Variable Nucleotide Tandem-Repeat Analysis Revealing a Unique Group of Leptospira interrogans Serovar Pomona Isolates Associated with California Sea Lions

Pathogenic Leptospira bacteria cause leptospirosis, one of the most widespread zoonotic diseases known. The presence of Leptospira-infected wildlife and domesticated animals poses a persistent public health threat. Leptospira bacteria usually gain access to new hosts by passage across mucous membranes or through skin abrasions, often from environmental sources, such as urine-contaminated water. After septicemia, the bacteria localize in the kidneys and are released back into the environment during urination, thereby providing a source of infection of naïve animals (9). Leptospira infection leads to one of two outcomes; maintenance hosts infected by selected serovars develop chronic infections with few clinical signs, whereas accidental infection of nonmaintenance hosts often results in an acute infection that may result in death through multiple organ failure (9).

Leptospirosis due to L. interrogans serovar Pomona may not follow the maintenance host-accidental host paradigm. Animal populations of the same species can exhibit either chronic or acute infections with serovar Pomona. Restriction fragment length polymorphism (RFLP) analysis of L. interrogans serovar Pomona isolates has revealed the presence of two major subtypes (5, 22) that can be further differentiated by HpaII digestion (2) or IS1500 hybridization (24) patterns. Specific clonal populations, rather than all members of serovar Pomona, may form stable associations with selected maintenance host species (2). To refine our understanding of serovar Pomona-host relationships, variable nucleotide tandem-repeat (VNTR) analysis was used to compare bacterial isolates obtained from different animal species. VNTR loci evolve independently and provide strong support for or against kinship among isolates.

L. interrogans strains (Table 1) were propagated in liquid EMJH medium at 29°C (8, 14). All of the strains (n = 63) included in this study, with the exception of L. interrogans serovar Copenhageni, were identified as L. interrogans serovar Pomona by RFLP analysis (22). Genomic DNA samples prepared as described previously (19) were used as templates for PCR amplification. All of the primers used, except P2447 (5′-CGGTTGCTTTAGGTGAAGCTGTCCAATG-3′) and P2498 (5′-TCATTCTCTCTGCGGTCATAGGCATCCC-3′), were described previously (16, 18, 20, 21). P2447 and P2498 were designed and used to amplify a single product from VNTR locus 23 because published primers for this locus generated two products per template. PCR products were separated by agarose gel electrophoresis and visualized as described (Fig. 1) (24), and product sizes were calculated by comparison to size standards (100-bp ladder and 1-kb ladder; Invitrogen, Carlsbad, CA) by using the linear regression function in Excel (Microsoft Corp., Redmond, WA).

PCR products for VNTR loci 4, 7, 10, 23, 27, 29, 30, 31, and 36 (16, 18, 20, 21) were assessed for all of the strains in this study. Published primers for the amplification of VNTR loci 9, 11, 19, and 50 failed to generate products from serovar Pomona DNA (data not shown) and were not used. The number of tandem repeats for each VNTR product was calculated from the predicted product length, less the length of unique sequence DNA, divided by the length of the repeating unit (Table 1). The number of tandem-repeat copies of representative PCR products at loci that varied among L. interrogans serovar Pomona isolates was confirmed by DNA sequence analysis as described previously (3). (Sequence data used in this study are available in GenBank under accession numbers EU938676 to EU938693.) Eight VNTR loci, 4, 7, 10, 23, 29, 30, 31, and 36, were used for comparison to L. interrogans serovar Copenhageni, which was used as an outgroup. VNTR loci 29, 30, and 36 were invariant among serovar Pomona isolates but were useful for distinguishing serovar Pomona from serovar Copenhageni. Serovar Pomona isolates varied at five VNTR loci, 4, 7, 10, 23, and 31 (Fig. 1). The kinship coefficient was calculated for each isolate based on VNTR data with Microsat version 1.5 (http://hpgl.stanford.edu/projects/microsat/) by using 6,435 bootstrap replications (the most allowed when using eight variables). The output was analyzed by neighbor joining using Neighbor in the PHYLIP software package (http://evolution.genetics.washington.edu/phylip.html), and the resulting dendrogram (Fig. 2) was drawn with TREEVIEW (17).

