Immune response of pigs to parenteral vaccination with an aromatic-dependent mutant of Salmonella typhimurium.

Cellular and humoral immune responses to parenteral vaccination with an aromatic-defined (aroA) Salmonella typhimurium and to oral challenge with the S. typhimurium parent strain were examined in pigs. The effectiveness of aroA S. typhimurium vaccination for prevention of clinical disease following challenge was also evaluated. A split litter model was utilized and analysis of variance was by least squares. The statistical model accounted for the effects of vaccination and litter. Parenteral vaccination of pigs with the aroA mutant induced a significant O-polysaccharide (O-ps) specific lymphocyte blastogenic response as well as a significant antibody response to O-ps, lipopolysaccharide and killed bacteria. The aroA strain was avirulent in pigs, was not shed in the feces and significantly reduced the severity of diarrhea following oral challenge.     

Antibody avidity in swine lymphocyte antigen-defined miniature pigs.

Antibody avidity to hen egg white lysozyme (HEWL) was measured by thiocyanate ion elution enzyme-linked immunosorbent assay (ELISA) in swine lymphocyte antigen (SLA) defined miniature pigs. Serum antibody avidity was evaluated on day 14 and 30 after primary (day 0) and secondary (day 14) immunizations in eight to ten week old miniature pigs previously typed for swine lymphocyte antigen genotype. The effect of SLA genotype, litter, and gender on anti-HEWL antibody avidity was determined by least squares. Antibody avidity varied amongst individuals. Antibody avidity maturation was observed as a mean rise in antibody avidity from primary response (0.89 +/- 0.64) to secondary response (1.23 +/- 0.54) (p < 0.0005). Overall, SLA genotype did not significantly influence antibody avidity or avidity maturation, but pigs of dd genotype had greater avidity maturation between primary and secondary responses than other genotypes. Litter effects significantly affected antibody avidity and maturation.     

Immune response of sows and their offspring to pseudorabies virus: serum neutralization response to vaccination and field virus challenge.

One month prior to breeding, sows were vaccinated with an attenuated pseudorabies virus vaccine or challenged with a field strain of pseudorabies virus. A third group of sows were not vaccinated or challenged before breeding. Pigs from these sows were vaccinated at 3, 6, or 12 weeks of age and challenged with virulent virus three weeks later. One pig from each litter served as an unvaccinated, unchallenged control. Serum neutralization titers of these pigs were monitored from birth until 22 weeks of age. Titers of the sows were monitored through breeding, gestation and farrowing. The maximum prefarrowing anti-pseudorabies virus titer in the field virus challenged sows occurred four weeks following challenge. A significant decline in titers occurred at farrowing. Titers rose from one week postfarrowing and then declined. Titers in the field virus infected sows were consistently two to threefold greater than those of the vaccinated sows. The maximum prefarrowing anti-pseudorabies virus titer in the vaccinated sows occurred six weeks following vaccination. The geometric mean titer in these sow's then decreased and increased for two weeks after farrowing. The results in the pigs can be summarized as follows: Pigs from control sows had a greater serological response following field virus challenge than following vaccination with a modified live virus. Pigs from control sows responded serologically to vaccination at 3, 6 and 12 weeks of age. Pigs from control sows which were challenged at 6, 9 and 15 weeks of age had similar antibody responses. Pigs from vaccinated sows had no increase in titer following vaccination at three and six weeks of age. Titers increased when these pigs were vaccinated at 12 weeks of age. There was no significant increase in mean titers of pigs from challenged sows following vaccination at 3, 6 and 12 weeks of age. Vaccinated pigs from control and vaccinated sows had a secondary response following challenge three weeks after vaccination.(ABSTRACT TRUNCATED AT 250 WORDS)     

Breed and swine lymphocyte antigen haplotype differences in agglutination titers following vaccination with B. bronchiseptica

