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Circulating serogroups of Leptospira in swine from a 7-year study in France (2011–2017)

Circulating serogroups of Leptospira in swine from a 7-year study in France (2011–2017) Background: Leptospirosis is a widespread zoonotic disease caused by pathogenic Leptospira and is responsible for significant economic porcine livestock losses. Knowledge of Leptospira serogroups and their distributions is important for evaluation of the relevance of leptospirosis management measures, including use of the prophylactic vaccine that was recently made available in France. A retrospective study was conducted to determine the relationships between different circulating Leptospira serogroups. Pigs from across France presenting clinical signs suggestive of leptospirosis were tested with the microagglutination test (MAT ) between 2011 and 2017. We used weighted averages to deter- mine serogroup distributions according to MAT results and considering cross-reactions. Results: A total of 19,395 pig sera, mostly from Brittany, were tested, and 22.7% were found to be positive for at least one Leptospira serogroup. Analysis of the 4,346 seropositive results for which the putative infective serogroup could be defined, revealed that two out of ten serogroups were much more frequent than the others: Australis (48.5%) and Icterohaemorrhagiae (38.2%). Other serogroups, including Autumnalis, Panama, Ballum, Tarassovi, Sejroe, Grippoty- phosa, Bataviae, and Pomona, were less common. Conclusions: Although diagnostic laboratory data cannot be extrapolated to infer the distribution of Leptospira serogroups at the nationwide scale in France, the analysis of such data can provide an overview of the relationship between circulating Leptospira serogroups in space and time. During the last decade, protection against the sero- groups Australis and Icterohaemorrhagiae would have prevented most of the clinical porcine leptospirosis cases in the large number of farms that we studied. In the future, epidemiological information related to circulating Leptospira serogroups should be extracted from data with a standardized approach for use in nationwide or international surveil- lance and prophylactic strategy support. Keywords: Leptospira, Pig, Reproductive failure, Microagglutination test, Australis, Icterohaemorrhagiae Introduction pathogenic Leptospira taxa, more than 300 serovars have Leptospirosis is a worldwide zoonotic disease of major been identified and classified into serogroups according importance caused by pathogenic spirochetes of the to their antigenic similarities [2]. genus Leptospira. To date, 38 pathogenic Leptospira spe- In both humans and other animals, Leptospira infec- cies have been described (comprising subclades 1 and 2, tion can cause mild or strong clinical signs or be asymp- previously referred to as pathogenic and intermediary tomatic. In swine, acute and chronic infections are Leptospira, respectively) [1]. Among the currently known described mainly with regard to reproductive impair- ments (abortion, stillbirth, and perinatal mortality) responsible for economic losses; however, deterioration *Correspondence: Florence.ayral@vetagro-sup.fr of the general condition, including haemorrhage, haema- These authors contributed equally: Jeanne Naudet and Florence Ayral turia, renal damage and death, has also been described VetAgro Sup, Université de Lyon, USC 1233, Marcy L’Etoile, France [3]. Asymptomatic carriage also appears to occur in pigs, Full list of author information is available at the end of the article © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Naudet et al. Porcine Health Management (2022) 8:15 Page 2 of 9 allowing for the undetected transmission and mainte- 19% and 26% of tested pigs were seropositive in Italy, nance of the bacteria on farms [4]. Germany and France [16–19]. However, the inclusion Transmission generally occurs by contact with urine- criteria (e.g., the inclusion or exclusion of pigs without contaminated water, in which the bacteria can survive for clinical suspicion of infection) and the serogroup pan- several months [5]. The main hosts of Leptospira bacteria els varied among studies, limiting reliable comparisons are rodents; however, other wild and domestic mammal of the results. species can also be involved in the transmission path- In addition, previous serological surveys do not ways [6, 7]. Because pig farming is mainly conducted report how the MAT results, including the cross sero- indoors in France, contamination is most likely via the reactivity results, were managed [16–19]. According introduction of an infected individual or through contact to previous studies, the interpretation of MAT results with commensal rodents. Furthermore, as close contact is subjective, and possible cross-reactions should be between animals can promote intraherd transmission considered [20, 21]. Chappel et  al. 2004 recommended of the bacteria, the regulation that has recently man- that in the presence of cross-reactions, the serogroup dated that pregnant sows be grouped together (Direc- associated with the maximum titre should be consid- tive 2008/120/EC) could lead to an increased number of ered the predominant serogroup. In addition, Miller Leptospira-infected sows. et al. (2011) showed that the values of a single dilution Two stages of leptospirosis generally occur. The first is titre vary among operators. Thus, a strict difference of the acute phase, which occurs in the first weeks of infec - two dilutions between the maximum titre and the oth- tion, when the hosts may show clinical signs [7]. The ers should be considered to account for this variabil- second is the immune phase, which generally occurs in ity and ensure that the putative infective serogroup is the second week of infection, when the host starts to pro- identified with sufficient evidence. As no consensus duce antibodies against Leptospira [7]. During the acute currently exists in the literature regarding the identi- phase, polymerase chain reaction (PCR) performed on fication of the predominant serogroup, we suggest the blood is highly sensitive and can rapidly detect patho- use of weighted averages to evaluate the importance of genic Leptospira species [8–10]; nonetheless, when treat- circulating serogroups from large sets of MAT surveil- ment is effective, negative results may occur. Culturing lance data. Weighted averages allow the assignment of is less advantageous than PCR for early diagnosis; the higher weights to certain observations considered more culture method is time-consuming, and the isolation of reliable or important than others in the computation of Leptospira is rare. The microagglutination test (MAT) is a the average [22]. serologic method that detects only antibodies indicating Given that several mammalian species may act as a past or current infection [11]. Nonetheless, the MAT reservoirs for swine leptospirosis, disease source con- is the immunological reference standard for experimen- trol is complex. Vaccination may be a promising way tal leptospirosis diagnosis by the World Organization for to reduce the health and economic consequences of Animal Health (OIE) and the World Health Organiza- Leptospira infections. Vaccines provide protection tion (WHO) [12, 13]. Another supportive immunological against homologous or closely related but not heter- test for the detection of antibodies is the enzyme-linked ologous serovars; therefore, particular attention should immunosorbent assay (ELISA) [14]. However, its diag- be given to identifying relevant serovar antigens for nostic accuracy has not been completely established [8]. vaccine development. In France, a swine vaccine that Among the Leptospira tests, the MAT has the advantage protects against the following serogroups has been of a higher sensitivity and the potential identification of commercialized since 2019: Icterohaemorrhagiae, a particular serogroup using available reference strains. Australis, Grippotyphosa, Pomona and Tarassovi. Cross-reactivity between serogroups frequently occurs However, a comprehensive survey of all serogroups in microagglutination testing and results from a lack of circulating in swine herds in France is required to specificity, especially from predominant nonspecific IgM assess the relevance of the inclusion of a given sero- antibodies at the onset of infection [15]. In these cases, group in a putative vaccine. The last such survey dates the MAT results involve titres directed against two or back to 2007 [16], and in light of the spatiotemporal more serogroups, thus preventing determination of the variation in serogroup distribution observed in domes- infecting serogroup. tic animals [23, 24], updated data are needed. Using a The burden of Leptospira on pig farms in Europe descriptive approach for the analysis of available lab- remains unknown. However, epidemiological informa- oratory data, our paper aims to report the results of tion has been extracted from laboratory MAT data for circulating Leptospira serogroups from a large dataset diagnostic purposes. According to these data, between obtained from swine herds in France. Naudet  et al. Porcine Health Management (2022) 8:15 Page 3 of 9 ensured that each individual was given the same weight Materials and methods (1.00) in all computations. For confirmatory diagnosis, 28,332 swine sera were col - More precisely, we applied the following criteria: lected by local veterinarians from pigs that showed (1) A single predominant serogroup was defined by clinical signs consistent with leptospirosis. The samples either a titre ≥ 1:100 against a single serogroup or a three- originated from throughout the country. The veterinar - fold or greater difference between the highest titre and ians requested the laboratory for MAT depending on the other titres. In both cases, a weight  value of 1.00, their