Alessia Galiero

Mycobacterium avium subsp. paratuberculosis (Map) is the agent of a chronic, progressive granulomatous enteritis in ruminants known as Johne’s disease or paratuberculosis. Nowadays, the interest regarding this pathogen is increasing not only because Map causes economic losses, but it has also been suggested as a potential risk factor for the development of human diseases such as Crohn disease, autoimmune diseases like type-1 diabetes, sarcoidosis, multiple sclerosis and Hashimoto’s thyroiditis. The aim of our study was to determine the presence of paratuberculosis in Garfagnana (Tuscany, Italy) where raw milk is not submitted to pasteurization, but it is directly sold to consumers or it is destined for cheese making without the employment of heat treatment. The survey was conducted in Garfagnana district where there are 17 herds which produce and market bovine milk for direct human consumption. Serum samples (n=162) were obtained form 16 herds and were analyzed performing ELISA ID screen® Paratuberculosis Indirect screening test (ID.VET, Montpellier, France) and positive samples were tested with ELISA ID screen® Paratuberculosis Indirect confirmation test (ID.VET, Montpellier, France), according to the instructions provided by the manufacturer. The analysis performed by ELISA revealed that the true seroprevalence was 29.1% at the herd level and 4.6% at the animal level. In our opinion, although ELISA is a very useful tool to screen herds for paratuberculosis, more studies should be carried out in our territory combining serological data with cultural and PCR analysis.

Johne’s disease (JD) is an economically important infectious disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). This study evaluated the differences in various hematological and biochemical parameters between healthy goats and goats with JD. Forty goats were chosen randomly from a herd endemic for JD. A complete physical examination was performed. Blood and fresh fecal samples were collected from each goat. A complete blood cell (CBC) count and a protein electrophoresis were performed. Polymerase chain reaction (PCR) on fecal samples was performed in order to divide goats into two groups: group A “positive PCR on feces”; and group B “control (negative).” A Student’s t test was performed for each parameter to verify differences between groups A vs B. Twenty goats were included in each group. Clinical signs likely related to JD were found in the history of 4/40 (10%) goats, while 36/40 (90%) goats were reported to be asymptomatic. CBC and electrophoresis values were within reference intervals in both groups. No differences were found for CBC parameters between the two groups. Values for alpha 1, beta, gamma globulins, and total protein (TP) were statistically higher in group A vs those in group B, while those for albumin and albumin/globulin (A/G) ratio were lower. An increase in TP, hypoalbuminemia, and hypergammaglobulinemia has been reported in group A, while no abnormalities were found concerning CBC. JD-positive goats seem to show earlier clinical pathological alternations than clinical signs. Protein electrophoresis may help the diagnosis of JD in asymptomatic goat herds, acting as an economical screening method.

Paratuberculosis disease is a chronic bacterial disease infection of ruminants of global relevance, caused by MAP (Mycobacterium avium subsp. paratuberculosis). The present study was conducted on the Garfagnina goat breed that is an Italian native goat population registered on the Tuscan regional repertory of genetic resources at risk of extinction. Forty-eight adult goats (27 serologically positive to MAP-positive and 21 serologically negative to MAP-negative) belonging to a single flock that had experienced annual mortalities due to MAP infection were identified and genotyped with the Illumina GoatSNP60 BeadChip. Diagnosis was achieved by serological tests, as well as post-mortem examination of affected animals. A genome-wide scan was then performed on the individual marker genotypes, in an attempt to identify genomic regions associated with MAP infection disease. Nine significant markers were highlighted and they were located within, or nearby, annotated genes. Two genes found in this study encode are linked to protein kinases that are among the most important enzymes involved in the immune response to Johne’s disease, and four genes are involved in the functions of the Golgi complex.

Mycobacterium avium paratuberculosis (Map) is a pathogen which causes a chronic progressive granulomatous enteritis known as paratuberculosis or Johne’s disease and it primarily affects wild and domestic ruminants. The aim of this research was to examine a flock which consisted of 294 goats and was located in Garfagnana district (Tuscany, Italy) performing ELISA tests, culture and IS900 PCR assay; direct diagnostic methods were carried out not only on bulk tank milk and cheese samples but also on individual milk and tissue specimens collected from nine subjects positive to ELISA tests. Out of 294 animals, 20 goats (6.8%) were positive to ELISA surveys. Bulk tank milk samples were negative to culture and to PCR assay carried out on the DNA extracted directly from them, while, with respect to cheese, Map was detected by culture in 2/12 (16.66%) cheeses ripened for 3–7 days, and by PCR in 2/12 (16.66%) cheeses ripened for 3–7 days and in 3/12 (25%) cheeses ripened for 45 days. Regarding individual milk samples, Map was detected by culture in 2/9 (22.22%) specimens and by PCR in 5/9 (55.55%) samples. Furthermore, Map was isolated from the intestine in 9/9 (100%) animals, from the mesenteric lymph nodes in 8/9 (88.88%) subjects, from the liver in 4/9 (44.44%) goats, from the spleen in 5/9 (55.55%) animals, while Map DNA was found in all the tissue samples analyzed. The results demonstrated the presence of paratuberculosis in a goat flock located in Garfagnana district (Tuscany, Italy).

The consumption of raw milk is currently increasing due to several beneficial aspects, such as nutritional qualities, taste, and health benefits. However, some authors highlight the potential risk associated with raw milk consumption. In Italy, while the absence of some pathogen microorganisms is set by the regional regulation DGR 381/2007, for other microorganisms, such as Leptospira, no recommendations are provided. Leptospira is not ascribed among classical milk pathogens; however, it can potentially be present in raw milk. The aim of this study was to evaluate the survival in raw milk of six serovars of Leptospira after storage at different temperatures (4 °C ± 2 °C, 20 °C ± 2 °C, and 30 °C ± 2 °C) for different incubation times (20 min, 45 min, 1 h, and 1 h and 30 min), in order to determine the potential risk for consumers. Moreover, the immediate effect of bovine, goat, and donkey raw milk on tested Leptospira serovars was visually evaluated. After incubation, all samples were subcultured in EMJH and incubated aerobically at 30 °C for 3 months. All inoculated media were weekly examined by dark-field microscope in order to assess Leptospira survival. Extemporary observation of strains’ behavior in milk allowed to detect an almost immediate motility loss, and no leptospires were detected by microscopic observations carried out weekly during the trial period. According to our results, it could be possible to exclude raw milk as a source of Leptospira infection for consumers.