Genetic differences in immune response to B. bronchiseptica after vaccination with a commercial B. bronchiseptica bacterin were investigated in 1,069 8-wk-old pigs. These pigs were from 65 litters born in the spring and 66 litters born in the fall of 1982 and were purebreds from the Chester White (n = 128), Duroc (n = 281), Hampshire (n = 143), Landrace (n = 309) and Yorkshire (n = 208) breeds. Each litter was raised separately. Individual pigs were vaccinated im at 4 and 6 wk of age with 2 ml of B. bronchiseptica bacterin. At 8 wk of age, 8 ml of blood were collected from each animal and serum prepared to determine agglutinating antibody titers against B. bronchiseptica bacterin by a bacterial agglutination method. In addition, lymphocytes were separated from 1 ml of heparinized blood and used to determine Swine Lymphocyte Antigen (SLA) haplotypes by using cytotoxic antibodies against the SLA complex. Antisera for 3 SLA haplotypes were made available by the National Institutes of Health. Results indicated that breed of pig (P less than .01) and dam of pig (P less than .01) affected the immune response of the pig after B. bronchiseptica vaccination. Higher immune response was also associated (P less than .05) with one of the SLA haplotypes tested. Heritability estimates for immune response following vaccination were .10 +/- .12 (half-sib) and .42 +/- .19 (full-sib). Results suggest that the relationship of the SLA complex to immune response in the pig and nonadditive genetic and maternal effects on immune response should be further investigated.     

The influence of maternal immunity on the efficacy of a classical swine fever vaccine against classical swine fever virus,genogroup 2.2, infection

In Thailand, where vaccination is routinely employed, there has been an increased incidence of chronic classical swine fever (CSF) outbreaks during the past decade. The major causative virus has been identified to be the moderate virulence, classical swine fever virus (CSFV) of the genogroup 2.2. An investigation was made into the efficacy of a CSF vaccine against this genogroup 2.2 challenge. Five-week-old pigs, grouped by their level of passive antibody titer were immunized with lapinized Chinese-strain CSF vaccine and challenged with CSFV genogroup 2.2, 13 days after vaccination. The group containing passive titers of lower than 64 at the time of immunization, had significantly higher number of CSFV-specific IFN-gamma secreting cells and was completely protected against the challenge. Interestingly, both cellular and antibody responses were inhibited in the pigs with the higher passive titer. Furthermore, following challenge, CSFV could be isolated from 50% of the pigs in this group. It was demonstrated that the CSF vaccine could induce complete protection in pigs, provided that the maternal derived titer at the time of vaccination was lower than 64. The result implied that an increase in CSFV outbreaks might be due to the inappropriate timing of vaccination as well as the nature of the CSFV genogroup 2.2.     

Immunologic reactions of pigs regrouped at or near weaning

Using 64 pigs, 2 experiments (32 pigs each) were conducted to evaluate the effects of regrouping nonlittermate pigs at weaning or 2 weeks after weaning on mitogen-induced lymphocyte blastogenesis, intradermal reactions to phytohemagglutinin, and primary antibody responses to sheep erythrocytes. Plasma cortisol concentrations were determined in all pigs and behavior of regrouped pigs was monitored. Compared with control values, plasma cortisol concentrations were higher in nonlittermate pigs regrouped at weaning (P less than 0.001) or 2 weeks after weaning (P less than 0.01). However, regrouping pigs at weaning or 2 weeks after weaning did not influence lymphocyte blastogenesis, phytohemagglutinin skin-test responses, or antibody titers to sheep erythrocytes. Plasma cortisol concentrations were not related to agonistic behavior in regrouped pigs or to lymphocyte blastogenic or phytohemagglutinin skin-test responses; however, higher plasma cortisol concentrations were related (P less than 0.05) to lower sheep erythrocyte antibody titers. These data indicate that regrouping nonlittermate pigs at weaning or 2 weeks after weaning is an acute stressor that does not detrimentally affect mitogen-induced lymphocyte blastogenesis, intradermal reactions to phytohemagglutinin, or primary antibody responses to sheep erythrocytes.     

Passive protection of calves against experimental infection with Salmonella typhimurium

