Evaluation of the innate immune response of caprine neutrophils against Mycobacterium avium subspecies paratuberculosis in vitro.

Neutrophils constitute an essential component of the innate immune response, readily killing most bacteria through phagocytosis, degranulation, and the release of neutrophil extracellular traps (NETs) among other mechanisms. These cells play an unclear role in mycobacterial infections such as Mycobacterium avium subspecies paratuberculosis (Map), the etiological agent of paratuberculosis, and its response is particularly understudied in ruminants. Herein, a wide set of techniques were adapted, or newly developed, to study the in vitro response of caprine neutrophils after Map infection. Immunofluorescence was used to demonstrate, simultaneously, chemotaxis, phagocytosis, degranulation, and NETs. The quantification of neutrophil phagocytic activity against Map at a 1:10 multiplicity of infection (MOI), through flow cytometry, showed values that varied from 4.54 to 5.63% of phagocyting neutrophils. By immunofluorescence, a 73.3 ± 14.5% of the fields showed NETs, and the mean release of DNA, attributable to NETosis, calculated through a fluorometric method, was 16.2 ± 3.5%. In addition, the RNA expression of TGF-ß, TNF and IL-1ß cytokines, measured through reverse transcription qPCR, was significantly higher in the two latter. Overall, neutrophil response was proportional to the number of bacteria. This work confirms that the simultaneous study of several neutrophil mechanisms, and the combination of different methodologies, are essential to reach a comprehensive understanding of neutrophil response against pathogens, demonstrates that, in vitro, caprine neutrophils display a strong innate response against Map, using their entire repertoire of effector functions, and sets the basis for further in vitro and in vivo studies on the role of neutrophils in paratuberculosis.

Local assessment of the immunohistochemical expression of Foxp3+ regulatory T lymphocytes in the different pathological forms associated with bovine paratuberculosis.

BACKGROUND: Mycobacterium avium subsp. paratuberculosis infected animals show a variety of granulomatous lesions, from focal forms with well-demarcated granulomas restricted to the gut-associated lymphoid tissue (GALT), that are seen in the initial phases or latency stages, to a diffuse granulomatous enteritis, with abundant (multibacillary) or scant (paucibacillary) bacteria, seen in clinical stages. Factors that determine the response to the infection, responsible for the occurrence of the different types of lesion, are still not fully determined. It has been seen that regulatory T cells (Treg) play an important role in various diseases where they act on the limitation of the immunopathology associated with the immune response. In the case of paratuberculosis (PTB) the role of Treg lymphocytes in the immunity against Map is far away to be completely understood; therefore, several studies addressing this subject have appeared recently. The aim of this work was to assess, by immunohistochemical methods, the presence of Foxp3+ T lymphocytes in intestinal samples with different types of lesions seen in cows with PTB. METHODS: Intestinal samples of twenty cows showing the different pathological forms of PTB were evaluated: uninfected controls (n = 5), focal lesions (n = 5), diffuse paucibacillary (n = 5) and diffuse multibacillary (n = 5) forms. Foxp3+ lymphocyte distribution was assessed by differential cell count in intestinal lamina propria (LP), gut-associated lymphoid tissue (GALT) and mesenteric lymph node (MLN). RESULTS: A significant increase in the number of Foxp3+ T cells was observed in infected animals with respect to control group, regardless of the type of lesion. However, when the different categories of lesion were analyzed independently, all individuals with PTB lesions showed an increase in the amount of Foxp3+ T lymphocytes compared to the control group but this increase was only significant in cows with focal lesions and, to a lesser extent, in animals with diffuse paucibacillary forms. The former showed the highest numbers, significantly different from those found in cows with diffuse lesions, where no differences were noted between the two forms. No specific distribution pattern was observed within the granulomatous lesions in any of the groups. CONCLUSIONS: The increase of Foxp3+ T cells in focal forms, that have been associated with latency or resistance to infection, suggest an anti-inflammatory action of these cells at these stages, helping to prevent exacerbation of the inflammatory response, as occurs in diffuse forms, responsible for the appearance of clinical signs.

Early response of monocyte-derived macrophages from vaccinated and non-vaccinated goats against in vitro infection with Mycobacterium avium subsp. paratuberculosis.

