Characterization of innate immune response to Brucella melitensis infection in goats with permissive or restrictive phenotype for Brucella intramacrophagic growth.

Caprine brucellosis is a chronic, world-wide distributed disease which causes reproductive failure in goats and Brucella melitensis, its causative agent, bears a great zoonotic potential. There is evidence suggesting that some cattle and pigs have an innate ability to resist Brucella infection, but this has not yet been investigated in goats. In this study, we compared caprine macrophages that exhibit extreme restriction and permissiveness to B. melitensis' intracellular growth in vitro. Monocyte derived macrophages (MDMs) from 110 female goats were cultured and challenged in vitro with B. melitensis 16 M. After initial screening, 18 donor goats were selected based on their macrophages ability to restrict or allow bacterial intracellular growth and some elements of humoral and cellular immunity were studied in depth. MDMs that were able to restrict the pathogen's intracellular growth showed enhanced bacterial internalization, although there were no differences between groups in the production of reactive oxygen and nitrogen intermediates following 48 h treatment with heat-killed B. melitensis. Moreover, there were no differences between groups in the level of antibodies reacting with keyhole limpet hemocyanin (natural antibodies, NAbs) or with Brucella LPS antigens (cross-reacting antibodies, CrAbs), although a strong positive correlation between individual levels of IgM NAbs and IgM CrAbs was detected. Altogether, these results represent an initial step in understanding innate primary host response to B. melitensis, and deciphering which mechanisms may determine a successful outcome of the infection in goats.

Association of TNF rs668920841 and INRA111 polymorphisms with caprine brucellosis: A case-control study of candidate genes involved in innate immunity.

Caprine brucellosis is an infectious, contagious zoonotic disease caused by Brucella melitensis. Multiple factors, including host genetics, can influence the outcome of the exposure to Brucella; and it is expected that genetic variants that affect the host innate immune response could have a key role in Brucella infection and pathogenesis. In this study, we evaluated if polymorphisms in innate immunity-related genes are associated with results of Brucella infection in goats. Nine polymorphisms within interferon gamma (IFNG), tumor necrosis factor (TNF), MyD88 innate immune signal transduction adaptor (MYD88), interleukin 10 (IL10) and IL-10 receptor subunit alpha (IL10RA) genes and two molecular markers (BMS2753 and INRA111) were resolved by PCR-capillary electrophoresis in samples from 81 seronegative and 61 seropositive goats for brucellosis. A heterozygous genotype at INRA111, a microsatellite near the VRK serine/threonine kinase 2 (VRK2) gene, was associated with absence of Brucella-specific antibodies in goats naturally exposed to the pathogen (P = .004). Conversely, variants in the TNF gene (rs668920841) and near the IFN gamma receptor 1 (IFNGR1) gene (microsatellite BMS2753) were significantly associated with presence of Brucella-specific antibodies at allelic (P = .042 and P = .046) and genotypic level (P = .012 and P = .041, respectively). Moreover, an in silico analysis predicted a functional role of the insertion-deletion polymorphism rs668920841 on the transcriptional regulation of the caprine TNF gene. Altogether, these results contribute to the identification of genetic factors that have a putative effect on the resistance / susceptibility phenotype of goats to Brucella infection.

Genetic analysis of the 3' untranslated region of the bovine SLC11A1 gene reveals novel polymorphisms.

Polymorphisms in microsatellites at the 3' untranslated region (3'UTR) of the SLC11A1 (solute carrier family 11 member A1) gene have been associated with natural resistance to Brucella abortus and Mycobacterium bovis infection in livestock species. Here, we carried out an individual genetic analysis of the two microsatellites present at the 3'UTR SLC11A1 gene in 254 Bos taurus purebred, 125 B. indicus purebred and 54 B. taurus × B. indicus crossbred cattle. The genotyping by capillary electrophoresis showed the presence of four alleles (157, 159, 161 and 163) for the first microsatellite (MS1) and six alleles (175, 177, 179, 181, 183 and 185) for the second microsatellite (MS2). The alleles 159 and 175 were the most frequent in all breeds analyzed. B. taurus showed the most homogeneous haplotype and genotype for both microsatellites, whereas B. indicus showed the most heterogeneous haplotype and genotype. Two novel variants (alleles 161 and 163) within the MS1 are reported as well as novel variants in MS2 in Holstein breed. The knowledge of the polymorphisms distribution in both microsatellites at the 3'UTR of the SLC11A1 gene in cattle breeds is useful for future experimental design to evaluate the association between reported genotypes and natural resistance to pathogens infection.

Comparative analysis of the early transcriptome of Brucella abortus--infected monocyte-derived macrophages from cattle naturally resistant or susceptible to brucellosis.

