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Pathogenic Leptospira species are the causal agents of Leptospirosis, are a widespread but often neglected zoonoses. Rodents are often implicated as the primary reservoir host. Human infection may be acquired by direct contact with infected animal urine or indirectly by contaminated soil or water. Multiple serovars have been identified using the reference Microscopic Agglutination Test (MAT). However, this is an ambiguous system of classification and serological diagnoses are not reliable for the confirmation of infection during the early acute phase. Molecular techniques have been developed to overcome these problems. This study aimed to determine if rodents in the UK are potential reservoirs of leptospires, by Real-Time PCR, using primers and probes targeting Leptospira genus specific 16S ribosomal RNA gene and confirming positives, using the pathogenic spp. specific LipL32 outer surface protein gene. Reactive samples were further subjected to conventional PCR to amplify DNA for sequencing. DNA extracts from kidney lysates (n=292), and two reference strains were included in this study. 19% (55 of 292) DNA samples tested were positive for 16S rRNA gene, 56% (31 of 55) were also positive for LipL32 gene. Conventional PCR and agarose gel electrophoresis detected five amplified products from three gene targets, which were sequenced. The phylogenetic cluster of positive samples showed they were closely related to L. borgpetersenii and L. interrogans or L. kirschneri. The detection of leptospiral DNA in 19% of rodent kidneys indicates that rodents pose a significant risk of leptospirosis for humans and other animals in the UK.
The genus Leptospira are highly motile spirochaetes belonging to the family Leptospiraceae, are about 6-20Âµm in length and 0.1Âµm in diameter (Adler and de la Pena Moctezuma, 2009). Initially, leptospires were classified into two species, Leptospira interrogans for pathogenic and Leptospira biflexa for non-pathogenic leptospires based on serology, with currently over 300 antigenic distinct serovars distributed into 29 serogroups that are antigenetically related (Doungchawee et al., 2007; Cerqueira et al., 2010b). Current genotypic classification based on DNA-DNA hybridization have identified twenty Leptospira species comprising pathogenic, intermediate and saprophytic species, with seven pathogenic species mainly L. interrogans, L. borgpetersenii, L. kirschneri, L. noguchi, L. alexanderi, L. santarosa and L. weilli, considered the main agents of leptospirosis (Slack et al., 2010; Nalam et al., 2010).
Leptospirosis is a neglected and an emerging zoonotic infectious disease of significant global public health concern. More than 500,000 severe cases are reported yearly, with mortality rate above 10% worldwide (Fraga et al., 2011). Prevalence is higher in tropical and subtropical but less common in temperate regions of the world such as the UK (see figure 2.1) (Health protection agency, 2009). Infections peak during summer and early autumn in temperate regions, but occur throughout the year with high incidence during rainfall season in the tropics. (Pavli and Maltezou, 2008).
Data source: Health protection agency, (2009).
Figure 2.1: Laboratory confirmed cases of Leptospirosis in the UK.
Cases of Leptospirosis reported in the UK between 1998 and 2009. Confirmed cases are highest in England and Wales and lowest in N. Ireland.
2.3 Clinical Infection
Disease manifestations ranges from an acute febrile illness with flu-like symptoms to the severe type of the disease called Weil's disease, with non-specific manifestations involving multiple organs thus making the diagnosis difficult and consequently, often under diagnosed (Ahmed et al., 2009). International travel and recreational activities such as fresh water swimming and rafting have been recognized as sources for exposure to Leptospira spp. in both tropical and temperate regions. While incidence associated with occupational exposure such as abattoir and sewer workers is decreasing, cases concurrent to recreational activities is increasing (Pavli and Maltezou, 2008).
Previous studies have identified a broad range of wild and domestic mammals and rodents as reservoirs and sources of leptospiral infection for human and other animals (Roczek et al., 2008; Adler and Moctezuma, 2009). Rodents, particularly rats and mice are the most significant and extensively dispersed agents that facilitate transmission of Leptospira infection (Pavli and Maltezou, 2008). These chronically infected maintenance host which are normally asymptomatic become colonized in the renal tubules by the leptospires and subsequently excrete spirochaetes via urine into the environment (Stern et al., 2010). Leptospires are able to survive in a broad range of moist environments including water and soil. Humans and animals become infected either by direct contact with infected animals urine or indirectly with water or soil contaminated by infected urine (Monahan et al., 2009).
