Immunopathogenesis of Chlamydia abortus infections in vaccinated and non-vaccinated ewes

Enzootic abortion of ewes (EAE) is caused by the obligate intracellular Gram-negative bacterium Chlamydia abortus. EAE is considered one of the most important causes of infectious abortion in sheep in many parts of the world. A necrosuppurative placentitis is typically associated with EAE. The primary function of the placenta is to provide fetal nutrition, therefore, anything that affects organ integrity may indirectly affect the viability of the fetus. The severity of placental lesions giving rise to abortion is a consequence of multiple interacting factors, in particular chlamydial growth, host immune responses and hormonal balance. A better understanding of these interactions may help to explain why EAE results in different pregnancy outcomes spanning: abortions, the birth of weak lambs that may die during the first days of their life or the birth of healthy lambs. Effective control of the disease is achieved through a combination of diagnosis, antibiotic therapy, flock management and vaccination. In the UK, vaccination is carried out using the live C. abortus 1B vaccine (vt) strain, which has been associated with abortion events in sheep flocks. The research hypothesis for this PhD thesis is that the distribution of C. abortus in the ovine placenta, pathological lesions and clinical outcomes of infection are not homogeneous within and between infected ewes and ewes vaccinated with the live 1B vaccine. This study aimed to increase knowledge of the pathogenesis of C. abortus in the placenta and uterus and its relationship with pregnancy outcome, thereby informing on improved diagnosis and control of the disease. The objectives were to: 1. Determine the relationship between phenotypical patterns of immune cell infiltration and the different outcomes of chlamydial infection in multifetal pregnancies. 2. Investigate the distribution and severity of lesions in placentas from wild-type (wt) C. abortus infected ewes and their relationship with pregnancy outcome. 3. Compare the distribution and severity of the lesions caused by the commercial C. abortus 1B vaccine strain with those resulting from a wt C. abortus infection. The uteri and placentas from experimental wt C. abortus -challenged twin-bearing ewes with different pregnancy outcomes (dead/dead, dead/live and live/live) were collected after parturition to address objective 1. Tissues were also collected from non-infected and EAE-free ewes as negative controls. All samples were analysed using a broad range of immune cell features, including cell surface antigens, T-cell transcription factors and cytokines. Elevated lymphocytes (cluster of differentiation (CD)4+, CD8+, gamma-delta T cells and natural killer cells), interleukin (IL)-10, tumour necrosis factor-alpha (TNF-α) and Forkhead box P3 (Foxp3) responses were observed by immunohistochemistry (IHC). Interferon-gamma and IL-17A increases were measured by in situ hybridisation. Additionally, a C. abortus major outer membrane protein (MOMP) antibody was used to detect Chlamydiae in the tissues to determine the relationship between pregnancy outcome, pathological lesion, and presence of immune cells. The level of T-helper (Th) and T-regulatory cell (Treg) features revealed statistically significant group effects, showing the important role that the balance of lymphocyte subsets may play in the different pregnancy outcomes in ewes. In addressing objective 2, several features associated with EAE, including placental lesions, length of gestation, lambing outcome, modified Ziehl-Neelsen scores, and the number of copies of C. abortus genomes were analysed, using the co-variables sex of the lamb (male or female) and type of gestation (single, multiple). One of the most significant parameters that was statistically associated with pregnancy outcome was the severity of placental lesions. For that reason, placentas presenting differences in the severity and distribution of lesions were examined and compared (specifically 0, 10, 25, 40, 50, 60, 85, 90 and 100% of the placental area affected). Where placentas exhibited gross lesions of between 0 and 20% of the placental surface, the difference was statistically significant between pregnancy and lamb birth weight. There were some variations in severity when the percentage of lesions increased from 25 % to 100%, but the statistical analysis of those differences was less significant. Vaccination with commercial vaccines containing the vt strain is a key strategy for controlling EAE in sheep flocks, but this strain has in some circumstances been associated with abortion. A pathological comparison was, therefore, made between placentas infected with the vt strain and EAE lesions caused by a wt strain, to address objective 3. Placentas collected from an EAE-free commercial sheep flock were assessed for gross pathological lesions and analysed for the presence of chlamydial DNA by real-time quantitative polymerase chain reaction (qPCR). Two placentas were observed to exhibit lesions consistent with EAE and were also the only two found positive by qPCR. Following isolation of the strain, PCR-restriction fragment length polymorphism (RFLP) and whole-genome sequence analyses, to distinguish vt from wt infections, confirmed the presence of only the vt strain. Comparative analyses were performed by histology and IHC for chlamydial labelling, to evaluate the difference between vt and wt strain placentas. The lesions caused by both, vt and wt strains were found to be indistinguishable. The results obtained from this study demonstrate that the distribution and severity of the placental lesions and chlamydial load are not uniform in both vt and wt infected ewes, confirming the research hypothesis. This study contributes relevant information about the pathogenesis of EAE that will help to inform the iterative improvement of control strategies. It is also a new starting point for future research into the rate of vertical transmission in vt and wt infected ewes, and the impact of this transmission on the reproductive performance of these intrauterine infected lambs in their adult life

