Neospora caninum tachyzoites attenuated through passage in tissue culture were tested for their ability to induce protective immunity against a lethal challenge dose of parasites. Balb/c mice were each inoculated with either 1x10(6) live virulent tachyzoites (Group 1) or 1x10(6) live attenuated tachyzoites (Group 2), while (Group 3) received a control inoculum. All mice were each challenged 28 days later with 5x10(6) virulent parasites. Histopathological lesions in the brains including necrosis and microgliosis were observed following post-mortem on day 28 post-challenge (p.c.) in 71% of Group 1 and 56% of Group 2. Immunohistochemistry (IHC) of these lesions showed tachyzoites and Neospora antigens to be associated with moderate brain lesions in 17% of Group 1, while in 11% of Group 2 N. caninum tissue cysts were detected, but these were not associated with lesions, Parasite DNA was detected by PCR in the brains of 86% of mice in Group 1 and 56% of mice in Group 2. Following challenge the mice in Group 3 showed high morbidity and 100% mortality within 17 days p.c. Positive IHC for N. caninum was seen in 88% of the Group 3 mice and parasite DNA was detected in all brain samples. This study shows that it is possible to protect against a lethal challenge of N. caninum through inoculation with attenuated or virulent tachyzoites. However, more severe pathology developed in mice initially inoculated with virulent parasites following a secondary challenge, compared to mice initially inoculated with attenuated parasites.

To determine whether prolonged in vitro passage would result in attenuation of virulence in vivo , Neospora caninum tachyzoites were passaged for different lengths of time in vitro and compared for their ability to cause disease in mice. Groups of Balb/c mice were inoculated intraperitoneally with 5×10 6 or 1×10 7 of low-passage or high-passage N. caninum tachyzoites. The mice were monitored for changes in their demeanour and body weight, and were culled when severe clinical symptoms of murine neosporosis were observed. Mice inoculated with the high-passage parasites survived longer ( P <0·05), and showed fewer clinical symptoms of murine neosporosis, compared to the mice receiving the low-passage parasites. The parasite was detected in the brains of inoculated mice using immunohistochemistry and ITS1 PCR. Tissue cysts containing parasites were seen in mice inoculated with both low-passage and high-passage parasites. When the in vitro growth rates of the parasites were compared, the high-passage parasites initially multiplied more rapidly ( P <0·001) than the low-passage parasites, suggesting that the high-passage parasites had become more adapted to tissue culture. These results would suggest that it is possible to attenuate the virulence of N. caninum tachyzoites in mice through prolonged in vitro passage.

Various existing serological tests were compared with a standard panel of 523 sera in a multicentred study across Europe. Well characterised sera from animals that were experimentally or naturally infected with Neospora caninum as well as sera from cattle deemed uninfected with N. caninum were provided by the participants of the study and analysed in several commercial (CHEKIT® Dr. Bommeli/Intervet, CIVTEST™ BOVIS NEOSPORA Hipra, Cypress Diagnostics C.V., Herd Check® IDEXX, Mastazyme™ MAST Diagnostics, P38-ELISA Animal Welfare and Food Safety GmbH (AFOSA)) as well as in-house assays (five ELISAs and one IFAT). Most tests showed a high level of agreement in the interpretation of the test results (positive or negative). A further distinct increase in agreement between tests was obtained after the application of standardised cut-offs offered by a two-graph receiver operating characteristic analysis. This procedure allows a standardised interpretation of results obtained with different tests used in independent, parallel seroepidemiological studies.

Optimised immunomagnetic separation methods to detect Cryptosporidium parvum and Escherichia coli O157 in UK shellfish are described. Whole tissue homogenates gave the best recoveries for C. parvum oocysts compared with gill or haemolymph extracts. The sensitivity of recovery from spiked samples was comparable to that achieved when processing water and varied from 12–34% in mussels, 48–69.5% in oysters and 30–65% in scallops. Maximum recovery of E. coli O157 was achieved by enriching in buffered peptone water supplemented with vancomycin at 42 °C. Increasing enrichment temperatures from 37 to 42 °C gave a significant increase in target number recovery. Implementation of these methods into monitoring programmes and end-product testing will enable shellfish producers to better assess product safety.

