Francesca Pedonese

Evaluation of dot enzyme-linked immunosorbent assay (dot-ELISA) for the serodiagnosis of canine leishmaniosis as compared with indirect immunofluorescence assay

A dot enzyme-linked immunosorbent assay (dot-ELISA) was developed and compared with a standard indirect immunofluorescence assay (IFA) for the rapid serodiagnosis of canine leishmaniosis. The two tests were used to examine sera from Leishmania infantum-infected and control dogs. Using the dot-ELISA, 137 of 149 sera (91.9%) from infected animals gave a clearly positive reaction, whereas in the standard IFA technique 147 (98.7%) were positive at a reciprocal titer of 40 or over (titer range 40-10,240). Control sera from 75 healthy dogs, not living in endemic areas, and 11 dogs with other diseases (babesiosis, cryptococcosis, ehrlichiosis, dermatitis, and chronic hepatitis) but Leishmania-negative were used to determine the specificity of the assays. All the sera were negative with IFA (100%), whereas using the dot-ELISA only 74 sera (86%) from controls gave a negative result. In the standard IFA no cross-reactivity was noted, in the dot-ELISA a weak cross-reaction was observed with a serum sample from a dog with babesiosis. The interpretation of dot-ELISA could be easily performed by visual inspection of the nitrocellulose disks. The most remarkable feature of dot-ELISA was the high sensitivity (91.9%) and positive predictive value (92.6%), even if the test showed a specificity lower than IFA. Because of its easy performance and high sensitivity, the dot-ELISA may be a useful test to be executed in the field for the diagnosis of canine leishmaniosis.

Efficacy of oral terbinafine in feline dermatophytosis due to Microsporum canis

Microsporum canis is the dermatophyte most commonly responsible for ringworm in cats. The purpose of this paper was to evaluate the in vivo efficacy of oral terbinafine (Lamisil; Sandoz) in the treatment of feline ringworm caused by M canis, and to consider this drug as an alternative to griseofulvin or imidazoles. Fifteen cats infected with M canis were treated orally once daily with 30 mg/kg of terbinafine over a 2-week period. All treated animals were checked for dermatophytes on the last day of treatment, a month later and 3 months after the last administration of the drug. Only 12 cats could be used in the whole trial and 11 of these (92%) showed a complete cure. Terbinafine could be an effective alternative to griseofulvin when fungal resistance or idiosyncrasic intolerance are shown and, compared with griseofulvin, could give a faster rate of cure and less relapses.

Seroprevalence, Detection of DNA in Blood and Milk, and Genotyping of Toxoplasma gondii in a Goat Population in Italy

Toxoplasma gondii is the causative agent of a major zoonosis with cosmopolitan distribution and is known to be transmitted mainly by the ingestion of undercooked or raw animal products. Drinking unpasteurized goats milk is a risk factor associated with human toxoplasmosis. However, very little is known about the excretion of DNA in goat milk. Aim of the present study was to determine the seroprevalence of T. gondii infection using a modified agglutination test (MAT), to detect T. gondii DNA by nested-PCR (n-PCR) in samples of blood and milk from seropositive goats, and to genotype DNA isolates using 11 molecular markers in 127 adult lactating goats from 6 farms in Italy. Positive MAT results were found in 60.6% of goats while 13% of blood and milk samples from seropositive goats were positive to n-PCR. A kappa coefficient of 1 indicated a perfect agreement between blood and milk n-PCR. Genetic characterization of isolates revealed the occurrence of genotype III (n = 7), genotype I (n = 1), and atypical genotypes with hints for genotype I (n = 2). Our results suggest that the risk of excretion of Toxoplasma tachyzoites might frequently occur in milk of seropositive goats testing positive to n-PCR on blood.