Distribution and severity of placental lesions caused by the Chlamydia abortus 1B vaccine strain in vaccinated ewes

Chlamydia abortus infects livestock species worldwide and is the cause of enzootic abortion of ewes (EAE). In Europe, control of the disease is achieved using a live vaccine based on C. abortus 1B strain. Although the vaccine has been useful for controlling disease outbreaks, abortion events due to the vaccine have been reported. Recently, placental pathology resulting from a vaccine type strain (vt) infection has been reported and shown to be similar to that resulting from a natural wild-type (wt) infection. The aim of this study was to extend these observations by comparing the distribution and severity of the lesions, the composition of the predominating cell infiltrate, the amount of bacteria present and the role of the blood supply in infection. A novel system for grading the histological and pathological features present was developed and the resulting multi-parameter data were statistically transformed for exploration and visualisation through a tailored principal component analysis (PCA) to evaluate the difference between them. The analysis provided no evidence of meaningful differences between vt and wt strains in terms of the measured pathological parameters. The study also contributes a novel methodology for analysing the progression of infection in the placenta for other abortifacient pathogens.EEA MercedesFil: Caspe, Sergio Gaston. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Caspe, Sergio Gaston. Moredun Research Institute; Reino UnidoFil: Caspe, Sergio Gaston. University of Edinburgh. Royal (Dick) School of Veterinary Studies; Reino UnidoFil: Palarea-Albaladejo, Javier. Biomathematics & Statistics Scotland; Reino UnidoFil: Underwood, Clare. Moredun Research Institute; Reino UnidoFil: Livingstone, Morag. Moredun Research Institute; Reino UnidoFil: Wattegedera, Sean Ranjan. Moredun Research Institute; Reino UnidoFil: Milne, Elspeth. University of Edinburgh. Royal (Dick) School of Veterinary Studies; Reino UnidoFil: Sargison, Neil Donald. University of Edinburgh. Royal (Dick) School of Veterinary Studies; Reino UnidoFil: Chianini, Francesca. Moredun Research Institute; Reino UnidoFil: Longbottom, David. Moredun Research Institute; Reino Unid

An applied printing immunoassay with recombinant Nc-SAG1 for detection of antibodies to neospora caninum in cattle

Neospora caninum is a protozoan parasite that causes an important reproductive disease in cattle. Neospora caninum surface antigen 1 (Nc-SAG1) is an immunodominant candidate for the development of a diagnostic reagent for neosporosis. The current study describes the development and evaluation of an antigen print immunoassay (APIA) with recombinant Nc-SAG1 for the detection of specific antibodies to N. caninum in cattle. The concordance between APIA and a commercial enzyme-linked immunosorbent assay (ELISA) was evaluated with 232 serum samples from experimentally and naturally infected cattle. Sixty-one (26.7%) samples were positive for antibodies to N. caninum by ELISA and 58 (25.4%) by APIA. The new assay had a sensitivity of 85% and a specificity of 96%. These results, along with the potential of APIA to evolve into a multiple antigen detection format, suggest that this method would be a reliable diagnostic test for detection of antibodies to N. caninum in cattle.Fil: Wilkowsky, Silvina Elizabeth. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Gimenez Bareiro, Guillermo. Universidad Nacional de Asunción; ParaguayFil: Mon, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar; ArgentinaFil: Moore, Dadin Prando. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Agencia de Extensión Rural Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Caspe, Gastón. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: Campero, Carlos. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: Fort, Marcelo. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: Romano, Maria Isabel. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

