Leptospira Reservoirs among Small Mammals in Sri Lanka

Abstract

Leptospirosis is a zoonotic disease with worldwide importance. In Sri Lanka, confirmed leptospirosis cases are ever increasing. Humans contract leptospirosis through Leptospira infected urine-contaminated environments. While all mammals are capable of being reservoirs for this disease, feral and peridomestic rodents are known to play an important role. The objective of this study was to identify small mammal species carrying Leptospira in Sri Lanka. Small mammals were captured from eight localities with high leptospirosis prevalence according to hospital records in Kurunegala district. Blood, urine and kidneys were tested for lipL32 gene of pathogenic Leptospira using quantitative polymerase chain reaction. Of the 131 small mammals belonging to six species collected, 14.5% had Leptospira positive blood, urine or kidney. Bandicota bengalensis, Mus cervicolor, Rattus rattus and Suncus murinus were Leptospira carriers. Leptospira was previously reported only from R. rattus and B. bengalensis from the country, M. cervicolor and S. murinus are new carrier reports for Sri Lanka. All sampled locations had infected small mammals, indicating that the reservoirs are widespread in the district. This data could be used to control the disease and to encourage the public to take preventative measures against infection.

Keywords: Leptospirosis; Suncus murinus; Murine rodents; Shrews

Abbreviations

qPCR: quantitative Polymerse Chain Reaction; ELISA: Enzyme Linked Immunosorbent Assay; MAT: Microscopic Agglutination Test

Introduction

Leptospirosis has become a major threat to human health world over. Though it is most common in tropical and subtropical regions, reports of disease incidents are ever increasing from the developed world as well [1,2]. Humans contract leptospirosis through Leptospiracontaminated environments, bacteria entering the body through abraded skin or mucous membranes. Although Leptospirosis can be treated effectively with antibiotics, delay in diagnosis may result in serious complications that even lead to death. Most commonly, Leptospira infections cause asymptomatic seroconversion, but may result in severe disease conditions with jaundice, renal failure, hemorrhage, refractory shock, and myocarditis [3]. Leptospirosis is known to be an occupational disease, commonly occurring among farmers, but reported from veterinarians, abattoir workers and fishermen [4]. The disease has spread to urban areas with unhealthy sanitary practices and poor garbage disposal facilities. Recreational activities such as water sports have also been recognized as a risk factor for this disease in the recent years [1,5].

In Sri Lanka, first confirmed case of leptospirosis was reported in 1959 [6]. Since then, confirmed cases were reported from many districts in the country [7]. Leptospirosis has been reported as a major public health problem from Kurunegala, Kandy, Matele, Rathnapura, Gampaha, Matara and Kegalle districts [7]. The last three outbreaks have been reported in 2003, 2008 and 2011, 2008 outbreak being the worst ever reported from the country and the second highest leptospirosis incidence in the world during recent years [8]. In 2011, disease outbreak also extended to Anuradhapura, a region that is relatively dry and where leptospirosis is never heard of before [9]. Peak incidence of the disease is reported to be associated with the rice paddy-harvesting seasons, wherein an increase in the rodent population is observed. The majority of patients are also farmers who had been exposed in the paddy fields [4].

Animal reservoir of leptospirosis is a key factor concerning the disease control and prevention, however little attention has been given to study the reservoir animals. While any mammal is capable of carrying Leptospira [10], animals that live in close association with humans such as rodents, wild boars and the domesticated animals such as dogs, cattle and pigs play a major role in transmitting the disease to humans [11,12,13]. Many species of murine rodents are recorded as carriers of this pathogen around the world [14,15]. Due to their wide distribution and high abundance in rural areas with farmlands and in urban areas with high density of human population, feral and peridomestic rodents are believed to be most important reservoirs. In Sri Lanka Leptospira is reported only from three small mammal species; murine rodents, B. bengalensis, R. rattus, and shrew Suncus sp. [12,16].

The objective of this study was to identify murine rodents and shrews carrying Leptospira in and around paddy fields in localities with reported high prevalence of leptospirosis in Kurunegala District.

