RNAseq reveals modulation of genes involved in fatty acid biosynthesis in chicken liver according to genetic background, sex, and diet.

Increases in chicken production are mainly due to specialised breeds. However, local breeds are of increasing importance, known for ability to adapt to the environment and unique products. Conventional poultry products contain lower levels of n-3 fatty acids (FAs) compared to those obtained from local breeds, therefore the aim of this study was to evaluate the modulation of expression of genes involved in long-chain polyunsaturated FA (PUFA) biosynthesis pathways according to genetic background, diet conditions, and sex. Animals from two local breeds and a commercial line were fed different diets: control and experimental diet (10% linseed supplementation). For each breed and diet group, both sexes were reared. The RNA was extracted from 36 liver samples and sequenced by RNAseq method. Bioinformatic analysis was carried out to find differentially expressed genes from comparisons between experimental groups. Results showed low impact of diet on differentially expressed genes related to FA biosynthesis, but linseed diet increased percentage of n-3 FAs of liver. Sex and genetic background determined the differential expression of genes related to long-chain PUFA biosynthesis. Specifically, females of local breeds shared 23 up-regulated genes when compared to their respective commercial line groups. Some of the shared genes had a role in de novo triglyceride biosynthesis (MTTPL and GPAM), and in de novo FA biosynthesis (ACACA and SCD) was detected. In conclusion, local breeds are able to better adapt to a diet rich in PUFA, by triggering certain transcriptomic shifts in the liver that allow birds to process the high PUFA content provided by diet.

Multifactorial Evaluation of Regrouping Effects on Performance and Welfare in Two Italian Dual-Purpose Chicken Breeds: Bianca di Saluzzo and Bionda Piemontese.

The aim of the present study was to investigate the ability of two Italian slow-growing poultry breeds (namely, Bionda Piemontese, BP, and Bianca di Saluzzo, BS) to cope with a stressful event, such as collective grouping, using a multifactorial approach. A total of 120 hens of BP and BS breeds were homogenously distributed, according to breed, in 12 pens (10 hens/pen; 6 pens/breed), from 18 to 49 weeks of age. At 50 weeks of age, hens were regrouped (Stressful Farm Practice, SFP), by removing separators, both in indoor and outdoor areas. At 49 weeks of age, 24 hens/breed were randomly selected for the evaluation of welfare (ethological and physiological) parameters, at different time points: T0: 1-week pre-SPF; T1: 1-week post-SFP; T2: 3-week post-SFP; T3: 5-week post-SFP. Egg production was recorded from 38 to 56 weeks of age. Grouping produced a social stress in both BS and BP-laying hens, which was expressed in terms of productive traits (reduction of oviposition), behavioral modification (worsening of plumage condition due to feather peaking and extension of the duration of the tonic immobility test) and physiological modification (increased heterophil/lymphocyte ratio and corticosterone metabolites in droppings). Both breeds reacted in a similar way; in fact, no differences were attributed to the breed. At the end of the observation period, the egg rate fully recovered, while the behavioral and physiological parameters partially recovered but failed to recover to those recorded prior to the stressful event.

A Practical Application of Genomic Predictions for Mastitis Resistance in Italian Holstein Heifers.

Heifers are a fundamental resource on farms, and their importance is reflected in both farm management and economy. Therefore, the selection of heifers to be reared on a farm should be carefully performed to select only the best animals. Genomic selection is available nowadays to evaluate animals in a fast and economic way. However, it is mainly used on the sire line and on performance traits. Ten farms were selected based on their 5-year records of average somatic cell count and evenly classified into high (>300,000 cells/mL) and low somatic cell count (<150,000 cells/mL). Genomic indexes (regarding both wellness and productive traits) were evaluated in 157 Italian Holstein heifers reared in the selected ten farms (90 from high-cells farms and 67 from low-cells ones). Linear mixed models were fitted to analyze the effects of the abovementioned genomic indexes on related phenotypes. Results have shown that farms classified into low somatic cell count had an overall better animal genomic pool compared to high somatic cell count ones. Additionally, the results shown in this study highlighted a difference in wellness genomic indexes in animals from farms with either a high or a low average somatic cell count. Applying genomic tools directly to heifer selection could improve economic aspects related to herd turnover.

Does Feeding Management Make a Difference to Behavioural Activities and Welfare of Horses Reared for Meat Production?

