Leptospirosis is a widespread zoonotic disease caused by infection with a pathogenic species of the genus Leptospira [1
]. It is a worldwide public health and veterinary problem involving many domestic and wild animal species. Dogs have been known to be hosts of pathogenic leptospires since 1931, when Klarenbeek and Schuffner first isolated leptospires from the urine of a dog affected by nephritis [2
]. Dogs are highly susceptible to infection, and they act as a sentinel species for the environmental risk to humans because of their high level of environmental exposure to pathogenic leptospires. Canine leptospirosis has been largely described worldwide [1
], and its seroprevalence varies in accordance with geographic location, as follows: 1.8% in Australia [4
], 7.3% in China [5
], 9.9% in Brazil [6
], 14.63% in Iran [7
], 17.1% in the USA [8
], 25.8% in Spain [9
] and 71.1% in India [10
]. In Italy, a 2002 survey conducted on kenneled dogs reported a seroprevalence of 29.4% [11
], and national data collected in 2010–2011, involving more than 3000 dogs, described a seropositivity of 29.9% [12
]. Nevertheless, these studies were not standardized, and some were partially biased by vaccine responses, the panel of serovars adopted for the Micro Agglutination Test (MAT) and sample collection, which was based on clinical suspects. In fact, a recent survey conducted in Italy on both ill and healthy dogs reported a lower seroprevalence of 8% [13
Canine leptospirosis has mainly been associated with serovars Canicola and Icterohaemorrhagiae, two serogroups included within the Leptospira interrogans
species. In Europe, protective vaccines for dogs against these serovars have been available for ~60 years [14
]; however, recently, leptospirosis’ epidemiological situation in dogs has changed, and clinical syndromes have been described in association with serovars not included in the traditional vaccines [1
]. Because Grippotyphosa and Bratislava have emerged as major causes of canine leptospirosis in Europe [12
], new canine Leptospira
vaccines containing antigens from up to four different serogroups, Canicola, Icterohaemorrhagiae, Australis and Grippotyphosa, have been introduced in the area [20
]. In particular, trivalent (serogroups Canicola, Icterohaemorrhagiae and Grippothyphosa) and tetravalent (serogroups Canicola, Icterohaemorrhagiae, Grippothyphosa and Australis) vaccines have been licensed in European countries.
To date, epidemiological studies have been mainly based on serological diagnoses, particularly MAT, which is based on determining the ability of serial dilutions of the tested serum to agglutinate live leptospiral serovars in vitro. Agglutination is assessed by darkfield microscopy and suggests exposure to a serovar belonging to the corresponding serogroup (but not necessarily to the serovar tested) [22
]. It is based on the specific antigenic determinants related to the structural heterogeneity of the lipopolysaccharide, and it is considered the diagnostic test of choice in dogs suspected of having leptospirosis [16
]. Despite the widespread use of MAT to diagnose leptospirosis in dogs, this assay has limitations linked to its indirect diagnostic nature, such as possible false-negative results for initial infections, cross-reactions and paradoxical reactions early during the course of the disease [22
], the variability of the selected antigenic panel and the subjective interpretation of the results. Furthermore, it generally does not discriminate between vaccinated and infected, increasing the difficulty of interpreting canine tests [26
]. In addition, making direct comparisons between different studies is complicated by the variability in cut-off MAT titers used.
Several direct molecular assays, such as real-time PCR targeting various leptospiral genes, have been developed to support serological methods [27
]. The diagnostic performances of different PCR assays are not equivalent [33
], but they have been very useful in confirming diagnoses at the early stages of infection, when antibody titers are at undetectable levels [34
], and in testing vaccinated patients because previous vaccination does not interfere with the PCR results [35
]. Positive PCR results indicate that leptospiral DNA is present in the sample, but negative blood or urine results do not rule out leptospirosis. In fact, leptospiremia is transient, and urinary shedding is delayed after acute infection and can be intermittent. Furthermore, having received an antibiotic treatment recently may affect the detection of leptospiral DNA [1
]. For this reason, PCR results should always be interpreted cautiously and in conjunction with MAT results, and they should take into account the clinical context. Furthermore, although its use is highly feasible, routine diagnostic PCR provides no information on the infecting serovar.
