A novel missense mutation responsible for factor VII deficiency in research Beagle colonies.

BACKGROUND: Canine factor VII (cFVII) deficiency, an autosomal recessive trait originally identified in research Beagles, is associated with a mild to moderate bleeding tendency. OBJECTIVE: Our aim was to identify and characterize the mutation causing cFVII deficiency. METHODS: In order to sequence the coding regions of the cFVII gene, we cloned the cFVII cDNA. Genomic DNA and plasma from FVII-deficient Beagles and obligate carriers were utilized. RESULTS: In all FVII-deficient dogs, we identified a single causative G to A missense mutation in exon 5, encoding the second epidermal growth factor-like domain, resulting in substitution of glycine 96 by glutamic acid, with plasma FVII coagulant activity of

Evaluation of colonoscopic allergen provocation as a diagnostic tool in dogs with proven food hypersensitivity reactions.

OBJECTIVE: To evaluate the colonoscopic allergen provocation (COLAP) test as a new tool for the diagnosis of IgE-mediated food allergy. METHODS: Oral food challenges as well as COLAP testing were performed in a colony of nine research dogs with proven immediate-type food allergic reactions. In addition, COLAP was performed in five healthy dogs. RESULTS: When compared with the oral challenge test, COLAP accurately determined 18 of 23 (73 per cent) positive oral challenge reactions (73 per cent) in dogs with food allergies and was negative in the healthy dogs. CLINICAL SIGNIFICANCE: The accuracy of this new test may be higher than that for gastric sensitivity testing. Therefore, COLAP holds promise as a new test to confirm the diagnosis of suspect IgE-mediated food allergy in dogs.

Importance of early allergen contact for the development of a sustained immunoglobulin E response in a dog model.

BACKGROUND: Immunoglobulin E (IgE)-mediated allergies are postulated to require early allergen contact and sensitization for the full development of sustained IgE levels. METHODS: Thirty-two Beagle dogs from seven litters selectively bred for their high IgE response were sensitized by subcutaneous injection of chicken ovalbumin (OVA), peanut extract and recombinant birch pollen allergen (Bet v 1). In half of the dogs from each litter, sensitization injections were started on the first day of life; the other half of the same litter was first sensitized at the age of 4 months. To evaluate whether early sensitization also predisposes the animals to IgE responses to other allergens later in life, we injected a recombinant timothy grass pollen allergen (Phl p 5) later on, at the age of 10-12 months. Allergen-specific serum IgE and IgG levels were evaluated with enzyme-linked immunosorbent assays. In addition, 21 dogs were challenged with aerosolized OVA to measure bronchoconstrictive changes in lung function. RESULTS: Early sensitized dogs developed significantly higher OVA-specific serum IgE levels than late sensitized dogs, in contrast to the IgG levels, which were lower in these dogs (p < 0.001). The increase in specific serum IgE and IgG following boosting remained different between the two groups for over a year. Titers of specific serum IgE and IgG were also different after sensitization with a new allergen injected later in life for the first time. Dynamic pulmonary compliance and resistance, both parameters for bronchoconstriction induced by OVA aerosol challenge, were also significantly higher in early sensitized dogs (for both parameters, p < 0.01). CONCLUSIONS: Contact with an allergen early in life is decisive for the development of sustained IgE levels and the development of IgE responses to additional allergens encountered later in life. Allergen avoidance during early life may have some preventive effect on IgE-mediated allergy in dogs.

Flea bite hypersensitivity: new aspects on the involvement of mast cells.

