A systematic review on leptospirosis in cattle: A European perspective

Leptospirosis is a zoonotic disease which is globally distributed and represents a classic One Health issue that demands a comprehensive understanding of the hosts, transmission paths, and risk factors of transmission. Bovine leptospirosis often results in economic losses through its severe impact on reproduction performance while it threatens human health at human-cattle-environment interfaces. However, a clear analysis of the disease characteristics in European cattle is currently lacking. The objective of this review was to summarise the current knowledge on the epidemiology of bovine leptospirosis in Europe. We conducted a systematic literature review, screening four electronic databases, and filtered articles published between 2001 and 2021, in English, German, and French. Sixty-two articles were ultimately included in the review. The seroprevalence of leptospirosis in cattle was remarkably variable among studies, probably reflecting local variations but also heterogeneity in the study designs, laboratory methods, and sample sizes. Risk factors positively associated with the disease were diverse, related to local, environmental, and climatic parameters as well as farming practices. The most reported circulating Leptospira serogroups in European cattle were Sejroe (58.5%), Australis (41.5%), Grippotyphosa (41.5%), Icterohaemorrhagiae (37.7%), and Pomona (26.4%), which have also been associated with human infections worldwide. Abortion (58.6%) and fertility disorders (24.1%) were the most frequently reported signs of leptospirosis in European cattle and were generally associated with chronic infections. This work highlights several research gaps, including a lack of harmonisation in diagnostic methods, a lack of large-scale studies, and a lack of molecular investigations. Given that predictions regarding the climatic suitability for leptospirosis in Europe suggest an increase of leptospirosis risk it is important to raise awareness among stakeholders and motivate an integrated One Health approach to the prevention and control of this zoonotic disease in cattle and humans.


Introduction
Leptospirosis is a neglected zoonotic disease, that is distributed worldwide [1]. The disease is estimated to affect 1.03 million people globally and cause 58,900 deaths annually [2]. Most outbreaks occur in tropical regions, but cases are also reported from temperate areas [3]. The causative bacterial agent, Leptospira spp., has been reported in a wide range of mammals worldwide [4]. Transmission occurs primarily through direct or indirect (i.e. via contaminated water or soil) contact with the urine of infected animals [3,5] although venereal transmission is also described [6]. The bacteria can enter the body through the mucous membranes or damaged skin [5]. Following a leptospiraemic phase, the bacteria can colonise various organs, especially the kidneys, from where they are then shed intermittently in the urine [4,5], or the genital tract, where they can be detected in the semen or vaginal discharge [6].
The phylogenetic classification, which organises Leptospira species based on DNA relatedness [7], currently acknowledges 68 species [7,8]. It coexists with the historical, serological classification, which recognises more than 300 serovars of Leptospira, grouped into serogroups [9] based on the expression of the surface-exposed lipopolysaccharide (LPS).
The gold standard for the detection of leptospiral antibodies is the microscopic agglutination test (MAT), which uses live cultures of Leptospira strains that are tested at different dilutions against patient (animal or human) serum [4]. The enzyme-linked immunosorbent assay (ELISA) is also widely used for the serological diagnostic of leptospirosis [5]. Isolation of the bacteria via culture is possible but not suitable for the diagnosis of acute leptospirosis due to the fastidious growth of the bacteria, which often requires specific culture media and might take several weeks to provide a positive result [9,10]. PCR-based strategies give faster results and demonstrate high sensitivity and specificity to detect Leptospira from urine, cerebrospinal fluid, or blood samples during the early stages of the disease [11] as well as in the urine, kidney, or genital tract of chronic animal carriers [12,13].
The environmental persistence of Leptospira and its epidemiology rely on the chronic renal colonisation of reservoir animals [14]. A reservoir may remain symptom-free while excreting the bacteria in its urine, either transiently or for its entire life [4,12]. Cattle are recognised as the maintenance host for serovar Hardjo (serogroup Sejroe), encompassing serovar Hardjobovis and Hardjoprajitno [5], and infection of cows with this serovar typically results in chronic infection and can lead to abortion, fertility disorders, and decrease in milk yield [4,5]. Therefore, the disease has an important economic impact due to both reproductive and non-reproductive losses to production [15]. Occurring at human-cattle-environment interfaces, bovine leptospirosis is considered an occupational zoonotic disease to e.g., farm workers and veterinarians [3] and represents a challenge for public and animal health.
Leptospirosis in cattle has been reported worldwide. Previous reviews referring to bovine leptospirosis, were conducted for Africa [16] and Latin America [17], however, to date, no work has summarised the knowledge on cattle leptospirosis from a European perspective, although essential to develop One Health strategies to prevent and manage outbreaks at human-cattle interfaces. To characterise the epidemiology of bovine leptospirosis in Europe, we conducted a systematic literature review and examined the extent and nature of the knowledge pertaining to this topic over a 20-year period, 2001-2021.

