Viviana Floridia

Coat colour is one of the most recognizable and traditionally selected traits in dog breeds. However, despite its apparent simplicity, it represents a biologically complex phenotype, shaped by the interaction of multiple genetic loci, modifier genes, and variable phenotypic expression, challenging standardized phenotypic classification. This study presents the first characterization of coat colour and texture in the Sicilian Mastiff, integrating pedigree records, photographic assessment, and genetic analyses. Breeder-declared pedigree coat colour was examined for 760 dogs to evaluate terminology and its consistency, while photographs from 197 individuals enabled standardized phenotypic classification. Fifty dogs, selected within a monitored subset, were genotyped for 40 coat-related variants using the MyDogDNA™ commercial test, with additional analysis of KRT71 curly-related variants in nine animals. Pedigree records included 115 terms for only 23 colour categories, revealing substantial inconsistency in terminology. Photographic evaluation identified considerably greater phenotypic diversity than registry data, showing that complex traits like brindling, ticking, melanistic masks, and white markings were frequently omitted or simplified; for example, only 8/64 Brindle black and tan dogs were identified as such in the registry. Genetic analyses confirmed segregation of alleles influencing eumelanin/pheomelanin distribution, dilution, white spotting, and hair texture—including both KRT71 variants contributing to the characteristic curly coat of the breed—, while no known variants associated with merle, harlequin, cocoa, or albinism were detected. Our findings could provide valuable support to the breed association in requesting that the Italian Kennel Club (ENCI) revise the breed standard terminology and coat colour recording process, including allowing updates during growth, to improve clarity and consistency. Finally, requiring photographic documentation for registration applications could improve the reliability of coat colour assessment.

In post-mortem genetic identification, teeth represent one of the most reliable sources of nuclear DNA when other biological samples are unavailable, owing to their ability to protect genetic material from autolysis, microbial degradation, and environmental insults. The present study describes a forensic veterinary case report involving a Staffordshire Bull Terrier litter in which the appearance of blue-coated offspring from a phenotypically black sire and a blue dam prompted parentage verification. The sire died before formal ante-mortem sampling could be carried out. In accordance with Regulation (EC) No. 1069/2009, Article 15, the carcass was interred in the owner’s garden. Before burial, two teeth were collected as the sole biological material available for identification by the official veterinarian, ensuring an unambiguous chain of custody from death certification to laboratory analysis. DNA extraction was performed using two independent protocols: Method A, a silica-membrane-based purification (QIAamp DNA Kit, Qiagen), applied to dental pulp, and Method B, a decalcification-based workflow (T-Bone Ex Kit) followed by DNA extraction using EZ2 Connect Instruments (Qiagen). Quantification of the DNA samples obtained were done by fluorometry. Short Tandem Repeat (STR) genotyping was performed using the Canine Genotypes™ Panel 1.1 by Applied Biosystems (Thermo Fisher Scientific, Waltham, MA, USA) targeting the 18 autosomal ISAG-recommended microsatellite loci. Parentage assignments were carried out against reference STR profiles obtained from peripheral blood of the dam and offspring. The two methods, considering a different biological matrix and different operating conditions, allowed us to obtain the STR profile, but Method B showed quality electropherograms, improving the confidence and interpretation of all the markers analyzed. The differences observed between the two approaches may reflect a combination of factors, including the different dental matrices analysed, the quantity and quality of the starting material, tooth identity and tissue preservation, and differences inherent to the extraction procedures themselves; therefore, in this case, the more complete and clearer STR profile obtained with Method B should be considered a case-specific observation rather than evidence of a direct effect of the extraction method. Overall, regardless of the DNA extraction method used, the results did not exclude the biological paternity of the blue-mantled offspring. Furthermore, this case report provides practical methodological guidance for DNA recovery from canine teeth and supports their use as a reliable source of post-mortem DNA for STR-based parentage testing.

Vector-borne diseases are endemic in Mediterranean regions, where dogs are frequently exposed to multiple pathogens throughout their lives. Although the immunological consequences of these infections have been widely investigated, less is known about the biochemical changes associated with natural pathogen exposure in clinically healthy autochthonous breeds. To characterize haematological and biochemical alterations associated with exposure to vector-borne pathogens in clinically healthy Mannara dogs living in an endemic Mediterranean area. Thirty-two clinically healthy Mannara dogs were sampled between September 2024 and January 2025. Antibodies against Anaplasma phagocytophilum, Ehrlichia canis, Leishmania infantum and Rickettsia spp. were determined by indirect immunofluorescence assay, whereas L. infantum DNA was assessed by real-time PCR. Complete blood count, serum biochemistry and serum protein electrophoresis were performed. Statistical analyses included parametric or non-parametric tests with false discovery rate correction. All dogs were negative for L. infantum DNA by PCR, although 37.5% were seropositive. Coinfections involving two or more pathogens were detected in 40.6% of the animals. Dogs seropositive for A. phagocytophilum showed significantly lower albumin concentrations and albumin/globulin ratios together with higher β2-globulin concentrations than seronegative dogs. Coinfected animals also exhibited increased β2- and γ-globulin fractions and reduced albumin concentrations. No significant differences were observed according to sex or age after multiple-comparison correction. Natural exposure to vector-borne pathogens was associated with consistent serum protein alterations in clinically healthy Mannara dogs despite the absence of detectable L. infantum parasitaemia. These findings are consistent with persistent antigenic stimulation associated with natural exposure to vector-borne pathogens and provide reference biochemical information for this autochthonous breed and support further studies investigating the biological significance of these alterations under natural exposure conditions.