A study was performed to test the effect of sensitization to flea antigen, followed by exposure to fleas on mast cells (MCs), their subtypes, and IgE+ cells. Biopsies were taken from flea-sensitized dogs (n=28) and non-sensitized dogs (n=5) that had been exposed to fleas. Control groups consisted of flea-sensitized (n=12) and non-sensitized dogs (n=9) that were not exposed to fleas. Biopsies, taken before, 24 and 72 h after local flea exposure, were stained with haematoxylin and eosin (H&E), toluidine blue, a double labelling technique for MC chymase and tryptase and anti-IgE. An intradermal test for flea antigen was performed and serum titres of allergen-specific IgE and IgG were measured. Significantly higher numbers (P<0.001) of double labelled MCs compared to toluidine blue stained MCs were detectable in flea-sensitized dogs independent of flea exposure. In contrast, in non-sensitized dogs, the number of toluidine blue stained MCs and the number of double labelled MCs did not differ. In flea-sensitized dogs after flea exposure the percentage of C-MC was significantly increased at day 1 (P<0.001) and day 3 (P<0.001), whereas the percentage of TC-MCs decreased significantly at day 1 (P<0.001) and day 3 (P<0.05). The percentage of T-MCs decreased (P<0.05 day 0 versus day 1; P<0.05 day 0 versus day 3). No significant difference was detectable after toluidine blue staining and staining for IgE+ cells between the groups nor between the MC density and the number of IgE+ cells. All flea-sensitized dogs had positive skin tests to flea antigen and high serum titres of flea-specific serum IgE and IgG antibodies. In non-sensitized dogs, these results were negative. Our data provide strong evidence for an upregulation of MC proteases during the process of sensitization and a generalized selective release of mast cell tryptase after exposure to the antigen.

Allergic pulmonary and ocular tissue responses in the absence of serum IgE antibodies (IgE) in an allergic dog model.

Allergen-specific serum IgE may be insensitive as a marker for IgE-mediated reactions at the mucosal level. Five of six atopic beagle dogs developed high ovalbumin (OVA)-specific serum IgE levels after sensitization. This study aimed to show that these dogs still express allergen-specific IgE at the pulmonary and ocular mucosal levels and in the skin even when corresponding serum IgE was below the detection limit. When serum IgE levels were negative, all dogs exhibited allergic reactions at the tissue level. Specifically, they displayed positive ocular reactions after an ocular OVA challenge. After airway challenge with aerosolized OVA, five out of six animals reacted with decreased compliance and increased resistance of the lungs. Furthermore, an eosinophilia in the bronchoalveolar lavage fluid (BALF) was observed. Four weeks after the last exposure to OVA, IgE-positive BALF cells were seen in all animals. Six weeks on, all dogs still displayed positive skin reactions to OVA. This indicates that not only skin testing but also detection of ocular and pulmonary allergic tissue reactions including cell-bound IgE in BALF can serve as more sensitive and lasting surrogate markers of hypersensitivity in the allergic dog model than detection of allergen-specific serum IgE levels.

The humoral immune response to recombinant nucleocapsid antigen of canine distemper virus in dogs vaccinated with attenuated distemper virus or DNA encoding the nucleocapsid of wild-type virus.

This study compared the humoral immune response against the nucleocapsid-(N) protein of canine distemper virus (CDV) of dogs vaccinated with a multivalent vaccine against parvo-, adeno-, and parainfluenza virus and leptospira combined with either the attenuated CDV Onderstepoort strain (n = 15) or an expression plasmid containing the N-gene of CDV (n = 30). The vaccinations were applied intramuscularly three times at 2-week intervals beginning at the age of 6 weeks. None of the pre-immune sera recognized the recombinant N-protein, confirming the lack of maternal antibodies at this age. Immunization with DNA vaccine for CDV resulted in positive serum N-specific IgG response. However, their IgG (and IgA) titres were lower than those of CDV-vaccinated dogs. Likewise, DNA-vaccinated dogs did not show an IgM peak. There was no increase in N-specific serum IgE titres in either group. Serum titres to the other multivalent vaccine components were similar in both groups.

Correlation between the clinical course of granulomatous meningoencephalomyelitis in dogs and the extent of mast cell infiltration.

The data from 20 dogs with histopathologically confirmed granulomatous meningoencephalomyelitis were reviewed in an attempt to identify clinical signs and morphological and cellular parameters, particularly the infiltration of mast cells, which might be associated with the clinical course of the disease. Thirteen of the dogs had the acute form of the disease and seven had the chronic form. Young to middle-aged, small breed female dogs were over-represented. Central vestibular signs were observed in six of the dogs with the acute disease. Analyses of cerebrospinal fluid revealed moderate to severe pleocytosis and high protein concentrations in all cases. Histopathological investigations revealed disseminated perivascular cuffs, large confluent granulomata, tissue necrosis, infiltration with neutrophils and a large number of mitotic cells in the dogs with either of the clinical forms of the disease. Tryptase-positive mast cells were observed in all the cases, but there were significantly larger numbers in the dogs with the acute form.