Pregnant cattle were inoculated with N. caninum strain NC-1 tachyzoites intravenously (iv) (group 1, n = 8) or subcutaneously (sc) (group 2, n = 8) at 70 days' gestation. Control animals (group 3; n = 8) received uninfected Vero cells iv. Two animals from each group were killed at 14, 28, 42 and 56 days post-inoculation (dpi). Fetal mortality was 100% and 50%, respectively, in groups 1 and 2 from 28 dpi. In group 1 foci of degenerative fetal placental villi were observed at 14 dpi, with clusters of N. caninum tachyzoites in the affected mesenchyme. There was also inflammation of maternal septal tissues, with necrotic cell debris and serum exudate at the interstitium. At 28 dpi pregnancy had ended and the fetal cotyledons had become detached from the maternal caruncles. Immunohistochemically, particulate N. caninum antigen was detected in the cotyledons. At 42 and 56 dpi, fetal tissues had disappeared, the caruncles were greatly reduced in size, and the uterine epithelium had been largely restored. In group 2, lesions were either severe or absent ("all or nothing" response). In one animal carrying a dead fetus at 28 dpi, placentitis was much more severe than that seen in group 1 at 14 dpi. Lesions contained neutrophils, eosinophils and N. caninum antigen. In animals carrying dead fetuses at 42 and 56 dpi, fetal remains were found and the cotyledons contained N. caninum antigen. Antigen was also detected in fetal tissues. No significant pathological changes were detected in group 2 animals carrying live fetuses or any animal in group 3. Thus, N. caninum administered iv or sc in early pregnancy resulted in rapid fetal death, with parasite-associated lesions in the placenta and fetus. Of the two inoculation routes, the intravenous induced the more acute placental lesions and greater mortality.

The humoral and cell-mediated immune responses of pregnant cattle and their fetuses were examined at intervals after infection with Neospora caninum tachyzoites at mid-gestation (day 140). All cattle seroconverted and interferon gamma was detected in supernatants of peripheral blood mononuclear cells stimulated with specific antigen. At day 14 post-inoculation (pi), specific cell proliferation responses were detected in the lymph node draining the site of inoculation and in the uterine lymph node. The peak response was recorded in the majority of maternal lymph nodes by day 28 pi and cells from the maternal retropharyngeal lymph node, which in part drains the central nervous system, showed no specific activity to N. caninum until day 42 pi. This changing pattern of immune responsiveness may reflect parasite invasion and development within different host tissues. Fetal lymph node cells showed mitogen responsiveness from day 14 pi (day 154 of gestation) and also showed N. caninum-specific cell proliferation and interferon-gamma responses by day 28 pi (day 168 of gestation). At day 42 pi, specific cell-mediated immune responses were not apparent; however, N. caninum-specific fetal IgG and IgM antibodies were detected.

Neospora caninum is an important cause of fetal loss in cattle but has also infrequently been shown to cause disease in sheep and goats. Experimental infection of pregnant sheep with N. caninum causes clinical and pathological changes very similar to those of neosporosis in cattle. An experiment in sheep was undertaken to examine whether infection with N. caninum before pregnancy conferred immunity to subsequent challenge with the parasite during pregnancy. Primary inoculation of NC1 tachyzoites subcutaneously, either before or during pregnancy, caused a significant temperature response in ewes, while those given a secondary challenge at 90 days gestation (dg) did not show such a response. Primary infection of 12 ewes during pregnancy resulted in the loss of all fetuses while a further 12 ewes inoculated with NC1 tachyzoites before mating and subsequently challenged with the same dose at 90 dg produced nine live and seven dead lambs. There were no fetal deaths in ewes only infected with Neospora before mating although there was serological evidence of vertical transmission in four of their clinically normal offspring while Neospora DNA was detected in the cerebrospinal fluid of a fifth healthy lamb. Thus an experimental primary infection with N. caninum during pregnancy killed all the fetuses while inoculation before pregnancy did not cause any mortality but did provide a degree of protection against subsequent challenge with Neospora during pregnancy.

Sheep immunized with Toxoplasma gondii (Toxovax) prior to pregnancy were tested for their ability to withstand a challenge at 90 days gestation with 107 Neospora caninum (NC1) tachyzoites. The antibody responses in sheep following immunization with T. gondii were specific for T. gondii whereas peripheral blood mononuclear cells responded to both T. gondii and N. caninum antigen in vitro. This suggested that there was induction of crossreactive immune recognition in the sheep, at least at the cellular level. Following challenge of sheep at mid-gestation with N. caninum, no febrile responses were recorded in the group of sheep which had previously received Toxovax while significant febrile responses were recorded in the group of sheep which received N. caninum challenge alone. Antibody responses to N. caninum developed in all sheep following N. caninum challenge and antibody responses to T. gondii were boosted in the group of sheep which had previously been immunized with Toxovax. No antibodies to T. gondii were observed in the sheep which received the N. caninum challenge alone. Peripheral blood mononuclear cells from both groups of sheep responded to T. gondii and N. caninum antigen in vitro and interferon gamma was present in the cell-free supernatant from activated cells. However despite evidence of the induction of crossreactive immunity between T. gondii and N. caninum, this was not sufficient to prevent foetal death. The group of sheep which had received Toxovax prior to pregnancy and the group of sheep which only received the N. caninum challenge experienced 100% foetal death compared with 0% in the unchallenged control group. Vaccination prior to pregnancy with Toxovax did protect against foetal death following oral challenge at 90 days with 2000 T. gondii oocysts which caused 100% foetal death in a control challenge group.

