Anti-Leishmania IgA in urine samples from dogs with clinical leishmaniasis

Recently, anti-Leishmania IgG has been detected in urine samples from Leishmania-infected dogs and its concentrations have been correlated with impairment of renal function. The presence and relationship with other anti-Leishmania Ig isotypes in urine have not yet been investigated. The current study analyzed the concentrations of anti-Leishmania IgA and IgG in sera (Ig-S) and urine (Ig-U) samples by ELISA in 64 untreated dogs with clinical leishmaniasis. All 64 serum samples tested were positive for anti-Leishmania IgG. Fifty of them (78.1%) were also positive for anti-Leishmania IgA. The results showed the presence of anti-Leishmania IgA-U in 38% of the 50 dogs that were positive for specific IgA-S. Thirty-eight of the 64 dogs positive for Leishmania-specific IgG-S (59.4%) were also positive for Leishmania-specific IgG in urine (IgG-U). The concentrations of anti-Leishmania IgA-U were significantly correlated with urine protein/creatinine (uP/C) ratio (ρ = 0.542; P < 0.001) and with serum biochemical parameters, such as γ-globulins, urea and creatinine. Goldmann–Witmer coefficient (C value) indicated that detection of specific IgA in urine samples from dogs with leishmaniasis might not only be due to impairment of filtration of the glomerular barrier but also be due to local production of this isotype, which might reflect a local immunological response to the presence of the parasite in the genitourinary tract. Anti-Leishmania IgG-U concentrations were highly correlated with uP/C ratio (ρ = 0.779; P < 0.001) and C value did not support in any case local production of this isotype. IgG isotype might be a more suitable and specific tool to evaluate renal damage due to the lower IgA-U sensitivity and correlation coefficients and evidence of IgA local production. However, dogs found positive for both Ig isotypes in urine presented significantly higher specific IgG-U concentrations and higher uP/C ratios than dogs found positive only for IgG-U, thus suggesting that the first group suffered more severe renal damage. This fact makes it necessary to evaluate the prognosis of dogs showing both anti-Leishmania IgA-U and IgG-U in future studies.     

Serum anti-trichostrongyle antibody responses of first and second season grazing calves

Serum anti-Ostertagia ostertagi and anti-Cooperia oncophora antibody responses were assessed in first season and second season calves grazing permanent paddocks. Calves without previous exposure to trichostrongyles were found to mount significant parasite-specific IgG1 antibody responses within two months of introduction to the pastures. A significant serum IgA response to O ostertagi and IgG2 responses to both O ostertagi and C oncophora antigens were also observed, but these responses were weaker. No consistent serum anti-trichostrongyle IgM responses were discernible in either age group. Second season grazing calves had significantly elevated IgG1, IgG2 and IgA antibody levels at turnout when compared to first season calves, but only IgA antibody levels against O ostertagi increased during the second grazing season. Comparison of serum antibody levels in first and second season calves grazed separately or together suggests that mixed grazing had no discernible effect on antigen priming.     

Monitoring the circadian rhythm of serum and salivary cortisol concentrations in the horse

Daily fluctuations of cortisol concentration in the blood or saliva have been repeatedly reported. However, several contradictions in the existing literature appear on this subject. The present study was performed to definitively establish options for testing adrenocortical function. To the best of our knowledge, this is the first study to evaluate parallel circadian rhythms in salivary and serum cortisol concentrations during a 24-h period. Twenty horses were examined under the same conditions. Blood and saliva samples were taken every 2 h for 24 h to determine the daily changes in cortisol concentrations of saliva and serum at rest and to determine the relationship between salivary and serum cortisol levels. Cosinor analysis of group mean data confirmed a significant circadian component for both serum and salivary cortisol concentrations (P < 0.001 in both cases). The serum cortisol circadian rhythm had an acrophase at 10:50 AM (95% CI, 10:00 AM–11:40 AM), a MESOR of 22.67 ng/mL, and an amplitude of 11.93 ng/mL. The salivary cortisol circadian rhythm had an acrophase at 10:00 AM (95% CI, 9:00 AM–11:00 AM), a MESOR of 0.52 ng/mL, and an amplitude of 0.12 ng/mL. We found a significant but weak association between salivary and serum cortisol concentrations; the Pearson correlation coefficient was 0.32 (P < 0.001). The use of salivary cortisol level as an indicator of hypothalamic-pituitary-adrenal axis activity may be warranted. However, the salivary cortisol levels are more likely to be correlated with free plasma cortisol than with the total plasma cortisol concentration.     

