Pork production is critically important to the national economy and food security of Vietnam, and despite major animal-disease outbreaks, the swine industry of Vietnam has achieved remarkably sustained growth in production and profitability over the last 30 to 40 years. Between 2000 and 2010, the total volume of pork production in Vietnam increased 114%.1 These increases were due to growth in pig stocks (approximately 3.6% annual growth in swine population between 2000 and 2010, from 23.0 to 49.3 million head), as well as increased efficiencies in production.2 Animal-health issues facing the industry include fatal epizootics of porcine viruses, endemic circulation of several notifiable diseases (eg, foot-and-mouth disease, classical swine fever), and additional pathogens that reduce efficiency and profitability, some of which may have zoonotic implications for human health (eg, influenza A viruses, Streptococcus suis, Salmonella serovars, Trichinella species, cysticercosis).2-5

Major epizootics of porcine high fever disease (PHFD) caused devastating losses to the Vietnamese swine sector in 2007-2010, impacting 53 of 63 provinces and resulting in more than 1,100,000 pigs destroyed in 2010 alone.6 The principle agent suspected in these outbreaks was porcine reproductive and respiratory syndrome virus (PRRSV). Although PRRSV was clearly a major driver of the explosive outbreaks, experimental studies using a Vietnamese isolate of PRRSV failed to reproduce the severe clinical syndromes seen in the field,7 suggesting possible co-infections or other co-factors contributing to the highly pathogenic phenotype. Among the agents suspected of involvement were porcine circovirus type 2 (PCV2), classical swine fever virus, and various bacterial agents (eg, Pasteurella multocida, S suis, Mycoplasma hyopneumoniae, Haemophilus parasuis, and Actinobacillus pleuropneumoniae).8 During the PHFD outbreaks of 2007-2010, PRRSV and PCV2 were detected in 80% and 90% of swine cases, respectively, submitted to the National Center for Veterinary Diagnostics, Hanoi, Vietnam.9 During 2009-2011, approximately 60% of PHFD outbreaks were confirmed positive for PRRSV, while the remaining 40% were negative for PRRSV but positive for PCV2 or other co-infecting agents.

Influenza A viruses (IAVs) circulating in pigs are of particular concern for the Mekong Delta region due to the endemic circulation of highly pathogenic avian influenza (HPAI) within domestic poultry populations,10 the frequency of mixed rearing of pigs and poultry in backyard farming operations,11 and the potential role of swine in the emergence of avian-swine-human reassortant viruses.12 Data from the Mekong Delta suggest that all three major lineages of IAVs in swine (classical swine H1N1, Eurasian avian-like swine H1N1, and North American triple reassortant viruses) co-circulate.13 Although neither HPAI H5N1 nor low pathogenic avian influenza viruses have been isolated yet from pigs in Vietnam, a novel human-swine reassortant H3N2 was detected in Vietnamese pigs in 2010.5 Studies of IAV in Vietnamese pigs have shown significant geographic variability in seroprevalence,14 from very low levels of circulation (3.1% positive) in semi-commercial farms in a remote northern province15 to 65% seropositive in intensive farms of the Red River Delta.16

Despite the critical imperatives for improved surveillance of swine diseases, the network for animal-disease reporting lacks resources, and veterinary laboratory diagnostics are rarely available, hence few samples are submitted for confirmatory analyses. The lack of baseline prevalence data is due in part to the logistical and technical challenges of sampling animals from small backyard operations; among Vietnamese households raising pigs, approximately 91% have fewer than 10 pigs, and only 6% have more than 30 pigs.11 Veterinary extension services are limited, and farmers are generally reluctant to restrain animals for collection of blood or nasal swabs. Oral fluids are a diagnostic specimen for detection of many human and veterinary pathogens, and are of increasing interest for routine surveillance activities.17-19 To the authors’ knowledge, oral-fluids-based surveillance has not been evaluated within the context of smallholder farming systems in Vietnam. We hypothesized that oral fluids would present a viable alternative to serum samples for routine surveillance and would assist in overcoming farmer reluctance to sampling, particularly of young piglets. We therefore evaluated the performance of individual and pen-based oral-fluids diagnostics for three of the most important porcine respiratory viruses, PRRSV, PCV2, and IAV, in a province of the Mekong Delta that had previously experienced outbreaks of PHFD.