Some methods of molecular typing, such as Multilocus Sequence Typing (MLST) [36
] and Multiple Loci Variable-number Tandem Repeat Analysis (MLVA) [37
], offer interesting epidemiological perspectives by providing a specific and unique barcode for the infecting Leptospira
through the analysis of specific fragments of particular bacterial loci. Until recently, the MLST technique was only applicable to isolated strains, because it required relatively large amounts of leptospiral DNA, making its direct use on clinical specimens impossible. This was a considerable limitation because culturing leptospires is challenging, time consuming and requires an equipped laboratory. Moreover, the isolation efficiency is very low and is dependent on the Leptospira
strain and the use of an antimicrobial treatment prior to collection. Fortunately, a fast and specific method for genotyping Leptospira
DNA directly from biological samples has been developed [38
], allowing the rapid identification of the pathogen without strain isolation and providing the opportunity to investigate all the circulating strains, not just those successfully isolated, which represent a small percentage of the spreading strains. Through the assignment of sequence types (STs), MLST permits objective comparisons between strains of Leptospira
infecting the same host in different geographic regions or different host species within the same geographic region, providing a helpful and powerful tool in investigating the epidemiology of leptospirosis. Therefore, knowledge of regional epidemiology, which can be reliably assessed only by the identification of locally prevalent strains, is necessary for understanding the infection and transmission chains and for maintaining up-to-date vaccination strategies.
The aims of this study were to provide the existent assessment of the genetic diversity in leptospires infecting dogs in Northeast Italy, highlight the need to update vaccine formulations to improve effective preventive measures and reduce the burden of leptospirosis among the canine population.
The absence of available molecular typing data and the complex genomic diversity of strains responsible for canine leptospirosis motivated us to genotype strains from symptomatic dogs in Northeast Italy, sampled between 2013 and 2019, using molecular methods.
Real-time PCR targeting the lipL32
] or rrs
(16S) gene [29
] was used to screen the infected dogs and genotyping was conducted to identify the leptospires responsible for the infections.
The isolation of Leptospira
strains was attempted, but only 15 out of 486 samples were successfully cultured, despite the immediate inoculation of the culture medium with samples. This confirmed that culturing Leptospira
is difficult [22
] and indicates the importance of increasing the tools available for genotyping strains directly from the DNA of biological samples.
The genotyping analysis, which was successfully performed on 15 isolates and 120 leptospiral DNAs, has increased our understanding of the epidemiological status of canine leptospirosis in Northeast Italy. It corroborated the serological evidence of the presence of high frequencies of strains from serogroup Icterohaemorrhagiae (ST17) and Australis (ST24 and ST198), but it also revealed the important roles of other serogroups, such as Pomona (ST117 and ST289) and Sejroe (ST155), as causative agents of the disease. Furthermore, this study revealed the presence of two recent genotypes, ST198 and ST289, which were discovered in hedgehogs and pigs, respectively, in Northern Italy. Thus, the results offer an interesting epidemiological perspective on the circulation of strains among different species and areas.
Among the identified genotypes, ST17, indicating the presence of L. interrogans
serogroup Icterohaemorrhagiae, was confirmed as the major cause of canine leptospirosis. Previous serological surveys have consistently shown Icterohaemorrhagiae as the most reactive serogroup in dogs suspected of having leptospirosis [13
] and interestingly, the serological profiles of human subjects suspected of having leptospiral infections also indicate that serovars Icterohaemorrhagiae and Copenhageni act as the main causative agents [47
The application of an epidemiological approach, which considered data from the National Reference Centre for Animal Leptospirosis of Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna (IZSLER) related to historical canine samples or belonging to other host species, provided insights into the infection’s transmission chain. Our findings corroborated the hypothesis that dogs are exposed to environmental contamination spread by rodents [49
], notably Rattus
spp., which act as the main reservoir host of Icterohaemorrhagiae/Copenhageni serovars worldwide [50
]. Although the inclusion of serogroup Icterohaemorrhagiae in vaccine formulations has led to its decreasing seroprevalence in Italy [46
], this serogroup is still a major cause of disease in dogs, owing to the ubiquitous nature of its maintenance host. The dog, in this case, represents a spill-over and can be used as an important sentinel species for human and other animal infections [51
