Paul V. Rossitto

Toxoplasma in Animals, Food, and Humans: An Old Parasite of New Concern

All hosts, including humans, can be infected by any one of the three forms of the parasite Toxoplasma gondii that correspond to three morphological stages: tachyzoite, bradyzoite, and sporozoite form. Felids are definitive hosts for T. gondii, which is an intracellular pathogen that infects a wide range of warm-blooded intermediate hosts. Toxoplasmosis is a disease where the interest of the diverse medical and veterinary specialties converge. Awareness needs to be increased that toxoplasmosis can induce clinical disease not only in immunocompromised patients or through congenital infections, but also in healthy patients. This is a review article that aims at illustrating why toxoplasmosis should be regarded a veterinary public health issue and how veterinary practitioners can contribute in controlling the infection.

Prevention of diseases caused by Staphylococcus aureus using the peptide RIP.

Staphylococcus aureus causes many diseases including cellulitis, keratitis, osteomyelitis, septic arthritis and mastitis. The heptapeptide RIP has been shown to prevent cellulitis in mice, which was induced by S. aureus strain Smith diffuse. Here we show that RIP can also significantly reduce the overall pathology and delay the onset of disease symptoms in several other models of S. aureus infections, including: keratitis (tested in rabbits against S. aureus 8325-4), osteomyelitis (tested in rabbits against S. aureus MS), mastitis (tested in cows against S. aureus Newbould 305, AE-1, and environmental infections) and septic arthritis (tested in mice against S. aureus LS-1). These findings substantiate that RIP is not strain specific in its inhibitory activity and that RIP is an effective inhibitor of bacterial pathology at multiple body sites following diverse routes and doses of administration. These findings strongly evidence the potential value of RIP as a chemotherapeutic agent.

Enterotoxin production by Staphylococcus aureus isolated from mastitic cows

Staphylococcus aureus is an important cause of mastitis in cows. The ability of S. aureus strains to produce one or more enterotoxins in milk and dairy products is linked to staphylococcal food poisoning. To determine whether staphylococci causing bovine mastitis could cause human foodborne intoxication, the production of staphylococcal enterotoxins A through D (SEA, SEB, SEC, and SED) by 160 S. aureus isolates was evaluated with the use of a reverse passive latex agglutination enterotoxin kit. All S. aureus strains were isolated over a 9-month period from 2,343 routine submissions of a composite quarter collection of individual mastitic cows at 18 dairy farms in the San Joaquin Valley in California. Prior to enterotoxin detection, isolates were grown by a method that enhances the in vitro synthesis of enterotoxin. Twenty-two of 160 S. aureus isolates produced enterotoxin. Seven produced SEC, 12 produced SED, and 3 produced both SEC and SED. None of the isolates produced SEA or SEB.

A 29K envelope glycoprotein of equine arteritis virus expresses neutralization determinants recognized by murine monoclonal antibodies

A panel of six neutralizing murine monoclonal antibodies (MAbs) to equine arteritis virus (EAV) was produced. The MAbs were characterized by Western immunoblotting assay and competitive ELISA. The six MAbs identify a single neutralization site on a 29K envelope glycoprotein. Deglycosylation of viral proteins prior to immunoblotting showed that the 29K protein is the glycosylated form of a 20K protein. Equine anti-EAV serum also strongly bound the 29K glycoprotein, as well as an unglycosylated protein of 17K. The equine antisera to EAV blocked the binding of a selected MAb to EAV, whereas normal equine serum did not. Two neutralization-resistant escape mutant (EM) variants of the EAV prototype were produced using MAb 6D10. The phenotypic properties of the EM viruses were characterized by neutralization and immunoblotting assays with two MAbs (6D10 and 5G11). The two MAbs failed to neutralize either EM virus, and they did not react in an immunoblot assay with any proteins of the EM viruses. In contrast, binding of the equine antiserum to viral proteins was equivalent with prototype and EM virus strains. These data clearly indicate that a 29K envelope glycoprotein expresses at least one neutralization determinant of EAV.

