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subsequent analysis. Genus identification Isolates were subjected to genus confirmation using the GCAT gene as described by Chacón et al. (2002) . The DNA amplification reaction was carried out in a PCR mixture that contained 1U Dream Taq polymerase
Reaction (RT-PCR) targeting ( uidA and lacY or ipaH and lacY ), (MALDI-TOF MS) using an analysis software (ClinPro Tools Bruker Daltonics) [ 10–12 ] and even the Whole Genome Sequencing (WGS) followed by bioinformatics tools such as k-mers or Single
confirmed using the VITEK-2 Compact system. The selected resistant isolates were then genotypically characterized for inclusion in the study by performing PCR to detect carbapenemase genes ( bla NDM , bla KPC , bla IMP , bla OXA-48 , and bla VIM
). The first aim of this study was to provide laboratory confirmation by polymerase chain reaction (PCR) screening for ORFV and perform molecular characterisation based on the partial B2L gene sequences in a goat flock affected by clinical orf disease
further nucleic acid isolation. This step was performed using the IndiSpin Pathogen Kit based on the product description and the QIAGEN QIAcube nucleic acid detection and isolation automata (Qiagen, Hilden, Germany). For the RT-PCR test, we used the
ability of the isolates to produce indole, urease, and ornithine decarboxylase [ 10 ]. Capsule detection and PCR-serotyping The capsular variants were detected by amplification of the bexB gene [ 11 ]. All encapsulated strains were subjected to a PCR
stored in a trypticase soy broth (TSB) medium containing 10% glycerol at −70 °C. Identification of E. coli ST131 clones and sat and hlyA virulence genes Genomic DNA was extracted by boiling method [ 18 ]. PCR amplification of the pabB gene was
samples were analysed by TaqMan type real-time polymerase chain reaction (PCR) assay targeting the multicopy IS1111 insertion element of the C. burnetii genome ( Loftis et al., 2006 ). The detection threshold of the PCR system was ∼0.1 CFU (Ct 36
as described previously ( Korczak et al., 2014 ). Sequencing of PCR products was performed by Macrogen Europe (Amsterdam, The Netherlands). Nucleotide sequences were aligned and compared using Geneious Prime software (version 2019.2.1; http
Samples from 45 dams (milk/colostrum, faeces, vaginal fluid and blood on days 171–177 of gestation and at parturition, and cotyledons at parturition) and their calves (blood collected before colostrum intake and weekly until days 29–35) were analysed to examine the vertical transmission of Coxiella burnetii and links between shedding and seropositivity. All calves were born C. burnetii seronegative. Only those born to seropositive dams seroconverted following colostrum intake. Logistic regression analyses indicated that the likelihood of dam seropositivity was 21 and 4.85 times higher for multiparous than for primiparous (65.6% vs. 8.3%, P = 0.006) and for prepartum shedding cows (75% vs. 38.2%, P = 0.03) compared to the remaining animals, respectively. In conclusion, the results of this study indicate no detectable precolostral antibody response in calves born from dams with cotyledons positive for C. burnetii by qPCR. In order to analyse the possibility of persistent infection due to immunotolerance to an early in utero infection, further studies will need to test for C. burnetii DNA. In addition, in the present study multiparous cows showed a significantly higher seroprevalence than primiparous cows and heifers, colostral antibodies were efficiently transferred to newborn calves, and there was a link between bacterial shedding on days 171–177 of gestation and Coxiella seropositivity of the dam.