Materials and methods

The survey did not require ethical review because the activities comprised part of periodic routine postvaccination monitoring, did not involve animal experimentation, and were implemented by the relevant animal-health authorities of the province.

The survey was implemented by the Subdepartment of Animal Health (SDAH) of Can Tho province within the context of periodic routine postvaccination monitoring. The survey was carried out in September 2011 in the Can Tho province of Southern Vietnam, located between latitudes 9°55'08″ and 10°19'38″ north and longitudes 105°13'38″ and 105°50'35″ east. With an area of 1409 km2, the province is home to approximately 1.2 million people and 5343 pig farms with approximately 126,000 pigs (2011 agricultural census). The province has a total of nine districts and 85 communes. Farms were selected at random (using coin toss and census lists of registered farms) from 21 communes within the eight districts that had a history of confirmed porcine reproductive and respiratory syndrome (PRRS) in 2010 as determined by the SDAH in Can Tho. The number of farms sampled was proportional to the number of farms in the study communes. The study aimed to collect up to six oral-fluids samples and up to 12 blood samples per farm. Farmer consent was obtained with financial compensation, as per standard SDAH practice. Samples were collected from individually confined and group-penned animals. For individually confined animals (sows, boars), one oral-fluids sample was collected per animal. For group-penned pigs (weaners and growers up to 50 weeks old), pen-based oral fluids were collected. Blood samples were collected only from pigs that contributed to oral-fluids collections (ie, were observed to actively chew on ropes).

Animal sampling method

The protocol for oral-fluids collection was first tested in a pilot study on a local farm. We selected locally produced, 100% cotton, 2-cm diameter woven rope, which was cut into 100-cm sections and unraveled for approximately 10 cm at one end. Ropes were tied to the railings of each pen, and pigs were allowed to chew for 20 minutes under continuous observation. The wet portion of the rope was inserted into a 1-litre re-sealable plastic bag and hand-wrung to extract the fluids; 2 mL was transferred to a cryovial and immediately flash frozen in a liquid nitrogen vapor-cooled Dewar dry shipper (-140°C) to ensure optimal conditions for subsequent virological testing. After completion of oral-fluids collections, pigs that had actively chewed were restrained by rope, and 6 to 8 mL of whole blood was collected by jugular venipuncture. Serum separation, aliquoting, and transfer to temporary storage at -20°C were performed within approximately 6 hours of collection. All sample collections were transferred to -80°C within 1 week of collection. Sample identification enabled linkage between serum and oral-fluids samples.

Sample processing

Serum samples were analyzed both individually and pooled for detection of viral pathogens. Pools were prepared by mixing 100 µL of each sample to reflect the same aggregates as the pen-based oral fluids. Nucleic acids (NA) were extracted from sera and oral fluids using 200 µL and the MagNA Pure 96 Viral NA small volume kit (Roche, Basel, Switzerland) and an automated extractor (Roche). Presence of polymerase chain reaction (PCR) inhibitors and NA quality control were assessed by spiking samples with an RNA internal extraction control (equine arterivirus) prior to extraction.20 The total RNA recovered (60 µL in nuclease-free water) was stored at -80°C until use. Real-time reverse transcriptase PCR (RT-PCR) was performed using primers and probes described for PRRSV21 and matrix gene of IAV,22 using SuperScriptIII Platinum One-Step Quantitative kits (Invitrogen, Carlsbad, California) performed in a 25-µL reaction mix on a Chromo4 real-time PCR machine (Biorad, Hercules, California). Molecular screening for influenza was limited to oral-fluids samples, because IAV is not known to cause viremia in swine. Oral fluids positive for IAV by matrix gene PCR were further tested using primer pairs for a swine-specific influenza A nucleoprotein (NP) gene and hemagglutinin subtyping primers for A/H1N1/pdm09, human H3, and avian H5 lineages (current US Centers for Disease Control subtyping primers). Additional testing was subsequently performed using pan-hemagglutinin23 and pan-neuraminidase24 primers, a 2× PCR enzyme mix as described for oral-fluids optimization,25 and products detected by conventional gel electrophoresis. Virus isolation for IAV-positive samples was attempted in embryonated chicken eggs (three eggs per sample) and concurrently for three serial passages in Madin-Darby canine kidney (MDCK) cells.22 For PCV2 detection, amplifications were performed using primers and probes26 that had been used in previous studies in southern Vietnam.27 The real-time PCV2 PCR was performed in a 25-µL format using TaqMan Universal PCR Master Mix (Applied Biosystems, Carlsbad, California) and Lightcycler480 (Roche).

