Abstract
Rat carcasses obtained from pest control interventions can potentially be used for an efficient surveillance of zoonotic diseases such as leptospirosis. To evaluate the performance of different laboratory methods for detection of pathogenic Leptospira spp., heart and kidney samples from wild Norway rats were analyzed by microscopic agglutination test (MAT, the gold standard), a commercial IgG enzyme-linked immunosorbent assay, and by an optimized quantitative PCR (secY qPCR, followed by sequencing). We found secY qPCR to be as sensitive as MAT for screening of Leptospira infection in pest control rats and selected secY qPCR for a larger screening of rats from urban and rural areas in central and southern Sweden. We identified secY qPCR positive rats from the cities Stockholm, Gothenburg, and Malmö, which were further confirmed by sequencing.
Introduction
Leptospirosis has been claimed to be the geographically most widespread bacterial zoonosis, causing disease in both humans and animals (Evangelista and Coburn 2010). Most of the human leptospirosis cases are found in tropical areas, whereas its prevalence in Europe is generally low (Dupouey et al. 2014). During 2016, a total of 783 clinical cases of leptospirosis were reported from countries within the European Union (ECDC 2017).
In most cases, the clinical symptoms of human leptospirosis are mild or even nonapparent. In severe cases, also known as Weil's disease, lung, liver, and/or kidney failure can develop, and ultimately lead to cardiovascular collapse (Bharti et al. 2003). Globally, there are estimated 1,030,000 cases and 58,900 deaths annually due to leptospirosis, but in western Europe, the number of deaths per 100,000 is estimated to be as low as 0.18 (Costa et al. 2015).
The gold standard for Leptospira diagnostics is the microscopic agglutination test (MAT), based on the use of dark-field microscopy (WHO 2003). MAT includes live bacterial strains and control antibodies, resulting in it being both time consuming and technically demanding. However, the MAT is the methodology most typically used to identify unique serovars of epidemiological and public health significance. Other diagnostic methods include enzyme-linked immunosorbent assay (ELISA) and PCR (Picardeau 2013). Quantitative PCR (qPCR) has been shown to be less sensitive to contamination as compared with earlier standard PCRs (Picardeau 2013), and can detect extremely low levels of leptospiral DNA (Ahmed et al. 2009).
Leptospira spp. are primarily carried by rats (Rattus norvegicus and R. rattus), but may also have other animal reservoirs (Bharti et al. 2003). Although rats that are infected by Leptospira serovar Icterohaemorrhagiae, or other Leptospira serovars, are symptom free, as chronic carriers they can excrete the bacteria in urine for up to 220 days (Thiermann 1981).
Both rat populations and rat extermination rates are presently believed to be increasing within Swedish cities (Håkan Kjellberg, Anticimex, personal communication). As the rat population grows, the probability of contact between humans and rats increases, and thus also the risk of infectious disease transmission (Meerburg et al. 2009).
If local authorities are able to efficiently screen rats for Leptospira, the improved knowledge on prevalences could constitute basis for actions to be taken, important for public health and domestic animals.
In this study we investigated whether the primary screening of Leptospira can be performed on rat carcasses obtained from regular pest control interventions, instead of directed captures of live rats (i.e., within specific research studies or targeted screening campaigns). We also compared the optimality of presently used molecular methods and serology for screening of rats from pest control interventions. Based on this evaluation, we optimized (e.g., various extraction methods, template loads, and primer concentrations) a qPCR and explored the occurrence of Leptospira spp. in Swedish urban and rural populations of the Norway rat (Rattus norvegicus).
Materials and Methods
Samples
Year 2006–2007
Twenty Norway rats were either shot, caught using snap traps, or cages and used as in earlier research studies (Backhans et al. 2011a, 2011b, 2012). Fourteen of these rats were caught in farms in the vicinity of Uppsala (Uppsala, Almunge, and Örsundsbro) and six of the rats were caught within Uppsala city. The rats were dissected on the same day and samples were stored at −80°C.