Eleven distinct groups of L. interrogans serovar Pomona isolates were detected by VNTR analysis and assigned a letter designation from A to K (Fig. 2). This method detected greater diversity between serovar Pomona isolates than did HpaII-generated RFLPs (which identified six distinct types) (2) but detected fewer differences than did an IS1500-based typing method (which detected 15 distinct types) (24). Most cattle isolates have either a common VNTR profile ([16/29 or 55%] in group K; Fig. 1) or a common HpaII-RFLP pattern (type 2) (2). In contrast, greater diversity among serovar Pomona isolates from pigs and wildlife was detected by VNTR analysis than by the HpaII-based method. The discriminative power of VNTR analysis and the ease with which results can be compared between laboratories suggest that this method will be suitable for standardization as a typing method for L. interrogans (16, 18, 20, 21).

A significant finding of this study was that all California sea lions isolates (n = 10) have common VNTR profiles, despite separation by time and geography, and are distinct from all other serovar Pomona isolates (group G, Fig. 1). California sea lion isolates are distinguished by the presence of 10 to 12 repeats at VNTR locus 4, compared to 3 to 7 repeats in all other isolates (Table 1). Isolate Po048, which is unique among California sea lion isolates at VNTR locus 4, was obtained in 1970 during the earliest reported leptospirosis outbreak along the California coast (23); other isolates obtained during subsequent outbreaks (1, 4, 13) were identical to each other. California sea lions are persistently exposed to serovar Pomona during nonoutbreak years (15). Intermittent exposure of California sea lions to the same L. interrogans serovar Pomona clonal population from another host species through accidental exposure over a 37-year period is unlikely (1, 4, 6, 7, 10-13, 23). We conclude that California sea lions are maintenance hosts of L. interrogans serovar Pomona. Persistent Leptospira infection of California sea lions is a potential public health threat; infected animals migrate and become stranded on beaches along the Pacific coast of North America (25) and may transmit disease to humans, pets, and wildlife. Identification of a unique marker (VNTR locus 4) associated with serovar Pomona isolates from California sea lion isolates should be useful for detecting marine mammal-terrestrial mammal disease transmission.

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FIG. 1.

Comparison of VNTR products used to differentiate L. interrogans serovar Pomona isolates. The diversity of PCR products observed among L. interrogans serovar Pomona isolates is shown for VNTR loci 4 (panel A, lanes 1 to 7), 10 (panel B, lanes 1 to 4), 23 (panel C, lanes 1 to 3), 7 (panel D, lanes 1 and 2), and 31 (panel E, lanes 1 to 3). Products were separated by agarose gel electrophoresis and visualized as described in the text. Migration of the corresponding products from L. interrogans serovar Copenhageni is shown for comparison (lane C in each panel). Panel A, products with 3, 4, 5, 6, 7, 10, and 12 repeats (lanes 1 to 7). Panel B, products with 10, 11, 12, and 13 repeats (lanes 1 to 4). Panel C, products with 2, 3, and 4 repeats (lanes 1 to 3). Panel D, products with 1 and 2 repeats (lanes 1 and 2). Pane E, products with 2, 3, and 4 repeats (lanes 1 to 3). The migration of 100-bp size markers is shown at the right, with alternate size markers labeled.

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FIG. 2.

Cluster analysis of L. interrogans serovar Pomona isolates by VNTR typing. Genetic relationships among serovar Pomona isolates are shown with L. interrogans serovar Copenhageni (Ic02001) used as the outgroup. A broken line is used to indicate the depth of the serovar Copenhageni branch. The host species from which each isolate was obtained is highlighted as indicated in the key provided. A reference marker for branch length (kinship coefficient, 0.1) is provided.

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