PRRS virus antibody detection was performed on serum and oral fluids using the HerdChek PRRS X3 (Idexx Laboratories, Westbrook, Maine), designed to detect Chinese, European, and North American lineages of PRRSV. Serum samples were processed according to manufacturer’s instructions, whereas oral-fluids processing was modified by decreasing the dilution (1:2 instead of 1:20) and using larger volumes (250 versus 100 µL) and longer incubation (16 hours versus 1 hour).28 Influenza antibody detection was performed using Influenza A Antibody Test (Idexx Laboratories, Westbrook, Maine), and both serum samples and oral fluids were processed identically following the manufacturer’s instructions.

Statistical analyses

The interquartile range (IQR) of pigs per farm was calculated. Diagnostic yields (numbers of positives) of oral-fluids versus serum samples (representing the same sampled animals) were compared using McNemar’s chi-square test; the kappa test was used to measure the level of agreement among tests.29 The following benchmarks were used for interpretation of kappa test results: 0 to < 0.01 = poor; 0.01 to 0.20 = slight; 0.21 to 0.40 = fair; 0.41 to 0.60 = moderate; 0.61 to 0.80 = substantial; 0.81 to 1 = almost perfect. For PRRSV antibody detection, results from individual serum samples (N = 313), pooled serum samples (N = 84), individual oral-fluids samples (N = 40), and pooled oral-fluids samples (N = 84) were stratified by PRRSV vaccination status and history of disease compatible with PRRS on the farm (abortion in sows and respiratory signs in weaners and growers). All comparisons were made using the chi-square test. Analyses were carried out using R software within the EpiR package (http://www.r-project.org/). Comparisons were considered significant at P < .05.

Results

Farm characteristics and sample collection

A total of 68 farms from 21 communes in eight districts were sampled. Of the 68 farms surveyed, 25 (36.8%) were small-scale household farms with three to five sows; 30 (44.1%) were medium size (six to 20 sows); six (8.8%) were larger commercial units (> 20 sows); and seven (10.3%) raised only growers or finishers. The median total number of pigs (all ages) per farm was 24.5 (IQR 16.0 to 75.4), which is representative of the median farm size in the province. Twenty-three farms (33.8%) reported a history consistent with PRRSV infection (abortion in sows or respiratory signs in piglets), as determined by SDAH, and 36 (52.9%) reported vaccination against PRRSV over the past 12 months. Other diseases for which vaccination was carried out included classical swine fever (79.3% of farms); pasteurellosis (69.8% of farms); salmonellosis (67.2% of farms); and foot-and-mouth disease (56.7% of farms).

A total of 124 oral-fluids samples were collected. These corresponded to 40 animals individually penned (gilts, sows, and boars) and 84 animals in pens with ≥ 8 individuals (mostly weaners, growers, and some gilts, range eight to 15) (n = 84 groups). Upon initial exposure to the ropes, most pigs engaged in active chewing. One pen was sampled from 35 farms (51.5%); two pens were sampled from 18 farms (26.5%); three pens were sampled from 10 farms (14.7%); and four to six pens were sampled from five farms (7.4%). Blood was collected from a total of 313 pigs, which were the same animals (individuals or groups) observed chewing the ropes and from which oral fluids were collected (40 from individually penned animals and 273 from 84 pens with eight to 15 animals each).

Virus detection by PCR in oral fluids and serum

Summary results for the tests performed in matched oral fluids and serum are presented in Table 1. Porcine circovirus type 2 DNA was detected in 54.8% and 61.3% of serum and oral-fluids samples, respectively, indicating that assay sensitivity did not differ significantly by specimen type (Table 2). Results of paired comparisons of oral-fluids and serum samples from individual pigs were more concordant (fair agreement) than those obtained with pooled oral-fluids and serum samples (Table 2). Estimates of overall farm-level, oral-fluids antibody prevalence for IAV and PRRSV did not differ (14.7%, 10 of 68 in each case); however, estimates for pathogen prevalence that were based on serum antibody were significantly different (IAV 23.5%, 16 of 68) and (PRRSV 8.8%, six of 68) (P < .5).

