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Clinical pathology endpoints are evaluated during the course of toxicity studies to monitor structural and functional changes in organs and tissues in response to administration or withdrawal of a test item. A variety of assays can be performed in a clinical pathology laboratory with body fluids using either clinical pathology analyzers or other instruments. Information from the basic assays in clinical pathology, including hematology, clinical chemistry, urinalysis and coagulation are used with other safety assessment endpoints to evaluate immunotoxicity. Non-routine clinical pathology variables, such as acute phase proteins, cytokines, complement, hormones, autoantibodies, and bone marrow cytology are also included in preclinical toxicity studies as supplementary assays to ensure adequate interpretation of test item-related effects on the immune system. This chapter will describe the clinical pathology assays that are most useful to assess immunotoxicity.

It has long been a quest in ruminants to understand how two very similar mycobacterial species, Mycobacterium avium ssp. paratuberculosis (MAP) and Mycobacterium avium ssp. avium (MAA) lead to either a chronic persistent infection or a rapid-transient infection, respectively. Here, we hypothesized that when the host immune response is activated by MAP or MAA, the outcome of the infection depends on the early activation of signaling molecules and host temporal gene expression. To test our hypothesis, ligated jejuno-ileal loops including Peyer's patches in neonatal calves were inoculated with PBS, MAP, or MAA. A temporal analysis of the host transcriptome profile was conducted at several times post-infection (0.5, 1, 2, 4, 8 and 12 hours). When comparing the transcriptional responses of calves infected with the MAA versus MAP, discordant patterns of mucosal expression were clearly evident, and the numbers of unique transcripts altered were moderately less for MAA-infected tissue than were mucosal tissues infected with the MAP. To interpret these complex data, changes in the gene expression were further analyzed by dynamic Bayesian analysis. Bayesian network modeling identified mechanistic genes, gene-to-gene relationships, pathways and Gene Ontologies (GO) biological processes that are involved in specific cell activation during infection. MAP and MAA had significant different pathway perturbation at 0.5 and 12 hours post inoculation. Inverse processes were observed between MAP and MAA response for epithelial cell proliferation, negative regulation of chemotaxis, cell-cell adhesion mediated by integrin and regulation of cytokine-mediated signaling. MAP inoculated tissue had significantly lower expression of phagocytosis receptors such as mannose receptor and complement receptors. This study reveals that perturbation of genes and cellular pathways during MAP infection resulted in host evasion by mucosal membrane barrier weakening to access entry in the ileum, inhibition of Ca signaling associated with decreased phagosome-lysosome fusion as well as phagocytosis inhibition, bias toward Th2 cell immune response accompanied by cell recruitment, cell proliferation and cell differentiation; leading to persistent infection. Contrarily, MAA infection was related to cellular responses associated with activation of molecular pathways that release chemicals and cytokines involved with containment of infection and a strong bias toward Th1 immune response, resulting in a transient infection.

Pellegrin A.O., Miranda K.L., Figueiredo J.F., Barbosa E.F. & Lage A.P. 2011. The use of enzyme-linked immunosorbent assay and immunoblotting for the detection of Campylobacter fetus immunoglobulins in the cervico-vaginal mucus of female cattle. Pesquisa Veterinaria Brasileira 31(3):247-254. Embrapa Pantanal, Rua 21 de Setembro 1880, Corumba, MS 79320-900, Brazil. E-mail: aiesca@cpap.embrapa.br An indirect enzyme-linked immunosorbent assay was developed to detect antigen- specific secretory IgA antibodies to Campylobacter fetus subsp. venerealis in bovine vaginal mucus with a protein extract of the Campylobacter fetus subsp. venerealis by the acid glycine extraction method. Mean optical density measurement (λ=450 nm) was 0.143±0.9. The most immunoreactive protein bands of the Campylobacter fetus subsp. venerealis or Campylobacter fetus subsp. fetus recognized by IgA in immunoblotting, using bovine vaginal mucus samples, migrate at 42.6 kDa. The protein that migrates at 93 kDa was recognized exclusively for C. fetus subsp. venerealis. A positive vaginal mucus sample of a cow from negative herd recognized antigens of C. jejuni subsp. jejuni e C. fetus subsp. fetus.