The 1B vaccine strain of Chlamydia abortus produces placental pathology indistinguishable from a wild type infection

Chlamydia abortus is one of the most commonly diagnosed causes of infectious abortion in small ruminants worldwide. Control of the disease (Enzootic Abortion of Ewes or EAE) is achieved using the commercial live, attenuated C. abortus 1B vaccine strain, which can be distinguished from virulent wild-type (wt) strains by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Published studies applying this typing method and whole-genome sequence analyses to cases of EAE in vaccinated and non-vaccinated animals have provided strong evidence that the 1B strain is not attenuated and can infect the placenta causing disease in some ewes. Therefore, the objective of this study was to characterise the lesions found in the placentas of ewes vaccinated with the 1B strain and to compare these to those resulting from a wt infection. A C. abortus-free flock of multiparous adult ewes was vaccinated twice, over three breeding seasons, each before mating, with the commercial C. abortus 1B vaccine strain (Cevac® Chlamydia, Ceva Animal Health Ltd.). In the second lambing season following vaccination, placentas (n = 117) were collected at parturition and analysed by C. abortus-specific real-time quantitative PCR (qPCR). Two placentas, from a single ewe, which gave birth to live twin lambs, were found to be positive by qPCR and viable organisms were recovered and identified as vaccine type (vt) by PCR-RFLP, with no evidence of any wt strain being present. All cotyledons from the vt-infected placentas were analysed by histopathology and immunohistochemistry and compared to those from wt-infected placentas. Both vt-infected placentas showed lesions typical of those found in a wt infection in terms of their severity, distribution, and associated intensity of antigen labelling. These results conclusively demonstrate that the 1B strain can infect the placenta, producing typical EAE placental lesions that are indistinguishable from those found in wt infected animals

Aislamiento de <i>Campylobacter fetus</i> en un rodeo de cría con mermas tacto-parición de la provincia Corrientes (Argentina)

En Argentina, una de las limitantes de la eficiencia reproductiva de los rodeos de cría bovina está dada por la alta incidencia de enfermedades de la reproducción. Alrededor del 50 % de las pérdidas se deben a enfermedades infecciosas. El objetivo del trabajo fue establecer el motivo de la merma tacto-parición registrada en un establecimiento de cría para carne y evaluar posibles asociaciones estadísticas. El rodeo de 1454 vientres de la raza Braford se encuentra en un campo de terraza media en la localidad de Sauce, Corrientes, Argentina. El servicio es estacionado (noviembre a marzo) con una tasa preñez del 77,4 %. El análisis serológico para Brucella abortus (Buffered Plate Antigen, BPA), Leptospira spp. (microaglutinación en tubo, MAT) y Neospora caninum (inmunofluorescencia indirecta, IFI) fue realizado a 33 toros, 93 hembras preñadas (HP) y 7 hembras diagnosticadas como preñadas que luego resultaron vacías (HA).Trabajo publicado en Cagliada, Maria del Pilar Lilia y Galosi, Cecilia Mónica (comps.). I Congreso de Microbiología Veterinaria. Libro de resúmenes. La Plata: Facultad de Ciencias Veterinarias, 2021.Facultad de Ciencias Veterinaria

Humoral and cellular immune response and transplacental transmission in cows experimentally infected with Neospora caninum NC-6 Argentina strain

