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Anti-Leishmania antibodies were determined by IFAT (immunofluorescence antibody test; cut-off value at a titre ≥64) in the 16 sera samples that were previously screened [15 (link)]. Briefly, an L. infantum MON-1 (MCAN/PT/05/IMT-373) suspension of 10⁷ promastigotes was used as antigen, and the anti-cat IgG (whole molecule)-FITC was used in a dilution of 1:20. A serum sample from a seropositive cat (IFAT titre 1204) was used as positive control, while the serum sample of a cat from a non-endemic country of leishmaniosis was used as negative control [17 (link)]. The detection of Leishmania DNA in the buffy coat was done using a nested PCR protocol with genus-specific primers targeting the SSU-rDNA gene [18 (link)]. A positive control containing L. infantum MON-1 (MHOM/PT/88/IMT-318) DNA and a negative control without DNA template were included in each amplification.
DNA amplicons were resolved by electrophoresis on 1.5% agarose gels stained with Green Safe Premium (Nzytech, Portugal), using a 100-bp DNA ladder as a molecular weight marker, and then visualized under UV illumination. Cats were considered infected by Leishmania if they tested positive for at least one of the techniques.

Identification of blood sources was conducted by the amplification of a 350 bp segment of the host mitochondrial cytB, using the modified vertebrate-universal specific primers (cytB1-F and cytB-2-R) on blood fed sand fly specimens [31 (link)]. The cytB PCR was carried out with 5 μL of extracted DNA in a final volume of 25 μL, using 12.5 μL of NZYTaq 2× Green Master Mix (Nyztech, Portugal) and 1.5 μL of each primer (10 pmol/μL). Amplification was performed as follows: one cycle at 94°C for 5 min, followed by 40 cycles consisting of denaturation at 94°C for 1 min, annealing at 55°C for 1 min and elongation at 72°C for 1 min, followed by final elongation at 72°C for 7 min [4 (link)] (S1 Table). Electrophoresis of PCR products was carried out in 1.5% agarose gel stained with 2.5 μL Greensafe premium^® (Nzytech, Portugal), using a 100 bp DNA ladder as a molecular weight marker and final amplicons were visualized under UV light.

The PCR technique was used to confirm the expression of the previously selected genes in spermatozoa RNA. As a positive control, a sample in which the presence of each gene was previously confirmed (human testis, human nasal ciliated cells (obtained by nasal brushing) or white blood cells (obtained from peripheral blood)) was included. The reaction mixture (20 μL) contained: 4 μL of FIREPol PCR Master Mix (Cat #. 04-12-00125, Solis BioDyne, Tartu, Estonia), 13 μL of DEPC treated water; 1 μL of each primer (Eurofins Genomics, Ebersberg, Germany) at 10 pmol/μL each, and 1 μL of cDNA at 40 pmol/μL. PCR conditions were optimized for each primer pair and was performed in a PCR T100 thermal cycler (BioRad, Hercules, CA, USA). PCR products were analyzed by 1.5% agarose gel electrophoresis; a mix of TAE 1x SeaKem LE Agarose (Lonza, Basel, Switzerland), and 5 μL/100 mL of GreenSafe Premium (MB13201, NZYTech).

A PCR Multiplex Campylobacter was performed as described by Yamazaki-Matsune and colleagues (2007) for Campylobacter genus confirmation and species identification, targeting C. jejuni, C. upsaliensis, C. coli, C. fetus, C. lari and C. hyointestinalis [16 (link)]. PCR products were analyzed by gel electrophoresis through a 1.5% (weight/volume) agarose (Agarose Ultrapure grade, NZYtech, Lisbon, Portugal) in 1× TBE buffer, stained with 1% GreenSafe (GreenSafe Premium, NZYTech).
All the isolates confirmed as Campylobacter spp. by the Multiplex PCR, but whose species could not be identified by this method, were submitted to identification by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (Vitek^® MS, Oxoid).

Plasmids were isolated from E. coli with either PureLink™ HiPure Plasmid Midiprep Kit (Invitrogen) or PureLink™ HiPure Plasmid Miniprep Kit (Invitrogen). PCR products and DNA fragments were purified with GFX™ PCR DNA and Gel Band Purification Kit (Cytiva).
The concentration and purity of nucleic acid solutions were estimated with NanoDrop ND‐1000 Spectrophotometer (Thermo Scientific), and their integrity was assessed by agarose gel electrophoresis.
To visualize agarose gel electrophoresed DNA molecules, gels containing GreenSafe premium (NZYTech) nucleic acid stain were imaged with ChemiDoc XRS+ Gel Imaging System (BIORAD). The 1 Kb Plus DNA Ladder (Invitrogen) was included in the agarose gels as a DNA weight marker.

DNA isolation and purification from the samples in TripleXtractor reagent (Grisp^®) was performed using the GRS Genomic DNA Kit-Broad Range (Grisp^®). DNA concentration and purity were assessed using the NanoDrop™ Lite spectrophotometer (Thermo Scientific, Waltham, MA, USA). The presence of the HPV16 early region was confirmed by the amplification of the HPV16 E7 gene, using the mouse β-globin gene as a control, through polymerase chain reaction (PCR) with specific primers for both genes (Table S1). The PCR reaction was performed in the GeneAmp^® PCR System 9700 thermal cycler (Applied Biosystems^®, Foster City, CA, USA), in a total volume of 25 μL with 1x PCR Buffer with dye, 0.4 mM dNTPs, 1.5 mM MgCl2, 0.3 μM concentrations of each primer, 0.5 U of Xpert Taq DNA Polymerase (Grisp^®) and 0.2 μg of genomic DNA. For both genes, the amplification conditions were the following: DNA denaturation at 95 °C for 5 min, followed by 35 cycles at 95 °C for 20 s, 55 °C for 30 s and 72 °C for 1 min, and a final extension at 72 °C for 5 min. For each PCR, the amplified fragments were analyzed by electrophoresis in 1.5% (w/v) agarose gels stained with GreenSafe Premium (NZYTech, Lisbon, Portugal) and visualized in GelDocXR (Bio-Rad Laboratories, Hercules, CA, USA).

Identification of the isolates as E. amylovora was carried out by PCR following the standard diagnostic protocols for E. amylovora as recommended by EPPO [59 (link)], using three pairs of chromosomal specific [60 (link)–62 (link)], and one pair of pEA29 specific primers [63 (link)] (Table 2). PCR reactions were carried out using a reaction mixture containing 1x DreamTaq Buffer with 2.0 mM MgCl₂ (Thermo Fisher Scientific, Waltham, Massachusetts, USA), 0.2 mM of dNTPs (GRiSP, Porto, Portugal), 0.2 μM of each primer, 1U of DreamTaq DNA Polymerase (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and 25 ng of DNA template. PCR cycling conditions for the chromosomal specific primers (G1-F+G2-R, FER1-F+FER1-R, and FER1-F+rgER2R) and for the plasmid specific primers (PEANT1+PEANT2) were the same as detailed by the EPPO standard diagnostic protocol [59 (link)]. PCR products were separated by electrophoresis in a 0.8% agarose gel stained with GreenSafe Premium (NZYTech, Lisbon, Portugal), with constant voltage (90V) in 1x Tris-EDTA (TE) buffer. The agarose gel was observed with a GelDoc™ (Bio-Rad Laboratories, California, USA).