Quantitative assessment of mast cells and expression of IgE protein and mRNA for IgE and interleukin 4 in the gastrointestinal tract of healthy dogs and dogs with inflammatory bowel disease.

OBJECTIVE: To characterize the mucosal IgE network in dogs affected with inflammatory bowel disease (IBD) and compare it with that for healthy dogs. ANIMALS: 9 healthy dogs and 20 dogs with IBD. PROCEDURE: In situ hybridization of mRNA specific for IgE and interleukin 4 (IL-4) and immunohistochemical analysis for IgE protein and 2 markers of mast cells (ie, tryptase and chymase) were performed on tissue sections obtained from the gastrointestinal (GI) tract and lymph nodes of dogs. RESULTS: Dogs with IBD had significantly more cells positive for IgE protein and more mast cells in the GI mucosa than healthy dogs. Despite this significant increase in number of cells positive for IgE, cells positive for IgE mRNA were rarely detected in the GI mucosa; most cells positive for IgE mRNA were found in mesenteric lymph nodes. Signal pattern of IL-4 mRNA was similar to that of IgE mRNA. CONCLUSIONS AND CLINICAL RELEVANCE: The increased numbers of cells positive for IgE and mast cells in dogs with IBD suggest hypersensitivity such as hypersensitivity to bacterial or dietary-derived antigens in the intestinal lumen. Future studies need to elucidate whether this represents a cause of inflammation or is a result of the inflammatory process of IBD.

Immunoglobulin-E-bearing cells in skin biopsies of horses with insect bite hypersensitivity.

The aim of the present study was to investigate, with immunohistochemistry and in situ hybridisation, if immunoglobulin-E (IgE) and mast cells are involved in the pathogenesis of insect bite hypersensitivity (IBH), an allergic dermatitis of horses. In tissue sections fixed in paraformaldehyde (PFA) for <24 h, significantly more IgE protein-bearing cells were found in the dermis and epidermis of acute and chronic IBH lesions than in skin biopsies from healthy horses (medians = 466, 236 and 110 cells/mm2, respectively; P < or = 0.01). More IgE-mRNA positive (+) cells were observed in the dermis of acute IBH lesions than in the dermis of healthy skin (median = 2.8 vs. 0.0 cells/mm2; P < or = 0.01). Significantly, more mast cells were detected with metachromatic (median = 160 vs. 62 cells/mm2; P < or = 0.001) and tryptase-specific stainings (median = 120 vs. 69 cells/mm2; P < or = 0.001) in the dermis of acute IBH biopsies compared to healthy skin. No chymase+ mast cells were found in any skin biopsy. IBH lesions fixed in PFA for >24 h were compared to dermatomycosis (DM) lesions; IBH biopsies contained a similar number of IgE-protein+ cells to DM biopsies (median = 249 vs. 192 cells/mm2; P = 0.08) but had significantly more IgE-mRNA+, metachromatic and tryptase+ mast cells than DM biopsies. This study suggests an involvement of IgE-mediated immune reactions in the pathogenesis of IBH as well as, sometimes, in dermatomycosis. Using double labelling, cells which expressed IgE protein and contained mast cell enzymes were detected.

Partial sequences of feline and caprine immunoglobulin epsilon heavy chain cDNA and comparative binding studies of recombinant IgE fragment-specific antibodies across different species.

Parts of the feline and caprine IgE epsilon heavy chain cDNA (third and fourth constant domains, IgEf3/4) were cloned, sequenced, and expressed to raise antibodies (Abs). The DNA and derived protein sequences of the feline recombinant IgEf (rIgEf) shared high homology with the analogous canine parts (81% at the nucleotide and 71% at the protein levels) and the caprine with the ovine ones (95%/84%), respectively. The polyclonal Abs raised in chickens against the feline and caprine rIgEf3/4 were subjected to a comparative binding study utilizing an ELISA including rIgEf and specific Abs to these rIgEf from dog and horse (rIgEf2 and rIgEf3/4) and sheep (rIgEf3/4). All but the ovine-specific rIgEf3/4 Ab were polyclonal, which had been raised in chickens, and bound to most applied rIgEf; the ovine-specific monoclonal mouse Ab recognized only in addition to ovine rIgEf3/4 the closely related caprine rIgEf3/4. Significant, positive correlations were detected between binding reactions of the polyclonal Abs in ELISA and percentage protein sequence homology (p<0.01). Thus, the newly described feline and caprine IgE nucleotide sequences and corresponding Abs represent useful tools for further species-specific and comparative allergy and disease-associated research.