Year 2011
Nine Norway rats were caught using snap traps in Anticimex Malmö for pest control purposes in central Malmö and kept frozen at −20°C after dissection.
Year 2014–2016
A total of 94 Norway rats were trapped for pest control purposes either by rodenticides or by snap traps in the town centers or in the periurban areas of Stockholm, Uppsala, Gävle, Gothenburg, Växjö, Västervik, and Malmö. Rats were also collected in rural areas outside Uppsala, Nynäshamn, Växjö, and Västervik. Since rats were not collected on a daily basis, the carcasses were decomposed to various levels. Carcasses were stored at −20°C until they were thawed in the laboratory at 4°C the day before dissection. Tissues were collected aseptically and stored at −80°C until analyzed.
Kidney and heart tissues were used for DNA extraction and serological methods. To be able to select an optimal screening method, a subset of the “pest control” rats was initially tested for Leptospira by indirect detection (ELISA and MAT) and direct detection (qPCR).
Leptospira reference strain
Leptospira DNA was obtained from a reference laboratory (KIT Biomedical Research, the Netherlands) and used as a standard for the optimization of the Leptospira qPCR. The species was Leptospira santarosai (strain 1342K) belonging to the serovar and serogroup Shermani (KIT code: KIT0004), originating from a spiny rat from Panama. This Leptospira species was chosen as it has not been reported to be present in Europe to the best of our knowledge.
DNA extraction
DNA was extracted from 123 individual rat kidneys using DNeasy Blood & Tissue kits (Qiagen, Sollentuna, Sweden) following the spin-column protocol provided by the kit. DNA was eluted in 100 μL (instead of 200 μL according to the original protocol) to increase the DNA concentrations. Kidney tissues from laboratory rats (strain Sprague Dawley) were used as negative controls (these rats had previously been used for education purpose with ethical permit C111/15). A positive control was created by spiking a noninfected laboratory rat kidney sample with the Leptospira reference strain before proceeding with the extraction protocol.
qPCR amplification and analysis
The qPCR system used in this study was based on a modified version of a previously described system (Ahmed et al. 2009). The primer pairs, SecYIVF (5′- GCG ATT CAG TTT AAT CCT GC -3′) and SecYIV (5′- GAG TTA GAG CTC AAA TCT AAG -3′), amplify a 202 bp fragment from the L. interrogans S10-spc-α locus (GenBank acc. no. AF115283.1) between locus positions 15744 and 15946 (Ahmed et al. 2009). The secY qPCR was performed on a CFX 96 Touch™ real-time system in conjunction with a C1000 Touch™ thermal cycler (Bio-Rad, Solna, Sweden) using the FAM channel. Optimization of the qPCR was performed using the L. santarosai strain 1342K (KIT Biomedical Research, the Netherlands).
The total reaction volume for each PCR was 25 μL, which consisted of 1 × SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Solna, Sweden) from 2 × stock. Primers were added to the reaction to make a final concentration of 400 nM. Nuclease-free sterile water (Qiagen, Sollentuna, Sweden) was used to bring the reaction volume to 25 μL and as a negative control. In this study, 6.5 μL of template was used for each reaction mix. The parameters for the amplification protocol was as follows: 3 initial minutes at 98°C proceeded by 40 cycles of amplification consisting of 5 s at 98°C then 15 s at 59°C. A melt curve parameter was set to immediately proceed after the amplification step as follows: temperature range set at 65°C to 95°C with plate reading set at 0.5°C increase increments every 5 s. Each sample was analyzed in triplicates.
Analyses of secY qPCR
Data collected from the secY qPCR was analyzed using the Bio-Rad CFX Manager (V3.1) software. If the Cq value of the reaction was <35, it was considered positive. If a Cq value >35 was obtained, it was considered positive only if accompanied by a positive melt curve between the ranges of 75°C and 85°C. Positive PCR products were sent to Macrogen Europe (The Netherlands) for sequencing. The sequence data were then analyzed in CLC Main Workbench V7.8.1 (Qiagen, Sollentuna, Sweden) and identified to species level using the BLAST program that compares sequences against the NCBI GenBank database.
