C. Pollera

Efficacy of vaccination on Staphylococcus aureus and coagulase-negative staphylococci intramammary infection dynamics in 2 dairy herds.

The aim of this study was to evaluate vaccine efficacy of a commercial vaccine (Startvac, Hipra Spain) aimed at reducing intramammary infections (IMI) with Staphylococcus aureus and coagulase-negative staphylococci under field conditions. During the 21-mo duration of the study, 1,156 lactations from 809 cows were enrolled in 2 herds. During the first phase of the trial, all cows that were due to calve were vaccinated until approximately 50% of cows in the milking herd were vaccinated (at ~6mo). At that point, when 50% vaccination coverage was reached, cows that were due to calve were randomly assigned to be vaccinated or left as negative controls. Cure rate, rate of new infection, prevalence, and duration of infections were analyzed. Vaccination resulted in a moderate reduction in incidence of new staphylococcal IMI and a more pronounced reduction in duration of IMI associated with reduction of the basic reproduction ratio of Staph. aureus by approximately 45% and of coagulase-negative staphylococci by approximately 35%. The utilization of vaccine in combination with other infection-control procedures, such as excellent milking procedures, treatment, segregation, and culling of known infected cattle, will result in an important reduction in incidence and duration of intramammary staphylococcal infections.

Effects of clioquinol on memory impairment and the neurochemical modifications induced by scrapie infection in golden hamsters

Prion protein (PrP) is a glycoprotein expressed on the surface of neurons and glial cells. Its pathological isoform (PrP(res)) is protease resistant, and involved in the pathogenesis of a number of transmissible encephalopathies (TSEs). One common feature of neurodegenerative diseases, including TSEs, is oxidative stress, which may be responsible not only for the dysfunction or death of neuronal cells, but also cognitive deficits. Clioquinol (5-chloro-7-iodo-8-quinolinol) chelates zinc and copper, which are involved in the deposition of amyloid plaques and acts as an antioxidant; increased lipid peroxidation has also been demonstrated in the early phases of PrP propagation. The aim of this study was to investigate the effects of clioquinol on the changes in motor and cognitive behaviours induced by scrapie infection, as well as its effects on oxidative stress and the neurotransmitters known to be involved in motor and cognitive functions. The results show that clioquinol counteracts the massive memory deficit induced by scrapie infection. This effect is not paralleled by neurochemical changes because the levels of all of the biogenic amines and their metabolites were reduced despite clioquinol treatment. The main biochemical change induced by clioquinol was a marked reduction in lipid peroxidation at all time points. The antioxidant effect of clioquinol can reduce functional impairment and thus improve memory, but clioquinol does not reduce PrP deposition or synapse loss, as indicated by the unchanged Western blot, histopathological and histochemical findings.

Evaluation of Anti-Prionic Activity of Clioquinol in an in vivo Model (Mesocricetus auratus)

C. Pollera1,∗, B. Lucchini1, E. Formentin1, S. Bareggi2, G. Poli1 and W. Ponti1 1Department of Veterinary Pathology, Hygiene and Public Health, Microbiology and Immunology Unit, Faculty of Veterinary Medicine, Centre of Excellence on Neurodegenerative Diseases, University of Milan, Italy; 2Department of Pharmacology, Chemoterapy and Medical Toxicology, School of Medicine, Centre of Excellence on Neurodegenerative Diseases, University of Milan, Italy ∗Correspondence: E-mail: claudia.pollera@unimi.it

Neurochemical and behavioural modifications induced by scrapie infection in golden hamsters.

Scrapie-infected hamsters were tested for spontaneous motor activity and passive avoidance at various times after infection. After testing, some animals were killed and their whole brains assayed for norepinephrine, dopamine, serotonin and their metabolites. The apparent rate of turnover was estimated in terms of metabolite/amine concentrations. After 70 days, there was a decrease in passive avoidance and dopamine and serotonin. Passive avoidance correlated with the apparent rate of turnover of dopamine, whereas motor activity correlated with that of serotonin and dopamine.

Pharmacokinetics and distribution of clioquinol in golden hamsters

Clioquinol (5‐chloro‐7‐iodo‐8‐quinolinol) is a zinc and copper chelator that can dissolve amyloid deposits and may be beneficial in Alzheimers disease. Prion diseases are also degenerative CNS disorders characterised by amyloid deposits. The pharmacokinetics and tissue distribution of drugs active against prions may clarify their targets of action. We describe the harmacokinetics of clioquinol in hamster plasma, spleen and brain after single and repeated oral or intraperitoneal administration (50 mg kg−1), as well as after administration with the diet. A single intraperitoneal administration led to peak plasma clioquinol concentrations after 15 min (Tmax), followed by a decay with an apparent half‐life of 2.20 ± 1.1 h. After oral administration, Tmax was reached after 30 min and was followed by a similar process of decay; the AUC0‐last was 16% that recorded after intraperitoneal administration. The Cmax and AUC values in spleen after a single administration were about 65% (i.p.) and 25% (p.o.) those observed in blood; those in liver were 35% (p.o.) those observed in blood and those in brain were 20% (i.p.) and 10% (p.o.) those observed in plasma. After repeated oral doses, the plasma, brain and spleen concentrations were similar to those observed at the same times after a single dose. One hour after intraperitoneal dosing, clioquinol was also found in the ventricular CSF. Clioquinol was also given with the diet; its morning and afternoon concentrations were similar, and matched those after oral administration. No toxicity was found after chronic administration. Our results indicate that clioquinol, after oral administration with the diet, reaches concentrations in brain and peripheral tissues (particularly spleen) that can be considered effective in preventing prion accumulation, but are at least ten times lower than those likely to cause toxicity.