Brucellosis is a worldwide zoonotic infectious disease that has a significant economic impact on animal production and human public health. We characterized the gene expression profile of B. abortus-infected monocyte-derived macrophages (MDMs) from naïve cattle naturally resistant (R) or susceptible (S) to brucellosis using a cDNA microarray technology. Our data indicate that (1) B. abortus induced a slightly increased genome activation in R MDMs and a down-regulated transcriptome in S MDMs, during the onset of infection, (2) R MDMs had the ability to mount a type 1 immune response against B. abortus infection which was impaired in S cells, and (3) the host cell activity was not altered after 12 h post-B. abortus infection in R MDMs while the cell cycle was largely arrested in infected S MDMs at 12 h p.i. These results contribute to an improved understanding of how host responses may be manipulated to prevent infection by brucellae.

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.

Early phase morphological lesions and transcriptional responses of bovine ileum infected with Mycobacterium avium subsp. paratuberculosis.

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 ss-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.

Long-term preservation of leptospiras by liquid nitrogen.

Liquid nitrogen freezing is recommended for long-term preservation of Leptospira serovars. However, there is no standard protocol to follow for this methodology. We herein report a simple procedure to preserve well-characterized Leptospira serovars unaltered for long-term storage in liquid nitrogen. Forty-three (43) leptospira strains, cryoprotected with 10% (v/v) glycerol were rapidly frozen in a dry-ice methanol bath and immediately submerged in liquid-nitrogen. Viability was retained in 100%, 93% and 83% of the frozen cultures after 6, 18 and 54 months, following freezing and storage in liquid nitrogen, respectively. Motility and agglutinability were not altered. These results demonstrate the usefulness of this protocol for long-term storage of genus Leptospira in liquid nitrogen.

Long-term preservation of leptospiras by liquid nitrogen / Conservación de leptospiras en nitrógeno líquido

Liquid nitrogen freezing is recommended for long-term preservation of Leptospira serovars. However, there is no standard protocol to follow for this methodology. We herein report a simple procedure to preserve well-characterized Leptospira serovars unaltered for long-term storage in liquid nitrogen. Forty-three (43) leptospira strains, cryoprotected with 10% (v/v) glycerol were rapidly frozen in a dry-ice methanol bath and immediately submerged in liquid-nitrogen. Viability was retained in 100%, 93% and 83% of the frozen cultures after 6, 18 and 54 months, following freezing and storage in liquid nitrogen, respectively. Motility and agglutinability were not altered. These results demonstrate the usefulness of this protocol for long-term storage of genus Leptospira in liquid nitrogen.

Se recomienda la congelación en nitrógeno líquido para el mantenimiento de cepas de leptospiras a largo plazo. Sin embargo, no existe para ello una metodología de trabajo estandarizada. En este trabajo se presenta y evalúa un protocolo simple para conservar inalteradas cepas de leptospiras en nitrógeno líquido durante largo tiempo. Cuarenta y tres (43) cepas de leptospiras crioprotegidas con glicerol al 10% (v/v) fueron rápidamente congeladas en un baño de metanol y hielo seco, e inmediatamente sumergidas en nitrógeno líquido. Fue posible recuperar el 100%, 93% y 83% de los cultivos congelados a los 6, 18 y 54 meses poscongelación, respectivamente, sin observarse alteración en la movilidad ni en la aglutinabilidad de las cepas recuperadas. Estos resultados demuestran la utilidad del protocolo presentado para conservar cepas del género Leptospira en nitrógeno líquido durante largos períodos de tiempo.

Occurrence and characteristics of CS31A antigen-producing Escherichia coli in calves with diarrhoea and septicaemia in Argentina.

CS31A is a K88-related non-fimbrial adhesin first described on Escherichia coli strains isolated from diarrhoeic and septicaemic calves. In this report, CS31A antigen was screened by immunological methods and confirmed by PCR among bovine E. coli isolates. In addition, CS31A-producing strains were characterized with respect to different fimbrial antigens, O-serogroup and other properties related to virulence. Faecal or tissue specimens of 100 diarrhoeic or septicaemic calves and 27 older cattle with different pathologies from 71 outbreaks or individual cases that occurred in Buenos Aires province, Argentina, were examined. CS31A + E. coli strains were isolated from 21 (21.0%) calves from 16 outbreaks or individual cases. No CS31A + E. coli was detected in samples from cattle more than 1 year old. Fimbriae F5, F41, F17a and F17b were not detected among the CS31A-producing strains. Three (14.3%) of the CS31A+ E. coli strains expressed the F17c fimbria. All of the 21 isolates exhibited at least one property of septicaemic strains (resistance to serum, production of aerobactin or colicins) but none of them demonstrated heat-stable enterotoxigenic activity. CS31A + E. coli isolates belonged to 10 serogroups, more commonly O8, O7, O17 and O21. The results obtained here confirm the worldwide distribution of CS31A antigen in bovine E. coli strains. However, CS31A + or CS31A + /F17c + E. coli were less frequently isolated than they were in North hemisphere countries.