Seroprevalence of leptospirosis and Japanese encephalitis in swine in ten provinces of Vietnam

Background Leptospirosis is an important zoonotic disease with a global distribution, affecting a wide range of mammalian animals and humans. Japanese encephalitis (JE) virus is the major vector-borne zoonotic disease in the Asia-Pacific region. The main objective of this study was to evaluate the seroprevalence of serovar-specific Leptospira and JE in swine from 10 provinces in Vietnam. Methods Samples were initially collected for swine influenza surveillance from March to April 2017 at large-scale farms (with at least 50 sows and/or 250 fattening pigs) with pigs that tested positive for influenza in the previous surveillance period (2015–16). Findings A total of 2,000 sera samples were analyzed from 10 provinces. Overall, the seroprevalence of leptospirosis was 21.05% (95% CI: 19.28–22.90) using a cut-off titer of ≥ 1:100. The apparent prevalence of JE was 73.45% (95% CI: 71.46–75.37) while the true prevalence was slightly higher (74.46%, 95% credible interval: 73.73–86.41). We found a relatively high presence of leptospirosis and JE in pigs kept on large farms. Prevalence was comparable with other studies suggesting opportunistic testing of samples collected for other surveillance purposes can be a valuable tool to better understand and prevent the potential transmission of these zoonotic diseases from pigs to people in Vietnam. Conclusion Our study provides evidence to veterinarians and animal health professionals for evidence-based practice such as diagnosis, vaccination and zoonotic control. Further investigation into the possible role of different domestic animals, wildlife species or environmental factors is needed to identify the potential risk factors and transmission routes in Vietnam.


Methods
Samples were initially collected for swine influenza surveillance from March to April 2017 at large-scale farms (with at least 50 sows and/or 250 fattening pigs) with pigs that tested positive for influenza in the previous surveillance period .

Findings
A total of 2,000 sera samples were analyzed from 10 provinces. Overall, the seroprevalence of leptospirosis was 21.05% (95% CI: 19.28-22.90) using a cut-off titer of � 1:100. The apparent prevalence of JE was 73.45% (95% CI: 71.46-75.37) while the true prevalence was slightly higher (74.46%, 95% credible interval: 73.73-86.41). We found a relatively high presence of leptospirosis and JE in pigs kept on large farms. Prevalence was comparable with other studies suggesting opportunistic testing of samples collected for other surveillance purposes can be a valuable tool to better understand and prevent the potential transmission of these zoonotic diseases from pigs to people in Vietnam.

Conclusion
Our study provides evidence to veterinarians and animal health professionals for evidencebased practice such as diagnosis, vaccination and zoonotic control. Further investigation PLOS

Introduction
Leptospirosis is an important zoonotic disease with a global distribution, affecting a wide range of mammalian animals and humans. Japanese encephalitis (JE) virus is the major vector-borne zoonotic disease in the Asia-Pacific region [1][2][3][4][5]. Leptospira spp. are maintained in a wide range of reservoir hosts, such as cattle, pigs, dogs, rats, raccoons, skunks and opossums [6][7][8][9][10]. Leptospirosis is caused by gram-negative bacteria, and more than 260 pathogenic serovars have been identified [11]. Pigs are considered to be reservoir hosts for serovars Bratislava, Pomona and Tarassovi Mitis, and also become infected with Icterohaemorrhagiae from rats and Hardjo from cattle [12][13][14]. JE virus is a flavivirus and the main cause of viral encephalitis in Asian countries, resulting in 13,600-20,400 deaths per year [15,16]. It is estimated that more than 3 billion people live in endemic areas [17]. . A JE vaccine was introduced in 1997 and is used for children under five years of age as part of Vietnam's national immunization program in 12 high-risk diseases in the northern region [4].
To our knowledge, only a few multi-site studies have been implemented to assess the seroprevalence of leptospirosis and JE in swine and these have covered in limited areas in Vietnam. Therefore, the main objective of this study was to evaluate the seroprevalence of serovar-specific Leptospira and JE in swine from 10 provinces in Vietnam.

Study locations and sampling
Samples were initially collected for swine influenza surveillance from March to April 2017 under the Department of Animal Health (DAH) and Food and Agriculture organization (FAO) of the United Nations. The Department selected 10 provinces (Ha Noi, Bac Ninh, Thai Binh, Bac Giang, Quang Ninh, Quang Ngai, Binh Duong, Dong Nai, Dong Thap and Soc Trang) (Fig 1). The sampling was targeted at large-scale farms (with at least 50 sows and/or 250 fattening pigs) with pigs that tested positive for influenza in the previous surveillance period (2015-16, 19 provinces: 3800 sera samples). In each province, 10 blood samples from piglets (4-8 weeks old) and fattening pigs (9-12 weeks old) and five blood samples from sows were collected from four-large scale farms, but the sex of the pigs was not recorded. Pigs with symptoms of influenza were prioritized for sampling.