Paratuberculosis is a disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). Vaccination is the most cost-effective control method. However, despite the fact that macrophages are the main target cells for this pathogen, the precise mechanisms behind the response of the macrophage to Map infection and how it is modified by vaccination are yet poorly understood. The aim of this study was to investigate the effect of Silirum® vaccination in the early immune response of caprine monocyte-derived macrophages (CaMØs). Peripheral blood mononuclear cells (PBMCs) were obtained from vaccinated and non-vaccinated goats, cultured in vitro until differentiation to macrophages and infected with Map. After a 24 h incubation, Map viability and DNA were assessed in culture by viable colony count and real time quantitative polymerase chain reaction (qPCR). In addition, Map phagocytosis and expression of IL-10, IL-12, IFN-γ, TNF-α, IL-17A, IL-1ß, iNOS, IL-6 and MIP-1ß were also evaluated through immunofluorescence labelling and reverse transcriptase qPCR (RT-qPCR), respectively. A significant reduction of Map viability was observed in both supernatants (P < 0.05) and CaMØs (P < 0.001) from the vaccinated group. Similarly, the percentage of infected CaMØs and the number of internalized Map by CaMØs (P < 0.0001) was higher in the vaccinated group. Finally, iNOS (P < 0.01) and IL-10 were significantly up-regulated in CaMØs from vaccinated goats, whereas only MIP-1ß was up-regulated in non-vaccinated animals (P < 0.05). These results show that vaccination modifies the immune response of CaMØs, suggesting that the phagocytosis and microbiocidal activity of macrophages against Map is enhanced after vaccination.

Characterization of Fetal Brain Damage in Early Abortions of Ovine Toxoplasmosis.

There is an unacknowledged clinical presentation of ovine toxoplasmosis characterized by early abortions and lesions of fetal leukoencephalomalacia. To investigate the pathogenesis of this condition, the extent and distribution of leukomalacia and the variations in the cell populations associated with it were characterized in 32 fetal brains from 2 previously published experimental studies of Toxoplasma gondii infection in pregnant sheep. Immunohistochemical labeling of ßAPP allowed for the detection of leukomalacia in 100/110 (91%) studied samples. There was no clear influence of the challenge dose or the area of the brain (frontal lobe, corpus callosum, midbrain, and cerebellum). In tissues with leukomalacia, there was loss of oligodendrocytes and increased number of astrocytes and microglia both in the areas of necrosis but also in the surrounding area. These findings were similar to those described in ovine experimental models (inflammation syndrome and hypoxic models) of periventricular leukomalacia in humans. Thus, a fetal inflammatory syndrome may be involved in the pathogenesis of early abortion in ovine toxoplasmosis. However, further studies are needed to determine the pathogenesis of this clinical presentation because placental thrombosis and resulting hypoxia could also be responsible for the leukomalacia.

Essential oil supplementation in milk replacers: short- and long-term impacts on feed efficiency, the faecal microbiota and the plasma metabolome in dairy calves.

Early supplementation with oregano essential oil (EO) in milk replacer (MR) may improve growth, immune responses, the microbiota and the metabolome in dairy calves during pre-weaning and in adulthood. Sixteen female dairy calves (3 days of age) were divided in two groups (n = 8/group): the control group (no EO) and the EO group (0.23 ml of EO in MR during 45 days). After weaning, calves were kept in a feedlot and fed ad libitum. The animals were weighed, and blood and faecal samples were collected on days 3 (T0), 45 (T1) and 370 (T2) to measure the biochemical profile and characterise peripheral blood mononuclear cells (PBMCs; CD4+, CD8+, CD14+, CD21+ and WC1+), the metabolome and microbiota composition. The EO group only had greater average daily weight gain during the suckling (EO supplementation) period (P = 0.030). The EO group showed higher average CD14+ population (monocytes) values, a lower abundance of Ruminococcaceae UCG-014, Faecalibacterium, Blautia and Alloprevotella and increased abundances of Allistipes and Akkermansia. The modification of some metabolites in plasma, such as butyric acid, 3-indole-propionic acid and succinic acid, particularly at T1, are consistent with intestinal microbiota changes. The data suggest that early EO supplementation increases feed efficiency only during the suckling period with notable changes in the microbiota and plasma metabolome; however, not all of these changes can be considered desirable from a gut health point of view. Additional research studies is required to demonstrate that EOs are a viable natural alternative to antibiotics for improving calf growth performance and health.

