Maternal and fetal immune response patterns in heifers experimentally infected with Neospora caninum in the second trimester of pregnancy--a descriptive study.

Fetal and maternal immune responses 3, 6 and 9 weeks post infection (wpi) were investigated in cows experimentally infected with Neospora caninum on day 110 of gestation. Descriptive analysis showed that the fetuses had lower percentages of spleen T cell subpopulations (CD3+, CD4+ and CD8+) at 6 wpi compared to 3 wpi and/or 9 wpi, with the lowest percentages observed in a dead fetus found upon euthanasia at that time. Increased expression of most cytokines over levels recorded at 3 and 9 wpi were found in fetuses that were alive at 6 wpi. Up-regulated Th1, Th2 and Treg expression was also observed at 6 wpi in the spleen and in the lymph nodes draining the placenta of the cows. At the placental level, while most cytokines were down-regulated from 6 wpi, up-regulation of IL-4 expression was observed at 6 wpi in the caruncle. Our results suggest that the immune response at 6 wpi was crucial for fetal survival in this model of bovine neosporosis.

Effects of gastrointestinal parasites on parasite burden, rectal temperature, and antibody titer responses to vaccination and infectious bovine rhinotracheitis virus challenge.

Thirty-three colostrum-deprived Holstein bull calves (initial BW of 131 ± 4 kg) were used to determine the effect of timing of anthelmintic administration relative to vaccination on antibody titer response to vaccine component antigens. When calves were at least 3 mo of age, they were sorted randomly into individual pens and assigned to 1 of 3 treatment groups, treatments consisted of 1) dewormed 2 wk before vaccination (DPV), 2) dewormed at the time of vaccination (DV), or 3) control, vaccinated but not dewormed (CONT). All calves were inoculated with infective larvae of brown stomach worms (Ostertagia ostertagi) and intestinal worms (Cooperia spp.) on d 1, 7, 10, 14, and 18 for a total dose of 235,710 infective larvae per calf. Calves (DPV and DV) were dewormed on d 21 or 35 with a 10% fenbendazole suspension at 5 mg/kg of BW. On d 35, all calves were vaccinated with a modified-live virus respiratory vaccine containing IBRV (infectious bovine rhinotracheitis virus), BVDV-1 (bovine viral diarrhea virus genotype 1), BVDV-2 (BVDV genotype 2), PI-3 (parainfluenza-3), and BRSV (bovine respiratory syncytial virus). During the 103-d experiment, weekly fecal egg counts, blood, and rectal temperatures were collected and health status was recorded daily. Blood samples were obtained weekly to determine serum neutralizing (SN) antibody titers to IBRV, BVDV-1, BVDV-2, and PI-3 and cytokine levels for IL-4, IL-6, TNF-α (tumor necrosis factor-α), and IFN-γ (interferon-gamma). There was a tendency (P < 0.09) for CONT calves to have greater IL-4 concentrations. By design, control calves had greater (P < 0.01) fecal egg counts during the experiment. All calves developed antibody titers to IBRV, BVDV-1, BVDV-2, and PI-3 by d 15 postvaccination. On d 88, all calves were challenged with IBRV and blood samples were obtained on d 88, 89, 90, 93, 95, 98, 99, and 103. All calves had increased rectal temperatures during the final 7 d of the IBRV challenge. However, the CONT group had greater (P < 0.01) rectal temperatures on each sampling day except d 90 compared with the DPV and DV treatments. Therefore, deworming before or at vaccination reduced parasite burden and decreased rectal temperature increase after an IBRV challenge. Deworming strategy had no effect on antibody response to vaccination or IBRV challenge.

Cytokine gene expression at the materno-foetal interface after experimental Neospora caninum infection of heifers at 110 days of gestation.