Literature search strategy
From 7 June to 26 August 2021, we performed a systematic literature search, using four electronic databases: Pubmed, Web of Science, Scopus, and CABI. The search queries included the following keywords "lepto*", "cattle" "cows," and "cow" (Supplementary material 1: Table 1). Additional papers were identified through internet-based search engines such as Google and Google Scholar and by handsearches of the references cited in the reviewed studies. We considered articles published between 1 January 2001 and the date of search (26 August 2021), thereby covering over 20 years.

Paper selection and screening
First, citation data, title and abstracts were compiled and deduplicated in Mendeley Reference Manager. Two reviewers (CS and ADL) independently screened all titles and abstracts. Titles/abstracts were selected if they contained qualitative and/or quantitative data on leptospirosis in cattle (e.g. data on clinical signs, prevalence, risk factors, circulating strains/serovars/serogroups) and if the study was conducted in a European country, as defined by the most common geographical definition of Europe, i.e. the land bordered by the Arctic Ocean to the north, the Atlantic Ocean to the west, the Mediterranean Sea to the south, and the Ural Mountains to the east. Case reports, outbreak description, epidemiologic surveys, reviews, or epidemiological reports were included.
Articles deemed eligible in the first round of screening were retrieved in full text format and reviewed independently by CS and ADL. Papers were excluded if the study was not pertaining to bovine leptospirosis, was performed outside Europe and/or was written in languages other than English, German, or French. Editorials, commentaries, book chapters, and conference proceedings were excluded. We also excluded papers dealing with immunology, vaccine strategy or efficacy, and diagnostic tools or methods. Disagreements between reviewers were discussed and resolved by consensus.

Data extraction and synthesis
Two reviewers (CS and ADL) extracted the following information from included papers: bibliographic information (including citation, type of paper, year of publication), study purpose and design, geographic location of the study, sampling unit (animal or herd), number of sampling unit(s) investigated, sample type(s), laboratory methods, positivity threshold, study period, reported prevalence and/or incidence, reported serogroup(s) and/or serovar(s) and/or genomospecies, clinical presentation, histological or necropsy findings, and production type (i.e. dairy, beef, mixed). For each serovar, when not provided, the serogroup was retrieved from the literature [18]. Moreover, we assessed the One Health-ness of each study by checking for the mention of the term "One Health" and examining whether each study explored compartments beyond cattle, such as human, environment, or other animals.
We also collected information on risk factors associated with Leptospira infection, i.e. investigated risk factor(s), assessed outcome(s), whether or not a statistical analysis was performed, relationships between the dependent variable and the risk factor (e.g. positive, negative, not evidenced), statistical model used, statistics reported and value, 95% confidence interval, and p-value. To obtain a more comprehensive understanding of the risk associated with Leptospira infection in European cattle, the risk factors were clustered into seven broader categories (Supplementary material 2: Appendix A). Finally, two additional independent reviewers (JS and JJ) performed a final curation and validation of the extracted data.

