RESUMEN
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.
Asunto(s)
Enfermedades de las Cabras , Mycobacterium avium subsp. paratuberculosis , Paratuberculosis , Animales , Neutrófilos , Paratuberculosis/microbiología , Cabras , Inmunidad InnataRESUMEN
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.
Asunto(s)
Enfermedades de los Bovinos , Granuloma , Mycobacterium avium subsp. paratuberculosis , Paratuberculosis , Animales , Bovinos , Enfermedades de los Bovinos/microbiología , Femenino , Granuloma/microbiología , Granuloma/veterinaria , Intestinos/patología , Linfocitos T ReguladoresRESUMEN
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.
Asunto(s)
Vacunas Bacterianas/administración & dosificación , Enfermedades de las Cabras/inmunología , Leucocitos Mononucleares/inmunología , Macrófagos/inmunología , Mycobacterium avium subsp. paratuberculosis/inmunología , Paratuberculosis/inmunología , Vacunación/veterinaria , Animales , Enfermedades de las Cabras/microbiología , Cabras , Paratuberculosis/microbiologíaRESUMEN
To better understand the epizootiology of caprine paratuberculosis in the North of Portugal, a cross-sectional study was conducted from 2014 to 2015. The seroprevalence and risk factors for Mycobacterium avium subsp. paratuberculosis (Map) seropositivity were evaluated. Antibodies against Map were determined by a commercial ELISA. In 936 sera tested from 56 goat herds, 120 (12.8%, 95% CI: 10.8-15.1%) goats and 34 (60.7%, 95% CI: 47.6-72.4%) herds were positive. Risk factors for seropositivity were investigated by logistic regression models. The odds of Map seropositivity were found to be higher for animals with clinical signs, OR = 5.1 (95% CI: 2.7-9.6%), animals belonging to herds with previous wasting disease, OR = 2.3 (95% CI: 1.1-4.8%), and accumulation of manure in the herd, OR = 3.1 (95% CI: 1.7-5.7%). The potential risk factors identified in this study support the current recommendations for the control of paratuberculosis in these and other animals.
RESUMEN
Early abortion in ovine toxoplasmosis has had limited investigation. This study evaluated the immune response in the placenta of sheep orally infected with Toxoplasma gondii and euthanized between 2 and 4 weeks postinfection. Toxoplasma infection of the placenta was only found at 4 weeks after infection. Parasitic debris in foci of necrosis were immunolabeled in the maternal caruncle, whereas well-preserved intracellular parasitic vacuole-like structures were found in trophoblasts of fetal cotyledon. Early abortions had increased macrophages in caruncular septa, whereas in later abortions the placentas containing the parasite had an increase of T lymphocytes and macrophages mainly in the fetal cotyledons. This study suggests that the immune response in both the fetal and maternal compartments of the placenta may contribute to the pathogenesis of ovine toxoplasmosis and that these responses differ between early and late presentations of the disease.
Asunto(s)
Aborto Veterinario , Macrófagos/patología , Enfermedades de las Ovejas/patología , Linfocitos T/patología , Toxoplasmosis Animal , Aborto Veterinario/parasitología , Aborto Veterinario/patología , Animales , Femenino , Inmunidad , Inmunohistoquímica/veterinaria , Necrosis/parasitología , Necrosis/patología , Placenta/inmunología , Placenta/patología , Embarazo , Ovinos , Toxoplasma/aislamiento & purificación , Toxoplasma/patogenicidad , Toxoplasmosis Animal/inmunología , Toxoplasmosis Animal/patologíaRESUMEN
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.