Fatal experimental equine arteritis virus infection of a pregnant mare: Immunohistochemical staining of viral antigens

polyclonal (C2 -93 ) antibodies against VP7 and control antibodies (4H4 against NS2 of BTV and M1 00 against pseudorabies virus) and normal bovine serum. The bluetongue immunoblot assay was useful as a c-ELISA confirmatory test as demonstrated by evaluation of 2 2 experimental and 29 field samples. Validation of this assay is in progress by testing large number of samples (300-500) with BTV-positive and-negative status. Nevertheless, immunoblot assay is a sensitive, specific, and rapid confirmatory assay that can be used to demonstrate group-specific antibodies against BTV in sera with c-ELISA inhibition results > 50% of threshold value. Acknowledgements. We thank Dr. A. Afshar for the supply of monoclonal anti-VP7 antibodies and Dr. C. Dubuc for polyclonal anti-BTV antisera and MTSN and ELISA test results. We thank Drs. B . Brooks and R. Heckert for their suggestions and comments.

Flow cytometric analysis of in vitro bluetongue virus infection of bovine blood mononuclear cells

Cultures of adherent and non-adherent bovine peripheral blood mononuclear (PBM) cells were inoculated with bluetongue virus (BTV) serotype 10. Some cultures of non-adherent cells were stimulated with interleukin 2 (IL-2) and concanavalin A for 24 h prior to virus inoculation. Cells were harvested at various intervals up to 72 h after inoculation. A panel of leukocyte differentiation antigen-specific monoclonal antibodies (MAbs), specific for bovine CD2, CD4 or CD8, monocytes and granulocytes, B cells, gamma delta T cells or the IL-2 receptor (IL-2r), was directly conjugated to fluorescein isothiocyanate, and a MAb specific for the BTV major core protein VP7 was directly conjugated to phycoerythrin. Cells were labelled with conjugated MAbs in single- and double-label immunofluorescence studies to identify specifically the BTV-infected cells in inoculated cultures. The viability of cells was determined by propidium iodide exclusion, and all analyses were done using flow cytometry. Productive infection of cultures of PBM cells was confirmed by virus titration. The data revealed a clear difference between subsets of bovine PBM cells in susceptibility to infection with BTV in vitro. Monocytes were readily infected with BTV, as were stimulated CD4+ cells, and infection was cytopathic to monocytes and stimulated lymphocytes. The proportion of infected cells decreased after 24 h and virus titres dropped markedly by 72 h in all cultures. CD4+ cells in cultures of unstimulated non-adherent cells inoculated with BTV showed increased expression of IL-2r. The possible relevance of these findings to the pathogenesis of BTV infection of cattle is discussed.

Effect of selected dairy starter cultures on microbiological, chemical and sensory characteristics of swine and venison (Dama dama) nitrite-free dry-cured sausages

The aim of this study was the evaluation of selected lactic acid bacteria (LAB) starter culture of dairy origin in the production of nitrite-free low-acid fermented venison (Dama dama) sausage (salame di daino) produced in a small-scale plant in Umbria (Italy), and their effect on microbiological, physico-chemical and sensorial properties of the products. Salame di daino was obtained with two different processes: with and without the addition of selected LAB starter cultures. Microbial counts of Enterobacteriaceae, coliform organisms and Pseudomonas spp. were lower in salami made with the addition of starter cultures. Staphylococcus aureus, Salmonella spp, and Listeria monocytogenes after the first week of ripening were only detected from control salami. Control salami were paler and harder, whereas those made with the addition of starter cultures were slightly saltier, juicier and in general more acceptable. Selected dairy-origin starter (SDS) cultures did prevent the growth of both indicators of food safety and of process hygiene and increased the acceptability of full-ripened salami.

Response of the regional lymph node to bluetongue virus infection in calves.