Their relatively large genome size and high degree of signal transduction genes present in leptospires has been linked to their capability to survive the varied conditions inside and outside the host, with exception of L. borgpetersenii serovar Hardjo, which is transmitted by direct contact as it appears to be losing genes necessary for survival outside the host while virulence and genes for survival in the host are retained (Matsunaga et al., 2007).
The need for a robust diagnostic method for the timely detection of Leptospira which will contribute to effective and patient management and unambiguous classification of leptospires have necessitated the development and validation of several PCR-based molecular methods such as real-time polymerase chain reaction (PCR) (Ahmed et al., 2009; Lourdault & Picardeau, 2009) and multi locus sequence typing (MLST) (Levette, 2007; Leon et al., 2010).
Real-time PCR is a rapid, sensitive and specific assay which allows the detection of amplified PCR products during the early phase of reaction and is less prone to contamination compared to conventional PCR. It is well documented that Real-time PCR assays are exceptionally useful for the early
diagnosis of leptospirosis (Slack et al., 2006; Bedir et al., 2010;) but serological methods are inappropriate during acute infection whilst antigen detection is valuable (Bedir et al., 2010). Assays can be performed targeting specific regions of genes including those that are pathogenic leptospira spp. Specific, such as LipL32 (outer membrane lipoprotein), LigA and LigB (immunoglobulin-like protein genes) and others that are leptospira genus specific such as 16S ribosomal RNA (small subunit of ribosome in prokaryotes) and secY (preprotein translocase) (Stoddard et al., 2009; Thaipadumpanit et al., 2011). The TaqMan real-time PCR based on specific sequencing of target gene such as 16S rRNA has been successfully used in the detection of Leptospira species (Slack et al., 2006).
Characterisation of Leptospira isolates has been attempted employing various serological and molecular typing methods. However, the previously used serogroups lack compatibility with molecular classification, since different serovars of pathogenic and non-pathogenic species fall under the same species (Galloway and Levett, 2008; Cerqueira and Picardeau, 2009).
Multi locus sequence typing (MLST) is a robust PCR based technique which allow the use of sequence polymorphisms of several target genes, mainly housekeeping genes for the unambiguous characterization of isolates and investigation of evolutionary relationships between interrelated bacteria (Ahmed et al., 2006). Hence, appropriate for classification of leptospires, population and epidemiological surveillance. MLST has been successfully applied for typing various bacterial pathogens including leptospires (Perez and Goarant, 2010). Several housekeeping gene have been validated in previous studies as ideal markers for use in an MLST typing system, because they can produce amplicons with heterogeneous sequences appropriate for phylogenetic analysis from all Leptospira species. These genes include, accA2 (Acetyl coenzyme A), gcvP (Glycine cleavage system P protein), ccmF (Cytochrome c biogenesis factor), czcA (Heavy-metal efflux pump), groEL (60-kDa chaperonin), polA (DNA polymerase 1), and recF (DNA replication and repair protein) (Leon et al., 2010) and secY (Preprotein translocase) (Ahmed et al., 2009).
Sequencing of 16S ribosomal RNA is the earliest universal target gene for molecular detection of bacteria species especially fastidious organisms such as leptospires. But its use is limited to species level due to insufficient distinguishing power of interrelated species (Morey et al., 2006; Fournier et al., 2007; Cerqueira et al., 2010a).
Hoke et al, (2008) showed in their studies that LipL32 is an extracellular matrix (ECM) interacting protein gene which is conserved in pathogenic Leptospira species but absent in non-pathogenic or intermediate species and that it is the most prominent outer membrane lipoprotein expressed during Leptospira infection. Hence, it is ideal for detecting pathogenic Leptospira species.
2.7 Project Aims
This study aimed at detecting the presence of Leptospira in UK rodents by real-time PCR and to speciate pathogenic isolates using MLST and to undertake phylogenetic analysis. The detection and characterisation of Leptospira in UK rodents will contribute to the improvement of leptospirosis prevention, control strategies and epidemiological surveillance.
3. Materials and Methods
3.1 Kidney samples
Kidney tissue lysates (n=292) from rodents caught at sites in northern England and Scotland (collected by veterinary students at Edinburgh university) and two reference strains namely Leptospira grippotyphosa Valbuzzi and Leptospira santarosai Georgia, supplied by the Leptospira reference laboratory at Hereford were provided.
3.2 DNA extraction
Kidney tissue lysates were thawed and genomic DNA was extracted from 100Âµl aliquot of each tissue lysates, using QIAcube automated machine with DNeasy Blood and Tissue Kits according to manufacturer's instructions (QIAGEN).