Horses reared for meat production are generally fed a diet rich in starch with the aim of maximizing production performances. This study evaluated the effects of two feeding management systems on horse welfare by analysing the relative time spent engaged in different behavioural activities. Nineteen Bardigiano horses aged 14.3 ± 0.7 months were randomly assigned to one of two group pens: one group was fed high amounts of starch-rich concentrates (HCG; n = 10), the other was fed a fibre-based diet (HFG; n = 9). Behavioural activities performed by each horse were video-recorded over a 96-h period. A scan sampling process (n = 144 scans/horse/day; total n of scans sampled = 10,368) was used, and the scans were analysed according to a specific ethogram. The mean frequency (%/24 h) spent exhibiting each behavioural activity was calculated to obtain the time budget. After checking for normality (Shapiro-Wilk test), Student's t tests (normally distributed data) and Mann-Whitney tests (not normally distributed data) were used to compare the time budgets of the two groups of horses (HCG vs. HFG). Principal Component Analysis (PCA) was applied to identify the components explaining the variability in behavioural activities between the two groups. K-means cluster analysis subsequently confirmed the PCA results. The behavioural activities associated with feeding horses a fibre-based diet correlated with better horse welfare compared with feeding horses a starch-based diet. Feeding horses a fibre-based diet resulted advantageous from both the welfare and economic perspective; it allowed horses to spend more time expressing feeding behaviours and reduced energy expenditure in the form of excitable, or "fizzy", behaviours.

Carcass Yields and Meat Composition of Male and Female Italian Slow-Growing Chicken Breeds: Bianca di Saluzzo and Bionda Piemontese.

The slaughter performance and meat quality of two native Italian chicken breeds, Bionda Piemontese (BP, n = 64) and Bianca di Saluzzo (BS, n = 64), were investigated. Two-way ANOVA, considering breed, sex, and their interaction, was used to compare the properties of birds slaughtered at 5, 6, 7, and 8 months of age. Subsequently, data were analyzed using one-way ANOVA and the Duncan test to evaluate the differences between slaughter ages. The BP breed produced a better carcass yield than BS at 5, 7, and 8 months of age (p < 0.05). Breast moisture and crude protein contents were influenced by gender, and were higher in males than in females (p < 0.05). By contrast, the crude fat content was higher in females than in males (p < 0.05). The saturated fatty acid content of breast meat increased as the birds aged in both breeds (p < 0.05). The polyunsaturated fatty acid content of both breast and thigh meat was higher in males than in females (p < 0.001 and p < 0.05, respectively). In general, slaughtering at 7 months was associated with the best slaughter and meat quality characteristics in both breeds. Moreover, from a nutritional point of view, the characteristics of the meat from male birds were preferable to those of meat from females.

Genetic Diversity of 17 Autochthonous Italian Chicken Breeds and Their Extinction Risk Status.

The preservation of genetic variability of autochthonous poultry breeds is crucial in global biodiversity. A recent report revealed small breed size and potential risk of extinction of all native Italian poultry breeds; therefore, a correct assessment of their genetic diversity is necessary for a suitable management of their preservation. In this work, we provided an overview of the contribution to poultry biodiversity of some Italian autochthonous breeds reared in conservation centers devoted to local biodiversity preservation. The level of genetic diversity, molecular kinship, inbreeding, contribution to overall genetic diversity, and rate of extinction of each breed were analyzed with a set of 14 microsatellite loci in 17 autochthonous chicken breeds. To evaluate genetic variability, total number (Na), and effective number (Ne) of alleles, observed (Ho) and expected (He) heterozygosity, and F (Wright's inbreeding coefficient) index were surveyed. The contribution of each analyzed breed to genetic diversity of the whole dataset was assessed using MolKin3.0; global genetic diversity and allelic richness contributions were evaluated. All the investigated loci were polymorphic; 209 alleles were identified (94 of which private alleles). The average number of alleles per locus was 3.62, and the effective number of alleles was 2.27. The Ne resulted lower in all breeds due to the presence of low-frequency alleles that can be easily lost by genetic drift, thus reducing the genetic variability of the breeds, and increasing their risk of extinction. The global molecular kinship was 27%, the average breed molecular kinship was 53%, and the mean inbreeding rate 43%, with a self-coancestry of 78%. Wright's statistical analysis showed a 41% excess of homozygous due to breed genetic differences (34%) and to inbreeding within the breed (9%). Genetic variability analysis showed that 11 breeds were in endangered status. The contribution to Italian poultry genetic diversity, estimated as global genetic diversity, and ranged from 30.2 to 98.5%. In conclusion, the investigated breeds maintain a unique genetic pattern and play an important role in global Italian poultry biodiversity, providing a remarkable contribution to genetic variability.

