Osmotic fragility of erythrocytes in clinically normal dogs and dogs infected with parasites

The osmotic fragility of the erythrocytes was measured in blood samples collected from randomly selected healthy and infected dogs at a dogs' rescue shelter. The dogs were classified into six groups on the basis of the final diagnoses from clinical, post mortem and laboratory findings. The minimum (less than 5 per cent) and maximum (more than 90 per cent) haemolysis of the erythrocytes of the clinically normal dogs (group 1), occurred in 0·60 per cent and 0·30 per cent solutions of sodium chloride (NaCI). For the non-anaemic hookworm-infected dogs (group 2a) the respective values were 0·8 per cent and 0·4 per cent NaCl, and for the anaemic hookworm-infected dogs (group 2b) they were 0·85 per cent and 0·5 per cent NaCl, respectively. The erythrocytes from dogs with Babesia canis (group 3), concurrent hookworm and B canis (group 4) and Ehrlichia canis infections (group 5) had minimum haemolysis in 0·75 per cent NaCl and maximum haemolysis at between 0·20 per cent and 0·35 per cent NaCI solutions. The derivative fragiligrams for groups 2a, 2b, 3 and 4 were shifted to the left, whereas the fragiligram for group 5 was similar to that for the clinically normal dogs (group 1). The left shift for the hookworm-infected dogs was due to the increased osmotic fragility of a minor sub-population of the erythrocytes, but for the dogs, infected with B canis major sub-populations of the erythrocytes had an increased osmotic fragility.     

Effect of dietary chloride content on the elimination of bromide by dogs

The effect of dietary chloride content (0·2, 0·4 and 1·3 per cent chloride on a dry matter basis) on the disposition of a single oral dose of bromide (14 mg kg⁻¹ was evaluated in normal beagles. Increasing the dietary chloride content from 0·2 to 1·3 per cent resulted in a significant decrease in the mean apparent elimination half-life from 69 ± 22 days to 24 ± 7 days. The mean area under the concentration curve ( ) for dogs fed 1·3 per cent chloride was significantly smaller than the for dogs fed 0·2 per cent chloride. Dietary chloride had no effect on the maximum serum concentrations (C_max) or on the time (T_max) to reach the maximum concentrations. The steady-state serum bromide concentrations predicted from the single dose data for daily doses of 14 mg kg⁻¹ of bromide were significantly lower in dogs fed 1·3 per cent chloride (310 ± 150 mg litre⁻¹) than in dogs fed 0·2 per cent chloride (1950 ± 1140 mg litre⁻¹). The predicted mean daily doses of bromide necessary to maintain serum levels within the therapeutic range for dogs fed 1·3 per cent chloride (43 ± 13 mg kg⁻¹) were almost twice as high as the dose estimated for dogs fed 0·4 per cent chloride (22 ± 3 mg kg⁻¹) and nearly three times as high as the dose estimated for dogs fed 0·2 per cent chloride (15 ± 4 mg kg^−l). These differences were statistically significant (P=0·002).     

Dynamics of Leishmania-specific immunoglobulin isotypes in dogs with clinical leishmaniasis before and after treatment

Concentrations of Leishmania-specific immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin A (IgA) isotypes were analyzed by enzyme-linked immunosorbent assay (ELISA) in 23 dogs naturally infected with Leishmania infantum before and 1 year after initiating drug therapy. Results showed a high expression and prevalence of Leishmania-specific IgG (176.4 +/- 89 ELISA units [EU]), IgM (105.3 +/- 95.5 EU), and IgA (153.6 +/- 98 EU) in dogs before treatment (median +/- interquartile range EU). One year after treatment was started, dogs were classified as responsive dogs (RDs; n = 13) or unresponsive dogs (UDs; n = 10) based on clinicopathologic findings. Both groups of dogs experienced a statistically significant decrease (P < .05) in Leishmania-specific IgG (RDs = 27%, UDs = 41%), IgM (RDs = 42%, UDs = 29%), and IgA (RDs = 56%, UDs = 46%). Concentrations of specific IgG and IgM were not different at diagnosis or after treatment between the 2 groups. However, the median value for Leishmania-specific IgA 1 year after treatment was significantly lower (P < .05) in RDs (60.8 +/- 67 EU) than in UDs (117 +/- 54 EU). Examination of our data indicates that both the IgA isotype, which is mostly produced by mucosal plasma cells, and the IgM isotype are increased in infected symptomatic dogs, as previously reported for IgG. These 3 isotypes decreased significantly 1 year after initiation of medical treatment.     