Asunto(s)
Aborto Veterinario/patología , Encéfalo/patología , Feto/patología , Enfermedades de las Ovejas/patología , Toxoplasmosis Animal/patología , Aborto Veterinario/parasitología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Astrocitos/patología , Femenino , Inmunohistoquímica/veterinaria , Leucoencefalopatías/veterinaria , Microglía/patología , Necrosis/patología , Necrosis/veterinaria , Embarazo , Ovinos , Toxoplasma/patogenicidadRESUMEN
Although it is known that gestation could influence the clinical course of ovine toxoplasmosis, the precise effect of the term of gestation when sheep are infected are yet mostly unknown. The aim of this study was to evaluate the peripheral and placental immune responses developed in pregnant sheep after experimental infection with Toxoplasma gondii at different times of gestation. Thirty-six pregnant sheep were allocated in different groups, orally inoculated with sporulated oocysts of T. gondii at early, mid and late gestation and culled within 30 days post-infection. The peripheral humoral and cytokine responses were evaluated, as well as the transcription of cytokines at the placenta. Serological analysis revealed that, regardless the term of gestation when infected, specific IgG against T. gondii were detected from day 8 post-infection and there was an early peripheral release of IFN-γ at the first week post-infection followed by a short peak of IL10 and TNF-α at the second week post-infection. There were no significant differences in this response between infected groups. At the placenta, a similar increase in transcription of IFN-γ, and TNF-α was found at the three terms of gestation, while IL-4 increased mainly at the first and second terms and IL-10 transcription was higher at the last term. While these findings show that both Th1 and Th2 cytokines play a key role in the pathogenesis of ovine toxoplasmosis and that placental and peripheral immune responses do not closely correlate, there seems to be no clear modulation of these responses along the gestation.
Asunto(s)
Inmunidad Humoral/inmunología , Placenta/inmunología , Enfermedades de las Ovejas/inmunología , Toxoplasma/fisiología , Toxoplasmosis Animal/inmunología , Animales , Anticuerpos Antiprotozoarios , Femenino , Edad Gestacional , Oocistos/fisiología , Embarazo , Ovinos , Enfermedades de las Ovejas/parasitología , Factores de Tiempo , Toxoplasmosis Animal/parasitologíaRESUMEN
Ovine herpesvirus 2 (OvHV-2) is one of the gammaherpesviruses in the genus Macavirus that can cause malignant catarrhal fever (MCF) in ungulates. Sheep are the adapted host for OvHV-2 and it is generally assumed that infection is not associated with disease in this species. However, cases of "polyarteritis nodosa" or idiopathic systemic necrotizing vasculitis reported in sheep are similar to vascular lesions in clinically susceptible species with MCF. Using a recently developed in situ hybridization (ISH) method, we were able to identify OvHV-2 nucleic acids within lesions and correlate the viral distribution with systemic necrotizing vasculitis in 9 sheep, including both naturally and experimentally OvHV-2-infected animals. ISH, combined with polymerase chain reaction and histology, identify OvHV-2 as the likely agent responsible for sporadic, MCF-like vascular disease in sheep.
Asunto(s)
Gammaherpesvirinae , Poliarteritis Nudosa/veterinaria , Enfermedades de las Ovejas/virología , Animales , Poliarteritis Nudosa/virología , Ovinos , Enfermedades de las Ovejas/patologíaRESUMEN
Endogenous transplacental transmission, which occurs during pregnancy as the result of reactivation of a latent infection in the dam, is the main mechanism of propagation of Neospora caninum within cattle herds. However, the importance of this propagation mechanism has not yet been evaluated in relation to ovine neosporosis. In this study, involving three generations of ewes naturally infected by N. caninum, we demonstrated that endogenous transplacental transmission may also be highly efficient in the ovine host since transmission of infection occurred in 96.6% of gestations and the congenital infection rate ranged between 66.7 and 93%. Nevertheless, parasite burdens decreased gradually in consecutive generations. Reactivation of latent infections had a strong impact on the pregnancy outcome, with high mortality rates recorded in the offspring of the two first generations of ewes (21.4-46.1%). Histological examination of the brain revealed that all aborted foetuses had characteristic lesions of neosporosis (necrotic glial foci) and a few parasite cysts, whereas most stillborn and newborn lambs that died shortly after birth had non-specific lesions (mild glial foci without necrosis) and parasite cysts were more frequent. Microsatellite analysis revealed scarce genetic variability in the N. caninum population, in accordance with a scenario in which infections were of a single origin and were exclusively maintained by clonal propagation through endogenous transplacental transmission.
