Alessia Galiero

Detection of Mycobacterium avium subsp. paratuberculosis in cheeses from small ruminants in Tuscany

Paratuberculosis is an infectious disease which affects mainly domestic and wild ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). Map has been associated with human diseases like Crohn disease, type-1 diabetes, sarcoidosis, multiple sclerosis and Hashimotos thyroiditis. The aim of this study was to determine the level of Map positivity of cheeses produced in Tuscany (Italy) as an indication of human exposure to the specific pathogen. Sampling was focused on artisanal cheeses produced without commercial starter culture from raw sheep or goat milk, on small-scale farms. Samples were tested by quantitative PCR (qPCR) and culture. Map DNA was detected in 4/7 (57.14%) goat, and in 14/25 (56%) sheep cheeses by qPCR, whereas cultivation produced a positive result in only one case. This corresponded to a goat cheese that had also reacted positively by qPCR and yielded a viable Type S (sheep) strain of Map. The Map load of the tested samples based on qPCR ranged from 6×10 to 1.8×10(4)Map cells/g of cheese. The results indicate on average 56.57% and 66.6% positivity of cheese samples and farms, respectively. Hence, the type of cheeses that were analyzed within the context of this study seem to constitute a considerable source of human exposure to Map; although the question remains of whether the Map cells were present in a viable form, since positive results were almost exclusively recorded by qPCR.

Identification of candidate genes for paratuberculosis resistance in the native Italian Garfagnina goat breed

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.

Lactobacillus plantarum and Streptococcus thermophilus as starter cultures for a donkey milk fermented beverage

Donkey milk is recently gaining attention due to its nutraceutical properties. Its low casein content does not allow caseification, so the production of a fermented milk would represent an alternative way to increase donkey milk shelf life. The aim of this study was to investigate the possibility of employing selected Streptococcus thermophilus and Lactobacillus plantarum isolates for the production of a novel donkey milk fermented beverage. Lysozyme resistance and the ability to acidify donkey milk were chosen as main selection parameters. Different fermented beverages (C1-C9) were produced, each with a specific combination of isolates, and stored at refrigerated conditions for 35days. The pH values and viability of the isolates were weekly assessed. In addition, sensory analysis was performed. Both S. thermophilus and L.plantarum showed a high degree of resistance to lysozyme with a Minimum Bactericidal Concentration>6.4mg/mL for 100% of S. thermophilus and 96% of L. plantarum. S. thermophilus and L. plantarum showed the ability to acidify donkey milk in 24h at 37°C, with an average ΔpH value of 2.91±0.16 and 1.78±0.66, respectively. Four L. plantarum and two S. thermophilus were chosen for the production of fermented milks. Those containing the association S. thermophilus/L. plantarum (C1-C4) reached a pH lower than 4.5 after 18h of fermentation and showed microbial loads higher than 7.00logcfu/mL until the end of the storage period. Moreover, comparing the microbial loads of samples containing both species and those containing S. thermophilus alone (C5), we highlighted the ability of L. plantarum to stimulate S. thermophilus replication. This boosted replication of S. thermophilus allowed to reach an appropriate pH in a time frame fitting the production schedule. This was not observed for samples containing a single species (C5-C9). Thus, L. plantarum strains seem to be good candidates in the production of a novel type of fermented milk, not only for their probiotic potential, but also for the enhancing effect on S. thermophilus growth.

Occurrence of Coxiella burnetii in goat and ewe unpasteurized cheeses: Screening and genotyping.

Q fever is a zoonosis caused by Coxiella burnetii which infects humans as well as several animal species; sheep, goats and cattle are the primary animal reservoir. The main route of human exposure to Coxiella burnetii is inhalation of contaminated aerosols from excreta, especially birth products, while the role of unpasteurized dairy products in the transmission of Q fever to humans remains still controversial. The aim of this work was to evaluate the presence of Coxiella burnetii in unpasteurized cheese samples (n=84) by PCR and to genotype the circulating strains by Multispacer sequence typing (MST) analysis. Coxiella burnetii DNA was detected in 27/84 (32.14%) cheeses and positivity rate of handicraft cheeses reached 17.24%, while positivity rate of non-handicraft cheeses reached 65.38%. In addition, the MST profile of Coxiella burnetii detected in 5 cheese samples have shown the circulation of ST12 and ST32 genotypes in Tuscany.

Mycobacterium avium subsp. paratuberculosis isolated from wild red deer ( Cervus elaphus ) in Northern Italy

Paratuberculosis (or Johnes disease) is an infectious disease which affects mainly ruminants and it is caused by Mycobacterium avium subsp. paratuberculosis (MAP). During a culling program (years 2011-2015) aimed at controlling the red deer (Cervus elaphus) population in Stelvio National Park (Italian Alps), where paratuberculosis was already described in this species, 382 tissue samples from the Lombardy Region and 102 fecal specimens from the Autonomous Province of Bolzano were analyzed by PCR. Of these, 77 samples (20.16%) from the Lombardy area and 19 specimens (18.63%) from the Bolzano area resulted PCR positive. The cultural test was carried out on PCR positive samples (n = 96), enabling the isolation of 19 MAP field strains which were genotyped using MIRU-VNTR typing and Short Sequence repeats (SSRs). Our results suggest that all isolates share an identical VNTR profile corresponding to the INMV1 genotype. The only variation was on the locus SSR2, but the utility of this last locus has already been questioned because of its instability. Overall, these data suggest a common clonal origin and host adaptation during the diffusion of paratuberculosis in this population. Finally, this profile is the same as that which has already been described in the cattle population in Northern Italy, suggesting a possible inter-species disease transmission pattern from wildlife to domestic ruminants and vice versa.

Evaluation of clinical pathology parameters in fecal PCR-positive or PCR-negative goats for Johne’s disease

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.