Louis C. Gasbarre

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.

Cryptosporidium parvum infection in bovine neonates: dynamic clinical, parasitic and immunologic patterns

Twenty-six experimentally infected calves were monitored daily for oocyst excretion. All began excreting oocysts 3-6 days p.i. Most calves (n = 23) excreted oocysts for 6-9 days, with a daily range from 4 x 10(2) to 4.15 x 10(7) oocysts g(-1) of faeces. Over half the calves excreted peak numbers of oocysts 6-8 days p.i. Diarrhoea, observed intermittently beginning as early as day 3 p.i., lasted 4-16 days and varied greatly in severity from calf to calf. In a second study, nine of 18 calves were orally inoculated with 5 x 10(6) oocysts between birth and 2 days of age and nine remained uninfected. Monoclonal antibodies for cell surface markers indicated substantial increases in CD4+ and CD8+ T cells in the intraepithelial lymphocyte population of the ilea of infected calves at 7-9 days of age. RT-PCR demonstrated increases in mRNA for interleukin-12 and interferon-gamma that correlated with increases in both CD4+ and CD8 + intraepithelial lymphocyte cells. Increased mRNA for interleukin-12 and interferon-gamma from lamina propria lymphocytes correlated with increased numbers of CD8+ cells. No changes were found in interleukin-2, interleukin-4 or interleukin-10 mRNA levels. However, interleukin-15 mRNA, possibly from epithelial cells contaminating intraepithelial lymphocytes, was decreased in infected calves and had a negative correlation with increases in CD4+ and CD8+ cells. No differences were detected in mRNA levels for cytokines from lymph node lymphocytes.

Effects of gastrointestinal nematode infection on the ruminant immune system.

Gastrointestinal (GI) nematodes of ruminants evoke a wide variety of immune responses in their hosts. In terms of specific immune responses directed against parasite antigens, the resulting immune responses may vary from those that give strong protection from reinfection after a relatively light exposure (e.g. Oesophagostomum radiatum) to responses that are very weak and delayed in their onset (e.g. Ostertagia ostertagi). The nature of these protective immune responses has been covered in another section of the workshop and the purpose of this section will be to explore the nature of changes that occur in the immune system of infected animals and to discuss the effect of GI nematode infections upon the overall immunoresponsiveness of the host. The discussion will focus primarily on Ostertagia ostertagi because this parasite has received the most attention in published studies. The interaction of Ostertagia and the host immune system presents what appears to be an interesting contradiction. Protective immunity directed against the parasite is slow to arise and when compared to some of the other GI nematodes, is relatively weak. Although responses that reduce egg output in the feces or increase the number of larvae undergoing inhibition may occur after a relatively brief exposure (3-4 months), immune responses which reduce the number of parasites that can establish in the host are not evident until the animals second year. Additionally, even older animals that have spent several seasons on infected pastures will have low numbers of Ostertagia in their abomasa, indicating that sterilizing immune responses against the parasite are uncommon. In spite of this apparent lack of specific protective immune responses, infections with Ostertagia induce profound changes in the host immune system. These changes include a tremendous expansion of both the number of lymphocytes in the local lymph nodes and the number of lymphoid cells in the mucosa of the abomasum. This expansion in cell numbers involves a shift away from a predominant classic T cell population (CD2 and CD3 positive), to a population where T cell percentages are decreased and B cells (immunoglobulin-bearing) and gamma-delta cells are increased. At the same time the expression of messenger RNAs for T cell cytokines (IL2, IL4, IL10 and gamma-interferon) is changed to that of increased expression of IL4 and IL10 and decreased expression of IL2 and perhaps of gamma-interferon. The reasons for these changes remain to be elucidated, but it is evident that the lack of protective immune responses is not the result of a poor exposure of the host to parasite products, or to the stomach being an immunoprivileged site. In fact, a superficial look at the responses elicited indicates that Ostertagia induces responses (the so-called TH2 mediated responses) that are widely considered to be the type of responses necessary for protection against GI nematodes. There are many factors that could lead to this apparent lack of immunity in the face of a strong stimulation of immune responses including: (1) the elicitation of suboptimal responses; (2) the failure of the abomasum to function as an efficient effector organ; (3) active evasion of the functional immune response by the parasite; and (4) that these classic responses are not protective in this particular ruminant-parasite system and that novel protective mechanisms may be required. The strong stimulation of the host gut immune system by Ostertagia and perhaps by other GI nematode infections, raises questions about the potential effects of such infections on the overall well-being of the host. A number of authors have indicated that Ostertagia infections may diminish the hosts ability to mount subsequent immune responses to antigenic challenges such as vaccination against other infectious organisms. In addition, recent studies have indicated that infections with GI nematodes may result in increased circulatory levels of stress-related hormo

