Characterization of Immune Cell Infiltration in the Placentome of Water Buffaloes (Bubalus bubalis) Infected with Neospora caninum During Pregnancy

Neospora caninum infection in cattle stimulates host immune responses, which may be responsible for placental damage leading to abortion. Susceptibility of water buffaloes (Bubalus bubalis) to neosporosis is not well understood, although vertical transmission and fetal death have been documented. The aim of this study was to characterize the immune response in the placentome of water buffalo following experimental infection in early gestation with the Nc-1 strain of N. caninum. Placentomes were examined by immunohistochemistry using antibodies specific for T-cell subsets, natural killer cells and CD79αcy cells. Placental inflammation was characterized by the infiltration of CD3+ and CD4+ T cells and T cells expressing the γδ T-cell receptor. The distribution of these cellular subsets in buffalo placentomes was similar to that previously described in cattle infected with N. caninum in early gestation, but the lesions were milder, which may explain the lower number of abortions observed in this species after infection.EEA BalcarceFil: Canton, German Jose. Moredun Research Institute; Reino Unido. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Konrad, Jose Luis. Universidad Nacional del Nordeste. Facultad de Veterinaria; ArgentinaFil: Moore, Prando Dadin. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Caspe, Sergio Gaston. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Palarea-Albaladejo, Javier. Biomathematics & Statistics Scotland; Reino UnidoFil: Campero, Carlos Manuel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Chianini, Francesca. Moredun Research Institute; Reino Unid

Interferon gamma responses to proteome-determined specific recombinant proteins: Potential as diagnostic markers for ovine Johne’s disease

Johne’s disease (JD), or paratuberculosis is a fatal enteritis of animals caused by infection with Mycobacterium avium subspecies paratuberculosis (Map). There may be a long subclinical phase with no signs of clinical disease. Diagnosis of JD is problematic and no test can reliably detect sub-clinical disease. Th1 responses to Map are believed to be activated first with a later switch to Th2 responses and progression to clinical disease. Detection of a cell-mediated response, indicated by interferon gamma (IFN-) produced in response to mycobacterial antigens, may give an early indication of infection. Crude extracts of Map (PPDj) have been used to detect the cell-mediated response, but more specific, quantifiable antigens would improve the test. Thirty Map-specific proteins were screened for their ability to raise a cell-mediated response in subclinically infected sheep. Four proteins were selected and tested using blood from subclinical animals and controls from a JD-free flock. Three proteins elicited IFN- levels which were higher in the subclinical group than in the control group, two were statistically significant. Thus these proteins have the ability to discriminate groups of infected and uninfected animals and may have use in diagnosis of JD

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

Experimental transmission of bovine spongiform encephalopathy to European red deer (Cervus elaphus elaphus)

<p>Abstract</p> <p>Background</p> <p>Bovine spongiform encephalopathy (BSE), a member of the transmissible spongiform encephalopathies (TSE), primarily affects cattle. Transmission is via concentrate feed rations contaminated with infected meat and bone meal (MBM). In addition to cattle, other food animal species are susceptible to BSE and also pose a potential threat to human health as consumption of infected meat products is the cause of variant Creutzfeldt-Jakob disease in humans, which is invariably fatal. In the UK, farmed and free ranging deer were almost certainly exposed to BSE infected MBM in proprietary feeds prior to legislation banning its inclusion. Therefore, although BSE has never been diagnosed in any deer species, a possible risk to human health remains via ingestion of cervine products. Chronic wasting disease (CWD), also a TSE, naturally infects several cervid species in North America and is spreading rapidly in both captive and free-ranging populations.</p> <p>Results</p> <p>Here we show that European red deer (<it>Cervus elaphus elaphus</it>) are susceptible to intra-cerebral (i/c) challenge with BSE positive cattle brain pool material resulting in clinical neurological disease and weight loss by 794–1290 days and the clinical signs are indistinguishable to those reported in deer with CWD. Spongiform changes typical of TSE infections were present in brain and accumulation of the disease-associated abnormal prion protein (PrP^d) was present in the central and peripheral nervous systems, but not in lymphoid or other tissues. Western immunoblot analysis of brain material showed a similar glycosylation pattern to that of BSE derived from infected cattle and experimentally infected sheep with respect to protease-resistant PrP isoforms. However, the di-, mono- and unglycosylated bands migrated significantly (p < 0.001) further in the samples from the clinically affected deer when compared to BSE infected brains of cattle and sheep.</p> <p>Conclusion</p> <p>This study shows that deer are susceptible to BSE by intra-cerebral inoculation and display clinical signs and vacuolar pathology that are similar to those of CWD. These findings highlight the importance of preventing the spread to Europe of CWD from North America as this may necessitate even more extensive testing of animal tissues destined for human consumption within the EU. Although the absence of PrP^din lymphoid and other non-neurological tissues potentially limits the risk of transmission to humans, the replication of TSE agents in peripheral tissues following intra-cerebral challenge is often limited. Thus the assessment of risk posed by cervine BSE as a human pathogen or for environmental contamination should await the outcome of ongoing oral challenge experiments.</p