Neospora caninum is a major cause of abortion in cattle, but the reasons why only some animals abort remain unclear. The immunological control of the parasite in the placenta or by the foetus could be the key to determining the mechanism of abortion and/or transplacental transmission to the foetus. In this study, cytokine gene expression, analysed by real-time RT-PCR, at the maternal (caruncle) and foetal placenta (cotyledon) of heifers infected at 110 days of gestation by intravenous inoculation of N. caninum tachyzoites was compared with the responses in uninfected heifers. Animals were euthanized 3 weeks after infection. Upregulated Th1, Th2 and T-regulatory (Treg) cytokine gene expression was observed in both the maternal and the foetal placenta in the infected group. In the caruncle of infected animals, the main changes included upregulation of IFN-γ, IL-12p40, IL-6 and IL-10. In the cotyledon, the main changes included upregulation of IFN-γ and downregulation of TGF-ß, being the later the only cytokine downregulated in the infected group. The observed cytokine expression pattern was associated with alive but transplacentally infected foetuses, suggesting that such cytokine pattern is beneficial to foetal survival, but could have a role in the transplacental transmission of the parasite.

A calcium-activated nucleotidase secreted from Ostertagia ostertagi 4th-stage larvae is a member of the novel salivary apyrases present in blood-feeding arthropods.

Apyrases (ATP-diphosphohydrolase) comprise a ubiquitous class of glycosylated nucleotidases that hydrolyse extracellular ATP and ADP to orthophosphate and AMP. One class of newly-described, Ca2+-dependent, salivary apyrases known to counteract blood-clotting, has been identified in haematophagous arthropods. Herein, we have identified a gene (Oos-apy-1) encoding a protein that structurally conforms to the Ca2+-activated apyrase from the bed bug, Cimex lectularius, by immunologically screening an Ostertagia L4 cDNA expression library. The expressed protein (rOos-APY-1) was biochemically functional in the presence of Ca2+ only, with greatest activity on ATP, ADP, UTP and UDP. Host antibodies to the fusion protein appeared as early as 14 days post-infection (p.i.) and increased through 30 days p.i. Immunohistochemical and Western blot analyses demonstrated that the native Oos-APY-1 protein is present in the glandular bulb of the oesophagus and is confined to the L4. A putative signal sequence at the N-terminus and near 100% identity with a Teladorsagia circumcincta L4 secreted protein is consistent with the native protein being secreted at the cellular level. Predicated upon substrate specificity, the native protein may be used by the parasite to control the levels of host extracellular nucleotides released by locally-damaged tissues in an effort to modulate immune intervention and inflammation.

Fetal death in cows experimentally infected with Neospora caninum at 110 days of gestation.

Neospora caninum is a major cause of abortion in cattle, but the reasons why some animals abort and not others remain unclear. Most of the N. caninum experimental primary infections in cattle late in gestation, after 120 days of pregnancy, result in birth of full-term congenitally infected fetuses. In the present study, the distribution of parasites and pathogenesis of infection in both dams and fetuses after inoculation with 10(7) culture derived tachyzoites of N. caninum NC-Illinois cattle strain at 110 days of gestation were analyzed at 3 weeks, 6 weeks and 9 weeks after infection (WAI) in eight Angus heifers. One dam from the group euthanized at 6 WAI had a dead fetus at necropsy. Extensive lesions were observed in the placenta and tachyzoites were detected in both the placenta and the fetus. The fetus was seropositive and had high IFN-gamma g production in fetal fluids. Another fetus, still alive when euthanized at 3 WAI, had severe lesions and high IFN-gamma production and a similar fate could have been expected if the experimental period would have been longer. Lesions in the placenta of the remaining six dams that had live fetuses at necropsy were mild. In those dams, the fetal and maternal placentas had not separated and contained focal areas of placentitis at the materno-fetal junction. Transplacental infection took place on all fetuses based on detection of parasitic DNA in fetal tissues. The present study shows that experimental N. caninum infection of naïve dams after 110 days of pregnancy can lead to fetal death. The results suggest that the severity of placental lesions and the strong IFN-gamma response in some fetuses, possibly as part of the immune response trying to control the high parasitemia, might, in fact, be the cause of their death.

Detection of germline and somatic copy number variations in cattle.