Neospora caninum is a recently recognized protozoan parasite which has been described as causing a neuromuscular paralysis in dogs and is emerging as a major cause of bovine infertility and abortion worldwide. The parasite is known to infect a range of warm blooded animals but the disease predominates in dogs and cattle. It is not yet known if N. caninum can infect and cause disease in people. The dog has recently been identified as the definitive host and the parasite may be transmitted through the ingestion of oocysts or congenitally from mother to fetus. N. caninum is known to infect red foxes (Vulpes vulpes) and coyotes (Canis latrans) and the role of wildlife species as reservoirs of infection requires further investigation. Little is known about the range of parasite genotypes within the environment or the variation in virulence between different strains. RAPD-PCR analysis of geographically distinct bovine and canine isolates has revealed little genetic variation. Epidemiological studies from different areas of the world have investigated the importance of N. caninum as an abortifacient agent and longitudinal studies have shown the high rate (approximately 80 %) of congenital transmission within infected herds. Information on the rates of repeat abortion due to neosporosis are less well defined however current estimates put this at 5 % suggesting that cattle may develop some form of protective immunity against N. caninum-induced abortion. Diagnosis of the disease is based upon detection of the parasite in the tissues, most commonly using immunohistochemistry with additional information provided by serology. However, although positive fetal serology is a strong indicator of exposure to the parasite, care should be taken in the interpretation of maternal serology. As we understand more about the epidemiology of neosporosis we are also better able to interpret the results of diagnostic tests. The mere presence of the parasite does not necessarily infer that this was the primary cause of abortion. CD4(+) T-cells, interferon gamma and macrophages have all been found to significantly inhibit multiplication of N. caninum tachyzoites. The nature of a protective immune response and its modulation in the pregnant animal is discussed.

This study aimed to evaluate the continuous flow centrifuge (CF centrifuge) for recovering Cryptosporidium oocysts from water under realistic environmental conditions. This was achieved by taking 100 l samples from a water tank with known seeding and turbidity levels. Samples were concentrated and purified using percoll discontinuous gradients and stained using monoclonal antibody on polycarbonate membrane filters. The maximum percent recoveries were achieved at a flow rate of 0.75 l/min and centrifuge speed of 2900 x g. When compared directly with cartridge filtration at a seeding level of 10 oocysts/l and low turbidity of 1 nephelometric turbidity unit (NTU), recoveries were 14.8 and 9.7% (n = 3) for the CF centrifuge and cartridge filtration respectively. At a higher turbidity of 5 NTU, recoveries were 13.0 and 9.0% respectively. These differences were not significant but the CF centrifuge sample is faster, simpler and safer to process. Its integration into flow cytometry is also evaluated. The recovery of Gini dia by CF centrifugation at 10 cysts/l and 5 NTU gave a mean recovery of 37.9%. (C) 2000 Elsevier Science Ltd. All rights reserved.

It has been reported in the literature that cattle are more resistant to toxoplasmosis than sheep. Congenital disease due to T. gondii infection is rarely reported in cattle whereas the parasite is a major cause of abortion and neonatal mortality in sheep. It is believed that sheep remain chronically infected for life. Undercooked meat from infected sheep is an important source of infection for man. In contrast cattle are thought to harbour fewer parasite tissue cysts which may not persist for the lifetime of the host. Therefore, cattle are believed to pose less of a risk for human infection. In this study we examined the presence of T. gondii within a range of tissues in sheep and cattle at 6 weeks and 6 months following oral infection with 103 or 105 sporulated oocysts of T. gondii. The presence of parasite was determined by bioassay in mice and using polymerase chain reaction (PCR). The results from this study show that T. gondii was more frequently and consistently detected in sheep, in particular within brain and heart tissues, whereas parasites were not detected in the samples of tissues taken from cattle. T. gondii was more frequently detected in sheep given the higher dose of T. gondii. Examination of tissues at either 6 weeks or 6 months after infection did not appear to affect the distribution of T. gondii. The polymerase chain reaction has more specificity and sensitivity when detecting the presence of T. gondii in large animals than histological detection.

