Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Urinary shedding of pathogenic Leptospira in stray dogs and cats, Algiers: A prospective study

  • Sara Zaidi,

    Roles Conceptualization, Investigation, Writing – original draft

    Affiliation Ecole Nationale Supérieure Vétérinaire, Alger, Algérie

  • Amar Bouam,

    Roles Data curation, Formal analysis, Writing – original draft

    Affiliation Aix Marseille Univ, IRD, MEPHI, IHU Méditerranée Infection, Marseille, France

  • Amina Bessas,

    Roles Data curation, Formal analysis, Investigation, Writing – original draft

    Affiliation Ecole Nationale Supérieure Vétérinaire, Alger, Algérie

  • Djamila Hezil,

    Roles Investigation

    Affiliation Ecole Nationale Supérieure Vétérinaire, Alger, Algérie

  • Hicham Ghaoui,

    Roles Investigation

    Affiliations Ecole Nationale Supérieure Vétérinaire, Alger, Algérie, Aix Marseille Univ, IRD, MEPHI, IHU Méditerranée Infection, Marseille, France

  • Khatima Ait-Oudhia,

    Roles Investigation

    Affiliation Ecole Nationale Supérieure Vétérinaire, Alger, Algérie

  • Michel Drancourt ,

    Roles Writing – original draft, Writing – review & editing

    michel.drancourt@univ-amu.fr

    Affiliation Aix Marseille Univ, IRD, MEPHI, IHU Méditerranée Infection, Marseille, France

  • Idir Bitam

    Roles Conceptualization, Supervision, Validation, Writing – original draft

    Affiliations Ecole Supérieure en Sciences de l'Aliment et des Industries Agroalimentaires (ESSAIA), El Harrach, Alger, Algérie, Aix Marseille Univ, IRD, VITROME, IHU Méditerranée Infection, Marseille, France

Urinary shedding of pathogenic Leptospira in stray dogs and cats, Algiers: A prospective study

  • Sara Zaidi, 
  • Amar Bouam, 
  • Amina Bessas, 
  • Djamila Hezil, 
  • Hicham Ghaoui, 
  • Khatima Ait-Oudhia, 
  • Michel Drancourt, 
  • Idir Bitam
PLOS
x

Abstract

Background

Leptospirosis is an important worldwide zoonosis. This disease is caused by pathogenic species of the genus Leptospira which are maintained in the environment via chronic renal infection of carrier animals which can be asymptomatic excretors of the organisms in their urines and become a source of infection for humans and other hosts. The prevalence of animal leptospirosis in Algiers, Algeria, is unknown.

Methodology/principal findings

Real-time PCR and standard PCR and sequencing were used to detect pathogenic Leptospira organisms in the urines of stray dogs and cats in Algiers. In the presence of appropriate controls, none of the 107 cat urine samples were positive while 5/104 (4.8%) canine urine samples (asymptomatic mixed-breed dogs, three females and two males) were positive in two real-time PCR assays targeting the rrs and hsp genes. The positivity of these samples was confirmed by partial PCR-sequencing of the rpoB gene which yielded 100% sequence similarity with Leptospira interrogans reference sequence. In this study, L. interrogans prevalence was significantly higher in dogs aged < one year (16.46% - 29.41%) than in adults (0%) (P value = 0.0001) and then in the overall dog population (2.68% - 4.8%) (P = 0.0007).

Conclusions/significance

These results suggest that dogs are maintenance hosts for zoonotic leptospirosis in Algiers, Algeria. To face this situation, effective canine vaccination strategies and raising public health awareness are mandatory. Further investigations incorporating a larger sample in more localities will be undertaken to document the epidemiology of urban animal leptospirosis in Algeria at large.

Introduction

Leptospirosis is a worldwide disease that affects wild and domestic animals and human populations. Affected persons are primarily farmers, fishermen, veterinarians and people working in sewers and slaughterhouses [1]. This zoonosis is caused by pathogenic spirochetes of the genus Leptospira which colonize the renal tubules where they reproduce before being excreted via the urines [2]. Infected urines or contaminated water are sources of leptospirosis infection and Leptospira can enter the body of mammalian hosts via lacerations in the skin, contacts with mucosa or conjunctiva and inhalation of aerosols [35]. Some host animals such as dogs may have an asymptomatic form or may suffer from a wide range of clinical manifestations, including hepatic and renal failure and severe pulmonary hemorrhage [6]. Asymptomatic and chronic carrier dogs can be maintenance hosts [7] acting as sources of infection and therefore cause a public health problem [8]. Formerly, it was thought that domestic cats were resistant to leptospirosis infection and many practitioners did not consider feline leptospirosis in the differential diagnosis of other diseases [9]. However, recently published reports on feline leptospirosis conclude that cats are exposed to Leptospira and may play a role in the epidemiology of this disease [1012].