DNA vaccine encoding nucleocapsid and surface proteins of wild type canine distemper virus protects its natural host against distemper.

Canine distemper virus (CDV), a member of the genus Morbillivirus induces a highly infectious, frequently lethal disease in dogs and other carnivores. Current vaccines against canine distemper consisting of attenuated viruses have been in use for many years and have greatly reduced the incidence of distemper in the dog population. However, certain strains may not guarantee adequate protection and others can induce post vaccinal encephalitis. We tested a DNA vaccine for its ability to protect dogs, the natural host of CDV, against distemper. We constructed plasmids containing the nucleocapsid, the fusion, and the attachment protein genes of a virulent canine distemper virus strain. Mice inoculated with these plasmids developed humoral and cellular immune responses against CDV antigens. Dogs immunized with the expression plasmids developed virus-neutralizing antibodies. Significantly, vaccinated dogs were protected against challenge with virulent CDV, whereas unvaccinated animals succumbed to distemper.

Domain mapping and comparative binding features of eight dog IgE-specific reagents in ELISA, immunoblots, and immunohistochemistry.

Eight dog IgE-specific reagents including monoclonal and polyclonal antibodies (Ab) and a cross-reactive alpha chain of the human high affinity IgE receptor were mapped to recombinant fragments of the second (IgEf2) and third/fourth (IgEf3/4) domains of the dog IgE heavy chain. In ELISA, five out of eight reagents reacted to solid-phase bound IgEf2, of which two polyclonal Ab bound in addition to IgEf3/4. All Ab which recognized at least one recombinant IgE fragment, also bound to IgE in ELISA, immunoblots, and immunohistochemistry. In contrast, only one monoclonal Ab, that did not bind to the recombinant IgE fragments, reacted with immunoblots of serum and immunohistochemistry. The alpha chain could only be applied to ELISA with serum IgE. Furthermore, there was a wide range of heat-lability of binding reactions. Comparative analysis of available dog IgE-specific reagents enables more in-depth functional studies on IgE-mediated phenomena in dogs, and helps to further establish the dog as an animal model for allergy research.

The AB blood group system in wild felids.

The blood type of 131 non-domesticated felids belonging to 26 felid species was surveyed in this study. Based upon a tube hemagglutination assay established for domestic cats, 80% of felids had type-A, 18% type-B, and 2% type-AB blood. Felids in the Puma group and African and Asian golden cats had blood type B, whereas all other species were found to have blood type A. Two cheetahs and one bobcat had type-AB blood. Red cell glycolipids analysed by high performance thin layer chromatography revealed a similar ganglioside pattern in wild cats as reported in domestic cats. Independent of the AB blood group system, incompatible blood crossmatch reactions were detected between different felid groups. In conclusion, wild felids display the same AB-erythrocyte antigens as domestic cats, and the same blood typing procedures can be applied for wild and domestic felids.

Influence of sex and age on serum total immunoglobulin E concentration in Beagles.

OBJECTIVE: To establish an ELISA for detection of serum total IgE concentration in dogs and to analyze IgE values in a dog colony. ANIMALS: 147 healthy Beagles (31 males and 116 females). PROCEDURE: 2 canine IgE-specific polyclonal antibodies elicited by 2 recombinant fragments of the epsilon chain in hens were used to develop a capture ELISA specific for serum total IgE concentration. The IgE values were calculated by comparing serum dose-response curves (1:50 to 1:6,400) with a reference serum pool assigned 100 relative ELISA units (REU). Results-Mean IgE concentration in female Beagles was 51.2 REU (range, 0 to 337.8 REU; median, 31.4 REU), whereas mean IgE concentration in male dogs was only 7.5 REU (range, 0 to 32.6 REU; mean, 3.6 REU). Distribution of IgE values was skewed; approximately 80% of dogs had IgE values < 50 REU. Analysis of natural logarithmically transformed IgE values indicated that sex and age significantly (P < 0.05) influenced IgE values; mean serum IgE values increased until the age of 4 years. Heritability estimates of IgE concentration indicated a trend toward a genetic influence. CONCLUSION: A reliable capture ELISA specific for canine IgE was developed. Serum total IgE values vary with age and sex in the sample population. CLINICAL RELEVANCE: Serum total IgE concentration can now be evaluated in various dog breeds and, subsequently, in dogs with IgE-mediated diseases provided that these significant influences are accounted for. Serum total IgE values may then prove to be of diagnostic value, similar to their use in human beings.