Experimental infection of sheep at mid-pregnancy with archetypal type II and type III Toxoplasma gondii isolates exhibited different phenotypic traits.

Toxoplasma gondii is a major cause of reproductive failure in small ruminants. Genotypic diversity of T. gondii strains has been associated with variations in phenotypic traits in in vitro and murine models. However, whether such diversity could influence the outcome of infection in small ruminants remains mostly unexplored. Here, we investigate the outcome of oral challenge in sheep at mid-pregnancy with 10 sporulated oocysts from three different T. gondii isolates belonging to archetypal II and III and selected according to their genetic and phenotypic variations shown in previous studies. Seventy-three pregnant sheep were divided in four groups: G1 infected with TgShSp1 isolate (type II, ToxoDB#3), G2 with TgShSp16 isolate (type II, ToxoDB#3), G3 with TgShSp24 isolate (type III, ToxoDB#2) and G4 of uninfected control sheep. Two different approaches were carried out within this study: (i) the outcome for the pregnancy after infection (n = 33) and (ii) the lesions and parasite tropism and burden at 14 and 28 days post infection (dpi) (n = 40). The onset of hyperthermia and seroconversion occurred one and two days later, respectively in G1 when compared to G2 and G3. However, sheep that suffered from reproductive failure, either by abortion, foetal dead at the time of euthanasia or stillbirth were similar among infected groups (50%, 40% and 47%, respectively). Histological lesions in placentomes and foetal tissues from euthanized animals from the second approach were only detected at 28 dpi and mainly in G1. At 14 dpi, T. gondii-DNA was only detected in G1 in the 11% of the placentomes. However, at 28 dpi the frequency of detection in placentomes was higher in G1 (96%) than in G2 and G3 (7% and 47%, respectively) besides in foetuses was lower in G2 (20%) than in G1 and G3 (100% and 87%, respectively). Regarding late abortions, stillbirths, and lambs of G1, G2 and G3, the frequency of microscopic lesions was similar between groups (79%, 78% and 67%, respectively) whereas T. gondii-DNA was evidenced in 100%, 55% and 100%, respectively. These recently obtained T. gondii isolates led to similar reproductive losses but intra- and inter-genotype variations in the rise of hyperthermia, dynamics of antibodies, frequency of lesions and parasite detection and distribution. Thus, the different phenotypic traits of the isolates could influence the outcome of the infection and mechanisms responsible for it, and further investigations are warranted.

Pathology of the Mammary Gland in Sheep and Goats.

The recognition of lesions of the mammary gland in small ruminants is a useful diagnostic procedure that can aid in the identification of several udder diseases. This article reviews the main pathological lesions in this organ in sheep and goats. Mastitis is, by far, the most commonly diagnosed change. Acute clinical mastitis is associated with bacterial infections, mainly Staphylococcus aureus or Mannheimia haemolytica. Lesions related to subclinical and chronic mastitis are also described, either as localized cases or as a part of systemic diseases such as contagious agalactia, maedi-visna or tuberculosis. Neoplasia is rare in the mammary gland of sheep and goats with sporadic mammary adenocarcinomas most commonly reported. Teat lesions, including those due to trauma, orf virus infection or papillomas, are predisposing factors for the subsequent development of mastitis.

Ovine Neosporosis: The Current Global Situation.

In the past 20 years, Neospora caninum infection in sheep has been reported in at least 31 countries worldwide from all sheep-rearing continents (Europe, Asia, the Americas, Africa, and Oceania), and its role as an abortifacient agent is becoming more evident. Most studies of ovine neosporosis have focused on its epidemiology, based primarily on serological analysis, with only a few studies investigating the actual presence of the parasite by PCR and/or IHC. Individual seroprevalence rates were highly variable between countries, and even between regions within the same country, ranging from 0.0% to 67.4% positive. Furthermore, most of the studies were not directly comparable due to differences in experimental designs, sample sizes, husbandry systems, ecological factors, and serological tests (e.g., IFAT, ELISA, MAT, Western blot). The latter, along with the scarcity of studies on the relevance of N. caninum as an abortifacient agent, may bias the perception of the importance of this disease. This review summarizes the situation of N. caninum infection in sheep using all available published studies describing natural ovine neosporosis. The epidemiology shows that ovine neosporosis is found worldwide, and it poses a relevant risk to the sustainability of sheep flocks.