Cooperia punctata: Effect on cattle productivity?

Cooperia spp. have become the most prevalent parasites in United States cow/calf operations as observed in the USDA NAHMS (National Animal Health Monitoring System) Beef Cow/Calf survey in 2008. This is at least in part due to the widespread use of macrocyclic lactones that have recently been shown to have a reduced activity against these parasites. The effects of Cooperia spp. on cattle productivity are largely unknown. This study was conducted to assess their effect upon cattle housed under conditions found in American feedlots. Two hundred yearling calves (average weight 460 lb/209 kg) were acquired from northwestern Arkansas and northeastern Oklahoma and were vaccinated and dewormed upon arrival at the feedlot. Animals were comingled and preconditioned for approximately one month, and were fed a standard growing ration throughout the study. Calves were randomly divided into two groups (n=80, infected and control) and each group was further divided into two replicate pens (n=40). Calves from the two infected pens were orally inoculated with a gavage of 1 × 10(5) and 0.825 × 10(5) infective larvae of a recent isolate of Cooperia punctata on day 0 and 14, respectively, with the two control pens receiving a similar volume of tap water. Data collected included biweekly fecal egg counts, daily individual feed consumption and weight gain over the 60-day test period. The presence of C. punctata (>99% of recovered worms) was confirmed by necropsy and recovery from the small intestine on days 35 and 60 post infection (PI) in a subset of animals. Egg counts were positive by day 14 PI and remained at numbers similar to values seen in field studies. The control group gained weight 7.5% more rapidly (p=0.02) than infected animals (3.24 lb/1.47 kg per day vs. 3.0 lb/1.36 kg per day, respectively). The Cooperia-infected calves also consumed 1.5 lb (0.68 kg) less dry feed per day than the control animals (p=0.02). These data suggest that C. punctata has a deleterious effect on both appetite and nutrient uptake or utilization. At necropsy (days 35 and 60), the draining mesenteric lymph nodes of infected animals were increased in size and the small intestinal mucosa was thickened and covered with a thick layer of mucus in the infected animals. The most prominent histological changes in the Cooperia- infected animals included a moderate increase in the number of intraepithelial lymphocytes and globule leukocytes, as well as aggregates of eosinophils within the lower lamina propria. The only significant difference was an increase in the goblet cell density at day 60. Anthelmintic sensitivity/resistance of the Cooperia isolate used was determined by treatment of one pen of infected calves with a macrocyclic lactone and the other pen with a benzimidazole at the completion of the study. The macrocyclic lactone treatment (n=40) did not remove the parasites (FECRT=8.8%), while treatment with a benzimidazole was very effective (FECRT=98.1%). This study demonstrated that C. punctata has a significant effect on cattle productivity, both reduced weight gain and decreased feed intake compared to controls.

Interferon-γ 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.

Cytokine profile induced by a primary infection with Ostertagia ostertagi in cattle

Changes that occur in the local draining lymph nodes including, changes in cell surface markers and cytokine gene expression were studied over the first 4 weeks of a primary, Ostertagia ostertagi infection of the abomasum. Cells recovered from the abomasal lymph nodes (ABLN) after infection showed a decrease in the percentage of CD3+ cells, and an increase in the percentage of IgM+ cells and cells bearing the TcR1 marker. These changes were coincident with an increase in the proportion of activated cells (II-2R). Analysis of mitogen-stimulated ABLN cells by RNase protection assay (RPA) showed a dramatic reduction in IL-2 and IFN-gamma transcription after infection. In addition, analysis of unstimulated ABLN cells by competitive RT-PCR showed a similar decrease in demonstrable levels of IL-2 mRNA, but IL-10, IL-4 and IFN-gamma mRNA levels were elevated.

