Large-scale investigation of the parameters in response to Eimeria maxima challenge in broilers.

Coccidiosis, a parasitic disease of the intestinal tract caused by members of the genera Eimeria and Isospora, is one of the most common and costly diseases in chicken. The aims of this study were to assess the effect of the challenge and level of variability of measured parameters in chickens during the challenge with Eimeria maxima. Furthermore, this study aimed to investigate which parameters are the most relevant indicators of the health status. Finally, the study also aimed to estimate accuracy of prediction for traits that cannot be measured on large scale (such as intestinal lesion score and fecal oocyst count) using parameters that can easily be measured on all animals. The study was performed in 2 parts: a pilot challenge on 240 animals followed by a large-scale challenge on 2,024 animals. In both experiments, animals were challenged with 50,000 Eimeria maxima oocysts at 16 d of age. In the pilot challenge, all animals were measured for BW gain, plasma coloration, hematocrit, and rectal temperature and, in addition, a subset of 48 animals was measured for oocyst count and the intestinal lesion score. All animals from the second challenge were measured for BW gain, plasma coloration, and hematocrit whereas a subset of 184 animals was measured for intestinal lesion score, fecal oocyst count, blood parameters, and plasma protein content and composition. Most of the parameters measured were significantly affected by the challenge. Lesion scores for duodenum and jejunum (P < 0.001), oocyst count (P < 0.05), plasma coloration for the optical density values between 450 and 490 nm (P < 0.001), albumin (P < 0.001), α1-globulin (P < 0.01), α2-globulin (P < 0.001), α3-globulin (P < 0.01), and ß2-globulin (P < 0.001) were the most strongly affected parameters and expressed the greatest levels of variation. Plasma protein profiles proved to be a new, reliable parameter for measuring response to Eimeria maxima. Prediction of intestinal lesion score and fecal oocyst count using the other parameters measured was not very precise (R2 < 0.7). The study was successfully performed in real raising conditions on a large scale. Finally, we observed a high variability in response to the challenge, suggesting that broilers' response to Eimeria maxima has a strong genetic determinism, which may be improved by genetic selection.

Fecundity of Cryptosporidium parvum is correlated with intracellular levels of the viral symbiont CPV.

Differences in the virulence and fecundity of Cryptosporidium parvum isolates have been observed by several researchers studying cryptosporidiosis. The purpose of the present study was to determine if there was a correlation between intracellular levels of the viral symbiont CPV in C. parvum and fecundity of two isolates of the parasite, namely C. parvum Beltsville (B) and C. parvum Iowa (I). Dairy calves infected with 10(6)C. parvum-B excreted 5-fold more oocysts compared with calves infected with the same number of C. parvum-I oocysts. The increased fecundity of the former strain was corroborated by semi-quantitative PCR assay of DNA isolated from cell cultures infected with either C. parvum-B or C. parvum-I. Quantitative reverse transcriptase-PCR analysis of viral RNA revealed a 3-fold greater number of CPV in C. parvum-B compared with C. parvum-I oocysts. These findings may indicate a role for CPV in fecundity and possibly virulence of C. parvum.

Gene expression and antimicrobial activity of bovine macrophages in response to Mycobacterium avium subsp. paratuberculosis.

We evaluated gene expression and antimicrobial responses of bovine monocyte-derived macrophages incubated with Mycobacterium avium subsp. paratuberculosis (M. a. ptb), the causative agent of Johne's disease. Gene expression was evaluated by the use of human noncompetitive high-density oligonucleotide microarrays. Bovine messenger RNA hybridized with 14.2-18.2% of the 12,600 oligonucleotide probe sets. When macrophages incubated with M. a. ptb were compared with nonactivated control macrophages, macrophages activated by addition of interferon-gamma and lipopolysaccharide, and macrophages incubated with Mycobacterium avium subspecies avium (M. a. a), 47, 79, and 27 genes, respectively, were differentially expressed. Differential expression of six of these genes was confirmed using reverse transcriptase polymerase chain reaction. Several functional assays were performed to evaluate the potential relevance of differentially expressed genes to host defense. Macrophages phagocytizing M. a. a had a greater capacity to kill the organisms and to acidify phagosomes and a greater degree of apoptosis than did macrophages incubated with M. a. ptb. The results of these studies indicate that multiple genes and metabolic pathways are differentially expressed by macrophages ingesting mycobacterial organisms. Although the intracellular fate of mycobacterial organisms appears to be dependent on a complex interaction between macrophage and organism, phagosome acidification and apoptosis may play central roles in organism survival.