Cows were vaccinated with formalin-killed Salmonella typhimurium approximately seven weeks and two weeks before parturition to investigate whether passive immunity could protect their calves against experimental S typhimurium infection. After birth the calves were left with their dam for 48 hours and then separated and fed cold, stored colostrum from their own dam for a further eight days. Oral challenge five days after birth with 10(8) S typhimurium did not result in the death of these calves even when they had absorbed little colostrum. Mortality was reduced to 22 per cent in calves which sucked from vaccinated dams and were then fed colostrum from unvaccinated cows and to 50 per cent in calves born to unvaccinated cows and later fed colostrum from vaccinated animals. Calves which sucked from a vaccinated dam and then received stored colostrum from the same cow excreted salmonellas for significantly shorter periods after challenge and were less often infected at necropsy 28 days after inoculation. Protection was not correlated with the levels of O or H agglutinating antibodies in serum, which were at a maximum 24 hours after sucking and then slowly declined. There was no evidence of an active antibody response in the serum. Measurement of the O and H response of cows after vaccination indicated that the vaccination schedule could be improved. The highest levels of agglutinating antibody were measured between two and three weeks after the first vaccination and there was only a minimal response to the second vaccination before parturition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Passive immunity to Pasteurella haemolytica A1 in dairy calves: effects of preparturient vaccination of the dams.

Dairy cows from five herds were assigned to receive a commercial Pasteurella haemolytica vaccine or no vaccine at all, administered at six and three weeks before parturition. Vaccination was associated with increased leukotoxin neutralizing serum antibody titers in the dams (p < 0.001), and with increased titers in colostrum (p < 0.001). Vaccination of dams also had a significant association with increased passive leukotoxin neutralizing antibody titers in their calves (p < 0.001). Vaccination was also associated with increased indirect agglutinating antibody titers in serum of the dams (p < 0.001). In the analysis of agglutinating antibody titers in colostral whey the interaction "vaccination*herd" was found to be significant (p < 0.001), indicating that the effects of vaccination on colostral titers were not consistent from herd to herd. The analysis was repeated, stratifying by herd. Vaccination was associated with increased agglutinating antibody titers in colostrum (p < 0.05) in three herds of the five in the study. In two of these three herds there were significant increases in passive neonatal titers associated with vaccination. In the remaining herd the mean IgG1 level in the calves was consistent with failure of passive transfer of immunoglobulins (IgG1 < 8.0 g/L). These results suggest that preparturient vaccination of dairy cows can induce modest increases in passive antibody titers to antigens of Pasteurella haemolytica in their calves, but the antigen of interest and the population being studied can affect the outcome.     

Protective efficacy of a live attenuated Mycoplasma hyopneumoniae vaccine with an ISCOM-matrix adjuvant in pigs

An attenuated Mycoplasma hyopneumoniae vaccine that requires intrathoracic administration is commercially available for use against mycoplasmal pneumonia in China. Given the limitations of such a route of administration, this study was undertaken to assess the capacity of an ISCOM-matrix adjuvant to enhance immunogenicity following intramuscular use. Immune responses in pigs following vaccination and subsequent intra-tracheal bacterial inoculation were examined using lymphocyte proliferation, serology and mucosal IgA in both nasal and saliva swabs.Vaccination induced clear lymphocyte proliferation, but only slight serum antibody responses although these were significantly increased following experimental infection. Mucosal IgA was not detected in either nasal or salivary secretions. Following bacterial challenge, animals vaccinated with the adjuvant-containing live vaccine exhibited less severe pulmonary lesions (median score 3.67) than unvaccinated pigs (median score 13.58). The degree of ciliary loss on the respiratory tract surface was reduced in vaccinated pigs compared with experimentally infected controls. The findings indicated that the adjuvant vaccine administered IM provided protection against experimentally induced mycoplasmal pneumonia and could have commercial potential.     

Study of immune function in inbred miniature pigs vaccinated and challenged with suid herpesvirus 1.

Specific immune responses of inbred miniature pigs following vaccination and challenge with suid herpesvirus 1 (SHV-1) were determined. Vaccination of swine with SHV-1 elicited both specific neutralizing antibody and lymphoproliferative responses. Moreover, pigs vaccinated with SHV-1 were fully protected against a lethal virus challenge. Pigs vaccinated with a recombinant (r) SHV-1 virus, followed by challenge with a virulent SHV-1, had lower percentages of circulating T- and B-lymphocytes, and showed a significant (P < or = 0.05) reduction in peripheral blood mononuclear cell (PBMC) antibody-dependent cell-cytotoxicity than control (noninfected, SHV-1 sero-negative) animals. From the 5th through the 8th week of postchallenge, rSHV-1 was isolated from 2 of 4 pigs. Presence of r-virus was indicative that PBMC were infectious in vivo. The rSHV-1, with beta-galactosidase activity, was only recovered from ConA- and IL-2-stimulated primary PBMC cocultivated with porcine kidney cells. Control pigs exposed to challenge SHV-1 elicited both specific neutralizing antibody and lympho-proliferative responses followed by subsequent infection. These infected pigs, compared to control pigs, had significantly (P < or = 0.05) lowered percentages of T- and B-lymphocytes, lowered T-cell mitogenic responses, variable PBMC counts, and lowered blood phagocytic cell function. When PBMC from control pigs were cultured and infected with SHV-1, the virus caused a significant (P < or = 0.05) suppression of T-cell proliferation and PBMC mitochondrial dehydrogenase and macrophage activities.     