Relative deficiency in IgA production by duodenal explants from German shepherd dogs with small intestinal disease

Matched samples of serum, saliva and tears were collected from four groups of dogs; two of the groups were German shepherd dogs (GSDs) either with (Group 1) or without (Group 4) a variety of small intestinal disorders; the remaining two groups were dogs of other breeds, again with (Group 2) or without (Group 3) small intestinal disease. Capture ELISAs were used to measure IgG, IgM, IgA and albumin concentrations within these samples; intestinal humoral immune status of clinical cases was assessed by quantifying immunoglobulin production from duodenal explant cultures.There were no significant differences in IgG, IgM or IgA concentrations in serum, saliva or tears between the different groups of dog. Moreover, no significant differences were noted between groups for IgG, IgM and IgA salivary and tear secretory indices. IgA production by 24-h explant cultures was significantly lower in GSDs compared with non-GSDs with small intestinal disease (groups 1 and 2, respectively), but the numbers of lamina propria IgA(+) plasma cells in duodenal biopsies were not different between groups. These results suggest that there may be a relative deficiency in local IgA secretion in GSDs with small intestinal enteropathies, which is not reflected in either serum IgA concentrations, or in secretion at unaffected mucosal sites. It remains to be determined whether such a deficiency is a breed-related primary defect, or whether it arises secondary to the pathological processes within the intestinal mucosa.     

Antibody reactivity to Cryptosporidium parvum in saliva of calves after experimental infection

Antibodies against Cryptosporidium parvum in the saliva and sera of three calves experimentally infected with this parasite were examined by an indirect immunofluorescence antibody test and immunoblotting. Salivary anti-C. parvum IgA antibody appeared on day 12 post-challenge and had a tendency to increase transiently between days 15 and 30 post-challenge. Salivary anti-C. parvum IgG antibody levels showed a gradual increase along with the change in IgA antibody levels during the infection. In contrast, serum anti-C. parvum IgA antibody levels showed only a slight increase between days 15 and 30 post-challenge. Serum anti-C. parvum IgG antibody levels rose on day 12 post-challenge and one calf maintained relatively high level up to the end of the experiment. In immunoblotting, an antigen with a molecular mass of 15 kDa was found to react strongly to salivary IgA antibody and a 27 kDa antigen to react to serum IgG antibody.     

Aspects of the humoral immune response to Staphylococcus intermedius in dogs with superficial pyoderma,deep pyoderma and anal furunculosis

Bacterial infection (pyoderma) of the canine skin is largely caused by Staphylococcus intermedius and may be a superficial or deep infection. Pyoderma may be a primary, idiopathic disease or secondary to a range of other dermatological disorders. In this study, the serum concentrations of IgG, IgA, antistaphylococcal IgG and antistaphylococcal IgA were measured by ELISA in normal dogs (n = 22), dogs with idiopathic deep pyoderma (n = 22), atopic dermatitis and superficial pyoderma (n = 24), atopic dermatitis without pyoderma (n = 25), flea bite dermatitis with superficial pyoderma (n = 8), pustular demodicosis (n = 8) and German shepherd dogs with anal furunculosis (n = 28). The serum IgG was significantly increased in dogs with atopy and superficial pyoderma (p < 0.001), and lower than normal in dogs with idiopathic deep pyoderma (p < 0.015). The concentration of serum IgA was significantly lower than normal in dogs with atopy uncomplicated by pyoderma (p < 0.015). The concentration of antistaphylococcal IgG in all clinical sera was significantly elevated (p < 0.001) when compared to normal dogs but concentrations of antistaphylococcal IgA were no greater than in normal dogs. Western blotting analysis for determination of the specificity of serum IgG antistaphylococcal antibody revealed that there were nine major epitopes. Discriminant analysis demonstrated that particular combinations of these epitopes were recognised more frequently by sera from dogs in different clinical groups.     