As a neglected tropical disease, leptospirosis has been increasingly observed in urban settlements, especially in slums in developing countries [6]. The prevalence of animal leptospirosis in Algiers, Algeria, is unknown. Only two studies were published about human leptospirosis in Algeria. These two serological investigations were conducted on patients of the Tizi-ouzou Hospital. The first one reported 48 cases of leptospirosis from 2006 to 2007 and the serogroup icterohaemorrhagiae was identified in 60% of cases [13]. In the second prospective study, 175 positive patients were diagnosed from 2005 to 2008, among the serovars identified, icterohaemorrhagiae and grippotyphosa were predominant [14].

The aim of the present work was to detect pathogenic Leptospira organisms in the urines of stray dogs and cats in Algiers.

Methods

Ethic statement

The study was submitted to and approved by the ethics committee and decision board (number 416/2017) of EPIC- H.U.P.E (EPIC: Entreprise publique à caractère industriel et commercial; H.U.P.E: Hygiène Urbaine et Protection de l’environnement) of Wilaya of Algiers (Ex: HURBAL). HURBAL was created in 1994 with a new status: EPIC-H.U.P.E under the register number: 16/00-0013132B00. EPIC- H.U.P.E is an institution affiliated with the Algerian Ministry of the Interior and the Local Government and the Algerian Ministry of Water Resources and Environment. In the context of the National Program for Rabies Control, EPIC- H.U.P.E captures stray dogs and cats in Algiers. Once captured, stray animals were housed in cages and euthanized after expiration of the seven day legal waiting time (in order to allow for owners to claim their pets), in compliance with the Algerian legislation for the protection of animals (Law 01/04/1994), which our protocol respected.

Study design and sampling

This study was designed to screen for the presence of Leptospira spp. organisms in stray cats and dogs in the region of Algiers, in the absence of any data available on that topic. Therefore, in this study, we aimed at collecting only the urines of the animals for the molecular detection of Leptospira spp. DNA. Urine specimens were aseptically collected between April 2017 and November 2017 were via cystocentesis from 211 stray animals (104 dogs and 107 cats). These animals were captured in the 57 municipalities of the region of Algiers. The sampling was realized in animal shelters with an average of seven animals sampled per week. The age of each animal was estimated, based on dentition and physical aspect. Information concerning sex, breed and clinical status was noted. Samples were stored at -20°C before being transported to the IHU Méditerranée Infection, Marseille, France, for PCR testing and culture was not performed. Up to 3 mL of each urine sample was centrifuged at 15,000 g for 20 minutes [15], the supernatant was discarded and the pellet was suspended in 200 μL of sterile phosphate-buffered saline solution (PBS, pH 7.2) [16].

DNA extraction

A total of 200 μL of DNA was extracted using the QIAamp Tissue Kit by QUIAGEN-BioRobot EZ1, according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Extracted DNA was stored at -20°C under sterile conditions until used in PCR assays.

Real time PCR

Extracted DNA was used in qPCR amplifications to detect pathogenic Leptospira organisms. The final qPCR reaction mixture consisted of 5 μL DNA with 15 μL of mix from the Roche PCR Kit (Roche Diagnostics, Meylan, France). The components of the final reaction mixture of these PCR assays are given in Table 1. A homemade plasmid containing sequences specific to Leptospira spp. was used as a positive control. Three negative controls were incorporated into each PCR run. Results were recorded as positive when the cycle threshold (Ct) was lower than 33. We performed real-time PCR (qPCR) with two systems (Table 2) in order to confirm the positivity of the samples according to current standards in microbiology. The first system targets a 88-pb fragment of the rrs gene coding for 16S rRNA of pathogenic Leptospira: 16S rRNA Forward (5’-CCCGCGTCCGATTAG-3’), 16S rRNA Reverse (5’-TCCATTGTGGCCGRACAC-3’) and 16S rRNA Probe (5’-CTCACCAAGGCGACGTCGGTAGC-3’) were analyzed as previously described [17]. The second system targets a 103-pb fragment of the hsp gene of L. interrogans: Lint_hsp_MB Forward (5’-CCCGCGTCCGATTAG-3’), Lint_hsp_MB Reverse (5’-TCCATTGTGGCCGRACAC-3’) and Lint_hsp_MB Probe (5’-CTCACCAAGGCGACGTCGGTAGC-3’) were analyzed as previously described [18]. The PCR cycling parameters for the qPCR were 5 min at 95°C followed by 39 cycles each consisting of 5 sec of denaturation at 95°C and 30 sec of annealing at 60°C.

thumbnail
Table 1. Concentration of components in the final reaction mixtures of the real-time polymerase chain reaction (qPCR) assays used in this study.