La infección por Neospora caninum es una importante causa de aborto en los bovinos. El objetivo de este estudio fue evaluar la respuesta inmune humoral y celular en bovinos y la posibilidad de transmisión transplacentaria durante una infección experimental con la cepa de N. caninum NC-6 Argentina. Se inocularon vacas preñadas de 65 días de gestación seropositivas (SP) y seronegativas (SN) a N. caninum por vía intravenosa con 108 taquizoítos de la cepa NC-6 o con PBS como control negativo.Facultad de Ciencias VeterinariasInstituto Nacional de Tecnología Agropecuari

Caracterización biológica y genética de la cepa de <i>Neospora caninum</i> NC-6 Argentina y aplicación práctica de la tipificación de microsatélites en infecciones experimentales en bovinos

La infección por Neospora caninum es una de las principales causas de abortos bovinos. Los objetivos de este trabajo fueron caracterizar genéticamente el aislamiento de N.caninum NC-6 Argentina utilizando el análisis de microsatélites y estudiar su comportamiento biológico mediante inoculaciones experimentales en bovinos preñados, evaluando la respuesta inmune producida y la ocurrencia de transmisión transplacentaria. Se inocularon vacas preñadas de 65 días de gestación, seropositivas y seronegativas a N. caninum, con 5 x 107 taquizoítos de la cepa NC-6 y se sacrificaron a los 108 +/- 2 días de gestación. Se tomaron muestras de sueros periódicamente y se les realizó inmunofluorescencia indirecta para anticuerpos. Se obtuvieron muestras de sangre los días 30 y 37, se estimularon in vitro con N. caninum y se analizó la producción de interferón gamma (IFNγ). Se tomaron muestras de órganos de las madres, las placentas y los fetos que fueron procesadas por histopatología, inmunohistoquímica y PCR para ADN de N. caninum Las muestras positivas se analizaron para la tipificación de los microsatélites. Los animales inoculados incrementaron significativamente los títulos de anticuerpos anti-N.caninum y la producción de IFNγ respecto a los controles. Una vaca seropositiva inoculada abortó, un feto del grupo seronegativo no fue viable y el resto de los fetos fueron viables pero presentaron lesiones. La PCR fue positiva en los fetos de las vacas seronegativas y en 2/3 fetos de las seropositivas. El análisis de microsatélites demostró que el ADN presente tenía un patrón idéntico a NC-6 Argentina. Éste es el primer reporte de una infección experimental de bovinos con la cepa de N. caninum aislada en Argentina. Esta cepa demostró su patogenicidad en animales seropositivos y seronegativos, fue capaz de atravesar la placenta y fue patógena para los fetos, el análisis de microsatélites demostró que la cepa hallada en las placentas era NC-6 Argentina.El presente trabajo ha sido galardonado con el Premio Mayor AAPAVET (Asociación Argentina de Parasitología Veterinaria) - Premio Anual Rioplatense 2010/2011 “Congreso Mundial de Parasitología Veterinaria” en la categoría Mejor trabajo original de investigación.Facultad de Ciencias Veterinaria

Alternatives for the serological assessment of foot-and-mouth disease vaccine immunity in buffaloes (Bubalus bubalis)

Buffaloes are compulsory vaccinated against foot-and-mouth disease virus (FMDV) in many countries as part of the official control programmes. Serological testing aimed to indirectly assess herd immunity is currently performed using the same Enzyme-linked immunosorbent assays (ELISAs) applied for bovine sera, assuming an agreement between the ELISA’s diagnostic results and those obtained using the virus neutralization test (VNT). Here we evaluated the accuracy of different ELISA tests to assess vaccine-induced antibodies against FMDV in buffalo’s sera classified according to their VNT titres. Currently used liquid-phase blocking ELISA yielded very low specificity, producing high titres for many samples with low VNT titres. To increase specificity, we developed an indirect ELISA using purified 140S viral particles and an avidity single-dilution ELISA, which includes a urea washing step after the incubation of the diluted serum sample, to detach weak binders. Combining these two high-throughput single-dilution tests, an excellent concordance with VNT was achieved. This is the first study analysing the diagnostic agreement of traditional and novel serological tests with VNT for the indirect assessment of antibodies against FMDV capsid proteins in buffalo serum samples