Asunto(s)
Coccidiosis/veterinaria , Transmisión Vertical de Enfermedad Infecciosa/veterinaria , Neospora , Complicaciones Parasitarias del Embarazo/veterinaria , Enfermedades de las Ovejas/parasitología , Animales , Anticuerpos Antiprotozoarios/sangre , Femenino , Inmunoglobulina G/sangre , Placenta , Embarazo , OvinosRESUMEN
Paratuberculosis (PTB), a chronic granulomatous enteritis produced by Mycobacterium avium subspecies paratuberculosis (MAP), is considered as one of the diseases with the highest economic impact in the ruminant industry. Vaccination against MAP is recommended during the first months after birth on the basis that protection would be conferred before the first contact with mycobacteria. However, little is known about the therapeutic effect of MAP vaccination in controlled experimental conditions. The current study was designed to evaluate the efficacy of vaccination before and after challenge with MAP in a rabbit infection model. The rabbits were divided into four groups: non-infected control (NIC, n = 4), infected control challenged with MAP (IC, n = 5), vaccinated and challenged 1 month after with MAP (VSI, n = 5) and challenged with MAP and vaccinated 2 months later (IVS, n = 5). The results from this study show a quick increase in IFN-γ release upon stimulation with bovine, avian and johnin PPD in animals vaccinated before MAP challenge. All vaccinated animals show an increased humoral response as seen by western blot and ELISA. The final bacteriology index (considering tissue culture and qPCR) shows that the IC group was the most affected. Vaccination after infection (IVS) produced the lowest bacteriology index showing significant differences with the IC group (p = 0.034). In conclusion, vaccination against MAP shows positive effects in a rabbit model. However, vaccination after infection shows a slightly stronger protective effect compared to vaccination before infection, suggesting a therapeutic effect. This feature could be applied to previously infected adult animals under field conditions.
Asunto(s)
Vacunas Bacterianas/uso terapéutico , Mycobacterium avium subsp. paratuberculosis/inmunología , Paratuberculosis/prevención & control , Animales , Carga Bacteriana/veterinaria , Vacunas Bacterianas/inmunología , Western Blotting/veterinaria , ADN Bacteriano/análisis , ADN Bacteriano/genética , Modelos Animales de Enfermedad , Electroforesis en Gel de Poliacrilamida/veterinaria , Ensayo de Inmunoadsorción Enzimática/veterinaria , Heces/química , Femenino , Inmunidad Celular/inmunología , Inmunidad Humoral/inmunología , Ensayos de Liberación de Interferón gamma/veterinaria , Paratuberculosis/inmunología , Paratuberculosis/microbiología , ConejosRESUMEN
Besides its importance in cattle, Neospora caninum may also pose a high risk as abortifacient for small ruminants. We have recently demonstrated that the outcome of experimental infection of pregnant sheep with 10(6) Nc-Spain7 tachyzoites is strongly dependent on the time of gestation. In the current study, we assessed peripheral and local immune response in those animals. Serological analysis revealed earlier and higher IFN-γ and IgG responses in ewes infected at early (G1) and mid (G2) gestation, when abortion occurred. IL-4 was not detected in sera from any sheep. Inflammatory infiltrates in the placenta mainly consisted of CD8+ and, to a lesser extent, CD4+ T cells and macrophages (CD163+). The infiltrate was more intense in sheep infected at mid-gestation. In the foetal mesenchyme, mostly free tachyzoites were found in animals infected at G1, while those infected in G2 displayed predominantly particulate antigen, and parasitophorous vacuoles were detected in sheep infected at G3. A similar pattern of placental cytokine mRNA expression was found in all groups, displaying a strengthened upregulation of IFN-γ and IL-4 and milder increases of TNF-α and IL-10, reminiscent of a mixed Th1 and Th2 response. IL-12 and IL-6 were only slightly upregulated in G2, and TGF-ß was downregulated in G1 and G2, suggestive of limited T regulatory (Treg) cell activity. No significant expression of TLR2 or TLR4 could be detected. In summary, this study confirms the pivotal role of systemic and local immune responses at different times of gestation during N. caninum infection in sheep.