https://doi.org/10.1371/journal.pone.0197068.t001

Standard PCR and sequencing

Samples that tested positive by qPCR were confirmed by standard PCR and sequencing in order to achieve a 100% specificity. The final standard PCR reaction mixture consisted of 5 μL of DNA with 15 μL of mix from the Roche PCR Kit (Roche Diagnostics). The components of the final reaction mixture of these PCR assays are given in Table 3. Samples were then confirmed by standard PCR using primers which amplified a 592-pb fragment of the rpoB gene: Lept 1900 Forward (5’-CCTCATGGGTTCCAACATGCA-3’) and Lept 2500 Reverse (5’-CGCATCCTCRAAGTTGTAWCCTT-3’), as described by La Scola et al., 2006 [19] (Table 2). The PCR cycling parameters for the standard PCR were 15 min at 95°C followed by 35 cycles of each consisting of 30 sec denaturation at 95°C, 30 sec annealing at 51°C and 6 min extension at 72°C in an ABI Thermocycler (Applied Biosystems Gene Amp PCR System 2700, Villebon sur Yvette, France). Negative controls were incorporated into each PCR run.

thumbnail
Table 3. Concentration of components in the final reaction mixtures of the standard polymerase chain reaction assay used in this study.

https://doi.org/10.1371/journal.pone.0197068.t003

The amplified PCR products were separated via gel electrophoresis using a 1.5% agarose gel stained with Sayber Safe (ThermoFisher, Paris, France). The DNA bands were visualized and photographed under ultraviolet light. PCR products were purified and sequenced with rpoB primers as described previously [19]. All obtained sequences were assembled and edited using ChromasPro (version 1.7.7). The sequences were then analyzed by Basic Local Alignment Search Tool (BLAST) and compared with sequences available in the GenBank database.

Statistical analyses

Statistical analyses were done by MEDCALC® online software https://www.medcalc.org/calc/comparison_of_proportions.php using the “N-1” Chi-squared test as recommended by Campbell., 2007 [20] and Richardson., 2011 [21]. The confidence interval was calculated according to the recommended method given by Altman et al., 2000 [22].

Results

Sample collection

From April 2017 to November 2017, a total of 104 stray dogs and 107 stray cats captured in Algiers, Algeria were sampled. These animals lived in urban areas, spending most of their time exclusively outdoors and did not receive any vaccine. Of the 104 dogs, 69/104 (66.34%) were males and 35/104 (33.65%) were females. The canine population consisted predominantly of mixed-breed dogs; other dogs belonged to the following races: German shepherd, American Staffordshire, shepherd crosses and Pit-bull. The dogs’ age ranged between 2 months and 11 years. Among the 107 cats, 66/107 (61.68%) were males and 41/107 (38.31%) were females. The cats were described as mostly belonging to mixed breeds, some belonging to European or Siamese crossbreeds. The 107 cats sampled were estimated to be under 5 years of age. All sampled animals were apparently healthy.

Real time PCR

qPCR targeting the 16S rRNA gene of pathogenic Leptospira and the hsp gene of L. interrogans revealed that none of the 107 urine samples of cats tested were positive while 5/104 (4.8%) dogs were positive. These five urine specimens were positive in the two qPCR systems (rrs and hsp). Using the Cts obtained from the 16S rRNA qPCR reactions and a calibration curve previously described for this system [23], we extrapolated the number of leptospira genomes per positive reaction (Table 4). Positive dogs were all very young, under one year of age. Three were females and two were males. All positive animals belonged to mixed-breeds (Table 4). In this study, L. interrogans prevalence was significantly higher in dogs aged < one year (5/17; 29.41%) than in adults (0/87; 0%) (P value = 0.0001, 95% CI: 12.73 to 53.13) and than in the overall dog population (5/104; 4.8%) (P = 0.0007, CI: 7.1929 to 48.2975). The sensitivity of our screening test based on the detection of 16S rRNA using qPCR was previously estimated to be of 56% [24], accordingly, the prevalence rate obtained here was estimated to be of 2.68%-4.8%. There was no significant difference regarding prevalence between males (3/69, 2.43% - 4.34%) and females (2/35, 3.19% - 5.71%) (p = 0.758).

thumbnail
Table 4. Information relative to animals detected positive for L. interrogans DNA in urine samples: Age, sex, race, number of genomes per positive pPCR reaction.

(The values were extrapolated from the calibration curve (21) using the Ct obtained from the 16S rRNA system).

https://doi.org/10.1371/journal.pone.0197068.t004

Standard PCR and sequencing

All five urine samples detected positive by real-time PCR were confirmed with gel-based PCR assay targeting the rpoB and subjected to sequencing analysis. The BLAST (www.ncbi.nlm.nih.gov/blast) analysis of the rpoB gene sequence from all samples, once compared with sequences available in the GenBank database, confirmed Leptospira infection. The BLAST analysis yielded a 100% sequence homology with L. interrogans homologous gene fragment (GenBank accession no. CP020414.1).