]. Interestingly, the IZSLER database revealed that Leptospira
ST17 occurs in different hosts (mouse, cat, hedgehog, horse, goat and cow), which indicates its ability to cause infection in a wide range of animal species. The sequencing of the lic12008
gene, which was recently reported by Santos et al. [54
] as useful in discriminating Icterohaemorrhagiae and Copenhageni serovars, allowed us to determine that the major portion (94%) of samples typed as ST17 represented serovar Icterohaemorrhagiae, and only a small portion (6%) represented serovar Copenhageni. The reliability of this molecular test for serovar discrimination is still unclear, and further studies are necessary, given that the serovar determinations of two isolates, as assessed by mAbs and by lic12008
sequencing, were not in agreement (Table 5
Three regularly vaccinated dogs were infected by L. interrogans
serogroup Icterohaemorrhagiae, which is included in all the commercial vaccine formulations, which, however, are commonly able to protect the animal against acute signs but may not prevent infection if the animal is exposed to high bacterial load [15
]. An evaluation of the anamnestic and prognostic data of these dogs revealed that they were borderline cases. The puppy, being in a vulnerable developmental period, showed symptoms even after it developed antibodies, but the protection provided by the vaccine may have played a key role in its complete recovery. The hunting dog, which may have been exposed to high bacterial loads that could be responsible for the disease manifestation, started therapy late, resulting in its death. In this case, the delayed treatment of leptospirosis may have allowed the escape of surviving bacteria to the bactericidal effect of antibiotics. The dog that commonly frequented the river and developed acute symptoms with severe hepatonephritis, responded to therapy and completely recovered, probably owing to an active and ready immune response previously stimulated by the vaccine. With the exception of the second case, in which the delay in receiving therapy may have been fatal, vaccination appears to have played a crucial role in protecting dogs from death, given that Icterohaemorrhagiae infections in dogs are often associated with clinical courses culminating in fatal outcomes [15
Interestingly, 13 dogs were infected by L. interrogans
ST198, a new genotype first revealed in hedgehogs of Northern Italy [41
] and continually serologically typed as belonging to serovar Australis and serogroup Australis. Very recently, Balboni et al. [57
] identified ST198 in a symptomatic dog, providing further evidence of its involvement in canine leptospirosis in Italy. The discovery of this genotype among dogs suggested two important findings: from an epidemiological point of view, a possible direct transmission between species or by indirect infection through a contaminated shared environment, and from a clinical point of view, its presence and ability to cause the clinical presentation of leptospirosis, which can be fatal in Italian dogs.
In this study, a novel genotype, called “ST17-like” because of its similarity to ST17, was discovered in a dog of 2019. Unfortunately, it was unsuitable for submission to BIGSdb because of its incomplete MLST profile. Further confirmation and investigations are required, including the evaluation of its presence in other dogs and hosts in the same area.
More than 7% of the genotyped samples were characterized as ST24 and found worldwide in isolates serologically typed as belonging to serovars Jalna and Bratislava, within serogroup Australis [58
]. Unfortunately, because the isolates were unavailable, they were not characterized using mAbs, and consequently, there is no data that can be used to define the specific infective serovars present in the sampled dogs. It is probable that these infections were largely caused by L. interrogans
serovar Bratislava, because data from the past 30 years indicate the widespread exposure of dogs to this serovar in Europe [11
]. This was confirmed by the isolation of this serovar from urine of infected dogs in Scotland [59
] and by its presence among hedgehogs [60
], pigs [12
] and wild boars [61
] in Italy. Bratislava is the serovar most commonly isolated from domestic animals and, for this reason, is most commonly used as the antigen to represent the Australis serogroup in MAT panels. Our study revealed that this strain is responsible for infections in several hosts other than dog, such as hedgehog, wild boar and horse, in agreement with previous serological studies [12
]. Interestingly, in this study, ST24 was also identified in a fox, indicating possible transmission through a contaminated shared environment.
The coverage of the vaccine for the Australis serogroup remains to be evaluated, not only because bivalent vaccines are still widely used but also because new emerging strains, such as Leptospira serovar Australis ST198, may represent a significant cause of canine leptospirosis in Italy. Because current tetravalent vaccines contain antigens of Australis Bratislava (ST24), new research is required to evaluate their ability to protect dogs against infection by Leptospira Australis ST198.