As a complement to the Bovine HapMap Consortium project, we initiated a systematic study of the copy numbervariation (CNV) within the same cattle population using array comparative genomic hybridization (array CGH). Oligonucleotide CGH arrays were designed and fabricated to cover all chromosomes with an average interval of 6 kb using the latest bovine genome assembly. In the initial screening, three Holstein bulls were selected to represent major paternal lineages of the Holstein breed with some maternal linkages between these lines. Dual-label hybridizations were performed using either Hereford L1 Dominette 01449 or L1 Domino 99375 as reference. The CNVs were represented by gains and losses of normalized fluorescence intensities relative to the reference. The data presented here, for the first time, demonstrated that significant amounts of germline and fewer somatic CNVs exist in cattle, that many CNVs are common both across diverse cattle breeds and among individuals within a breed, and that array CGH is an effective tool to systematically detect bovine CNV. Selected CNVs have been confirmed by independent methods using real-time (RT) PCR. The strategy used in this study, based on genome higher-orderarchitecture variation, is a powerful approach to generating resources for the identification of novel genomic variation and candidate genes for economically important traits.

Establishment of Neospora caninum antigen-specific T cell lines of primarily CD4 T cells.

Neosporosis is an important cause of pregnancy loss in cattle worldwide. Protective immunity against Neospora caninum infection may include both cell-mediated (CMI) and humoral immune responses. This study was to establish short-term antigen-specific T cell lines composed of primarily CD4(+)T cells from peripheral blood lymphocytes (PBL) of infected cows, which may be used to identify immunodominant antigens for the development of N. caninum vaccines. Crude N. caninum tachyzoite antigen was prepared from in vitro derived N. caninum tachyzoites. Multiple T cell lines were established and maintained for 11 weeks by weekly re-stimulation with N. caninum antigen and antigen-presenting cells. All cell lines responded highly to antigen between weeks 3 and 11. Phenotypically, these cells were composed primarily of CD4(+)T cells between weeks 2-8, with a gradual expansion of gamma/delta(+)T cells thereafter. The results indicate that N. caninum-specific T cell lines can be established and maintained without exogenous T cell growth factors and may be used to identify N. caninum antigens. This research will enhance our understanding of bovine CMI to neosporosis and may facilitate development of a proven neosporosis vaccine.

Inhibition of bovine T lymphocyte responses by extracts of the stomach worm Ostertagia ostertagi.

Lowered immune responses during bovine ostertagiosis have been reported in both in vivo and in vitro assay systems. In the present study we have employed three different life cycle stages of the nematode Ostertagia ostertagi to determine if products of this economically important parasite inhibit in vitro proliferation of Con A-stimulated cells from uninfected animals. We have demonstrated an inhibitory effect upon the growth of Con A-stimulated lymphocytes after addition of fourth stage larval (L4) soluble extract (L4SE) to the cultures. In contrast, extracts from the third stage larvae (L3) had little or no inhibitory activity. The suppressive products were also shown to be secreted by the late L4. The suppressive activity is reversible if the L4 products are removed from culture. There is no immediate effect on proliferating cells and the L4SE must be in culture for 24-48 h before suppression is observable. The L4SE caused slight but not statistically significant decreases in the percentage of T cells and increases in B cell percentages in cultures when compared with cultures stimulated with Con A alone. No changes were seen in percentage of cells positive for markers for CD4, CD8, gammadelta T cells, or monocytes/macrophages as a consequence of the addition of L4SE. In contrast, there was a strong and significant reduction in the expression of the IL-2 receptors in cells cultured in the presence of the worm extract. There was no evidence of either necrosis or apoptosis resulting from the presence of L4 products in culture. The expression of messenger RNA for interleukin-2, -4, -13, tumor necrosis factor-alpha (TNF-alpha), and gamma-interferon (gamma-IFN) was decreased when L4SE was included in cultures of Con A-stimulated cells compared to cultures stimulated with Con A only. In contrast, messenger RNA expression of transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) was increased in cells growing in the presence of L4 products. The potential role of these cytokines during ostertagiosis is discussed.

AAVP Symposium: new approaches in the study of animal parasites.