Total serum IgE and IgA antibody levels in healthy dogs of different breeds and exposed to different environments.

Total serum immunoglobulin (Ig) E and A levels were analysed in 233 healthy dogs as basis for comparison with atopic dogs in future studies. They were measured by ELISA in a group of non- colonised dogs of various breeds (group A) and three groups of colonised dogs including one German Shepherd and two Beagle kennels (groups B-D). IgE levels from non-colonised dogs were significantly higher than the ones of German Shepherds and Beagles C (P<0.05). IgA levels were alike in all groups except for the German Shepherds which displayed the lowest levels. Age and sex were not identified as common significant cofactors for IgE and IgA levels in all groups and IgE levels correlated negatively with IgA only in non-colonised dogs. In conclusion, IgE and IgA levels seem to be mainly influenced by genetic background. Thus use of total serum IgE as a diagnostic tool in the atopic dogs required extensive family data and therefore appears most suitable for research purposes within specific, well defined dog populations.

Characterization of two dog IgE-specific antibodies elicited by different recombinant fragments of the epsilon chain in hens.

Two recombinant [His]6-tagged fragments of the canine immunoglobulin E (IgE) heavy chain (second domain: IgEf2 and third and fourth domains: IgEf3/4) were cloned, expressed in Escherichia coli (E. coli) as [His]6-tagged proteins, and affinity-purified over nickel-nitrilotriacetic acid columns. The recombinant proteins were used to immunize hens. The raised and affinity-purified chicken antibodies (Ab) isolated from egg yolk exhibited specific binding to the respective recombinant canine IgE fragment (IgEf) on immunoblots and displayed high titers against the IgEf in ELISA. Immunoblotting of canine serum separated by PAGE under native conditions with the IgEf2- and IgEf3/4-specific Ab resulted in staining of a protein of approximately 180 kilodaltons (kD). The IgEf3/4-specific Ab further recognized an 80 kD protein in IgEf3/4-specific Ab affinity-enriched dog serum separated under denaturing conditions. In an ELISA for the detection of antigen-specific IgE in dog serum, reduced binding of the IgEf-specific Ab was observed after heat treatment of the dog serum. The reactivity of both of the raised chicken Ab was only present in postimmune reagents and could only be inhibited by preincubation with the IgEf used for immunization and not with dog immunoglobulin G, E. coli extract, or with a nonrelevant recombinant [His]6-tagged protein. In immunohistochemistry, the IgEf3/4-specific Ab specifically recognized cells in paraffin-embedded tissue sections of lymph nodes. Furthermore, both of the IgEf-specific Ab elicited positive immediate type 1 skin reactions in dogs. Semiquantitative assessment of total serum IgE in dogs was developed using IgEf2-specific Ab as coating reagent and the biotinylated IgEf3/4-specific Ab as developing Ab in ELISA. In conclusion, both IgEf-specific Ab recognize native dog IgE with the advantages that they are directed against different and known constant domains of the IgE molecule, and that they can be used for immunohistochemistry on paraffin-embedded tissue. The two dog IgE-specific Ab could initiate clinical research on the involvement of immediate-type hypersensitivity reactions in dogs.

[Convulsions in relation to polycythemia: literature review and case description]. / Krampfanfälle im Zusammenhang mit Polyzythämie: Literaturübersicht und Fallbeschreibung.

