Indirect ELISA for diagnosis of Brucella ovis infection in rams / ELISA indireto para diagnóstico da infecção por Brucella ovis em carneiros

Brucella ovis is a major cause of epididymitis in sexually mature rams, resulting in subfertility, infertility, and economic losses for the sheep industry worldwide. The aim of this study was to develop an indirect ELISA (iELISA) using recombinant proteins, namely rBoP59 and rBP26, as antigens for serological diagnosis of B. ovis infection. The BoP59 and BP26 recombinant proteins were expressed in E. coli and purified by affinity chromatography. Antigenicity was tested by Western blot and iELISA. Standardization of iELISA was performed with 500ng and 1µg BoP59 and rBP26 per well, testing serum from uninfected and experimentally infected rams. rBP26 was effective in distinguishing positive from negative rams. The rBP26 iELISA developed in this study is the first to use a completely purified rBP26 as antigen resulting in high sensitivity (100%) and specificity (90.2%), and an overall accuracy equal to 1.0...

Brucella ovis é uma das principais causas de epididimite em carneiros sexualmente maduros, resultando em subfertilidade e infertilidade e consequentes perdas econômicas para a ovinocultura em todo o mundo. O objetivo deste estudo foi desenvolver ELISA indireto (ELISAi), utilizando como antígeno proteínas recombinantes BoP59r e BP26r para diagnóstico da infecção por B. ovis. BoP59r e BP26r foram expressas em E. coli e purificadas por cromatografia de afinidade e a antigenicidade testada por Western blot e ELISAi. A padronização do ELISAi foi realizada testando 500 ng e 1 µg de BoP59r e BP26r por poço e soros de carneiros infectados e não infectados. A BP26r foi eficiente em diferenciar ovinos negativos de positivos. O ELISAi com BP26 desenvolvido neste estudo foi o primeiro a utilizar BP26 completamente purificada como antígeno, resultando em elevada sensibilidade (100%) e sensibilidade (90,2%), com acurácia igual a 1,0...

The mucosal inflammatory response to non-typhoidal Salmonella in the intestine is blunted by IL-10 during concurrent malaria parasite infection.

Coinfection can markedly alter the response to a pathogen, thereby changing its clinical presentation. For example, non-typhoidal Salmonella (NTS) serotypes are associated with gastroenteritis in immunocompetent individuals. In contrast, individuals with severe pediatric malaria can develop bacteremic infections with NTS, during which symptoms of gastroenteritis are commonly absent. Here we report that, in both a ligated ileal loop model and a mouse colitis model, malaria parasites caused a global suppression of gut inflammatory responses and blunted the neutrophil influx that is characteristic of NTS infection. Further, malaria parasite infection led to increased recovery of Salmonella enterica serotype Typhimurium from the draining mesenteric lymph node (MLN) of mice. In the mouse colitis model, blunted intestinal inflammation during NTS infection was independent of anemia but instead required parasite-induced synthesis of interleukin (IL)-10. Blocking of IL-10 in coinfected mice reduced dissemination of S. Typhimurium to the MLN, suggesting that induction of IL-10 contributes to development of disseminated infection. Thus IL-10 produced during the immune response to malaria in this model contributes to suppression of mucosal inflammatory responses to invasive NTS, which may contribute to differences in the clinical presentation of NTS infection in the setting of malaria.

The quest for a true One Health perspective of brucellosis.

One Health is an interdisciplinary collaboration that aims at mitigating risks to human health arising from microorganisms present in non-human animal species, which have the potential to be transmitted and cause disease in humans. Different degrees of scientific collaboration and sectoral integration are needed for different types of zoonotic diseases, depending on the health and associated economic gains that can be expected from a One Health approach. Indeed, mitigating zoonotic risks related to emerging diseases with pandemic potential is different from mitigating risks related to endemic zoonotic diseases like brucellosis. Likewise, management of brucellosis at the wildlife-livestock interface in wildlife conservation areas is in essence different from mitigating transmission of a given Brucella species within its preferential host species, which in turn is different from mitigating the spillover of a given Brucella species to non-preferential host species, humans included. Brucellosis economic models often oversimplify and/or wrongly assess transmission between reservoir hosts and spillover hosts. Moreover,they may not properly value non-market outcomes, such as avoidance of human disease, consumer confidence and conservation biology issues. As a result, uncertainty is such that the economic predictions of these models can be questionable. Therefore, understanding the infection biology of Brucella species is a prerequisite. This paper reviews and highlights important features of the infection biology of Brucella species and the changing epidemiology of brucellosis that need to be integrated into a true One Health perspective of brucellosis.