Asunto(s)
Coccidiosis/veterinaria , Neospora , Complicaciones Parasitarias del Embarazo/veterinaria , Enfermedades de las Ovejas/parasitología , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígenos de Protozoos/sangre , Coccidiosis/inmunología , Citocinas/genética , Citocinas/metabolismo , Femenino , Regulación de la Expresión Génica/inmunología , Inmunoglobulina G/sangre , Macrófagos , Placenta/metabolismo , Embarazo , Complicaciones Parasitarias del Embarazo/inmunología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Ovinos , Enfermedades de las Ovejas/inmunología , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMEN
The relation between gestational age and foetal death risk in ovine toxoplasmosis is already known, but the mechanisms involved are not yet clear. In order to study how the stage of gestation influences these mechanisms, pregnant sheep of the same age and genetic background were orally dosed with 50 oocysts of Toxoplasma gondii (M4 isolate) at days 40 (G1), 90 (G2) and 120 (G3) of gestation. In each group, four animals were culled on the second, third and fourth week post infection (pi) in order to evaluate parasite load and distribution, and lesions in target organs. Ewes from G1 showed a longer period of hyperthermia than the other groups. Abortions occurred in all groups. While in G2 they were more frequent during the acute phase of the disease, in G3 they mainly occurred after day 20 pi. After challenge, parasite and lesions in the placentas and foetuses were detected from day 19 pi in G3 while in G2 or G1 they were only detected at day 26 pi. However, after initial detection at day 19 pi, parasite burden, measured through RT-PCR, in placenta or foetus of G3 did not increase significantly and, at in the third week pi it was lower than that measured in foetal liver or placenta from G1 to G3 respectively. These results show that the period of gestation clearly influences the parasite multiplication and development of lesions in the placenta and foetus and, as a consequence, the clinical course in ovine toxoplasmosis.
Asunto(s)
Enfermedades de las Ovejas/parasitología , Toxoplasma/fisiología , Toxoplasmosis Animal/parasitología , Factores de Edad , Animales , Femenino , Feto/parasitología , Feto/patología , Edad Gestacional , Placenta/parasitología , Placenta/patología , Embarazo , Ovinos , Enfermedades de las Ovejas/patología , Toxoplasmosis Animal/patologíaRESUMEN
Mycobacterium tuberculosis complex, Mycobacterium avium, and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection of Mycobacterium genus, M. avium subspecies, and M. tuberculosis complex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial (n = 38) and nonmycobacterial (n = 28) strains, tissues spiked with different CFU amounts of three mycobacterial species (n = 57), archival clinical samples (n = 233), and strains isolated from various hosts (n = 147). The minimum detectable DNA amount per reaction was 50 fg for M. bovis BCG and M. kansasii and 5 fg for M. avium subsp. hominissuis. When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identify M. avium and M. tuberculosis complex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses.
Asunto(s)
Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Mycobacterium avium/aislamiento & purificación , Mycobacterium tuberculosis/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Tuberculosis/diagnóstico , Tuberculosis/veterinaria , Humanos , Sensibilidad y EspecificidadRESUMEN
Neospora caninum is considered one of the main causes of abortion in cattle, yet recent studies have also emphasised its relevance as an abortifacient in small ruminants. In order to gain deeper insight into the pathogenesis of ovine neosporosis, pregnant ewes were intravenously inoculated with 10(6) tachyzoites of the Nc-Spain7 isolate at days 40, 90 or 120 of gestation. Infection during the first term resulted in the death of all foetuses between days 19 and 21 post-infection, showing mainly necrotic lesions in foetal liver and the highest parasite DNA detection and burden in both placenta and foetal viscera. After infection at day 90, foetal death was also detected in all ewes, although later (34-48 days post-infection). In this group, lesions were mainly inflammatory. Foetal livers showed the lowest frequency of lesions, as well as the lowest parasite detection and burden. All ewes infected at day 120 delivered viable lambs, although 3 out of 9 showed weakness and recumbency. Neospora DNA was detected in all lambs but one, and parasite burden was similar to that observed in day 90 group. Lesions in this group showed more conspicuous infiltration of inflammatory cells and higher frequency in foetal brain and muscle when compared to both previous groups. These results highlight the crucial role that the stage of gestation plays on the course of ovine neosporosis, similar to that reported in bovine neosporosis, and open the doors to consider sheep as a valid model for exogenous transplacental transmission for ruminant neosporosis.
