Assessment of microRNA profiles in small extracellular vesicles isolated from bovine colostrum with different immunoglobulin G concentrations.

The consumption of bovine colostrum by newborn calves during the first days of life is essential to ensure the transfer of passive immunity. In addition to critical IgG, colostrum also contains non-IgG biomolecules, including microRNA (miRNA). The present study investigated the profiles of miRNA in small extracellular vesicles (sEV) isolated from bovine colostrum with high (256.5 ± 5.7 mg/mL, mean ± standard deviation, n = 4) and low (62.8 ± 3.6 mg/mL, n = 4) concentrations of IgG. Different combination of sEV extraction methods and bioinformatic pipelines (miRDeep2 and sRNAbench) for miRNA analysis were evaluated. Results showed that miRCURY exosome Cell/Urine/CSF and miRNeasy Mini kits yielded the highest RNA concentration. The miRNA-seq data analysis showed miRDeep2 yielded more comprehensive miRNAome compared with sRNAbench (527 versus 392 unique miRNA), whereas 389 shared miRNA were identified using both approaches. The profiles of top 50 miRNA were the same using both approaches, and their abundance contributed to 91.7% and 94.3% of total abundance of miRNA using miRDeep2 and sRNAbennch, respectively. These core miRNA were predicted to target 2,655 genes, which regulate 78 KEGG (Kyoto Encyclopedia of Genes and Genomes) level-3 pathways including PI3K-Akt and MAPK signaling pathway, axon guidance, and focal adhesion. The expression profiles of sEV-associated miRNA were similar between high- and low-IgG colostrum samples, despite the fact that the abundance of miR-27a-3p was higher in colostrum with high concentrations of IgG. In conclusion, a core miRNAome in bovine colostrum may play a role in regulating health and developmental stages in neonatal calves, independent of IgG concentration.

Delayed-type hypersensitivity skin test against Neospora caninum in heifers with undetectable specific antibodies.

Delayed-type hypersensitivity (DTH) has been used in human and veterinary medicine as a skin testing for evaluating in vivo cell-mediated immune responses (CMIR). Whereas CMIR is a key process to control intracellular pathogens, its value at identifying cattle exposed to the abortigenic intracellular coccidian parasite Neospora caninum is unknown. In this work, we have evaluated a DTH skin testing in cattle exposed to N. caninum and still seronegative. Female calves were experimentally sensitized by subcutaneous (SC) inoculation with live tachyzoites of N. caninum (NC-Argentina LP1) in sterile phosphate-buffered saline (PBS) (group A; n: 8) whereas other calveswere mock-sensitized with PBS (group B; n: 6). Two DTH skin tests were performed by intradermal inoculation with a soluble lysate of N. caninum tachyzoites (NC-Argentina LP1) in the neck region at 60d and 960 d after sensitization. Skinfold thickness at the intradermal inoculation site was measured at 0, 24, 48 h post each DTH skin test and skin biopsies taken for microscopic evaluation. Specific N. caninum antibodies kinetics was evaluated all throughthe experiment. We found that whereas N. caninum specific antibodies remained below the ELISA cut-off, a distinctive skinfold thickness increase was detected in sensitized animals (group A) at the DTH skin test site, showing induration, swelling and inflammatory infiltration. Mock sensitized animals (group B) showed no skinfold thickness growth and lacked specific antibody response. Thus, N. caninum DTH skin testing could be a useful diagnostic tool for the detection of CMIR during N. caninum infection in non-humoral responders.

Cathelicidin bovine myeloid antimicrobial peptide (BMAP) 28 is involved in the inflammatory response against alpha-herpesviruses in the bovine nervous system.

The role of the local innate immune response in the neuropathogenesis of bovine herpesvirus (BoHV) type 1 and 5 remains largely unknown. This study determined the gene transcriptional expression of relevant bovine cathelicidins, TNFα and IFNß in the nervous system of experimentally-infected cattle during the different stages of BoHV-1 and BoHV-5 infectious cycle. We studied the modulation of bovine myeloid antimicrobial peptide (BMAP) 27 and 28 by alpha-herpesviruses during acute infection of the central nervous system (CNS). However, BMAP28 was the main cathelicidin modulated. BoHV-5 supressed BMAP28 expression mainly in frontal cortex and cervical medulla whereas BoHV-1 slightly induced the expression of cathelicidins in the olfactory and posterior cortex. The differences in the regulation of the innate response are likely related to distinct replication rates of both alpha-herpesviruses in the CNS. During latency and reactivation, BoHV-1 and -5 decreased BMAP28 and BMAP27 expression, accompanied by high levels of TNFα and IFNß transcripts in the posterior brain region and medulla during BoHV reactivation. In terms of cytokines, a remarkably overexpression of IFNß was induced by BoHV-5 (133.8-fold). In trigeminal ganglion (TG) both alpha-herpesviruses induced cathelidicins gene expression at all stages of the infection cycle, while only acute BoHV-5 infection increased TNFα (129-fold) mRNA levels. This study suggests that the pronounced downregulation of BMAP28 in BoHV-5-acutely-infected CNS is due to a decreased immune stimulation during viral infection, favouring its establishment in the CNS with a low replication rate until latency. Thus, cathelicidins, together with IFNß and TNFα, are differentially regulated by BoHV-5 and BoHV-1 infections and this regulation is dependent on the stage of virus infection in the bovine nervous system.