Characterization of Brucella abortus infection of bovine monocyte-derived dendritic cells.

Brucella abortus is a Gram negative facultative intracellular pathogen of cattle, and an important zoonosis in humans worldwide. Previous studies have shown that dendritic cells (DC) from humans and mice are highly permissive for Brucella survival and proliferation. Impairment of DC activation and maturation by Brucella infection has also been reported in these two species. The aim of this study was to characterize infection of bovine DC with B. abortus. Monocyte-derived DC (mdDC) were cultured from bovine peripheral blood mononuclear cells (PBMC) using the recombinant bovine cytokines IL-4 and GM-CSF. The resulting mdDC were DEC205(+), MHC class II(hi). Approximately 70% of the cultured cells were DEC205(+), MHC II(+). MdDC were infected with B. abortus strain 2308 at an MOI of 1 and 100. Parallel infection experiments were performed in monocyte derived macrophages (mdM) isolated from the same subjects. Bacteria were successfully killed by mdDC by 24 hours post infection (PI) with high and low dose of B. abortus, bacteria persisted in mdM infected with a high dose. Expression of IL-1b, IL-6, IL-10, IL-12p40, IFNγ, iNOS and TNFα in B. abortus infected and LPS stimulated mdDC at 6 and 24 hours PI were evaluated using RT-qPCR. At 6 hours PI all transcripts were increased over control cells and significantly less IL-10, IL-12p40, and IFNγ were expressed in mdDC infected with B. abortus compared to LPS stimulation. Evaluation of mdDC cultures by flow cytometry was performed. Flow cytometric analysis of infected and LPS stimulated mdDC 24 hours PI showed expression of CD80 and CD86 was impaired in two of the three animals analyzed. MHC class II expression was equivocal between the groups. From these results we conclude that cultured bovine mdDC are not permissive for intracellular proliferation of B. abortus, and infected mdDC exhibit some signs of maturational and activational impairment.

Morphologic and cytokine profile characterization of Salmonella enterica serovar typhimurium infection in calves with bovine leukocyte adhesion deficiency.

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 alpha in the presence of neutrophil influx in CD18+ calves. In contrast, the proinflammatory cytokines interleukin 1beta and tumor necrosis factor alpha were poorly correlated with the presence or absence of tissue neutrophils.

Analysis of ten Brucella genomes reveals evidence for horizontal gene transfer despite a preferred intracellular lifestyle.

The facultative intracellular bacterial pathogen Brucella infects a wide range of warm-blooded land and marine vertebrates and causes brucellosis. Currently, there are nine recognized Brucella species based on host preferences and phenotypic differences. The availability of 10 different genomes consisting of two chromosomes and representing six of the species allowed for a detailed comparison among themselves and relatives in the order Rhizobiales. Phylogenomic analysis of ortholog families shows limited divergence but distinct radiations, producing four clades as follows: Brucella abortus-Brucella melitensis, Brucella suis-Brucella canis, Brucella ovis, and Brucella ceti. In addition, Brucella phylogeny does not appear to reflect the phylogeny of Brucella species' preferred hosts. About 4.6% of protein-coding genes seem to be pseudogenes, which is a relatively large fraction. Only B. suis 1330 appears to have an intact beta-ketoadipate pathway, responsible for utilization of plant-derived compounds. In contrast, this pathway in the other species is highly pseudogenized and consistent with the "domino theory" of gene death. There are distinct shared anomalous regions (SARs) found in both chromosomes as the result of horizontal gene transfer unique to Brucella and not shared with its closest relative Ochrobactrum, a soil bacterium, suggesting their acquisition occurred in spite of a predominantly intracellular lifestyle. In particular, SAR 2-5 appears to have been acquired by Brucella after it became intracellular. The SARs contain many genes, including those involved in O-polysaccharide synthesis and type IV secretion, which if mutated or absent significantly affect the ability of Brucella to survive intracellularly in the infected host.

