Immune Response In Lambs Research Articles

Immunization with DNA vaccines encoding different mycobacterial antigens elicits a Th1 type immune response in lambs and protects against Mycobacterium avium subspecies paratuberculosis infection

Paratuberculosis, or Johne's disease, is a disease of domestic and wild ruminants that culminate with a chronic enteritis caused by Mycobacterium avium subsp. paratuberculosis. The aim of this work was to evaluate the type of immune response, Th1 or Th2, induced by DNA vaccinations in lambs of Sarda breed. Twenty-five lambs, serum negative for M. paratuberculosis, were selected at birth from equally serum negative mothers. The lambs were inoculated at 5 months of age with three different mycobacterial antigens cloned into a mammalian expression vector as fusion protein with the enhanced green fluorescent protein (pEGFP-N1). The animals were divided in five groups containing each five lambs. Each group was vaccinated as following (A: physiological solution; B: Gudair™; C: p-85A-Mav; D: p-85A-BCG; E: p-Hsp65). Immune response was evaluated by measuring the expression of INF-γ (Th1 type response) and IL-10 (Th2 type response) by real-time PCR. Gene expression was estimated by comparing the results with that of β-actin. INF-γ expression level was increased in lambs vaccinated with plasmids codifying mycobacterial antigens, in particular with p-Hsp65, in comparison with the controls suggesting stimulation of a Th1 immune response similar to that supported by natural infection of M. paratuberculosis. Moreover, animals were infected orally with live M. paratuberculosis. Three months after vaccination and again INF-γ and IL-10 expression was evaluated in order to verify in vivo the protection level of the vaccines. Plasmids p-85A-BCG and p-Hsp65 seem to elicit a stronger protective immune response against M. paratuberculosis by evaluating the expression level of INF-γ and evaluating the presence of M. paratuberculosis and animal cell organ damage post-mortem.

Effect of gradual transition from maternal to reconstituted milk on behavioural, endocrine and immune responses of lambs

The possibility of reducing the stress associated with artificial rearing through a gradual transition from maternal to reconstituted milk was assessed in thirty Comisana lambs. Animals were assigned to a control dam-suckled group (Suckled) and two test groups. Eighteen to twenty-four hours after parturition, test lambs were separated from their dams: 10 subjects were offered a commercial milk substitute (Sub), whereas the other ten (Mix) were subjected to a gradual transition from maternal to reconstituted milk over ten days. Test-lambs had free access to milk 3 h a day. In vivo cell-mediated immune response of lambs to phytohemagglutinin percutaneous injection was evaluated at 5, 15 and 25 days of age. At 10 and 20 days of age, all lambs were subjected to 15 min isolation tests, during which latency time to move, duration of movement and number of bleats were recorded. Blood samples were collected immediately before the test, 15 and 60 min after in order to assess the effect of differential rearing on cortisol response of animals at different ages. In addition, weight gains of all lambs and milk intakes of test lambs were recorded during the first 35 days of age at weekly intervals. Sub showed a lower immune response ( P<0.01) and, at 10 days of age, increased levels of plasma cortisol ( P<0.001) as well as a reduced duration of movement ( P<0.001) and a longer latency time to move ( P<0.001) compared to Suckled and Mix. There were no significant differences between Suckled and Mix for both immune, cortisol and behavioural responses. Mix lambs consumed more milk ( P<0.006) and had higher weight gains ( P<0.001) compared to Sub lambs. However, Mix lambs gained less weight on average ( P<0.05) than Suckled lambs. Our results suggest that a gradual transition from maternal to reconstituted milk is effective in stimulating milk consumption and minimising the detrimental effects of artificial rearing on behavioural, endocrine and immune responses of lambs.

Vaccination with glutaraldehyde-fixed bovine respiratory syncytial virus (BRSV)-infected cells stimulates a better immune response in lambs than vaccination with heat-inactivated cell-free BRSV

The lamb model was used to investigate the possible protective effects of vaccination with inactivated viral antigens against experimental infection with bovine respiratory syncytial virus. Two groups of eight lambs were vaccinated with either glutaraldehyde-inactivated cell-associated virus or heat-inactivated cell-free virus and subsequently challenged with live virus, along with a group of naive lambs. The virus was shed for significantly longer periods, and the virus titres in nasal secretions were significantly higher in the group of naive lambs than in the two groups of vaccinated lambs. The period of virus-shedding in nasal secretions and virus titres was significantly lower ( p<0.01) in the group of lambs immunized with the cell-associated preparation. The same antigen stimulated better cellular immune responses as measured by virus-specific cytotoxicity or by virus-specific lymphocyte proliferation. However, priming with inactivated vaccines had no significant effect on lymphocyte responses to phytohaemagglutinin, which was found to be significantly reduced ( p<0.01) following challenge with live virus.

Transmission of ovine herpesvirus 2 in lambs.

