Canine Macrophage Research Articles

Cytotoxic effect of jasmonate and methyl jasmonate on a canine macrophage tumor cell line

Cancer is one of the leading causes of death in dogs, a fact that has boosted the investigation for new more specific antitumor drugs. This study evaluated the antitumor activity of jasmonates in the canine macrophage cell line DH82 (ATCC # CRL-10389) isolated from a case of malignant histiocytoma. The activities of methyl jasmonate and jasmonic acid were compared to that of doxorubicin by the MTT assay. Methyl jasmonate resulted in the highest inhibition of cell growth (82.2%), followed by doxorubicin (80.7%) and jasmonic acid (36.5%). More detailed studies regarding the action of jasmonates on animal health are necessary, but the present results indicate methyl jasmonate as an alternative to the development of drugs for the treatment of canine oncologies.

Adenosine and ATP Affect LPS-Induced Cytokine Production in Canine Macrophage Cell Line DH82 Cells

Macrophages are essential for controlling the majority of infections, and are mediators of natural immunity. During infection, lipopolysaccharide (LPS) stimulates macrophages to produce pro-inflammatory cytokines. Adenosine and ATP released into the extracellular space by immunological stimuli have been shown to regulate various immune functions. More recently, it has been shown adenosine and ATP have a critical role on the physiological negative feedback mechanism for limitation and termination of tissue-specific and systemic inflammatory responses. It was useful and meaningful to gain information about interaction between LPS, which generates the inflammation, and adenosine and ATP, which terminate the inflammation. We evaluate effects of adenosine and ATP on the production of cytokines related to inflammation in canine macrophage cell line DH82 cells. Adenosine and ATP respectively increased the production of IL-10 without affecting the production of IL-6, TNF-α and IL-12 in DH82 cells. In addition, adenosine and ATP prevented the production of LPS-induced IL-6, TNF-α and IL-12 in DH82 cells. In contrast, adenosine and ATP potentiated LPS-induced IL-10 production in DH82 cells. Moreover, adenosine, but not ATP inhibited LPS-induced expression of TLR4 in DH82 cells. These results suggest that conditions related to increased adenosine and/or ATP may play an important role in the inflammatory reactions.

Endothelin-1 production by the canine macrophage cell line DH82: Enhanced production in response to microbial challenge

Endothelin-1 (ET-1) is a potent vasoconstrictive peptide which plays an important role in regulating mammalian cardiovascular development and homeostasis. Originally identified as a factor released by vascular endothelial cells, ET-1 is now recognized as a product of numerous cells and tissues with demonstrated involvement in an array of physiological and pathological processes. An area of great interest is the production of ET-1 by mononuclear cells (monocytes and macrophages) and its role in inflammation. We report that the canine macrophage cell line, DH82, constitutively secretes both ET-1 and its biologically inactive precursor big ET-1. The production of both peptides was increased following stimulation with lipopolysaccharide (endotoxin) from gram-negative bacteria. ET-1 production was also increased in response to stimulation with intact and viable gram-positive and gram-negative bacteria. In addition to producing ET-1, DH82 cells express transcripts encoding two receptors for the ET-1 peptide (ET A and ET B receptors) and an enzyme involved in the conversion of big ET-1 to ET-1. The constitutive secretion of ET-1 and the expression of ET A and ET B receptors may be related to the malignant origin of this cell line. Our results are the first report of ET-1 production by a canine cell line and provide the basis for further investigation into the role of ET-1 during infection and inflammation.

Priming Effects of Lipopolysaccharide and Inflammatory Cytokines on Canine Granulocytes

Granulocytes play a pivotal role in natural immunity. Under inflammatory conditions, granulocytes are universally primed by several agents, such as endotoxins and inflammatory cytokines. Primed granulocytes exert potent adhesiveness, chemotaxis, phagocytosis and reactive oxygen species (ROS) production, effectively eliminating invading agents. Reactivity against priming agents is known to vary with species; however, there have been few reports on the effects of priming agents on canine granulocytes. In the present study, we assayed the priming effects of lipopolysaccharide (LPS), recombinant canine tumor necrosis factor-alpha (rcTNF-alpha) and recombinant canine granulocyte macrophage colony-stimulating factor (rcGM-CSF) on canine granulocyte function in vitro. Isolated recombinant canine were primed with various concentrations of LPS, rcTNF-alpha and rcGM-CSF, and CD11b expression was assayed. Furthermore, actin polymerization, phagocytosis and ROS production were then assayed at primer concentrations where enhancement of CD11b expression was observed. LPS did not enhance canine granulocyte function. Phagocytosis and actin polymerization were not enhanced by priming agents; however, rcTNF-alpha and rcGM-CSF enhanced CD11b expression and ROS production in canine granulocytes. These results suggest that priming effects are mainly reflected in CD11b expression and ROS production, with rcGM-CSF and rcTNF-alpha having a priming effect similar to that observed in humans.