The following three papers are a very small window onto the types of research being pursued by members of the American Association of Veterinary Parasitologists. They are related by the fact that newer areas in the biology of parasites and their hosts are discussed. The first paper by Dr. Tom Klei, gives a brief view of the interactions between host and parasite of the fascinating organism Wolbachia, a parasite of parasites. The second paper by Dr. Gloria Solano-Aguilar addresses the use of probiotics to alter the host­parasite interface and influence host resistance. The final paper by Dr. Lou Gasbarre outlines an example of integration of the genomics revolution into Veterinary Parasitology. While the subjects are diverse, they demonstrate the vitality of the AAVP.

Cytokine gene expression in dams and foetuses after experimental Neospora caninum infection of heifers at 110 days of gestation.

Neospora caninum is a major cause of abortion in cattle. An essential role for Th1 cytokines, such as IFN-gamma and IL-12 in protective immunity against N. caninum in murine models has been indicated. However, little is known about immunity to Neospora in pregnant cattle where a considerable level of immunomodulation may exist. In this study, the immune response of heifers infected early in the second trimester of pregnancy by intravenous inoculation of N. caninum tachyzoites was compared with immune responses in uninfected pregnant heifers. Animals were killed 3 weeks after infection. No abortion was observed in any infected dam, however, transplacental infection was shown to have already taken place. Infection with N. caninum during pregnancy induced significant immune responses in both dams and their foetuses. Infected dams showed significant changes in lymphocyte subpopulations compared with uninfected pregnant animals and these changes were compartmentalized. Increased levels of T lymphocytes were observed in the infected foetuses. Cytokine gene expression analysed by real time RT-PCR showed increased expression of both Th1 and Th2 cytokines in N. caninum infected animals. This cytokine expression could have a role in the transplacental transmission of the parasite and/or mediate tissue damage.

Limited effect of recombinant porcine interleukin-12 on porcine lymphocytes due to a low level of IL-12 beta2 receptor.

The cytokine interleukin-12 (IL-12) is a key molecule in the regulation of CD4 + T cell development and specifically potentiates T helper 1 responses in mouse and man. However, biological effects mediated by IL-12 have not been well defined in pigs. Herein, recombinant porcine IL-12 (rPoIL-12) was expressed in a swine poxvirus system as a biologically active heterodimer and used to stimulate bovine or swine lymphoblast cells. After 3 days of incubation, only bovine blasts were responsive to the rPoIL-12 treatment as monitored by cell proliferation in several independent trials. Similarly, i.m. administration of rPoIL-12 in the hind leg of 3-week-old pigs indicated a reduction in the number of interferon-gamma (IFN-gamma) producing lymphocytes isolated from inguinal lymph nodes. The porcine IL-12R beta2 (IL-12Rbeta2) sequence was cloned and results generated by reverse transcriptase polymerase chain reaction (RT-PCR) demonstrated that the expression of IL-12R on porcine blasts as measured by the relative levels of IL-12Rbeta2 mRNA was less than that in bovine blasts and are in agreement with the reduced proliferation response of swine blast cells to rPoIL-12 treatment. Real time PCR analysis demonstrated that after PBMC stimulation, bovine blasts had an 11-fold increase in IL-12Rbeta2 mRNA levels while porcine blasts had almost no change. These data support a mechanism for IL-12 stimulation in swine inconsistent with that observed in conventional models.

Genomic tools to improve parasite resistance.