ability to recognize the clinical expression of the associated with a high certainty of incrimination, was disease, which may vary among individuals, and consent assigned to the predominant serogroup. of the owners. Thus, the collection of samples from farms (2) Equally predominant serogroups were defined with potential leptospirosis varied according to clini- based on the detection of titres ≥ 1:100 against two or cal signs, veterinarian experience and owner consent. three serogroups with equal titres or a less than three- In addition, because the sensitivity of the MAT may be fold difference between the highest titre and the next low in the early infection stage or for specific serogroup highest titre. The presence of two or three predominant infections [7, 25], samples from several pigs are recom- serogroups most likely resulted from cross reactions [26]. mended to increase the sensitivity of the diagnosis at u Th s, weight values of 0.50 or 0.33 were allotted to the farm level. However, no consensus regarding the number two or three predominant serogroups, respectively. of individuals to be included for confirmatory analysis is (3) Serological profiles with more than three predomi - available, which lead to variability in the number of con- nant serogroups were considered uninformative and tributions per farm. A farm was defined as the group of were removed from the data analysis, as these profiles animals from which pig serum was sampled. It is thus likely resulted from cross-reactive IgM antibodies at the an epidemiological unit. Altogether, the available data onset of infection [15, 21, 27]. resulted from convenience sampling, allowing descriptive The frequency of each serogroup was computed as a statistical analysis. weighted average by summing the numbers of positive Serum samples were analysed by the Laboratoire des swine sera per serogroup, weighting by the values defined Leptospires (Marcy-l’Étoile, France) between October above, and dividing by the total number of positive sera 2007 and April 2017 using the MAT to detect antibod- [22]. ies against a large panel of serogroups. Over the ten-year A seropositive farm was a farm in which at least one duration of the survey, the panel of serogroups varied individual tested positive. Each seropositive farm was slightly. However, 19,395 sera sampled between Janu- associated with one or more serogroups depending on ary 2011 and January 2017 were tested for the following the MAT profile obtained from each tested pig. The sero - serogroups, with their serovars in parentheses: Australis groups computed at the farm level included all seroreac- (munchen, australis, and bratislava), Autumnalis (autum- tive serogroups regardless of the number of pigs reactive nalis and bim), Ballum (ballum), Bataviae (bataviae), against each serogroup. For instance, a farm with two Icterohaemorrhagiae (icterohaemorrhagiae and copen- pigs that were seroreactive against the serogroup Austra- hageni), Grippotyphosa (grippotyphosa and vanderhoe- lis would be associated with that serogroup. A farm with doni), Panama (panama and mangus), Pomona (pomona one pig seroreactive against the serogroup Australis and and mozdok), Sejroe (sejroe, saxkoebing, hardjo, and one pig seroreactive against the serogroups Icterohaem- wolffi) and Tarassovi (tarassovi). This test was performed orrhagiae and Panama would be associated with the sero- using a serum dilution series from 1:100 to 1:6400, and a groups Australis, Icterohaemorrhagiae and Panama. titre ≥ 1:100 was considered suggestive of a past or cur- To describe the spatial serogroup distribution, main- rent infection. land France was studied at the “département” scale To avoid the limitations of cross-reactions and the (France is divided into 96 “départements”, administrative subjective nature of MAT interpretation at the indi- units similar to counties in the United States). The tested vidual level [21], we used a weighted average approach. pigs in each “département” were visualized in Quantum Weighted averages allowed us to assign higher weights to GIS (QGIS version 2.18) with the background map from certain observations considered more reliable than others IGN GEOFLA . Open access pig density data were used in the computation of the average [22]. Here, we assumed for pig distribution mapping [28]. that a previously or currently infected individual would To explore the temporal variability of the serogroups, be associated with MAT seroreactivity directed against a the numbers of swine sera positive for a given serogroup single serogroup or a combination of two or three sero- weighted by the values defined above were retrieved for groups because of possible cross-reactions. Therefore, each year. These counts were analysed using general - we used the number of serogroups detected in the com- ized estimated equations (GEEs) for three reasons [29]. bination of serogroups as the weight. These weights also Naudet et al. Porcine Health Management (2022) 8:15 Page 4 of 9 First, GEE models were developed from generalized lin- ear models to accommodate correlated data [30]. In our study, the primary  sampling unit was the farm and so some correlations among the counts of Leptospira circu- lating  serogroups  were expected  at the farm level. Sec- ond, we were not interested in obtaining estimates for a particular farm but rather in describing the year-to-year variability among the relative numbers of serogroups at the scale of all farms (marginal models). Third, a limita - tion of GEE models is that they rely on the asymptotic normality of estimators for inferences [31]. With 1,114 farms sampled from 2011 to 2016 (2017 was discarded because the sampling was not complete for this year, Fig. 1 The distribution of Leptospira serogroups among 4,346 with only ten farms sampled), we are confident that swine samples. Australis (AUS), Autumnalis (AUT ), Ballum (BAL), this approximation was reasonable for the data analy- Icterohaemorrhagiae (ICT ), Panama (PAN) sis. Because the data were count data, we used a log link function with a Poisson distribution. The model also included the number of seropositive individuals per farm predominant serogroups were Australis and Icterohaem- as an offset variable. We followed the methods of Hardin orrhagiae, which had frequencies of 48.5% and 38.2%, & Hilbe [30], who recommended an unstructured work- respectively, followed by Autumnalis (6.1%), Panama ing correlation matrix for complete datasets with few (5%), Ballum (1.2%), Tarassovi (0.5%), Sejroe (0.2%), Grip- observations (three herein) by panels. PROC GENMOD potyphosa (0.2%), Bataviae (0.1%), and Pomona (< 0.1%) was used to fit the GEE models (SAS Institute Inc. 2012. (Fig. 1). SAS/STAT Software, Version 9.4. Cary, NC). The numbers of tested animals were higher in four “départements” of the Western France (n = 14,509, or 75% of the samples) than in those of the other regions. Results As shown in Fig.  2 (top right), Western France includes Of the 19,395 tested swine sera, 4,398 (22.7%) were posi- the vast majority of pigs reared in the country. Seroposi- tive for at least one serogroup, including 2,608 that were tivity against the predominant serogroups Australis and positive for a single serogroup, 1,410 that were positive Icterohaemorrhagiae was retrieved in 55 and 52 of the 68 for two serogroups and 328 that were positive for three tested “départements”, respectively (Fig. 2). serogroups. Using the three criteria defined above, 52 Sera originated from a total of 2325 farms across MAT results (1.2% of the positive results) were sero- France, among which 1124 (48%) had at least one sero- reactive against more than three serogroups and were positive individual. These farms were distributed in therefore excluded from the data analysis. Laboratory 87% (n = 59/68) of the tested “départements” in main- analysis using only the sera for which a single serogroup land France. The serogroups obtained at the farm level was unambiguously identified would have been based are shown in Table  1. More than 97.5% of the farms on 2,608 positive samples (59.3% of the samples testing (n = 1,087) were associated with at least Australis and/or positive), whereas our data analysis was based on 4,346 Icterohaemorrhagiae, and 50% (n = 561) were exclusively positive samples (98.8% of the samples testing positive), associated with Australis and/or Icterohaemorrhagiae. corresponding to a 66% increase in sample size. The Naudet  et al. Porcine Health Management (2022) 8:15 Page 5 of 9 Fig. 2 The spatial distribution of the swine tested and the seropositivity against Australis, Icterohaemorrhagiae per “département” (administrative district) in mainland France Table 1 Number of farms having pigs with MAT profiles including Australis, Icterohaemorrhagiae and/or other serogroups Leptospira serogroups OnlyAustralis Only Onlyothers Australis and Australisand Icterohaemorrhagiae Australis, Icterohaemorrhagiae Icterohaemorrhagiae others and others Icterohaemorrhagiae and others Num- 134 94 27 333 66 51 409 ber of farms The temporal distribution of the dataset, including chi-square = 0.61). The relative counts of serogroups seropositive vs. seronegative pigs and farms, is displayed varied widely from year to year, as shown by the signifi - in Table  2. Considering the temporal variability of the cant interactions (generalized score for serogroup×year: serogroups among years (Table  3), the fit of the model χ2 (DF = 10) = 203.79, p < 0.0001). The relative counts of was adequate, as shown by the result that only seven Australis and Icterohaemorrhagiae fluctuated from year Pearson residuals were larger than 3 (6 were less than to year but nevertheless represented approximately 80% 3.84 and one was 8.82, out of 3,342 observations) and of infected individuals each year. The correlations among that there was no overdispersion (normalized Pearson the serogroups