Development of maternal and foetal immune responses in cattle following experimental challenge with Neospora caninum at day 210 of gestation

14 páginas, 2 tablas, 6 figuras.Bartley, Paul M. et al.This study examined the immunological responses of pregnant cattle and their foetuses following an experimental challenge with live Neospora caninum tachyzoites at day 210 of gestation. Animals were bled prior to and weekly throughout the experiment and sacrificed at 14, 28, 42 and 56 days post inoculation (dpi). At post mortem examination, samples of lymph nodes and spleen were collected from both dam and foetus for immunological analysis. Subcutaneous (sc) inoculation over the left prefemoral (LPF) lymph node of pregnant cattle at day 210 of gestation, led to the vertical transmission of parasites by 14 dpi, however no foetal deaths were observed in the infected animals. Foetuses from infected dams mounted Neospora-specific humoral and cell-mediated immune (CMI) responses by 14 dpi. These responses involved anti-Neospora IgG, antigen-specific lymphocyte proliferation, and the production of the cytokines IFN–γ, interleukin (IL)-4 and IL-10. There was also evidence of innate immunity during the response against Neospora from infected dams, with statistically significant (p < 0.05) increases in mean expression of toll like receptors (TLR)-2 on 56 dpi in maternal spleen, LPF, right prefemoral (RPF), left uterine (LUL) and right uterine (RUL) lymph nodes and TLR-9 in retropharyngeal (RLN), LPF and RPF lymph nodes from 28 dpi. Statistically significant (p < 0.05) increases in mean TLR-9 were detected in spleen samples from foetuses of infected dams, compared to the foetuses from control animals. Our results show that vertical transmission of the parasite occurred in all infected dams, with their foetuses showing effective Neospora-specific cell mediated, humoral and innate immune responses.The authors would like to thank The Scottish Government's Rural and Environment Science and Analytical Services Division (RESAS) for supporting this work and the Bioservices Division of the Moredun Research Institute for care and management of the animals.Peer Reviewe

Shedding of Cryptosporidium in calves and dams – evidence of re-infection and shedding of different gp60 subtypes

One of the most common causes of calf diarrhoea is the parasite Cryptosporidium parvum. Two longitudinal studies were carried out on a dairy farm Scotland to determine the prevalence of Cryptosporidium species and subtypes in a group of calves and to determine whether dams were a possible source of calfhood infection. Fecal samples were collected from 25 calves from birth to 12 months in the first year. In the second year, fecal samples were collected from pregnant cows (n = 29) and their calves (n = 30) from birth to 6 months. The samples were tested for Cryptosporidium and speciated. Cryptosporidium parvum-positive samples were subtyped by GP60 fragment analysis. All calves in both studies shed Cryptosporidium during the study period. Cryptosporidium parvum was the predominant species detected in calves ⩽6 weeks of age and at 6 months of age, C. bovis and C. ryanae were detected in calves older than 4 weeks of age but ⩽6 months of age. The prevalence of Cryptosporidium was higher in younger animals than in older animals. GP60 subtyping revealed two subtypes in calves on this farm (IIaA15G2R1 and IIaA19G2R1) that differed in frequency by age. Adult cattle also shed C. parvum, of four gp60 genotypes

Development of a sensitive method to extract and detect low numbers of Cryptosporidium oocysts from adult cattle faecal samples:results from the MRC ALL97 clinical trial

AbstractCryptosporidium transmission studies to date have concluded that adult cattle are not a significant source of oocysts contributing to clinical cryptosporidiosis in calves on farm. However current methods of sample processing have been optimised for calf faecal samples and may be less sensitive when used on adult samples due to lower numbers of oocysts and larger size of samples. A modified and novel method of oocyst extraction and concentration was developed and applied in an experiment involving spiking adult cattle faecal samples with known concentrations of Cryptosporidium oocysts. The results showed an increased sensitivity of detection from 100oocysts/g of faecal sample using conventional protocols to 5oocysts/g using the newly developed method. As it is important to be able to accurately assess the contribution of adult ruminants to the transmission of Cryptosporidium, both on farm and in the environment, the development of the techniques described here is likely to make an important contribution to Cryptosporidium transmission studies in future and in subsequent control strategies aimed at the reduction of Cryptosporidium infection in calves on farm

Selection of Neospora caninum antigens stimulating bovine CD4+ve T cell responses through immuno-potency screening and proteomic approaches

