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Brockmeier S.L.,Virus and Prion Diseases Research Unit | Register K.B.,Ruminant Diseases and Immunology Research Unit | Kuehn J.S.,Iowa State University | Nicholson T.L.,Virus and Prion Diseases Research Unit | And 4 more authors.
PLoS ONE | Year: 2014

Haemophilus parasuis is the cause of Glässer's disease in swine, which is characterized by systemic infection resulting in polyserositis, meningitis, and arthritis. Investigation of this animal disease is complicated by the enormous differences in the severity of disease caused by H. parasuis strains, ranging from lethal systemic disease to subclinical carriage. To identify differences in genotype that could account for virulence phenotypes, we established the virulence of, and performed whole genome sequence analysis on, 11 H. parasuis strains. Virulence was assessed by evaluating morbidity and mortality following intranasal challenge of Caesarean-derived, colostrum-deprived (CDCD) pigs. Genomic DNA from strains Nagasaki (serotype 5), 12939 (serotype 1), SW140 (serotype 2), 29755 (serotype 5), MN-H (serotype 13), 84-15995 (serotype 15), SW114 (serotype 3), H465 (serotype 11), D74 (serotype 9), and 174 (serotype 7) was used to generate Illumina paired-end libraries for genomic sequencing and de novo assembly. H. parasuis strains Nagasaki, 12939, SH0165 (serotype 5), SW140, 29755, and MN-H exhibited a high level of virulence. Despite minor differences in expression of disease among these groups, all pigs challenged with these strains developed clinical signs consistent with Glässer's disease between 1-7 days post-challenge. H. parasuis strains 84-15995 and SW114 were moderately virulent, in that approximately half of the pigs infected with each developed Glässer's disease. H. parasuis strains H465, D74, and 174 were minimally virulent or avirulent in the CDCD pig model. Comparative genomic analysis among strains identified several noteworthy differences in coding regions. These coding regions include predicted outer membrane, metabolism, and pilin or adhesin related genes, some of which likely contributed to the differences in virulence and systemic disease observed following challenge. These data will be useful for identifying H. parasuis virulence factors and vaccine targets. Source

Sacco R.E.,Ruminant Diseases and Immunology Unit | McGill J.L.,Ruminant Diseases and Immunology Unit | Palmer M.V.,Infectious Bacterial Diseases Research Unit | Lippolis J.D.,Ruminant Diseases and Immunology Unit | And 2 more authors.
Viruses | Year: 2012

Respiratory syncytial virus (RSV) is the most common viral cause of childhood acute lower respiratory tract infections. It is estimated that RSV infections result in more than 100,000 deaths annually worldwide. Bovine RSV is a cause of enzootic pneumonia in young dairy calves and summer pneumonia in nursing beef calves. Furthermore, bovine RSV plays a significant role in bovine respiratory disease complex, the most prevalent cause of morbidity and mortality among feedlot cattle. Infection of calves with bovine RSV shares features in common with RSV infection in children, such as an age-dependent susceptibility. In addition, comparable microscopic lesions consisting of bronchiolar neutrophilic infiltrates, epithelial cell necrosis, and syncytial cell formation are observed. Further, our studies have shown an upregulation of pro-inflammatory mediators in RSV-infected calves, including IL-12p40 and CXCL8 (IL-8). This finding is consistent with increased levels of IL-8 observed in children with RSV bronchiolitis. Since rodents lack IL-8, neonatal calves can be useful for studies of IL-8 regulation in response to RSV infection. We have recently found that vitamin D in milk replacer diets can be manipulated to produce calves differing in circulating 25-hydroxyvitamin D3. The results to date indicate that although the vitamin D intracrine pathway is activated during RSV infection, pro-inflammatory mediators frequently inhibited by the vitamin D intacrine pathway in vitro are, in fact, upregulated or unaffected in lungs of infected calves. This review will summarize available data that provide parallels between bovine RSV infection in neonatal calves and human RSV in infants. © 2012 by the authors; licensee MDPI, Basel, Switzerland. Source