The role of neutrophils in the pathogenesis of Salmonella enterica Typhimurium–induced ruminant and human enteritis and diarrhea has yet to be characterized with in vivo models. To address this question, the in vivo bovine ligated ileal loop model of nontyphoidal salmonellosis was used in calves with the naturally occurring bovine leukocyte adhesion deficiency (BLAD) mutation whose neutrophils are unable to extravasate and infiltrate the extravascular matrix. Data obtained from 4 BLAD Holstein calves homozygous for BLAD (CD18–), 1 to 5 weeks of age, were compared with 4 controls, age-matched Holstein calves negative for BLAD (CD18+). Morphologic studies revealed that infection of CD18– calves with S Typhimurium resulted in no significant tissue infiltration by neutrophils, less tissue damage, reduced luminal fluid accumulation, and increased bacterial invasion, when compared with CD18+ calves. Ultrastructurally, lesions in enterocytes induced by S Typhimurium infection in CD18– calves—including attachment and disruption of the brush border, apical membrane ruffling formation, and cellular degeneration—were similar to the ones reported in the literature for CD18– calves. Study of cytokine gene expression by quantitative real-time polymerase chain reaction revealed that early stages of acute infection (4-8 hours postinfection) were associated with increased interleukin 8 gene expression in the absence of tissue influx of neutrophils in CD18– calves, whereas later stages of infection (12 hours postinfection) were associated with increased expression of growth-related oncogene α in the presence of neutrophil influx in CD18+ calves. In contrast, the proinflammatory cytokines interleukin 1β and tumor necrosis factor α were poorly correlated with the presence or absence of tissue neutrophils.

The hematopoietic sites of juvenile rats change as the animals advance from PND 0 to 42. At PND 0 and 1, hematopoiesis occurs primarily in the liver and spleen. Hepatic hematopoiesis gradually decreases from PND 7 through 28 and reaches adult levels by PND 35. Splenic hematopoiesis is marked from PND 0 to between PND 7 and 14, after which it gradually decreases to PND 42. Bone marrow hematopoiesis is minimal at PND 0 and 1 both in the femur and sternum. By PND 7 the femur and sternum marrow cavities are heavily populated by hematopoietic cells which persist beyond PND 42. In the marrow, an increased proportion of lymphocytes is noted mainly from PND 14 to 42. The goal of this chapter is to describe postnatal hematopoiesis, including both intramedullary and extramedullary hematopoiesis (EMH), from PND 0 to 42.

Salmonella enterica serotype Typhi is a strictly human adapted pathogen that does not cause disease in nonprimate vertebrate hosts, while Salmonella enterica serotype Typhimurium is a broad-host-range pathogen. Serotype Typhi lacks some of the proteins (effectors) exported by the invasion-associated type III secretion system that are required by serotype Typhimurium for eliciting fluid secretion and inflammation in bovine ligated ileal loops. We investigated whether the remaining serotype Typhi effectors implicated in enteropathogenicity (SipA, SopB, and SopD) are functionally exchangeable with their serotype Typhimurium homologues. Serotype Typhi elicited fluid accumulation in bovine ligated ileal loops at levels similar to those elicited by a noninvasive serotype Typhimurium strain (the sipA sopABDE2 mutant) or by sterile culture medium. However, introduction of the cloned serotype Typhi sipA , sopB , and sopD genes complemented the ability of a serotype Typhimurium sipA sopABDE2 mutant to elicit fluid secretion in bovine ligated ileal loops. Introduction of the cloned serotype Typhi sipA , sopB , and sopD genes increased the invasiveness of a serotype Typhimurium sipA sopABDE2 mutant for human colon carcinoma epithelial (HT-29 and T84) cells and bovine kidney (MDBK) cells. Translational fusions between the mature TEM-1 β-lactamase reporter and SipA or SopD demonstrated that serotype Typhi translocates these effectors into host cells. We conclude that the inability of serotype Typhi to cause fluid accumulation in bovine ligated ileal loops is not caused by a functional alteration of its SipA, SopB, and SopD effector proteins with respect to their serotype Typhimurium homologues.