Asunto(s)
Coccidiosis/veterinaria , Transmisión Vertical de Enfermedad Infecciosa/veterinaria , Neospora/fisiología , Enfermedades de las Ovejas/transmisión , Animales , Coccidiosis/parasitología , Coccidiosis/transmisión , ADN Protozoario/genética , Femenino , Feto/parasitología , Neospora/genética , Placenta/parasitología , Embarazo , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Ovinos , Enfermedades de las Ovejas/parasitología , Factores de TiempoRESUMEN
BACKGROUND: Rabbits are susceptible to infection by different species of the genus Mycobacterium. Particularly, development of specific lesions and isolation of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis, both subspecies of the M. avium complex, has been reported in wildlife conditions. Although, rabbit meat production worldwide is 200 million tons per year, microbiological data on this source of meat is lacking and more specifically reports of mycobacterial presence in industrially reared rabbit for human consumption have not been published. To this end, we sought mycobacteria by microbiological and histopathological methods paying special attention to Mycobacterium avium subsp. paratuberculosis in rabbits from commercial rabbitries from the North East of Spain. RESULTS: M. avium subsp. paratuberculosis was not detected either by culture or PCR. However, Mycobacterium avium subsp. avium was detected in 15.15% (10/66) and Mycobacterium avium subsp. hominissuis was detected in 1.51% (1/66) of gut associated lymphoid tissue of sampled animals by PCR, whereas caecal contents were negative. 9% (6/66) of the animals presented gross lesions suggestive of lymphoid activation, 6% (4/66) presented granulomatous lesions and 3% (2/66) contained acid fast bacilli. Mycobacterial isolation from samples was not achieved, although colonies of Thermoactinomycetes sp. were identified by 16s rRNA sequencing in 6% (4/66) of sampled animals. CONCLUSIONS: Apparently healthy farmed rabbits that go to slaughter may carry M. avium subspecies in gut associated lymphoid tissue.
Asunto(s)
Tracto Gastrointestinal/microbiología , Tejido Linfoide/microbiología , Mycobacterium avium/clasificación , Conejos/microbiología , Tuberculosis/veterinaria , Animales , Mycobacterium avium/aislamiento & purificación , Tuberculosis/microbiologíaRESUMEN
BACKGROUND: Up to 0.5% of churra lambs from two genetically related flocks showed congenital skin lesions of variable severity, jeopardizing the life of the lambs in the most severe cases. HYPOTHESIS/OBJECTIVES: The primary objective of this study was to classify the type of congenital epithelial disease suffered by these animals, based on the description of the macroscopic skin defects, the histological and ultrastructural changes and the hereditary nature of the condition. ANIMALS: Thirty affected newborn lambs from two genetically related flocks were studied. Three additional lambs acquired from two other flocks, which had no grossly apparent skin lesions and had died of infectious diseases, were studied as unaffected control animals. METHODS: Histological and ultrastructural examinations of skin and oral mucosa samples were performed. Pedigree analyses were used to investigate genealogical relationships. RESULTS: Generalized severe junctional epidermolysis bullosa with congenital absence of skin was described in all lambs studied and an autosomal recessive mode of inheritance was identified. CONCLUSIONS AND CLINICAL IMPORTANCE: The pathological findings and mode of inheritance in these lambs are similar to an inherited epidermolysis bullosa subtype of humans, which has not been reported previously in veterinary medicine.