Effects of Lidocaine Hydrochloride on Canine Granulocytes, Granulocyte CD11b Expression and Reactive Oxygen Species Production

Lidocaine hydrochloride (Lido) is widely used for analgesia in veterinary medicine; however, in humans, it has been suggested that Lido attenuates granulocyte functions, such as adhesion and reactive oxygen species (ROS) production. Thus, Lido may affect canine granulocyte function; however, there have been no reports on the effects of Lido on canine granulocyte function. Thus, we studied the effects of Lido on canine granulocyte CD11b expression and ROS production. We further studied the effects of Lido on the priming of canine granulocyte CD11b expression and ROS production by recombinant canine granulocyte macrophage colony stimulating factor (rcGM-CSF). Isolated granulocytes were incubated with 3, 30 or 300 microg/ml Lido, or with Lido followed by priming with 5 ng/ml rcGM-CSF. CD11b was detected by the immune fluorescent antibody method, and the mean fluorescence intensity (MFI) was assayed by flow cytometry. ROS production was assessed by the peak time (PT) of ROS production and area under the luminol reaction curve (AUC), which represents total ROS production quantity against opsonized zymosan stimuli. Only 300 microg/ml Lido (tissue level observed by regional block) significantly attenuated both the MFI of CD11b and its enhancement by rcGM-CSF. Moreover, at this concentration, the AUC and its enhancement by rcGM-CSF were significantly attenuated by Lido; in contrast, Lido did not affect PT. In conclusion, Lido suppressed granulocyte adhesion to the endothelium and antiseptic capability by suppressing CD11b expression and/or ROS production. Particular care should thus be exercised when performing regional anesthesia block using Lido.

Effect of Recombinant Lactobacillus Expressing Canine GM-CSF on Immune Function in Dogs

Many Lactobacillus strains have been promoted as good probiotics for the prevention and treatment of diseases. We engineered recombinant Lactobacillus casei, producing biologically active canine granulocyte macrophage colony stimulating factor (cGM-CSF), and investigated its possibility as a good probiotic agent for dogs. Expression of the cGM-CSF protein in the recombinant Lactobacillus was confirmed by SDS-PAGE and Western blotting methods. For the in vivo study, 18 Beagle puppies of 7 weeks of age were divided into three groups; the control group was fed only on a regular diet and the two treatment groups were fed on a diet supplemented with either 1 x 10(9) colony forming units (CFU)/day of L. casei or L. casei expressing cGM-CSF protein for 7 weeks. Body weight was measured, and fecal and blood samples were collected from the dogs during the experiment for the measurement of hematology, fecal immunoglobulin (Ig)A and IgG, circulating IgA and IgG, and canine corona virus (CCV)-specific IgG. There were no differences in body weights among the groups, but monocyte counts in hematology and serum IgA were higher in the group receiving L. casei expressing cGMCSF than in the other two groups. After the administration of CCV vaccine, CCV-specific IgG in serum increased more in the group supplemented with L. casei expressing cGM-CSF than the other two groups. This study shows that a dietary L. casei expressing cGM-CSF enhances specific immune functions at both the mucosal and systemic levels in puppies.