Knowledge about the origin and identity of the microbial products recognized by the innate immune system is important for understanding the pathogenesis of inflammatory diseases. We investigated the potential role of Salmonella enterica serotype Typhimurium fimbriae as pathogen-associated molecular patterns (PAMPs) that may stimulate innate pathways of inflammation. We screened a panel of 11 mutants, each carrying a deletion of a different fimbrial operon, for their enteropathogenicity using the calf model of human gastroenteritis. One mutant (csgBA) was attenuated in its ability to elicit fluid accumulation and GROalpha mRNA expression in bovine ligated ileal loops. The mechanism by which thin curled fimbriae encoded by the csg genes contribute to inflammation was further investigated using tissue culture. The S. Typhimurium csgBA mutant induced significantly less IL-8 production than the wild type in human macrophage-like cells. Purified thin curled fimbriae induced IL-8 expression in human embryonic kidney (HEK293) cells transfected with Toll-like receptor (TLR) 2/CD14 but not in cells transfected with TLR5, TLR4/MD2/CD14 or TLR11. Fusion proteins between the major fimbrial subunit of thin curled fimbriae (CsgA) and glutathione-S-transferase (GST) elicited IL-8 production in HEK293 cells transfected with TLR2/CD14. Proteinase K treatment abrogated IL-8 production elicited in these cells by GST-CsgA, but not by synthetic lipoprotein. GST-CsgA elicited more IL-6 production than GST in bone marrow-derived macrophages from TLR2+/+ mice, while there was no difference in IL-6 secretion between GST-CsgA and GST in macrophages from TLR2-/- mice. These data suggested that CsgA is a PAMP that is recognized by TLR2.

The frequency of bovine genital campylobacteriosis (BGC) in 157 cows from nine herds from the microregion of Varginha - Minas Gerais - Brazil was evaluated. Farmers use either artificial insemination and natural breeding after two insemination procedures or natural breeding. The diagnosis of BGC was performed by the direct fluorescent antibody test (DFAT) using vaginal mucus. All studied herds showed C. fetus infected animals and, of 157 animals, 40 (25.5%) were positive in DFAT: 7 (26.9%) from herds with natural breeding and 33 (25.1%) from herds with both artificial insemination and natural breeding after the second-to-third insemination. The high frequency of BGC found in this study shows that this disease is present among herds which have reproductive problems and the use of natural breeding after the second-to-third unsuccessful insemination could be a risk factor for the disease.

The Salmonella enterica serotype Typhimurium (S. Typhimurium) genome contains 13 putative fimbrial operons termed agf (csg), fim, pef, lpf, bcf, saf, stb, stc, std, stf, sth, sti and stj. Evidence for in vitro expression of fimbrial proteins encoded by these operons is currently only available for agf, fim and pef. We raised antisera against putative major fimbrial subunits of S. Typhimurium, including AgfA, FimA, PefA, LpfA, BcfA, StbA, StcA, StdA, StfA, SthA and StiA. Elaboration of StcA on the bacterial surface could be detected by flow cytometry and immunoelectron microscopy after expression of the cloned stcABCD operon from a heterologous T7 promoter in Escherichia coli. To study the expression of fimbrial antigens in S. Typhimurium by flow cytometry, we constructed strains carrying deletions of agfAB, pefBACDI, lpfABCDE, bcfABCDEFG, stbABCD, stcABC, stdAB, stfACDEFG, sthABCDE or stiABCDE. Using these deletion mutants for gating, expression of fimbrial antigens was measured by flow cytometry in cultures grown in vitro or in samples recovered 8 h after infection of bovine ligated ileal loops with S. Typhimurium. FimA was the only fimbrial antigen expressed by S. Typhimurium after static growth in Luria-Bertani (LB) broth. Injection of static LB broth cultures of S. Typhimurium into bovine ligated ileal loops resulted in the expression of BcfA, FimA, LpfA, PefA, StbA, StcA, StdA, StfA and StiA. These data show that in vivo growth conditions drastically alter the repertoire of fimbrial antigens expressed in S. Typhimurium.

Recent studies on the molecular pathogenesis of Salmonella enterica serotype Typhimurium-induced enterocolitis using tissue culture models and the neonatal calf model have led to an improved understanding of key events occurring during the complex series of host-pathogen interactions leading to