Asunto(s)
Epidermólisis Ampollosa de la Unión/veterinaria , Enfermedades de las Ovejas/congénito , Anomalías Cutáneas/veterinaria , Animales , Epidermólisis Ampollosa de la Unión/genética , Epidermólisis Ampollosa de la Unión/patología , Femenino , Masculino , Microscopía Electrónica de Transmisión/veterinaria , Linaje , Ovinos , Enfermedades de las Ovejas/genética , Enfermedades de las Ovejas/patología , Piel/patología , Piel/ultraestructura , Anomalías Cutáneas/genética , Anomalías Cutáneas/patologíaRESUMEN
The two main genotypes of recognized isolates of Mycobacterium avium subsp. paratuberculosis (Map) are cattle (C) and sheep (S) strains. An experimental infection was conducted to establish the effect of Map strain on the pathogenesis of ovine paratuberculosis. Twenty-four out of thirty 1.5-month-old Assaf lambs were divided into 4 groups of 6 and infected orally with three low passage field isolates, two of S- (22G and the pigmented Ovicap49) and one of C- (764) type, and the reference K-10 strain (C type). The remaining six animals were unchallenged controls. Animals were euthanized at 150 and 390 days post-infection (dpi). Throughout the experiment, the peripheral immune response was assessed and histological and molecular (PCR) studies were conducted on samples of intestine and related lymphoid tissue. Specific antibody and IFN-γ production was significantly higher in animals infected with the C strains, while no consistent IFN- γ responses were observed in the S-type strain infected groups. A positive intradermal skin test response was detected in all infected groups. Lambs infected with S-type strains had granulomatous lesions restricted to the lymphoid tissue with no differences in the lesion intensity over time. In both C-type strain groups, lesions were more severe at 150 dpi while at 390 dpi lesions, characterized by well-demarcated granulomas with fibrosis, decreased in severity. Only infected lambs were positive to PCR. These results suggest that the strain of Map has a strong influence over the immune and pathological responses developed by the host. Lesions induced by C-type strains in lambs show a regressive character and tend to decrease as the infection progresses.
Asunto(s)
Inmunidad Celular , Mycobacterium avium subsp. paratuberculosis/fisiología , Paratuberculosis/inmunología , Paratuberculosis/patología , Enfermedades de las Ovejas/inmunología , Enfermedades de las Ovejas/patología , Animales , Ensayo de Inmunoadsorción Enzimática/veterinaria , Interferón gamma/sangre , Ensayos de Liberación de Interferón gamma/veterinaria , Intestinos/inmunología , Pruebas Intradérmicas/veterinaria , Tejido Linfoide/inmunología , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculosis/virología , Reacción en Cadena de la Polimerasa/veterinaria , Distribución Aleatoria , Ovinos , Enfermedades de las Ovejas/virologíaRESUMEN
After oral administration of ewes during mid gestation with 2000 freshly prepared sporulated oocysts of T. gondii isolate M4, abortions occurred between days 7 and 11 in 91.6% of pregnant and infected ewes. Afterwards, a further infection was carried out at late gestation in another group of sheep with 500 sporulated oocysts. Abortions happened again between days 9 and 11 post infection (pi) in 58.3% of the infected ewes. Classically, abortions in natural and experimental ovine toxoplasmosis usually occur one month after infection. Few experimental studies have reported the so-called acute phase abortions as early as 7 to 14 days after oral inoculation of oocysts, and pyrexia was proposed to be responsible for abortion, although the underline mechanism was not elucidated. In the present study, all placentas analysed from ewes suffering acute phase abortions showed infarcts and thrombosis in the caruncullar villi of the placentomes and ischemic lesions (periventricular leukomalacia) in the brain of some foetuses. The parasite was identified by PCR in samples from some placentomes of only one sheep, and no antigen was detected by immunohistochemical labelling. These findings suggest that the vascular lesions found in the placenta, and the consequent hypoxic damage to the foetus, could be associated to the occurrence of acute phase abortions. Although the pathogenesis of these lesions remains to be determined, the infectious dose or virulence of the isolate may play a role in their development.