Peripheral IFN-É£ Production after Blood Stimulation with Different Mycobacterial Antigens in Goats Vaccinated against Paratuberculosis.

Vaccination can be an efficient method for the control of paratuberculosis in ruminants. However, the official tuberculosis control tests cross-interfere with the animals vaccinated against paratuberculosis. In order to test and compare new antigens that could solve this problem, the production of interferon-gamma (IFN-γ) in peripheral blood at different post-vaccination days in experimental kids and adult goats, in field conditions, using the avian and bovine purified protein derivative (PPD), the johnin, two peptide cocktails of Mycobacterium bovis (PC-EC and PC-HP) and the antigens VK 055 and VK 067 of Mycobacterium avium subspecies paratuberculosis (Map) has been analyzed in vitro. The non-specific production of IFN-γ was observed after blood stimulation with the PC-EC and PC-HP cocktail in any sample from vaccinated animals, whereas it was detected when bovine PPD was used. These results support the possible use of these new Mycobacterium bovis antigens in the in the differentiation of animals vaccinated against paratuberculosis or infected with tuberculosis by improving the specificity of bovine PPD. In contrast, the two Map antigens tested in this study did not improve the sensitivity of johnin or avian PPD in the detection of vaccinated or Map-infected goats.

Effects of Paratuberculosis Vaccination at Different Ages in a Dairy Goat Herd: A 2-Year Follow-Up.

Vaccination could be considered as an effective method for paratuberculosis control, although controversial, with a need for investigation in some aspects. The objective of this study was to evaluate the effect of vaccination, depending on the age of the animals, on their immune response, the reduction of paratuberculosis cases, mortality and culled animals in a commercial dairy herd. Goats from three different ages were immunized with the inactivated Gudair® vaccine. Peripheral antibody and IFN-γ output were evaluated for 21 months post-vaccination (mpv) and intradermal skin tests (IDSTs) for tuberculosis, with avian- and bovine-purified protein derivatives (PPD), were carried out at 6 and at 18 mpv to evaluate the humoral and cellular immune peripheral responses, respectively. The number of dead or culled animals, regardless of the reason, was also monitored and the causes of death determined by pathological examination. A significant increase in the production of IFN-γ was observed in all the vaccinated groups when the blood samples were stimulated with avian PPD, from 3 mpv to 18 mpv, and with bovine PPD, between 3 and 21 mpv. Moreover, serum antibody levels increased between 3 and 21 mpv in all vaccinated groups. The highest levels were found in animals vaccinated at 5 months, and the lowest in adult individuals. No positive reactants to tuberculosis were found by intradermal skin test. No animal losses associated with clinical paratuberculosis were detected in any of the groups. The number of total culled animals was significantly lower in the vaccinated than in the unvaccinated groups, especially on 1.5-month-old vaccinated kids. These results suggest that vaccination of paratuberculosis, especially in young animals, could induce heterologous protection.

Assessment of Paratuberculosis Vaccination Effect on In Vitro Formation of Neutrophil Extracellular Traps in a Sheep Model.

Vaccination of domestic ruminants against paratuberculosis has been related to homologous and heterologous protective effects that have been attributed to the establishment of a trained immune response. Recent evidence suggests that neutrophils could play a role in its development. Therefore, we propose an in vitro model for the study of the effect of paratuberculosis vaccination on the release of neutrophil extracellular traps (NETs) in sheep. Ovine neutrophils were obtained from non-vaccinated (n = 5) and vaccinated sheep (n = 5) at different times post-vaccination and infected in vitro with Mycobacterium avium subsp. paratuberculosis (Map), Staphylococcus aureus (SA), and Escherichia coli (EC). NETs release was quantified by fluorimetry and visualized by immunofluorescence microscopy. Typical NETs components (DNA, neutrophil elastase, and myeloperoxidase) were visualized extracellularly in all infected neutrophils; however, no significant percentage of extracellular DNA was detected in Map-infected neutrophils compared with SA- and EC-infected. In addition, no significant effect was detected in relation to paratuberculosis vaccination. Further assays to study NETs release in ovine neutrophils are needed. Preliminary results suggest no implication of NETs formation in the early immune response after vaccination, although other neutrophil functions should be evaluated.