Influenza viral RNA was identified by matrix gene detection in one oral-fluids sample from penned growers (0.8%, one of 124). This sample was confirmed positive using NP primers designed to detect all contemporary swine influenza lineages, and primers for the HA of A/H1N1/pdm09. Virus isolation attempts in eggs and MDCK cells were unsuccessful and depleted the original sample volume. Subsequent attempts to generate amplicons from stored RNA extractions using pan-HA and pan-NA primers did not yield quality sequence reads, and above-threshold cycle threshold (Ct) values for internal RNA controls suggested poor sample quality.

All 124 oral-fluids samples tested negative for PRRSV by RT-PCR, whereas two serum samples tested positive (one pooled sample from a pen of growers with Ct value = 30 and one individually tested sow serum sample with Ct value = 24). The farm with a single pen of PRRSV-positive growers was a relatively large operation (100 sows, total > 400 pigs) and reported prior use of PRRSV vaccine, although the farmer could not specify the manufacturer. These growers also tested positive for PRRSV antibody in the corresponding pooled oral-fluids sample, but not in the pooled serum sample. The PRRSV PCR-positive sow was from a household with two sows and six piglets, and the farmer reported no PRRSV vaccination. The sow tested negative by ELISA for PRRSV antibody in both oral-fluids and serum samples.

Antibody detection by ELISA in oral fluids and serum

Antibody detection for IAV and PRRSV in oral-fluids versus serum samples is presented in Table 3. Overall, antibody testing for IAV was more sensitive for serum than for oral-fluids samples, and there was moderate agreement between the sample types. In individually tested pigs, there was a larger differential in antibody prevalence between serum and oral-fluids samples. For pooled samples, sensitivities of the sample types did not differ for IAV antibody detection. This pattern was similar for PRRSV antibody detection; prevalence of PRRSV antibody detection was greater in serum samples than in oral-fluids samples, and antibody prevalence was greater when individual samples were tested rather than pools. There was fair to moderate agreement between oral-fluids samples and serum samples in all cases.

PRRSV ELISA testing results by age, vaccination status, and history of disease on farms

Comprehensive ELISA testing of the 313 individual serum samples yielded an overall PRRSV seropositivity of 29.1% (24.0% to 34.1%). Older pigs had a greater probability of testing seropositive (Figure 1). Overall PRRSV seropositivity in vaccinated versus unvaccinated farms was 48.6% (17 of 35) and 12.1% (four of 33), respectively. The highest rates of PRRSV seropositivity were found among farms that had vaccinated for PRRSV and had a history of PRRSV disease (67.6%; 95% CI, 68.4%-40.0%), and the lowest for farms with no history of PRRSV or vaccination (6.7%; 95% CI, 1.9%-11.4%; P < .001, χ2). No statistical differences in rate of seropositivity were observed between samples from unvaccinated farms with and without history of PRRSV disease (8.7% versus 6.7%; P = .99, χ2) (Figure 2). Pooled oral-fluids samples from unvaccinated farms with no history of PRRSV had an unusually high prevalence of seropositivity (26.9%; 95% CI, 9.9%-44.0%).

Discussion

Our virological and serological analyses confirm endemic co-circulation of PRRSV, PCV2, and IAV within one southern province of Vietnam. We report low levels of PRRSV viremia and IAV shedding in oral fluids, and high levels of both viremia and shedding in oral fluids for PCV2. Antibody detection was more sensitive in serum samples than in oral fluids for both IAV and PRRSV, and there was fair to moderate concordance between the two sample types. Regarding diagnostic efficacy for molecular screening, oral-fluids samples yielded promising results for PCV2 and IAV, but no detections of PRRSV. Detection of PCV2 viral DNA was comparable in oral-fluids and serum samples. In the older pigs, PCV2 was detected significantly more often in oral-fluids samples than in serum samples, indicating prolonged shedding of PCV2 from the respiratory tracts of mature pigs (in contrast to resolution of systemic viremia).