Antibody dynamics in BRSV-infected Danish dairy herds as determined by isotype-specific immunoglobulins

Using specific ELISAs, antibody levels of four different isotypes to bovine respiratory syncytial virus (BRSV) were determined in calves, following experimental BRSV infection.Most calves experienced an increase in the specific IgM and IgG1 titres about 6-10 days after infection with BRSV. The IgM titre was transient showing positive titres for only 5-10 days, while specific IgG1 was present for a longer time. IgA was detected concomitantly with IgM but at a lower level. Production of IgG2 anti-BRSV antibodies was detected from 3 weeks after infection.In two closed herds, repeated blood samplings were performed on young stock to analyse maternal immunity. The passively transferred antibodies were mainly of the IgG1 isotype and the half-life of IgG1 to BRSV was estimated to be 26.6 days. One of the herds had an outbreak of enzootic pneumonia, diagnosed to be caused by BRSV. Furthermore, another herd with acute BRSV was followed by weekly blood samples in six calves; in both herds IgM and IgG1 was detected shortly after the appearance of clinical signs. Serum samples from 50 Danish dairy herds (453 samples) were tested for immunoglobulins of the isotypes IgG1, IgG2 and IgM. The presence of antibodies to BRSV was widespread and more than 54% of the samples had BRSV antibodies of both the IgG1 and IgG2 isotypes indicating a high herd prevalence to BRSV. Test samples from two herds out of 50 were free from all isotypes to BRSV.     

Investigations on the nature and pathogenicity of circulating antikeratinocyte antibodies in dogs with pemphigus foliaceus

In humans with pemphigus foliaceus (PF), pathogenic autoantibodies are principally of IgG4 subclass and they cause superficial vesiculation when injected into neonatal mice. The objectives of this study were to determine the isotypes of circulating antikeratinocyte antibodies in dogs with PF, to assess whether serum antikeratinocyte antibody titres decreased during successful treatment, and to study whether such antibodies were pathogenic in passive transfers. Using indirect immunofluorescence with neonatal mouse skin substrates, circulating antikeratinocyte IgG antibodies were detected in 36 of 44 dogs with PF (82%). Serum autoantibodies belonged predominantly to IgG4 (three of 44; 80%) and IgG1 (30 of 44; 68%) subclasses. Antikeratinocyte IgG antibodies were detected in 16 of 20 normal dogs (80%), and these antibodies were IgG1 (16 of 20, 80%) but rarely IgG4 (two of 20; 10%) isotypes. In four dogs, IgG4 antikeratinocyte antibody titres decreased concomitantly to lesions nearing or reaching complete remission. In contrast, IgG or IgG1 titres remained stable or increased when lesions abated. Antikeratinocyte antibodies targeted mainly intercellular autoantigen(s) in the stratum granulosum, while in fewer dogs, such antibodies bound to cytoplasmic basal antigen(s). Intradermal injections of PF or pemphigus vulgaris (PV) IgG into neonatal mice caused subgranular or suprabasal acantholytic vesiculation without granulocyte infiltration, respectively. Similar transfers of normal dog IgG did not cause vesiculation. These observations suggest that antikeratinocyte IgG4 antibodies could be relevant to disease pathogenesis. Importantly, canine PF or PV IgG appear to be pathogenic when transferred passively into mice, causing vesiculation at epidermal levels similar to those of the natural disease.     