Occurrence of Vancomycin-Resistant Enterococci in Foods of Animal Origin Purchased in Tuscany

Enterococci are ubiquitous microorganisms, commonly found in plants, insects and wild animals, and are normal residents of the gastrointestinal tract of both animals and humans. They frequently occur in large numbers in certain foods, especially those of animal origin. Over the last 10 years, enterococci have emerged as major nosocomial pathogens, thus representing an increasingly important problem for public health, in relation to their ability to acquire resistance to some antimicrobial agents of relevance to human therapy, such as glycopeptides, particularly vancomycin. In this regard, the food chain represents a possible source of vancomycin-resistant enterococci for humans, mainly as a result of the use of avoparcin, a vancomycin-like glycopeptide, as a feed additive for growth promotion of farm animals, as undoubtedly established by previous studies (Bates, 1997; Mc Donald et al., 1997). Although the utilization of avoparcin in animal husbandry has been banned since 1997, VRE have recently been isolated from foods of different typology (Giraffa et al., 2000; Knijff et al., 2002), therefore investigation into the frequency of recovery of VRE at this level is still recommendable. Thus, the present survey was carried out with the aim of evaluating the occurrence of VRE in foods of animal origin purchased in Tuscany, considering that these microorganisms could play a relevant role both directly as potential pathogens for humans, as well as in relation to their capability to spread antibiotic resistance genes to other pathogenic bacteria, such as Staphylococcus aureus.

Caciotta della Garfagnana cheese: selection and evaluation of autochthonous mesophilic lactic acid bacteria as starter cultures

The aim of this study was to isolate, identify and select, with respect to acidification and proteolytic activities, the autochthonous mesophilic lactic acid bacteria (LAB) present in milk and Caciotta della Garfagnana, a cheese produced either with raw or thermised cow’s milk in small dairies and family plants of Garfagnana (Tuscany), to obtain LAB strains with attributes suitable to be employed as starter cultures in this type of cheese, particularly when thermised milk is used to control spoilage microflora. Samples of raw milk, curd and cheese were collected from three representative farmers of the production area and used to isolate autochthonous LAB. Phenotypic and genotypic (species-specific PCR assay) identification of isolated LAB was done. Twenty-eight strains of LAB isolated from milk, curd and cheese were screened for acidifying and proteolytic activities. LAB strains with the better attributes were used as mesophilic starter cultures in technological trials: experimental cheeses manufactured with the addition of autochthonous LAB and control cheeses were compared for LAB and pH evolution. Experimental cheeses presented a significant increase in the mesophilic lactic acid microflora up to 14 days of ripening and significantly lower pH values up to seven days of ripening. The use of wild selected mesophilic lactic acid bacteria, together with thermisation of milk, for the Caciotta della Garfagnana looks very promising and could help to both standardise the production and improve quality and traditional characteristics of this type of cheese.

Fatty acid and microbiological profile of the meat (longissimus dorsi muscle) of wild boar (Sus scropha scropha) hunted in Tuscany

Abstract The aim of this work is to study the influence of gender, age and hunting period on the fatty acid composition of the longissimus dorsi muscle excised from 42 wild boars hunted on a farm located in Tuscany. A sub-sample of 22 muscles was used for the study of the microbiological profile of wild boar meat. The results show that gender had an effect only on C16:1 n7, which was higher in females (p ≤ .05). Slaughtering age influenced more parameters, thus resulting in a higher content of C6:0, C18:2, C20:1 and n6/n3 ratio in meat from adults (p ≤ .05) and in a higher percentage of C16:0 (p ≤ .05) in meat from young animals. The hunting month greatly influenced the meat quality because, in addition to the single fatty acids, it significantly modified the MUFA, PUFA, PUFA/SFA and n6/n3 ratio, underlining the great difference between meat of wild boar shot in October – November vs December – January. Ether extract was higher in sub-adults than in young animals (p ≤ .01) but was not influenced by gender and hunting month. Regarding the microbiological analyses, Salmonella spp. was only detected in one sample, while Yersinia enterocolitica was not detected at all. Data on the presence of pathogenic bacteria confirmed health risk for the consumer comparable to that associated with meat obtained from farm animals, while data on microbial loads (total aerobic mesophilic and psychrotrophic counts, Enterobacteriaceae, Escherichia coli) did not highlight any specific criticality.

