Toxoplasma gondii is a zoonotic pathogen defined by three main clonal lineages (types I, II, III), of which type II is most common in Europe. Very few data exist on the prevalence and genotypes of T. gondii in the UK. Wildlife can act as sentinel species for T. gondii genotypes present in the environment, which may subsequently be transmitted to livestock and humans. DNA was extracted from tissue samples of wild British carnivores, including 99 ferrets, 83 red foxes, 70 polecats, 65 mink, 64 badgers and 9 stoats. Parasite DNAwas detected using a nested ITS1 PCR specific for T. gondii, PCR positive samples were subsequently genotyped using five PCR-RFLP markers. Toxoplasma gondii DNA was detected within all these mammal species and prevalence varied from 6.0 to 44.4% depending on the host. PCR-RFLP genotyping identified type II as the predominant lineage, but type III and type I alleles were also identified. No atypical or mixed genotypes were identified within these animals. This study demonstrates the presence of alleles for all three clonal lineages with potential for transmission to cats and livestock. This is the first DNA-based study of T. gondii prevalence and genotypes across a broad range of wild British carnivores.

Samples of brain and other tissues were collected from 99 ferrets (Mustela furo), 83 red foxes (Vulpes vulpes), 70 European polecats (Mustela putorius), 65 American mink (Neovison vison), 64 Eurasian badgers (Meles meles) and 9 stoats (Mustela erminea), from around Great Britain. DNA was extracted from approximately 1g of tissue and tested by specific nested ITS1 PCR for Neospora caninum. The results from the PCR demonstrated that Neospora specific DNA was detected in all species of wild carnivorans with the exception of the stoats (0/9). Neospora DNA positive samples were detected in: polecats 18.6% (13/70), badgers 10.9% (7/64), ferrets 10.1% (10/99), foxes 4.8% (4/83) and mink 4.6% (3/65). In the badgers N. caninum DNA positive samples were found in brain (n=2), liver (n=2) and neck muscle (n=3). Selected positive ITS1 DNA sequences were submitted to Genbank. Sequence UKwildlife1 (accession number JX857862) was found in two badgers, whilst UKwildlife2 and UKwildlife3 (accession numbers JX857863 and JX857864 respectively) were found in ferrets, all three sequences demonstrated point mutations at a single base, while sequence UKwildlife4 (accession number JX857865) was found in all the species that tested positive and showed complete identity when compared against published reference sequences for: N. caninum (Nc Liverpool isolate, EU564166). Our data shows that almost all the wild carnivoran mammal species tested are intermediate hosts for N. caninum and are therefore capable of acting as reservoirs of infection for other species. These species could also act as useful sentinel species, demonstrating the presence of the parasite in particular geographical and environmental locations.

Summary Balb/c mice were inoculated intraperitoneally (i.p.) with either 5 × 10 6 live virulent (group 1) or 5 × 10 6 live attenuated (group 2) tachyzoites, or Vero cells (group 3). Animals were killed at 0, 14, 28 and 42 days post‐inoculation (p.i.), with the remaining mice receiving a lethal challenge on day 48 p.i. Serum, spleen and brain samples were collected post‐mortem to examine humoral and cell‐mediated immune responses as well as pathological lesions and to quantify parasite loads. On day 14 p.i. group 2 (attenuated) demonstrated statistically significant ( P < 0·001) lower levels of mean morbidity and weight loss, while also showing significantly ( P = 0·01) higher levels of splenocyte proliferation and IFN‐γ production ( P = 0·003), compared to group 1 (virulent). Histology of brain samples showed milder lesions and a lower incidence of positive immunohistochemistry, demonstrating tachyzoites and tissue cysts, and statistically significant ( P = 0·03) lower mean burdens of parasite DNA in group 2 (attenuated) compared to group 1 (virulent). All mice in group 2 were protected following challenge on day 48 p.i. whereas naïve control mice succumbed to the challenge. No mice from group 1 (virulent) survived beyond day 24 p.i. so they were not included in the challenge.