were negative (Table, 4). Naudet et al. Porcine Health Management (2022) 8:15 Page 6 of 9 Table 2 Features of the dataset (n = 19,343) and distribution of Australis and Icterohaemorrhagiae, with frequencies of the seropositive and seronegative pigs and farms over time from 48.5% and 38.2%, respectively. These high frequencies 2011 to 2017. suggested that among the 4,346 seropositive pigs, most associated Leptospira infections originated from only two Year 2011 2012 2013 2014 2015 2016 2017 serogroups out of the ten detected in this study. Total tested samples 795 2770 3089 3965 3715 4846 163 The observed predominant seroreactivity against Number of seroposi- 190 550 597 988 664 1320 37 Leptospira serogroups Australis and Icterohaemorrha- tive pigs giae in pigs was consistent with previous data reported Number of seronega- 605 2,220 2,492 2,977 3,051 3,526 126 in France [16]. This suggests the absence of any changes tive pigs in the epidemiological context of Leptospira strain dis- Total tested farms 100 333 347 497 459 568 21 tribution. A recent study in Italy that included pigs with Number of tested 70 197 126 236 173 312 10 positive farms clinical suspicion of leptospirosis was implemented using Number of tested 30 136 221 261 286 256 11 an eight-serovar panel; seven serovars were the same as negative farms those in our panel, and their study identified Australis Minimum sample 1 1 1 1 1 1 1 and Pomona as the most frequently detected serogroups size per farm [19]. However, caution is warranted in interpreting this Maximum sample 31 42 31 35 31 31 17 consistency because the previous study do not report size per farm how the MAT results with cross-seroreactivity were Median sample size 7 7 8 7 7 8 8 per farm managed, and the approach may have differed from that Median number of 2 2 4 4 3 4 3.5 in the present study. These observations support the need seropositive samples for a standardized approach for MAT surveillance data per seropositive farm analysis. Total tested pigs is the sum (in bold) of the two lines below (tested positive and Previous studies have described a poor correlation negative). Total tested farms is the sum (in bold) of the two lines below (positive and negative) between the presence of antibodies and the carrier state [25, 32]. Among 22 Australis-infected sows, six had titres above 1:100, which is the limit of positivity recommended Table 3 Weighted number of pigs with MAT profiles including by the OIE for screening and diagnosis [12, 25]. Our data Australis, Icterohaemorrhagiae and/or other serogroups between 2011 and 2017 underestimated the number of seroreactive samples against the serogroup Australis. However, we assume that Leptospira Years Total Serogroups the underestimation is the same among farms and over 2011 2012 2013 2014 2015 2016 2017 time, leading to a lower relative importance of the sero- Australis 86.67 224.17 242.50 343.50 376.17 802.33 24.00 2,099.33 group Australis in our results. Icterohaemor- 82.83 231.67 286.67 529.33 205.17 310.33 5.50 1,651.50 Regardless of the method used, the simple counts rhagiae excluding any MAT result with cross reaction or the Others 20.50 94.17 67.83 115.17 82.67 207.33 7.50 595.17 weighted average method, the results revealed predomi- 190 550 597 988 664 1320 37 4346 nance of the serogroups Australis and Icterohaemorrha- Total giae. The weighted average method allowed a consistent interpretation of the results among data and the inclu- Table 4 Estimated correlations between serogroups from the sion of 66% more MAT results. The weighted average unstructured working correlation matrix used with the generalized approach was thus useful for providing a more com- estimating equation model prehensive overview of the results. In addition, it is an Australis Icterohaemorrhagiae Others opportunity to standardize output of laboratory MAT results and to compare them among laboratories. Australis 1.00 − 0.66 − 0.43 Australis and Icterohaemorrhagiae remained predomi- Icterohaemorrhagiae − 0.66 1.00 − 0.26 nant in the farm-level analysis. This predominance was Others − 0.43 − 0.26 1.00 observed over time, even though the relative proportion of Australis and Icterohaemorrhagiae serogroups varied from one year to another. For anonymity reasons, it was Discussion not possible to identify farms that were potentially sam- Employing a retrospective approach, our study shows pled multiple times during the study period; however, that antibodies against pathogenic Leptospira serogroups this number of farms was assumed negligible consid- were frequently detected on French pig farms. In addi- ering that our sampling was limited to 10% of the total tion, two serogroups were detected much more fre- pig farms in France (n = 22,000) [33]. In addition, the quently than the others among seropositive pigs included, Naudet  et al. Porcine Health Management (2022) 8:15 Page 7 of 9 number of contributions (number of sampled pigs per limited by the low specificity and sensitivity of the MAT farm) varied from one farm to another and may have had [38]. According to our results, which showed that more an impact on the results. Nonetheless, the contribution than 50% of the seropositive pigs were exposed to Aus- variation is unlikely to change the predominance of Aus- tralis and/or Icterohaemorrhagiae, management options tralis and Icterohaemorrhagiae. preventing such infections could have greatly reduced the Regarding MAT, a previous experimental infection burden of the disease among the pigs. study showed that Leptospira-specific IgG (titres above As the serogroups Australis and Icterohaemorrhagiae 1:100) could be detected for more than 100 days [34]. are also pathogenic to humans, infected pig popula- Even when a threshold of positivity of 1:100 is applied, tions represent a potential cause of occupational disease, recently infected, chronically infected or previously especially for breeders or slaughterhouse staff [39, 40]. exposed individuals cannot be clearly distinguished [4]. Following a One Health approach, leptospirosis manage- However, because we were chiefly interested in the dis - ment in pig populations may contribute to the protection tribution of the Leptospira serogroups at a large temporal of human populations and to the mitigation of Leptospira scale, any evidence of past infection was relevant to the persistence and transmission in the country. description of the serogroup distribution in pigs. In addi- tion, the serogroups Australis and Icterohaemorrhagiae Conclusions were predominant in each year from 2011 to 2016 in our The analysis of data from diagnostic laboratories is useful dataset, which suggests that the misclassification of past for obtaining an indication of the circulating Leptospira or current infections would have limited effects on our serogroups in a region or a country and over time. Our results. results suggested that over the study period, most Lepto- Most of the samples were obtained from Brittany, spira infections in swine in France originated from only which is an intensive pig farming area compared with two serogroups, Australis and Icterohaemorrhagiae, out other regions in France. However, the serogroup distri- of the ten used for the laboratory analysis. This informa - bution was similar among regions. The samples analysed tion should be considered to support future prophylactic originated from swine herds with clinical suspicion of measures. leptospirosis; thus, we can conclude that throughout In Europe, numerous laboratories have published France, herds with reproductive disorders that were interpreted MAT results. However, there is no consen- exposed to Leptospira were most likely exposed to the sus regarding serovars to be included in the panel, the Australis and Icterohaemorrhagiae serogroups. Among positive threshold and serogroup determination in the the pig isolates related to the serogroup Australis, some case of cross-reactions, leading to the incomparability of were more likely to be associated with disease [35], and results among studies. Therefore, this is an appeal to vet - Australis exposure in pigs should be seriously considered erinary laboratories in Western Europe to standardize their as a potential cause of reproductive failure. A broader approach to MAT surveillance data analysis. Based on our survey including nonsymptomatic herds could help clar- results and previously published results, future MAT sur- ify the relationship between Leptospira serogroup expo- veillance data analysis in Western Europe should be sup- sure and reproductive failure. However, given the low ported by microagglutination testing with a common set MAT titres found in infected sows and boars from farms of serovars belonging to the serogroups Australis, Ballum, where reproductive failure had occurred and where the Bataviae, Icterohaemorrhagiae, Grippotyphosa, Panama, serovar bratislava had been recovered from aborted sows, Pomona, Sejroe and Tarassovi and using a weighted aver- foetuses and boars, this may not be of value for the Aus- age approach to determine the serogroup distributions and tralis serogroup. considering cross-reactions. Herd management of leptospirosis may rely on ani- mal reservoir control. Rattus norvegicus is a frequent Abbreviations commensal rodent in livestock buildings and a selective MAT: Microagglutination test; AUS: Australis.; AUT : Autumnalis; BAT: Bataviae; carrier of Leptospira of the serogroup Icterohaemorrha- GRI: Grippotyphosa; ICT: Icterohaemorrhagiae; PAN: Panama; POM: Pomona; giae [36, 37]. Thus, rats may play a role to some extent PYR: Pyrogenes; TAR: Tarassovi; SJ: Sejroe. in Icterohaemorrhagiae pig exposure, and this exposure Acknowledgements could be reduced through rat management. As no selec- The authors thank the technicians for their technical support in laboratory tive carriers of Leptospira of the serogroup Australis have procedures and microagglutination testing between 2007 and 2017. We are also grateful to VetAgro Sup for funding the study. been described among commensal rodents, pigs them- selves could be the main reservoir host in the context Authors’ contributions of pig farming, as suggested by Ellis [4]. Leptospirosis FA made substantial contributions to the concept and design of the study, interpreted the results and critically revised the manuscript. LC and JC management through the culling of Leptospira carriers is Naudet et al. Porcine Health Management (2022) 8:15 Page 8 of 9 contributed to the concept and design of the study and critically revised the 9. Picardeau M, Bertherat E, Jancloes M, Skouloudis AN, Durski K, Harts- manuscript. JC was involved in the spatial data analysis. LC was involved in keerl RA. Rapid tests for diagnosis of leptospirosis: current tools and the analysis of temporal variability. JN wrote the first draft of the manuscript emerging technologies. Diagn Microbiol Infect Dis. 2014;78(1):1–8. and constructed the figures. AK provided the data from the Laboratoire des 10. Musso D, La Scola B. Laboratory diagnosis of leptospirosis: a challenge. Leptospires. All authors read and approved the final manuscript. 