In vitro binding and survival assays of Leishmania parasites to peripherical blood monocytes and monocyte-derived macrophages isolated from dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi

BackgroundThere are a few works considering the characterization of canine monocyte-derived macrophages as well as a standardized procedure for isolation, culture, and infection of these cells with Leishmania. We have performed several modifications in order to improve the canine monocyte-derived macrophage cultures. In addition, we have done a comparative study between monocytes and monocyte-derived macrophages from dogs naturally and experimentally infected with L. chagasi.ResultsIn the presence of exogenous serum, opsonized Leishmania promastigotes binds better to monocytes/macrophages than without serum. Otherwise, this binding occurs due to the strict correlation between the opsonized biologic particles with the third receptor of the complement (CR3-CD11b/CD18). In fact, our assays with CD11b confirmed the importance of this receptor for canine cells and the L. chagasi experimental system. Moreover, monocytes obtained from naturally infected dogs have shown a higher number of monocytes bounded to promastigotes. The experimental results regarding survival have shown that promastigote forms of opsonized L. chagasi were more infective, because we found higher numbers of promastigotes bound to the different cells. As a consequence, after forty-eight hours of binding, higher numbers of amastigotes appeared inside monocyte-macrophages.ConclusionThese studies have given support to continue comparative studies involving canine monocytes, monocyte-derived macrophages and peritoneal macrophages. Since we have standardized the canine cell culture, we are looking forward to determining the phenotypic properties of these cells before and after L. chagasi infection using flow cytometry.

Effect of dehydroepiandrosterone on lipopolysaccharide-induced interleukin-6 production in DH82 cultured canine macrophage cells

Objective This study examined the effect of dehydroepiandrosterone (DHEA) on lipopolysaccharide (LPS)-stimulated interleukin-6 (IL-6) production in the DH82 canine macrophage cell line. Study design Cultured DH82 cells were stimulated with varying concentrations of LPS with or without DHEA for various times. Supernatant IL-6 levels were measured by enzyme-linked immunosorbent assay, and cellular cytoplasmic IκBα protein expression measured by Western blot analysis. Results LPS dose-dependently stimulated IL-6 production ( p = 0.016). Cells stimulated with 20 μg LPS showed a time-dependent increase of IL-6 concentration up to 10 h post-treatment ( p = 0.007). Co-treatment of DH82 cells with 20 μg LPS and various concentrations of DHEA for 14 h showed that up to 10 μM DHEA dose-dependently decreased the IL-6 concentration ( p = 0.007). Also, addition of 20 μM DHEA to DH82 cells with 20 μg LPS time-dependently decreased the IL-6 concentration for up to 14 h post-treatment ( p = 0.018). Stimulation of cultured DH82 cells with 20μg LPS significantly decreased cellular cytoplasmic IκBα expression, beginning at 30 min post-treatment and persisting to at least 2 h post-treatment ( p = 0.012). However, co-treatment of cells with 20 μg LPS and 20 μM DHEA abrogated this effect until 2 h post-treatment. Conclusions DHEA decreased the IL-6 concentration in the supernatant of LPS-stimulated DH82 cells by inhibiting the sequestration of IκBα, which is necessary for the activation of nuclear factor-kappa B. These findings provide new insights into the immunomodulatory effects of DHEA.

Intravenous Infusion of Liposomal Clodronate Blocks Clearance of Opsonized Erythrocytes in Dogs.

Immune-Mediated Hemolytic Anemia (IMHA) is a spontaneously occurring disease of man and dogs in which immunoglobulin-coated erythrocytes are destroyed in the spleen. Immunosuppressive therapies and splenectomy have been previously used to combat premature destruction of erythrocytes. Liposomal clodronate has been previously shown to be a potent anti-macrophage agent when administered intravenously in a murine model. Splenic macrophages were selectively depleted through the induction of apoptosis following phagocytosis of liposomes containing clodronate. We hypothesized that such an approach might be useful in blocking clearance of erythrocytes in dogs with spontaneous IMHA.Clodronate (dichloromethyl bisphosphonate) was formulated in liposomes (phosphatidyl choline, cholesterol and mannose), according to a previously published technique. The liposomal clodronate was evaluated in vitro for activity using canine and murine histiocytic cell lines, as assessed by cell killing and blocking of erythrophagia. Cell killing was monitored using microscopic evaluation of cell change, MTT and by propidium iodide staining. Blocking of erythophagia was monitored using flow cytometry. In vivo in normal dogs, increasing doses of liposomal clodronate were administered intravenously and evaluated to determine efficacy in blocking erythrocyte clearance and toxicity. Twenty-four hours post infusion, dye-labeled erythrocytes were opsonized using rabbit anti-canine erythrocyte antibody. Erythrocytes clearance was monitored using flow cytometry. The safe and effective dose of liposomal clodronate was determined to be 0.5 ml/kg administered IV via CRI over a 90 minute time period. After administration of intravenous liposomal clodronate in normal dogs, clearance of opsonized erythrocytes was nearly completely blocked at the 0.5 ml/kg dose. In vitro assays indicated that liposomal clodronate induced death of canine macrophage and dendritic cell lines. Five dogs with spontaneous IMHA were treated once with liposomal clodronate at the 0.5 ml/kg dose. Though erythrocyte clearance was not completely blocked, the drug was well-tolerated and all 5 dogs survived to leave the hospital. These studies suggest that liposomal clodronate may be an effective agent for temporary suppression of erythrocyte destruction in IMHA. Additional studies, including evaluation of higher doses of liposomal clodronate, are warranted in dogs with spontaneous IMHA.