Since our study implementation and sample processing, a number of published investigations have highlighted the need to specifically tailor diagnostic assays for the oral-fluid matrix30-32 and have thoroughly evaluated the use of oral fluids for monitoring herd health. Panyasing et al31 document modifications to an influenza blocking NP ELISA similar to those described by Kittawornrat et al33 for PRRS ELISA, with reportedly better results; these modifications were not used in the present study. Our failure to find high concordance between assay results for oral fluids and serum for all three pathogens (in particular for PRRSV) are not consistent with the recent reports and may reflect important technical deficiencies in our sample processing. Our results may also reflect inherent variability or bias when evaluating diagnostic protocols using relatively small sample sizes or populations with overall low prevalence of viral shedding.

The oral-fluids screening for IAV yielded one positive (one of 124; 0.8%), and subtyping by PCR confirmed that the sample was positive for hemagglutinin of A/H1N1/pdm09. Because we were unable to confirm the partial HA or NA amplicons by sequencing and did not sequence internal gene fragments, it remains unclear whether the detected virus was similar to pH1N1 currently circulating in people, or was an independent lineage or mixed virus. We anticipate further reports from government swine-surveillance activities that will clarify the complex situation of co-circulating reassortant subtypes in the region. The fact that IAV virus isolation from oral fluids was not successful suggests the presence of virus-inactivating factors within saliva (such as IAV antibodies), dilution effects in saliva, or sample degradation that impaired infectivity but did not entirely degrade RNA. Current swine surveillance programs continue to focus exclusively on use of nasal-swab specimens, and it remains to be seen whether optimization protocols for oral-fluids virus isolations will be accepted in the Vietnam context.

Conventional individual testing of serum samples by PRRSV ELISA revealed the expected age-dependent increase in PRRSV seropositivity, as well as a significant relationship between seropositivity, PRRS vaccination status, and history of PRRSV disease on farms. The observed PRRSV seropositivity in approximately 12% of unvaccinated farms that did not report PRRSV disease might reflect asymptomatic seroconversion to wild-type field virus, inaccuracy in reporting PRRSV vaccination status, secondary transmission of live attenuated vaccine virus, or all three. The JAX-1 vaccine (based on an attenuated virus of the highly pathogenic Chinese lineage) has been licensed for use in Vietnam since 2008, but was not used in Can Tho province during the time of the survey collections in 2011. The vaccines used on the survey farms at that time were commercial vaccines from Singapore, Germany, and Spain that were based on North American or European lineages of PRRSV, and would not have been detected by the RT-PCR used for screening. Thus, the two RT-PCR-positive detections from one sow and one pen of growers indicate asymptomatic infections with circulating wild-type virus.

Although the infrastructure and laboratory capacity for swine-disease surveillance in Vietnam is limited, government authorities regularly engage in vaccination campaigns for high-priority diseases, and postvaccination monitoring activities afford an opportunity to conduct cross-sectional surveys of viral prevalence. In general, field-based investigations face challenges in obtaining farmer consent for blood collection from pigs, particularly from piglets. Because oral-fluids collections are perceived as posing little or no risk to livestock health, large numbers of diagnostic samples can be easily collected at low cost by staff with limited animal-handling experience. It might be particularly productive to implement oral-fluids collections for case clusters of swine with clinical respiratory disease. We conclude that oral-fluids collection shows promise for future field research on respiratory porcine viruses in Vietnam. However, widespread implementation will require standardization of field sampling techniques and careful adoption of optimized and validated diagnostic assays.

Implications

•  PRRSV, IAV, and PCV2 are endemic in swine farms of the Mekong Delta, with moderate levels of PRRSV and IAV transmission and nearly ubiquitous PCV2 circulation.

•  Oral fluids provide comparable sensitivity to serum for molecular detection of PCV2.

•  Oral-fluids screening can provide an acceptable surrogate for serum samples to estimate overall exposure to porcine respiratory viruses and may prove particularly useful in the context of developing countries.

Acknowledgements

We thank the staff of the SDAH of Can Tho and students of Can Tho University veterinary science department for their assistance in sample collections. This research was supported by the VIZIONS grant of the Wellcome Trust Major Overseas Programme.

Conflict of interest

None reported.

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