Pharmacological inhibition of Mannheimia haemolytica lipopolysaccharide and leukotoxin-induced cytokine expression in bovine alveolar macrophages.

The inflammatory cytokines tumor necrosis factor-alpha (TNFalpha), interleukin-1 beta (IL-1beta), and interleukin-8 (IL-8) are believed to contribute to the pathogenesis of lung injury in bovine pneumonic mannheimiosis (BPM) caused by Mannheimia (Pasteurella) haemolytica. Inflammatory cytokines may, therefore, represent therapeutic targets to be modulated for the purpose of treating or preventing this important disease of cattle. The purpose of this study was to evaluate the ability of six pharmacological agents to suppress the expression of TNFalpha, IL-1beta, and IL-8 genes and proteins in bovine alveolar macrophages (AM) exposed to M. haemolytica lipopolysaccharide (LPS) and leukotoxin (LktA) in vitro. The compounds tested included dexamethasone (DEX), tetrahydropapaveroline (THP), pentoxifylline (PTX), rolipram (ROL), SB203580 (SB), and thalidomide (THL). Cytokine expression was induced by the addition of purified M. haemolytica LPS and LktA to AM cell cultures following pretreatment with inhibitor compounds. Secretion of TNFalpha, IL-1beta, and IL-8 proteins into the cell culture supernatant was measured using enzyme-linked immunosorbent assays, and steady-state accumulation of cytokine-specific mRNA was measured by northern blot analysis. Dose-dependent inhibition of cytokine secretion occurred in response to pretreatment of AM with DEX (TNFalpha, IL-1beta, IL-8), THP (TNFalpha, IL-1beta, IL-8), PTX (TNFalpha, IL-1beta, IL-8), ROL (TNFalpha, IL-1beta), and SB (TNFalpha, IL-8). Significant dose-dependent inhibition of cytokine mRNA expression occurred in response to pretreatment with DEX (TNFalpha, IL-1beta, IL-8), THP (TNFalpha, IL-1beta, IL-8), and PTX (TNFalpha). DEX was the most effective inhibitor by far; pretreatment with this compound yielded greater than 95% inhibition of cytokine gene and protein expression over a broad range of concentrations. These findings demonstrate that DEX, THP, PTX, ROL, and SB are capable of suppressing inflammatory cytokine secretion by bovine AM in vitro. If pulmonary cytokine secretion may be similarly inhibited in vivo, anti-cytokine therapy may represent a novel strategy for the management of BPM.

Sequence analysis of the nucleocapsid and phosphoprotein genes of avian pneumoviruses circulating in the US.

Avian pneumovirus (APV) has recently been described as the cause of a new respiratory syndrome in turkey flocks in the United States. We here describe the complete sequence of the nucleocapsid (N) and phosphoprotein (P) genes of this emerging APV (APV/US). Our results show 59 and 61% nucleotide sequence identity of the APV/US N gene with N genes of previously described European APV subgroups A and B, respectively. The P gene of APV/US showed only 53% nucleotide sequence identity with the ortholog from APV subgroup A. Phylogenetic analyses of both N and P genes clearly demonstrate that the APV/US lineage is evolutionarily related but distinct from European APVs. Moreover, sequence analysis of the N and P genes from two laboratory adapted isolates of APV/US (APV/MN-1a and APV/MN-1b) and from ten clinical samples from APV-infected turkeys suggests only modest level of amino acid divergence in the N (0-0.3%) and P (0-1.4%) proteins. Taken together, the results of this study indicate support that APV/US represents a new subgroup (subgroup C) of APV and show that there is limited heterogeneity in the N and P genes of APV/US isolates.