Emprego do propofol, isofluorano e morfina para a anestesia geral de longa duraçäo em bezerros / Use of propofol, isoflurane and morphine for prolonged general anesthesia in calves

Foram estudadas características da bioquímica do sangue, da pressäo arterial e da freqüência de pulso de 12 bezerros mantidos sob anestesia por 13 horas, utilizando-se propofol para a induçäo e isofluorano para manutençäo, associados à administraçäo de morfina intra-tecal. Os valores de freqüência de pulso, pressäo arterial e glicemia apresentaram pequenas variaçöes e se mantiveram próximos dos valores de referência para bezerros anestesiados. Ao longo do período de anestesia houve aumento significativo, mas discreto, do hematócrito, hemoglobina, pCO2, CO2 total, bicarbonato e potássio. O pH do sangue, pO2, Na+ e Ca++ apresentaram reduçöes significativas. Este protocolo anestésico foi seguro para a manutençäo de bezerros anestesiados por período prolongado.

Pathogenesis of Salmonella-induced enteritis.

Infections with Salmonella serotypes are a major cause of food-borne diseases worldwide. Animal models other than the mouse have been employed for the study of nontyphoidal Salmonella infections because the murine model is not suitable for the study of Salmonella-induced diarrhea. The microbe has developed mechanisms to exploit the host cell machinery to its own purpose. Bacterial proteins delivered directly into the host cell cytosol cause cytoskeletal changes and interfere with host cell signaling pathways, which ultimately enhance disease manifestation. Recently, marked advances have been made in our understanding of the molecular interactions between Salmonella serotypes and their hosts. Here, we discuss the molecular basis of the pathogenesis of Salmonella-induced enteritis.

Pathogenesis of Salmonella-induced enteritis

Infections with Salmonella serotypes are a major cause of food-borne diseases worldwide. Animal models other than the mouse have been employed for the study of nontyphoidal Salmonella infections because the murine model is not suitable for the study of Salmonella-induced diarrhea. The microbe has developed mechanisms to exploit the host cell machinery to its own purpose. Bacterial proteins delivered directly into the host cell cytosol cause cytoskeletal changes and interfere with host cell signaling pathways, which ultimately enhance disease manifestation. Recently, marked advances have been made in our understanding of the molecular interactions between Salmonella serotypes and their hosts. Here, we discuss the molecular basis of the pathogenesis of Salmonella-induced enteritis

Morphologic and molecular characterization of Salmonella typhimurium infection in neonatal calves.

The host response to Salmonella plays a major role in the outcome of infection. The present study was undertaken to further characterize Salmonella typhimurium infection in neonatal calves at both the morphologic and the molecular level using the ligated ileal loop model. Eight 4-5-week-old male Holstein calves underwent laparotomy, and loops were prepared in the ileum. The loops were either inoculated with an S. typhimurium strain pathogenic for cattle or injected with sterile LB broth as control. Samples for histology, transmission and scanning electron microscopy, and RNA extraction were collected at various time points between 5 minutes and 12 hours postinfection. Invasion of both M cells and enterocytes began at 15 minutes postinfection. No specific cell type was the main target for invasion. Intracellular bacteria were observed in the lamina propria after 1 hour postinfection. A severe acute neutrophilic response was associated with invasion of the Peyer's patches. Upregulated expression of CXC chemokines (interleukin [IL]-8, growth-related oncogenes, [GRO] alpha and gamma, and granulocyte chemotactic protein [GCP]2) was detected by reverse transcription polymerase chain reaction beginning at 1 hour postinfection. Expression of proinflammatory (IL-1beta, IL-18, and tumor necrosis factor [TNF]alpha) and anti-inflammatory (IL-10, IL-IRa, and IL-4) cytokines was also assessed. A marked increase in expression of IL-1beta was observed, whereas the profile of expression of IL-18 and TNFalpha did not change after infection. Upregulation of IL-1Ra and IL-4 but not of IL-10 was observed. These findings indicate that infection of bovine ligated ileal loops with S. typhimurium results in an acute neutrophilic inflammatory response that is associated with the upregulation of CXC chemokines (IL-8, GROalpha and gamma, and GCP2), IL-1beta, IL-IRa, and IL-4.

Salmonella-induced cell death is not required for enteritis in calves.