The natural genetic variability of the ruminant immune system provides a feasible means to control gastrointestinal (GI) parasite infection without anthelmintics. However, the paradigm of traditional selection has not been effectively applied to the moderately heritable traits of parasite resistance (h approximately equal to 0.3) due to the difficulty and expense of gathering accurate phenotypes in a commercial production setting. These characteristics make host traits related to GI nematode infection ideal candidates for genomics-based research. To initiate explanation of important allelic differences, economic trait loci (ETL) are being identified and mapped using a resource population of Angus cattle segregating for GI nematode resistance and susceptibility to the two most common nematode parasites of US cattle, Ostertagia ostertagi and Cooperia oncophora. The population is composed of five generations of half-sib progeny with complete phenotypic records produced from controlled infections. To detect the genomic locations of the three distinct phenotypic traits being expressed (innately immune, acquired immune, and immunologically non-responsive), genotypes have been generated for DNA markers (N=199) spaced at regular intervals (approximately 20cm intervals) throughout the entire genome (3000cm). Although initial ETL detection may be limited by half-sib family size, the unique structure of this population provides additional statistical power for refining map position of potential ETL. After allele frequency and contribution to phenotype are determined in this population, marker tests associated with ETL most beneficial for controlling parasite infection can be accurately used for selection. Comparative map and functional genomic information from humans and other species of biomedical importance will be utilized in further investigations to elucidate the genes underlying ETL.

Gastrointestinal nematodes of cattle in the northeastern US: results of a producer survey.

A questionnaire covering management practices and producer perception of the effects of gastrointestinal nematode infections was sent to dairy and beef producers in the northeastern US. The mailing list was derived from membership in grazing groups and attendance at grazing events. A final total of 474 responses were suitable for analyses. These responses covered 14 states, but for the purpose of analysis were broken into five groups: New England (NE), Vermont (VT), New York (NY), Pennsylvania (PA), and south and west (S and W) of Pennsylvania. Two-thirds of the responses were from dairy producers. The average number of animals for the farms was 50 cows, 27 heifers, and 20 calves. The average acreage used for grazing was 70 acres, and about two-thirds of the responses used rotational grazing for at least the cows. About one-half of the rotational grazers had been practicing rotational grazing for more than 5 years. Most rotational programs for cows involved a daily rotation, but the rotational interval for other age groups was longer. There was a difference of about 2 months (5.25-7.27) in the length of the grazing season as one moved from New England to south and west of Pennsylvania. Parasite control practices varied greatly by location and animal class. Most producers used anthelmintics one to two times per year, but 10-30% of responses said they did not deworm their cattle. The most common time to deworm was in the spring, and the second most common time was the fall. Between 10 and 20% of respondents reported deworming as a response to decreased productivity or body condition. The use of anthelmintics increased as the location moved from New England to south and west of Pennsylvania. Producer perception of parasite effects was closely related to their anthelmintic use, and also increased as the location moved to the south, and is most likely the result of the increased length of the grazing season. Of producers who ascribed estimated a cost of the parasite, the majority estimated this cost to be between US$ 5 and 20 per animal per year.

Mouse cytokine profiles associated with Brucella abortus RB51 vaccination or B. abortus 2308 infection.

This study indicated that mice immunized with Brucella abortus RB51 bacteria and subsequently challenged with B. abortus 2308 were protected from reinfection. After vaccination, both Th1 and Th2 cytokine patterns were observed. Of those, the early production of gamma interferon seems to have the prominent role in inducing an immunologically based protection.

Role of the bovine immune system and genome in resistance to gastrointestinal nematodes.