Clinical signs and histo-pathological findings associated with bovine leptospirosis in Europe
Twenty-nine studies (46.8%), from 13 different countries, described clinical signs and/or histo-pathological findings associated with Leptospira infection in cattle. Clinical signs of leptospirosis differed largely with respect to the age of the animal, with acute forms of the disease typically affecting calves and foetuses, while chronic forms are generally observed in adults (Table 3). We also evidenced that clinical signs depicted in dairy versus beef cattle were slightly different (Supplementary material 3: Fig. 3).
The main risk factors statistically and positively associated with Leptospira infection in cattle included: i) environmental factors, such as the geographic location of the animal (or herd) [33,37,40,41,61], exposure to flooding [72], the season spring [56], and access to pasture [40]; ii) herd management practices, such as the herd size [30,32,36,37,[39][40][41] (although some studies found no statistical relationship [33,55,63]), rearing calves off farm, co-grazing of calves and cows, housing the calves later in the year [39], segregating heifers and cows at calving [40], and the percentage of primiparous cows in the herd [39]; iii) factors related to biosecurity, such as the employment of agricultural contractors untrained on biosecurity [39], the purchase of animals [32,36,54] (although two studies did not confirm this result [30,40]), the movement of cattle onto and off the farm [39], and the use of a stock bull [40]: iv) comorbidity with infectious diseases (e.g. past or co-occurring infection with BVD, bovine herpes virus 1 (BHV-1), Salmonella) [30,32,33,38]; v) clinical conditions, such as a recent history of abortion [52] (especially icteric abortion [71,81]); and vi) individual factors, such as age and sex (which showed inconsistent effects on the risk of infection by Leptospira [41,58,60]), breed [41], or type of production [63], with dairy herds being significantly more at risk of infection with Leptospira serovar Copenhageni, Grippotyphosa, and Tarassovi than beef herds [63]. Finally, factors related to the study design (e.g. date of sampling) may have an impact on the seroprevalence of Leptospira [61] (although, date of sampling might be a confounding factor reflecting the influence of e.g. the season, weather, or specific conditions at time of sampling) (Fig. 3, Supplementary material 3: Fig. 4).
In contrast, minimising the number of visitors in farms [39] or increasing the percentage of wetland grazed [40] were shown to Colours represent the risk categories. The y-axis represents the number of times a risk factor was tested across the 18 studies; x-axes represent the production type, risk category, and statistical association between the risk factor and the presence of Leptospira. Association may be positive or negative; "significant" means that the association is statistically significant, but no direction is provided. decrease significantly the probability of infection. Interestingly, a previous Leptospira infection was not reported as a risk factor for subsequent infection [40]. Finally, although not tested statistically, the presence of rodents [48,50], the use of pig manure for the cow grazing pasture [60], extreme weather conditions [75], and access to natural water sources [29,60] were also noted as risk factors of infection in cattle. Surprisingly, herds where cleaning of drenching equipment was performed were more likely to test positive for antibodies to Leptospira serovar Hardjo [39], as were cattle vaccinated against the BVD virus [36]. One possible reason for these associations is that the cleaning of drenching equipment and vaccination might have been operated in response to the presence of the bacteria or other infectious diseases in the herd [39].