Modulation of cathelicidins, IFNß and TNFα by bovine alpha-herpesviruses is dependent on the stage of the infectious cycle.

Production of antimicrobial peptides cathelicidins, interferons and cytokines is an important feature in airway epithelial host defense. The innate immune response to alpha-herpesvirus infection at the sites of primary replication has not been fully studied. Thus, the aim of this study was to determine the expression of innate immune components, cathelicidins, IFNß, TNFα and TNF receptors (TNFRI and TNFRII) during acute infection and reactivation of bovine herpesvirus type 1 (BoHV-1) and 5 (BoHV-5) in the respiratory tract and lymphoid tissue of their natural host. We found that BoHV infection modulates mainly the expression of BMAP28, a key cathelicidin in cattle. It was downregulated by both viruses in retropharyngeal lymph nodes of acutely infected-calves, and it was accompanied by a lower expression of IFNß, TNFα and TNFRI. BoHV-5 showed a pronounced role in the downregulation of BMAP28, even in nasal mucosa and lung. However, during reactivation, BoHV-5 upregulated both BMAP28 and IFNß in retropharyngeal lymph nodes. Acute replication induced also TNFα mRNA and protein synthesis, and expression of TNFRI and II was positively regulated during both acute infection and reactivation, particularly in the trachea. Moreover, BMAP27 was detected during BoHV-1 reactivation suggesting a potential role at this stage. Thus, cathelicidins are implicated in alpha-herpesvirus infections of the bovine respiratory system and the response is distinct during BoHV-1 and BoHV-5 acute infection and reactivation. This demonstrates that these viruses modulate differentially the components of innate immune response, possibly influencing their pathogenesis. This study provides an initial pilot analysis of factors that might be implicated in alpha-herpesvirus infection of the bovine respiratory system.

Characteristic pro-inflammatory cytokines and host defence cathelicidin peptide produced by human monocyte-derived macrophages infected with Neospora caninum.

Neospora caninum is a coccidian intracellular protozoan capable of infecting a wide range of mammals, although severe disease is mostly reported in dogs and cattle. Innate defences triggered by monocytes/macrophages are key in the pathogenesis of neosporosis, as these cells are first-line defenders against intracellular infections. The aim of this study was to characterize infection and innate responses in macrophages infected with N. caninum using a well-known cell model to study macrophage functions (human monocyte THP-1 cells). Intracellular invasion of live tachyzoites occurred as fast as 4 h (confirmed with immunofluorescence microscopy using N. caninum-specific antibodies). Macrophages infected by N. caninum had increased expression of pro-inflammatory cytokines (TNFα, IL-1ß, IL-8, IFNγ). Interestingly, N. caninum induced expression of host-defence peptides (cathelicidins), a mechanism of defence never reported for N. caninum infection in macrophages. The expression of cytokines and cathelicidins in macrophages invaded by N. caninum was mediated by mitogen-activated protein kinase (MEK 1/2). Secretion of such innate factors from N. caninum-infected macrophages reduced parasite internalization and promoted the secretion of pro-inflammatory cytokines in naïve macrophages. We concluded that rapid invasion of macrophages by N. caninum triggered protective innate defence mechanisms against intracellular pathogens.

Immunization with inactivated antigens of Neospora caninum induces toll-like receptors 3, 7, 8 and 9 in maternal-fetal interface of infected pregnant heifers.