2013;(46):245–52. 11. Levett PN. Leptospirosis. Clin Microbiol Rev. 2001;14(2):296–326. Funding 12. Anonymous. Leptospirosis. In: OIE Terrestrial Manual 2021. 8e Ed. 2018. This work was supported by VetAgro Sup, Marcy L’Etoile, France [Agriculture p. 1833. Ministry Funding]. 13. Anonymous. Human leptospirosis: guidance for diagnosis, surveil- lance and control. [Internet]. WHO Library Cataloguing-in-Publication Availability of data and materials Data; 2003. Available from: https:// apps. who. int/ iris/ bitst ream/ handle/ The datasets used and/or analysed during the current study are available from 10665/ 42667/ WHO_ CDS_ CSR_ EPH_ 2002. 23. pdf? seque nce= 1& isAll the corresponding author upon reasonable request. owed=y. 14. Padilha BCR, Simão HQ, Oliveira TL, Hartwig DD. The use of ErpY-like recombinant protein from Leptospira interrogans in the develop- Declarations ment of an immunodiagnostic test for swine leptospirosis. Acta Trop. 2019;193:31–4. Ethics approval and consent to participate 15. Levett PN. Leptospirosis. Clin Microbiol Rev. 2001;14(2):296–326. The present research does not include experimental data, and all laboratory 16. André-Fontaine G. Leptospirosis in domestic animals in France: analyses were performed on data from previous examinations of swine for serological results from 1988 to 2007. Rev Sci Tech Int Off Epizoot. routine diagnosis. The Ethical Committee was consulted, and it confirmed that 2016;35(3):913–23. approval was not required in this case. 17. Bertelloni F, Cilia G, Turchi B, Pinzauti P, Cerri D, Fratini F. Epidemiology of leptospirosis in North-Central Italy: fifteen years of serological data Consent for publication (2002–2016). Comp Immunol Microbiol Infect Dis. 2019;1:65:14–22. Not applicable. 18. Strutzberg-Minder K, Tschentscher A, Beyerbach M, Homuth M, Kreienbrock L. Passive surveillance of Leptospira infection in swine in Competing interests Germany. Porc Health Manag [Internet]. 2018 Mar 27 [cited 2020 May The authors declare that they have no competing interests. 10];4. Available from: https:// www. ncbi. nlm. nih. gov/ pmc/ artic les/ PMC58 72537/. Author details 19. Tagliabue S, Figarolli BM, D’Incau M. Serological surveillance of 1 2 VetAgro Sup, Université de Lyon, USC 1233, Marcy L’Etoile, France. Université Leptospirosis in Italy: two year national data (2010- 2011). Vet Ital. Lyon, INRAE, VetAgro Sup, UMR EPIA, 69280 Marcy L’Etoile, France. Université 2016;(52(2)):129–39. Clermont Auvergne, INRAE, VetAgro Sup, UMR EPIA, 63122 Saint-Genès- 20. Chappel RJ, Goris M, Palmer MF, Hartskeerl RA. Impact of proficiency Champanelle, France. UMR ASTRE, CIRAD, INRAE, Univ Montpellier, testing on results of the microscopic agglutination test for diagnosis of 34398 Montpellier, France. Laboratoire des Leptospires, VetAgro Sup, Marcy Leptospirosis. J Clin Microbiol. 2004;42(12)(1):5484–8. L’Etoile, France. 21. Miller MD, Annis KM, Lappin MR, Lunn KF. Variability in results of the microscopic agglutination test in dogs with Clinical Leptospi- Received: 17 August 2021 Accepted: 14 March 2022 rosis and Dogs Vaccinated against Leptospirosis. J Vet Intern Med. 2011;25(3):426–32. 22. Sokal RR, Rohlf FJ. Biometry: the principles and practice of statistics in biological research. 3rd ed. New York : W.H. Freeman; 1995. Available from: https:// trove. nla. gov. au/ work/ 10262 434. References 23. Ayral F, Bicout DJ, Pereira H, Artois M, Kodjo A. Distribution of leptospira 1. Vincent AT, Schiettekatte O, Goarant C, Neela VK, Bernet E, Thibeaux R, serogroups in cattle herds and dogs in France. Am J Trop Med Hyg. et al. Revisiting the taxonomy and evolution of pathogenicity of the 2014;91(4)(1):756–9. genus Leptospira through the prism of genomics. PLoS Negl Trop Dis. 24. Stokes JE, Kaneene JB, Schall WD, Kruger JM, Miller R, Kaiser L, et al. 2019;13(5):e0007270. Prevalence of serum antibodies against six Leptospira serovars in 2. Levett PN. Systematics of Leptospiraceae. In: Adler B, editor. Leptospira healthy dogs. J Am Vet Med Assoc. 2007;230(11):1657–64. and Leptospirosis. Berlin, Heidelberg: Springer; 2015. p. 11–20. (Current 25. Ellis WA, McParland PJ, Bryson DG, Thiermann AB, Montgomery J. Isola- Topics in Microbiology and Immunology). Available from: https:// doi. tion of leptospires from the genital tract and kidneys of aborted sows. org/ 10. 1007/ 978-3- 662- 45059-8_2. Vet Rec. 1986 Mar;15(11):294–5. 118. 3. Bolin CA. Diagnosis of leptospirosis in swine. Swine Health Prod Off J 26. Kusum M, Boonsarthorn N, Biaklang M, Sina U, Sawanpanyalert P, Am Assoc Swine Pract USA. 1994; Available from: http:// agris. fao. org/ Naigowit P. Comparison of leptospiral serovars identification by serol- agris- search/ search. do? recor dID= US951 9052. ogy and cultivation in northeastern region, Thailand. J Med Assoc Thail 4. Ellis WA. Animal leptospirosis. Curr Top Microbiol Immunol. Chotmaihet Thangphaet. 2005;88(8):1098–102. 2015;387:99–137 27. Lupidi R, Cinco M, Balanzin D, Delprete E, Varaldo PE. Serological 5. Andre-Fontaine G, Aviat F, Thorin C. Waterborne Leptospirosis: survival follow-up of patients involved in a localized outbreak of leptospirosis. J and preservation of the virulence of pathogenic Leptospira spp. in Clin Microbiol. 1991;29(4):805–9. fresh water. Curr Microbiol. 2015;71(1):136–42. 28. Gilbert M, Nicolas G, Cinardi G, Van Boeckel TP, Vanwambeke S, Wint 6. Ayral F, Djelouadji Z, Raton V, Zilber A-L, Gasqui P, Faure E, et al. WGR, et al. Global pigs distribution in 2010 (5 minutes of arc). Harvard Hedgehogs and mustelid species: major carriers of pathogenic Lepto- Dataverse; 2018 [cited 2021 Dec 16]. Available from: https:// datav erse. spira, a survey in 28 animal species in France (20122015). PLoS ONE. harva rd. edu/ datas et. xhtml? persi stent Id= doi:https:// doi. org/ 10. 7910/ 2016;28(9):e0162549. DVN/ 33N0JG. 7. Ellis WA. Animal leptospirosis. In: Leptospira and leptospirosis. B. Adler 29. Liang K-Y, Zeger SL. Longitudinal data analysis using generalized linear (3rd). Berlin, Heidelberg, Germany: Springer; 2015. p. 99–137. models. Biometrica. 1986;76:13–22. 8. Niloofa R, Fernando N, de Silva NL, Karunanayake L, Wickramasinghe 30. Hardin JW, Hilbe JM. Generalized estimating equations, 2nd ed. New H, Dikmadugoda N, et al. Diagnosis of Leptospirosis: comparison York: Chapman and Hall/CRC; 2013. 277 p. between microscopic agglutination rest, IgM-ELISA and IgM rapid 31. Agresti A. Categorical data analysis. Hoboken: Wiley; 2002. 710 p. immunochromatography test. PLoS ONE. 2015;18(6):e0129236. Naudet  et al. Porcine Health Management (2022) 8:15 Page 9 of 9 32. Ellis WA, McParland PJ, Bryson DG, Cassells JA. Boars as carriers of lepto- spires of the Australis serogroup on farms with an abortion problem. Vet Rec. 1986;118(20):563. 33. Roguet C. Evolution des modèles d’exploitations porcines en France sous l’effet des contraintes économiques et réglementaires depuis 2008. Journées Recherche Porcine. 17AD;49:277–82. 34. Ballard SA, Adler B, Millar BD, Chappel RJ, Jones RT, Faine S. The immunoglobulin response of swine following experimental infec- tion with leptospira interrrogans serovar pomona. Zentralblatt Für Bakteriol Mikrobiol Hyg 1 Abt Orig Med Mikrobiol Infekt Parasitol. 1984;256(4):510–7. 35. Ellis WA, Montgomery JM, Thiermann AB. Restriction endonuclease analysis as a taxonomic tool in the study of pig isolates belonging to the Australis serogroup of Leptospira interrogans. J Clin Microbiol. 1991;29(5):957–61. 36. Ayral F, Zilber A-L, Bicout DJ, Kodjo A, Artois M, Djelouadji Z. Distribu- tion of Leptospira interrogans by multispacer sequence typing in Urban Norway Rats (Rattus norvegicus): a survey in France in 2011–2013. PLOS ONE. 2015;10(10):e0139604. 37. Thiermann AB. The Norway rat as a selective chronic carrier of Lepto- spira icterohaemorrhagiae. J Wildl Dis. 1981;17(1):39–43. 38. Ellis WA. 03.01.12_Leptospirose. In: Manual of diagnostic tests and vaccines for terrestrials animals. OIE; 2018. p. 503–16. Available from: http:// www. oie. int/ filea dmin/ Home/ fr/ Health_ stand ards/ tahm/3. 01. 12_ LEPTO. pdf. 39. Mirambo MM, Mgode GF, Malima ZO, John M, Mngumi EB, Mhamphi GG, et al. Seropositivity of Brucella spp. and Leptospira spp. antibod- ies among abattoir workers and meat vendors in the city of Mwanza, Tanzania: a call for one health approach control strategies. PLoS Negl Trop Dis. 2018;12(6):e0006600. 40. Thornley CN, Baker MG, Weinstein P, Maas EW. Changing epidemi- ology of human leptospirosis in New Zealand. Epidemiol Infect. 2002;128(1):29–36. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? 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Circulating serogroups of Leptospira in swine from a 7-year study in France (2011–2017)