Toxoplasma gondii is a significant cause of abortion in sheep. Infection is picked up from the environment and if initiated during pregnancy may cause fetal mortality. Infected sheep remain persistently infected with tissue cysts in brain and muscle (meat), and are also immune and would not be expected to abort again. The live tachyzoite vaccine (Toxovax) protects against abortion and this allows the suggestion that it may also reduce or prevent tissue cyst development in muscle. If this were so it raises the question of whether the vaccine could be used to make meat safer for human consumption.

A serial examination of three groups of cattle infected intravenously (iv) (Group 1, n=8) or subcutaneously (sc) (Group 2, n=8) with live Neospora caninum tachyzoites or with VERO cells (Group 3, n=8) at 70 days’ gestation was carried out and the nature of the inflammatory responses in the placenta and the presence of parasite antigen were analysed. Immune cells expressing CD3, CD4, CD8, gamma delta (γδ) T-cell receptors (TCR), CD79α cytoplasmic (cy) (B cells) and NKp46 [natural killer (NK) cells] antigens were identified immunohistochemically and cells expressing mRNA for interferon-γ (IFN-γ) were labelled by in-situ hybridization.Intravenous inoculation caused mortality in all fetuses from 28 days post-inoculation (dpi) onwards. Subcutaneous inoculation caused mortality in 50% of the animals by 28dpi. Pathological changes in the placenta consisted of necrosis of fetal placental villi, necrosis and inflammation in adjacent areas of the maternal septum and inflammation at the base of the maternal caruncle. The inflammatory infiltrate consisted mainly of CD3+ lymphocytes, dominated by CD4+ and γδ TCR+ cells, with CD8+ cells present to a lesser extent. The results from the control group indicated fewer NK cells than those occurring in the placenta of human beings or mice. Infiltration of CD4+ cells and NKp46+ cells was observed in the caruncular base and septa 14 days after infection, whereas infiltration of γδ TCR+ cells was observed from 28 dpi onwards. To our knowledge this is the first report on the presence and distribution of NK cells in the bovine placenta. Maternal inflammatory cells expressing mRNA for IFN-γ were identified in animals inoculated with parasites iv or sc at 14 and 28 dpi, respectively. In the sc-inoculated dams with live fetuses at 28, 42 and 56dpi, there was no evidence of parasite antigen, infiltration of immune cells or production of IFN-γ, suggesting that the parasite had not reached the placenta.The exact cause of fetal death was not established. Tissue destruction by the parasite may have occurred; in addition, there may have been a T helper 1 (Th-1) immune response to the neospora infection at the materno–fetal interface, resulting in infiltrations of CD4T cells, γδ T cells and NK cells and the subsequent production of IFN-γ. It is possible that a pro-inflammatory Th-1 response early in gestation protects the dam by eliminating the parasite; however, it may lead to destruction of the placental tissues themselves and thus be incompatible with fetal survival.

To investigate the potential role of endogenous transplacental transmission of Toxoplasma gondii, 31 seropositive ewes presumed to be persistently infected with the parasite and 15 seronegative ewes were mated and monitored throughout pregnancy and lambing. Antibody titres were determined in precolostral sera from the liveborn lambs and in thoracic fluid from the dead lambs. A PCR for the B1 gene of T gondii was applied to the placentas from all the ewes and to the brains of the stillborn lambs. Samples of brain, lung, liver, spleen and heart from the dead lambs were examined by histopathology. No evidence of toxoplasmosis was detected by histopathology or PCR in any of the samples, but low titres of antibody to T gondii were detected in two liveborn, healthy offspring of a seropositive ewe by the immunofluorescent antibody test (3.2 per cent of pregnancies and 4.1 per cent of lambs in the seropositive group). Antibody to specific antigens of T gondii was demonstrated in sera from these two lambs by Western blotting.

The consensus view is that ovine toxoplasmosis results from a primary infection in pregnant sheep, following ingestion of sporulated oocysts contaminating the environment. Recent research has suggested that vertical transmission from persistently infected ewes to the foetus occurs more frequently than previously thought. Our studies fully support the consensus view, although it may be that in exceptional circumstances vertical transmission can occur with greater frequency.