Evaluation of species-specific polyclonal antibodies to detect and differentiate between Neospora caninum and Toxoplasma gondii

Neosporosis and toxoplasmosis are major causes of abortion in livestock worldwide, leading to substantial economic losses. Detection tools are fundamental to the diagnosis and management of those diseases. Current immunohistochemistry (IHC) tests, using sera raised against whole parasite lysates, have not been able to distinguish between Toxoplasma gondii and Neospora caninum. We used T. gondii and N. caninum recombinant proteins, expressed in Escherichia coli and purified using insoluble conditions, to produce specific polyclonal rabbit antisera. We aimed to develop species-specific sera that could be used in IHC on formalin-fixed, paraffin-embedded (FFPE) tissue sections to improve the diagnosis of ruminant abortions caused by protozoa. Two polyclonal rabbit sera, raised against recombinant proteins, anti–Neospora-rNcSRS2 and anti–Toxoplasma-rTgSRS2, had specificity for the parasite they were raised against. We tested the specificity for each polyclonal serum using FFPE tissue sections known to be infected with T. gondii and N. caninum. The anti–Neospora-rNcSRS2 serum labeled specifically only N. caninum–infected tissue blocks, and the anti–Toxoplasma-rTgSRS2 serum was specific to only T. gondii–infected tissues. Moreover, tissues from 52 cattle and 19 sheep previously diagnosed by lesion profiles were tested using IHC with our polyclonal sera and PCR. The overall agreement between IHC and PCR was 90.1% for both polyclonal anti-rNcSRS2 and anti-rTgSRS2 sera. The polyclonal antisera were specific and allowed visual confirmation of protozoan parasites by IHC, but they were not as sensitive as PCR testing.</p

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

Phenotypic characterisation of the cellular immune infiltrate in placentas of cattle following experimental inoculation with Neospora caninum in late gestation

Abstract Despite Neospora caninum being a major cause of bovine abortion worldwide, its pathogenesis is not completely understood. Neospora infection stimulates host cell-mediated immune responses, which may be responsible for the placental damage leading to abortion. The aim of the current study was to characterize the placental immune response following an experimental inoculation of pregnant cattle with N. caninum tachyzoites at day 210 of gestation. Cows were culled at 14, 28, 42 and 56 days post inoculation (dpi). Placentomes were examined by immunohistochemistry using antibodies against macrophages, T-cell subsets (CD4, CD8 and γδ), NK cells and B cells. Macrophages were detected mainly at 14 days post inoculation. Inflammation was generally mild and mainly characterized by CD3+, CD4+ and γδ T-cells; whereas CD8+ and NK cells were less numerous. The immune cell repertoire observed in this study was similar to those seen in pregnant cattle challenged with N. caninum at early gestation. However, cellular infiltrates were less severe than those seen during first trimester Neospora infections. This may explain the milder clinical outcome observed when animals are infected late in gestation.The authors acknowledge the Scottish Government’s Rural and Environment Science and Analytical Services Division (RESAS), UK, and Instituto Nacional de Tecnología Agropecuaria (INTA), Argentina, for funding this study and Dr Alex Schock from Animal Health and Veterinary Laboratories Agency and Prof. Gary Entrican from Moredun Research Institute for useful and constructive discussion.Peer Reviewe

Characterization of the immune cell response in the placentas from cattle following experimental inoculation with Neospora caninum throughout pregnancy