The pattern of acquisition of ovine herpesvirus 2 (OHV-2) infection in lambs was examined by a competitive-inhibition enzyme-linked immunosorbent assay and PCR. Newborn lambs (n = 118) did not exhibit antibody at birth. Viral DNA in peripheral blood leukocytes was detected in only 3% (n = 77) of newborn lambs before suckling. After nursing, viral DNA was sporadically present in about 10 to 20% of lambs until about 3 months of age. Thereafter, strong DNA signals began to appear in increasing numbers of lambs, reaching 100% by 5.5 months of age. Viral DNA in nasal secretions began to be detectable in about 30% of lambs at 5.5 months of age, achieved significant levels in 88% of lambs by 7.5 months of age, and then declined. The kinetics of the humoral immune response in lambs paralleled those of viral DNA in nasal secretions but did not parallel its presence in blood leukocytes. In the experiment to define the time of infection of OHV-2 in lambs, all five lambs separated from the flock at 2.5 months of age remained uninfected until the termination of the experiment at 1 year of age. In contrast, lambs weaned at 2.5 months of age and returned to the flock had become infected at 3.5 months of age. Weaning and separation from the flock at 3.5 months of age did not prevent infection. The study showed that OHV-2 infection does not commonly occur in perinatal lambs and that OHV-2-free sheep can be established by separation of lambs at the proper time, which has important implications for potential control measures.

The effect of continuous drug exposure on the immune response of lambs challenged with drug-susceptible or drug-resistant nematode larvae.

Groups of lambs either with or without controlled-release albendazole (ABZ) capsules (CRCs) were challenged twice weekly for 6 weeks with either drug-susceptible or drug-resistant Ostertagia circumcincta and Trichostrongylus colubriformis. Groups with and without CRCs remained unchallenged as controls. There was minimal establishment of drug-susceptible parasites of either species in those lambs with CRCs. However, drug-resistant parasites of both species established adult worm burdens in the presence of the capsules. The humoral immune response, as measured by the serum antilarval (L3) antibody (Ab) titre, was pooled for weeks 4-6 and compared for each group. With the exception of anti-T. colubriformis Ab in group 2, anti-L3 Ab titres were significantly higher in all the parasite-challenged groups as compared to the control animals. Also, with the exception of anti-resistant O. circumcincta Ab levels in the CRC-treated animals (group 5), no significant difference was observed in Ab titres between the four groups challenged with either resistant or susceptible larvae. The results demonstrate the inability of CRCs to prevent establishment of drug-resistant parasites and that immune stimulation is not inhibited by the capsules.

Suppression of immune response in lambs during treatment with the beta-adrenergic agonist clenbuterol.

The effect of the beta-adrenergic agonist clenbuterol on immune function was examined in sheep. Twenty ewe lambs were housed indoors, with food and water available on an ad libitum basis, and immunized against somatostatin (SRIF) using a SRIF-ovalbumin conjugate. Ten of the lambs were also treated with clenbuterol (400 micrograms/kg) each day; 10 controls were not treated. After 5 wk of treatment (with booster injections of the SRIF conjugate each fortnight), the lambs were bled and then slaughtered for carcass composition. The lambs that received immunization alone produced significant antibody titers against SRIF, whereas 9 of the 10 clenbuterol-treated lambs produced no significant, specific antibody response. There was no effect of clenbuterol treatment on liver, thymus, spleen, kidney, kidney fat, or biceps femoris weight compared with those lambs that were only immunized. These results indicate that treatment with clenbuterol may inhibit humoral antibody response to infection.

Influence of artificial rearing on behavioral and immune response of lambs

The influence of artificial rearing at different ages was evaluated in Comisana lambs. Animals were assigned to three test groups (2d, 15d and 28d) on the basis of age (2, 15 or 28 days, respectively) at the time of their separation from the ewe, and three corresponding control non-separated groups (2c, 15c and 28c). On the day of separation from their dam, animals were injected i.m. with 250 μg of keyhole limpet hemocyanin in complete Freund's adjuvant. Antibody titers and surface leukocyte antigens were determined at weekly intervals. Group 2d showed a lower antibody response than the corresponding control group ( F = 8.0, P < 0.01), whereas groups 15d and 28d did not differ from the control. No significant difference between test and control groups was found in the proportion of cells bearing different surface antigens. Furthermore, lambs were subjected to a behavioral test (isolation from tactile and visual contact with other animals). Younger animals showed reduced duration of movement (162.8 vs. 272.1 and 221.1 s for 2d, 15d and 28d, respectively; U = 21.0, P < 0.01 and U = 30.5, P < 0.05, respectively; Mann-Whitney U test) and enhanced latency time (15.8 vs. 3.4 and 2.3 s for 2d, 15d and 28d, respectively; U = 28.0, P < 0.01 and U = 14.5, P < 0.001, respectively; Mann-Whitney U test) in comparison with older animals. Cortisol concentration was evaluated immediately before the separation from the mother and 15, 45, 90, 150, and 225 min after. An increased cortisol level was observed in group 2d 15 min after removal from the dam (LSD: P < 0.001), whereas the other two groups did not show any significant increase. In addition, during the first 28 days of age all the animals were weighed at birth and at weekly intervals. When the animals were removed from the mother either at 2 or 15 days of age, the daily weight gain decreased significantly in comparison with the corresponding control groups (107.1 vs. 157.1 g for 2d and 2c, respectively, F = 4.8, P < 0.05; 37.3 vs. 126.8 g for 15d and 15c, respectively, F = 5.8, P < 0.05). In conclusion, early separation from the ewe affects humoral immune response and post-separation performance of lambs as a possible consequence of a reduced ability of young animals to cope with emotional and nutritional stresses.