The expression and distribution of dense granule proteins in the enteric (coccidian) forms of Toxoplasma gondii in the small intestine of the cat. Experimental Parasitology 91, 203-211. The expression and location of the dense granule proteins (GRA1-6 and NTPase) in the merozoite and during asexual and sexual development of Toxoplasma gondii in the small intestine of the cat (definitive host) was examined by immuno-light and electron microscopy. This was compared with that of tachyzoites and bradyzoites present in the intermediate host. It was found that the merozoite contained the characteristic apical organelles plus a few large dense granules. By immunocytochemistry, dense granules in merozoites were negative for GRA proteins 1 to 6 in contrast to both tachyzoites and bradyzoites in which dense granules were positive for all six proteins. The GRA proteins were associated with the parasitophorous vacuole (PV) during tachyzoite and bradyzoite development but were absent from the PV of the enteric stages. However, the merozoite dense granules were positive for NTPase, which was similar to the tachyzoite while this antigen was down regulated in the bradyzoite. The apparent release of the NTPases into the PV formed by merozoites was also similar to that described for the tachyzoite, possibly reflecting the relative metabolic activity of the various stages. This study shows that the majority of GRA proteins have a similar stage-specific expression, which is independent of NTPases expression. These observations are consistent with T. gondii having a different host parasite relationship in the enteric forms, which does not involve the GRA proteins 1-6.

The apicomplexan parasite Cryptosporidium parvum invades and multiplies primarily in the brush border cells of the intestinal mucosa causing in AIDS patients a severe diarrhoea that represents a significant contributing factor leading to death. Morphological analysis indicates that the invasion machinery of C. parvum is similar to the apical complex of other parasites of the phylum Apicomplexa. We provide here evidence indicating that C. parvum also shares with these parasites a molecule crucial for the invasion of host cells. We have cloned a 3894 bp-long C. parvum cDNA encoding a protein characterised by sequence and structural similarities with members of the thrombospondin (TSP) family previously described in apicomplexan parasites of the genera Toxoplasma, Eimeria and Plasmodium. This novel C. parvum molecule, the TSP-related adhesive protein of Cryptosporidium-1 (TRAP-C1), is encoded by a single copy gene containing no introns. TRAP-C1 is localised in the apical end of C. parvum sporozoites and is structurally related to the micronemal proteins MIC2 of Toxoplasma and Etp100 of Eimeria, which are involved in host-cell attachment and/or invasion. The identification of TRAP-C1 sheds new light on the molecules possibly involved in the invasion process of intestinal cells by C. parvum. We have also analysed the sequence variation of TRAP-C1 among C. parvum isolates and in the closely related species C. wrairi.

Three groups of eight pregnant sheep were inoculated with tachyzoites of the NC1 isolate of Neospora caninum at 45 (group 1), 65 (group 2) or 90 (group 3) days' gestation. A further six animals (group 4) served as controls. Fourteen of the infected ewes developed a fever, which in two cases was biphasic. In six ewes in group 1, the fetuses died and were resorbed, and in the other two the fetuses were aborted. In group 2, one ewe resorbed her fetus, six aborted dead fetuses and one produced a live lamb. In group 3, six ewes aborted and two produced one live and one stillborn lamb each. Thus, the stage of gestation influenced the outcome of infection. All but one of the ewes “seroconverted”, as shown by enzyme-linked immunosorbent assay, and 10 of 13 fetal sera examined by an indirect immunofluorescent antibody test were positive. The polymerase chain reaction was also used to detect DNA of N. caninum in aborted tissues. Immunohistochemical examination showed that the parasite had invaded the placentas of all cases examined, displaying an apparent predilection for fetal chorionic epithelium and fetal placental blood vessels, as well as inducing thrombosis in some maternal caruncular blood vessels. Organisms were associated with fetal vasculitis, focal degeneration and inflammation of the chorioallantois, and widespread, severe focal necrosis in the placentome. Characteristic lesions were seen in the fetal brains, in addition to focal leucomalacia, though to be due to anoxia resulting from the placental damage. The six control sheep in group 4 remained clinically healthy and produced normal uninfected lambs.