Effects of Ovine Monocyte-Derived Macrophage Infection by Recently Isolated Toxoplasma gondii Strains Showing Different Phenotypic Traits.

Ovine toxoplasmosis is one the most relevant reproductive diseases in sheep. The genetic variability among different Toxoplasma gondii isolates is known to be related to different degrees of virulence in mice and humans, but little is known regarding its potential effects in sheep. The aim of this study was to investigate the effect of genetic variability (types II (ToxoDB #1 and #3) and III (#2)) of six recently isolated strains that showed different phenotypic traits both in a normalized mouse model and in ovine trophoblasts, in ovine monocyte-derived macrophages and the subsequent transcript expression of cytokines and iNOS (inducible nitric oxide synthase). The type III isolate (TgShSp24) showed the highest rate of internalization, followed by the type II clonal isolate (TgShSp2), while the type II PRU isolates (TgShSp1, TgShSp3, TgShSp11 and TgShSp16) showed the lowest rates. The type II PRU strains, isolated from abortions, exhibited higher levels of anti-inflammatory cytokines and iNOS than those obtained from the myocardium of chronically infected sheep (type II PRU strains and type III), which had higher levels of pro-inflammatory cytokines. The present results show the existence of significant intra- and inter-genotypic differences in the parasite-macrophage relationship that need to be confirmed in in vivo experiments.

Influence of Heterologous and Homologous Vaccines, and Their Components, on the Host Immune Response and Protection Against Experimental Caprine Paratuberculosis.

Vaccination against paratuberculosis, a chronic disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map), has been considered as the most effective control method. However, protection is incomplete, and the mechanisms operating in the response of the animals to vaccination are not fully understood. Therefore, this study analyzed the immune response and the effects on protection against Map infection, elicited by paratuberculosis (Silirum®) and tuberculosis (heat-inactivated M. bovis [HIMB]) vaccines and their components in a caprine experimental model. Fifty goat kids were divided into 10 groups (n = 5) according to their vaccination (Silirum®, HIMB and nonvaccinated), immunization (inactivated bacteria or adjuvant), and/or infection. Oral challenge with Map was performed 45 days postvaccination/immunization (dpv), and animals were euthanized at 190 dpv. Peripheral immune response and proportion of lymphocyte subpopulations were assessed monthly by enzyme-linked immunosorbent assay and flow cytometry analysis, respectively. Local immune response, proportion of tissue lymphocyte subpopulations, Map detection (polymerase chain reaction), and histological examination were conducted in gut-associated lymphoid tissues. All infected groups developed paratuberculosis granulomatous lesions despite vaccination or immunization. The Silirum® and HIMB-vaccinated groups showed a considerable lesion reduction consistent with a significant peripheral cellular and humoral immune response. Besides, a lower number of granulomas were observed in groups immunized with inactivated bacteria and adjuvants in comparison to nonvaccinated and infected group. However, despite not being significant, this reduction was even higher in adjuvant immunized groups, which developed milder granulomatous lesion with no detectable peripheral immune responses associated with immunization. No changes in the peripheral and local proportion of lymphocyte subsets or local immune response were detected in relation to either vaccination/immunization or infection. Despite that paratuberculosis and tuberculosis vaccination showed a partial and cross-protection against Map infection, respectively, only histological examination could assess the progression of infection in these animals. In addition, the pattern observed in the reduction of the lesions in adjuvant immunized groups suggests the possible involvement of a nonspecific immune response that reduces the development of granulomatous lesions.

Optimized in vitro isolation of different subpopulation of immune cells from peripheral blood and comparative techniques for generation of monocyte-derived macrophages in small ruminants.