From the Semen Collection Method to the Hatchlings: The Use of Cryopreserved Sperm from Pheasants Fed an Antioxidant-Enriched Diet.

A widely used approach to preserving genetic diversity in birds involves the cryopreservation of semen. In this process, cells are subjected to physical and chemical stresses, but not all cell species respond equally. Many studies have been published on the freezing-thawing of sperm cells from a wide variety of domestic and wild species, on issues ranging from the sperm quality to different protocols, fertilisation success rates, etc. Nevertheless, very little information is available on the common pheasant. To fill this gap, the aim of this study was to describe the pheasant semen collection method, evaluate some qualitative parameters of sperm from males fed an antioxidant-enriched diet, and to test the in vivo fertilising capacity of the cryo-preserved semen. The freezing protocol employed involved pellets thawed by the hotplate method. Dimethylacetamide was used as a cryoprotectant at a final concentration of 6%. A total of six AIs were performed at 3-4-day intervals on a total of 40 females with doses of 35 × 106 of normal live thawed sperm. Males receiving the enriched diet produce more abundant and concentrated ejaculates. Freeze-thawed sperm lost 85% of their initial mobility, and diet influenced neither sperm mobility nor viability. The enriched diet did improve the number of normal freeze-thawed cells and was associated with a lower sperm fracture incidence. Regardless of the dietary group, frozen-thawed sperm resulted in a fertility rate of 30%, with 8-9 chicks hatching for every 100 eggs incubated.

The climatic and genetic heritage of Italian goat breeds with genomic SNP data.

Local adaptation of animals to the environment can abruptly become a burden when faced with rapid climatic changes such as those foreseen for the Italian peninsula over the next 70 years. Our study investigates the genetic structure of the Italian goat populations and links it with the environment and how genetics might evolve over the next 50 years. We used one of the largest national datasets including > 1000 goats from 33 populations across the Italian peninsula collected by the Italian Goat Consortium and genotyped with over 50 k markers. Our results showed that Italian goats can be discriminated in three groups reflective of the Italian geography and its geo-political situation preceding the country unification around two centuries ago. We leveraged the remarkable genetic and geographical diversity of the Italian goat populations and performed landscape genomics analysis to disentangle the relationship between genotype and environment, finding 64 SNPs intercepting genomic regions linked to growth, circadian rhythm, fertility, and inflammatory response. Lastly, we calculated the hypothetical future genotypic frequencies of the most relevant SNPs identified through landscape genomics to evaluate their long-term effect on the genetic structure of the Italian goat populations. Our results provide an insight into the past and the future of the Italian local goat populations, helping the institutions in defining new conservation strategy plans that could preserve their diversity and their link to local realities challenged by climate change.

Overview of Native Chicken Breeds in Italy: Conservation Status and Rearing Systems in Use.

The most reared species of farm animal around the world is the chicken. However, the intensification of livestock systems has led to a gradual increase in the concentration of a limited number of breeds, resulting in substantial erosion to the genetic pool. The initial step of an 'animal conservation program' entails establishing the actual conservation statuses of the breeds concerned in a defined area; in this case, in Italy. To this end, a survey of breeds was performed by means of a census questionnaire divided into two parts. The first part collected information on breeds, breeders, housing facilities, and management aspects, the results of which are presented here. The second part of the questionnaire regarded chicken products and their markets, and these data will be reported in a second paper. The breed status of six chicken breeds was shown to be exceptionally worrying, with total numbers ranging from just 18 to 186 birds. Population sizes exceeding 1000 birds was identified for just four breeds, the maximum being 3400. Some improvements in status were noted in relation to breeds which had been the subject of conservation efforts in the past. The two most common breeds reported are the Bionda Piemontese, a double-purpose breed, and the Livorno egg-laying hen. Collo Nudo Italiano, Millefiori Piemontese, Pollo Trentino, and Tirolese chicken breeds and the Castano Precoce turkey breed were not listed by breeders at all. The most reported turkey breeds are the Bronzato Comune and the Ermellinato di Rovigo. The population sizes of native Italian poultry breeds were shown to be generally poor. Italian poultry farmers and the population at large are largely ignorant about indigenous poultry breeds. Thus, promoting the virtues of Italian breeds would help their conservation by encouraging breeders to rear these birds and consumers to buy their products. The identification of strategies to facilitate access to pure breed birds is essential, and will require the collaboration of university research centers, public entities, and breeders. The results presented in this paper constitute the initial part of a more complex conservation program.