Evaluation of antibody response and nonspecific lymphocyte blastogenesis following inoculation of a live attenuated bluetongue virus vaccine in goats

OBJECTIVE: To evaluate vaccine safety, antibody response, and nonspecific lymphocyte blastogenesis following inoculation of a commercial monovalent live attenuated bluetongue virus (BTV) serotype 2 vaccine in goats. ANIMALS: 12 nonpregnant and nonlactating Saanen goats. PROCEDURE: 6 goats were inoculated with the monovalent live attenuated BTV serotype 2 vaccine, which has been widely used in Italy during the proceding 2 years. The other 6 goats were unvaccinated and represented negative controls. Nonspecific lymphocyte blastogenesis was evaluated 14 and 7 days before and 7, 21, and 49 days after vaccination by measuring DNA synthesis in peripheral blood mononuclear cells (PBMCs) stimulated with phytohemagglutinin, concanavalin-A, and pokeweed mitogen. On the same days as lymphocyte blastogenesis, blood samples were taken to determine serum concentrations of anti-BTV antibodies. RESULTS: During the 7 weeks following vaccination, PBMCs obtained from vaccinated goats had a significantly decreased response to mitogens in terms of DNA synthesis, compared with PBMCs from the same goats before vaccination. Conversely during the experiment, no significant change was found in the response of the PBMCs obtained from unvaccinated goats. Starting from 21 days after vaccination, serum from vaccinated goats had anti-BTV antibodies. No anti-BTV antibodies were detected in the serum from unvaccinated goats. CONCLUSIONS AND CLINICAL RELEVANCE: Inoculation of goats with the monovalent live attenuated BTV serotype 2 vaccine described herein resulted in a profound depression of nonspecific lymphocyte blastogenesis, which might compromise the resistance of vaccinated goats to pathogens.     

Resistance to fecal shedding of salmonellae in pigs and chickens vaccinated with an aromatic-dependent mutant of Salmonella typhimurium.

The purpose of this study was to evaluate the effectiveness of an aromatic-dependent mutant of Salmonella typhimurium as a parenteral vaccine for prevention of fecal shedding of Salmonella spp. Pigs and chickens were vaccinated IM, with 1 x 10(9) and 1 x 10(8) organisms, respectively, followed by a second identical vaccination 2 weeks later. Salmonella organisms were not detected by analysis of fecal or cloacal swab specimens from any animal after vaccination. Deleterious side effects were not noticed after vaccination. Pigs were challenge-inoculated PO with 1 x 10(12) virulent S typhimurium 1 week after the second vaccination. Chickens were challenge-inoculated PO with 3 x 10(8) organisms of either S enteritidis or the virulent parent strain of S typhimurium 3 weeks after the second vaccination. Vaccinated pigs shed Salmonella spp significantly less frequently than did nonvaccinated pigs. Vaccinated chickens challenge-inoculated with either S enteritidis or S typhimurium also shed Salmonella less frequently than the corresponding nonvaccinated control birds; however, the difference was not significant.     

Interferon induction in swine lymphocyte antigen-defined miniature pigs

Interferon was induced in two groups of swine lymphocyte antigen (SLA)-defined miniature pigs with polyinosinic: polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose. The group 1 pigs were low antibody-response phenotypes (SLA^a/a, SLA^a/c, SLA^c/c), and the group 2 pigs were high antibody-response phenotypes (SLA^d/d, SLA^d/g, SLA^g/g). Six hours after induction the antiviral titres were not influenced by the SLA group, but higher titres were observed in females. Higher antiviral titres were found in group 2 pigs before treatment and 24 hours after treatment, and higher titres were found in female pigs. The antiviral titres before and after treatment were also influenced by the sire. Group 2 pigs had lower total leucocyte counts before treatment, and there was a significant reduction in leucocyte numbers in both groups six hours after induction, due mainly to a large reduction in lymphocyte counts.     