Antibiotic treatment of the hard tick Ixodes ricinus: Influence on Midichloria mitochondrii load following blood meal

Midichloria mitochondrii is the most prevalent symbiont of the hard tick Ixodes ricinus, present in 100% of eggs and adult females of wild ticks. This bacterium is intracellular, and is the only known symbiont able to invade the mitochondria of the host cells. However, the role that M. mitochondrii plays in the host metabolism has yet to be elucidated. Multiple lines of evidence indicate the possibility of transmission of this bacterium to the vertebrate host during the tick blood meal. In order to investigate the role of M. mitochondrii in the biology of the tick host, we performed an antibiotic treatment on Ixodes ricinus individuals, with the aim of reducing/eliminating the symbiont, and to potentially observe the dynamic of bacterial infection in the tick host. We microinjected engorged adult females of I. ricinus with tetracycline, and we allowed the resulting larvae to feed on gerbils treated with the same antibiotic. The amount of M. mitochondrii was evaluated at different stages of the experiment using molecular techniques. In addition we evaluated the presence/absence of the symbiont DNA in the blood of gerbils used for the larval feeding. The performed treatments did not allow to eliminate the symbiont population from the host tick, however it allowed to reduce the multiplication that occurs after the larval blood meal. These results open the way for future experiments, using different antibiotic molecules, different administration methods and antibiotic administration on subsequent tick stages, to fulfill the goal of eliminating M. mitochondrii from the host I. ricinus, a major step in our understanding of the impact of this bacterium on ticks.

In vivo Model for the Evaluation of Molecules Active Towards Transmissible Spongiform Encephalopathies

W. Ponti1*, M. Sala2, C. Pollera1, D. Braida 2, G. Poli1 and S. Bareggi2 1Dipartimento di Patologia Animale, Igiene e Sanita Pubblica Veterinaria, Sezione di Microbiologia e Immunologia; 2Dipartimento di Farmacologia Medica, Chemioterapia e T ossicologia, Milano. Centro di Eccellenza per le Malattie Neurodegenerative *Correspondence: Dipartimento di Patologia Animale, Igiene e Sanita Pubblica Veterinaria, Sezione di Microbiologia e Immunologia. V ia Celoria 10. 20133 Milan, Italy E-mail: wilma.ponti@unimi.it

Identification of virulence factors in 16S-23S rRNA intergenic spacer genotyped Staphylococcus aureus isolated from water buffaloes and small ruminants

Staphylococcus aureus is an important human and animal pathogen, and is regarded as an important cause of intramammary infection (IMI) in ruminants. Staphylococcus aureus genetic variability and virulence factors have been well studied in veterinary medicine, especially in cows as support for control and management of IMI. The aim of the present study was to genotype 71 Staph. aureus isolates from the bulk tank and foremilk of water buffaloes (n=40) and from udder tissue (n=7) and foremilk (n=24) from small ruminants. The method used was previously applied to bovine Staph. aureus and is based on the amplification of the 16S-23S rRNA intergenic spacer region. The technique applied was able to identify different Staph. aureus genotypes isolated from dairy species other than the bovine species, and cluster the genotypes according to species and herds. Virulence gene distribution was consistent with genotype differentiation. The isolates were also characterized through determination of the presence of 19 virulence-associated genes by specific PCR. Enterotoxins A, C, D, G, I, J, and L were associated with Staph. aureus isolates from buffaloes, whereas enterotoxins C and L were linked to small ruminants. Genes coding for methicillin resistance, Panton-Valentine leukocidin, exfoliative toxins A and B, and enterotoxins B, E, and H were undetected. These findings indicate that RNA template-specific PCR is a valid technique for typing Staph. aureus from buffaloes and small ruminants and is a useful tool for understanding udder infection epidemiology.

Plasma Noradrenalin as Marker of Neuroinvasion in Prion Diseases

Pollera, C., Bondiolotti, G., Formentin, E., Puricelli, M., Mantegazza, P., Bareggi, S., Poli, G. and Ponti, W., 2007. Plasma noradrenalin as marker of neuroinvasion in prion diseases. Veterinary Research Communications, 31(Suppl. 1), 249–252

Evaluation of clioquinol activity towards transmissible spongiform encephalopathies (TSE) in cellular models and cell-free systems

Neuropathogenesis of TSE is due to a misfolding process of the cellular prion protein (PrPsen) which is converted into the pathological isoform (PrPres). This mechanism induces the disease because of the loss of PrPsen function and because of PrPres accumulation and toxicity. One of the hypotheses about the cellular function of PrP is the transport activity of Cu++ ions, whose segregation reduces the neurotoxic effects of copper itself (Harris, 2003). PrPsen to PrPres conversion impairs the protective mechanism after and thus Cu++ cannot be internalised and accumulates in the extracellular space, contributing to neuronal death. In fact, in scrapie–infected mice a significant increase in copper ions has been found in brain tissues during the synptomatic phase of the disease (Brown, 2004). The use of metallic ion chelators, such as Clioquinol, an antibiotic used for enteric diseases and able to cross the hematoencephalic barrier, may decrease the toxic effects of copper and solubilize amyloid plaques, by reducing the conformational stability of PrPres. In experimental models of Alzheimer’s disease, whose pathogenic mechanism is similar to TSE, Clioquinol reduces β-amyloid deposits by 50%, acting in the mechanisms of ionamyloid interaction (Nitzan et al., 2003). In the current work the Clioquinol activity on PrPres has been evaluated using in vitro models of TSE, consisting of cell cultures and cell-free systems.