Overview of Native Chicken Breeds in Italy: Small Scale Production and Marketing.

The intensive use of high-performing strains in poultry production has led to the extinction of several autochthonous chicken breeds and, consequently, loss of genetic variability. Interest in saving biodiversity is growing rapidly and has become a major objective worldwide. The aim of this study was to shed light on the production trends of native Italian poultry breeds and the related market. A questionnaire, which asked about the production cycles, the number of animals and table eggs produced per year and their retail prices was completed by 121 breeders across Italy. The surveyed breeders were divided into two categories: breeders conducting an agrozootechnical farm, referred to as 'farmers' (F); and breeders keeping chickens as backyard poultry, referred to as 'fancy breeders' (FB). Analysis of the data acquired indicated that animals were mainly slaughtered between 6 and 12 months of age, with F processing more animals per year. The same production trend was observed for table eggs. The recorded retail prices of native chicken products were higher than those for conventional products, but similar to those reported for valuable niche poultry products, such as the Poulet de Bresse in France and organic eggs. Knowledge about these highly valuable markets should be used to encourage the use of local breeds in alternative poultry farming and help protect biodiversity.

Identification of SNPs Associated with Somatic Cell Score in Candidate Genes in Italian Holstein Friesian Bulls.

Mastitis is an infectious disease affecting the mammary gland, leading to inflammatory reactions and to heavy economic losses due to milk production decrease. One possible way to tackle the antimicrobial resistance issue stemming from antimicrobial therapy is to select animals with a genetic resistance to this disease. Therefore, aim of this study was to analyze the genetic variability of the SNPs found in candidate genes related to mastitis resistance in Holstein Friesian bulls. Target regions were amplified, sequenced by Next-Generation Sequencing technology on the Illumina® MiSeq, and then analyzed to find correlation with mastitis related phenotypes in 95 Italian Holstein bulls chosen with the aid of a selective genotyping approach. On a total of 557 detected mutations, 61 showed different genotype distribution in the tails of the deregressed EBVs for SCS and 15 were identified as significantly associated with the phenotype using two different approaches. The significant SNPs were identified in intergenic or intronic regions of six genes, known to be key components in the immune system (namely CXCR1, DCK, NOD2, MBL2, MBL1 and M-SAA3.2). These SNPs could be considered as candidates for a future genetic selection for mastitis resistance, although further studies are required to assess their presence in other dairy cattle breeds and their possible negative correlation with other traits.

Genome-Wide SNP Analysis Reveals the Population Structure and the Conservation Status of 23 Italian Chicken Breeds.

The genomic variability of local Italian chicken breeds, which were monitored under a conservation plan, was studied using single nucleotide polymorphisms (SNPs) to understand their genetic diversity and population structure. A total of 582 samples from 23 local breeds and four commercial stocks were genotyped using the Affymetrix 600 K Chicken SNP Array. In general, the levels of genetic diversity, investigated through different approaches, were lowest in the local chicken breeds compared to those in the commercial stocks. The level of genomic inbreeding, based on runs of homozygosity (FROH), was markedly different among the breeds and ranged from 0.121 (Valdarnese) to 0.607 (Siciliana). In all breeds, short runs of homozygosity (ROH) (<4 Mb in length) were more frequent than long segments. The patterns of genetic differentiation, model-based clustering, and neighbor networks showed that most breeds formed non-overlapping clusters and were clearly separate populations, which indicated the presence of gene flow, especially among breeds that originated from the same geographical area. Four genomic regions were identified as hotspots of autozygosity (islands) among the breeds, where the candidate genes are involved in morphological traits, such as body weight and feed conversion ratio. We conclude that the investigated breeds have conserved authentic genetic patterns, and these results can improve conservation strategies; moreover, the conservation of local breeds may play an important role in the local economy as a source of high-quality products for consumers.

Antimicrobial Effects of Black Soldier Fly and Yellow Mealworm Fats and Their Impact on Gut Microbiota of Growing Rabbits.