Lipopolysaccharide enhances cytolysis and inflammatory cytokine induction in bovine alveolar macrophages exposed to Pasteurella (Mannheimia) haemolytica leukotoxin.

Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) and lipopolysaccharide (LPS) are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Previous studies have characterized in vitro responses of bovine alveolar macrophages (AMs) to Lkt and LPS. Activation of AMs with Lkt or LPS causes induction of proinflammatory cytokines, and Lkt causes cytolysis of AMs at higher concentrations. Since AMs are exposed to both of these bacterial virulence factors during disease, previous studies may have underestimated the possibility of functional interactions between Lkt and LPS. The purpose of this study was to characterize the effect of simultaneous exposure to both Lkt and LPS on AM cytolysis and proinflammatory cytokine expression. Using cellular leakage of lactate dehydrogenase as an indirect measure of cytolysis, we studied AM responses to Lkt alone, LPS alone and Lkt+LPS. We found that 80-200 pg/ml LPS, which does not itself cause cytolysis, synergistically enhanced the cytolysis induced by 2-5 Lkt units (LU)/ml Lkt. Northern blot analysis demonstrated that synergism between Lkt and LPS resulted in increased levels of IL-8 mRNA, and that the kinetic patterns of TNF-alpha and IL-8 mRNA expression induced by Lkt+LPS differed from those induced by each agent separately. Finally, the WEHI 164 (clone 13) bioassay was used to show that Lkt/LPS synergism resulted in enhanced secretion of biologically active TNF-alpha. These results provide direct evidence of synergism between Lkt and LPS in AM cytolysis and inflammatory cytokine expression. Additional studies to characterize the molecular basis of this phenomenon are indicated.

Pulmonary expression of tumor necrosis factor alpha, interleukin-1 beta, and interleukin-8 in the acute phase of bovine pneumonic pasteurellosis.

Inflammatory cytokines are suspected to contribute to the pathogenesis of bovine pneumonic pasteurellosis (BPP) through neutrophil recruitment, leukocyte activation, and the induction of a broad array of soluble inflammatory mediators. An in vivo experimental model of BPP was used to characterize the pulmonary expression kinetics of tumor necrosis factor alpha (TNFalpha), interleukin-1 beta (IL-1beta), and interleukin-8 (IL-8) genes and proteins during the acute phase of disease development. Cytokine expression in bronchoalveolar lavage (BAL) fluid, BAL cells, and pneumonic lung parenchyma was quantitated by northern blot analysis, enzyme-linked immunosorbent assay (ELISA), and in situ hybridization at 2, 4, 8, 16, and 24 hours after endobronchial inoculation of Pasteurella (Mannheimia) haemolytica. Expression of TNFalpha, IL-1beta, and IL-8 was significantly increased in the airways and lung lesions of infected calves as compared with mock-infected controls. Although kinetic patterns varied, peak levels of cytokine mRNA occured within 8 hours postinfection (PI), and peak cytokine concentrations occurred within 16 hours PI. In all samples, IL-8 was expressed to the greatest extent and TNFalpha was least expressed. Expression of TNFalpha was restricted to alveolar macrophages. Alveolar and interstitial macrophages produced IL-1beta and IL-8 in the first 4 hours; bronchial and bronchiolar epithelial cells were also significant sources of IL-8 during this period. By 8 hours PI, neutrophils were the dominant source of both IL-1beta and IL-8. These findings demonstrate a spatial and temporal association between pulmonary expression of inflammatory cytokines and acute lung pathology, supporting the hypothesis that cytokines contribute to inflammatory lung injury in BPP.

Regulation of expression of major histocompatibility antigens by bovine macrophages infected with Mycobacterium avium subsp. paratuberculosis or Mycobacterium avium subsp. avium.

Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium are antigenically and genetically very similar organisms; however, they differ markedly in their virulence for cattle. We evaluated the capacity of bovine macrophages infected with M. avium subsp. paratuberculosis or M. avium subsp. avium to express major histocompatibility complex (MHC) class I and class II antigens on their surface and to interact with primed autologous lymphocytes. Our results indicate that infection of bovine macrophages with M. avium subsp. paratuberculosis promoted the downregulation of MHC class I and class II molecules on the macrophage surface within 24 and 12 h, respectively. Alternatively, MHC class II expression by M. avium subsp. avium-infected macrophages was not detected until 24 h after infection, and the magnitude of the decrease was smaller. Decreased MHC class I expression by M. avium subsp. avium-infected macrophages was not detected. Unlike M. avium subsp. paratuberculosis-infected macrophages, M. avium subsp. avium-infected macrophages upregulated MHC class I and class II expression after activation by gamma interferon or tumor necrosis factor alpha. Further, M. avium subsp. avium-infected macrophages were lysed by primed autologous lymphocytes, whereas M. avium subsp. paratuberculosis-infected macrophages were not. Overall, the results support the hypothesis that the difference in the virulence of M. avium subsp. paratuberculosis and M. avium subsp. avium for cattle is dependent on a difference in the capacity of the organisms to suppress mycobacterial antigen presentation to T lymphocytes.

Cryptosporidium parvum genome project.

A lack of basic understanding of parasite biology has been a limiting factor in designing effective means of treating and preventing disease caused by Cryptosporidium parvum. Since the genomic DNA sequence encodes all of the heritable information responsible for development, disease pathogenesis, virulence, species permissiveness and immune resistance, a comprehensive knowledge of the C. parvum genome will provide the necessary information required for cost-effective and targeted research into disease prevention and treatment. With the recent advances in high-throughput automated DNA sequencing capabilities, large-scale genomic sequencing has become a cost-effective and time-efficient approach to understanding the biology of an organism. In addition, the continued development and implementation of new software tools that can scan raw sequences for signs of genes and then identify clues as to potential functions, has provided the final realization of the potential rewards of genome sequencing. To further our understanding of C. parvum biology, we have initiated a random shotgun sequencing approach to obtain the complete sequence of the IOWA isolate of C. parvum. Our progress to date has demonstrated that sequencing of the C. parvum genome will be an efficient and costeffective method for gene discovery of this important eukaryotic pathogen. This will allow for the identification of key metabolic and immunological features of the organism that will provide the basis for future development of safe and effective strategies for prevention and treatment of disease in AIDS patients, as well as immunocompetent hosts. Moreover, by obtaining the complete sequence of the C. parvum genome, effective methods for subspecific differentiation (strain typing) and epidemiologic surveillance (strain tracking) of this pathogen can be developed.

Estimating viability of Cryptosporidium parvum oocysts using reverse transcriptase-polymerase chain reaction (RT-PCR) directed at mRNA encoding amyloglucosidase.

The purpose of the present study was to determine if reverse transcriptase-polymerase chain reaction (RT-PCR) directed at mRNA encoding the enzyme amyloglucosidase (CPAG) could serve as a indicator for C. parvum oocyst viability. Oocysts were stored for 1-11 months in the refrigerator and at monthly intervals extracted for total RNA for RT-PCR analysis. An aliquot of these C. parvum oocysts was inoculated into neonatal mice which were necropsied 4 days later for ileal tissue that was analyzed by semi-quantitative PCR to determine the level of parasite replication. The CPAG RT-PCR assay detected RNA from as few as 10(3) C. parvum oocysts. An effect of storage time on both RT-PCR signal and mouse infectivity was observed. RNA from oocysts stored for 1-7 months, unlike oocysts stored for 9 or 11 months, contained CPAG mRNA that was detectable by RT-PCR. A gradual decrease in the RT-PCR signal intensity was observed between 5 and 7 months storage. The intensity of RT-PCR product from oocysts and the signal from semi-quantitative PCR of ileal tissue DNA from mice infected with these same aged oocysts were comparable. The RT-PCR assay of CPAG mRNA in cultured cells infected with viable C. parvum oocysts first detected expression at 12 h with highest expression levels observed at 48 h post-infection. These results indicate that CPAG RT-PCR may be useful for differentiating viable from non-viable C. parvum oocysts and for studying the expression of the gene for amyloglucosidase in vitro.