Trabajo presentado al 2nd International Meeting on Apicomplexan Parasites in Farm Animals (Kusadasi, Turquía, 31 octubre al 2 noviembre, 2013).Despite Neospora caninum (NC) being a major cause of bovine abortion worldwide, its pathogenesis is not completely understood. Evidence of immune mediated placental pathology has been reported as being responsible for compromising pregnancy probably due to the adverse effect of an exacerbated Th1 response at the maternal-foetal interface. Different clinical outcomes are known to follow experimental infections at different stages of gestation, with foetal death being the most common finding during early gestation infections, and the birth of live congenitally infected calves upon infection at mid or late gestation. The aim of our studies was to characterise placental immune responses following experimental infection during pregnancy. Cows were infected with NC tachyzoites at day 70, 140 and 210 of pregnancy and culled at 14, 28, 42 and 56 days post inoculation. Placentomes were examined by immunohistochemistry using antibodies against macrophages, T-cells (CD3, CD4, CD8, ¿¿TCR), NK and B cells and by in situ hybridization to characterize cytokine expression (IL-12, IFN-¿, TNF-¿ and IL-4). Inflammation was mainly characterised by the presence of CD3+, CD4+ and ¿¿ T-cells during the three time points. In early gestation inflammation was generally moderate to severe and mainly characterized by infiltration of IL-12, IFN-¿ and TNF-¿ expressing cells. This infiltration was more pronounced in the samples of placentome collected from dams carrying a dead foetus or one that had aborted, compared with the mothers carrying live foetuses at the time of sampling. In contrast, the infiltration of CD3+, CD4+, CD8+ and ¿¿ T-cells and Th1 cytokine expressing-cells was less evident following NC infection at mid gestation and scarce during infection at late gestation. These findings may partially explain the milder clinical outcome observed when animals are infected with NC at mid or late gestation.Peer Reviewe

Susceptibility to scrapie and disease phenotype in sheep: cross-PRNP genotype experimental transmissions with natural sources

<p>Abstract</p> <p>It has long been established that the sheep <it>Prnp</it> genotype influences the susceptibility to scrapie, and some studies suggest that it can also determine several aspects of the disease phenotype. Other studies, however, indicate that the source of infection may also play a role in such phenotype. To address this question an experiment was set up in which either of two different natural scrapie sources, AAS from AA₁₃₆ Suffolk and VVC from VV₁₃₆ Cheviot sheep, were inoculated into AA₁₃₆, VA₁₃₆ and VV₁₃₆ sheep recipients (<it>n</it> = 52). The immunohistochemical (IHC) profile of disease-associated PrP (PrP^d) accumulation in the brain of recipient sheep was highly consistent upon codon 136 homologous and semi-homologous transmission, but could be either similar to or different from those of the inoculum donors. In contrast, the IHC profiles were highly variable upon heterologous transmission (VVC to AA₁₃₆ and AAS to VV₁₃₆). Furthermore, sheep of the same <it>Prnp</it> genotype could exhibit different survival times and PrP^d profiles depending on the source of infection, and a correlation was observed between IHC and Western blot profiles. It was found that additional polymorphisms at codons 112 or 141 of AA₁₃₆ recipients resulted in a delayed appearance of clinical disease or even in protection from infection. The results of this study strongly suggest that the scrapie phenotype in sheep results from a complex interaction between source, donor and recipient factors, and that the <it>Prnp</it> genotype of the recipient sheep does not explain the variability observed upon codon 136 heterologous transmissions, arguing for other genetic factors to be involved.</p

Transgenic Mouse Bioassay : Evidence That Rabbits Are Susceptible to a Variety of Prion Isolates

Interspecies transmission of prions is a well-established phenomenon, both experimentally and under field conditions. Upon passage through new hosts, prion strains have proven their capacity to change their properties and this is a source of strain diversity which needs to be considered when assessing the potential risks associated with consumption of prion contaminated protein sources. Rabbits were considered for decades to be a prion resistant species until proven otherwise recently. To determine the extent of rabbit susceptibility to prions and to assess the effects of passage of different prion strains through this species a transgenic mouse model overexpressing rabbit PrPC was developed (TgRab). Intracerebral challenges with prion strains originating from a variety of species including field isolates (ovine SSBP/1 scrapie, Nor98- scrapie; cattle BSE, BSE-L and cervid CWD), experimental murine strains (ME7 and RML) and experimentally obtained ruminant (sheepBSE) and rabbit (de novo NZW) strains were performed. On first passage TgRab were susceptible to the majority of prions (Cattle BSE, SheepBSE, BSE-L, de novo NZW, ME7 and RML) tested with the exception of SSBP/1 scrapie, CWD and Nor98 scrapie. Furthermore, TgRab were capable of propagating strain-specific features such as differences in incubation periods, histological brain lesions, abnormal prion (PrPd) deposition profiles and proteinase-K (PK) resistant western blotting band patterns. Our results confirm previous studies proving that rabbits are not resistant to prion infection and show for the first time that rabbits are susceptible to PrPd originating in a number of other species. This should be taken into account when choosing protein sources to feed rabbits