Vector-borne diseases (VBDs) are major zoonotic threats in Mediterranean regions, where dogs act as reservoirs for pathogens such as Anaplasma phagocytophilum, Ehrlichia canis, Leishmania infantum, and Rickettsia rickettsii. This study evaluated biochemical and haematological profiles associated with single and multiple vector-borne infections in an autochthonous Sicilian dog breed naturally exposed to endemic pathogens.Thirty-two clinically healthy Mannara dogs were studied between September 2024 and January 2025. Serological detection of pathogens was performed by IFAT, and L. infantum DNA was assessed by real-time PCR. Complete blood count, serum biochemistry, and protein electrophoresis were analyzed. Statistical tests were applied according to data distribution, with false discovery rate (FDR) correction.None of the dogs tested positive for L. infantum DNA, although 37.5% were seropositive. Coinfections were detected in 40.6% of animals, mainly in adults. Dogs seropositive for A. phagocytophilum showed significantly higher β2-globulin levels and lower albumin and albumin/globulin (A/G) ratios compared with seronegative dogs (p < 0.05, FDR-adjusted). Coinfected animals exhibited increased β2- and γ-globulin fractions and reduced albumin and A/G ratios. Effect sizes indicated moderate to strong differences between groups. Sex and age were not associated with the evaluated parameters.These findings indicate a biochemical profile consistent with sustained but balanced immune activation in clinically healthy dogs exposed to multiple pathogens, in the absence of detectable L. infantum infection. This pattern may reflect controlled exposure rather than active infection. However, results should be interpreted cautiously due to the limited sample size and lack of functional immunological data.

Background: Progressive retinal atrophy (PRA) was diagnosed in four related male English Cocker Spaniels (ECS) between the ages of 3 and 4 years. All affected dogs were born to clinically unaffected parents that had tested negative for known PRA-causing variants through commercial vendors. Pedigree analysis revealed that the mothers of the affected dogs were paternal half-siblings, and their father had been reported as visually impaired, suggesting an X-linked recessive mode of inheritance. Results: To identify the underlying genetic cause, we performed homozygosity mapping, identifying six candidate regions, and whole-genome sequencing. A single high-impact coding variant—a 1-bp deletion in exon 36 of the CACNA1F gene on X chromosome (UU_Cfam_GSD_1.0 NC_049260.1 g.42,516,353del) —was identified. This variant is predicted to cause a frameshift and premature stop codon (XM_038587436.1, c.4,481del, XP_038443364.1:Phe1482LeufsTer8) and was found in a hemizygous state in all affected males and heterozygous in their mothers, while being absent in fathers as well as unaffected control dogs. A specifically designed PCR test was developed and applied to a broader cohort of 92 related and unrelated ECS dogs, confirming the segregation of this variant. Conclusions: This study identified a novel segregating CACNA1F variant, likely to be responsible for an X-linked recessive form of PRA in the ECS. Given that CACNA1F is already associated with retinal disorders in other mammals, including humans, this finding adds to its relevance across species. This study highlights the importance for breeders of regular health examinations for late-onset conditions and prompt reporting of hereditary disorders of unknown cause to enable rapid identification of their genetic basis. Development and implementation of specific genetic screening tests are recommended to assess the frequency of this variant in ECS and other populations, inform breeding strategies, and prevent further dissemination of this deleterious variant.

Background: Vector-borne diseases (VBDs) are the major zoonotic threats in Mediterranean regions, where dogs act as key reservoirs for pathogens such as Anaplasma phagocytophylum, Ehrlichia canis, Leishmania infantum, and Rickettsia rickettsii. Understanding how these pathogens affect the host physiology is crucial to identifying biochemical patterns potentially associated with a phenotype compatible with resistance to L. infantum infection. This study analyzed biochemical and haematological profiles associated with single and multiple vector-borne infections in an autochthonous Sicilian dog breed naturally exposed to endemic pathogens. Methods: Thirty-two clinically healthy Mannara dogs were studied in Sicily between September 2024 and January 2025. Serological detection of A. phagocytophylum, E. canis, L. infantum, and R. rickettsii antibodies were performed using IFAT, and L. infantum DNA was assessed by real-time PCR. Complete blood count, serum biochemistry, and protein electrophoresis were performed. Statistical analysis was performed using parametric or non-parametric tests, followed by false discovery rate (FDR) correction. Results: None of the dogs tested were positive for L. infantum DNA, although 37.5% showed specific antibodies for this pathogen. Coinfections with two or more pathogens occurred in 40.6% of animals, mainly in adults. Dogs seropositive for A. phagocytophylum showed significantly higher β2-globulin levels and lower albumin and albumin/globulin (A/G) ratios than uninfected dogs (p-value < 0.05, FDR-adjusted). Coinfected dogs displayed elevated β2- and γ-globulin fractions and reduced albumin and A/G ratios compared with non-infected dogs (p-value < 0.05, FDR-adjusted). Sex and age do not affect the biochemical or haematological parameters evaluated. Conclusions: Mannara dogs (an autochthonous breed of the Mediterranean basin) showed biochemical evidence of chronic but balanced immune activation, despite the absence of active L. infantum infection. The decrease in A/G ratio and the increase in β2- and γ-globulins suggest sustained humoral stimulation compatible with a putative phenotype associated with resistance to L. infantum. Continuous exposure to multiple vector-borne pathogens may promote an immune-trained phenotype conferring resilience against L. infantum infection.