Bluetongue virus (BTV) infection of cattle is characterized by prolonged cell-associated viremia. In an effort to further evaluate the antiviral response of BTV-infected cattle, the role of the regional lymph node (LN) in the immune response of calves to BTV was characterized. Calves were inoculated with BTV in the skin of the neck in an area drained by the superficial cervical LN. Calves were euthanized at regular intervals after inoculation and both BTV-challenged and contralateral (control) superficial cervical LNs were harvested. In addition, some calves had cannulation of the superficial cervical efferent lymphatics prior to inoculation. Lymphocyte subpopulation analysis was done on LN cell suspensions and lymph cells using a panel of cell-specific monoclonal antibodies. There was a significant increase in the proportion of B cells in the challenged LN after inoculation as compared with the control LN. In addition, BTV-specific antibodies were detected in efferent lymph plasma from the challenged LN in one cannulated calf by 9 days after inoculation (DAI), as determined by competitive enzyme-linked immunosorbent assay, whereas BTV-specific antibodies were not detected in serum from this calf through 12 DAI. Analysis of lymph cells revealed a sustained increase in cell output from the challenged LN due to an increase in lymphoblasts and CD8+ T cells. In contrast, the cell output from the control LN dropped markedly by 8 DAI and there was no significant increase in any specific cell population. Double label analysis characterized lymphoblasts as activated CD8+ cells, as determined by expression of MHC Class II antigens (CD8+ MHC II+). These cells were only transiently present as CD8+ MHC II+ cells were not identified in lymph from the challenged LN at 14 DAI. Few CD8+ MHC II+ cells were identified at any time in lymph from the control LN or in lymph from a mock infected calf. The data indicate that B cell proliferation in the challenged LN and release of activated CD8+ cells from this LN were specific responses to BTV infection. The rapid expansion of activated CD8+ T cells indicates that these cells may limit early viral spread. It is concluded that the regional LN draining inoculated skin is critical to the immune response of calves to BTV infection.

Identification of four distinct neutralizing epitopes on bluetongue virus serotype 10 using neutralizing monoclonal antibodies and neutralization-escape variants

Neutralizing sites on bluetongue virus serotype 10 (BTV-10) were investigated with a panel of seven neutralizing monoclonal antibodies (MAbs). Each MAb was coupled to agarose beads and tested against the other MAbs and a nonneutralizing control MAb in a competitive immueprecipitation assay. In addition, neutralization-escape viral variants of BTV-10 were identified and cloned by selecting individual plaques that formed in the presence of neutralizing MAbs. Four antigenic variants were isolated, each under the selective pressure of a different MAb. Parental BTV-10 and the four antigenic variants were compared by microneutralization assay using the MAb panel. The panel of neutralizing MAbs was subdivided into four groups on the basis of these assays, indicating that at least four distinct neutralizing epitopes exist on the BTV-10 virion. These epitopes are individually defined by representative MAbs 034, 039, 041, and 045, and the results indicate that the four epitopes are interacting sites within a single antigenic domain on BTV-10 outer capsid protein VP2. This conclusion is further supported by the fact that a double-site neutralization-escape variant designated DE34/39 (sequentially produced against MAbs 034 and 039) was not neutralized by any MAb of the panel.

Efficacy of Ultraviolet (UV-C) Light in a Thin-Film Turbulent Flow for the Reduction of Milkborne Pathogens.

Nonthermal technologies are being investigated as viable alternatives to, or supplemental utilization, with thermal pasteurization in the food-processing industry. In this study, the effect of ultraviolet (UV)-C light on the inactivation of seven milkborne pathogens (Listeria monocytogenes, Serratia marcescens, Salmonella Senftenberg, Yersinia enterocolitica, Aeromonas hydrophila, Escherichia coli, and Staphylococcus aureus) was evaluated. The pathogens were suspended in ultra-high-temperature whole milk and treated at UV doses between 0 and 5000 J/L at a flow rate of 4300 L/h in a thin-film turbulent flow-through pilot system. Of the seven milkborne pathogens tested, L. monocytogenes was the most UV resistant, requiring 2000 J/L of UV-C exposure to reach a 5-log reduction. The most sensitive bacterium was S. aureus, requiring only 1450 J/L to reach a 5-log reduction. This study demonstrated that the survival curves were nonlinear. Sigmoidal inactivation curves were observed for all tested bacterial strains. Nonlinear modeling of the inactivation data was a better fit than the traditional log-linear approach. Results obtained from this study indicate that UV illumination has the potential to be used as a nonthermal method to reduce microorganism populations in milk.