Gastrointestinal nematode infections of cattle remain a constraint on the efficient raising of cattle on pasture throughout the world. Most of the common genera of parasites found in cattle stimulate an effective level of protective immunity in most animals within the herd after the animals have been on pasture for several months. In contrast, cattle remain susceptible to infection by Ostertagia for many months, and immunity that actually reduces the development of newly acquired larvae is usually not evident until the animals are more than 2 years old. This prolonged susceptibility to reinfection is a major reason that this parasite remains the most economically important GI nematode in temperate regions of the world. Although, animals remain susceptible to reinfection for a prolonged period of time, there are a number of manifestations of the immune response that result in an enhanced level of herd immunity. These include a delay in the development time of the parasites, an increase in the number of larvae that undergo an inhibition in development, morphological changes in the worms, stunting of newly acquired worms, and most importantly a reduction in the number of eggs produced by the female worms. The overall result of these manifestations of immunity is a reduction in parasite transmission within the cattle herd. The immune mechanisms responsible for these different types of functional immunity remain to be defined. In general, GI nematode infections in mammals elicit very strong Th2-like responses characterized by high levels of Interleukin 4 (IL4), high levels of IgG1 and IgE antibodies, and large numbers of mast cells. In cattle, the most extensively studied GI nematode, in regards to host immune responses, is Ostertagia ostertagi. In Ostertagia infections, antigens are presented to the host in the draining lymph nodes very soon after infection, and within the first 3-4 days of infection these cells have left the nodes, entered the peripheral circulation, and have homed to tissues immediately surrounding the parasite where they become established. The immune response seen in the abomasum is in many ways are similar to that seen other mammalian hosts, with high levels of expression of IL4 in the draining lymph nodes and in lymphocytes isolated from the mucosa. But unlike a number of other systems, lymphocyte populations taken from Ostertagia infected cattle seem to be up-regulated for a number of other cytokines, most notably Interferon (IFN, implying that in Ostertagia infections, the immune response elicit is not simply a stereotypic Th2 response. In addition, effector cell populations in the tissues surrounding the parasites, are not typical, inferring the Ostertagia has evolved means to suppress or evade protective immune mechanisms. Studies have also demonstrated that the number of nematode eggs/gram (EPG) in feces of pastured cattle is strongly influenced by host genetics and that the heritability of this trait is approximately 0.30. In addition, EPG values are not "normally" distributed and a small percentage of a herd is responsible for the majority of parasite transmission. This suggests that genetic management of a small percentage of the herd can considerably reduce overall parasite transmission. A selective breeding program has been initiated to identify the host genes controlling resistance/susceptibility to the parasites. The best indicator of the number of Cooperia infecting a host is the EPG value, while Ostertagia is best measured by serum pepsinogen levels, weight gain, and measures of anemia. Other phenotypic measures are either not significantly associated with parasite numbers or are very weakly correlated. In addition, calves can be separated into three types: (1) Type I which never demonstrates high EPG values, (2) Type II which shows rises in EPG values through the first 2 months on pasture which then fall and remain at levels associated with Type I calves, and (3) Type III calves which maintain high EPG levels. The approximate percentage of these calves is 25:50:25 respectively. Because these cattle are segregating for traits involved in resistance and susceptibility to GI nematodes, this resource population is being used to effectively detect the genomic locations of these Economic Trait Loci (ETL). For relational analysis between phenotype and genome location, over 80,000 genotypes have been generated by PCR amplification, and marker genotypes have been scored to produce inheritance data. The marker allele inheritance data is currently being statistically analyzed to detect patterns of co-segregation between allele haplotype and EPG phenotypes. Statistical power of this genome-wide scan has been strengthened by including genotypic data from the historic pedigree. In our herd, paternal half-sib families range from 5-13 progeny/sire, and extensive marker genotypes are available from ancestors of the population most of which are paternally descended from a single founding sire. Once ETL have been identified the next will be to refine ETL map resolution in attempt to discover the genes underlying disease phenotypes. Accurate identification of genes controlling resistance will offer the producer several alternatives for disease control. For a non-organic producer, the small percentage of susceptible animals can be targeted for drug administration. This approach would reduce both the cost of anthelmintics used and the odds for selection of drug resistant mutants, because the selective agent (drug) would not be applied over the entire parasite population. A second treatment option would be based on correcting a heritable immunologic condition. In this case, susceptible animals could be the targets for immunotherapy involving vaccines of immunomodulation. A final option would be genetic selection to remove susceptible animals from the herd. Producers with a high degree of risk for parasite-induced production losses, such as organic producers of producers in geographic areas with environmental conditions favorable to high rates of transmission would benefit the most from this strategy. In contrast, producers at low risk could take a more conservative approach and select against susceptibility when other factors were equal.

A multiplex PCR assay for differentiating economically important gastrointestinal nematodes of cattle.