Levamisole and its influence on the immune response of lambs.

Ten parasite-free lambs were drenched with 8 mg/kg of levamisole on days 0 and 28 and were injected with human erythrocytes and ovalbumin one day after each drench. Ten other antigen-injected lambs were not drenched with anthelmintic as controls. Lymphocytes from the control and drenched lambs were cultured in vitro with RPMI 1640 plus 5% fetal calf serum (FCS), with 50% autologous serum only, with concanavalin A (Con A) or with phytohaemagglutinin (PHA). Decreased blastogenesis was observed in cells from the drenched lambs cultured in the presence or absence of mitogen and was most obvious when 50% autologous serum was used, particularly with PHA, and when lymphocytes were collected 3 and 7 days after the first and 3 days after the second antigen injection. There were no significant changes in antibody titres between the groups. Decreased serum complement activity was seen 3 days after the second antigen injection in the drenched lambs. Although there was a significant reduction in the serum insulin-like growth factor I levels 4 days after each levamisole drench, the drenched lambs gained significantly more weight than the non-drenched control lambs.

The immunologic response to experimental infection with bovine respiratory syncytial virus in lambs.

An experimental model was designed to study the humoral immune response in lambs experimentally infected with bovine respiratory syncytial virus (BRSV), 25 Merino lambs of both sexes, with a live weight of 17 +/- 3 kg, received an intratracheal inoculation of 20 ml. saline solution containing 1.26 x 10(6) TCID50 BRSV (strain NMK-7) per ml. Blood samples for serological analysis were collected from the jugular vein of the animals on days 0, 1, 3, 5, 7, 9, 11 and 15 post inoculation. Serum proteins were determined by electrophoresis, and a qualitative and quantitative analysis of immunoglobulins (Ig) was carried out using immunoelectrophoresis (IE) and light nephelometry, respectively. Serum anti-BRSV antibody levels were tested by enzyme-linked immunoabsorbent assay (ELISA). Lambs were necropsied 1, 3, 7, 11 and 15 postinoculation days (p.i.d.), and samples were taken for immunohistochemical analysis. IgA, IgG and IgM were labelled on paraffin-embedded lung sections using an avidin-biotin-peroxidase complex. Results reflected an increase in gamma fractions in experimental animals with respect to controls. IE analysis of these sera revealed a polyclonal increase in immunoglobulins. Nephelometric examination showed an increase in serum IgA, IgG an IgM levels up to 7 p.i.d., coinciding closely with high titres for humoral virus antibodies. After 9 p.i.d., titres and immunoglobulin levels both fell. Immunoglobulin labelling in formol-fixed and paraffin-embedded lung tissue samples detected IgG and IgA, mainly in bronchial, bronchiolar and alveolar epithelia. IgM, however, was not detected in the lungs of infected animals.

Immune responses of lambs experimentally infected with bovine respiratory syncytial virus and Pasteurella haemolytica

The immune responses of lambs experimentally infected with bovine respiratory syncytial virus (RSV) and Pasteurella haemolytica were compared with lambs infected with bovine RSV or Pasteurella haemolytica alone. Superinfection with P. haemolytica aggravated the reduction in the number of CD5+, CD4+ and LCA p220+ (B) lymphocytes caused by bovine RSV, but it had no effect on the neutralizing antibodies against P. haemolytica cytotoxin. Serum samples obtained from lambs experimentally infected with bovine RSV and P. haemolytica had significantly lower amounts of neutralizing antibodies to bovine RSV than those obtained from lambs infected with bovine RSV alone.

The influence of colostral antibodies on the immunization of lambs against adenoviruses

The effect of colostral antibodies on the immune response in lambs following adenovirus vaccination was studied. Young lambs of different ages, born from vaccinated ewes, were vaccinated with an inactivated and aluminium hydroxide-adsorbed sheep adenovirus vaccine. In a group of lambs vaccinated at 5–7 days of age, the titre of humoral antibodies declined in parallel to unvaccinated controls. In lambs vaccinated at 24–35 days of age, antibody titres to adenovirus stabilised and persisted after an initial fall. Cell mediated immunity, as measured by blastogenic responses of circulating lymphocytes, was stimulated in both groups, but higher numbers of IgG-producing cells became evident in the group of lambs vaccinated at 24–35 days of age.