Resistance against the membrane attack complex of complement induced in porcine endothelial cells with a Gal alpha(1-3)Gal binding lectin: up-regulation of CD59 expression.

Endothelial cells (EC) play central roles in vascular physiology and pathophysiology. EC activation results in proinflammatory activities with production of cytokines and expression of adhesion molecules. However, we have shown before in a model of xenotransplantation that prolonged stimulation of porcine EC with human anti-porcine IgM natural Abs can activate the cells to become resistant against cytotoxicity by the membrane attack complex of complement (MAC). Now we report the major characteristics of induction and maintenance of resistance elicited in porcine EC with Bandeiraea simplicifolia lectin that binds terminal gal alpha(1-3)gal. Lectin-treated cells underwent little or no cytotoxicity and PGI2 release when exposed to MAC. Induction of resistance required incubation of the EC with lectin for 4 h but was not fully manifested until 16 h later. Most of the initially bound lectin remained on the cell surface for >60 h. EC-bound lectin did not inhibit binding of IgM natural Abs or activation and binding of C components, including C9, but a C-induced permeability channel of reduced size was present. Induction of resistance required protein synthesis, developed slowly, and was associated with up-regulation of expression of mRNA for the MAC inhibitor CD59 and membrane-associated CD59 protein. Resistance lasted at least 3 days, and the cells regained normal morphology and were metabolically active. This induced resistance may have a physiologic counterpart that might be amenable to pharmacologic manipulation in vascular endothelium pathophysiology.

Chromosome mapping in Cryptosporidium parvum and establishment of a long-range restriction map for chromosome VI.

We used contour-clamped homogeneous electric field (CHEF) gel electrophoresis and Southern blot hybridization to analyze the molecular karyotype of Cryptosporidium parvum and establish the chromosomal location of 12 single copy genes. In agreement with previous studies, the molecular karyotype of C. parvum was found to consist of partially co-migrating chromosomes ranging in size from 0.97 to 1.55 Mb and segregating into five distinct electrophoretic bands. Hybridization results allowed the definition of a linkage group comprised of five distinct loci located on chromosome VI. Southern hybridization and restriction analysis of total C. parvum chromosomes or isolated chromosome VI using gene-specific probes and an oligonucleotide specific for C. parvum telomeres allowed the development of a long-range restriction map of chromosome VI.

A random survey of the Cryptosporidium parvum genome.

Cryptosporidium parvum is an obligate intracellular pathogen responsible for widespread infections in humans and animals. The inability to obtain purified samples of this organism's various developmental stages has limited the understanding of the biochemical mechanisms important for C. parvum development or host-parasite interaction. To identify C. parvum genes independent of their developmental expression, a random sequence analysis of the 10.4-megabase genome of C. parvum was undertaken. Total genomic DNA was sheared by nebulization, and fragments between 800 and 1,500 bp were gel purified and cloned into a plasmid vector. A total of 442 clones were randomly selected and subjected to automated sequencing by using one or two primers flanking the cloning site. In this way, 654 genomic survey sequences (GSSs) were generated, corresponding to >320 kb of genomic sequence. These sequences were assembled into 408 contigs containing >250 kb of unique sequence, representing approximately 2.5% of the C. parvum genome. Comparison of the GSSs with sequences in the public DNA and protein databases revealed that 107 contigs (26%) displayed similarity to previously identified proteins and rRNA and tRNA genes. These included putative genes involved in the glycolytic pathway, DNA, RNA, and protein metabolism, and signal transduction pathways. The repetitive sequence elements identified included a telomere-like sequence containing hexamer repeats, 57 microsatellite-like elements composed of dinucleotide or trinucleotide repeats, and a direct repeat sequence. This study demonstrates that large-scale genomic sequencing is an efficient approach to analyze the organizational characteristics and information content of the C. parvum genome.