Neospora caninum is an obligate parasite and a major cause of abortion in cattle. Pregnancy failures appear to be associated with weak innate defences on the maternal-fetal interface during infection with N. caninum. Herein, we studied the gene expression of Toll-like receptors (TLRs) in pregnant heifers immunized with different vaccine formulations against N. caninum before mating and then challenged the heifers with live N. caninum on day 70 of gestation. TLR7 and TLR8 expression was upregulated in the placental caruncle of infected-pregnant heifers previously exposed to live N. caninum as immunogen. However, TLR7 and 8 expression in both placenta and caruncle as well as, TLR3 and 9 expression in caruncle were upregulated when heifers were previously immunized with inactivated soluble whole antigens and recombinant NcSAG1, NcHSP20 and NcGRA7 proteins. All dams were carrying viable fetuses when they were culled at day 104 of gestation. Upregulation of TLR7 and IFNγ expression was detected in fetal spleen when their mothers where previously vaccinated with soluble antigens and recombinant NcSAG1, NcHSP20 and NcGRA7 proteins. These studies demonstrate that soluble or recombinant NcSAG1, NcHSP20 and NcGRA7 antigens induce key TLRs expression at the maternal-fetal interface, probably triggering damaging inflammatory cellular immune responses associated with abortion. Previous infection with N. caninum seems to attenuate the innate immune response at the maternal-fetal interface, which could favour pregnancy maintenance and perpetuation of the disease. This finding represents novel information on how N. caninum vaccination and infection modulate TLRs expression at the placenta and fetal spleen, the possible role in the pregnancy outcomes and transplacental transmission of the protozoa.

Toll-like receptors 3, 7 and 8 are upregulated in the placental caruncle and fetal spleen of Neospora caninum experimentally infected cattle.

Innate immune responses at the maternal-fetal interface are key in the pathogenesis of Neospora caninum, an obligate parasite that causes abortion in cattle. Herein, we determined the gene expression of endosomal Toll-like receptors (TLRs) in the placenta and fetuses from both non-infected pregnant heifers and pregnant heifers intravenously challenged with live tachyzoites of N. caninum on day 70 of gestation. On day 104 of pregnancy, mRNA expression of TLRs 3 and 8, as well as that of TLRs 7 and 9, was high in the spleen of fetuses from N. caninum-infected heifers. Gene expression levels of endosomal TLRs were also detectable in the placenta and the maternal caruncle from infected heifers, being TLRs 3, 7 and 8 particularly upregulated, mostly in the caruncle. Basal TLR levels were higher in fetal spleen than in placental tissues. This study provides novel information on how innate TLR responses are induced at the maternal-fetal interface of cattle in response to intracellular N. caninum.

Colonic MUC2 mucin regulates the expression and antimicrobial activity of ß-defensin 2.

In this study we identified mechanisms at the colonic mucosa by which MUC2 mucin regulated the production of ß-defensin in a proinflammatory milieu but functionally protected susceptible bacteria from its antimicrobial effects. The regulator role of MUC2 on production of ß-defensin 2 in combination with the proinflammatory cytokine interleukin-1ß (IL-1ß) was confirmed using purified human colonic MUC2 mucin and colonic goblet cells short hairpin RNA (shRNA) silenced for MUC2. In vivo, Muc2(-/-) mice showed impaired ß-defensin mRNA expression and peptide localization in the colon as compared with Muc2(+/-) and Muc2(+/+) littermates. Importantly, purified MUC2 mucin abrogated the antimicrobial activity of ß-defensin 2 against nonpathogenic and enteropathogenic Escherichia coli. Sodium metaperiodate oxidation of MUC2 removed the capacity of MUC2 to stimulate ß-defensin production and MUC2's inhibition of defensin antimicrobial activity. This study highlights that a defective MUC2 mucin barrier, typical in inflammatory bowel diseases, may lead to deficient stimulation of ß-defensin 2 and an unbalanced microbiota that favor the growth of ß-defensin-resistant microbes such as Clostridium difficile.

Comparative histopathology and antibody responses of non-Tritrichomonas foetus trichomonad and Tritrichomonas foetus genital infections in virgin heifers.

The potential pathogenicity of non-Tritrichomonas foetus trichomonads (NTfTs) recently isolated from the prepuce of virgin bulls is not known. The purpose of this study was to determine the ability of these NTfTs to cause disease in the female reproductive tract relative to T. foetus. Forty-four virgin heifers were experimentally infected intravaginally with either one of two NTfTs (Pentatrichomonas hominis or Tetratrichomonas spp.), T. foetus, or sterile media and cultured weekly from 0 time until slaughter at 8 weeks. Serum and vaginal antibody responses during infection were assessed, and the reproductive tracts were histologically examined, scored, and compared based on numbers of neutrophils, eosinophils, lymphocytes, and plasma cells as well as the qualitative appearance of the reproductive tract. The NTfTs did not persist in the reproductive tract, while T. foetus persisted for at least 6-8 weeks. Further, no vaginal IgA response to infection was found in NTfT-infected and control heifers, but a vaginal IgA response was present in the T. foetus-infected group. Heifers infected with NTfT or controls showed little mucosal inflammatory response compared to T. foetus-infected heifers. Among the trichomonads studied, persistent infection by T. foetus alone seems responsible for uterine inflammatory lesions usually associated with pregnancy loss. The NTfTs studied in this work only transiently infected the vagina and were associated with strictly mild inflammatory changes, which probably do not cause significant disease, i.e., pregnancy loss.