A cross-protection experiment in pigs vaccinated with Haemophilus parasuis serovars 2 and 5 bacterins, and evaluation of a bivalent vaccine under laboratory and field conditions.

Cross-protection between Haemophilus parasuis serovars 2 and 5 was examined in pigs using a bacterin based vaccine, and subsequently the safety and efficacy of a bivalent vaccine were evaluated. Upon intratracheal challenge of a serovar 2 or 5 strain, pigs immunized with a monovalent vaccine were protected against challenge with a homologous serovar strain, but not with a heterologous serovar strain. Immunization with a bivalent vaccine containing both serovars 2 and 5 bacterins conferred protection in pigs against lethal challenge with each of the serovar strains. A total of 86 pigs from two SPF herds were injected with the bivalent vaccine intramuscularly twice at a four-week interval. No adverse reactions following the vaccination were observed. On day 7 after the second vaccination, vaccinated and non-vaccinated control pigs from herd A were transferred to herd B, where Glasser's disease had broken out. Pigs in the control group developed clinical signs of the disease, and 6 of 8 (75%) pigs died until slaughter, in contrast with only 4 of 46 (9%) pigs in the vaccinated group. In herd C, where there was no outbreak of Glasser's disease, complement fixation antibody titer was raised only in the vaccinated group. A challenge experiment on days 20 and 79 after the second vaccination showed that only the vaccinated pigs were protected. From these findings, the safety and efficacy of the bivalent vaccine were confirmed under laboratory and field conditions.     

Weaning pigs at an early age decreases cellular immunity

An experiment involving 118 pigs was conducted to evaluate the influence of weaning pigs at four different ages on in vivo and in vitro cell-mediated immunity. One-half of each litter was weaned at 2, 3, 4 or 5 wk of age; the other one-half remained with the sow as nonweaned controls. Phytohemagglutinin skin-test responses were determined on all pigs. Blastogenic responses of mitogen-stimulated lymphocyte cultures were determined before and after weaning. The intradermal response to phytohemagglutinin was reduced (P less than .001) in pigs weaned when 2 or 3 wk old and was suppressed (P less than .05) in those weaned when 4 wk old. In vivo cellular immunity was not altered by weaning in 5-wk-old pigs. The capability of lymphocytes to undergo blastogenesis in response to phytohemagglutinin was decreased in pigs weaned at 2 and 3 wk of age (P less than .001 and P less than .01, respectively). Pokeweed mitogen-stimulated blastogenesis was lower (P less than .01) in pigs weaned at 2 wk of age. Mitogen-stimulated lymphocyte blastogenesis was similar (P greater than .10) in control pigs and those weaned when 5 wk old. These data suggested that weaning pigs when younger than 5 wk old causes physiological changes detrimental to cellular immune reactivity. Those changes could alter disease susceptibility in young pigs.     

The Immune Response to Microsporum canis Induced by a Fungal Cell Wall Vaccine*

Abstract— An experimental infection model was used to assess induction of specific immunity against Microsporum canis in cats with an M. canis cell wall vaccine preparation. Kittens 8–9 weeks old (n= 12) received five doses of either vaccine or placebo at biweekly intervals. Specific immunity was monitored via plasma anti-dermatophyte antibody titers and lymphocyte blastogenesis (LB) to dermatophyte antigens. After vaccination, cats were challenged with viable M. canis spores, and lesion development was monitored. Vaccinated cats developed higher anti-dermatophyte IgG, but not IgM, titers than controls, beginning after the second dose of vaccine (P < 0.001). During the vaccination period, specific cellular immunity as measured by LB was absent in control cats, but developed to a limited degree in vaccinated cats (P < 0.05). After challenge with 10⁵ fungal spores per cat, both control and vaccinated cats developed active infections. The vaccine appeared to induce an antibody titer quantitatively similar to that produced by infection, but less measured cellular immunity than was seen with infection and recovery. These results suggest that induction of high titers of serum IgG or IgM antibody against Microsporum canis is not protective against challenge exposure.     

Q fever (Coxiella burnetii) investigations in dairy cattle: persistence of antibodies after vaccination.