A multiplex polymerase chain reaction (PCR) test was developed for identifying gastrointestinal (GI) nematodes that commonly infect cattle. This assay was developed using adult-derived genomic DNA and shown capable of discriminating parasite eggs from the feces of experimentally-infected animals at both the species and genus levels. Sequence data from internal (ITS) and external (ETS) transcribed spacers of the ribosomal DNA (rDNA) repeats as well as the 3'-end of the small subunit rDNA and 5'-end of the large subunit rDNA were used to generate five primer sets which, when used simultaneously in a multiplex PCR, produce a unique electrophoretic DNA banding pattern characterized by a single DNA fragment for Ostertagia ostertagi (257bp), Haemonchus placei (176bp), Oesophagostomum radiatum (329bp), Trichostrongylus colubriformis (243bp) and Cooperia oncophora (151bp). In a similar manner, the constructed primer sets amplified DNA from Ostertagia lyrata, Haemonchus contortus, Trichostrongylus axei, Cooperia surnabada and Cooperia punctata. With respect to H. contortus, a closely migrating doublet was generated suggesting size heterogeneity in the ETS which is consistent with multiple rDNA repeat units within this species. PCR analyses using mixtures of monospecifically-purified nematode eggs indicated a sensitivity of less than 0.5 egg-DNA equivalent per species. Although, not designed as a quantitative technique, relative PCR signal intensities corresponded to relative egg burdens within the DNA samples from mixed species of eggs.

Interferon-gamma and interleukin-4 mRNA expression by peripheral blood mononuclear cells from pregnant and non-pregnant cattle seropositive for bovine viral diarrhea virus.

The acceptance of the fetal allograft by pregnant women and mice seems to be associated with a shift from a Th 1 dominated to a Th 2 dominated immune response to certain infectious agents. The goal of this study was to examine cytokine expression in peripheral blood mononuclear cells (PBMCs) from cattle immune to bovine viral diarrhea virus (BVDV) to determine whether pregnancy also has an influence on the type of immune response in this species. Forty-six heifers and cows between 14 months and 13 years of age were included in this study. Twenty-four were seropositive and 22 seronegative for BVDV. Eleven of the seropositive animals and 11 of the seronegative animals were in the eighth month of gestation, the remaining animals were virgin heifers. PBMC from these animals were analyzed for Interferon (IFN)-gamma and Interleukin (IL)-4 mRNA expression by real-time RT-PCR after stimulation with a non-cytopathic strain of BVDV. Additionally, an ELISA was performed to measure IFN-gamma in the supernatants of stimulated cell cultures. In BVDV seropositive animals, IFN-gamma mRNA levels were significantly higher than in BVDV seronegative animals and there was a significant positive correlation between the changes in IFN-gamma and IL-4 mRNA expression. There was, however, no significant difference in IFN-gamma and IL-4 mRNA levels between pregnant and non-pregnant animals. These results are inconsistent with BVDV inducing a Th1 or Th2 biased immune response. Furthermore, a shift in the cytokine pattern during bovine pregnancy was not evident.

Oral infection of calves with Neospora caninum oocysts from dogs: humoral and cellular immune responses.

Neospora caninum has been identified as a major cause of abortion in cattle in a number of countries throughout the world. Until the recent demonstration that dogs can serve as a definitive host of this parasite, it was not possible to study the infection in cattle orally exposed to oocysts. The aim of this study was to investigate the potential of N. caninum oocysts to infect calves, and to define initial immune responses that arise after oral infection. Seven calves were fed approximately 10(4)-10(5) N. caninum oocysts, three calves served as uninfected controls. Before infection, all calves were serologically negative for anti-Neospora antibodies and the calves were non-reactive to Neospora antigen in an in vitro lymphocyte proliferation assay. Peripheral blood lymphocytes from inoculated calves were able to mount in vitro proliferative responses to crude N. caninum antigen extract as early as 1 week p.i. Within 2 and 4 weeks p.i., Neospora-specific IgG1 and IgG2 antibodies were detected by IFAT and ELISA in serum from infected calves but not from sham-infected calves. The continued presence of reactive cells in the blood, spleen and mesenteric, inguinal, bronchial lymph nodes was seen as late as 2.5 months p.i., and parasite DNA was detected in the brain and spinal cord of the infected animals by PCR, indicating that the cattle were infected by oral inoculation of N. caninum oocysts collected from dogs, and that the animals were systematically sensitised by parasite antigen.