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Abstract

Background: Leptospirosis is a widespread zoonotic disease caused by pathogenic Leptospira and is responsible for significant economic porcine livestock losses. Knowledge of Leptospira serogroups and their distributions is important for evaluation of the relevance of leptospirosis management measures, including use of the prophylactic vaccine that was recently made available in France. A retrospective study was conducted to determine the relationships between different circulating Leptospira serogroups. Pigs from across France presenting clinical signs suggestive of leptospirosis were tested with the microagglutination test (MAT ) between 2011 and 2017. We used weighted averages to deter- mine serogroup distributions according to MAT results and considering cross-reactions. Results: A total of 19,395 pig sera, mostly from Brittany, were tested, and 22.7% were found to be positive for at least one Leptospira serogroup. Analysis of the 4,346 seropositive results for which the putative infective serogroup could be defined, revealed that two out of ten serogroups were much more frequent than the others: Australis (48.5%) and Icterohaemorrhagiae (38.2%). Other serogroups, including Autumnalis, Panama, Ballum, Tarassovi, Sejroe, Grippoty- phosa, Bataviae, and Pomona, were less common. Conclusions: Although diagnostic laboratory data cannot be extrapolated to infer the distribution of Leptospira serogroups at the nationwide scale in France, the analysis of such data can provide an overview of the relationship between circulating Leptospira serogroups in space and time. During the last decade, protection against the sero- groups Australis and Icterohaemorrhagiae would have prevented most of the clinical porcine leptospirosis cases in the large number of farms that we studied. In the future, epidemiological information related to circulating Leptospira serogroups should be extracted from data with a standardized approach for use in nationwide or international surveil- lance and prophylactic strategy support. Keywords: Leptospira, Pig, Reproductive failure, Microagglutination test, Australis, Icterohaemorrhagiae Introduction pathogenic Leptospira taxa, more than 300 serovars have Leptospirosis is a worldwide zoonotic disease of major been identified and classified into serogroups according importance caused by pathogenic spirochetes of the to their antigenic similarities [2]. genus Leptospira. To date, 38 pathogenic Leptospira spe- In both humans and other animals, Leptospira infec- cies have been described (comprising subclades 1 and 2, tion can cause mild or strong clinical signs or be asymp- previously referred to as pathogenic and intermediary tomatic. In swine, acute and chronic infections are Leptospira, respectively) [1]. Among the currently known described mainly with regard to reproductive impair- ments (abortion, stillbirth, and perinatal mortality) responsible for economic losses; however, deterioration *Correspondence: Florence.ayral@vetagro-sup.fr of the general condition, including haemorrhage, haema- These authors contributed equally: Jeanne Naudet and Florence Ayral turia, renal damage and death, has also been described VetAgro Sup, Université de Lyon, USC 1233, Marcy L’Etoile, France [3]. Asymptomatic carriage also appears to occur in pigs, Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Naudet et al. Porcine Health Management (2022) 8:15 Page 2 of 9 allowing for the undetected transmission and mainte- 19% and 26% of tested pigs were seropositive in Italy, nance of the bacteria on farms [4]. Germany and France [16–19]. However, the inclusion Transmission generally occurs by contact with urine- criteria (e.g., the inclusion or exclusion of pigs without contaminated water, in which the bacteria can survive for clinical suspicion of infection) and the serogroup pan- several months [5]. The main hosts of Leptospira bacteria els varied among studies, limiting reliable comparisons are rodents; however, other wild and domestic mammal of the results. species can also be involved in the transmission path- In addition, previous serological surveys do not ways [6, 7]. Because pig farming is mainly conducted report how the MAT results, including the cross sero- indoors in France, contamination is most likely via the reactivity results, were managed [16–19]. According introduction of an infected individual or through contact to previous studies, the interpretation of MAT results with commensal rodents. Furthermore, as close contact is subjective, and possible cross-reactions should be between animals can promote intraherd transmission considered [20, 21]. Chappel et  al. 2004 recommended of the bacteria, the regulation that has recently man- that in the presence of cross-reactions, the serogroup dated that pregnant sows be grouped together (Direc- associated with the maximum titre should be consid- tive 2008/120/EC) could lead to an increased number of ered the predominant serogroup. In addition, Miller Leptospira-infected sows. et al. (2011) showed that the values of a single dilution Two stages of leptospirosis generally occur. The first is titre vary among operators. Thus, a strict difference of the acute phase, which occurs in the first weeks of infec - two dilutions between the maximum titre and the oth- tion, when the hosts may show clinical signs [7]. The ers should be considered to account for this variabil- second is the immune phase, which generally occurs in ity and ensure that the putative infective serogroup is the second week of infection, when the host starts to pro- identified with sufficient evidence. As no consensus duce antibodies against Leptospira [7]. During the acute currently exists in the literature regarding the identi- phase, polymerase chain reaction (PCR) performed on fication of the predominant serogroup, we suggest the blood is highly sensitive and can rapidly detect patho- use of weighted averages to evaluate the importance of genic Leptospira species [8–10]; nonetheless, when treat- circulating serogroups from large sets of MAT surveil- ment is effective, negative results may occur. Culturing lance data. Weighted averages allow the assignment of is less advantageous than PCR for early diagnosis; the higher weights to certain observations considered more culture method is time-consuming, and the isolation of reliable or important than others in the computation of Leptospira is rare. The microagglutination test (MAT) is a the average [22]. serologic method that detects only antibodies indicating Given that several mammalian species may act as a past or current infection [11]. Nonetheless, the MAT reservoirs for swine leptospirosis, disease source con- is the immunological reference standard for experimen- trol is complex. Vaccination may be a promising way tal leptospirosis diagnosis by the World Organization for to reduce the health and economic consequences of Animal Health (OIE) and the World Health Organiza- Leptospira infections. Vaccines provide protection tion (WHO) [12, 13]. Another supportive immunological against homologous or closely related but not heter- test for the detection of antibodies is the enzyme-linked ologous serovars; therefore, particular attention should immunosorbent assay (ELISA) [14]. However, its diag- be given to identifying relevant serovar antigens for nostic accuracy has not been completely established [8]. vaccine development. In France, a swine vaccine that Among the Leptospira tests, the MAT has the advantage protects against the following serogroups has been of a higher sensitivity and the potential identification of commercialized since 2019: Icterohaemorrhagiae, a particular serogroup using available reference strains. Australis, Grippotyphosa, Pomona and Tarassovi. Cross-reactivity between serogroups frequently occurs However, a comprehensive survey of all serogroups in microagglutination testing and results from a lack of circulating in swine herds in France is required to specificity, especially from predominant nonspecific IgM assess the relevance of the inclusion of a given sero- antibodies at the onset of infection [15]. In these cases, group in a putative vaccine. The last such survey dates the MAT results involve titres directed against two or back to 2007 [16], and in light of the spatiotemporal more serogroups, thus preventing determination of the variation in serogroup distribution observed in domes- infecting serogroup. tic animals [23, 24], updated data are needed. Using a The burden of Leptospira on pig farms in Europe descriptive approach for the analysis of available lab- remains unknown. However, epidemiological informa- oratory data, our paper aims to report the results of tion has been extracted from laboratory MAT data for circulating Leptospira serogroups from a large dataset diagnostic purposes. According to these data, between obtained from swine herds in France. Naudet  et al. Porcine Health Management (2022) 8:15 Page 3 of 9 ensured that each individual was given the same weight Materials and methods (1.00) in all computations. For confirmatory diagnosis, 28,332 swine sera were col - More precisely, we applied the following criteria: lected by local veterinarians from pigs that showed (1) A single predominant serogroup was defined by clinical signs consistent with leptospirosis. The samples either a titre ≥ 1:100 against a single serogroup or a three- originated from throughout the country. The veterinar - fold or greater difference between the highest titre and ians requested the laboratory for MAT depending on the other titres. In both cases, a weight  value of 1.00, their ability to recognize the clinical expression of the associated with a high certainty of incrimination, was disease, which may vary among individuals, and consent assigned to the predominant