Kinetics of foot-and-mouth disease vaccine-induced antibody responses in buffaloes (Bubalus bubalis): Avidity ELISA as an alternative to the virus neutralization test

The role of water buffaloes in foot-and-mouth disease (FMD) epidemiology as one of the major hosts of the virus that can develop persistent asymptomatic infection highlights the importance of sustaining surveillance on the antibody response elicited by vaccination in these animals. There is gap in the knowledge on how serological assays that measure antibodies against capsid proteins perform with buffalo samples and which would be the most reliable test to substitute the virus neutralization test (VNT) a cumbersome and low-throughput tool for field surveillance. Alternatively, the liquid-phase blocking sandwich ELISA (LPBE) is commonly used. Previous data from our laboratory demonstrated that the vaccine-induced antibodies assessed by the LPBE yielded low specificity with buffaloes’ samples. In contrast, a single-dilution avidity ELISA (AE) aimed to detect high-avidity antibodies against exposed epitopes, combined with an indirect ELISA (IE) to assess IgG levels, produced more reliable results. Here we analyzed for the first time the kinetics of the antibodies induced by vaccination in two different buffalo herds (n = 91) over 120 days using AE, IE, LPBE, and the VNT. Kinetics were similar in the different assays, with an increase of antibodies between 0- and 14-days post-vaccination (dpv) which were maintained thereafter. VNT and AE results were concordant (Kappa value = 0.76), and both assays revealed a decay in the antibody response in calves with maternal antibodies at 90 and 120 dpv, which was not evidenced by the LPBE. These results show that kinetics of antibody responses to FMD vaccination are similar in buffalo and cattle, and support the use of indirect ELISA assays, in particular Avidity ELISA, as alternatives to the VNT for vaccine-immunity monitoring irrespectively of the animal’s passive or active immune status.Fil: Sala, Juan Manuel. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes. Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Mansilla, Florencia Celeste. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología e Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Miraglia, Maria Cruz. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología e Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Caspe, Sergio Gastón. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes. Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Pérez Filgueira, Daniel Mariano. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología e Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Capozzo, Alejandra Victoria. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología e Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Virología e Innovaciones Tecnológicas; Argentin

Leptospirosis: una enfermedad latente

En medicina veterinaria se entiende como enfermedad reproductiva, aquella que imposibilita o dificulta la fecundación, el mantenimiento de una gestación completa o la obtención de una cría con posibilidades de vida, o bien aquella enfermedad que afecta los parámetros reproductivos propios del sistema de producción que se maneje (Anderson, 2007). Durante el ciclo reproductivo del bovino se pueden presentar diversas pérdidas prenatales y posnatales: en el servicio, en la concepción, durante el período embrionario, fetal y neonatal (Morrel, 2010).Aunque nuestro país sufre importantes pérdidas por enfermedades que afectan la reproducción de los bovinos,sólo se conocen el 33% de las causas abortigénicas (Mooreet al., 2001). El objetivo del presente trabajo fue determinar asociaciones entre precipitaciones y presentación de casos positivos a Leptospira interrogans en cualquiera de sus serovares presentes en la provincia de Corrientes-Argentina. La presentación de mayores precipitaciones facilitaría la diseminación de la enfermedad, debido a que la bacteria no sobrevive mucho tiempo en el medio ambiente sin las condiciones adecuadas siendo sensible a la desecación. Sin embargo, según nuestros resultados, no hay evidencias deque este factor por sí solo, pueda ser determinante para la transmisión de la enfermedad bajo nuestras condiciones.Fil: Della Rosa, Paola. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes. Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Berecochea, F.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes. Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Sala, Juan M.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes. Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Morel, Victoria Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes. Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Biotti, Graciela. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes. Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Bevans, Walter. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes. Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Gómez, Sebastián. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes. Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Caspe, Sergio Gastón. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes. Estación Experimental Agropecuaria Mercedes; Argentin