The immune response and persistence of antibodies were investigated in dairy cattle vaccinated with formalin-inactivated phase (ph) I Coxiella burnetii vaccine agglutinating antibody geometric mean titer (GMT) of 193.2 at 1 month after vaccination compared to a GMT of 2.0 for nonvaccinated calves. The agglutinating antibodies gradually decreased in vaccinated cattle, but the GMT remained approximately 4 times higher than that for the nonvaccinated group for at least 20 months. Results of serotests at 2 months after revaccination indicated a rapid increase in the GMT to 177.0 with agglutinating titers between 1:64 and 1:512.     

Cross-protection studies between feline infectious peritonitis and porcine transmissible gastroenteritis viruses

Cross-protection studies between the feline infectious peritonitis (FIP) and the porcine transmissible gastroenteritis (TGE) viruses were conducted in cats, pigs and pregnant gilts. Cats vaccinated with TGE virus developed neutralizing antibodies against TGE virus and low titer antibody against FIP virus detected by an indirect fluorescent antibody technique but were not protected against a virulent FIP virus challenge. Baby pigs and pregnant gilts vaccinated with FIP virus did not develop detectable antibodies to TGE virus. Nevertheless, it appeared that vaccination of swine with FIP virus conferred some immunity against TGE virus infection. Seventeen-day-old pigs vaccinated with two doses of FIP virus had a 67% survival rate following a virulent TGE virus challenge, and 75% of the 3-day-old pigs suckling either FIP or TGE-virus-vaccinated gilts survived virulent TGE virus infection in contrast to 0% survival of baby pigs suckling unvaccinated gilts.     

Development and duration of protection against salmonellosis in mice and sheep immunised with live aromatic-dependent Salmonella typhimurium.

The aim of this investigation was to determine the development and duration of protection in mice or sheep immunised with aromatic-dependent (aro-) Salmonella typhimurium strain CS332, by either parenteral or oral routes. Immunisation of mice by the intraperitoneal or sheep by the intramuscular routes was found to impart protection against oral challenge with the virulent parent S typhimurium strain CS94 as early as seven days after immunisation. In contrast, when immunisation was carried out by the oral route, protection was not evident until three weeks after immunisation. Regardless of the route of immunisation, mice were still partially protected at three months and were fully susceptible at six months after immunisation. In sheep, protection persisted for six months but not 12 months after immunisation. Only parenterally immunised mice and sheep developed high ELISA and, or, agglutinating antibody titres, and cutaneous delayed-type hypersensitivity (DTH) at three weeks after immunisation. Although both antibody and DTH were detectable three months after immunisation of mice with aro- S typhimurium strain CS332, none was detected at six months. Antibody measured by agglutination and ELISA was detectable six months after immunisation in sheep, although no DTH was evident. At 12 months after immunisation low levels of anti-LPS antibody (measurable by ELISA only) were detected in sheep immunised by the intramuscular route.     

Changes in blastogenic responses of lymphocytes and delayed type hypersensitivity responses after vaccination in dogs.

To clarify the immunologic effects of vaccination in dogs, we monitored total leukocyte and lymphocyte counts, humoral antibody responses, blastogenic responses of lymphocyte, and delayed type hypersensitivity (DTH) responses after vaccination. Mixed vaccines were administered on day 0 except for canine parvovirus (CPV) vaccine which was readministered on day 21. The puppy and adult dogs had a significant decrease in leukocyte and lymphocyte counts on day 7. The puppies showed a significant increase in the blastogenesis of lymphocytes after each vaccination, whereas the adult dogs had no significant changes. However, the adult dogs were divided into two groups, high responders and low responders in blastogenesis of lymphocytes. The dogs with higher or lower response in SI values on day 0 tended to show decrease or increase after the first vaccination, respectively. Since almost all dogs developed high titers of humoral antibody, it is considered that vaccination acts in an immunomodulative fashion. DTH responses to phytohemagglutinin (PHA) and CPV vaccine monitored at 0, 3, and 8 weeks after the first vaccination produced strong reactions, in particular those to CPV vaccine rose significantly after vaccination and maintained the higher responses for at least 2 months. These results suggest that DTH responses to PHA and CPV vaccine are helpful to monitoring non-specific and specific immune functions in vivo, therefore, DTH could be used as simple and rapid immunologic tests in canine practice.