Discussion

In Algeria, the exact morbidity and the mortality due to leptospirosis are unknown. In 1975, a study based on serology reported seven cases in a military group in Algiers’ suburbs [25]. Two more recent studies reported cases of leptospirosis among hospitalized patients in the region of Tizi-Ouzou, located 100 km east of the capital Algiers. The investigation of 48 patients from 2006 to 2007 in the rural area of Tala-Athmane revealed that they were living in close contact to two garbage dumps invaded by rodents, the cases were confirmed serologically by the microagglutination test (MAT) and more than 60% (n = 29) were from the serogroup icterohaemorrhagiae [13]. A second prospective study conducted from 2005 to 2008 in the same region reported 173 cases among hospitalized patients, the cases were confirmed serologically with a predominance the of serovars icterohaemorrhagiae and grippotyphosa [14].

However, the prevalence of this zoonosis in reservoirs is totally unknown in Algeria as the only report of it is the observation of Leptospira organisms in histological sections of the liver in dogs presenting with severe jaundice, subcutaneous hemorrhages and acute nephritis [26]. For this pioneering study of animal leptospirosis in Algeria, we used urines in which leptospiral DNA can be found much longer than in blood [2729]. The need for a rapid diagnosis of leptospirosis has led to the development of numerous PCR assays, which appeared to have more applicability in determining zoonotic risks [30]. This method is rapid, sensitive, specific and robust and many PCR assays were developed to detect universal Leptospira genes such as gyrB, rrs and secY genes or genes restricted to pathogenic species such as lipL32, lfb1, ligA and ligB2 [5]. In this study we aimed at retrospectively confirming the positive detection of Leptospira spp. DNA by targeting two different molecular targets. We chose the rrs and hsp as genus-level targets of identification and rpoB sequencing for the identification at the species level of pathogenic leptospira.

We confirmed the presence of L. interrogans in the urines of stray dogs in Algiers using qPCR and standard PCR-sequencing targeting universal and pathogen-related genes. We observed an overall prevalence of 2.68% - 4.8% and a high prevalence of 29.41% in the specific population of young dogs aged < one year. All animals were apparently healthy, indicating asymptomatic carriers. These dogs were always outside, in contact with garbage and small rodents which were likely sources of infection. Crowding animals in unsanitary quarters is associated with a high prevalence of infection since animals may acquire the disease through contact with urines from infected dogs or infected rodents [31]. Despite the fact that we did not attempt to isolate Leptospira spp. to confirm the role of stray dogs as reservoirs, present data indicate that stray dogs would indeed be good sentinels to know which serovars/groups are circulating in the rodent populations.

Subclinical, latent leptospirosis in dogs has regularly been reported and can also be observed in unsteady vaccinated animals [32]. In addition, there are data suggesting that clinically asymptomatic dogs can be chronic carriers, shedding Leptospira via urines into the environment [30, 33, 34]. Many epidemiological studies were conducted worldwide on the renal carriage of leptospirosis in dogs using molecular tools [8, 14, 3443], showing a prevalence between 0.2% and 22% worldwide Fig 1). Some other studies have not found the presence of pathogenic Leptospira species in dogs in the USA (0/100) and in Egypt (0/25), but only the presence of antibodies as an evidence of the exposure to the disease [44] [45]. This may be due to the minute amount of Leptospira DNA in the blood and urine specimens of infected dogs. Therefore, a highly sensitive PCR platform is required to obtain an accurate diagnosis and an innovative approach must be adopted to maximize sample DNA input in the PCR or by increasing the volume of urine for DNA extraction. This could be achieved by high-speed sedimentation of a milliliter volume of urines and performing DNA extraction from the complete sediment, as performed in the present study [46]. Positive dogs in our study were very young dogs (under one year of age). However, it has been shown in the USA that dogs aged between 4 and 6.9 years and between 7 and 10 years faced a significantly higher risk of being infected than dogs under one year of age [47]. In a study conducted in Reunion Island, only adult dogs were positive for leptospirosis [48]. The difference in age range can be explained by the fact that the young stray dogs of our study were not vaccinated against leptospirosis and exposed at an early age to the bacterium in their environment. Furthermore, the canine population of two previous studies [47, 48] was composed of domestic dogs with different risk factors and exposure to other maintenance host species.

thumbnail
Fig 1. Molecular detection of Leptospira spp. DNA in the urines of dogs, worldwide.