Discussion
Despite the apparent growing risk, the impact on cattle and human health, and the potential severe economic losses related to bovine leptospirosis, this review shows that there is a lack of comprehensive data on Leptospira infection in cattle in the European region, with studies published from 18 European countries only. To some extent, the geographic distribution of the publications included in this review reflects the distribution of the bovine population in Europe, with a large proportion of studies conducted in the United Kingdom, Ireland, and France.
The occurrence of leptospirosis in European cattle exhibited significant variation, both between and within countries. This variability may be influenced by several factors, including the geographical scale of the study (national, regional, or at farm level), geographic location, and study design (e.g. case investigation versus retrospective study, sample size, laboratory methods). Knowledge of the (sero)prevalence of bovine leptospirosis in a geographic region is essential for veterinary practitioners, but also medical doctors, to include or exclude the disease in their differential diagnoses and reduce under-and misdiagnoses. However, the prevalence of the bacteria in cattle in Europe remains largely elusive since many studies involved a relatively small number of animals while clinical case reports represented one quarter of the included studies.
This review demonstrates that MAT is the prevailing technique used to confirm leptospirosis in cattle across Europe, despite significant heterogeneity in laboratory procedures. Notably, the MAT cut-off values displayed significant variations, indicating a lack of consensus over which titre should be used for a positive result. In addition, the MAT antigen panel was found to be extremely variable between studies, which prevents accurately mapping serogroup distribution in Europe. Furthermore, intrinsic limitations of the method, i.e. possible crossreactivity between serogroups, does not allow the identification of the infecting serogroup with absolute certitude [85] and generally, the agglutinating serogroup with the highest MAT titres is the only one reported. These drawbacks narrow our understanding of the epidemiology of the circulating serogroups.
To provide comparable data at European level, harmonised protocols to investigate, diagnose, and report cases of leptospirosis in cattle are necessary. For example, cattle-specific cut-off values for the MAT should be defined. Likewise, a pan-European consensus is needed regarding the minimum panel of serogroups to be included in the MAT, which could further be regionally optimised with locally isolated strains [86], as recommended by, the World Organisation for Animal Health (WOAH) [85]. Harmonised protocols could further help identifying possible associations between infecting serogroups and clinical signs in cattle.
This review demonstrates that the serogroups Sejroe, Australis, Grippotyphosa, Icterohaemorrhagiae, and Pomona are the most reported ones in cattle in Europe. Yet, those serogroups were the most used in the MAT panels across reviewed studies, which may bias the overall epidemiological picture. Nevertheless, this finding supports previous observations, e.g. from South America [17] Africa [16], Malaysia [87], and New Zealand [88]. These serogroups have also been associated with human cases globally [16,89,90], stressing the need for further research on the transmission potential of the bacteria at human-cattleenvironment interfaces, that would address the disease through a One Health lens.
As described in tropical and sub-tropical areas [91,92], New Zealand [93], and Australia [94], the most recognised and reported clinical signs of bovine leptospirosis in European cattle are abortions and fertility disorders. Therefore, in case of abortion events in European cattle, leptospirosis should be considered as a differential diagnosis among other, more classical, infectious causes of abortion, i.e. Brucella spp., Neospora caninum, Coxiella burnetii, BVD virus, BHV-1, or Salmonella enterica serotype Dublin [95]. However, clinical signs can vary widely, making a field diagnosis challenging and emphasising the importance to rely on standardised laboratory tests.
This review provides a comprehensive overview of the risk factors of Leptospira infection in cattle in Europe despite the heterogeneity of the study designs, which challenges the comparison of the results. The most important risk factors of bovine leptospirosis were related to biosecurity measures and the environment. Local or even regional conditions influence the presence of Leptospira in the environment as well as hostbacteria interactions. Several factors related to the soil and water pH, temperature, or composition of the environmental microbiome may determine the possibility of persistence of Leptospira outside its animal host [96]. Climatic conditions in Europe are becoming increasingly suitable for the survival and transmission of water-and rodent-borne diseases, including leptospirosis [97]. Extreme weather events compounding the impact of changes in land use (especially urbanisation) intensify the direct and indirect contacts between leptospires, humans, and animal hosts [89], therefore increasing the risk for public and animal health. Overall, this review evidences the multiplicity and complexity of the risk factors associated with Leptospira exposure in cattle. These findings should motivate local and national health authorities, veterinarians, and farmers to implement integrated disease prevention and control measures at farm or regional level.
Research on cattle leptospirosis in Europe suffers several data and research gaps. Remarkably, we did not find any paper reporting a successful isolation of a Leptospira strain from naturally-infected cattle in Europe between 2001 and 2021. Although the recovery of Leptospira from field samples is extremely challenging, the isolation of local strains from infected animals, humans, or the environment is essential to optimise the MAT panel for the serological diagnosis of leptospirosis in humans and animals [85,98] but also to delineate relevant One Health interventions [99]. We highlighted a lack of molecular data on Leptospira strains circulating in cattle. The isolation and molecular characterisation of Leptospira from cattle and their environment would advance knowledge on the epidemiology, ecology, and pathogenesis of the bacteria, and would have practical applications in the prevention, surveillance, and control of the disease in both animals and humans. More efforts are needed in this direction. This review also stresses a lack of large-scale studies, necessary for drawing representative conclusions and achieving sufficient statistical power, and points out that dairy herds are disproportionally more frequently investigated compared to beef herds, leading to a data gap regarding the clinical signs, prevalence, and impact of the disease in beef cattle. We also demonstrated that the zoonotic and One Health aspect of bovine leptospirosis is largely neglected in Europe, highlighting a potential gap in understanding and addressing the epidemiological link between cattle, human, and the broader ecological context. Additionally, we noted a lack of data on the risk related to artificial insemination and a limited investigation of rodents as a source of Leptospira infection in cattle herds. Finally, in the last 20 years, no study investigated bovine genital leptospirosis (BGL) in Europe, although studies from Brazil have evidenced a relatively high prevalence of the disease [13] and point toward the recognition of BGL as a distinct syndrome [6].

Conclusions
Research on bovine leptospirosis is generally under-resourced while the disease is globally neglected [100], including in Europe, where a limited number of countries have investigated and reported the disease. Considering the veterinary and public health importance of leptospirosis as well as its economic impact, it is crucial to raise awareness among stakeholders, including farmers, veterinarians, and other health professionals, in areas where the disease is not (yet) endemic. This is especially important in Europe where this zoonotic disease is (re-) emerging in humans, but also in animals. Moreover, intensification of livestock farming in certain regions of Europe, concomitant with an increasing trend toward herd grazing outdoors in other areas will also certainly play a major role in the future regional incidences of leptospirosis in cattle, probably increasing regional contrasts. Local studies are essential to advance our understanding of the epidemiology of bovine leptospirosis and therefore develop and implement relevant, locally-adapted prevention and control strategies. Nevertheless, an overview of the epidemiology of the disease at continental scale, as presented here, can yield novel insights into its epidemiological features in a One Health context.

Funding
This study was funded by the federal state of Lower Austria (Land Niederösterreich) (WST3-F-5033836/001-2020). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Declarations of interest: none.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability
All relevant data are within the manuscript and its Supporting Information files.