51 SHORT COMMUNICATION Cat scratch disease (CSD) is a zoonotic disease mainly caused by an aerobic and pleomorphic Gram‑negative bacterium, Bartonella henselae belonging to α sub‑group of Proteobacteria, family Bartonellaceae. Cat acts as the principal reservoir of B. henselae which is transmitted among cats mainly by the flea Ctenocephalides felis (Bouhsira et al. 2013, Fabbi et al. 2004, Greco et al. 2019, Iannino et al. 2018, Pinna Parpaglia et  al. 2007). Bartonella clarridgeiae is another agent of CSD, though rare (Capitta et  al. 2010, Greco et al. 2019). Humans mainly acquire infection through an infected cat scratch or bite and can exhibit an acute febrile lymphadenopathy. Immunosuppressed individuals may show hepatic and/or splenic peliosis and also bacillary angiomatosis (Breitschwerdt 2008, Breitschwerdt et  al. 2010, Capitta et  al. 2010, Fabbi et al. 2004a, Iannino et al. 2018, Pinna Parpaglia et al. 2007, Zobba et al. 2009). Naturally‑infected cats with B.  henselae are usually healthy carrier and can be bacteremic for weeks to months/years, representing an important reservoir for Bartonella (Breitschwerdt 2008, Chomel et  al. 2006, Fabbi et  al. 2004b). Young cats (<  1  year) can develop a stronger bacteremia than older cats, as well as the street cats compared to pet cats (Chomel et al. 2006). Thus, infected cats may represent a risk to human health. Moreover, positive cats are considered the most important vehicle for the spread of the disease (Ebani et al. 2012, Fabbi et al. 2004 a, b, Pinna Parpaglia et al. 2007, Zobba et al. 2009). Bartonella infections in symptomatic cats should be confirmed by culturing the organism from blood or tissues but some molecular diagnostic approaches have been developed during the last years. In particular, real‑time PCR assays are commonly used to detect Bartonella spp. Since Bartonella genus shows high genetic variability, a universal method is difficult to develop, so different conventional PCR and real‑time PCR methods, targeting different portions of the genome, have been proposed. For example, gene gltA is a common genetic target for Bartonella detection and is considered a reliable tool for distinguishing genotypes (La Scola et  al. 2003, Tapp et  al. 2003). However, this test has showed high cross‑reactivity with other bacteria, such as Ehrlichia spp. (Colborn et al. 2010). The immunofluorescence antibodiy test (IFAT) is the most used serological diagnostic tool for detecting Bartonella exposure. This study aimed at detecting Bartonella infection in cats from Abruzzo region. During 2014, a total of fifty‑two samples of whole blood and sera were Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale', Teramo, Italy *Corresponding author at: Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale', Campo Boario, 64100 Teramo, Italy. Tel.: +39 0861 332471, e‑mail: a.angioni@izs.it. Keywords Abruzzo, Bartonella henselae, Bartonella clarridgeiae, Cat, IFAT, Real‑time PCR. Summary Cat scratch disease (CSD) is a zoonotic disease, caused predominantly by Bartonella henselae and transmitted to humans through a scratch or bite of the cat. Cat represents the principal reservoir and healthy carrier of Bartonella, which is mainly transmitted, among cats, by the flea Ctenocephalides felis. During 2014, fifty‑two samples of whole blood and sera were collected randomly from cats in Abruzzo region and were examined by real‑time PCR and IFAT tests, respectively. Seven samples out of fifty‑two (13.5%) resulted positive for Bartonella  spp. in both tests, while six specimens (11.5%) resulted real‑time PCR negative but IgG positive; thirty‑nine were instead both real‑time PCR and IFAT negative (75%). Sequence analysis of a fragment of DNA identified B. henselae and B.  clarridgeiae in four and in two real‑time PCR positive samples, respectively. Salvatora Angela Angioni*, Luigina Di Gialleonardo, Marco Di Domenico, Daniele Giansante, Manuela Tittarelli and