An 8-year-old male neutered Labrador Retriever was referred to the University of Wisconsin Veterinary Medical Teaching Hospital with a presumptive diagnosis of leukemia. Hematologic abnormalities included normal neutrophil count with a left shift, monocytosis, eosinophilia, thrombocytopenia, and circulating immature mononuclear cells. Bone marrow was effaced by immature hematopoietic cells of various morphologic appearances. In addition, large multinucleated cells were observed frequently. Flow cytometric analysis of nucleated cells in blood revealed 34% CD34(+) cells, consistent with acute leukemia. By immunocytochemical analysis of cells in blood and bone marrow, some mononuclear cells expressed CD18, myeloperoxidase, and CD11b, indicating myeloid origin; some, but not all, large multinucleated cells expressed CD117 and CD42b, the latter supporting megakaryocytic lineage. The diagnosis was acute myeloblastic leukemia without maturation (AML-M1). To identify genetic aberrations associated with this malignancy, cells from formalin-fixed paraffin-embedded bone marrow were analyzed cytogenetically by multicolor fluorescence in situ hybridization (FISH). Co-localization of bacterial artificial chromosome (BAC) containing BCR and ABL was evident in 32% of cells. This confirmed the presence of the canine BCR-ABL translocation or Raleigh chromosome. In people, the analogous translocation or Philadelphia chromosome is characteristic of chronic myelogenous leukemia (CML) and is rarely reported in AML. BCR-ABL translocation also has been identified in dogs with CML; however, to our knowledge this is the first report of AML with a BCR-ABL translocation in a domestic animal.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of chronic enteritis in ruminants (Johne's disease) and a possible etiopathologic agent in human Crohn's disease. The host-pathogen interaction in this chronic disease has largely depended on the randomly collected static lesions studied in subclinically or clinically infected animals. We have established and utilized the neonatal calf ligated ileal loop model to study the early temporal host changes during MAP infection. After inoculation of ligated ileal loop with MAP, samples were analyzed for bacterial invasion, histologic and ultrastructural morphologic changes, and gene expression at several times (0.5–12 hours) postinfection. Our results indicate that MAP invades the intestinal mucosa as early as 0.5 hour postinoculation. Distribution and migration of neutrophils, monocytes/macrophages, and goblet cells were confirmed by histopathology, scanning and transmission electron microscopy. Coincident with the morphologic analysis, we measured by real-time polymerase chain reaction gene expression of various cytokines/chemokines that are involved in the recruitment of mononuclear and polymorphonuclear leukocytes to the site of infection. We also detected expression of several other genes, including intestinal-trefoil factor, profilin, lactoferrin, and enteric β-defensin, which may play significant roles in the early MAP infection. Thus, the calf ligated intestinal loop model may be used as a human disease model to understand the role of MAP in the pathogenesis of Crohn's disease.

The invasion-associated type III secretion system (T3SS-1) of S. Typhimurium is required to initiate and sustain an acute inflammatory response in the intestine. We investigated the relationship of S. Typhimurium T3SS-1-induced IL-8 expression and invasion with intracellular Ca(2+) mobilization in HeLa cells. Compared to the sipAsopABDE2 mutant, strains carrying a mutation in sipA, or mutations in sopABDE2 induced higher levels of IL-8 and greater bacterial internalization despite the fact that these mutants elicited similarly low intracellular concentrations of Ca(2+). Likewise, complemented sipAsopABDE2 mutant with sopE2 did not affect intracellular Ca(2+) concentrations or IL-8 expression, but significantly increased bacterial internalization. Treating HeLa cells with the calcium chelator BAPTA-AM or with D-BAPTA-AM, a derivative with greatly reduced Ca(2+) chelating activity, yielded strong evidence that BAPTA-AM does not affect invasion and inhibits IL-8 secretion by a calcium-dependent mechanism. These findings suggest that, although wild-type S. Typhimurium-induced IL-8 expression and bacterial internalization in HeLa cells coincides with increased cytosolic Ca(2+), the differing levels of IL-8 and invasion induced by strains carrying different effector proteins are unrelated to levels of intracellular Ca(2+).

The role of Salmonella typhimurium type III secretion system (T3SS-1)-translocated proteins in chemokines' expression and protein phosphorylation was investigated in HeLa cells. Infection of HeLa cells with S. typhimurium activated IL-8 and GRO-α expression at higher levels than infection with a S. typhimurium sipAsopABDE2 mutant, confirming that T3SS-1-secreted proteins are required to fully induce chemokine expression in HeLa cells. A S. typhimurium sipAsopABDE2 mutant complemented with sipA or a strain carrying a chromosomal copy of sipA (sopABDE2 mutant) activated chemokines at significantly higher levels than a S. typhimurium sipAsopABDE2 mutant. However, extracellular addition of recombinant SipA failed to induce IL-8 expression. Phosphorylation analyses revealed that S. typhimurium induced a twofold increase in the phosphorylation of B23, CREB1, ERK1, JUN, p38MAPK, and NR1. JUN and p38MAPK were phosphorylated by S. typhimurium carrying a chromosomal copy of sipA (sopABDE2 mutant) while none was more than twofold phosphorylated in cells infected with the S. typhimurium sipAsopABDE2 mutant. Treating cells with JUN and p38MAPK inhibitors significantly decreased IL-8 expression in sopABDE2 mutant infected cells. These data indicate that S. typhimurium SipA induces expression of CXC chemokines through phosphorylation of IL-8-transcription regulatory proteins, JUN and p38MAK.