Biogenic Amines Content of Four Types of “Pecorino” Cheese Manufactured in Tuscany

Biogenic amines content of four types of Tuscan ewes’ milk “pecorino” cheese was evaluated using HPLC-UV analysis. All cheeses were manufactured in the same dairy factory with different combinations of milk (raw or pasteurized) and type of ripening. Total biogenic amines and tyramine levels of a raw milk “pecorino” ripened five months, partly in a traditional cave, were significantly higher than those of a pasteurized milk “pecorino” with a similar ripening; and of a two months raw milk “pecorino” ripened in the dairy plant. No statistical significant difference was found when comparing total biogenic amines and tyramine contents of the same five month ripened raw milk “pecorino” with a pasteurized milk “pecorino” ripened six months, partly in a traditional “fossa.” In raw milk cave-ripened and “fossa”-ripened cheeses, total biogenic amines exceeded 1000 mg/kg. In cheeses manufactured with raw milk and/or in particular ripening environments, specific hygienic cares are needed to limit biogenic amines formation.

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.

Antimicrobial activity of four essential oils against pigmenting Pseudomonas fluorescens and biofilmproducing Staphylococcus aureus of dairy origin

Essential oils (EOs) are mixtures of secondary metabolites of plant origin with many useful properties, among which the antimicrobial activity is also of interest for the food industry. EOs can exert their antimicrobial potential both directly, in food products and active packaging, and indirectly, as sanitizing and anti-biofilm agents of food facility surfaces. Aim of this research was to evaluate the antimicrobial activity of four EOs (bergamot, cinnamon, manuka and thyme) against Pseudomonas fluorescens and Staphylococcus aureus isolated from milk and dairy products. The chemical composition of EOs was evaluated by Gas Chromatography-Mass Spectrometry analysis. Minimum Inhibitory Concentration values were determined by a microplate method against 9 Ps. fluorescens from marketed mozzarella with blue discoloration defect, and 3 biofilm-producing S. aureus from milk. Reference ATCC strains were included. Pigment production activity by Ps. fluorescens was assessed both in culture and in cheese. EOs of manuka (leptospermone 23%) and thyme (carvacrol 30%, pcymene 20%, thymol 15%) showed the highest antimicrobial activity against S. aureus, MIC values were 0.012%-0.024% and 0.024% v/v, respectively; meanwhile EOs from thyme and cinnamon (cinnamaldehyde 55%) exhibited the best activity against Ps. fluorescens with MIC values of 0.098%-0.195% and 0.195%-0.391% v/v, respectively. The antimicrobial activity of these EOs is promising and they could be exploited in the dairy production chain.

Microbiological and physicochemical profile of traditional Salsiccia toscana during storage

The aim of this study was to evaluate the evolution during refrigerated storage of the main microbiological and physicochemical parameters of a traditional Italian fresh sausage, salsiccia toscana. The following parameters were analyzed at Days 0, 6 and 9 of storage: total aerobic mesophilic and psychrotrophic counts, Brochothrix thermosphacta, lactic acid bacteria, Pseudomonadaceae, Enterobacteriaceae, Escherichia coli, Micrococcaceae, yeasts and moulds, Salmonella spp., Listeria monocytogenes; pH, Aw, moisture, protein, total lipids, ash, fatty acid composition, Thiobarbituric Acid Reactive Substances, colour measurement. The results revealed a statistically significant increase in the main microbiological parameters between Days 0 and 9 of refrigerated storage, with total bacterial counts exceeding 107 CFU/g after nine days and Brochothrix thermosphacta representing the main spoilage microorganism. Among physicochemical parameters, discolouration was noted during the time of storage, with a significantly paler colour on the surface of sausages with casings after nine days of storage. Considering that the initial bacterial counts were higher than 105 CFU/g for Brochothrix thermosphacta, Pseudomona - daceae, lactic acid bacteria and Enterobac - teriaceae, and that the product is traditionally purchased loose without any packaging, it is fundamental to improve the good manufacturing practices, particularly measures to control processing temperatures.