The IgG antibody responses of sheep, goats and cattle inoculated subcutaneously with live Neospora caninum tachyzoites of the NC1 isolate were analysed by Western blotting. Antibodies were detected against a wide range of NC1 tachyzoite antigens (6.5 to 80 kDa). The dominant antibody responses were directed against proteins at 36.5-38, 45.5-48.5, 52-53.5, 58, 58.5, 59.5, 60.5, 62, 63.5, 64, 66.5, 67, 67.5, 68.5 and 69.5 kDa, with sera from all three species. These sera were also used to probe blots of Toxoplasma gondii antigen and, while a number of protein bands were recognized, there was no consistency within or between animal species. The IgG antibody responses of sheep, goats and cattle orally infected with T. gondii oocysts of the M3 isolate were analysed by the same methods. Antibodies were detected to a range of S48 toxoplasma tachyzoite antigens (11 to 83 kDa). The dominant antibody responses were directed against proteins at 11, 16-17, 21.5, 22.5-23.5, 26-28.5, 32-35, 49.5, 50.5, 53, 54.5, 60.5 and 61 kDa, with sera from all three species. These sera were also used to probe blots of N. caninum antigen; antibody responses to numerous antigens were detected but showed little consistency within or between animal species.

Modified Ziehl-Neelsen (MZN), auramine-phenol (A-P) and fluorescein isothiocyanate-labelled (FITC-labelled) monoclonal antibody (MAb) techniques were compared for detection of Cryptosporidium parvum oocysts in cat faecal specimens inoculated with known numbers of C. parvum oocysts. Of the three techniques, the FITC-labelled MAb technique detected more oocysts than the MZN and A-P techniques (P < 0.05), but A-P was more efficient than MZN (P < 0.05). Comparison of sucrose flotation, zinc sulphate (ZnSO4) flotation and formol-ether (F-E) sedimentation techniques revealed that F-E was the most efficient of the three (P < 0.05) for concentration of C. parvum oocysts from cat faecal specimens. On average, the F-E technique recovered 37% of oocysts from the original sample, whereas the sucrose and ZnSO4 flotation techniques recovered 33% and 11%, respectively. The findings of this study suggest that MZN and A-P staining are both useful for screening C. parvum oocysts in cat faecal materials containing 10(6) oocysts or more, but FITC-labelled MAb should be used when the number of oocysts is low. Also, the F-E sedimentation technique is recommended for concentrating oocysts in cat faecal specimens.

A clinical and post mortem survey of domestic and feral cats in the Glasgow area revealed that 19 of 235 (8.1 per cent) were infected with Cryptosporidium species. More kittens than adults were infected (P less than 0.01), and of 51 of the cats which had diarrhoea, four also had cryptosporidium infection. Of seven domestic cats with cryptosporidium infection, two were also positive for feline immunodeficiency virus. There was no significant difference between the prevalence of cryptosporidium infection in domestic and feral cats. Cryptosporidium oocysts were detected in faecal and mucosal impression smears stained with auramine-phenol and modified Ziehl-Nielsen techniques. Endogenous developmental stages of cryptosporidium were found in the microvillus region of enterocytes of eight of 19 positive cats in sections stained with haematoxylin and eosin. The results suggest that cryptosporidium infection is common among young and newborn kittens, and that the disease is usually asymptomatic.

Enteric cryptosporidiosis was studied in the small intestine of five-day-old sucking mice after infection with 10(6) Cryptosporidium parvum oocysts. It was shown that excystation and the majority of subsequent endogenous stages occurred predominantly in the ileum. During the first three days of infection the number of merozoites collected in ileal washings increased over 100-fold to approximately 10(6) merozoites per mouse on the third day. In contrast to control mice, wash fluid from infected mice contained numerous strands of dislodged mucus. Estimates of mucus in the ileal washings of infected mice were similar to those made in controls until day 4 after infection when they increased and remained high throughout the remainder of the experiment. This study describes a method whereby ileal mucus washings from C parvum infected infant mice could be used as a rich source of merozoites.

Enteric cryptosporidiosis was studied in colostrum-deprived lambs each infected at five days old with 10(6) oocysts. The prepatent period was three to five days and faecal oocyst concentration fell below detectable levels by day 16 after infection. Specific IgA, the only isotype detected by immunofluorescent assay in faecal extracts from infected lambs, was first evident on day 10 and titres continued to rise until day 16 of infection in association with declining oocyst output. Specific IgM and IgG antibodies were first detected in serum seven days after infection. No specific antibody was detected in uninfected control lambs. Immunoblotting methods showed that serum antibody and faecal IgA had similar profiles of antigen recognition. Antigens with approximate molecular weights of 180,000, 23,000 and 15,000 were consistent features on immunoblots performed with convalescent sera and faecal extracts. The results suggest that specific IgA in intestinal secretions has an important role in immunity to cryptosporidiosis.