The Capra Comune di Sicilia (CCS), also known as Mascaruna, is a Sicilian local goat population first described in 1870 and is currently the focus of a recovery project aimed at its characterization and formal ethnic recognition. To elucidate the ancestral genetic components and selection trajectories of the CCS population, we genotyped 78 CCS goats using the Goat 60 K SNP BeadChip, integrated with genotype data from 1,920 individuals representing 66 goat breeds of Mediterranean and African origin. CCS exhibited relatively high heterozygosity (0.408), and moderate levels of inbreeding (0.04), an estimated effective population size of 185. Genetic ancestry analysis revealed gene flow from Maltese, Girgentana, Rossa Mediterranea and Saanen populations, alongside evidence of putative Greek ancestry shared with most Mediterranean breeds in our dataset, reflecting Sicily’s profound historical and cultural ties with Greece. To better understand the evolutionary trajectories of the CCS population and to explore the potential contribution of Greek goat ancestry, we investigated selection signatures using iHS and ROH analyses. We identified 76 and 31 SNPs intercepting 38 and 12 genes, respectively, under putative selection. Subsequently, we applied XP-nSL and ROH analyses using Greek populations as ancestral references, identifying 21 and 431 SNPs associated with four and 157 genes, respectively, under putative selection. Overall, these selection signature analyses highlighted genes under positive selection related to traits such as milk and meat production, body size and growth, fertility, coat colour, fat deposition, and ear and horn development. Our findings shed light on the historical and genetic distinctiveness of the CCS population, emphasizing its uniqueness and providing critical insights into its genetic background. This information is essential for supporting informed efforts to formally recognize CCS as a distinct and valuable breed.

Background: Local livestock breeds play a pivotal role in maintaining agricultural sustainability, conserving biodiversity, and preserving cultural heritage. These breeds often possess unique genetic characteristics tailored to their specific environments. The Cinisara is a dual-purpose local cattle breed of Podolian origin, primarily farmed in western Sicily, Italy. However, reports of spurious crossbreeding with cosmopolitan breeds aimed at improving the breed productivity exist. To assess the conservation status and ongoing selective pressures on this unique breed, we genotyped 71 unrelated Cinisara cattle (CIN_A) at 65k SNPs, and extended the dataset with publicly available genotype data of 30 Cinisara individuals sampled 20 years ago (CIN_B). We also included 194 individuals from seven cattle breeds, including the Podolica (POD) breed and the cosmopolitan Holstein (HOL) and Brown Swiss (BRW) breeds. We assessed the genetic diversity, population structure, and determined the extent of introgression from cosmopolitan breeds into Cinisara using local ancestry inference. Results: Population structure analyses confirmed the Cinisara’s Podolian lineage and revealed significant HOL and BRW introgression. While both Cinisara populations, CIN_A and CIN_B, displayed broadly comparable genetic diversity to larger breeds, CIN_B showed reduced heterozygosity and increased inbreeding. CIN_A exhibited higher introgression, suggesting ongoing crossbreeding. Local ancestry was inferred using POD, HOL, and BRW references. CIN_A showed about 258/257 HOL/BRW introgressed SNPs, intercepting 186/131 genes and 1,584/1,772 QTLs. CIN_B had approximately 256/254 HOL/BRW introgressed SNPs, intercepting 218/184 genes and 547/437 QTLs. Predominantly, these regions overlapped with milk production QTLs, but some intercepted genes linked to unique Cinisara traits, like milk quality and climate adaptation, potentially altering breed typicality. Notably, CIN_B shows a potentially higher relative BRW contribution, while CIN_A shows a higher HOL contribution. Conclusion: Our findings align with the reports of crossbreeding with cosmopolitan breeds to enhance the production performance of Cinisara, and reflect breeding choices such as a reduction in BRW crossing or a preference for HOL. This raises significant concerns regarding the preservation of local breeds, livestock biodiversity, and their cultural and economic value, and highlights the importance of developing informed breeding strategies that balance production improvements with the conservation of genetic heritage.