Knowledge about the origin and identity of the microbial products recognized by the innate immune system is important for understanding the pathogenesis of inflammatory diseases. We investigated the potential role of Salmonella enterica serotype Typhimurium fimbriae as pathogen-associated molecular patterns (PAMPs) that may stimulate innate pathways of inflammation. We screened a panel of 11 mutants, each carrying a deletion of a different fimbrial operon, for their enteropathogenicity using the calf model of human gastroenteritis. One mutant (csgBA) was attenuated in its ability to elicit fluid accumulation and GROalpha mRNA expression in bovine ligated ileal loops. The mechanism by which thin curled fimbriae encoded by the csg genes contribute to inflammation was further investigated using tissue culture. The S. Typhimurium csgBA mutant induced significantly less IL-8 production than the wild type in human macrophage-like cells. Purified thin curled fimbriae induced IL-8 expression in human embryonic kidney (HEK293) cells transfected with Toll-like receptor (TLR) 2/CD14 but not in cells transfected with TLR5, TLR4/MD2/CD14 or TLR11. Fusion proteins between the major fimbrial subunit of thin curled fimbriae (CsgA) and glutathione-S-transferase (GST) elicited IL-8 production in HEK293 cells transfected with TLR2/CD14. Proteinase K treatment abrogated IL-8 production elicited in these cells by GST-CsgA, but not by synthetic lipoprotein. GST-CsgA elicited more IL-6 production than GST in bone marrow-derived macrophages from TLR2+/+ mice, while there was no difference in IL-6 secretion between GST-CsgA and GST in macrophages from TLR2-/- mice. These data suggested that CsgA is a PAMP that is recognized by TLR2.

A comparative study of the indirect haemagglutination (IHA), immunofluorescence (IFAT) and immunoenzymatic (ELISA) tests was carried out to determine the prevalence of Toxoplasma gondii antibodies in goats. One hundred seventy-four serum samples were obtained from four goat herds from the region of Uberlândia, State of Minas Gerais. The distribution of the animals, according to their origin, was as follow: 71 from herd I; 39 from herd II; 37 from herd III; and 27 from herd IV. Serum samples were analyzed by IHA, IFAT and ELISA, considering the reactivity of the serum samples at dilution > or = 1:64 as cut off titer for the three tests. A global seroprevalence of 18.4% was observed, with significantly higher positivity rate in the herd II (66.7%) and older animals (> 36 months). A high and significant positive correlation was found between the titers obtained by the IHA versus IFAT, IHA versus ELISA, and ELISA versus IFAT. Therefore, it can be concluded that the three analyzed tests have shown to be highly concordant and appropriate for epidemiological surveys of Toxoplasma infection in goats. Although the seroprevalence of T. gondii infection in goats is relatively low in this region as compared to other regions of the country, adequate management might be useful and essential to control the infection in the goat herds.

Survival and persistence of Mycobacterium avium subsp. paratuberculosis (MAP) in the intestinal mucosa is associated with host immune tolerance. However, the initial events during MAP interaction with its host that lead to pathogen survival, granulomatous inflammation, and clinical disease progression are poorly defined. We hypothesize that immune tolerance is initiated upon initial contact of MAP with the intestinal Peyer's patch. To test our hypothesis, ligated ileal loops in neonatal calves were infected with MAP. Intestinal tissue RNAs were collected (0.5, 1, 2, 4, 8 and 12 hrs post-infection), processed, and hybridized to bovine gene expression microarrays. By comparing the gene transcription responses of calves infected with the MAP, informative complex patterns of expression were clearly visible. To interpret these complex data, changes in the gene expression were further analyzed by dynamic Bayesian analysis, and genes were grouped into the specific pathways and gene ontology categories to create a holistic model. This model revealed three different phases of responses: i) early (30 min and 1 hr post-infection), ii) intermediate (2, 4 and 8 hrs post-infection), and iii) late (12 hrs post-infection). We describe here the data that include expression profiles for perturbed pathways, as well as, mechanistic genes (genes predicted to have regulatory influence) that are associated with immune tolerance. In the Early Phase of MAP infection, multiple pathways were initiated in response to MAP invasion via receptor mediated endocytosis and changes in intestinal permeability. During the Intermediate Phase, perturbed pathways involved the inflammatory responses, cytokine-cytokine receptor interaction, and cell-cell signaling. During the Late Phase of infection, gene responses associated with immune tolerance were initiated at the level of T-cell signaling. Our study provides evidence that MAP infection resulted in differentially regulated genes, perturbed pathways and specifically modified mechanistic genes contributing to the colonization of Peyer's patch.