Sensitivity and specificity of culture and PCR of smegma samples of bulls experimentally infected with Tritrichomonas foetus.

The sensitivity (Se) and specificity (Sp) of different testing schemes were estimated for detecting Tritrichomonas foetus (T. foetus) in smegma samples from experimentally infected bulls. Culture and polymerase chain reaction (PCR) on smegma samples were evaluated alone and in parallel testing. Mature dairy bulls (n=79) were intrapreputially inoculated with T. foetus (n=19); Campylobacter (C.) fetus venerealis (n=13); both T. foetus and C. fetus venerealis (n=11); Tetratrichomonas spp. (n=9); C. fetus fetus (n=8); or were not inoculated (n=19). For each bull, smegma samples were collected for 6 week post-inoculation and tested for T. foetus by In Pouch TF culture and PCR. Most T. foetus-inoculated bulls became infected, according to culture (86.7%), PCR (90.0%), and both tests together (93.3%). In T. foetus-inoculated bulls, both tests combined in parallel on a single sample had a Se (78.3%) and Sp (98.5%) similar to two cultures (Se 76.0%, Sp 98.5%) or two PCR (Se 78.0%, Sp 96.7%) sampled on consecutive weeks. The PCR on three consecutive weekly samples (Se 85.0%, Sp 95.4%) and both tests applied in parallel on three consecutive weekly samples (Se 87.5%, Sp 95.6%) were similar to the current gold-standard of six weekly cultures (Se 86.7% and Sp 97.5%). Both tests used in parallel six times had the highest Se (93.3%), with similar Sp (92.5%). Tetratrichomonas spp. were only sporadically detected by culture or PCR. In conclusion, we have proposed alternative strategies for T. foetus diagnostics (for the AI industry), including a combination of tests and repeat testing strategies that may reduce time and cost for bull surveillance.

Lectin binding patterns and immunohistochemical antigen detection in the genitalia of Tritrichomonas foetus-infected heifers.

Heifers inoculated intra-vaginally with Tritrichomonas foetus were examined after long-term infection (70 days) and short-term infection (20 days) by lectin-histochemical, immunohistochemical and cultural techniques. The organism was recovered from the genital tract and T. foetus antigens were detected immunohistochemically in the lumina of uterine glands and cytoplasm of vaginal subepithelial macrophages. An increase of galactosylated residues (galactose and N-acetyl galactose), binding to PNA, was observed in the genital epithelium (vagina, uterus and oviduct) from infected animals. In the oviductal epithelium of short- but not long-term infected heifers, mannose (binding to Con A) was detected, suggesting that the persistent presence of T. foetus and its virulence factors or inflammatory processes result in a change in the glycoproteins of the epithelial surface. The findings have implications for the adhesion of T. foetus to cells and for the pathogenesis of bovine trichomonosis.

Effect of two commercial vaccines to Campylobacter fetus subspecies on heifers naturally challenged.

Efficacy of two commercial vaccines containing Campylobacter fetus subspecies on heifers naturally challenged by service with an infected bull was tested. Sixteen heifers were vaccinated parentally two times with 3 weeks as interval, eight with commercial vaccine A and the other eight with commercial vaccine B. Eight other heifers were used as unvaccinated controls. Forty days after the first vaccine dose, the heifers were served by an infected bull during 60 days. Measure of systemic immune response and identification of the microorganism from genital secretions by culture and immunofluorescent antibody test (IFAT) were done. Vaccinated and control heifers had a poor reproductive performance (pregnancy rates were 2/8, 3/8 and 0/8 in groups A, B and C, respectively) and were infected by both methods during breeding time and after it. Moreover, one heifer in the groups B and C remained infected until 300 days post-breeding time. Neither vaccinated nor control heifers had an important increment of systemic antibody level. Only, they had a slight increment of antibody level after the breeding period and it may be because of natural stimulus by the infected bull during the copula. Culture and IFAT yielded high correlation on identification of C. fetus subspecies.

Experimental infection with Tritrichomonas suis in heifers.

Nine heifers were intravaginally challenged with 9.3x10(6) Tritrichomonas suis reference strains. Vaginal mucus and serum samples were collected weekly 4 weeks post-inoculation. Vaginal mucus was cultured for T. suis and sera was tested by ELISA against whole cell antigens for T. suis and Tritrichomonas foetus. All vaginal mucus cultures were T. suis-negative during the experiment. ELISA values for both antigens were similar and differences were not significant (P>0.05). Positive control serum samples from one heifer vaccinated against T. foetus showed anti-T. suis ELISA values. We concluded that T. suis intravaginal inoculation induced a low level of serum immune response in heifers measured by ELISA and both protozoa probably share a common antigen. However, under the experimental conditions of this trial, colonization of the heifers' genital tract was not possible in any of the nine animals.