3.3 Real-time PCR analysis
A real-time PCR previously published, that used TaqMan probes targeting either 16S rRNA or LipL32 was evaluated with modifications (Thaipadunpanit et al., 2011). Both assays were conducted using specific primers and probes for each gene. For 16S rRNA gene, rrsF (5'-CCCGCGTCCGATTAG-3'), rrsR (5'-TCCATTGTGGCCGRACAC-3') and rrsP FAM-CTCACCAAGGCGATCGGTAGC-BHQ1. For LipL32, F (5'-TCGCTGAAATRGGWGTTCGT-3'), R (5'-CGCCTGGYTCMCCGATT-3'), and FAM-ATTTCCCCAACAGGCG-BHQ1. DNA amplification was performed in a total volume of 25 Âµl. The reaction mixture consisted of 2.5 Âµl 10x PCR buffer, 2.5 Âµl dNTP's, 2.5 Âµl MgCL2 50Mm, 2.5 Âµl of each oligonucleotide primer, 2.5 Âµl probe, 0.375 Âµl ROX 1Mm, 0.15 Âµl of 5 units/Âµl Taq polymerase, 7.48 Âµl of autoclaved distilled H2O and 2 Âµl of template DNA. 2 Âµl of each reference strain was used as positive controls and 2 Âµl of distilled water was used instead of DNA template for negative controls. DNA amplification was performed using Stratagene mx3000 Real-time thermo cycler with amplification conditions consisting of initial denaturation at 95°C for 10 min, followed by 40 cycles of 2 steps at 95°C for 15 s, and 60°C for 60 s.
For quality control, PCRs were performed using only the reference strains and distilled water prior to actual PCR reactions. This ensured the assay was working properly including all reagents.
3.4 MLST analysis
For MLST, seven genes recF, accA2, ccmF, czcA, gcvP, groEL and polA, previously validated by Leon et al (2010) were evaluated with the addition of secY gene making a total of eight genes used. Conventional PCRs were carried out on 31, LipL32 positive samples using PCR thermo cycler (Bio-Rad), with the following protocol parameters. Initial denaturation at 94°C for 5 min, followed by 45 cycles of 94°C for 30 s, 60°C for 45 s and 72°C for 60 s. And final extension at 72°C for 10 min. 5 Âµl of each amplified products were mixed with 1 Âµl of 6x gel loading buffer (Fermentas) and were separated on 1% agarose gel in TAE by electrophoresis, stained with 10 Âµl (1:10,000 dilution) safe view (NBS Biologicals Ltd) and viewed under UV light. Both strands of each five amplified products detected by agarose gel electrophoresis were then sequenced at Queen Mary Hospital, genome centre.
3.5 Phylogenetic analysis
A sequenced DNA fragment of each gene was used to construct phylogenetic tree. Phylogenetic analyses were perform with MEGA version 5. The neighbour-joining method was utilized for sequence comparison and the maximum composite likelihood method was used for computing the evolutionary distance the associated taxa were tested with bootstrap 1000 replications (Tamura et al., 2011).
4.1 Analysis of Real-time PCR assays
19% (55 of 292) DNA samples tested were positive for real-time PCR assay targeting 16S rRNA gene (Figure 4.1A), 56% (31 of 55) were positive while targeting LipL32 gene (Figure 4.1B). Positive samples were estimated based on cycle threshold (ct) values. Ct is the point at which cycle crosses threshold. Ct values â‰¤30 were taken as positive while ct values >30 were excluded.
Figure 4.1. Quantitative real-time PCR analysis of DNA samples.
A: targeting 16S ribosomal RNA gene and B: targeting LipL32 gene.
4.1.1 Cycle threshold value of real-time PCR positive samples
Cycle threshold (ct) is the cycle at which the DNA sample reaches a fluorescent intensity above background. The lower the ct value the higher the DNA content of the sample. From the real-time PCR assays, 55 samples were positive out of the 292 samples tested using 16S rRNA gene and the 55 positive samples were confirmed with LipL32 gene, which gave 31positives. The positive samples from each assay and their ct values are represented in table 4.1.1 below.
Table 4.1.1: Ct value of positives samples from Real-time PCR assay using 16S rRNA Gene and LipL32 Gene.
Sample identification Code
16S rRNA Gene
The results shows that DNA samples with low cycle threshold (ct) values (12.79-19.68) except one with 27.08 ct were positive for LipL32 assay. The ct values of samples positive for LipL32 assay generally have a lower ct value in the 16S rRNA assay. This indicates the presences of more DNA in the samples used for 16S rRNA assay, which was the first assay performed.