Enzyme-linked immunosorbent assay
Samples were prepared for ELISA through the homogenization of rat hearts. A small piece of heart tissue (∼5 × 4 mm) was put in 500 μL PBS, and the samples were ran in TissueLyser II (Qiagen, Sollentuna, Sweden) for 1.5 min (20.000 Hz) to extract heart tissues. The dilution of the heart lysis was assumed to be ∼1:10. The rat Leptospira IgG ELISA kit including antigens prepared from serogroup Icterohemorrhagiae and serogroup Grippotyphosa (cat. no.: MBS036971; Mybiosource, San Diego, CA) was used as described by the manufacturers. Positive controls, negative controls, and blank controls were analyzed in duplicates. A total of 17 of 34 (50%) of the samples did not have enough volume to be run in duplicates.
Microscopic agglutination test
Samples were analyzed by MAT as previously described (Boqvist et al. 2012, Strand et al. 2015). Five different Leptospira serovars: L. borgpetersenii serovar ballum strain Mus 127, L. interrogans serovar Bratislava strain Jez Bratislava, serovar Icterohaemorrhagiae strain Kantorowicz, L. kirschneri serovar Grippotyphosa strain Duyster, and one domestic strain Mus 2A (isolated from a mouse Mus musculus caught in a Swedish pig herd, closely related to L. borgpetersenii serovar Sejroe and serovar Istrica) were included in the analyses. The threshold titer was set to 1:100.
Statistical methods
Differences in proportions of positive samples by each test were compared by McNemar's chi-square (χ2) test for paired data. For the evaluation of sensitivities and specificities, the MAT was used as the gold standard.
Results
Comparison of laboratory methods
Heart lysis or blood samples from a total of 18 rats were initially tested by the MAT and all were found negative. From the analyzed rat heart lysis, 41.2% (14/34) were tested as positive by the ELISA. Leptospira DNA was found in 11.9% (5/42) of the analyzed kidney samples by secY qPCR, and later confirmed by sequencing as L. interrogans.
By combining the already described results with the results obtained by the MAT in an earlier study on wild Norway rats from Sweden (Strand et al. 2015), a total of 32 rat samples were subjected to both the MAT and ELISA analyses (Tables 1 and 2). The number of samples found positive by the ELISA was significantly higher than the samples detected by the MAT: 40.6% versus 12.5%, respectively (McNemar's χ2 = 4.267; p = 0.0389). Only one sample (3.1%) was found positive both by ELISA and by the MAT. Similarly, only one sample was found positive both by ELISA and by secY qPCR (2.9%). The results obtained by ELISA and secY qPCR, respectively, were found to be significantly different from each other (McNemar's χ2 = 6.667; n = 34, p = 0.0098). The results obtained by the MAT did not significantly differ from those found by the secY qPCR (Table 2) (McNemar's χ2 = 0.500; n = 40, p = 0.4795). By considering the MAT as the reference test (gold standard), the sensitivity and the specificity of the ELISA were calculated as 12.5% and 40.6%, respectively, and as 80% and 97.1%, respectively, for the qPCR (Table 2).
Summary of All Rats Included in This Study
Location of capture, sampling year, habitat, and detection method for detecting Leptospira nucleic acids or Leptospira antibodies are indicated. Gray areas indicate MAT data used from a previous study (Strand et al. 2015).
ELISA, enzyme-linked immunosorbent assay; MAT, microscopic agglutination test; NT, not tested; qPCR, quantitative PCR.
Results of ELISA and secY qPCR as Compared with MAT for Detection of Leptospira
Thirty-two rat samples were subjected to both MAT and ELISA analyses, and 40 rats were subjected to both MAT and secY qPCR.