Materials and Methods

Small mammals were trapped from eight sites in Kurunegala District (Table 1). Traps were placed in and around paddy fields. Sampling was carried out for 4-6 days at each collection site. A sample of voided urine (500-1200 μL/ 100-500 μL from M. cervicolor and S. murinus) and a sample of blood (100-300 μL) from saphenous vein were collected from each small mammal captured. Ethical clearance for sample collection and handling was obtained from the ethical clearance committee of the Postgraduate Institute of Science, Peradeniya, Sri Lanka. DNA was extracted from blood cell pellets, collected by centrifugation at 12000rpm for 10min, using Wizard® Genomic DNA Purification Kit, according to the manufacture’s protocol with few modifications. Volume of each blood pellet was adjusted to 300μl by adding PBS. Following modifications were done to the original protocol; Step 3: After adding cell lysis solution samples were centrifuged at 14,000rpm for 1min.; Step 6: samples were incubated at 80°C for 5min. in nuclei lysis solution; Step 15: samples were kept at room temperature over night to rehydrate the DNA after adding DNA rehydration solution; Step 16: DNA Samples were stored at -20°C. Sample processing and DNA Extraction from blood and urine samples were done within one week of collection. Urine samples were pelleted by centrifugation at 12000rpm for 20min at room temperature, pellet was washed once with PBS. DNA was extracted from the pellet using “QIAamp DNA Mini Kit®” (Qiagen Sciences, Maryland, USA) according to manufacturer’s protocol for “Isolation of bacterial DNA from biological fluids”. Two hundred and forty two BP fragment of lipL32 gene present only in pathogenic Leptospira spp. [17] were amplified in Step one Real Time PCR system (Applied Biosystems) using Go Taq probe qPCR master mix (Promega) and Taq Man probe. Primers and probe used were; Forward-48F (5’ -AAG CAT TAC CGC TTG TGG TG-3’), Reverse- 286R (5’ -GAA CTC CCA TTT CAG CGA TT-3’) and the Probe- 189P (FAM-5’ -AA AGC CAG GAC AAG CGC CG-3’ -BHQ1). Kidneys were collected only from accidental kills. DNA from kidneys was also extracted employing the same method used for blood pellet. Quantitative PCR mixture consisted of 13μl of Go taq probe qPCR master mix, 2μl of 6.25μM forward and reverse primer and 1μl of 2μM probe, 4.5μl of nuclease free water and 2.5μl of template. Final volume of the PCR mixture was 25μl. The thermal profile of the assay composed of initial holding temperature of 60°C for 30s for Pre PCR read and 95°C for 5min for heat activation, 45 cycles of amplification at 95°C for 15sec and 60°C for 1min and final post PCR read stage of 60°C for 30s. The reactions were performed in Step one Real Time PCR System (Applied Bio systems, USA) and analyzed using Step One software version 2.2.2. All samples were tested in duplicates and an additional run was performed for samples with one positive or one negative result. Samples, which gave positive results with Ct values less than 40 were considered positive and the smaller Ct values of the two positives were used in analysis. A standard curve was generated using tenfold dilution series of 200μl DHL vaccine (Merial) extracted and eluted to 200μl. According to the standard curve we report positives with 88.1% efficiency, R2=0.99, slope=-3.6, Y intercept=40.248.

  

    
Locality
    
Small mammal species
    
Ct values for positive samples
  
  

    
Blood
    
Urine
    
Kidney
  
  

    
Kiwlegedara (07˚23’N, 80˚12’E,    elevation 75m)
    
Bandicota bengalensis
    
 
    
36.046
    
 
  
  

    
Suncus murinus
    
 
    
35.886
    
 
  
  

    
Malliyagoda (07˚24’N, 80˚28’E,    elevation 170m)
    
B. bengalensis
    
 
    
35.198
    
 
  
  

    
S. murinus
    
 
    
27.845
    
18.531
  
  

    
Udawela (07˚33’N, 80˚02’E,    elevation 40m)
    
Mus cervicolor
    
34.694
    
 
    
35.1
  
  

    
Rattus rattus
    
34.861
    
 
    
 
  
  

    
 
    
34.937
    
 
    
 
  
  

    
 
    
 
    
27.106
    
 
  
  

    
 
    
 
    
27.943
    
 
  
  

    
Polgahawela (07˚19’N, 80˚17’E,    elevation 75m)
    
R. rattus
    
35.084
    
 
    
 
  
  

    
Ipalawa (07˚34’N, 80˚27’E,    elevation 145m)
    
R. rattus
    
 
    
35.106
    
 
  
  

    
 
    
 
    
36.835
    
 
    
 
  
  

    
Bogollagama (07˚47’N, 80˚10’E,    elevation 80m)
    
R. rattus
    
 
    
25.67
    
 
  
  

    
 
    
 
    
 
    
31.826
    
 
  
  

    
Herathgama (07˚52’N, 80˚25’E,    elevation 155m)
    
R. rattus
    
 
    
35.612
    
 
  
  

    
 
    
 
    
33.662
    
 
    
 
  
  

    
Minhettiya (07˚35’N, 80˚18’E,    elevation 100m)
    
S. murinus
    
 
    
32.208
    
33.321
  
  

    
 
    
 
    
 
    
23.571
  
  

    
R. rattus
    
 
    
 
    
33.559
  

Table 1:  Positive Blood, urine and kidney samples collected from eight localities in Kurunegala district.