Germany, 3/200 (1.5%) [8], the USA, 41/500 (8.2%) [14], Teheran (Iran), 33/150 (22%) [34], Canada, 11% (11/100) [35], Sicily (Italy), 5/64 (7.8%) [36], Ireland, 37/525 (7.05%) [37], Porto Alegre City (Brazil), 36/253 (14.2%) [38], Switzerland, 1/20 (5%) [39], Colombia 2/54 (3.7%)[40], New Caledonia, 1/13 (7.6%) [41], Brazil, 26/131 (19.8%) [42], Switzerland, 1/408 (0.2%) [43], Algiers (Algeria), 5/104 (2.68% - 4.8%) [Present work].

https://doi.org/10.1371/journal.pone.0197068.g001

In the present work, all sampled cats were qPCR-negative for L. interrogans. Some studies yielded a seroprevalence between 4% and 30% in different countries including Australia [49], Scotland [50], Greece [51], Iran [52], Spain [53], Canada [54], Taiwan [11], Chile [10], Colombia [55] and Brasil [56]. Some other studies found DNA sequences of Leptospira in urine samples indicating a renal carriage of leptospirosis in cats. In Taiwan, DNA of pathogenic Leptospira was detected in 67.8% (80/118) of the urine samples of cats including 71 stray cats and nine household cats [11]. In Canada, DNA of Leptospira was detected in the urines of sick cats (6/113) and healthy cats (2/125), corresponding to a prevalence of 8/238 (3.3%) [12]. In Germany, urine samples from 7/215 (3.2%) cats were PCR-positive [57]. In Quebec, PCRs on urines detected urinary excretion in 3.2% of the 250 cats sampled [58]. These results suggest that cats may have a role in the transmission of leptospirosis, as a reservoir or as an accidental host. The role of cats in the transmission of leptospirosis should be reevaluated, as it might in fact be underestimated.

In conclusion, this study demonstrates the high number of leptospiral carriers among asymptomatic young dogs. Improving the awareness of dog owners and the prevention of canine leptospirosis could be a valuable asset for human leptospirosis prevention [59]. The prevalence of leptospirosis in countries where dogs are correctly vaccinated remains high, this is mainly due to the difference in epidemiology between the different serovars and the absence of cross-protection in a vaccine, thus, a more effective vaccine needs to be developed [60]. The implementation of a surveillance system for canine leptospirosis, using dogs as sentinels for human risk assessment, could also provide a valuable tool for estimating and in turn minimize the risk for humans [61]. Further studies on leptospirosis in other animals and other regions in Algeria should be considered to clarify the status of this disease in our country. Also, further studies in the region of Algiers and other regions of Algeria will have to determine the serotypes of circulating leptospira in order to refine the epidemiology of leptospirosis in Algeria.

Acknowledgments

The authors would like to thank Mr. Schiff Lyès and veterinarians of HURBAL for their participation in providing samples. AB benefits a PhD grant from the Fondation Méditerranée Infection, Marseille, France. This work was supported by the French Government under the «Investissements d’avenir» (Investments for the Future) program managed by the Agence Nationale de la Recherche (ANR, fr: National Agency for Research), (reference: Méditerranée Infection 10-IAHU-03). The authors acknowledge Magdalen Lardière and Olga Cusack for their expert assistance in editing the manuscript.