serogroup. of the owners. Thus, the collection of samples from farms (2) Equally predominant serogroups were defined with potential leptospirosis varied according to clini- based on the detection of titres ≥ 1:100 against two or cal signs, veterinarian experience and owner consent. three serogroups with equal titres or a less than three- In addition, because the sensitivity of the MAT may be fold difference between the highest titre and the next low in the early infection stage or for specific serogroup highest titre. The presence of two or three predominant infections [7, 25], samples from several pigs are recom- serogroups most likely resulted from cross reactions [26]. mended to increase the sensitivity of the diagnosis at u Th s, weight values of 0.50 or 0.33 were allotted to the farm level. However, no consensus regarding the number two or three predominant serogroups, respectively. of individuals to be included for confirmatory analysis is (3) Serological profiles with more than three predomi - available, which lead to variability in the number of con- nant serogroups were considered uninformative and tributions per farm. A farm was defined as the group of were removed from the data analysis, as these profiles animals from which pig serum was sampled. It is thus likely resulted from cross-reactive IgM antibodies at the an epidemiological unit. Altogether, the available data onset of infection [15, 21, 27]. resulted from convenience sampling, allowing descriptive The frequency of each serogroup was computed as a statistical analysis. weighted average by summing the numbers of positive Serum samples were analysed by the Laboratoire des swine sera per serogroup, weighting by the values defined Leptospires (Marcy-l’Étoile, France) between October above, and dividing by the total number of positive sera 2007 and April 2017 using the MAT to detect antibod- [22]. ies against a large panel of serogroups. Over the ten-year A seropositive farm was a farm in which at least one duration of the survey, the panel of serogroups varied individual tested positive. Each seropositive farm was slightly. However, 19,395 sera sampled between Janu- associated with one or more serogroups depending on ary 2011 and January 2017 were tested for the following the MAT profile obtained from each tested pig. The sero - serogroups, with their serovars in parentheses: Australis groups computed at the farm level included all seroreac- (munchen, australis, and bratislava), Autumnalis (autum- tive serogroups regardless of the number of pigs reactive nalis and bim), Ballum (ballum), Bataviae (bataviae), against each serogroup. For instance, a farm with two Icterohaemorrhagiae (icterohaemorrhagiae and copen- pigs that were seroreactive against the serogroup Austra- hageni), Grippotyphosa (grippotyphosa and vanderhoe- lis would be associated with that serogroup. A farm with doni), Panama (panama and mangus), Pomona (pomona one pig seroreactive against the serogroup Australis and and mozdok), Sejroe (sejroe, saxkoebing, hardjo, and one pig seroreactive against the serogroups Icterohaem- wolffi) and Tarassovi (tarassovi). This test was performed orrhagiae and Panama would be associated with the sero- using a serum dilution series from 1:100 to 1:6400, and a groups Australis, Icterohaemorrhagiae and Panama. titre ≥ 1:100 was considered suggestive of a past or cur- To describe the spatial serogroup distribution, main- rent infection. land France was studied at the “département” scale To avoid the limitations of cross-reactions and the (France is divided into 96 “départements”, administrative subjective nature of MAT interpretation at the indi- units similar to counties in the United States). The tested vidual level [21], we used a weighted average approach. pigs in each “département” were visualized in Quantum Weighted averages allowed us to assign higher weights to GIS (QGIS version 2.18) with the background map from certain observations considered more reliable than others IGN GEOFLA . Open access pig density data were used in the computation of the average [22]. Here, we assumed for pig distribution mapping [28]. that a previously or currently infected individual would To explore the temporal variability of the serogroups, be associated with MAT seroreactivity directed against a the numbers of swine sera positive for a given serogroup single serogroup or a combination of two or three sero- weighted by the values defined above were retrieved for groups because of possible cross-reactions. Therefore, each year. These counts were analysed using general - we used the number of serogroups detected in the com- ized estimated equations (GEEs) for three reasons [29]. bination of serogroups as the weight. These weights also Naudet et al. Porcine Health Management (2022) 8:15 Page 4 of 9 First, GEE models were developed from generalized lin- ear models to accommodate correlated data [30]. In our study, the primary  sampling unit was the farm and so some correlations among the counts of Leptospira circu- lating  serogroups  were expected  at the farm level. Sec- ond, we were not interested in obtaining estimates for a particular farm but rather in describing the year-to-year variability among the relative numbers of serogroups at the scale of all farms (marginal models). Third, a limita - tion of GEE models is that they rely on the asymptotic normality of estimators for inferences [31]. With 1,114 farms sampled from 2011 to 2016 (2017 was discarded because the sampling was not complete for this year, Fig. 1 The distribution of Leptospira serogroups among 4,346 with only ten farms sampled), we are confident that swine samples. Australis (AUS), Autumnalis (AUT ), Ballum (BAL), this approximation was reasonable for the data analy- Icterohaemorrhagiae (ICT ), Panama (PAN) sis. Because the data were count data, we used a log link function with a Poisson distribution. The model also included the number of seropositive individuals per farm predominant serogroups were Australis and Icterohaem- as an offset variable. We followed the methods of Hardin orrhagiae, which had frequencies of 48.5% and 38.2%, & Hilbe [30], who recommended an unstructured work- respectively, followed by Autumnalis (6.1%), Panama ing correlation matrix for complete datasets with few (5%), Ballum (1.2%), Tarassovi (0.5%), Sejroe (0.2%), Grip- observations (three herein) by panels. PROC GENMOD potyphosa (0.2%), Bataviae (0.1%), and Pomona (< 0.1%) was used to fit the GEE models (SAS Institute Inc. 2012. (Fig. 1). SAS/STAT Software, Version 9.4. Cary, NC). The numbers of tested animals were higher in four “départements” of the Western France (n = 14,509, or 75% of the samples) than in those of the other regions. Results As shown in Fig.  2 (top right), Western France includes Of the 19,395 tested swine sera, 4,398 (22.7%) were posi- the vast majority of pigs reared in the country. Seroposi- tive for at least one serogroup, including 2,608 that were tivity against the predominant serogroups Australis and positive for a single serogroup, 1,410 that were positive Icterohaemorrhagiae was retrieved in 55 and 52 of the 68 for two serogroups and 328 that were positive for three tested “départements”, respectively (Fig. 2). serogroups. Using the three criteria defined above, 52 Sera originated from a total of 2325 farms across MAT results (1.2% of the positive results) were sero- France, among which 1124 (48%) had at least one sero- reactive against more than three serogroups and were positive individual. These farms were distributed in therefore excluded from the data analysis. Laboratory 87% (n = 59/68) of the tested “départements” in main- analysis using only the sera for which a single serogroup land France. The serogroups obtained at the farm level was unambiguously identified would have been based are shown in Table  1. More than 97.5% of the farms on 2,608 positive samples (59.3% of the samples testing (n = 1,087) were associated with at least Australis and/or positive), whereas our data analysis was based on 4,346 Icterohaemorrhagiae, and 50% (n = 561) were exclusively positive samples (98.8% of the samples testing positive), associated with Australis and/or Icterohaemorrhagiae. corresponding to a 66% increase in sample size. The Naudet  et al. Porcine Health Management (2022) 8:15 Page 5 of 9 Fig. 2 The spatial distribution of the swine tested and the seropositivity against Australis, Icterohaemorrhagiae per “département” (administrative district) in mainland France Table 1 Number of farms having pigs with MAT profiles including Australis, Icterohaemorrhagiae and/or other serogroups Leptospira serogroups OnlyAustralis Only Onlyothers Australis and Australisand Icterohaemorrhagiae Australis, Icterohaemorrhagiae Icterohaemorrhagiae others and others Icterohaemorrhagiae and others Num- 134 94 27 333 66 51 409 ber of farms The temporal distribution of the dataset, including chi-square = 0.61). The relative counts of serogroups seropositive vs. seronegative pigs and farms, is displayed varied widely from year to year, as shown by the signifi - in Table  2. Considering the temporal variability of the cant interactions (generalized score for serogroup×year: serogroups among years (Table  3), the fit of the model χ2 (DF = 10) = 203.79, p < 0.0001). The relative counts of was adequate, as shown by the result that only seven Australis and Icterohaemorrhagiae fluctuated from year Pearson residuals were larger than 3 (6 were less than to year but nevertheless represented approximately 80% 3.84 and one was 8.82, out of 3,342 observations) and of infected individuals each year. The correlations among that there was no overdispersion (normalized Pearson the serogroups were negative (Table, 4). Naudet et al. Porcine Health Management (2022) 8:15 Page 6 of 9 Table 2 Features of the dataset (n = 19,343) and distribution of Australis and Icterohaemorrhagiae, with