Study areas, rodents and sampling
Blood samples were collected from September 2019 to February 2021 from 102 rodents in different cities of the province of Valencia (39°99 28′12.864″N, 0°22′36.48″W), an area on the east coast of the Iberian Peninsula with a high incidence of Leishmaniasis in dogs (50-100/1000 dogs/year) [20, 21]. Based on epidemiological studies, 17.1% of dogs are seropositive in the Valencian community in Spain [21, 22]. However, the prevalence of human cutaneous leishmaniasis has been detected in the Valencia region 
Prior to sampling, information about each animal was obtained regarding age, cohabitation with a dog, lifestyle (indoor, outdoor, or mixed) and sex, and a complete physical examination was performed to determine health status (patient vs. healthy). Whenever possible, 1 ml of blood was collected aseptically by cranial venipuncture from each ferret. The collected volume was evenly divided between a sterile blood collection tube (for serum) and a second tube containing the anticoagulant ethylenediaminetetraacetic acid (EDTA) (for molecular analysis). EDTA-separated blood and serum were stored at −20 °C until processing. Routine laboratory tests, such as complete blood count and biochemistry profile, were not performed.
In total, 102 customer-owned rodents were sampled. One sample was obtained from 94 ferrets. The other eight customer-owned rodents were tested on an additional one (n = 5 to two (n = 3) Different times during the study period. A total of 113 serum samples were included.
Diagnostic serological tests
Detection of specific antibodies was performed using two in-house serological methods: enzyme-linked immunosorbent assay (ELISA) and western blot (WB).
revealing of L. infantum Antibodies by quantitative ELISA
ELISA was performed on all sera as previously described, with some modifications . Briefly, each plate was coated with 20 μg/ml of crude antigen obtained from L. infantum Promastigote (MHOM/MON-1/LEM 75) forms in 0.1 M carbonate/bicarbonate solution (pH 9.6) and incubated overnight at 4 °C. Then, 100 μl of cat serum diluted 1:200 in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST) and 1% skimmed dry milk (PBST-M), was then added to each well. The plates were incubated for 1 hour at 37 °C in a humid chamber. They were then washed, and 100 μl of horseradish peroxidase-conjugated protein A (Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:8000 in PBST-M was added. The plates were incubated for 1 hour at 37 °C in the humid chamber and washed again with PBST and PBS as described above. Substrate solution (ortho-phenylenediamine) and fixed peroxide substrate buffer (Thermo Fisher Scientific, Waltham, MA, USA) were added to each well and developed for 20 ± 5 min at room temperature in the dark. The reaction was stopped by adding 2.5 MH .2So4 for each well. Absorbance values at 492 nm were read in an automatic microELISA reader (Multiskan ELISA Reader, Labsystems, Midland, Canada).
As a positive (titrated) control, each panel included serum from a ferret from Spain diagnosed with leishmaniasis, confirmed by a positive farm, and as a negative control, serum from an uninfected healthy ferret. The same positive control serum was used for all assays and panels, with consistent inter-assay variability< 10%. Plates with an inter-assay variation of >10% was eliminated. All samples and controls were run in duplicate. The cut-off was set at 0.180 optical density (OD) (mean + 3 standard deviations of values from 30 inland ferrets from northern Spain), and results above this value were considered positive.
revealing of L. infantum Antibodies by WB
opposite-leishmaniasis Antibodies were detected by WB using a complete . antigen L. infantum promastigotes (MHOM/FR/78/LEM75 zymodeme MON-1), as described by Alcover et al. , with some modifications. The protocol used at the World Bank is based on the technique described by Issa et al. With a sensitivity of 95.8% and a specificity of 100% in dogs. Furthermore, specificity was analyzed including a group of healthy cats (n = 20) from a non-endemic area (Switzerland) with a value of 100%. Antigen electrophoresis was performed on 0.1% sodium dodecyl sulfate (SDS)-15% polyacrylamide with molecular mass protein standards (low band standard; Bio-Rad, Hercules, CA, USA) on a Mini-Gel AE-6400 Dual Mini. Tile kit (Ato, Bunkyo-ku, Japan).
Gels were run at 100 V for 1 hour at room temperature. Polypeptides were transferred onto nitrocellulose plates (0.45 mm pore size, HAWP 304 FO; Millipore, Bedford, MA, USA), which were blocked with 20 mM Tris, 0.13 mM NaCl, pH 7.6 (TS), and 5% skimmed milk overnight at 4°C.
Leaves were washed in TS and inserted into a multiscreen device (Mini-PROTEAN II; Bio-Rad, Hercules, CA, USA). Sera were diluted 1:200 in TS-1% skimmed milk and 0.2% Tween 20. Then 500 μl of each sample was introduced into each channel of the multiscreen device and incubated for 2 h at 37 °C. Bound immunoglobulins were developed by incubation with a 1:1000 dilution of protein conjugate A peroxidase (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h. After the leaves were washed three times with TST and one last time with TS, color was developed using 4-chloro-1-naphthol substrate (Thermo Fisher Scientific, Waltham, MA, USA). Based on our experience and the literature, a serum sample was considered positive when immune activity against L. infantum The 14 and/or 16 kDa antigen fraction was observed, and was not determined when molecular weight bands of 18, 20, 24, 28, 30, 36, 38 and 46 kDa appeared, as previously reported. .
revealing of L. infantum DNA by qPCR
DNA was extracted from 200 μl of mammalian blood using a high-purity PCR template preparation kit (Roche Applied Science, Mannheim, Germany), which allows genomic DNA to be quickly and easily isolated from a variety of material samples. All extractions were performed following the manufacturer’s instructions, and multiplex PCR templates were obtained in minutes using high-purity spin columns.
detection and appreciation leishmaniasis kDNA was performed by amplifying the kinetoplast minicircle DNA sequence by qPCR . Each amplification was performed in triplicate in 10 μl reaction mixture containing 1× iTaq Supermix with ROX (Bio-Rad, Hercules, CA, USA), 15 pmol of Leim1 direct primer (5′-CTT TTC TGG TCC). TCC GGG TAG G- 3′), 15 pmol of the reverse primer Leim2 (5′-CCA CCC GGC CCT ATT TTA CAC CAA-3), 50 pmol of the Leim3-labeled TaqMan probe (5′- FAM-TTT TCG CAG AAC GCC CCT) ACC CGC TAMRA-3′), and 2.5 μl of the DNA sample.
An ABI Prism 7900 HT thermocycler (Applied Biosystems, Waltham, MA, USA) was used at 94 °C and 55 °C over 40 cycles using a FAM reagent. A non-template control in each run was used as a qPCR negative control. 10-fold dilution sequencing of DNA from prostegotes (MHOM/ES/04/BCN-61, L. infantum)It was used for calibration (serial dilution of 105 Parasites / ml up to 10−3 parasites/ml), allowing a standard curve to be plotted. qPCR was considered positive when the threshold cycle (Ct) was less than 40 and when amplification was detected in all replicates. [3, 24, 25].
The data collected for the entire population was analyzed using descriptive statistics. associations between L. infantumThe registered variables were analyzed. The significance of this difference was assessed using the Chi-square or Fisher exact test. Values s0.05 was considered significant. The gramogram SPSS v.22 (IBM Corporation, Armonk, NY, USA) was used.
#Epidemiological #Survey #Infantile #Leishmaniasis #Domestic #Ferret #Mustella #Botorius #Foro #Area #Endemic #Leishmaniasis #Dogs #Serology #PCR #Parasites #vectors