Pasteurella haemolytica leukotoxin and endotoxin induced cytokine gene expression in bovine alveolar macrophages requires NF-kappaB activation and calcium elevation.

In bovine alveolar macrophages (BAMs), exposure to leukotoxin (Lkt) and endotoxin (LPS) from Pasteurella haemolytica results in expression of inflammatory cytokine genes and intracellular calcium ([Ca2+]i) elevation. Leukotoxin from P. haemolytica interacts only with leukocytes and platelets from ruminant species. Upregulation of cytokine genes in different cells by LPS involves activation of the transcription factor NF-kappaB (NF-kappaB), resulting in its translocation from the cytoplasm to the nucleus. Using immunocytochemical staining and confocal imaging, we studied whether NF-kappaB activation represents a common mechanism for the expression of multiple cytokine genes in BAMs (Lkt-susceptible cells) stimulated with Lkt and LPS. Bovine pulmonary artery endothelial cells and porcine alveolar macrophages were used as nonsusceptible cells. The role of Ca2+ and tyrosine kinases in NF-kappaB activation and inflammatory cytokine gene expression was studied, since an inhibitor of tyrosine kinases attenuates LPS-induced [Ca2+]i elevation in BAMs. The results are summarized as follows: (a) Lkt induced NF-kappaB activation and [Ca2+]i elevation only in BAMs, while LPS effects were demonstrable in all cell types; (b) chelation of [Ca2+]i blocked NF-kappaB activation and IL-1beta, TNFalpha, and IL-8 mRNA expression; and (c) tyrosine kinase inhibitor herbimycin A blocked expression of all three cytokine genes in BAMs stimulated with Lkt, while only the expression of IL-1beta was blocked in BAMs stimulated with LPS. We conclude that cytokine gene expression in BAMs requires NF-kappaB activation and [Ca2+]i elevation, and Lkt effects exhibit cell type- and species specificity.

Differential mRNA display cloning and characterization of a Cryptosporidium parvum gene expressed during intracellular development.

Differential mRNA display was used to detect differences in gene expression between mock-infected and Cryptosporidium parvum-infected human adenocarcinoma cells. A reproducible band present only in C. parvum-infected cells, ddHC-10 was isolated and cloned. Northern blot analysis was used to confirm the differential expression of the HC-10 mRNA. As differential mRNA display does not differentiate between parasite and host mRNAs, Southern blot analysis was used to demonstrate that ddHC-10 represented a C. parvum gene. Northern blot analysis demonstrated that HC-10 mRNA is expressed by sporozoites prior to invasion of host cells. Screening of a C. parvum genomic library identified 2 different genomic clones, HC-10-13C and HC-10-6C. The combined genomic sequence contained a predicted open reading frame of 2,952 base pairs (bp), coding for a protein of 984 amino acids with a predicted molecular weight of approximately 106 kDa. Reverse transcription polymerase chain reaction mapping of the HC-10 transcript demonstrated that the HC-10 gene lacks introns, and the approximately 4,789-bp mRNA contains relatively large 5' (approximately 1,390-bp) and 3' (approximately 440-bp) untranslated regions. The predicted polypeptide contained a high proportion of polar amino acids, with the most abundant amino acids being serine (10.5%), threonine (9.8%), and cysteine (7.6%). The C-terminal region of the predicted polypeptide is characterized by a threonine-rich region containing multiple repeats of the sequence TTTTRP. This repeat motif is similar to that found in the mucin-like genes of vertebrates and lower eukaryotes that have been shown to play important roles in cell-cell interactions in multicellular organisms and invasion of host cells by unicellular parasites.