Salmonella enterica serovar Typhimurium causes cell death in bovine monocyte-derived and murine macrophages in vitro by a sipB-dependent mechanism. During this process, SipB binds and activates caspase-1, which in turn activates the proinflammatory cytokine interleukin-1beta through cleavage. We used bovine ileal ligated loops to address the role of serovar Typhimurium-induced cell death in induction of fluid accumulation and inflammation in this diarrhea model. Twelve perinatal calves had 6- to 9-cm loops prepared in the terminal ileum. They were divided into three groups: one group received an intralumen injection of Luria-Bertani broth as a control in 12 loops. The other two groups (four calves each) were inoculated with 0.75 x 10(9) CFU of either wild-type serovar Typhimurium (strain IR715) or a sopB mutant per loop in 12 loops. Hematoxylin and eosin-stained sections were scored for inflammation, and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells were detected in situ. Fluid accumulation began at 3 h postinfection (PI). Inflammation was detected in all infected loops at 1 h PI. The area of TUNEL-labeled cells in the wild-type infected loops was significantly higher than that of the controls at 12 h PI, when a severe inflammatory response and tissue damage had already developed. The sopB mutant induced the same amount of TUNEL-positive cells as the wild type, but it was attenuated for induction of fluid secretion and inflammation. Our results indicate that serovar Typhimurium-induced cell death is not required to trigger an early inflammatory response and fluid accumulation in the ileum.

Salmonella enterica serovar typhimurium induces cell death in bovine monocyte-derived macrophages by early sipB-dependent and delayed sipB-independent mechanisms.

It was previously demonstrated that Salmonella enterica serovar Typhimurium induces cell death with features of apoptosis in murine macrophages. Mice infected with Salmonella serovar Typhimurium develop systemic disease without diarrhea, whereas the infection in cattle and in humans is localized and characterized by diarrhea. Considering these clinical disease expression differences between mice and cattle, we investigated whether serovar Typhimurium is cytotoxic for bovine macrophages. Macrophages infected with serovar Typhimurium grown in the logarithmic phase quickly underwent cell death. Macrophages infected with stationary-phase cultures or with a mutant lacking sipB underwent no immediate cell death but did develop delayed cytotoxicity, undergoing cell death between 12 and 18 h postinfection. Both pathways were temporarily blocked by the general caspase inhibitor Z-VAD-Fmk and by the caspase 1 inhibitor Z-YVAD-Fmk. Comparisons of macrophages from cattle naturally resistant or susceptible to intracellular pathogens indicated no differences between these two genetic backgrounds in terms of susceptibility to serovar Typhimurium-induced cell death. We conclude that Salmonella serovar Typhimurium induces cell death in bovine macrophages by two distinct mechanisms, early sipB-mediated and delayed sipB-independent mechanisms.

Animal models of Salmonella infections: enteritis versus typhoid fever.

The most common disease syndromes caused by Salmonella serotypes in humans, typhoid fever and enteritis, can be modeled using Salmonella enterica serotype Typhimurium infections in mice and calves, respectively. This article reviews murine typhoid and bovine enteritis and discusses strengths, limitations and distinctive features of these animal models.

Competitive exclusion of Salmonella enteritidis by Salmonella gallinarum in poultry.

Salmonella Enteritidis emerged as a major egg-associated pathogen in the late 20th century. Epidemiologic data from England, Wales, and the United States indicate that S. Enteritidis filled the ecologic niche vacated by eradication of S. Gallinarum from poultry, leading to an epidemic increase in human infections. We tested this hypothesis by retrospective analysis of epidemiologic surveys in Germany and demonstrated that the number of human S. Enteritidis cases is inversely related to the prevalence of S. Gallinarum in poultry. Mathematical models combining epidemiology with population biology suggest that S. Gallinarum competitively excluded S. Enteritidis from poultry flocks early in the 20th century.

Identification of genes required for chronic persistence of Brucella abortus in mice.

The genetic basis for chronic persistence of Brucella abortus in lymphoid organs of mice, cows, and humans is currently unknown. We identified B. abortus genes involved in chronic infection, by assessing the ability of 178 signature-tagged mutants to establish and maintain persistent infection in mice. Each mutant was screened for its ability to colonize the spleens of mice at 2 and 8 weeks after inoculation. Comparison of the results from both time points identified two groups of mutants attenuated for chronic infection in mice. The first group was not recovered at either 2 or 8 weeks postinfection and was therefore defective in establishing infection. Mutants in this group carried transposon insertions in genes involved in lipopolysaccharide biosynthesis (wbkA), in aromatic amino acid biosynthesis, and in type IV secretion (virB1 and virB10). The second group, which was recovered at wild-type levels 2 weeks postinfection but not 8 weeks postinfection was able to establish infection but was unable to maintain chronic infection. One mutant in this group carried a transposon insertion in a gene with homology to gcvB of Mycobacterium tuberculosis, encoding glycine dehydrogenase, an enzyme whose activity is increased during the state of nonreplicating persistence. These results suggest that some mechanisms for long-term persistence may be shared among chronic intracellular pathogens. Furthermore, identification of two groups of genes, those required for initiating infection and those required only for long-term persistence, suggests that B. abortus uses distinct sets of virulence determinants to establish and maintain chronic infection in mice.

