate was used as a negative control.After a final wash, 50 µl of TMB was added to each well and IP: 54.70.40.11On: Sat, 08 Dec 2018 02:07:22

Table 1 .
Detection of ASFV across sampling periods using PCR and serological diagnostic assays

Table 2 .
Summary of ASFV diagnostic results from sentinel pigs sacrificed after the end of the longitudinal survey and additional samples from slaughter slabs within the study site NB, The specific organs that tested positive in the study site sacrificed and slaughter slab sentinel pigs in this study are comprehensively summarized in TableS1.*The slaughter slab pigs were not tested using the Zsak et al. assay, due to the lack of an ASFV-specific Taqman probe.<1), while the E75CV1 and warthog sera had an IgG:IgM ratio >1.The IgG:IgM ratios are useful in distinguishing responses from a recent/acute infection and wellestablished responses, with acute responses having a low (<1) ratio compared with well-established responses (>1).The measurement of IgM antibodies as well as the IgG:IgM ratio may be a criterion in epidemiological studies to discriminate acutely infected animals and animals with wellestablished responses that are potentially protective.The other notable feature of the UG64/2013 serum was the high level of the IgA response.
Fig. 1.ASFV p62 isotype-specific antibody responses.IgG, IgA, IgM specific anti ASFV p62 responses and responses to an irrelevant species isotype control are shown.The columns show sera from a field seropositive animal (UG64/2013), a monkey CV1 cell line attenuated E75 virus vaccinated pig (E75CV1), Ol Pejeta warthogs (warthogs 1 and 2) and an ASFV-naïve pig.The IgG:IgM ratio in the respective sera is also shown.Downloaded from www.microbiologyresearch.orgby IP: 54.70.40.11On: Sat, 08 Dec 2018 02:07:22 serum ( King et al. and Zsak et al. qPCR assays relative to UPL and conventional PCR support this contention.However, none of the additional positives identified by the modified qPCR assays were confirmed by viral isolation and all exhibited high C t values.