Detection and genotyping of Leptospira spp . from the kidneys of a seemingly healthy pig slaughtered for human consumption

Introduction: Leptospirosis is a zoonotic disease caused by pathogenic Leptospira spp. Leptospirosis is maintained in an environment due to chronic kidney infection of a wide variety of domestic, peridomestic and wild reservoir mammals. In this study the role of pigs in maintenance of leptospires on the Caribbean island of St. Kitts was investigated. Methodology: The condemned kidneys of 60 pigs slaughtered at a St. Kitts abattoir were screened by a quantitative-PCR for the presence of Leptospira spp. Positive samples were genotyped using a six-gene based multilocus sequence typing scheme. Results: Leptospiral DNA was detected in the kidneys of one of the 60 pigs. Multilocus sequence typing identified the infecting species to be L. interrogans. Conclusions: Detection of this zoonotic pathogen in the kidneys of a seemingly healthy pig raises concerns regarding the subclinical carriers of the disease among the island’s swine population.


Introduction
Leptospirosis is a highly prevalent zoonotic disease caused by pathogenic Leptospira species.Leptospirosis affects over 150 species of animals, including humans, dogs, cattle, horses and pigs.The pathogen persists in the kidneys of reservoir animals and is shed in the urine.Susceptible hosts acquire the infection following exposure to infected animal urine or Leptospira-contaminated water or soil [1,2].The Caribbean region is considered to be an endemic area for leptospirosis with a high incidence rate [3].. Recently, we showed that one-fifth of tested water sources on the island of St. Kitts were contaminated with leptospiral DNA [4].Pigs are important source of meat on the island with approximately 2000 pigs slaughtered annually for local consumption.Pigs are primarily raised by landless livestock farmers who own a small number of animals and use traditional management practices, which include outdoor rearing, grazing and slaughtering of the older pigs.To understand the role pigs might play in maintenance of leptospirosis on the island, we collected condemned kidneys from 60 pigs slaughtered over a period of one year at a St. Kitts abattoir and screened for the presence of leptospiral DNA.

Methodology
Kidneys were condemned on the basis of the macroscopic criteria proposed by Baker et al. [5] namely the presence of foci of pale or red discoloration scattered throughout the cortical surface.A part of each kidney was fixed in 10% neutralbuffered formalin, routinely processed and embedded in paraffin wax, cut at 5μm and stained with hematoxylin and eosin (HE) for histological evaluation.DNA was isolated from unfixed, frozen or fresh kidney tissue (20mg) using a DNeasy Blood and Tissue Mini-Kit (Qiagen, Valencia, USA), following the manufacturer's directions.
To genotype the infecting Leptospira spp., a sixgene multilocus sequence typing (MLST) scheme based on adk, icdA, secY, rrs2, lipL41, and lipL32 genes was used [8].These loci were PCR amplified from the Leptospira in the positive sample, designated SKIT_AV_Po, using Phusion High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA), and previously described primers and thermal conditions [8,9].PCR products from icdA, secY, rrs2, lipL41, and lipL32 genes were gel extracted using QIAquick gel extraction kit (Qiagen, Valencia, USA) and sequenced in a commercial facility (Davis Sequencing, Davis, USA).Nucleotide sequences deposited in the GenBank database have accession numbers KM211315, KM211316, KM211317, KM211318, KM211319.Phylogenetic analyses were performed by the Neighbor-Joining method (10) using MEGA (v6.06) software.The trees were statistically supported by bootstrapping with 1000 replicates, and phylogenetic distances were measured by the Kimura two-parameter model.

Results and Discussion
Out of 114 swine kidney samples screened, two were positive by this assay.The two positive samples, RL7C and RL7M had a Ct value of 23.12 and 26.5, and contained 1.5x10 5 and 1.8x10 4 genome units/20mg of kidney tissue, respectively.Both these samples came from the cortical and medullary regions of the same kidney.Grossly, the affected kidney was characterized by poorly defined, multifocal areas of pale-tan discoloration, ranging from 2 to 5 mm in size, scattered throughout the cortex ('white-spotted' appearance) [2].Microscopically, sections of the kidney revealed multiple foci of perivascular and interstitial lymphoplasmacytic inflammatory cell infiltration within the cortex and a relatively prominent focus of lymphoplasmacytic tubulointerstitial nephritis near the cortico-medullary junction (Figure 1).
For genotyping of the positive sample, we were able to PCR amplify all but the adk gene.Primers for the adk gene non-specifically amplified a porcine gene, which suggests that the described primers and/or thermal conditions for PCR amplification of adk gene from porcine samples need further standardization.All five genes showed 100% identity with L. interrogans in BLAST searches using the National Center for Biotechnology Information server (http://www.ncbi.nlm.nih.gov/BLAST/).By phylogenetic analysis, the icdA, lipl32, lipl41, rrs2 and secY genes of leptospiral strain SKIT_AV_Po clustered very closely with cognate genes of L. interrogans strains [Supplemental Fig. S1 (A)-(F)].In all the five genes, L. interrogans strains detected in humans in the Andaman and Nicobar Islands, India, appeared to be the nearest neighbor of strain SKIT_AV_Po.This is the first report of identification of a circulating species of Leptospira on St. Kitts.Although subclinical infections are common in pigs, the detection of this zoonotic pathogen in the kidneys of an animal destined for human consumption raises concerns about its public health implications.One of the short-comings of this study was that we did not use a host housekeeping gene as a control for PCR inhibitors, which may have resulted in some false negative results.

Conclusions
Detection of this zoonotic pathogen in the kidneys of a seemingly healthy pig raises concerns regarding the subclinical carriers of the disease among the island's swine population.Larger multi-species studies are required to estimate the true prevalence of the disease, and the roles of wild and domestic animals in the transmission cycle of leptospirosis on the island.

Figure 1 .
Figure 1.A and B. Multifocal areas of discoloration scattered throughout the renal cortex.C and D. Multiple foci of perivascular and interstitial lymphoplasmacytic inflammatory cell infiltration within the cortex and a relatively prominent focus of lymphoplasmacytic tubulo-interstitial nephritis near the cortico-medullary junction, 20× and 40×, HE.