SspA is required for lethal Salmonella enterica serovar Typhimurium infections in calves but is not essential for diarrhea.

Salmonella pathogenicity island 1 (SPI-1) encodes virulence determinants, which are important for enteropathogenicity in calves. To determine whether the Salmonella enterica serovar Typhimurium SPI-1 effector proteins SspA and SptP are important for enteropathogenicity, strains lacking these proteins were tested during oral infection of calves. Calves infected with a sptP mutant or its isogenic parent developed diarrhea and lethal morbidity. In contrast, calves infected with an sspA mutant developed diarrhea, which resolved within 10 days but did not result in mortality. The sspA mutant was recovered from bovine intestinal tissues at numbers similar to those obtained for its isogenic parent and caused marked intestinal lesions. Thus, the severity of pathological changes caused by serovar Typhimurium strains or their ability to cause diarrhea were not predictive of their ability to cause lethal morbidity in calves. We conclude that factors other than or in addition to bacterial colonization, intestinal lesions, or electrolyte loss contribute to lethal morbidity in calves infected with serovar Typhimurium.

Identification of a putative Salmonella enterica serotype typhimurium host range factor with homology to IpaH and YopM by signature-tagged mutagenesis.

The genetic basis for the host adaptation of Salmonella serotypes is currently unknown. We have explored a new strategy to identify Salmonella enterica serotype Typhimurium (S. typhimurium) genes involved in host adaptation, by comparing the virulence of 260 randomly generated signature-tagged mutants during the oral infection of mice and calves. This screen identified four mutants, which were defective for colonization of only one of the two host species tested. One mutant, which only displayed a colonization defect during the infection of mice, was further characterized. During competitive infection experiments performed with the S. typhimurium wild type, the mutant was defective for colonization of murine Peyer's patches but colonized bovine Peyer's patches at the wild-type level. No difference in virulence between wild type and mutant was observed when calves were infected orally with 10(10) CFU/animal. In contrast, the mutant possessed a sixfold increase in 50% lethal morbidity dose when mice were infected orally. The transposon in this mutant was inserted in a 2.9-kb pathogenicity islet, which is located between uvrB and yphK on the S. typhimurium chromosome. This pathogenicity islet contained a single gene, termed slrP, with homology to ipaH of Shigella flexneri and yopM of Yersinia pestis. These data show that comparative screening of signature-tagged mutants in two animal species can be used for scanning the S. typhimurium genome for genes involved in host adaptation.

Salmonella typhimurium leucine-rich repeat proteins are targeted to the SPI1 and SPI2 type III secretion systems.

Salmonellae encode two virulence-associated type III secretion systems (TTSS) within Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2). Two Salmonella typhimurium genes, sspH1 and sspH2, that encode proteins similar to the Shigella flexneri and Yersinia species TTSS substrates, IpaH and YopM, were identified. SspH1 and SspH2 are proteins containing leucine-rich repeats that are differentially targeted to the SPI1 and SPI2 TTSS. sspH2 transcription was induced within RAW264.7 macrophages, and was dependent upon the SPI2-encoded regulator ssrA/ssrB. In contrast, sspH1 transcription is independent of SPI2, and is not induced after bacterial phagocytosis by eukaryotic cells. Infection of eukaryotic cells with strains expressing a SspH2-CyaA fusion protein resulted in SPI2 TTSS-dependent cAMP increases. In contrast, SspH1-CyaA-mediated cAMP increases were both SPI1 and SPI2 TTSS dependent. sspH2-like sequences were found in most Salmonella serotypes examined, whereas sspH1 was detected in only one S. typhimurium isolate, indicating that the copy number of sspH genes can be variable within Salmonella serotypes. S. typhimurium deleted for both sspH1 and sspH2 was not able to cause a lethal infection in calves, indicating that these genes participate in S. typhimurium virulence for animals.