These patterns were interpreted as it can be markers of the phase of FeMV infection [2,29]

These patterns were interpreted as it can be markers of the phase of FeMV infection [2,29]. One carcass tested positive by qPCRFeMV from kidney, urinary bladder, and submandibular lymph nodes. Antibodies against FeMV were detected in 14.5% (28/193) cats. We followed up 27 cats (13 FeMV positive cats) and documented in some cases urine shedding after up to 360 days. Older and foundling cats and cats living in rescue catteries, were more frequently infected with FeMV. A significant correlation between FeMV and higher serum creatinine values or low urine specific gravity was found. FeMV positivity was significantly associated Namitecan with retroviral contamination, and the presence of some clinical indicators apart from CKD clinicopathological markers. Our study highlights the possibility of a link between FeMV exposure and CKD and a general impairment of feline health. genus, family = 31) when recent or current IV fluid treatment at the time of sampling was reported, urinalysis, sCr and/or SDMA evaluations were missing, or pyuria, bacteriuria, and hematuria Namitecan were detected by urinalysis. 2.5. Molecular Detection of FeMV RNA and Other Relevant Feline Pathogens in Cats Nucleic acids were purified from EDTA-blood samples using the High Pure Viral Nucleic Acid Kit (Roche Life Science, Roche Diagnostics, Monza, Italy) and from urine and Namitecan tissue using the QiAamp Viral RNA Mini kit (Qiagen, Hilden, Germany). All samples were tested by a quantitative RT-PCR targeting a 76-bp region of the P/V/C gene for detection of FeMV RNA (qPCRFeMV) previously developed by the study group at IZSAM (Istituto Zooprofilattico Sperimentale dellAbruzzo e del Molise) [37]. Namitecan Primer sequences were 5- GGGATCCAGAGGGTAACCT -3 (FeMVrt-F), and 5-CCGGCCATTAATCTCTGAA -3 (FeMVrt-R), while the FeMVrt TaqMan probe sequence was FAM-5-TATTCGAAAGCGATGATGATGAAAACCATTA-3-TAMRA. The reactions was optimized using the Quantitect Probe RT-PCR Kit (Qiagen, Hilden, Germany) as follows: the 25 L reaction volume Rabbit polyclonal to IQCA1 contained 5 L of purified RNA, 12.5 L of 2 QuantiTect Probe RT-PCR Grasp Mix, 0.25 L of QuantiTect RT Mix, 1.5 L of each primers (final con-centration 600 nM), 0.625 L of probe (final concentration of 250 nM), and nuclease-free water up to final volume. The assay was carried out on a 7900HT Fast Real Time System cycler (Applied Biosystem, Waltham, MA, USA) using the following thermal profile: 1 cycle of reverse tran-scription at 50 C for 30 min, 1 cycle of PCR initial activation step at 95 C for 15 min followed by 45 cycles of 94 C for 30 s and 55 C for 1 Namitecan min. EDTA-blood samples and tissues of carcasses were tested for feline coronavirus (FCoV) (Dual IPC-TaqVetTM, LSI, Lissieu, France), feline immunodeficiency computer virus (FIV) (gag protein gene-genesig? Advanced Kit, Rownhams, UK) and feline leukemia computer virus (FeLV) (U3 region LTR- genesig? Advanced Kit, Rownhams, UK). DNAs were tested for feline parvovirus (FPV) as previously explained [38]. 2.6. Indirect Immunofluorescence Assay for Anti-FeMV Antibodies and Enzyme-Linked Immunosorbent Assay for Anti-FIV Antibodies Detections The presence of anti-FeMV antibodies (Abs) in serum samples was evaluated by indirect immunofluorescence (IIF) following the procedure recently explained by the study group at IZSAM [26]. In addition, serum samples were tested in enzyme-linked immunosorbent assay (ELISA) for the presence of anti-FIV antibodies (Abdominal muscles, Pet Chek FIV Antibody test kit; IDEXX Laboratory, Westbrook, ME, USA). 2.7. Histopathological Examination Histopathological examination was performed by the Histology section of IZSAM. Hematoxylin and eosin staining (HE) and immunohistochemistry (IHC) were performed on formalin fixed paraffin embedded kidney tissue of qPCRFeMV positive carcasses following the procedure recently explained by the study group at IZSAM [26]. 2.8. Sequencing and Phylogenetic Characterization All positive samples by qPCRFeMV were also tested by a RT-PCR targeting a 401-bp portion of the L protein encoding gene sequence of FeMV with primers FeMV fwd 5- AAGTATCCTTCAAACACCGAGT -3, and FeMV rev 5- TTGAGTAACTCCAAGATGAGGG- 3, designed by the study group at IZSAM by multiple alignments of the FeMV L encoding gene sequences available online [26]. The reaction was optimized using the QIAGEN OneStep RT-PCR kit (Qiagen, Hilden, Germany).