Evaluation of a dot ELISA kit for measuring immunoglobulin M antibodies to canine parvovirus and distemper virus

A dot ELISA for the detection of immunoglobulin M (IgM) antibodies to canine distemper virus (CDC) and canine parvovirus (CPV) was assessed. The titres of IgM antibodies to CDV and CPV in 100 dogs were measured by the Immunocomb ELISA kit and compared with the results derived from the immunofluorescence assay (IFA). There was a strong correlation between the results of the dot ELISA technique and the IFA (P < 0.001). The dot ELISA kit was also used to assess the changes in the levels of immunoglobulin G (IgG) and IgM antibodies to CPV and CDV in 10 puppies vaccinated with a polyvalent vaccine. High levels of IgM antibodies to CPV were first detected seven days after they were vaccinated, and after nine days all the pups had high titres of IgG antibodies to CPV. High levels of IgM antibodies to CDV were detected after nine days and the highest average titres were recorded after 12 days. IgG antibodies to CDV were present from nine days after vaccination.     

Four-layer enzyme immunoassay (EIA) detection of differences in IgG, IgM and IgA antibody response to Aujeszky's disease virus in infected and vaccinated pigs

The use of the four-layer enzyme immunoassay (EIA) for the detection of IgG, IgM and IgA antibodies against Aujeszky's disease virus in blood and oropharyngeal swabs of infected and vaccinated pigs is described. Mean antibody titres obtained using the four-layer EIA were 6.1 and 3829 times higher compared with the indirect enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) test, respectively. The VN test detected mainly IgG antibodies, while the IgM antibodies did not react. Using the EIA, the first antiviral antibodies in sera were demonstrated on Days 5-7 after infection or vaccination. Up to the 7th day, demonstrable antibodies were almost exclusively of the IgM class. In infected pigs high titres of IgM antibodies were still detected on Day 18, while in vaccinated animals they were absent by this time. Antibodies of the IgG class appeared in infected pigs sooner (Day 7) than in vaccinated pigs (Day 10) and reached higher mean titres. Antibodies of the IgA class were demonstrable from Day 10 only in samples from infected pigs. Similar antibody dynamics and distribution were detected in oropharyngeal swabs, except that the IgG and IgM titres were roughly 100 times lower than in sera. However, titres of IgA antibodies in oropharyngeal swabs were two times higher than in sera. The greatest differences between both groups of animals were recorded on Day 18; in the infected pigs, IgG, IgM and IgA antibodies were present in sera and oropharyngeal swabs at that time, while in vaccinated pigs only IgG antibodies were demonstrable. The effect of infection and vaccination on the pattern of the immune response as well as the importance of the detection of individual immunoglobulin classes for the specificity of the enzyme immunoassay are discussed.     

Viral antibodies in bovine fetuses in Argentina

In order to establish the prevalence of viral infections of the bovine fetus in Argentina, a serological survey for antibodies against viral agents currently affecting cattle in this country was conducted. Antibodies against foot-and-mouth disease virus (FMDV), bovine herpesvirus-1 (BHV-1), bovine leukaemia virus (BLV), bovine rotavirus (BRV), bovine coronavirus (BCV), bovine viral diarrhoea virus (BVDV) and parainfluenza-3 (PI-3) were investigated in a total of 315 fetal serum samples. Conventional techniques were used: indirect immunofluorescence (FMDV, BHV-1, BVDv and BCV), radial immunodiffusion (BLV), ELISA (BRV) and haemagglutination inhibition (PI-3). Antibodies against BHV-1, BVDV and PI-3 were detected in samples from fetuses in the second and third trimester of gestation, with a prevalence of 1·21 per cent (two of 165), 2·03 per cent (four of 197) and 5·08 per cent (nine of 177), respectively. Either antibodies or non-antibody factors able to bind to BRV and Bcv antigens were detected with a prevalence of 2·44 per cent (five of 205) and 4·54 per cent (five of 110), respectively. In addition, 14·68 per cent of non-specific inhibitors of PI-3 mediated haemagglutination were found. No seropositives against FMDV and BLV were detected.     