Polycythemia--characterized by an excessive number of erythrocytes--is a rare disease in the dog with a chronic progressive course and unspecific symptoms. There are several forms: a primary, a secondary adequate or a secondary inadequate polycythemia. The clinical workup is done step by step and after stabilization of critical patients, the remaining therapy must address the primary cause. We report on a five year old male Leonberger dog suffering from secondary, inadequate polycythemia. He was presented with apathy, gait disturbances and disorientation. On the basis of the diagnostic workup a pathological process in the kidneys was postulated. Initially focal seizures became generalized later, most probably because of formation of a forebrain thrombus with secondary hypoxia. Even after emergency treatment the general state deteriorated. The course indicated possible sepsis. Because of the critical picture with secondary complications and the poor prognosis, the dog was euthanised. The histopathological results showed T-cell renal lymphoma and secondary injury to the forebrain.

Chicken antibodies to a recombinant fragment of the equine immunoglobulin epsilon heavy-chain recognising native horse IgE.

An equine immunoglobulin E (IgE) heavy-chain cDNA fragment (CH3-CH4, nucleotides 1132 to 1592) was cloned, expressed in Escherichia coli as a fusion protein with a [His]6-tag and purified over a Ni-NTA column. The recombinant protein was used to immunise hens. Testing of the raised egg yolk immunoglobulin G (IgG) in Western-blot and ELISA revealed high titres against the recombinant equine IgE fragment (reqIgEf). The reqIgEf-specific IgG was successfully affinity-purified on an unconventional affinity matrix: the [His]6-tagged recombinant IgE fragment was bound to Ni-NTA agarose and used to adsorb specific immunoglobulins. In Western-blot of ammonium sulphate precipitated horse serum and bronchoalveolar lavage fluid, separated by SDS-PAGE under denaturing-reducing conditions, the raised antibodies reacted with a protein of approximately 80 kDa. A reaction of the reqIgEf-specific IgG was seen with a 190-200 kDa band when the same horse serum or bronchoalveolar fluid (BALF) was separated under non-reducing conditions. These reactions could be inhibited by preincubation of the immune IgG with reqIgEf, while preincubation with horse IgG did not inhibit the reaction. Antibody-affinity chromatography of horse serum with the reqIgEf-specific chicken IgG resulted in an enrichment of the 80 kDa protein in denaturing Western-blot. Determination of the amino acid composition of this protein and comparison with the equine IgE heavy- chain sequence strongly indicates that the 80 kDa band corresponds to the heavy chain of the horse IgE. The reqIgEf-specific chicken IgG was further characterised in an ELISA for the detection of allergen-specific horse IgE. It was demonstrated that heating IgE positive horse sera at 54 degrees C for 10 min drastically diminished the recognition by the reqIgEf-specific chicken IgG. The reaction is inhibitable by preincubation with reqIgEf in a concentration dependent manner. In addition, preincubation with horse IgG, a nonrelevant [His]6-tagged protein or 2% equine colostrum had no influence on the reqIgEf-specific chicken IgG binding characteristic. This antibody recognising horse IgE will be useful for further studies on the pathogenesis of equine allergic diseases.

Blood type AB in the feline AB blood group system.

OBJECTIVE: To assess the genetics, frequency, and biochemistry of the AB blood type in cats. ANIMALS: Domestic shorthair and purebred cats in a breeding colony and privately owned catteries and blood samples from a large feline blood typing laboratory. PROCEDURES: Samples from cats with blood type AB were selected from the feline blood typing laboratory at the university. Breeding experiments and family studies were used for the genetic analysis of cats with blood type AB. Simple slide hemagglutination assays were used to type cats. Hemagglutination assays, flow cytometry, and ganglioside analysis by high-performance thin layer chromatography were applied to characterize the AB antigens. RESULTS: Type AB was rare (13/9,239 cats; 0.14% frequency) in cats of the United States and Canada. Type AB occurred only in breeds in which type B was also detected. Cats with type-AB blood express biochemical features of type-A and type-B antigens. Genetic analyses of families with blood type-AB cats are consistent with the hypothesis of 3 alleles: A, B, and AB. The AB allele is recessive to the A allele, but dominant over the B allele. There may be an additional genetic mechanism responsible for the inheritance of blood type AB in cats. CONCLUSION: Blood type AE is an extremely rare and separately inherited type in the feline AB blood group system. CLINICAL RELEVANCE: Kittens with type-AB blood born to queens with type-B blood are at similar risk for neonatal isoerythrolysis as kittens with type-A blood because anti-A alloantiserum from blood type-B queens recognizes AB red blood cells. Furthermore, cats with type-AB blood are best transfused with type-AB or type-A blood.