Infection of a canine macrophage cell line with Leishmaniainfantum: determination of nitric oxide production and anti-leishmanial activity

We have previously shown that resistance to Leishmania infantum in dogs is associated with a Th1 type of immune response. In this study, we use a canine macrophage cell line (030-D) that can readily be infected with this protozoan parasite. Our aim is to further characterize the effector mechanisms involved in killing of Leishmania parasite in dogs. We observed that activation of 030-D cells by incubation with a supernatant derived from a Leishmania-specific T cell line containing IFN-γ, TNF-α and interleukin-2 (IL-2) resulted in enhanced nitric oxide (NO) production by these cells. In addition, we observed enhanced anti-leishmanial activity of infected 030-cells after activation. Both, NO production and anti-leishmanial activity were abrogated by addition of L-N G-nitroargininemethyl ester ( L-NAME), an analogue of L-arginine. Thus, NO play an important role in the anti-leishmanial activity of these canine macrophages. We propose the infection of the 030-D cell line as a good in vitro model to further investigate parasite-host cell interactions in dogs, a natural host of Leishmania parasites.

Attempted transmission of Ehrlichia chaffeensis among white-tailed deer by Amblyomma maculatum.

A deer was needle-exposed intravenously to Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) in canine macrophage (DH82) cells and 7 days later was infested with laboratory-reared Amblyomma maculatum (Koch) (Acari:Ixodidae) nymphs for acquisition feeding. After molting, the adult ticks were allowed to feed on a naive deer. The organism was reisolated from the needle-exposed deer by cell culture and E. chaffeensis DNA was detected in the deer's blood by PCR. Similar isolation/recovery techniques were used for the tick-exposed deer and no evidence of infection was found. Although these findings must be considered as preliminary owing to inadequate controls, the data suggest that A. maculatum is probably not a suitable vector for E. chaffeensis.

In vitro and in vivo enhancement of canine pulmonary alveolar macrophage cytotoxic activity against canine osteosarcoma cells.

The combination of chemotherapy with immunotherapy may offer an advantage over either therapy alone and provide a greater potential for total tumor eradication. Monocyte/macrophage-mediated tumor cell killing is a major mechanism of the host's defense against primary and/or metastatic neoplasia. We evaluated the tumoricidal activity against canine osteosarcoma cells of canine pulmonary alveolar macrophages (PAM) exposed in vitro to two recombinant canine (rc) cytokines (rcTNF alpha and rcIFN gamma). We also evaluated the in vivo tumoricidal activity of PAM from dogs treated with the macrophage activator, liposome-encapsulated muramyl tripeptide-phosphatidyl-ethanolamine (L-MTP-PE) alone or in combination with doxorubicin (DOX). This study demonstrated that rcTNF alpha and rcIFN gamma significantly enhance in vitro canine PAM cytotoxicity against canine osteosarcoma cells, and that PAM from dogs treated with DOX + L-MTP-PE have enhanced cytotoxic activity against osteosarcoma cells when compared to dogs treated with DOX or L-MTP-PE alone. These findings support the rationale for combining a chemotherapy agent with an immunotherapy agent for the treatment of metastatic disease, and suggest a role for TNF alpha and IFN gamma as agents for stimulating the antitumor activity of macrophages.