References

  1. 1. Brown PD, McKenzie M, Pinnock M, McGrowder D. Environmental risk factors associated with leptospirosis among butchers and their associates in Jamaica. Int J Occup Environ Med. 2011; 2(1):47–57. pmid:23022818
  2. 2. Levett PN. Leptospirosis. Clin Microbiol. 2001;14(2):296–326.
  3. 3. Baron S. Medical microbiology. University of Texas Medical Branch at Galveston; 1996. 1273 p.
  4. 4. Monahan AM, Miller IS, Nally JE. Leptospirosis: risks during recreational activities. J Appl Microbiol. 2009;107(3):707–16. pmid:19302325
  5. 5. Musso D, La Scola B. Laboratory diagnosis of leptospirosis: A challenge. J Microbiol Immunol Infect. 2013; 46(4):245–52. pmid:23639380
  6. 6. Ko AI, Goarant C, Picardeau M. Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen. Nat Rev Microbiol. 2009; 7(10):736–47. pmid:19756012
  7. 7. Muñoz-Zanzi C, Mason MR, Encina C, Astroza A, Romero A. Leptospira contamination in household and environmental water in rural communities in southern Chile. Int J Environ Res Public Health. 2014;11(7):6666–80. pmid:24972030
  8. 8. Llewellyn J-R, Krupka-Dyachenko I, Rettinger AL, Dyachenko V, Stamm I, Kopp PA, et al. Urinary shedding of leptospires and presence of Leptospira antibodies in healthy dogs from Upper Bavaria. Berl Munch Tierarztl Wochenschr. 2016;129(5–6):251–7. pmid:27344919
  9. 9. Arbour J, Blais M-C, Carioto L, Sylvestre D. Clinical leptospirosis in three cats (2001–2009). J Am Anim Hosp Assoc. 2012; 48(4):256–60. pmid:22611217
  10. 10. Azócar-Aedo L, Monti G, Jara R. Leptospira spp. in Domestic Cats from Different Environments: Prevalence of Antibodies and Risk Factors Associated with the Seropositivity. Anim an open access J from MDPI. 2014; 4(4):612–26.
  11. 11. Chan K-W, Hsu Y-H, Hu W-L, Pan M-J, Lai J-M, Huang K-C, et al. Serological and PCR detection of feline leptospira in southern Taiwan. Vector Borne Zoonotic Dis. 2014;14(2):118–23. pmid:24359421
  12. 12. Rodriguez J, Blais M-C, Lapointe C, Arsenault J, Carioto L, Harel J. Serologic and urinary PCR survey of leptospirosis in healthy cats and in cats with kidney disease. J Vet Intern Med. 2014; 28(2):284–93. pmid:24417764
  13. 13. Afiri M, Amara Khorba A, Ait Kaid D. Renal manifestations of leptospirosis: 88 cases. Med Sante Trop. 2013;23(2):234–5.
  14. 14. Afiri M, Toudeft F, Ait KD. Leptospirosis epidemic: 48 cases. Med Sante Trop. 2013;23(2):234.
  15. 15. Branger C, Blanchard B, Fillonneau C, Suard I, Aviat F, Chevallier B, et al. Polymerase chain reaction assay specific for pathogenic Leptospira based on the gene hap1 encoding the hemolysis-associated protein-1. FEMS Microbiol Lett. 2005; 243(2):437–45. pmid:15686847
  16. 16. Harkin KR, Roshto YM, Sullivan JT. Clinical application of a polymerase chain reaction assay for diagnosis of leptospirosis in dogs. J Am Vet Med Assoc. 2003; 222(9):1224–9. pmid:12725309
  17. 17. Smythe LD, Smith IL, Smith GA, Dohnt MF, Symonds ML, Barnett LJ, et al. A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp. BMC Infect Dis. BioMed Central; 2002; 2:13.
  18. 18. Appelt S, Armougom F, Le Bailly M, Robert C, Drancourt M. Polyphasic analysis of a middle ages coprolite microbiota, Belgium. PLoS One. Public Library of Science; 2014; 9(2):e88376. pmid:24586319
  19. 19. La Scola B La, Bui LTM, Baranton G, Khamis A, Raoult D. Partial rpoB gene sequencing for identification of Leptospira species. FEMS Microbiol Lett. 2006; 263(2):142–7. pmid:16978348
  20. 20. Campbell I. Chi-squared and Fisher–Irwin tests of two-by-two tables with small sample recommendations. Stat Med. 2007;26(19):3661–75. pmid:17315184
  21. 21. Richardson JTE. The analysis of 2 × 2 contingency tables-Yet again. Stat Med. John Wiley & Sons, Ltd.; 2011;30(8):890–890. pmid:21432882
  22. 22. Altman DG, Machin D, Bryant TN, Gardner MJ (Eds) (2000) Statistics with confidence, 2nd ed. BMJ Books. (p. 49).
  23. 23. Emily J. Viau, Alexandria B. Boehm. Quantitative PCR-based detection of pathogenic Leptospira in Hawai'ian coastal streams. Journal of Water and Health. 2011; 9(4):637–646.
  24. 24. Thaipadunpanit J, Chierakul W, Wuthiekanun V, et al. Diagnostic Accuracy of Real-Time PCR Assays Targeting 16S rRNA and lipl32 Genes for Human Leptospirosis in Thailand: A Case-Control Study. PLoS ONE. 2011;6(1):e16236. pmid:21283633
