Study area
The study was conducted in Trishal upazila (subdistrict) of Mymensingh district in Bangladesh. Trishal has a land area of 339 sq km, with a population of 3.7 million. The annual incidence of kala-azar in Trishal ranges from 21 to 26 per 10,000 people per year [16].
Sample-size
Results of a previous study with domestic and wild animals in Sudan showed that 21.4% had seropositivity against anti-L. donovani antibodies in cows [14]. However, in the absence of a similar study in the Indian subcontinent, we assumed that cattle might show 10% of seropositivity in our study. Based on this assumption, we calculated that 138 cattle would be required for our study [precession 5% and 95% confidence interval (CI)]. Sample-size was calculated using Windows® version of the Epi Info 3.2.2 software.
Sample collection from cattle
Blood samples were collected from cattle during August-September 2008. At the beginning, past (within last 3 months) and active (treatment ongoing or awaiting for treatment) VL and post-kala-azar dermal leishmaniasis (PKDL) patients were identified from the records of Trishal Upazila Health Complex, Mymensingh. A research team, consisting of an experienced veterinary doctor or a veterinary assistant, visited the study houses, enumerated domestic cattle (Bos indicus), and collected five mL of blood from the jugular vein from randomly-selected one from each study house. After collection, three mL of blood was transferred to an EDTA containing tube for Buffy coat separation, and the remaining two mL was transferred to a sterile test tube for serum separation. Relevant information on age, sex, body condition score, etc. and any changes in behaviour within the past six months of the selected cattle were recorded. Their physical condition was scored in the scale of 1-5 (with 0.5 fractions between 2 scores) which represent worst to best physical condition on the basis of bony prominence and deposition of subcutaneous fat [17]. The blood samples were transported to the nearby field office for Buffy coat and serum separation. The processed samples were then preserved at -20°C before transferring these to the Parasitology Laboratory of ICDDR, B in Dhaka for further serological and molecular investigations.
Ethical consideration
The study was approved by the Research Review Committee and the Animal Experimentation Ethics Committee of ICDDR, B. Consent was obtained from the owners of the cattle before the collection of blood sample.
Laboratory investigation
Enzyme-linked immunosorbent assay
For the qualitative detection of antibodies (IgG, IgM, and IgA) against L. donovani in the serum samples, the RecombiLISA Leishmania Ab Test (CTK Biotech Inc., San Siego, USA) was performed. The RecombiLISA Leishmania Ab Test comprises two key components: (a) solid microwells pre-coated with recombinant L. donovani antigens and (b) liquid conjugates composed of recombinant L. donovani antigens conjugated with horse reddish peroxidase (HRP-Leish Ag conjugates). The test was performed by incubating serum and HRP-Leish Ag conjugate in wells of the plate. Antibodies (IgG, IgM, or IgA) to L. donovani in the specimens would react to the L. donovani antigens coated onto the plate well by making complex with the HRP-Leish Ag conjugates. Unbounded conjugates were then removed by washing. After adding substrate and stop solution, the change in color to yellow indicates Ab-positive, and then the absorbance was measured by spectrophotometer at 450 nm absorbance. Duplicate wells were run for each sample.
Interpretation of ELISA results
The ELISA results were interpreted according to the instructions of the manufacturer. Briefly, a cut-off value was obtained from the mean optical density (OD) value of the negative controls + 0.1. Then an OD ratio was calculated for each sample by dividing the mean OD value by the cut-off value. Sample with OD ratio of ≥ 1.00 was considered to be positive, and the OD ratio of < 1.00 was considered to be negative. Serum samples collected from four cattle from a non-endemic area were used as negative controls in each test. Repeated tests were performed with serum samples with a positive result along with an equal number of randomly-selected negative serum samples and negative controls to make a precise conclusion. However, no discrepancy was found in the repeat tests.
Direct agglutination test
The DAT was performed with the serum samples which were found to be positive in the ELISA test, along with a similar number of ELISA-negative samples. Commercially-available DAT antigen (Koninklijk Instituut voor de Tropen, Amsterdam, the Netherlands) was used following the protocol described by Harith et al. [18]. Duplicate wells were run for each of the serum dilution. Heat-inactive fetal bovine serum was used along with physiologic saline and DAT antigen as negative control [19]. An agglutination titer of 1:3200 was considered positive in this study [14].
PCR
DNA from Buffy coat was extracted using QIAamp® DNA blood mini kit (QIAGEN, Düsseldorf, Germany). Nested PCR (Ln PCR) was performed to amplify the ssu-rRNA gene with some modifications of the protocol previously described by Cruz et al. [20]. The first PCR was specific for the order Kinetoplastida using the primer R221 and R332 with a product size of 603 base pair. The second PCR was specific for Leishmania genus using the diluted (1:50) first PCR product using the primers R223 and R333, with the product size of 358 base pair. Briefly, in the first reaction, 2 μL of the extracted DNA, 15 pmol of primers (Kinetoplastida-specific) R221 and R332 were used in 25 μL PCR mix containing 0.2 mM dNTP, 2 mM MgCl2, 5 mM KCl, 75 mM Tris-HCl (PH 9.0), 0.001% bovine serum albumin, 2.0 mM (NH4)2SO4 and 2.5 U of Taq polymerase. For the second reaction, 1 μL of a 1/50 dilution of the first PCR product was used as a template in the presence of 15 pmol of primer R223 and 15 pmol of primer R333 which are Leishmania genus-specific. Reaction conditions are the same as the first PCR. Amplification products were visualized on 1.5% agarose gel using a 100 bp DNA ladder after staining with ethidium bromide (0.1 mg/mL). DNA from in-house-cultured L. donovani was used as a positive control in the PCR assay.
Loop-mediated isothermal amplification
LAMP was performed followed the protocol described by Takagi et al. [21]. In brief, the LAMP reaction was performed in 25 μL of reaction mixture containing 40 pmol each of FIP and BIP primers, 5 pmol each of F3 and B3c primers, 1.4 mM each of deoxynucleoside triphosphate, 0.8 M betaine, 20 mM Tris-HCl, pH 8.8, 10 mM KCl, 10 mM (NH 4)2 SO4, 8 mM MgSO4, 0.1% TritonX-100, 8 units of Bst DNA polymerase large fragment (New England Biolabs, Ipswich, MA), and 2 μL of sample DNA. The mixture was incubated at 65°C for 50 minutes in a heat block. After incubation, the turbidity was examined visually.
Data analysis
Data were computed in the Microsoft Excel software. Frequencies and the association between different variables (chi-square test with Yate's correction and Fisher's exact test) were analyzed using the SPSS software (version 11.5). A p value of < 0.05 was considered significant.
