Multiple-locus variable-number tandem repeat analysis and clinical characterization of Leptospira interrogans canine isolates

Abstract

Canine leptospirosis occurs worldwide; however, information on the relationship between Leptospira serotypes/genotypes and virulence in dogs remains limited. We investigated the molecular characteristics of Leptospira interrogans canine isolates belonging to three serogroups using multiple-locus variable-number tandem repeat analysis (MLVA) and the effects of each serotype/genotype on the clinical characteristics of leptospirosis in dogs. MLVA using 11 loci of the three major L. interrogans serogroups in Japan, Australis (32 strains from 21 dogs), Autumnalis (12; 7) and Hebdomadis (66; 39), revealed more divergent genetic heterogeneity within each serogroup than multilocus sequence typing (MLST), and they formed two, three and five clusters (CLs), respectively. Lethal infections were caused by all Leptospira serogroup isolates (70.3 % with Hebdomadis, 83.3 % with Australis and 100 % with Autumnalis) or Leptospira isolates belonging to all the CLs (57.1-100 %) without any significant differences. A significant difference in hyperaemia and haemorrhage of mucus membrane was observed between serogroups Australis and Autumnalis (P50.03). Leptospira isolates of Australis CL2 caused no hyperaemia and haemorrhage from mucus membrane, whereas those of Australis CL1, Autumnalis CL3 and Hebdomadis CL1 and CL3 did (P,0.05). Significant differences in creatinine (Cre) levels were observed between serogroups Australis and Hebdomadis (P50.02). In addition, significant differences in blood urea nitrogen levels were observed between serogroups Australis and Hebdomadis (P50.004) and Australis and Autumnalis (P50.02). Based on MLVA types, a significant difference in Cre levels was observed between Hebdomadis CL1 and CL4 (P50.0018). Our results indicated that MLVA had a higher discriminatory power and was more concordant with serotyping than MLST. Although all Leptospira serotypes and genotypes caused lethal infections in dogs, the L. interrogans serogroup Australis strains were more likely to cause severe kidney damage than Autumnalis and Hebdomadis, which may be more critical to the outcome of infected dogs than haemorrhage. Our results also suggest that the virulence mechanisms and target organs in dogs may differ by Leptospira genotype.

Genetic relatedness of Leptospira interrogans serogroup Autumnalis isolated from humans, dogs, and mice in Japan

“…Multi-locus sequence typing is a highly reliable and reproducible method for the molecular typing of pathogenic Leptospira species [ 6 ]. Multiple-locus variable-number tandem repeat analysis (MLVA) has an excellent discrimination power in L. interrogans and its results are concordant with those from serotyping [ 7 , 8 ]. In our previous studies, we isolated L. interrogans serogroup Autumnalis from large Japanese field mice ( Apodemus speciosus ) and dogs in Miyazaki, Kagoshima, Saga, Hokkaido, Aomori, Akita, Fukushima, and Nagano Prefectures, Japan [ 9 , 10 ].…”

“…The serogroup of both isolates was identified as Autumnalis using the microscopic agglutination test and a panel of 18 antisera [ 9 ]. To investigate the genetic relatedness among human, murine, and canine isolates of L. interrogans serogroup Autumnalis, MLVA was performed for 11 loci [ 7 ] on the two human isolates, and their profiles were compared with those of the murine and canine isolates. The sizes of the amplicons were converted to repeat copy numbers for analysis using a categorical multi-state coefficient and unweighted pair group method with arithmetic averages (UPGMA) as a clustering algorithm with BioNumerics software version 7.6 (Applied Maths, Belgium).…”

“…There are several reports of possible dog-to-human transmission of L. interrogans serovar Canicola, indicating the important role of dogs in zoonotic transmission of leptospirosis [ 14 – 18 ]. However, the widespread use of bivalent vaccines containing serovar Canicola is likely to decrease the circulation of serovar Canicola among dogs in the U.S., Europe, and Japan [ 7 , 19 , 20 ]. Other Leptospira species and serovars have been detected in asymptomatic dogs, but it remains unclear whether these dogs serve as the maintenance host of other genotypes/serotypes and facilitate zoonotic transmission to humans [ 14 , 15 ].…”

“…In our previous studies, we isolated L. interrogans serogroup Autumnalis from large Japanese field mice ( Apodemus speciosus ) and dogs in Miyazaki, Kagoshima, Saga, Hokkaido, Aomori, Akita, Fukushima, and Nagano Prefectures, Japan [ 9 , 10 ]. MLVA performed on the mouse and dog isolates demonstrated that they are of different genotypes (MLVA type) [ 7 , 8 ].…”

Identification of Serogroups Australis and Icterohaemorrhagiae in Two Dogs with a Severe Form of Acute Leptospirosis in Italy

“…DNA in a variety of biological samples and to diagnose leptospirosis in dogs. Although molecular tests showed low diagnostic sensitivity as they might give a large number of false negative results [11], different techniques have been adopted for the typing of Leptospira strains by analyzing the bacterial genome or its specific regions [12][13][14][15]. In particular, the multi-locus sequence typing (MLST) technique is able to characterize the genetic profile of Leptospira strains by sequencing and analyzing specific fragments of some bacterial house-keeping genes, thus identifying specific sequence types (STs).…”