Cesare Cammà Survey of Bartonella species in cats from Abruzzo region, Italy Veterinaria Italiana 2020, 56 (1), 51‑54. doi: 10.12834/VetIt.1884.10006.2 Accepted: 11.12.2019 | Available on line: 24.04.2020 52 Veterinaria Italiana 2020, 56 (1), 51‑54. doi: 10.12834/VetIt.1884.10006.2 Detection of Bartonella spp. in cats from Abruzzo Angioni et al. the amount of DNA that could be detected by Probit Analysis with 95% of sensitivity. Amplification efficiency was calculated from slope of the standard curve using the following formula: E = (10‑1/slope ‑ 1) x 100 (Vaerman et al. 2004). Specificity was assessed by testing nucleic acid from closely genetically related bacterial species, or different species that may infect cats including Anaplasma phagocytophilum, Borrelia afzelii, Rickettsia helvetica, Rickettsia monacentis, Rickettsia slovaca and Coxiella burnetii. The intra‑assay variance (repeatability or short‑term precision) was determined by testing three replicates of the 10‑fold serial DNA dilutions (equal to 3.074 to 3.072 copy of DNA/reaction) in the same run. Similarly, the inter‑assay variance (reproducibility or long‑term precision) was determined by running triplicates of the DNA dilutions (3.072 to 3.071 copy of DNA/reaction) in three different runs and on separate days. These replicates were used to determine the mean and standard deviation of CT values and the coefficient of variation. The reaction mix (20 µl) contained 2 µl of extracted nucleic acid, 10 µl Go Taq Probe qPCR MMix 2x (Promega), primers and probe at a final concentration of 800 nM and 250 nM, respectively (Table  I). Real‑time PCR was performed on Quantstudio 7 Flex Systems Istrument (Life Technologies) with the thermal condition showed in Table I. In order to confirm and characterize the results of real‑time PCR a partial region of the 23S gene was sequenced (Parra et al. 2016). Obtained sequences were aligned using Clustal V (DNAStar, Madison, WI). Each sequence was compared to other homologous regions present in Genbank by Blast search tool (https://blast.ncbi.nlm. nih.gov/Blast.cgi). Seven blood samples out of fifty‑two (13.5%) tested positive for Bartonella spp. IFAT revealed IgG for Bartonella spp. in all real‑time PCR positive cats, while IgM were identified in two cats. Six specimens were real‑time PCR negative, IgM negative and IgG positive (11.5%) with values ranging from 1:40 to 1:160, whereas thirty‑nine collected randomly from cats in Abruzzo region and were examined at IZSAM by IFAT and real‑time PCR tests. B. henselae Houston1 (ATCC 49882) was cultivated in Brucella broth (BBL Microbiology System Cockeysville, MD, USA) supplemented with haemin (250 µg/ml) and 8% Fildes solution, and incubated at 37 °C in 5% CO 2 . Log ‑phase cultures were centrifuged (4,500 g, 30 min), washed three times in PBS, pH  7.2 and inoculated into a 25‑cm2 flask (Corning, NY, USA) containing L929 cells (ECACC 85011425) and Dulbecco minimal essential medium (Gibco, Introvigen, Grand Island, NY, USA) supplemented with 2 mM L‑glutamine (200  mM) and 10% (v/v) fetal bovine serum (Gibco) and maintained at 37 °C with 5% CO 2 . L929 cells infected with B. henselae were harvested by centrifugation at 4,500 g for 30 min and washed twice with PBS. The final pellets were resuspended in PBS and 10 µl were added onto each well of 18‑well slides (GSG Robotix Italy). Slides were air‑dried for at least 1 h, fixed with cold acetone for 10  min and used immediately or stored at ‑ 20 °C until use (Capitta et al. 2010, Zobba et al. 2009). Bartonella spp. IgM and IgG antibodies were determined by IFAT: 10 µl of serum diluited (1:40 to 1:640) in PBS was placed on the B.  henselae L929 slide and incubated at 37 °C in a humidified chamber for 30  min. After two washes in PBS (10 min) and one rinse with distilled water the slides were incubated for 30 min at 37 °C with fluorescein isothiocyanate‑labelled goat anti cat IgM (Bethyl Laboratories Inc‑ Montgomery, Texas) and IgG immunoglobuline (Sigma‑Aldrich, St. Louis, MO, USA), diluted in PBS 1% Evans Blue (Sigma‑Aldrich, St. Louis, MO, USA). The slides washed and dried as described above, were examined with a fluorescence microscope. The titre of <  1:40 was considered negative, a titre of ≥  1:40 was considered positive for both classes of antibodies (Capitta et  al. 2010, Zobba et al. 2009). A quantitative PCR method was developed to detect Bartonella spp. DNA in blood cat samples. The highly conserved region of nuoG gene (André et  al. 2016, Colborn et  al. 2010, Diaz et  al. 2012) was selected to design primers and probe by Primer Express software (ThermoFisher Scientific). Total genomic DNA was extracted from 300  µl of whole blood samples or from B.  henselae strain by Maxwell® 16 Instrument using Maxwell R 16 Blood DNA Purification kit (Promega), according to the manufacturer’s instructions. The limit of detection (LOD) of real‑time PCR assay was determined using 10‑fold serial dilutions of DNA extracted from B.  henselae (ATCC‑49882D‑5). Standard curve was generated and used to calculate Table I. Primers and probe sequences and thermal condition. nuoG_60_F 5’-GCGGCATAATTCGCATAACC-3’ nuoG_60_R 5’-CACTTGGCAGTGCTATCCGTATT-3’ nuoG_P 5’-FAM-ACGACCCCGGCTAT-3’- MGB Temperature Time 95°C 2 min 1 cycle 95°C 15 sec 45 cycles 60°C 1 min 53 Angioni et al. Detection of Bartonella spp. in cats from Abruzzo Veterinaria Italiana 2020, 56 (1), 51‑54. doi: 10.12834/VetIt.1884.10006.2 two different Bartonella species: four samples were identified as B. henselae and two as B. clarridgeiae. One positive sample was not identified as probably due to the lower concetration of DNA. This survey demonstrated correlation between serological and virological results. Reasonably, serological positive individuals without evidence of bacterial DNA in the bloodstream might be related with the presence of past infections. However non bacteremic cats may still represent a risk for humans given that reinfections might occurr (Fabbi et al. 2004a). It is generally accepted that the IFAT test should be performed standalone only when the prevalence of bacteric cats is high (40‑60%). However, when the transmission to humans (for example immune‑depressed individuals) is a risk, virological and serological tests should be performed in order to get a complete health status of a cat (Fabbi et al. 2004b). In this perspective, the novel molecular method which has been developed in this study, is useful to detect Bartonella infected cats and thus for prevalence studies in feline populations. Acknowledgments The authors would like to thank Alessandra Leone and Liana Teodori for providing the cell line used to prepare the IFAT antigen. samples resulted negative by both assays (75%). Results are summarized in Table II. The amplification efficiency was 100.4% (slope = ‑ 3.312; R2 = 0.997). The LOD, determined as the lowest dilution of DNA that could be detected with 95% sensitivity, was equivalent to 0.32 genome copies. The coefficient of variation, determined for intra‑ and inter‑assay repeatability, showed mean values of 0.33% and 2.16%, respectively. No amplification signal was obtained from a panel of different bacterial species tested to evaluate the specificity of the molecular assay. The sequence analysis showed the presence of Table II. Serological and real-time PCR results. No. of Cats IgG IgM Real-time PCR 1 1/160 Neg POS 2 1/320 Neg POS 3 1/160 Neg POS 4 1/320 Neg POS 5 1/80 1/40 POS 6 1/40 1/40 POS 7 1/160 Neg POS Total positive 7/7 2/7 7/7 POS = positive; Neg = negative; No = number. 54 Veterinaria Italiana 2020, 56 (1), 51‑54. doi: 10.12834/VetIt.1884.10006.2 Detection of Bartonella spp. in cats from Abruzzo Angioni et al. André M.R., Dumler J.S., Herrera H.M., Gonçalves L.R., de Sousa K.C., Scorpio D.G., de Santis A.C., Domingos I.H., de Macedo G.C. & Machado R.Z. 2016. 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