This study aimed to evaluate the in vitro antimicrobial activities of two types of insect fats extracted from black soldier fly larvae (HI, Hermetia illucens L.) and yellow mealworm larvae (TM, Tenebrio molitor L.) and their effects as dietary replacement of soybean oil (S) on cecal fermentation pattern, and fecal and cecal microbiota in rabbits. A total of 120 weaned rabbits were randomly allotted to three dietary treatments (40 rabbits/group) -a control diet (C diet) containing 1.5% of S and two experimental diets (HI diet (HID) and TM diet (TMD)), where S was totally substituted by HI or TM fats during the whole trial that lasted 41 days. Regarding the in vitro antimicrobial activities, HI and TM fats did not show any effects on Salmonella growth. Yersinia enterocolitica showed significantly lower growth when challenged with HI fats than the controls. The insect fat supplementation in rabbit diets increased the contents of the cecal volatile fatty acids when compared to the control group. A metataxonomic approach was adopted to investigate the shift in the microbial composition as a function of the dietary insect fat supplementation. The microbiota did not show a clear separation as a function of the inclusion, even if a specific microbial signature was observed. Indeed, HI and TM fat supplementation enriched the presence of Akkermansia that was found to be correlated with NH3-N concentration. An increase in Ruminococcus, which can improve the immune response of the host, was also observed. This study confirms the potential of HI and TM fats as antibacterial feed ingredients with a positive influence on the rabbit cecal microbiota, thus supporting the possibility of including HI and TM fats in rabbit diets.

Growth Performance Analysis of Two Italian Slow-Growing Chicken Breeds: Bianca di Saluzzo and Bionda Piemontese.

Bianca di Saluzzo (BS) and Bionda Piemontese (BP) are two Italian chicken breeds, mainly reared for meat production, primarily in antibiotic-free farming. However, technical information on their growth pattern is still missing. At hatching, 150 unsexed chicks of each breed were weighed, labeled, and reared in indoor pens up to 8 w of age. At 8 w of age, the chicks were separated by sex and randomly transferred to growing pens with access to an external paddock (15 birds/pen; 4 pens/sex for each breed). The body weight (BW) was recorded biweekly for each bird, from hatching to 32 w of age. In order to identify an improvement strategy, the objectives of our study were to analyze the growth pattern of these birds using the Gompertz mathematical model and compare results with other chicken breeds. Polymorphism of the PAX7 gene was also analyzed to test its association with growth traits. Both BS and BP are close to unselected native breeds and, among the Italian local poultry, they are confirmed to be slow-growing birds with an intermediate size between heavy and light chicken breeds. Regarding the PAX7 gene, two alleles were found, F and G, and showed an association with the actual BW in the BP females from 14 w of age onwards. The G allele always exhibited a more favorable effect than the F allele. In small size poultry population, a delicate balance between preservation of biodiversity and performance improvement should be considered. Consequently, the most proper way could be an approach based on a mating scheme to keep inbreeding under control, increase growth rate, and improve commercial maturity.

Effect of N-Methylacetamide Concentration and Thawing Rate on Chicken Sperm Quality after Cryopreservation.

In seeking alternative cryoprotectants to glycerol for a reference chicken semen freezing procedure, the aim of the present study was to compare the effect of two concentrations of N-Methylacetamide (MA) and two thawing rates on the quality of frozen-thawed semen. Semen samples were diluted in Lake pre-freezing extender, including 0.1 M trehalose in presence of 6% or 9% MA, loaded into straws, frozen in nitrogen vapors, and stored in liquid nitrogen. The following thawing treatments were used: 5 °C for 100 s and 38 °C for 30 s. Sperm quality (cell membrane integrity, motility and kinetic parameters) was assessed before and after cryopreservation. The decrease of MA concentration from 9 to 6% improved sperm quality after freezing/thawing and this effect was dependent on thawing temperature. Decreasing the MA concentration from 9 to 6% improved the proportion of undamaged membrane, motile, and progressive motile sperm recovered after thawing at 5 °C for 100 s; in contrast, no effect of the MA concentration was observed thawing at 38 °C for 30 s. Therefore, the treatment with 6% MA and thawing at 5 °C for 100 s has given the best cryoprotective action. These results contribute to improve the efficacy of the current chicken semen cryopreservation procedures.

Predicting the impact of selection for scrapie resistance on PRNP genotype frequencies in goats.