Infection with the protozoan parasite Neospora caninum is thought to be a major cause of reproductive failure in cattle worldwide. Cattle infected with the parasite are three to seven times more likely to abort compared to uninfected cattle. The parasite may be transmitted to cattle through the ingestion of oocysts that are shed in the faeces of acutely infected dogs (definitive host of N. caninum) or by congenital infection from mother to foetus via the placenta. Interestingly, transplacental transmission can occur over consecutive pregnancies and congenitally infected heifers can transmit the parasite to their own offspring. This repeated vertical transmission observed in naturally infected cattle suggests that cattle do not easily develop effective immunity to the parasite, presenting a significant challenge to the development of a control strategy based on vaccination.Neosporosis is a disease of pregnancy and studying the bovine maternal and foetal immune responses during pregnancy will help us to understand the change in the balance between the parasite and the host that may result in disease of the foetus. Studies in nonpregnant cattle and in murine models of infection have shown the importance of T-helper 1-type immune responses involving pro-inflammatory cytokines, such as IFN gamma and IL-12, in limiting intracellular multiplication of the parasite. During pregnancy, changes occur in the immune system allowing the mother to accept the foetal allograft. Research in other species has stressed the crucial role of T-helper 2-type cytokines at the materno-foetal interface in maintaining the pregnancy and regulating the potentially damaging effect of Th-1 responses. Studies in cattle have shown that cell proliferation and IFN gamma responses may be significantly down-regulated around mid-gestation. This may mean that cattle are less able to cope with N. caninum infection at this time and are more likely to transmit the parasite to the foetus. Another important factor is the gestational age and hence immuno-competence of the foetus at the time of infection. Early in gestation, N. caninum infection of the placenta and subsequently the foetus usually proves fatal, whereas infection occurring in mid to late pregnancy may result in the birth of a congenitally infected but otherwise healthy calf. Studies of foetal immune responses have shown that at 14 weeks of gestation, lymphocytes only respond to mitogen, while by 24 weeks (mid-gestation), they respond to antigen by proliferating and releasing IFN gamma.Clearly, there are several factors influencing the outcome of N. caninum infection in pregnancy: the timing, quantity and duration of parasitaemia, the effectiveness of the maternal immune response and the ability of the foetus to mount an immune response against the parasite. The challenge is to design a vaccine that will prevent foetal infection by N. caninum. This is likely to involve a fine balancing act with the immune system that will allow intervention in a manner that will tip the host-parasite balance in favour of the host without compromising the pregnancy. (c) 2005 Elsevier B.V. All rights reserved.

The purpose of this study was to establish the prevalence of antibodies to Toxoplasma gondii and Neospora caninum in a random sample of red foxes from around the UK. Lung fluid from over 500 foxes was examined using an indirect fluorescent antibody test. Reciprocal titres of specific antibodies to T. gondii or N. caninum ranged from < 1:16 to 1:1024. A titre of 1:128 or greater was deemed indicative of exposure to the parasite. One hundred and eleven (20%) of the 549 foxes tested were seropositive to T. gondii, and only five (0.9%) were seropositive to N. caninum. No correlation could be made between positive samples and geographical distribution, as sample numbers varied greatly between regions. The results of this study indicate that red foxes of the UK have more exposure to T. gondii than to N. caninum in their environment.