  25. 25. Aubry P, Bordahandy R, Ferah T, Mailloux M, Thomas J. An anademy of ictero-hemorrhagic leptospirosis in a military group in Algeria. Bull Soc Pathol Exot Filiales. 1975;68(4):370–6. pmid:1243751
  26. 26. Donatien A, Gayot G, Poul J. Leptospirosis of the dog in Algeria. Arch l’Institut Pasteur d’Algerie Inst Pasteur d’Algerie. 1951;29(1):20–4.
  27. 27. Bal AE, Gravekamp C, Hartskeerl RA, De Meza-Brewster J, Korver H, Terpstra WJ. Detection of leptospires in urine by PCR for early diagnosis of leptospirosis. J Clin Microbiol. 1994;32(8):1894–8. pmid:7989538
  28. 28. Brown PD, Gravekamp C, Carrington DG, van de Kemp H, Hartskeerl RA, Edwards CN, et al. Evaluation of the polymerase chain reaction for early diagnosis of leptospirosis. J Med Microbiol. 1995; 43(2):110–4. pmid:7629850
  29. 29. Villumsen S, Pedersen R, Borre MB, Ahrens P, Jensen JS, Krogfelt KA. Novel TaqMan® PCR for detection of Leptospira species in urine and blood: pit-falls of in silico validation. J Microbiol Methods. 2012;91(1):184–90 pmid:22750039
  30. 30. Harkin KR, Roshto YM, Sullivan JT, Purvis TJ, Chengappa MM. Comparison of polymerase chain reaction assay, bacteriologic culture, and serologic testing in assessment of prevalence of urinary shedding of leptospires in dogs. J Am Vet Med Assoc. 2003;222(9):1230–3. pmid:12725310
  31. 31. Scanziani E, Origgi F, Giusti AM, Iacchia G, Vasino A, Pirovano G, et al. Serological survey of leptospiral infection in kennelled dogs in Italy. J Small Anim Pract. 2002;43(4):154–7. pmid:11996391
  32. 32. Klopfleisch R, Kohn B, Plog S, Weingart C, Nöckler K, Mayer-Scholl A, et al. An emerging pulmonary haemorrhagic syndrome in dogs: similar to the human leptospiral pulmonary haemorrhagic syndrome Vet Med Int. Hindawi Limited;2010;2010:928541. pmid:21274452
  33. 33. Brown CA, Roberts AW, Miller MA, Davis DA, Brown SA, Bolin CA, et al. Leptospira interrogans serovar grippotyphosa infection in dogs. J Am Vet Med Assoc. 1996;209(7):1265–7. pmid:8837647
  34. 34. Zakeri S, Khorami N, Ganji ZF, Sepahian N, Malmasi A-A, Gouya MM, et al. Leptospira wolffii, a potential new pathogenic Leptospira species detected in human, sheep and dog. Infect Genet Evol. 2010;10(2):273–7. pmid:20074666
  35. 35. Cai HY, Hornby G, Key DW, Osuch MR, Maxie MG. Preliminary study on differentiation of Leptospira grippotyphosa and Leptospira sejroe from other common pathogenic leptospiral serovars in canine urine by polymerase chain reaction assay. J Vet Diagn Invest. 2002;14(2):164–8. pmid:11939341
  36. 36. Vicari D, Percipalle M, Concetta LM, Li Vecchi L, Curro V, Vitale M, et al. Evidence of canine leptospirosis in kennels in Sicily, by PCR method. Rev Cubana Med Trop. 2007;59(1):61–2. pmid:23427421
  37. 37. Rojas P, Monahan AM, Schuller S, Miller IS, Markey BK, Nally JE. Detection and quantification of leptospires in urine of dogs: a maintenance host for the zoonotic disease leptospirosis. Eur J Clin Microbiol Infect Dis. 2010;29(10):1305–9. pmid:20559675
  38. 38. Oliveira ST, Messick JB, Welker Biondo A, Pires A, Santos D, Stedile R, et al. Exposure to Leptospira spp. in Sick Dogs, Shelter Dogs and Dogs from an Endemic Area: Points to Consider. Acta Sci Vet. 2012;40(403):1056–1056.
  39. 39. Fraune CK, Schweighauser A, Francey T. Evaluation of the diagnostic value of serologic microagglutination testing and a polymerase chain reaction assay for diagnosis of acute leptospirosis in dogs in a referral center. J Am Vet Med Assoc. 2013;242(10):1373–80. pmid:23634681
  40. 40. Calderón A, Rodríguez V, Máttar S, Arrieta G. Leptospirosis in pigs, dogs, rodents, humans, and water in an area of the Colombian tropics. Trop Anim Health Prod. 2014;46(2):427–32. pmid:24254419
  41. 41. Gay N, Soupé-Gilbert M-E, Goarant C. Though not Reservoirs, Dogs might Transmit Leptospira in New Caledonia. Int J Environ Res Public Health. 2014;11(4):4316–25. pmid:24747539
  42. 42. Sant’anna R, Vieira AS, Grapiglia J, Lilenbaum W. High number of asymptomatic dogs as leptospiral carriers in an endemic area indicates a serious public health concern. Epidemiol Infect. 2017;145(9):1852–4. pmid:28367783