Seroprevalence of antibodies to Neospora caninum in Belgian dogs

Sera from 300 dogs from Ghent and Antwerp were tested for antibodies to Neospora caninurn using an Indirect fluorescent antibody test. Overall, 11 per cent (995 to 13 per cent; confidence interval of 95 per cent) of dogs were seropositive, at titres of 1:50 to 1:800. No sex or breed differences were detected, but there was an Increase In seropositivity with age.     

Rotavirus-associated diarrhoea in buffalo calves in Sri Lanka

Faecal samples from 150 buffalo calves, one to 150 days old, located in various districts of Sri Lanka, were examined for group A rotavirus antigen by a screening enzyme linked immunosorbent assay (ELISA). Positive samples were confirmed by the blocking ELISA. In the calves studied 27·3 per cent were diarrhoeic, and the rest were non-diarrhoeic but were in contact with the animals showing diarrhoea. Antigen was detected in 36·6 per cent of the diarrhoeic animals and in 11·9 per cent of the nondiarrhoeic animals. There was a strong association between the presence of antigen in faeces and diarrhoea in these animals (ξ² = 46·98; P<0·001). Of the 146 serum samples examined for antirotaviral antibodies, by the blocking ELISA at a single serum dilution (1:20) against a constant dose of antigen (8 units), 68·5 per cent were positive indicating a widespread infection with the virus in the population studied. This is the first record of the detection of rotavirus and its association with diarrhoea in buffalo calves in Sri Lanka.     

Sequence of the canine c-yes proto-oncogene

Cloning of the canine yes oncogene was attempted from a c library derived from a healthy canine spleen using a human c-yes-1 probe. The nucleotide and amino acid sequences revealed that the canine yes gene contained an open reading frame consisting of 539 amino acids. Its product had a molecular mass of 60,368 Daltons and showed 95·9 per cent and 90·4 per cent homology with human and chick p61^c-yes, respectively. Moreover, the product had a myristylation signal, src homology region (SH) 3, SH2, and tyrosine kinase domains showing 98·8 per cent and 96·0 per cent homology with those of human beings and chickens, respectively. These findings indicate that the products of the canine yes gene may have non-receptor-type tyrosine kinase activity on the cell membrane, as is the case in human and chick p61^c-yes     

Systemic and local immune response of cows to intramammary infection with Staphylococcus aureus

The association between Staphylococcus aureus chronic mammary gland infection and the resulting immune response expressed by the production of specific IgG and IgA antibodies in blood and milk was studied in Israeli Holstein cows. Specific antibodies of the IgG class were detected in sera of 82.6 per cent of the cows chronically infected by S aureus, while in 17.4 per cent no such antibodies could be detected. Specific IgG antibodies to S aureus were neither detected in sera of cows free of mammary infection nor in those infected with different coagulase-negative staphylococci (CNS) such as S intermedius, S chromogenes or S haemolyticus. In milk, specific IgG antibodies to S aureus were detected only in cows with positive serology. The end point dilutions in the milk were 5 to 30 per cent of that of blood from the same cow. No significant difference in IgG titres was found in the same cow if the quarter was infected with S aureus or not. Specific antibodies to S aureus of the IgA class could not be detected in the sera of any of the cows included in this study. In milk, a specific IgA antibody was detected only in the samples from the S aureus infected quarters in which S aureus was isolated at the time of the experiment. In the same cow, quarters infected by S aureus were found to have a significantly higher IgA titre (P < 0.0001) than that of the non-infected ones.     

Brucella abortus-specific immunoglobulin isotypes in serum and vaginal mucus from heifers vaccinated with Brucella abortus salt-extractable proteins and challenge exposed with virulent Brucella organisms