Peripheral blood from healthy sheep (n = 3) and goats (n = 3) were employed to establish an efficient method for simultaneous isolation of peripheral blood mononuclear cells (PBMCs) and neutrophils and to standardize protocols for monocyte purification and generation of monocyte-derived macrophages (MDMs). In both species, a significantly enriched population of PBMCs, with higher purity and number of cells determined by flow cytometry, was achieved when processing through a density gradient a mixture of buffy-coat and red blood cell layer (RBC) in comparison to the use of just the buffy-coat (p < 0.05). Neutrophils could be subsequently isolated from the layer, located underneath PBMCs fraction with significant higher purity rates, higher than 85 % determined by flow cytometry, than those obtained with protocols without density gradients (< 60 %) (p < 0.05). This technique would allow the isolation of both cell populations from the same sample of blood. A pure cell population of monocytes, CD14+ cells, was purified from PBMCs when using immunomagnetic columns, which allow for 17 % (nº monocytes/nº PBMCs) of yield and high percentages of expression of CD14+ (88 %), MHC-II+ (91.5 %) and CD11b+ (94 %) established by flow cytometry. On the other hand, the classical and non-expensive purification of monocytes from PBMCs based on the adherence capacity of the former, allowed significantly lower yield of monocytes (4.6 %), with percentages of surface markers expression that dropped to 35 %, 65 % and 55 %, respectively (p < 0.001), suggesting the isolation of a mixed population of cells. The addition of GM-CSF to the culture, at concentration from 25 to 125 ng/mL, enhanced proportionally the number of MDMs generated compared to the absence of supplementation or the use of autologous serum from 5% to 20 %. However, purification of monocytes through the adherence method achieved higher yields of MDMs than those isolated through immunomagnetic columns in both species (p < 0.001). Under the conditions of this study, the use of centrifugation in density gradients allow for the simultaneous purification of PBMCs and neutrophils, with high purity of both populations, from the same sample of blood. The isolation of monocytes could be subsequently achieved through two different methods, i.e. based on immunomagnetic columns or adherence. The preference between both methods would depend on the necessities of the experiment, the initial sample with high purity of monocytes or a final population of MDMs required.

Virulence in Mice of a Toxoplasma gondii Type II Isolate Does Not Correlate With the Outcome of Experimental Infection in Pregnant Sheep.

Toxoplasma gondii is an apicomplexan parasite that infects almost all warm-blooded animals. Little is known about how the parasite virulence in mice extrapolates to other relevant hosts. In the current study, in vitro phenotype and in vivo behavior in mice and sheep of a type II T. gondii isolate (TgShSp1) were compared with the reference type II T. gondii isolate (TgME49). The results of in vitro assays and the intraperitoneal inoculation of tachyzoites in mice indicated an enhanced virulence for the laboratory isolate, TgME49, compared to the recently obtained TgShSp1 isolate. TgShSp1 proliferated at a slower rate and had delayed lysis plaque formation compared to TgME49, but it formed more cyst-like structures in vitro. No mortality was observed in adult mice after infection with 1-105 tachyzoites intraperitoneally or with 25-2,000 oocysts orally of TgShSp1. In sheep orally challenged with oocysts, TgME49 infection resulted in sporadically higher rectal temperatures and higher parasite load in cotyledons from ewes that gave birth and brain tissues of the respective lambs, but no differences between these two isolates were found on fetal/lamb mortality or lesions and number of T. gondii-positive lambs. The congenital infection after challenge at mid-pregnancy with TgShSp1, measured as offspring mortality and vertical transmission, was different depending on the challenged host. In mice, mortality in 50% of the pups was observed when a dam was challenged with a high oocyst dose (500 TgShSp1 oocysts), whereas in sheep infected with the same dose of oocysts, mortality occurred in all fetuses. Likewise, mortality of 9 and 27% of the pups was observed in mice after infection with 100 and 25 TgShSp1 oocysts, respectively, while in sheep, infection with 50 and 10 TgShSp1 oocysts triggered mortality in 68 and 66% of the fetuses/lambs. Differences in vertical transmission in the surviving offspring were only found with the lower oocyst doses (100% after infection with 10 TgShSp1 oocysts in sheep and only 37% in mice after infection with 25 TgShSp1 oocysts). In conclusion, virulence in mice of T. gondii type II isolates may not be a good indicator to predict the outcome of infection in pregnant sheep.