Neospora caninum is recognised worldwide as a major cause of bovine infectious abortion. There is a real need to develop effective strategies to control infection during pregnancy which may lead to either abortion or congenital transmission. Due to the intracellular nature of the parasite, cell-mediated immune (CMI) responses involving CD4+ve, CD8+ve, γ/δ TCR+ve T cells and NK cells, as well as production of IFN-γ, are thought to be important for protective immunity. In this study we applied a combination of proteomic and immunological approaches to identify antigens of N. caninum that are recognized by CD4+ve T cell lines derived from infected cattle. Initially, N. caninum tachyzoite Water Soluble Antigens (NcWSA) were fractionated by size-exclusion HPLC and then screened for immune-potency using CD4+ve T cell lines. LC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass spectrometry) was employed to catalogue and identify the proteins comprising three immunologically selected fractions and led to the identification of six N. caninum target proteins as well as sixteen functional orthologues of Toxoplasma gondii. This approach allows the screening of biologically reactive antigenic fractions by the immune cells responsible for protection (such as bovine CD4+ve cells) and the subsequent identification of the stimulating components using tandem mass spectrometry

Increased Toxoplasma gondii positivity relative to age in 125 Scottish sheep flocks; evidence of frequent acquired infection

Toxoplasma gondii seroprevalence was determined in 3333 sheep sera from 125 distinct sheep flocks in Scotland, with the majority of flocks being represented by 27 samples, which were collected between July 2006 and August 2008. The selected farms give a representative sample of 14 400 sheep holdings identified in the Scottish Government census data from 2004. Overall T. gondii seroprevalence, at individual sheep level, was determined to be 56.6%; each flock tested, had at least a single positive animal and in four flocks all ewes tested positive. The seroprevalence of sheep increased from 37.7% in one year old stock to 73.8% in ewes that were older than six years, showing that acquired infections during the life of the animals is frequent and that environmental contamination by T. gondii oocysts must be significant. The median within-flock seroprevalence varied significantly across Scotland, with the lowest seroprevalence of 42.3% in the South and the highest seroprevalence of 69.2% in the far North of Scotland and the Scottish Islands, while the central part of Scotland had a seroprevalence of 57.7%. This distribution disequilibrium may be due to the spread and survival of oocysts on pasture and lambing areas. A questionnaire accompanying sampling of flocks identified farms that used Toxovax®, a commercial vaccine that protects sheep from abortion due to T. gondii infection. Only 24.7% of farmers used the vaccine and the vaccine did not significantly affect the within flock seroprevalence for T. gondii. The implications for food safety and human infection are discussed

Molecular detection of Toxoplasma gondii in water samples from Scotland and a comparison between the 529bp real-time PCR and ITS1 nested PCR

Waterborne transmission of Toxoplasma gondii is a potential public health risk and there are currently no agreed optimised methods for the recovery, processing and detection of T. gondii oocysts in water samples. In this study modified methods of T. gondii oocyst recovery and DNA extraction were applied to 1427 samples collected from 147 public water supplies throughout Scotland. T. gondii DNA was detected, using real time PCR (qPCR) targeting the 529bp repeat element, in 8.79% of interpretable samples (124 out of 1411 samples). The samples which were positive for T. gondii DNA originated from a third of the sampled water sources. The samples which were positive by qPCR and some of the negative samples were reanalysed using ITS1 nested PCR (nPCR) and results compared. The 529bp qPCR was the more sensitive technique and a full analysis of assay performance, by Bayesian analysis using a Markov Chain Monte Carlo method, was completed which demonstrated the efficacy of this method for the detection of T. gondii in water samples

Differences in virulence and oocyst shedding profiles in lambs experimentally infected with different isolates of Cryptosporidium parvum

A wide spectrum of disease severity associated with cryptosporidiosis has been described, ranging from asymptomatic to fatal in both human and animal hosts. The reasons for the variations in severity are likely to be multifactorial, involving environmental, host and parasite factors. This paper describes two experimental infection trials in lambs, a symptomatic host for the parasite, to investigate variation in the clinical manifestations following infection with two distinct isolates of Cryptosporidium parvum. In the first experiment, groups of naïve lambs were challenged with one of two isolates (CP1 or CP2) at ​&lt; ​1 week of age, to test the effect of the isolates on disease outcome. In a second experiment one group of lambs challenged at &lt; 1 week of age (CP1) was then re-challenged with the same isolate at 6 weeks of age (CP1), while a second group was challenged for the first time at 6 weeks of age (CP1). This experiment examined age-related disease symptoms, oocyst shedding and the effect of prior exposure to the parasite on a subsequent homologous challenge. The two isolates were associated with significant differences in the demeanour of the animals and in the numbers of oocysts shed in the faeces. There were also differences in the duration and severity of diarrhoea, though these were not significant. The age of the lamb, at the time of a primary challenge (&lt;1 week or 6 weeks), also resulted in differences in clinical outcomes, with younger lambs showing more severe clinical disease than the older lambs (feeding profiles and presentation of diarrhoea), while older lambs showed virtually no signs of infection but still produced large numbers of oocysts

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