Asunto(s)
Aborto Veterinario/patología , Enfermedades de las Ovejas/patología , Trombosis/veterinaria , Toxoplasma/fisiología , Toxoplasmosis Animal/patología , Aborto Veterinario/inmunología , Aborto Veterinario/parasitología , Animales , Femenino , Ensayos de Liberación de Interferón gamma/veterinaria , Enfermedades Placentarias/inmunología , Enfermedades Placentarias/parasitología , Enfermedades Placentarias/patología , Enfermedades Placentarias/veterinaria , Reacción en Cadena de la Polimerasa/veterinaria , Embarazo , Ovinos , Enfermedades de las Ovejas/inmunología , Enfermedades de las Ovejas/parasitología , Trombosis/inmunología , Trombosis/parasitología , Trombosis/patología , Toxoplasma/aislamiento & purificación , Toxoplasmosis Animal/inmunología , Toxoplasmosis Animal/parasitologíaRESUMEN
This work studies the influence of Neospora caninum intra-species diversity on abortion outcome, infection dynamics in terms of parasite dissemination and peripheral-local immune responses in pregnant cattle. Animals were intravenously inoculated at day 70 of pregnancy with 107 tachyzoites of two isolates showing marked differences in virulence in vitro and in pregnant mouse models: Nc-Spain7, a high virulence isolate, and Nc-Spain8, a low-to-moderate virulence isolate. After inoculation, pregnancy was monitored, and dams were culled when foetal death was detected. Foetal mortality occurred in all infected heifers between days 24 and 49 post-infection (pi), however, it was detected sooner in Nc-Spain7-infected animals (median day = 34) than those inoculated with Nc-Spain8 (median day = 41) with a trend towards significance (P < 0.11). Similar histological lesions were observed in placentomes and in most of the foetuses from the two infected groups. However, parasites were more frequently detected in the placenta and foetuses by PCR and in the foetal brain by immunohistochemistry in Nc-Spain7-infected animals. Specific antibodies were detected starting at day 13 post-infection in all infected cattle, with higher IgG levels in Nc-Spain7-infected group. IFN-γ and IL-4 profiles also varied between infected groups in PBMC stimulation assays. Infected animals showed significant increases in their cytokine mRNA levels (IFN-γ, IL-4, IL-10, IL-12p40 and TNF-α) in the caruncle at time of foetal death. Differences between the infected groups were also observed for cytokine profiles. These results demonstrate the influence of the N. caninum isolate on foetal death outcome, infection dynamics and immune responses in cattle.
Asunto(s)
Aborto Veterinario/parasitología , Enfermedades de los Bovinos/parasitología , Coccidiosis/veterinaria , Inmunidad Innata , Neospora/fisiología , Neospora/patogenicidad , Complicaciones Parasitarias del Embarazo/veterinaria , Aborto Veterinario/epidemiología , Aborto Veterinario/fisiopatología , Animales , Bovinos , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/fisiopatología , Coccidiosis/inmunología , Coccidiosis/parasitología , Coccidiosis/fisiopatología , Citocinas/inmunología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Feto/parasitología , Feto/patología , Técnica del Anticuerpo Fluorescente Indirecta/veterinaria , Inyecciones Intravenosas/veterinaria , Neospora/genética , Carga de Parásitos/veterinaria , Placenta/parasitología , Placenta/patología , Embarazo , Complicaciones Parasitarias del Embarazo/inmunología , Complicaciones Parasitarias del Embarazo/parasitología , Complicaciones Parasitarias del Embarazo/fisiopatología , VirulenciaRESUMEN
At present, bovine neosporosis is an important worldwide concern because of its wide geographic distribution and economic impact. Abortion is the main clinical sign of bovine neosporosis in both dairy and beef cattle. Ruminant challenge models are critical to evaluate potential vaccine candidates to help tackle bovine neosporosis and to study pathogenesis and host responses to infection. Several research groups have developed ruminant models of Neospora caninum infection independently of others, resulting in a high degree of variability due to the use of different species of animals, breeds, strains/isolates of N. caninum, doses, routes and times of inoculation. Standardization is greatly needed to advance research in a more collaborative, timely and efficient manner. In the absence of widely accepted international guidelines, this manuscript serves to summarize and discuss the different models and parameters currently in use. Parameters essential for the development of non-pregnant and pregnant ruminant models are outlined and the main knowledge gaps are identified. This information could act as the basis to develop a consensus for international standard guidelines for ruminant models of neosporosis that would be helpful for researchers in this field worldwide.