4.2 Agarose gel Electrophoresis
1% agarose gel electrophoresis detected five amplified products of the 31 positive samples amplified by conventional PCR, from three gene targets namely, secY (from 3 samples), ccmF and czcA (from a single sample).
4.3 Phylogenetic analysis of individual gene Loci
The phylogenetic analysis of positive samples was used for species discrimination. All nucleotide positions with gaps and omitted data were eliminated from the tree constructions. The results showed that positive samples were closely related to L. interrogans (Figure 4.3A), L. kirschneri (Figure 4.3B) and L. borgpetersenii (Figure 4.3C). The ccmF and czcA gene loci were amplified from a single positive sample and should have shown closeness to the same specie. The discrepancy in this result may be due to bias through data availability and insufficient sequences deposited on Genbank, because effort was made to use all available ccmF and czcA species sequences in the nucleotide database in constructing the Phylogenetic trees (http://www.ncbi.nlm.nih.gov/nuccore?term=leptospira).
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Figure 4.2. Phylogenetic analysis based on individual loci sequences.
A: ccmF gene loci, B: czcA gene loci and C: secY gene loci. Amplified products are designated AMO33454 for ccmF and czcA, and AMO470504, AMO49754 and AMO571504 for secY respectively. Accession numbers are followed by specie and serovar names.
The detection and classification of Leptospira have been mainly by serological methods including the reference microscopic agglutination test (MAT). These methods are not reliable for the confirmation of clinical suspected cases at the early acute stage of infection since they depend on the detection of antibodies which become detectable about 7 days after disease onset and the problem of cross-reaction is incumbent (Salaun et al., 2006; Bedir et al., 2010). Direct visualisation of leptospires in blood samples by dark-field microscopy cannot be relied upon because it cannot classify the organism but only confirm the presences of spirochaetes and isolation of leptospires is both low yield and time consuming, taking several weeks to months (Ahmed et al., 2009). Various molecular methods including pulsed-field gel electrophoresis (Galloway and Levett, 2008), DNA-DNA hybridization (Chang et al., 2008), have been employed in typing Leptospira isolates but limitations such as high level background, the need for large quantity of DNA, low reproducibility and discriminating power have been eminent (Salaun et al., 2006; Leon et al., 2010).
Various PCR based molecular methods which are useful for the timely detection and unambiguous characterization of Leptopsira isolates including MLST (Ahmed et al., 2006) and real-time PCR (Ahmed et al., 2009), have been developed to overcome the shortcomings encountered with the preceding methods. In addition, sequence-based methods allow collection of results into databases which can be compared between laboratories (Fournier et al., 2007).
Real-time PCR (TaqMan) assay was evaluated in this study for detecting the presence of Leptospira spp. in UK rodents. For 16S rRNA assay, 19% (55 of 292) DNA samples tested revealed presence of Leptospira spp. using 16S rRNA gene sequence as a screening test for Leptospira species. These were confirmed using LipL32 gene which revealed 56% (31 of 55) to be pathogenic leptospires. The difference seen between the two assays is that 16S rRNA gene assay screens for Leptospira species while LipL32 gene assay is predictive to be specific for pathogenic spp. as it is not present in intermediate or non-pathogenic species (Thaipadunpanit et al., 2011). The results also showed that the diagnostic sensitivity and specificity of LipL32 assay was higher compared to the 16S rRNA assay 56% (31 of 55) versus 19% (55 of 292). The results also revealed that DNA samples with low cycle threshold (ct) values (12.79-19.68) except one with 27.08 ct (table 4.1) were positive for LipL32 assay and hence pathogenic. MLST was attempted to speciate positive samples giving five amplified products from three out of eight gene targets namely secY, ccmF and czcA gene loci. Phylogenetic analysis of sequences showed close relationship with L. interrogans (figure 4.2A), L. kirschneri (figure 4.2B) and L. borgpetersenii (figure 4.2C).
The housekeeping genes used for MLST in this study were validated by previous studies as robust markers for genotyping of pathogenic Leptospira species including L. interogans and L. kirschneri (Leon et al., 2010). They also noted that one strain, L. kirschneri serogroup Grippotyphosa was classified in the L. interogans group. This is evident in this study since ccmF and czcA showed close relationshp with L. interogans and L. kirschneri respectively. However, ccmF and czcA gene loci were amplified from a single positive sample and should have shown closeness to the same specie. The discrepancy in this result may be due to bias through data availability and insufficient sequences deposited on Genbank. Candidate genes such as LipL32 were excluded from MLST since studies by Haake et al. (2004) revealed that horizontal transfer of outer membrane protein genes is concurrent with Leptospira species.