Up-regulation of inducible nitric oxide synthase mRNA in dogs experimentally infected with Borrelia burgdorferi

The up-regulation of the inducible nitric oxide synthase (iNOS) mRNA was determined by RT-PCR in 25 tissues each from 22 specific pathogen-free (SPF) dogs experimentally infected with Borrelia burgdorferi by tick exposure and from five uninfected control dogs. Using primers specific for a homologous region of the human and canine iNOS sequence, and canine macrophage mRNA, we isolated and partially sequenced canine iNOS. A sequence of 1775 bases was obtained and primers specific for canine iNOS mRNA constructed to investigate the expression of iNOS in dog tissues in response to infection with B. burgdorferi. In 12 out of 22 dogs infected with B. burgdorferi, acute lameness occurred within 55–82 days after infection whereas the other 10 dogs showed no or only mild clinical signs despite persistent infection up to Day 175. The numbers of iNOS mRNA-positive tissues in dogs with acute lameness were significantly higher than in dogs without lameness, while uninfected dogs showed only negligible iNOS expression. Dogs with acute lameness also had higher numbers of borrelia-positive tissues as well as higher scores in histopathological evaluations than infected dogs without lameness. Our results show that the expression of iNOS mRNA is related to the number of B. burgdorferi-positive tissues and the severity of inflammation as assessed by histopathology. These results implicate an up-regulation of the iNOS mRNA as part of the host's immune response to borrelia infection and a possible role for NO in the pathogenesis of canine Lyme arthritis.

Experimental transmission of Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) among white-tailed deer by Amblyomma americanum (Acari: Ixodidae).

Ehrlichia chaffeensis Anderson, Dawson & Wilson, causative agent of human (predominantly monocytic) ehrlichiosis, was successfully transmitted experimentally by Amblyomma americanum (L.) to white-tailed deer, Odocoileus virginianus (Zimmerman). Deer were needle-exposed intravenously to E. chaffeensis in tissue-culture canine macrophage (DH82) cells, and 11 d later were exposed to laboratory-reared A. americanum larvae, nymphs, and adults for acquisition feeding. Three months after this feeding, naive deer and dogs were exposed to recently molted nymphs and adults. Attempted reisolation of the pathogen by way of tissue culture was successful from one needle-exposed deer but not from the tick-exposed deer or dogs. Based on serologic evidence and polymerase chain reaction data, both nymphal and adult ticks transmitted E. chaffeensis to naive deer but not to dogs.

Immunophenotypic and Ultrastructural Evidence of the Langerhans Cell Origin of the Canine Cutaneous Histiocytoma

Canine cutaneous histiocytoma (CCH), a histiocytic benign, dermal, self-healing tumor in the young dog, and epidermal Langerhans cells (LC) are thought to be related. In this study, we used immunohistochemical staining and transmission electron microscopy for 5 fresh CCH and 17 fixed tumors, to examine if, on the basis of their immunophenotype and their ultrastructural morphology, these tumor cells originate as LCs. The immunophenotype of CCH: canine CD11a, 11c, 18, 45, MHC II positive and ACM1, human CD14 negative, was different from canine macrophage immunophenotype but very similar to the canine LC phenotype. Furthermore, we have described ultrastructural markers in CCH cells for the first time: these consist of coated vesicles, regularly laminated bodies, pleiomorphic inclusions, paracrystalline structures, and deep invaginations of the plasma membrane, usually observed in congenital self-healing histiocytosis, a human LC tumor, or occasionally observed in human LC. The occurrence of such immunophenotype and ultrastructural markers confirmed the common lineage of LCs and CCH cells.

Isolation and continuous culture of Neorickettsia helminthoeca in a macrophage cell line

Experimental evidence is presented supporting the development of a system for the isolation and propagation of a Neorickettsia sp. in a continuous canine macrophage cell line (DH82). To isolate a Neorickettsia sp. pathogenic to the canine species, three naive dogs were fed metacercaria-encysted kidneys of salmon caught in a river where infection of metacercariae with Neorickettsia helminthoeca has been circumstantially known for decades. Clinically, the classic course of salmon poisoning disease developed in all of the dogs. Parasitemia began on day 8 to 11 postinfection, when the dogs developed a febrile peak, and continued until euthanasia. At necropsy, characteristic gross and microscopic lesions of the disease were present. A Neorickettsia sp. was also isolated from liver and spleen samples of these animals. The isolates have been continuously propagated and passed in DH82 cells for more than 6 months. Electron microscopic examination confirmed that the rickettsial organisms multiplied in the membrane-bound compartment of DH82 cells and that they morphologically closely resembled rickettsia belonging to the genus Ehrlichia. An indirect fluorescent antibody test using Neorickettsia organisms cultured in DH82 cells showed that all dogs seroconverted 13 to 15 days postinfection. Finally, inoculation of the cell-cultured Neorickettsia organisms into a naive dog reproduced clinically typical salmon poisoning disease which was of greater severity and had a more rapid time course than that in the dogs from which the original isolation was made. On the basis of the clinical and pathologic responses of the dogs in our study, we believe that virulent N. helminthoeca was isolated and cultured in a continuous cell line.