  43. 43. Delaude A, Rodriguez-Campos S, Dreyfus A, Counotte MJ, Francey T, Schweighauser A, et al. Canine leptospirosis in Switzerland-A prospective cross-sectional study examining seroprevalence, risk factors and urinary shedding of pathogenic leptospires. Prev Vet Med. 2017;141:48–60. pmid:28532993
  44. 44. Fink JM, Moore GE, Landau R, Vemulapalli R. Evaluation of three 5’ exonuclease-based real-time polymerase chain reaction assays for detection of pathogenic Leptospira species in canine urine. J Vet Diagn Invest. 2015;27(2):159–66. pmid:25776541
  45. 45. Felt SA, Wasfy MO, El-Tras WF, Samir A, Rahaman BA, Boshra M, et al. Cross-species surveillance of Leptospira in domestic and peri-domestic animals in Mahalla City, Gharbeya Governorate, Egypt. Am J Trop Med Hyg. 2011;84(3):420–5. pmid:21363980
  46. 46. Xu C, Loftis A, Ahluwalia SK, Gao D, Verma A, Wang C, et al. Diagnosis of canine leptospirosis by a highly sensitive FRET-PCR targeting the lig genes. Dellagostin OA, editor. PLoS One. 2014;9(2):e89507. pmid:24586833
  47. 47. Ward MP, Glickman LT, Guptill LE. Prevalence of and risk factors for leptospirosis among dogs in the United States and Canada: 677 cases (1970–1998). J Am Vet Med Assoc. 2002:53–8.
  48. 48. Desvars A, Naze F, Benneveau A, Cardinale E, Michault A. Endemicity of leptospirosis in domestic and wild animal species from Reunion Island (Indian Ocean). Epidemiol Infect. 2013;141(6):1154–65. pmid:22998941
  49. 49. Dickeson D, Love DN. A serological survey of dogs, cats and horses in south-eastern Australia for leptospiral antibodies. Aust Vet J. 1993;70(10):389–90. pmid:8257320
  50. 50. Agunloye CA, Nash AS. Investigation of possible leptospiral infection in cats in Scotland. J Small Anim Pract. 1996;37(3):126–9. pmid:8683955
  51. 51. Mylonakis ME, Bourtzi-Hatzopoulou E, Koutinas AF, Petridou E, Saridomichelakis MN, Leontides L, et al. Leptospiral seroepidemiology in a feline hospital population in Greece. Vet Rec. 2005;156(19):615–6. pmid:15879545
  52. 52. Jamshidi S, Akhavizadegan M, Maazi N, Ali AG, Bokaie S. Serologic study of feline leptospirosis in Tehran, Iran J Microbiol 2009; 1:32–36.
  53. 53. Millán J, Candela MG, López-Bao JV, Pereira M, Jiménez MA, León-Vizcaíno L. Leptospirosis in wild and domestic carnivores in natural areas in Andalusia, Spain. Vector Borne Zoonotic Dis. 2009;9(5):549–54. pmid:18973450
  54. 54. Lapointe C, Plamondon I, Dunn M. Feline leptospirosis serosurvey from a Quebec referral hospital. La Rev Vet Can. 2013;54(5):497–9.
  55. 55. Ramírez-Ramírez MM, León-Castañeda OM, Rodriguez-Morales AJ. Leptospirosis in an urban setting: cases diagnosed at a private medical center of Western Colombia, 2008–2012. Recent Pat Antiinfect Drug Discov. 2015;10(1):59–63. pmid:25873099
  56. 56. Dos Santos LF, Guimarães MF, de Souza GO, da Silva IWG, Santos JR, Azevedo SS, et al. Seroepidemiological survey on Leptospira spp. infection in wild and domestic mammals in two distinct areas of the semi-arid region of northeastern Brazil. Trop Anim Health Prod. 2017;49(8):1715–22. pmid:28861677
  57. 57. Weis S, Rettinger A, Bergmann M, Llewellyn JR, Pantchev N, Straubinger RK, et al. Detection of Leptospira DNA in urine and presence of specific antibodies in outdoor cats in Germany. J Feline Med Surg. 2017;19(4):470–6. pmid:26927819
  58. 58. Davis MA, Evermann JF, Petersen CR, VancerSchalie J, Besser TE, Huckabee J, et al. Serological survey for antibodies to Leptospira in dogs and raccoons in Washington State. Zoonoses Public Health. 2008;55(8–10):436–42. pmid:18631236
  59. 59. Renaud C, Andrews S, Djelouadji Z, Lecheval S, Corrao-Revol N, Buff S, et al. Prevalence of the Leptospira serovars bratislava, grippotyphosa, mozdok and pomona in French dogs. Vet J. 2013;196(1):126–7. pmid:23141966
  60. 60. Hartmann K, Egberink H, Pennisi MG, Lloret A, Addie D, Belák S, et al. Leptospira species infection in cats: ABCD guidelines on prevention and management. J Feline Med Surg. 2013:576–81. pmid:23813819
  61. 61. Langston CE, Heuter KJ. Leptospirosis. A re-emerging zoonotic disease. Vet Clin North Am Small Anim Pract. 2003;33(4):791–807. pmid:12910744