Screening of urban and rural populations by secY qPCR
Since the results of the secY qPCR correlated well with the results obtained by the MAT, we chose the secY qPCR as the screening method for the larger sample material.
Of 123 Norway rats screened by the secY qPCR, 53 (43.0%) were females and 58 (47.2%) were males, whereas the remaining 12 (9.8%) were of unidentified gender. Ninety (73.2%) of the rats were collected from urban environments, 26 (21.1%) from rural environments, and the remaining 7 (5.7%) were of unidentified origin.
All six (4.8%) rats that were found positive by the secY qPCR originated from urban settings of Stockholm (3), Gothenburg (2), and Malmö (1) (Table 3). Two of the six positive rats were females and four were males. Five of the six positive rats were captured in 2014, and one was captured in 2016. Rats that were collected in 2006–2007 or 2011 were not identified as positive. We confirmed the positive rats by sequencing, and all six were identified as L. interrogans (data not shown).
Occurrence of Rats in Identified Geographic Areas of Sweden, Divided in Urban and Rural Areas and Years Sampled
In total, 123 rats were analyzed by secY qPCR, but only the geographically localized rats are described here (n = 116). The secY-positive samples were confirmed by sequencing. Number of positive/total number of tested.
Cities defined here as localities with >100,000 inhabitants.
Sampled year 2011.
Sampled years 2006 and 2007.
Sampled year 2006.
Discussion
Of the three laboratory methods compared, we found that the secY qPCR and the MAT displayed the most similar results when the samples from rat carcasses (killed for pest control purposes) were analyzed. By using the MAT, the infecting serovar is identified, which is important from an epidemiological viewpoint. In contrast, the secY qPCR does not require a panel of carefully selected bacterial strains like the MAT, but amplifies most known pathogenic leptospiral strains (Ahmed et al. 2009). Both the sensitivity and the specificity of the commercial ELISA were found to be low in this study when compared with the secY qPCR. It is possible that some of the samples, only found seropositive by ELISA, may have cross-reacted with other (unknown) serovars, which were not detected by the MAT panel or by the secY qPCR. However, as these samples were found negative by both the MAT and qPCR, this explanation is rather unlikely. It is more likely that several of the ELISA-positive samples were caused by general unspecific reactions, as reported earlier (Faine 1999).
To our current knowledge, beside our own earlier study (Strand et al. 2015), very few studies have attempted to use rats obtained from pest control interventions for Leptospira identification and surveillance (Runge et al. 2013, Heuser et al. 2016). The vast majority of studies of this nature use live capture methods and euthanize the rats at a laboratory (Webster et al. 1995, Amaddeo et al. 1996, Collares-Pereira et al. 1997, de Faria et al. 2008, Krojgaard et al. 2009, Backhans et al. 2012, Ayral et al. 2015a). Our study showed that it is feasible to use pest control rats for the purpose of Leptospira screening. However, the efficiency and viability of this method on larger number of samples remain to be further explored. Should the use of pest control rat carcasses prove to be just as viable for screening Leptospira as using live-captured rats, a novel and highly cost-efficient method for long-term screening and surveillance of Leptospira in regional rat populations can potentially be utilized. Interested government and research agencies could incentivize or compel private eradication companies to provide a percentage of all exterminated rats along with corresponding collection data to an appropriate laboratory for the screening of Leptospira. Any positive Leptospira sequences could then be made available to the public through an electronic database, potentially providing a wide range of scientists, epidemiologists, ecologists, and public health officials with invaluable data on the spread and prevalence of Leptospira within rat populations of the region in question. Rat carcasses from pest control interventions may also constitute a promising means to obtain surveillance samples also for other zoonotic pathogens. By using the secY qPCR on rat carcasses, poorer countries would be able to establish a surveillance network, providing them with data that can help stipulate where eradication efforts should be focused on.