frequencies of the seropositive and seronegative pigs and farms over time from 48.5% and 38.2%, respectively. These high frequencies 2011 to 2017. suggested that among the 4,346 seropositive pigs, most associated Leptospira infections originated from only two Year 2011 2012 2013 2014 2015 2016 2017 serogroups out of the ten detected in this study. Total tested samples 795 2770 3089 3965 3715 4846 163 The observed predominant seroreactivity against Number of seroposi- 190 550 597 988 664 1320 37 Leptospira serogroups Australis and Icterohaemorrha- tive pigs giae in pigs was consistent with previous data reported Number of seronega- 605 2,220 2,492 2,977 3,051 3,526 126 in France [16]. This suggests the absence of any changes tive pigs in the epidemiological context of Leptospira strain dis- Total tested farms 100 333 347 497 459 568 21 tribution. A recent study in Italy that included pigs with Number of tested 70 197 126 236 173 312 10 positive farms clinical suspicion of leptospirosis was implemented using Number of tested 30 136 221 261 286 256 11 an eight-serovar panel; seven serovars were the same as negative farms those in our panel, and their study identified Australis Minimum sample 1 1 1 1 1 1 1 and Pomona as the most frequently detected serogroups size per farm [19]. However, caution is warranted in interpreting this Maximum sample 31 42 31 35 31 31 17 consistency because the previous study do not report size per farm how the MAT results with cross-seroreactivity were Median sample size 7 7 8 7 7 8 8 per farm managed, and the approach may have differed from that Median number of 2 2 4 4 3 4 3.5 in the present study. These observations support the need seropositive samples for a standardized approach for MAT surveillance data per seropositive farm analysis. Total tested pigs is the sum (in bold) of the two lines below (tested positive and Previous studies have described a poor correlation negative). Total tested farms is the sum (in bold) of the two lines below (positive and negative) between the presence of antibodies and the carrier state [25, 32]. Among 22 Australis-infected sows, six had titres above 1:100, which is the limit of positivity recommended Table 3 Weighted number of pigs with MAT profiles including by the OIE for screening and diagnosis [12, 25]. Our data Australis, Icterohaemorrhagiae and/or other serogroups between 2011 and 2017 underestimated the number of seroreactive samples against the serogroup Australis. However, we assume that Leptospira Years Total Serogroups the underestimation is the same among farms and over 2011 2012 2013 2014 2015 2016 2017 time, leading to a lower relative importance of the sero- Australis 86.67 224.17 242.50 343.50 376.17 802.33 24.00 2,099.33 group Australis in our results. Icterohaemor- 82.83 231.67 286.67 529.33 205.17 310.33 5.50 1,651.50 Regardless of the method used, the simple counts rhagiae excluding any MAT result with cross reaction or the Others 20.50 94.17 67.83 115.17 82.67 207.33 7.50 595.17 weighted average method, the results revealed predomi- 190 550 597 988 664 1320 37 4346 nance of the serogroups Australis and Icterohaemorrha- Total giae. The weighted average method allowed a consistent interpretation of the results among data and the inclu- Table 4 Estimated correlations between serogroups from the sion of 66% more MAT results. The weighted average unstructured working correlation matrix used with the generalized approach was thus useful for providing a more com- estimating equation model prehensive overview of the results. In addition, it is an Australis Icterohaemorrhagiae Others opportunity to standardize output of laboratory MAT results and to compare them among laboratories. Australis 1.00 − 0.66 − 0.43 Australis and Icterohaemorrhagiae remained predomi- Icterohaemorrhagiae − 0.66 1.00 − 0.26 nant in the farm-level analysis. This predominance was Others − 0.43 − 0.26 1.00 observed over time, even though the relative proportion of Australis and Icterohaemorrhagiae serogroups varied from one year to another. For anonymity reasons, it was Discussion not possible to identify farms that were potentially sam- Employing a retrospective approach, our study shows pled multiple times during the study period; however, that antibodies against pathogenic Leptospira serogroups this number of farms was assumed negligible consid- were frequently detected on French pig farms. In addi- ering that our sampling was limited to 10% of the total tion, two serogroups were detected much more fre- pig farms in France (n = 22,000) [33]. In addition, the quently than the others among seropositive pigs included, Naudet  et al. Porcine Health Management (2022) 8:15 Page 7 of 9 number of contributions (number of sampled pigs per limited by the low specificity and sensitivity of the MAT farm) varied from one farm to another and may have had [38]. According to our results, which showed that more an impact on the results. Nonetheless, the contribution than 50% of the seropositive pigs were exposed to Aus- variation is unlikely to change the predominance of Aus- tralis and/or Icterohaemorrhagiae, management options tralis and Icterohaemorrhagiae. preventing such infections could have greatly reduced the Regarding MAT, a previous experimental infection burden of the disease among the pigs. study showed that Leptospira-specific IgG (titres above As the serogroups Australis and Icterohaemorrhagiae 1:100) could be detected for more than 100 days [34]. are also pathogenic to humans, infected pig popula- Even when a threshold of positivity of 1:100 is applied, tions represent a potential cause of occupational disease, recently infected, chronically infected or previously especially for breeders or slaughterhouse staff [39, 40]. exposed individuals cannot be clearly distinguished [4]. Following a One Health approach, leptospirosis manage- However, because we were chiefly interested in the dis - ment in pig populations may contribute to the protection tribution of the Leptospira serogroups at a large temporal of human populations and to the mitigation of Leptospira scale, any evidence of past infection was relevant to the persistence and transmission in the country. description of the serogroup distribution in pigs. In addi- tion, the serogroups Australis and Icterohaemorrhagiae Conclusions were predominant in each year from 2011 to 2016 in our The analysis of data from diagnostic laboratories is useful dataset, which suggests that the misclassification of past for obtaining an indication of the circulating Leptospira or current infections would have limited effects on our serogroups in a region or a country and over time. Our results. results suggested that over the study period, most Lepto- Most of the samples were obtained from Brittany, spira infections in swine in France originated from only which is an intensive pig farming area compared with two serogroups, Australis and Icterohaemorrhagiae, out other regions in France. However, the serogroup distri- of the ten used for the laboratory analysis. This informa - bution was similar among regions. The samples analysed tion should be considered to support future prophylactic originated from swine herds with clinical suspicion of measures. leptospirosis; thus, we can conclude that throughout In Europe, numerous laboratories have published France, herds with reproductive disorders that were interpreted MAT results. However, there is no consen- exposed to Leptospira were most likely exposed to the sus regarding serovars to be included in the panel, the Australis and Icterohaemorrhagiae serogroups. Among positive threshold and serogroup determination in the the pig isolates related to the serogroup Australis, some case of cross-reactions, leading to the incomparability of were more likely to be associated with disease [35], and results among studies. Therefore, this is an appeal to vet - Australis exposure in pigs should be seriously considered erinary laboratories in Western Europe to standardize their as a potential cause of reproductive failure. A broader approach to MAT surveillance data analysis. Based on our survey including nonsymptomatic herds could help clar- results and previously published results, future MAT sur- ify the relationship between Leptospira serogroup expo- veillance data analysis in Western Europe should be sup- sure and reproductive failure. However, given the low ported by microagglutination testing with a common set MAT titres found in infected sows and boars from farms of serovars belonging to the serogroups Australis, Ballum, where reproductive failure had occurred and where the Bataviae, Icterohaemorrhagiae, Grippotyphosa, Panama, serovar bratislava had been recovered from aborted sows, Pomona, Sejroe and Tarassovi and using a weighted aver- foetuses and boars, this may not be of value for the Aus- age approach to determine the serogroup distributions and tralis serogroup. considering cross-reactions. Herd management of leptospirosis may rely on ani- mal reservoir control. Rattus norvegicus is a frequent Abbreviations commensal rodent in livestock buildings and a selective MAT: Microagglutination test; AUS: Australis.; AUT : Autumnalis; BAT: Bataviae; carrier of Leptospira of the serogroup Icterohaemorrha- GRI: Grippotyphosa; ICT: Icterohaemorrhagiae; PAN: Panama; POM: Pomona; giae [36, 37]. Thus, rats may play a role to some extent PYR: Pyrogenes; TAR: Tarassovi; SJ: Sejroe. in Icterohaemorrhagiae pig exposure, and this exposure Acknowledgements could be reduced through rat management. As no selec- The authors thank the technicians for their technical support in laboratory tive carriers of Leptospira of the serogroup Australis have procedures and microagglutination testing between 2007 and 2017. We are also grateful to VetAgro Sup for funding the study. been described among commensal rodents, pigs them- selves could be the main reservoir host in the context Authors’ contributions of pig farming, as suggested by Ellis [4]. Leptospirosis FA made substantial contributions to the concept and design of the study, interpreted the results and critically revised the manuscript. LC and JC management through the culling of Leptospira carriers is Naudet et al. Porcine Health Management (2022) 8:15 Page 8 of 9 contributed to the concept and design of the study and critically revised the 9. Picardeau M, Bertherat E, Jancloes M, Skouloudis AN, Durski K, Harts- manuscript. JC was involved in the spatial data analysis. LC was involved in keerl RA. Rapid tests for diagnosis of leptospirosis: current tools and the analysis of temporal variability. JN wrote the first draft of the manuscript emerging technologies. Diagn Microbiol Infect Dis. 2014;78(1):1–8. and constructed the figures. AK provided the data from the Laboratoire des 10. Musso D, La Scola B. Laboratory diagnosis of leptospirosis: a challenge. Leptospires. All authors read and approved the final manuscript. 