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.

Genetic control of immunity to gastrointestinal nematodes of cattle.

Previous studies have indicated that host genetics significantly affects the number of gastrointestinal nematode eggs per gram (epg) in the feces of calves during their first grazing season. An entire calf crop of approximately 190 animals was monitored monthly until weaning to verify these earlier results, and to begin to discern the basis for this phenomenon. A significant genetic effect on fecal epg values was not observed until calves had been on pasture for 2-3 months, and was demonstrable until late in the grazing season when the effect was lost. The loss of a genetic effect coincided with the appearance of significant numbers of the more highly fecund nematode species Haemonchus placei and Oesophagostomum radiatum, and with an apparent increase in Ostertagia ostertagi transmission, indicating that the observed genetic control of epg values may be species specific, dose dependent or both. Calves were selected from the population, and grouped according to their epg phenotype over the grazing season as either high or low epg calves. Postmortem examination of some of these calves indicated that worm burdens in the low epg calves were 60% of those of the high epg calves. Experimental challenge inoculation of the remaining calves indicated that: (1) challenge with Cooperia oncophora resulted in low epg calves harboring worm numbers that were 65% of those of high epg calves; (2) challenge with O. ostertagi resulted in similar numbers of worms in both groups, but the fecundity of worms in the low epg groups was significantly lower (P less than 0.05) than in the high epg group. Analysis of serum anti-Ostertagia antibody levels in the grazing calf population showed rises in serum IgG1, IgG2, IgM and IgA antibody levels during the grazing season. Peak serum IgG2 and IgG1 anti-Ostertagia antibody levels were found to be significantly affected by host genetic factors while IgA and IgM levels were not under such control.

Ostertagia ostertagi: isolation and partial characterization of somatic and metabolic antigens.

One metabolic (ES) and two somatic extracts (AS and MS) were prepared from Ostertagia ostertagi. Partial characterization of the three preparations was accomplished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot techniques. In immunoblots, AS showed a low number of bands that reacted weakly with sera collected from infected calves. MS reacted strongly with the homologous sera, and a sharp group of bands appeared from 12 to 14 kDa, increasing in intensity as the infection progressed. ES showed a group of strongly immunogenic bands in the range of 16-22 kDa. The three preparations were also tested for reactivity with specific anti-Ostertagia antibodies in an enzyme-linked immunosorbant assay (ELISA). The ELISA results showed that all three worm extracts contained antigen epitopes recognized by circulating antibody in sera taken from O. ostertagi-infected calves. The strongest response was seen when antibodies of the IgG1 isotype were reacted with MS and ES. When sera taken from O. radiatum-infected calves were used both somatic extracts showed high levels of cross-reactivity.

Influence of host genetics upon antibody responses against gastrointestinal nematode infections in cattle

Previous studies have shown that the number of gastrointestinal nematode eggs released per gram of feces (EPG) of calves is strongly influenced by host genetics. The purpose of this study was to determine if host genetics also influenced immune recognition of parasite antigens in these same calves. Serum samples were taken at monthly intervals from calves during their first grazing season, from approximately 4 months after the onset of calving and were continued until weaning. Serum samples were analyzed for antibodies against Ostertagia ostertagi, Haemonchus placei, Cooperia oncophora, and Oesophagostomum radiatum. Significant rises in antibodies of the IgG1 class were seen against Ostertagia ostertagi, H. placei, and C. oncophora. In addition, rises in anti-Ostertagia antibodies of the IgG2 and IgM isotypes were also noted. During periods of elevated antibody responses, the sire of the individual calves was found to influence significantly the level of circulating antibody. The heritability of serum anti-parasite antibody levels was demonstrated to be between 70 and 80%, depending upon the time and antibody isotype. The antibody levels did not appear to be correlated with parasite EPG values. These results indicate that the ability of calves to recognize parasite antigens is strongly influenced by genetic factors, and that the genetic factors which control antibody responses may differ from those controlling EPG values.