Strains typed as ST117, indicating the presence of L. kirschneri
serovar Mozdok, play an important role in causing leptospirosis in dogs. Epidemiological studies have identified this serovar across Europe [17
], in dogs [19
], small rodents [65
], in cattle and pigs [68
]. It has been involved in human cases and in canine infections in Cuba [64
], making it a relevant risk to public health. Clinically, dogs infected with Leptospira
belonging to this serogroup experience a severe disease characterized by lethargy, fever, lack of appetite, diffuse hemorrhage and renal and liver failure [70
]. Serovar Pomona, included in the same serogroup, has emerged in the USA as cause of clinical symptoms in dogs [51
], and it has been added to vaccine formulations. In Italy, in 2002, Scanziani and colleagues reported some serological positivity of dogs to Pomona serogroup [11
], and recently, Bertelloni found an increasing incidence of Pomona among dogs in the north central area of Italy [13
]. In 2010, Ellis [17
] had proposed the usefulness of including other serovars, such as Pomona, in the vaccine formulation, but he also believed that additional clinical, cultural and serological studies were needed to support their inclusion. Here, the obtained data agree with previous serological data and support the inclusion of a Pomona serogroup strain, especially one belonging to serovar Mozdok, in dog vaccines in Europe.
Interestingly, five dogs were infected by leptospires genotyped as ST289, a new genotype recently found for the first time by IZSLER laboratory in a pig of 2014, living in Northern Italy [42
]. It was typed using core genome MLST as very similar, but not identical, to L. kirschneri
serovar Mozdok, within serogroup Pomona. The discovery of this genotype in Italian dogs suggested its ability to cause clinical manifestations in dogs and the possible direct transmission among animals or an indirect passage, possibly in a herd context. More studies should be performed to clarify the role of this new strain in causing canine leptospirosis, but our findings support the inclusion of serogroup Pomona in vaccine formulations
In the present study, ST155 was found in six dogs. It characterizes L. borgpetersenii
serogroup Sejroe, but the serovar status was not deducible from the MLST profile. Nevertheless, the international database BIGSdb [58
] reported that isolates typed as ST155 belong to serovars Polonica and Saxkoebing. Using the information from our database, ST155 was compatible with both serovar Sejroe and serovar Polonica, but not with serovar Saxkoebing. In fact, the reference strains serovar Sejroe strain Topino 1, serovar Sejroe strain M24 (Figure 2
) and serovar Polonica strain 493 Poland (not reported) were previously typed as ST155, while the reference strain serovar Saxkoebing strain Mus 24 was typed as ST219 (Figure 2
). Strains serotyped as Sejroe were previously described as being causative agents of canine leptospirosis worldwide [72
], and interestingly, this strain was found in an asymptomatic dog in Brazil [74
]. The minimum spanning tree, color-coded by animal host, revealed the shared presence of these STs among dogs, horses and mice.
Our results indicated that the use of the serovar of the serogroup Sejroe strain in MAT analyses to test local canine populations should be reconsidered. In fact, the MAT panel commonly contains serovar Hardjo (ST152), but the inclusion of serovar Sejroe (ST155) would be more useful for diagnosing canine leptospirosis in our area.
Serogroup Canicola, historically included in commercial vaccine formulations, was not found in this work, and this was in agreement with previous serological studies that describe a decline in the prevalence of this serogroup in European countries, possibly because of the widespread protection resulting from the vaccination of the reservoir host [76
], i.e., dog. The risk of stopping vaccinations against host-adapted Leptospira
in its target host is that its prevalence may rapidly increase once the population’s immunity falls; therefore, the vaccination of dogs against serovar Canicola should continue [17
Serovar Grippotyphosa is maintained by a number of small rodent species in mainland Europe [77
]. Seroprevalence studies have indicated Grippotyphosa infections in several European countries, including Italy [11
], and consequently, it has been included in commercial vaccines. However, in this molecular survey, this strain was not detected among the sampled canine population nor in other species.
The geographical map indicated a widespread distribution of the identified genotypes throughout the considered area, except for ST289, mostly concentrated in Emilia-Romagna region and ST117, found only in Veneto region. Nevertheless, these differences might be attributed to the variability in the numbers of genotyped samples rather than a geographic-dependent diversity in ST circulation. Proper investigations will determine the presence of specific environmental niches for canine leptospirosis or particular risk factors, but the common practice of owners traveling with their pets for recreational activities, such as hiking and hunting, often out of their town or province, should be taken into consideration during risk evaluation. Therefore, when planning dog vaccinations or investigating a suspected clinical case, all of the identified genotypes should be considered epidemiologically relevant.