In this study, the use of skimmed milk flocculation (SMF) to simultaneously concentrate viruses, bacteria and protozoa was evaluated. We selected strains of faecal indicator bacteria and pathogens, such as Escherichia coli and Helicobacter pylori. The viruses selected were adenovirus (HAdV 35), rotavirus (RoV SA-11), the bacteriophage MS2 and bovine viral diarrhoea virus (BVDV). The protozoa tested were Acanthamoeba, Giardia and Cryptosporidium. The mean recoveries with q(RT)PCR were 66% (HAdV 35), 24% (MS2), 28% (RoV SA-11), 15% (BVDV), 60% (E. coli), 30% (H. pylori) and 21% (Acanthamoeba castellanii). When testing the infectivity, the mean recoveries were 59% (HAdV 35), 12% (MS2), 26% (RoV SA-11) and 0.7% (BVDV). The protozoa Giardia lamblia and Cryptosporidium parvum were studied by immunofluorescence with recoveries of 18% and 13%, respectively. Although q(RT)PCR consistently showed higher quantification values (as expected), q(RT)PCR and the infectivity assays showed similar recoveries for HAdV 35 and RoV SA-11. Additionally, we investigated modelling the variability and uncertainty of the recovery with this method to extrapolate the quantification obtained by q(RT)PCR and estimate the real concentration. The 95% prediction intervals of the real concentration of the microorganisms inoculated were calculated using a general non-parametric bootstrap procedure adapted in our context to estimate the technical error of the measurements. SMF shows recoveries with a low variability that permits the use of a mathematical approximation to predict the concentration of the pathogen and indicator with acceptable low intervals. The values of uncertainty may be used for a quantitative microbial risk analysis or diagnostic purposes.

Cysticidal treatment of neurocysticercosis, an infection of humans and pig brains with Taenia solium, results in an early inflammatory response directed to cysts causing seizures and focal neurological manifestations. Treatment-induced pericystic inflammation and its association with blood brain barrier (BBB) dysfunction, as determined by Evans blue (EB) extravasation, was studied in infected untreated and anthelmintic-treated pigs. We compared the magnitude and extent of the pericystic inflammation, presence of EB-stained capsules, the level of damage to the parasite, expression of genes for proinflammatory and regulatory cytokines, chemokines, and tissue remodeling by quantitative PCR assays between treated and untreated infected pigs and between EB-stained (blue) and non stained (clear) cysts. Inflammatory scores were higher in pericystic tissues from EB-stained cysts compared to clear cysts from untreated pigs and also from anthelmintic-treated pigs 48 hr and 120 hr after treatment. The degree of inflammation correlated with the severity of cyst wall damage and both increased significantly at 120 hours. Expression levels of the proinflammatory genes for IL-6, IFN-γ, TNF-α were higher in EB-stained cysts compared to clear cysts and unaffected brain tissues, and were generally highest at 120 hr. Additionally, expression of some markers of immunoregulatory activity (IL-10, IL-2Rα) were decreased in EB-stained capsules. An increase in other markers for regulatory T cells (CTLA4, FoxP3) was found, as well as significant increases in expression of two metalloproteases, MMP1 and MMP2 at 48 hr and 120 hr post-treatment. We conclude that the increase in severity of the inflammation caused by treatment is accompanied by both a proinflammatory and a complex regulatory response, largely limited to pericystic tissues with compromised vascular integrity. Because treatment induced inflammation occurs in porcine NCC similar to that in human cases, this model can be used to investigate mechanisms involved in host damaging inflammatory responses and agents or modalities that may control damaging post treatment inflammation. Neurocysticercosis is caused by infection of the brain with the larval (cyst) stage of the tape worm Taenia solium in humans and pigs. Antiparasitic drug treatment is compromised by worsening of neurological symptoms during therapy due to reactive inflammation triggered by the dying parasite. The immune mechanisms that cause this inflammation are poorly understood. In this study, we investigated the nature of inflammation after treatment in pigs naturally infected with T. solium cysts. Evans blue dye injected into infected pigs marks areas in the brain where the normally impermeable capillaries have become more permeable, allowing damaging cells and molecules to leak out into the brain. By microscopy and measurement of gene expression for inflammation-inducing immune mediators, we show that inflammation in the brain tissues around cysts is more severe with increased vessel leakage. Furthermore, the levels of these mediators increased after antiparasitic drug treatment. A significant implication of these findings is that it may be possible to inhibit the inflammation around parasites using drugs or biologics that inhibit these inflammatory pathways and, thereby, reduce local brain damage during treatment. These observations may also be applicable to other inflammatory conditions that affect the brain.