2013;(46):245–52. 11. Levett PN. Leptospirosis. Clin Microbiol Rev. 2001;14(2):296–326. Funding 12. Anonymous. Leptospirosis. In: OIE Terrestrial Manual 2021. 8e Ed. 2018. This work was supported by VetAgro Sup, Marcy L’Etoile, France [Agriculture p. 1833. Ministry Funding]. 13. Anonymous. Human leptospirosis: guidance for diagnosis, surveil- lance and control. [Internet]. WHO Library Cataloguing-in-Publication Availability of data and materials Data; 2003. Available from: https:// apps. who. int/ iris/ bitst ream/ handle/ The datasets used and/or analysed during the current study are available from 10665/ 42667/ WHO_ CDS_ CSR_ EPH_ 2002. 23. pdf? seque nce= 1& isAll the corresponding author upon reasonable request. owed=y. 14. Padilha BCR, Simão HQ, Oliveira TL, Hartwig DD. The use of ErpY-like recombinant protein from Leptospira interrogans in the develop- Declarations ment of an immunodiagnostic test for swine leptospirosis. Acta Trop. 2019;193:31–4. Ethics approval and consent to participate 15. Levett PN. Leptospirosis. Clin Microbiol Rev. 2001;14(2):296–326. The present research does not include experimental data, and all laboratory 16. André-Fontaine G. Leptospirosis in domestic animals in France: analyses were performed on data from previous examinations of swine for serological results from 1988 to 2007. Rev Sci Tech Int Off Epizoot. routine diagnosis. The Ethical Committee was consulted, and it confirmed that 2016;35(3):913–23. approval was not required in this case. 17. Bertelloni F, Cilia G, Turchi B, Pinzauti P, Cerri D, Fratini F. Epidemiology of leptospirosis in North-Central Italy: fifteen years of serological data Consent for publication (2002–2016). Comp Immunol Microbiol Infect Dis. 2019;1:65:14–22. Not applicable. 18. Strutzberg-Minder K, Tschentscher A, Beyerbach M, Homuth M, Kreienbrock L. Passive surveillance of Leptospira infection in swine in Competing interests Germany. Porc Health Manag [Internet]. 2018 Mar 27 [cited 2020 May The authors declare that they have no competing interests. 10];4. Available from: https:// www. ncbi. nlm. nih. gov/ pmc/ artic les/ PMC58 72537/. Author details 19. Tagliabue S, Figarolli BM, D’Incau M. Serological surveillance of 1 2 VetAgro Sup, Université de Lyon, USC 1233, Marcy L’Etoile, France. Université Leptospirosis in Italy: two year national data (2010- 2011). Vet Ital. Lyon, INRAE, VetAgro Sup, UMR EPIA, 69280 Marcy L’Etoile, France. Université 2016;(52(2)):129–39. Clermont Auvergne, INRAE, VetAgro Sup, UMR EPIA, 63122 Saint-Genès- 20. Chappel RJ, Goris M, Palmer MF, Hartskeerl RA. Impact of proficiency Champanelle, France. UMR ASTRE, CIRAD, INRAE, Univ Montpellier, testing on results of the microscopic agglutination test for diagnosis of 34398 Montpellier, France. Laboratoire des Leptospires, VetAgro Sup, Marcy Leptospirosis. J Clin Microbiol. 2004;42(12)(1):5484–8. L’Etoile, France. 21. Miller MD, Annis KM, Lappin MR, Lunn KF. Variability in results of the microscopic agglutination test in dogs with Clinical Leptospi- Received: 17 August 2021 Accepted: 14 March 2022 rosis and Dogs Vaccinated against Leptospirosis. J Vet Intern Med. 2011;25(3):426–32. 22. Sokal RR, Rohlf FJ. Biometry: the principles and practice of statistics in biological research. 3rd ed. New York : W.H. Freeman; 1995. Available from: https:// trove. nla. gov. au/ work/ 10262 434. References 23. Ayral F, Bicout DJ, Pereira H, Artois M, Kodjo A. Distribution of leptospira 1. Vincent AT, Schiettekatte O, Goarant C, Neela VK, Bernet E, Thibeaux R, serogroups in cattle herds and dogs in France. Am J Trop Med Hyg. et al. Revisiting the taxonomy and evolution of pathogenicity of the 2014;91(4)(1):756–9. genus Leptospira through the prism of genomics. PLoS Negl Trop Dis. 24. Stokes JE, Kaneene JB, Schall WD, Kruger JM, Miller R, Kaiser L, et al. 2019;13(5):e0007270. Prevalence of serum antibodies against six Leptospira serovars in 2. Levett PN. Systematics of Leptospiraceae. In: Adler B, editor. Leptospira healthy dogs. J Am Vet Med Assoc. 2007;230(11):1657–64. and Leptospirosis. Berlin, Heidelberg: Springer; 2015. p. 11–20. (Current 25. Ellis WA, McParland PJ, Bryson DG, Thiermann AB, Montgomery J. Isola- Topics in Microbiology and Immunology). Available from: https:// doi. tion of leptospires from the genital tract and kidneys of aborted sows. org/ 10. 1007/ 978-3- 662- 45059-8_2. Vet Rec. 1986 Mar;15(11):294–5. 118. 3. Bolin CA. Diagnosis of leptospirosis in swine. Swine Health Prod Off J 26. Kusum M, Boonsarthorn N, Biaklang M, Sina U, Sawanpanyalert P, Am Assoc Swine Pract USA. 1994; Available from: http:// agris. fao. org/ Naigowit P. Comparison of leptospiral serovars identification by serol- agris- search/ search. do? recor dID= US951 9052. ogy and cultivation in northeastern region, Thailand. J Med Assoc Thail 4. Ellis WA. Animal leptospirosis. Curr Top Microbiol Immunol. Chotmaihet Thangphaet. 2005;88(8):1098–102. 2015;387:99–137 27. Lupidi R, Cinco M, Balanzin D, Delprete E, Varaldo PE. Serological 5. Andre-Fontaine G, Aviat F, Thorin C. Waterborne Leptospirosis: survival follow-up of patients involved in a localized outbreak of leptospirosis. J and preservation of the virulence of pathogenic Leptospira spp. in Clin Microbiol. 1991;29(4):805–9. fresh water. Curr Microbiol. 2015;71(1):136–42. 28. Gilbert M, Nicolas G, Cinardi G, Van Boeckel TP, Vanwambeke S, Wint 6. Ayral F, Djelouadji Z, Raton V, Zilber A-L, Gasqui P, Faure E, et al. WGR, et al. Global pigs distribution in 2010 (5 minutes of arc). Harvard Hedgehogs and mustelid species: major carriers of pathogenic Lepto- Dataverse; 2018 [cited 2021 Dec 16]. Available from: https:// datav erse. spira, a survey in 28 animal species in France (20122015). PLoS ONE. harva rd. edu/ datas et. xhtml? persi stent Id= doi:https:// doi. org/ 10. 7910/ 2016;28(9):e0162549. DVN/ 33N0JG. 7. Ellis WA. Animal leptospirosis. In: Leptospira and leptospirosis. B. Adler 29. Liang K-Y, Zeger SL. Longitudinal data analysis using generalized linear (3rd). Berlin, Heidelberg, Germany: Springer; 2015. p. 99–137. models. Biometrica. 1986;76:13–22. 8. Niloofa R, Fernando N, de Silva NL, Karunanayake L, Wickramasinghe 30. Hardin JW, Hilbe JM. Generalized estimating equations, 2nd ed. New H, Dikmadugoda N, et al. Diagnosis of Leptospirosis: comparison York: Chapman and Hall/CRC; 2013. 277 p. between microscopic agglutination rest, IgM-ELISA and IgM rapid 31. Agresti A. Categorical data analysis. Hoboken: Wiley; 2002. 710 p. immunochromatography test. PLoS ONE. 2015;18(6):e0129236. Naudet  et al. Porcine Health Management (2022) 8:15 Page 9 of 9 32. Ellis WA, McParland PJ, Bryson DG, Cassells JA. Boars as carriers of lepto- spires of the Australis serogroup on farms with an abortion problem. Vet Rec. 1986;118(20):563. 33. Roguet C. Evolution des modèles d’exploitations porcines en France sous l’effet des contraintes économiques et réglementaires depuis 2008. Journées Recherche Porcine. 17AD;49:277–82. 34. Ballard SA, Adler B, Millar BD, Chappel RJ, Jones RT, Faine S. The immunoglobulin response of swine following experimental infec- tion with leptospira interrrogans serovar pomona. Zentralblatt Für Bakteriol Mikrobiol Hyg 1 Abt Orig Med Mikrobiol Infekt Parasitol. 1984;256(4):510–7. 35. Ellis WA, Montgomery JM, Thiermann AB. Restriction endonuclease analysis as a taxonomic tool in the study of pig isolates belonging to the Australis serogroup of Leptospira interrogans. J Clin Microbiol. 1991;29(5):957–61. 36. Ayral F, Zilber A-L, Bicout DJ, Kodjo A, Artois M, Djelouadji Z. Distribu- tion of Leptospira interrogans by multispacer sequence typing in Urban Norway Rats (Rattus norvegicus): a survey in France in 2011–2013. PLOS ONE. 2015;10(10):e0139604. 37. Thiermann AB. The Norway rat as a selective chronic carrier of Lepto- spira icterohaemorrhagiae. J Wildl Dis. 1981;17(1):39–43. 38. Ellis WA. 03.01.12_Leptospirose. In: Manual of diagnostic tests and vaccines for terrestrials animals. OIE; 2018. p. 503–16. Available from: http:// www. oie. int/ filea dmin/ Home/ fr/ Health_ stand ards/ tahm/3. 01. 12_ LEPTO. pdf. 39. Mirambo MM, Mgode GF, Malima ZO, John M, Mngumi EB, Mhamphi GG, et al. Seropositivity of Brucella spp. and Leptospira spp. antibod- ies among abattoir workers and meat vendors in the city of Mwanza, Tanzania: a call for one health approach control strategies. PLoS Negl Trop Dis. 2018;12(6):e0006600. 40. Thornley CN, Baker MG, Weinstein P, Maas EW. Changing epidemi- ology of human leptospirosis in New Zealand. Epidemiol Infect. 2002;128(1):29–36. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? 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Journal

Porcine Health Management – Springer Journals

Published: Apr 4, 2022

Keywords: Leptospira; Pig; Reproductive failure; Microagglutination test; Australis; Icterohaemorrhagiae

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