Laboratory analysis
The sera were extracted after centrifugation and stored at -20˚C until transportation in a cool box to the National Center for Veterinary Diagnosis (NCVD) in Hanoi. For leptospirosis, the MAT was used to assess the positivity of samples. Two-fold serial dilution was used after an initial 1:100 dilution, and the end-point (1:1600) was the highest dilution of serum that shows at least 50% agglutination of live leptospires compared to the control sample. We considered a sample leptospirosis positive if the MAT was � 1:100 for at least one of the 16 serovars (Table 1). For JE, enzyme-linked immunosorbent assay (ELISA) was used to detect JE virusspecific antibodies in swine serum. We followed the manufacturer's instructions of JE Ab

Data analysis
We estimated the true prevalence (TP) of JE from apparent prevalence (AP) by adjusting for the diagnostic test sensitivity and specificity using a Bayesian framework The first 1,000 samples of the three MCMC chains were discarded as a burn-in period and the following 10,000 iterations were used for posterior inference. The AP was calculated based on the proportion of positive samples with a 95% Clopper-Pearson/Exact confidence interval (CI). The TP was estimated based on the posterior median value with a 95% credible interval. Statistical significance of differences between prevalence estimates was assessed by examining whether the two 95% CIs overlap. The outputs from the three chains were assessed visually using MCMC trace-plots, posterior density distribution plots, Brooks-Gelman-Rubin (BGR) plots, and auto-correlation plots using the CODA package. All data were entered in Microsoft Excel 2016 and analyzed using R (version 3.5.2). QGIS (Quantum GIS development Team 2018. QGIS version number 3.0.1) was used to generate the map.

Ethics approval and consent to participate
The study was approved by the Hanoi Medical University Institutional Review Board (HMU IRB: no. 00003121), Vietnam.

Japanese encephalitis (JE)
Overall, the AP of JE was 73.45% (95% CI: 71.46-75.37) while the TP was slightly higher (74.46%, 95% credible interval: 73.73-86.41) ( Table 4). Dong Thap had the highest seroprevalence followed by Thai Binh whereas Binh Dung showed the lowest seroprevalence. Overall, the AP showed slightly higher than the TP except for Binh Duong province. The seroprevalence in sows (82.27%, 95% CI: 78.15-85.92) was significantly higher than that in animals of other age groups (Fig 4). The BGR plots showed that our chains have converged for TP, sensitivity and specificity, respectively ( Fig 5).