The European Union has implemented breeding programmes to increase scrapie resistance in sheep. A similar approach can be applied also in goats since the K222 allele provides a level of resistance equivalent to that of ARR in sheep. The European Food Safety Authority stated that breeding for resistance could be offered as an option for Member States to control classical scrapie in goats. We assessed the impact of different breeding strategies on PRNP genotype frequencies using a mathematical model that describes in detail the evolution of K222 in two goat breeds, Chamois Coloured and Saanen. Different patterns of age structure and replacement rate were modelled as factors affecting response to selection. Breeding for scrapie resistance can be implemented in goats, even though the initial K222 frequencies in these breeds are not particularly favourable and the rate at which the resistant animals increase, both breeding and slaughtered for meat production, is slow. If the goal is not to achieve the fixation of resistance allele, it is advisable to carry out selection only until a desired frequency of K222-carriers has been attained. Nucleus selection vs. selection on the overall populations is less expensive but takes longer to reach the desired output. The programme performed on the two goat breeds serves as a model of the response the selection could have in other breeds that show different initial frequencies and population structure. In this respect, the model has a general applicability.

Polymorphism Analysis of Ch1 and Ch2 Genes in the Siberian Cat.

Cats are usually spreaders of allergens that are critical for sensitive people; the Siberian cat is a breed supposed to be low level allergenic, according to some breeders' statements. The sequence of the two genes, namely Ch1 and Ch2, that code for the allergen Fel d 1, the major allergen responsible for outbreaks of allergy symptoms, is not yet known in the Siberian cat, and finding this was the aim of our investigation. Notably, our work is the first survey of the genetic structure of these genes in Siberian cats. The comparison of the sequences of Siberian cats, non-Siberian cats, and sequences present in the National Center for Biotechnology Information database revealed a considerable number of mutations; some of those detected in the Siberian cat, due to their position in exon regions, could affect the Fel d 1 allergenic properties. Therefore, further investigations are recommended to assess if the identified mutations can be responsible for a reduced-allergen synthesis and can be used as markers for selection of low level allergenic cats.

Distinguishing industrial meat from that of indigenous chickens with molecular markers.

The aim of investigation was to evaluate a traceability system to detect industrial chicken meat among indigenous products, considering issues that could affect assignment accuracy. The dataset included 2 Italian indigenous meat breeds, namely Bionda Piemontese (2 ecotypes) and Bianca di Saluzzo, one broiler line, and 3 layer lines. Assignment tests were performed using a standard panel of 28 microsatellite loci. To evaluate effects of inbreeding and substructure on assignment accuracy, a simulated dataset was prepared. Broilers and layers belong to homogeneous populations and never enter the clusters of indigenous breeds. Ambiguity or misallocation are expected between the Bionda ecotypes and between the 2 indigenous breeds, but it is unlikely that niche products provided by Bionda and Bianca will compete with one another. Non-random mating reduces accuracy, but only populations having weak genetic differentiation are involved, namely those that are less interesting to discriminate. The dataset can be used as a reference population to distinguish commercial meat from indigenous meat with great accuracy. Misallocations increase as number of loci decreases, but only within or between the indigenous breeds. A subpanel of the most resolving 14 loci keeps sufficient informative content to provide accuracy and to correctly allocate additional test samples within the reference population. This analytical tool is economically sustainable as a method to detect fraud or mislabeling. Adoption of a monitoring system should increase the value of typical products because the additional burden of molecular analyses would improve commercial grade and perception of quality.

Development and validation of a microsatellite marker-based method for tracing infections by Microsporum canis.

BACKGROUND: Microsporum canis is a dermatophyte fungus harbored by cats and dogs and is frequently transmitted to humans. Molecular tools able to discriminate fungal isolates at the strain level would prove extremely useful for confirming the route of infection, thus contributing to optimization of prophylaxis and hygienic regimens. OBJECTIVE: To develop and validate a microsatellite marker-based method for use in tracking infections by M. canis. METHODS: Primers were designed against sequences flanking the microsatellites individuated by a BLAST search using the nucleotide sequence information assembled by the M. canis CBS 113480 genome project. The PCR conditions were standardized and fragment analysis was performed using a genetic analyzer. The resolving power of the markers was investigated on 26 unrelated M. canis strains while the reproducibility of the technique and the stability of the markers were evaluated on a single strain subcultured in time as well as on 36 strains isolated from nine outbreak episodes. RESULTS: Eight markers were recognized as being the most polymorphic within the set of M. canis strains isolated from unrelated distant hosts, with a total of 22 multilocus genotypes, which corresponded to a genotypic diversity of 97%. Repeated tests on subcultures of M. canis reference strain CBS 113480 always yielded the same results. Identical multilocus genotypes were obtained for all the isolates from each outbreak episode. CONCLUSION: The high resolving power and reproducibility of the markers that were identified support the potential of these tools to detect sources and routes of infection by M. canis.