Wu Q.,National Centers for Coastal Ocean Science | Prager K.C.,University of California at Los Angeles | Prager K.C.,U.S. National Institutes of Health | Prager K.C.,Marine Mammal Center | And 7 more authors.
Diseases of Aquatic Organisms | Year: 2014

Several real-time PCR assays are currently used for detection of pathogenic Leptospira spp.; however, few methods have been described for the successful evaluation of clinical urine samples. This study reports a rapid assay for the detection of pathogenic Leptospira spp. in California sea lions Zalophus californianus using real-time PCR with primers and a probe targeting the lipL32 gene. The PCR assay had high analytic sensitivity-the limit of detection was 3 genome copies per PCR volume using L. interrogans serovar Pomona DNA and 100% analytic specificity; it detected all pathogenic leptospiral serovars tested and none of the non-pathogenic Leptospira species (L. biflexa and L. meyeri serovar Semaranga), the intermediate species L. inadai, or the non-Leptospira pathogens tested. Our assay had an amplification efficiency of 1.00. Comparisons between the real-time PCR assay and culture isolation for detection of pathogenic Leptospira spp. in urine and kidney tissue samples from California sea lions showed that samples were more often positive by real-time PCR than by culture methods. Inclusion of an internal amplification control in the real-time PCR assay showed no inhibitory effects in PCR negative samples. These studies indicated that our real-time PCR assay has high analytic sensitivity and specificity for the rapid detection of pathogenic Leptospira species in urine and kidney tissue samples. © Inter-Research 2014. Source

Prager K.C.,University of California at Los Angeles | Prager K.C.,U.S. National Institutes of Health | Alt D.P.,Infectious Bacterial Diseases Research Unit | Buhnerkempe M.G.,University of California at Los Angeles | And 7 more authors.
Aquatic Mammals | Year: 2015

Stranded California sea lions (Zalophus cali- fornianus) along the California coast have been diagnosed with leptospirosis every year since at least the 1980s. Between September 2010 and November 2011, we followed 14 stranded California sea lions that survived to release and evaluated antibiotic efficacy in eliminating lep-tospiruria (urinary shedding of leptospires). Leptospiruria was assessed by real-time PCR of urine and urine culture, with persistence assessed using longitudinally collected samples. Serum chemistry was used to assess recovery of normal renal function. Microscopic agglutination testing (MAT) was performed to assess serum anti-Lepto-spira antibody titers, and the MAT reactivity pat-terns were consistent with L. interrogans serovar Pomona infection frequently observed in this population. Animals were initially treated for 6 to 16 d (median = 10.5; mean = 10.8) with antibiot-ics from the penicillin family, with some receiving additional antibiotics to treat other medical condi-tions. All urine cultures were negative; therefore, the presence of leptospiruria was assessed using PCR. Leptospiruria continued beyond the initial course of penicillin family antibiotics in 13 of the 14 sea lions, beyond the last antibiotic dose in 11 of the 14 sea lions, beyond recovery of renal func-tion in 13 of the 14 sea lions, and persisted for at least 8 to 86 d (median = 45; mean = 46.8). Five animals were released with no negative urine PCR results detected; thus, their total shedding duration may have been longer. Cessation of leptospiruria was more likely in animals that received antibiot-ics for a greater duration, especially if coverage was uninterrupted. Real-time PCR results indicate that an antibiotic protocol commonly used to treat leptospirosis in rehabilitating California sea lions does not eliminate leptospiruria. It is possible that antibiotic protocols given for a longer duration and/or including other antibiotics may be effec-tive in eliminating leptospiruria. These results may have important human and animal health implications, especially in rehabilitation facilities, as Leptospira transmission may occur through contact with animals with persistent leptospiruria. Source

Maggioli M.F.,Infectious Bacterial Diseases Research Unit | Maggioli M.F.,Iowa State University | Palmer M.V.,Infectious Bacterial Diseases Research Unit | Vordermeier H.M.,UK Veterinary Laboratories Agency | And 4 more authors.
Journal of Visualized Experiments | Year: 2015

Effector and memory T cells are generated through developmental programing of naïve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-γ. Interferon-γ release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-γ ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture. © 2015 Journal of Visualized Experiments. Source

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