Among the Swedish urban rats, 5 out of 30 (16.7%) were recently found positive for Leptospira by the MAT, whereas in another Swedish study, 1 of 51 (2%) was found positive by hap1 PCR in rats caught mainly from farms (Backhans et al. 2012). In U.K. farms, only 1% of 259 Norway rats were found positive by the MAT (Webster et al. 1995). In cosmopolitan European cities, in contrast, the prevalence can be very high. A prevalence of 53.0% was found in the Norway rat population in Copenhagen through secY PCR (Krojgaard et al. 2009), and 48.6% in Rome through isolation (Amaddeo et al. 1996). Using PCR with rpoBprimers, a prevalence of 42.9% was found in Lyon (Ayral et al. 2015b).
Different detection methods have been used in these studies making direct comparisons impossible; however, the Leptospira prevalence seems generally to be higher in urban than in farm rats. It is possible that rats in city environments have more contact with infected bodies of water; other possible factors include denser rat populations in cities, different competition and aggression behavior, different rat species can be infected at various rates, and different feeding behaviors.
Rats are reported to be more and more common in Swedish cities (Håkan Kjellberg, and Anticimex, personal communication) yet only three cases of human leptospirosis acquired within the country have been reported in Sweden during the past 10 years (Public Health Agency of Sweden 2018). Similarly, severe human leptospirosis is rare in Sweden and in most developed nations (Traxler et al. 2014, ECDC 2017). This could be explained by the strong sanitation infrastructures that are in place to prevent the spread of Leptospira spp. However, when the sanitation is limited, as, for example, in Bangladesh, the number of human cases of leptospirosis accedes 10 per 100,000 capita (Victoriano et al. 2009). In addition, Sweden and many other developed countries do not currently have the optimal climate for leptospiral survival in stagnant water; however, this could change in the future due to climate change (Lau et al. 2010). In July 2011, a heavy rainfall gave rise to flooding in the capital of Denmark, Copenhagen (Wójcik et al. 2012). Five human cases of leptospirosis were notified and one of the patients died (Kjelsø et al. 2011, Wójcik et al. 2012). These types of extreme weather events are projected to escalate with increasing climate change (Patz et al. 2005), which, in turn, can also increase the cases of human leptospirosis within northern Europe (Lau et al. 2010). If the rat population continues to grow, as exemplified in the largest cities in Sweden, the probability of contacts between humans and rats increases, and thus also the risk for infectious disease transmission (Meerburg et al. 2009). We, therefore, suggest improved surveillance of Leptospira to be focused on rats in urban areas also in Europe.
Conclusion
Our study demonstrated the relevance of using secY qPCR on rat carcasses obtained from pest control interventions to detect Leptospira in rat populations. Our method needs neither directed rat-trapping efforts nor the complex time- and labor-consuming MAT technique, which requires specifically trained personnel. We thereby suggest that secY qPCR-based analyses of pest control rats can be used to improve Leptospira surveillance. If the prevalence of Leptospira in rats turns out to be high in a specific area, the MAT is one of the techniques that can be used to identify serovars for epidemiological purpose.
Knowledge of the geographic range of Leptospira will improve the awareness of leptospirosis and will aid in potential prevention efforts. Currently, we have found Leptospira spp. in Norway rats in the three largest Swedish cities, Stockholm, Gothenburg, and Malmö, but not in rats in smaller towns or rural areas, corroborating previous findings in other studies. In-depth studies are needed to clarify prevalence levels.
Footnotes
Acknowledgments
We thank Hanna Lindberg, Emma Lundin, Per Eriksson, Olivia Borg, Rebecca Hansson, and Frida Wennerholm for assistance with laboratory work. We are grateful to Tove Hoffman for discussions of the project. We also thank Thomas Persson Vinnersten, Håkan Kjellberg, and coworkers at Anticimex. This study was supported by the Swedish Research Council (grant no. 2017-05807).
Disclosure Statement
No competing financial interests exist.