Serologic Diagnosis of Human Ehrlichiosis Using Two Ehrlichia canis Isolates

Ehrlichia canis or a closely related rickettsial organism has been implicated serologically and morphologically as the causative agent of human ehrlichiosis in the United States. Although E. canis has been serially propagated in primary canine monocytes, only a limited quantity of antigen is obtained by this method. A continuous canine macrophage cell line, DH82, supports the growth of a new isolate of E. canis established from the whole blood of a carrier dog in Oklahoma. Serologic comparison of the Oklahoma isolate in the continuous canine cell line with a Florida isolate in commercial antigen slides revealed 100% specificity and 87.5% sensitivity.

The effect of beryllium compound solubility on in vitro canine alveolar macrophage cytotoxicity

Pulmonary alveolar macrophage cells (PAM), obtained by bronchopulmonary lavage of Beagle dogs, were exposed in vitro to beryllium oxide (BeO) particles calcined at either 500 or 1000°C or to beryllium sulfate (BeSO 4). Cell viability was determined by trypan blue dye exclusion after 20 h in culture. The most toxic material tested was BeSO 4, followed by BeO calcined at 500°C, then BeO calcined at 1000°C. An in vitro dissolution technique was used to measure the relative solubility of the BeO particles. The BeO prepared at 500°C exhibited greater solubility compared with BeO prepared at 1000°C. This study ex- tends previous work by examining the effects of beryllium compounds on canine PAM, and by relating PAM cytotoxicity with measured values of beryllium compound solubility.

Heterogeneity in immunologic functions among canine alveolar macrophage subfractions.

Canine alveolar macrophages (AM) were examined for the presence of subpopulations with differnt immunologic functions. Lavaged bronchoalveolar cells (BAC) from normal beagle dogs were separated into 3 density subfractions by centrifugation through discontinuous gradients of Percoll solution. After incubated on plastic plates, each of adherent BAC subfractions recovered was identified as AM by morphologic criteria and phagocytic capacity of latex beads. These adherent BAC subfractions were then incubated with lipopolysaccharides (LPS) and/or silica for testing their capacity to produce interleukin 1 (IL-1). Il-1 activity of culture supernatants was greater both in the lowest and intermediate density subfractions, but much lower in the highest density subfraction. When each of adherent BAC subfractions was incubated with autologous nylon wool-passed lymphocytes from tracheobronchial lymph nodes in the presence of a lectin mitogen, phytohemagglutinin (PHA), proliferative responses of lymphocytes were prominently enhanced by increasing cell numbers of adherent BAC subfractions. On the contrary, another mitogen, concanavalin A (Con A)-induced lymphocyte responses were suppressed by addition of both of the lowest and intermediate density subfractions, whereas only the highest density subfraction enhanced lymphocyte response. The suppressive effect of adherent BAC subfractions on Con A-induced responses was, however, inhibited by addition of indomethacin, prostaglandin synthesis inhibitor. These results indicate the presence of functional subpopulations among canine AM with respect to IL-1 production and accessory cell, function for mitogen-induced lymphoproliferation.

Infection of canine mononuclear leucocytes by measles virus: possible mechanism of protection from canine distemper.

Measles virus was shown to be infectious to canine lymphocytes from peripheral blood as well as from different lymphoid tissues, and the same held true for canine macrophage cultures prepared from peripheral blood. The susceptibility of these leucocytes to measles virus was comparable with that of canine distemper virus. These observations supported the suggestion that interference with canine distemper virus by measles could be a possible mechanism of the heterotypic immunity observed in dogs.