Serum and vaginal Brucella-specific immunoglobulin isotypes (IgG1, IgG2, IgM, and IgA), obtained from 62 crossbred beef heifers vaccinated with Brucella abortus salt-extractable proteins and subsequently challenge exposed with B abortus S2308, were studied. Brucella-specific IgG antibodies and Brucella-specific immunoglobulin isotypes were quantitated by a fluorometric immunoassay. Serum and vaginal immunoglobulin responses were evaluated as a method of distinguishing infected from noninfected heifers. Rivanol precipitation, complement-fixation, buffered-antigen brucellosis tests and an ELISA were performed on sera. For immunoglobulin isotypes, vaccinated heifers had mean antibody responses higher than baseline mean antibody responses for at least 31 weeks after vaccination. After challenge exposure, significant differences (P greater than 0.05) were not detected between mean antibody responses of vaccinated and nonvaccinated heifers. Vaginal Brucella-specific antibody responses did not correlate with protection from disease. Vaginal Brucella-specific IgM was detected only at the time of abortion. Vaginal IgA appeared specific for identification of virulent B abortus infection. All serotests appeared adequate in distinguishing baseline titers from titers of heifers that had aborted and were considered bacteriologic culture-positive. Results of serotests neither consistently distinguished vaccinates from challenge-exposed cattle nor distinguished heifers that were challenge exposed, had aborted, and were considered bacteriologic culture-positive adequately from heifers that were challenge-exposed, had not aborted, and were considered bacteriologic culture-negative. Brucella-specific IgA appeared to be the most effective in distinguishing vaccinated heifers from challenge- exposed heifers and heifers that were challenge exposed and had aborted, from heifers that were challenge exposed and had not aborted. Brucella-specific serum IgA was detected up to 13 weeks after abortion.     

Profile of anti-Leishmania antibodies related to clinical picture in canine visceral leishmaniasis

This research investigated the profile of anti-Leishmania antibodies in different clinical forms of canine visceral leishmaniasis (CVL). Naturally infected dogs were divided into two groups: subclinical dogs (SD, n=10) and clinical dogs (CD, n=68). Non-infected dogs (ND, n=7) comprised the negative control group. The humoral response was evaluated by the profile of total IgG, IgG1, IgG2, IgM, IgA and IgE, determined by ELISA. Infected animals showed increased levels of total IgG, IgA and IgE in addition to IgG1 and IgG2 in groups SD and CD, when compared with group ND. Furthermore, it was observed that IgG2 and IgM were correlated with symptomatology, while total IgG, IgG1 and IgA were negatively correlated and IgE showed no correlation. It follows that serum levels of IgG2 anti-Leishmania are correlated with typical clinical signs of disease. Furthermore the determination of specific anti-Leishmania antibodies could be an important tool in monitoring CVL clinical picture.     

Serological responses of adult dogs to revaccination against distemper, parvovirus and rabies

Serum antibody titres to canine distemper virus (CDV), canine parvovirus (CPV) and rabies were measured in dogs that had not been revaccinated annually and compared with the titres in a control group of regularly vaccinated animals; 83 per cent (171 of 207) of the dogs vaccinated against CDV one or more years earlier had serum neutralising antibody titres equal to or greater than 16; 64 per cent (136 of 213) of the dogs vaccinated against CPV one or more years earlier had haemagglutination inhibiting titres equal to or greater than 80; and 59 per cent (46 of 78) of the dogs vaccinated against rabies two or more years earlier had serum neutralising antibody titres equal to or greater than 0.5 iu/ml. Three weeks after a single booster vaccination the dogs' antibody titres against CDV had increased above the threshold level in 94 per cent of the dogs, against CPV in 68 per cent, and against rabies in 100 per cent.     

Quantitation of bovine immunoglobulins in culture fluids by use of sandwich radioimmunoassay with monoclonal antibodies

Bovine immunoglobulin isotype-specific murine monoclonal antibodies were used in sandwich radioimmunoassays to detect and quantitate bovine IgG1, IgG2, IgM, and IgA in culture fluids. The concentrations of bovine immunoglobulins in unknown samples were extrapolated from standard curves generated with bovine monoclonal immunoglobulins. The lowest detection limits for the bovine immunoglobulin isotypes ranged from 65 to 270 ng/ml.     