As suggested by Victoria et al. (2008), the result shows that secY gene located within the S10-spc-Î± locus and encodes preprotein translocase for Leptospira, is a particularly robust housekeeping gene for phylogenetic typing of leptospires as it was amplified from three DNA samples while ccmF and czcA were both amplified from one DNA sample. The non amplification of the other housekeeping genes notably aacA, recF, gcvP, groEL and polA, from any sample may be due to the fact that the positive samples may not be L. interogans or L. kirschneri spp, since the primers used in targeting this genes have been validated for genotyping of pathogenic L. interogans and L. kirschneri spp. (Leon et al., 2010). These genes may be less sensitive compared to secY, ccmF and czcA and also, kidney tissue samples are not free of inhibitors.
The most encountered Leptospira species in human infections is L. interrogans, other pathogenic leptospires mostly implicated in animal infections including L. borgpetersenii and L. kirschneri, are also associated with human infections and the L. interrogans and L. kirschneri species are the most closely related (Salaun et al., 2006; Slack et al., 2010). The L. borgpetersenii has a more reduced coding density and is about 700kb smaller than L. interrogans (Adler and de la Pena Moctezuma, 2009). L. borgpetersenii is thought to be undergoing genetic decay due to impairment in genes required for survival in the environment, resulting in reduced transmission potentials to mainly host-to-host contact (Bulach et al., 2006; Matsunaga et al., 2007).
Though the incidence of leptospirosis in the UK is considered low as there are generally about 50-60 cases annually in England and Wales, that is about one per million of population yearly (Health protection agency, 2009). Recent studies have reported an increasing incidence of cases in both tropical and temperate regions attributed to international travel and recreational activities (Pavli and Maltezou, 2008; Stern et al., 2010). Leptospira infection is perhaps unrecognised in this group or since the incubation period of the disease can take up to 21 days, the link between water exposure and symptoms might not always be apparent and consequently, a delayed diagnosis.
Several cases of Leptospira infection following return from rafting and canoeing trips where leptospirosis is endemic have been reported. In one case, two adventure travellers who almost drowned while rafting in Thailand were both admitted with aseptic meningitis on returning to the UK, and diagnosis showed that they were infected with different serovars of Leptospira (Monahan et al., 2009). There was also the case of an athlete named Andy Holmes who died in 2010 from confirmed case of Weil's disease following exposure to freshwater in Lincolnshire, UK (Kathryn, 2010).
This study confirms the presences of significant levels of leptospires as supported by these confirmed clinical cases of leptospirosis in the UK, and necessitate the need for further studies to identify and characterize the infecting serovars by molecular typing methods since classification by serology depends on constantly altering surface antigenic properties (Haake et al., 2004). The work by Slack et al, (2009) also noted a discrepancy in global culture collections and pointed out the need for reclassification of leptospires. This will enhance timely case detection and therapeutic management, outbreak investigation and use of vaccine in animals which mainly induces humoral immunity that is serovar specific.
If time had permitted and the necessary resources available, culturing of the kidney samples would have been an added advantage of confirming the positive results. As noted by Kawanishi et al. (2011), culture techniques remain indispensible to microbiological research as it allows stable preservation and proliferation of microorganisms. Also, the real-time PCR assays could have been performed at least twice to validate the results and primers which have been validated to amplify various species of Leptospira and not particular species, should have been used.
The lack of associated large burden of human infection may either be related with low pathogenicity for humans or poor detection of cases as a result of the lack of clinical hallmarks of infection. The detection of leptospiral DNA in 19% of rodents indicates a significant reservoir that pose a risk of leptospirosis for humans and other animals. This study will increase the awareness of leptospirosis and contribute to the improvement of prevention and control measures and epidemiological surveillance.
I am most grateful to God who in his infinite mercy gave me the wisdom and grace to bring this project to completion. My appreciation goes to my project supervisor, Dr Sally Cutler for her technical support and expert suggestions throughout this study. Many thanks to the entire Laboratory staff for providing the materials when needed. I also acknowledge Ifeyinwa Maduka for her contribution during the experiments and Emeka Igboanuzue for his help. Finally, I acknowledge my husband Mr Jackson David-Mack for his immense support throughout this study.