To investigate the pathogenesis of bovine neosporosis, 14 pregnant cattle were each inoculated subcutaneously with either 10(7) or 5 x 10(8) Neospora caninum (strain NC1) tachyzoites at 140 days' gestation. Serial necropsies were then carried out over an 8-week period. In the placenta, Neospora DNA and histopathological changes were observed in samples taken 14 days post-inoculation (dpi), with focal necrosis of maternal caruncular septa and fetal placental villi, serum leakage, and a maternal and fetal inflammatory response. At subsequent samplings, pathological changes in the placenta showed signs of resolution. No parasitaemia was detected in the dams in the two weeks following inoculation. In the fetus, Neospora DNA was detected at 14 dpi, and histopathological changes in the fetal central nervous system at 28 and 42 dpi consisted of small foci of necrosis and inflammation. Resolution of placental lesions during the experiment indicated that the disease was being controlled, and fetal infection, although established, did not appear to be progressing to a fatal outcome. The two doses of tachyzoites produced similar results, but the higher dose elicited earlier and more extensive lesions in the placenta and fetus. Control animals remained negative for all parameters recorded. It is concluded that in bovine neosporosis the placenta plays a central role in the pathogenesis and epidemiology of the infection, and that while primary tissue destruction by the parasite may endanger the fetus, the maternal and fetal inflammatory responses may also be damaging.

In this study we were interested to determine whether infection of cattle prior to pregnancy would afford any protection to the foetus if the dams were challenged with Neospora caninum at mid-gestation. The experiment comprised four groups of cattle: group 1, uninfected controls; group 2, inoculated with N. caninum tachyzoites 6 weeks prior to mating and then challenged with N. caninum at mid-gestation; group 3, naive cattle challenged with N. caninum at mid-gestation and group 4 were infected with N. caninum prior to mating and left unchallenged throughout pregnancy. Positive cell-mediated and humoral immune responses to N. caninum were recorded in groups 2 and 4 prior to pregnancy and in groups 2, 3 and 4 following challenge at mid-gestation. However there was a marked down regulation of the cell-mediated immune response in all groups around mid-gestation. There was a significant increase in rectal temperature response in animals in group 3 compared to group 2 following challenge but no other clinical symptoms of disease were recorded and all cattle proceeded to calving. At calving, pre-colostral blood samples were negative for antibodies to N. caninum in all the calves born to dams in groups 1, 2 and 4. In contrast, all the calves born to dams in group 3 had high levels of specific antibody to N. caninum indicating that they had been exposed to the parasite in utero. At post-mortem N. caninum DNA was detected in CNS, thymus and placental cotyledon samples in calves from group 3. All tissue samples from calves in the other 3 groups were negative for N. caninum DNA with the exception of one calf from group 2 where specific DNA was detected in a sample of spinal cord. These results suggest that the immune response generated in the dams in group 2 prior to pregnancy had protected against vertical transmission of the parasite following challenge at mid-gestation.

Sera from 258 healthy and sick domestic and feral cats were screened for specific anti-Cryptosporidium antibodies using an indirect immunofluorescence antibody test (IFA). Sera were positive for IgG, IgM and IgA antibodies in 192 (74%), 84 (32%) and 67 (26%) samples, respectively. Antibody was not detected at dilutions of 1:10 and 1:20 or greater in any of eight specific pathogen-free kittens. IgM and IgA antibody classes were more prevalent in sick than in healthy domestic cats. The presence of IgM and/or IgA antibodies indicated early infection. However, these antibody classes were present in sera from cats either positive or negative for Cryptosporidium infection by faecal examination. Pronounced polar fluorescence was observed in the sporozoites in positive samples under fluorescence microscopy. The higher prevalence of specific anti-Cryptosporidium antibodies and the absence of Cryptosporidium oocysts in faecal samples from some IFA-positive animals suggests that detection of these antibodies in sera from cats could be helpful for the diagnosis of feline cryptosporidiosis.

The phenomenon of age-related resistance to infection with Cryptosporidium parvum has been well characterized in rodent models, and its existence has been demonstrated in calves. To determine whether this is a genuine age effect in a fully susceptible animal model or the result of infection with related pathogens inducing a nonspecific immunity, and to examine several parameters associated with severity of clinical diseases, lambs maintained in a parasite-free environment were infected with C. parvum oocysts at increasing ages. A marked decrease in the severity of clinical symptoms was observed as the age at infection increased, though the kinetics of both fecal and serum antibody responses were similar in all age groups, suggesting that mechanisms other than humoral response may play an important role in the development of age-related resistance. This study demonstrates the first experimental evidence for age-related resistance to ovine cryptosporidiosis and examines parameters which may influence the acquisition of resistance to infection.