Prevalence of Brucella abortus and Brucella canis antibodies in dogs in Nigeria

Serum samples collected from dogs brought for routine physical examination, vaccination and other complaints at the Small Animal Clinic of Ahmadu Bello University, Zaria, Nigeria were tested for Brucella abortus and Brucella canis antibodies. Ninety-five (38-2 per cent) of 249 dogs studied were positive for B. abortus agglutinins by the Rose Bengal plate test (RBPT) but none was sero-positive by the standard agglutination test (SAT). The antibody prevalence for B. canis by the SAT was 28-6 per cent for 224 dogs tested. Exotic breeds of dogs had a prevalence of 34-9 per cent for B. canis agglutinins while 28-1 per cent of local dogs were sero-positive. Twenty-two per cent of dogs older than 2 years were sero-positive compared to a prevalence of 33-3 per cent found amongst dogs younger than 1 year. A similar B. canis infection rate was observed amongst male (29-6 per cent) and female (26-7 per cent) dogs.     

Comparison of media and conditions of incubation for the quantitative culture of Malassezia pachydermatis from canine skin

Fifty-five swab-wash specimens from dogs were cultured at 32°C on Sabouraud's dextrose agar either with or without 1 per cent Tween 80, Ushijima's medium A and modified Dixon's and Leeming's media. The counts of Malassezia pachydermatis were not significantly different after three or seven days of incubation. Colony counts on contact plates were significantly greater after incubation for seven days on Sabouraud's dextrose (P<0·001) and modified Dixon's agars (P<0·05) than after three days at 26°C, but not at 32°C. Counts on Sabouraud's or modified Dixon's agars after incubation at 32°C and 37°C were not significantly different. When compared with aerobic culture, an atmosphere containing 5 to 10 per cent carbon dioxide significantly (P<0·01) increased the frequency of isolation and colony counts on Sabouraud's dextrose agar but not on modified Dixon's agar.     

Leishmania-specific isotype levels and their relationship with specific cell-mediated immunity parameters in canine leishmaniasis

In the current retrospective study, Leishmania infantum-specific IgG, IgA and IgM levels were determined by ELISA in 106 untreated dogs with clinically-patent leishmaniasis (Sx) and in 171 clinically healthy dogs (Asx) from Spain to investigate the relationship between these Ig isotypes and clinical status. In addition, we studied if different Leishmania-specific humoral pattern exists between Asx dogs with and without cellular mediated immunity (CMI). Fifty-six dogs were assessed by means of lymphoproliferation assay (LPA), interferon production (IFN) and leishmanin skin test (LST), 71 dogs by means of both LPA and IFN and 44 only by LST. Both Sx and Asx dogs produced Leishmania-specific IgG, IgA and IgM antibodies, however the levels and proportion of positive dogs for each Ig isotype were significantly higher in Sx than in Asx ones (P<0.001). Analysis of immunological profiles determined for each cellular technique (positive and negative cellular response for each technique combined with positive or negative specific humoral response) showed that Asx dogs constituted a high heterogeneous group. No correlations were observed between CMI tests and specific IgG or IgM levels. However, a significant inverse correlation was demonstrated between specific IgA levels and LPA response (Spearman's r=-0.220; P=0.035). In general, the low correlation detected between CMI tests and isotype levels might indicate that the immune response is not strongly polarized in the majority of Asx dogs. Additionally, this study suggests that dogs developing T-cell response are probably able to avoid the dissemination of the parasite at least to mucosal surfaces and, as a consequence, to produce low or background specific IgA levels. Further studies are needed to investigate the relationship between specific IgA and parasite load, especially at mucosal site.     

Total and relative deficiency of gut mucosal IgA in German shepherd dogs demonstrated by faecal analysis

The concentration of immunoglobulins in faecal extracts was investigated as a method of assessing the production of immunoglobulins by the gut mucosa of 137 dogs. There were significant correlations between the concentrations in faecal extracts and the concentrations produced in duodenal organ cultures. Seventy-six German shepherd dogs had significantly lower median immunoglobulin A (IgA) concentrations in their faecal extracts than 63 controls of various breeds. Sixteen of the German shepherd dogs had IgA concentrations below the 95 per cent confidence limit of the control population and six had no demonstrable faecal IgA. The faecal concentrations of immunoglobulin G and albumin were significantly higher in the German shepherd dogs than in the controls, but their immunoglobulin M concentrations were similar.