Discussion
A nation-wide study on seroprevalences of leptospirosis and JE in pigs was conducted in Vietnam. Overall, the seroprevalence (21.05%; 95% CI: 19.28-22.90) of leptospirosis was higher than that reported by Lee et al. (8.17%) [36]. A possible explanation is that pig samples from that study were randomly collected from slaughterhouses and it may be that healthy or less visually ill pigs were more likely to be sent for slaughter. Other studies conducted in the early These study sites were mostly in the Mekong Delta, close to river areas, so that pigs in these areas may have more opportunities exposure. However, in our study, samples from three provinces located in the Mekong Delta had a lower seroprevalence [(Soc Trang (10.0%), Dong Thap (11.5%) and Dong Nai (19.0%)] than other provinces. Therefore, further investigation is necessary to assess the association between environmental factors and leptospirosis.  [40,41]. Therefore, there is a possibility of leptospirosis transmission from wild boars to domesticated pigs. Further study is needed to better understand the role of wild animals in the transmission of leptospirosis in Vietnam. Serovars Pyogenes and Panama were newly detected in our study, which have been observed in cattle in other countries [42][43][44]. Interestingly, significantly higher prevalence of serovar Pyrogenes was detected in Quang Ngai. According to the Vietnam General  statistics office, Quang Ngai has the third largest cattle population in the country (approximately 277,000), after Nghe An and Gia Lai (all central areas) [45], where was only included from the central region for our study. Therefore, it would be worthwhile to conduct an epidemiological investigation on the circulation of serovar Pyrogenes in the central region, where the largest cattle population is raised in Vietnam. Moreover, Icterohaemorrhagiae and Javanica were observed that rat may play an important role in the transmission to pigs [46]. One study suggests that older pigs have a higher chance of exposure to Letospira which is consistent with our result [23]. In humans, leptospirosis is one of 28 notifiable diseases in Vietnam. A total of 85 cases were officially recorded between 2008 and 2015. The low number of notifications is due to lack of public awareness and diagnostic facilities in rural areas resulting in underreporting. Therefore, it is important to raise public awareness, especially among high-risk groups (e.g. agriculture/livestock farmers, mining workers, veterinarians and slaughterhouse workers) who are more likely to come in contact with infected animals and contaminated water or soil. Overall, the seroprevalence (73.45%) of JE in pigs was similar to that in neighboring countries (Laos and Cambodia [65-75%]) [47,48], but higher than that in some other Asian countries (Indonesia, Nepal and Taiwan) [49][50][51][52]. In Vietnam, pigs were not vaccinated against JE virus mainly due to economic reasons, implying that positivity was due to natural infection. Furthermore, seroprevalence (82%) in pigs older than two months showed that detected antibodies were caused by virus infections rather than maternal immunity (one study found that maternal antibodies disappeared after two months [53]). Therefore, we can conclude that JE is widespread in the pig population across the country. In Southeast Asia, it is well documented that JE is endemic and a public health concern. Current pig production practices may pose a risk for JE, because people live very close to pigs, increasing exposure to mosquitoes with JE virus infection. In Vietnam, small-scale backyard pig production accounts for 70-75% of the total production [54].
The average seroprevalence from five northern provinces was 75.1% (95% CI: 72.42-77.78) which was significantly higher than the previous study (60.4% among 641 pig sera), sampling conducted in the northern part of Vietnam from 2009 to 2010 [31]. However, there is no evidence that JE is increasing over time in Vietnam. Over the last decade, in Vietnam, livestock production is undergoing a major change, a moving toward intensive systems and large production scales [55]. Therefore, a possible explain is the growth of intensive pig farms which provides more opportunities to encounter mosquitoes with JE virus infection.
The potential limitation is that sero-prevalences of leptospirosis and JE were likely to be higher than healthy pigs as our sampling population was targeted to large-scale farms with previously tested positive for influenza surveillance. Some studies have suggested that viral infections in animals and humans induce long-lasting immunosuppression in hosts [56,57]. Due to a random error with our measuring instrument, the TP of JE may be under/overestimated. In addition, our prior estimates for the sensitivity and specify of the JE virus test were obtained from the company protocol, and it is not clear to what extent these estimates are appropriate for the local, Vietnamese context.
In humans, most cases are in unvaccinated children under 14 years of age [58]. JE vaccine was first introduced in 1997 through the national immunization program for children aged 1-5 years and 85% of districts were covered in 2013. Currently, there are no routine pig national surveillance programs for JE virus. Therefore, it is difficult to assess the potential role of pigs in the transmission cycle to humans as well as the impact of the national immunization program.
We found less discrepancy between them as sensitivity and specificity are relatively high (> 95%) as well as a higher proportion of positive sample. A Bayesian approach provides an opportunity to combine prior information with investigated data and estimate values for both prevalence and diagnostic characteristics of tests [59]. If a diagnostic test with less than 100% sensitivity and specificity is used to estimate the prevalence of a disease, our results would be biased. Therefore, a Bayesian approach was used to estimate the TP from AP. In general, Bayesian models are sensitive to the selection of the priors, so we employed a Jeffreys prior to minimize the influence of prior to the posterior distribution as no prior information was available for JE prevalence in our study area. Our study was the first attempt to estimate the TP for JE in Vietnam, which showed how a Bayesian approach can be used to better estimate the prevalence of animal diseases.
This study shows the added value of opportunistic use of samples collected for one purpose in providing valuable information on other diseases. An advantage is the large sample size and wide cover. A disadvantage is potential bias linked with sample collection from large farms with previous influenza detection and preference given to apparently sick pigs.

Conclusions
We found a relatively high presence of leptospirosis and JE in pigs kept on large farms. Prevalence was comparable with other studies suggesting opportunistic testing of samples collected for other surveillance purposes can be a valuable tool to better understand and prevent the potential transmission of these zoonotic diseases from pigs to people in Vietnam. Our study provides evidence to veterinarians and animal health professionals for evidence-based practice such as diagnosis, vaccination and zoonotic control. Further investigation into the possible role of different domestic animals, wildlife species or environmental factors is needed to identify the potential risk factors and transmission routes in Vietnam.