Serum and mucosal antibody responses against Mycoplasma hyopneumoniae following intraperitoneal vaccination and challenge of pigs with M hyopneumoniae

Pigs were immunised intraperitoneally when six weeks old and again at about 10 weeks old with killed Mycoplasma hyopneumoniae antigen prepared in an oil adjuvant. The pigs were challenged with live M hyopneumoniae (Beaufort strain) at between 11 and 15 weeks old. Antigen specific antibody levels for both IgG and IgA classes in serum and respiratory tract secretion were monitored over time. In serum anti-M hyopneumoniae antibody was detected shortly after the second intraperitoneal vaccination and was largely IgG. In respiratory tract secretion the response was observed after challenge, and was primarily IgA. Anti-M hyopneumoniae antibody-containing cells and their immunoglobulin class specificity were monitored in lung and tracheal lamina propria. In lung the majority of anti-M hyopneumoniae-containing cells were IgG, whereas in the tracheal lamina propria the majority were IgA. These results are discussed in terms of the use of intraperitoneal vaccination for the control of M hyopneumoniae infection.     

Circadian variations in salivary chromogranin a concentrations during a 24-hour period in dogs

The purpose of this study was to determine if salivary chromogranin a secretion in dogs exhibits a circadian rhythm. Saliva sampling was performed during three different sessions occurring in three nonconsecutive 24-h periods. Sixteen healthy adult beagle dogs (8 males and 8 females) were moved to a sampling room and housed individually in cages. Saliva samples were obtained every 4 h from 12:00 p.m. to 12:00 p.m. the following day. In the interest of habituation, saliva was obtained hourly from each dog 3 h before the experiment was started. Salivary chromogranin A concentrations were measured using an enzyme-linked immunosorbent assay. No circadian rhythm was detected for salivary chromogranin A secretion, and no differences in salivary chromogranin A concentrations measured every 4 h were demonstrated during the 24-h cycle in dogs.     

Comparative efficacy of an injectable vaccine and an intranasal vaccine in stimulating Bordetella bronchiseptica-reactive antibody responses in seropositive dogs

OBJECTIVE: To compare antibody responses to intranasal and SC Bordetella bronchiseptica vaccines in seropositive dogs. DESIGN: Randomized controlled study. ANIMALS: 40 young adult Beagles vaccinated against B bronchiseptica. PROCEDURE: Dogs were randomly assigned to 1 of 4 groups (intranasal vaccine, SC vaccine, intranasal and SC vaccines, no vaccine) and vaccinated on day 0. Serum and salivary B bronchiseptica-reactive antibody responses were measured on days 0 through 7, 10, 14, 21, and 28. RESULTS: Dogs that were vaccinated with the SC vaccine, alone or in combination with the intranasal vaccine, had a significant increase in serum concentration of B bronchiseptica-reactive IgG beginning on day 5 and persisting through day 28. Dogs that were vaccinated with the intranasal vaccine alone had a significant increase in serum concentration of B bronchiseptica-reactive IgG beginning on day 10 and persisting through day 28, but serum IgG concentration in these dogs was significantly less than concentration in dogs that received the SC vaccine. Neither vaccine had a demonstrable effect on salivary concentrations of B bronchiseptica-reactive IgA or IgG. On day 10, all vaccinated groups had significantly higher serum IgA concentrations than did unvaccinated control dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the SC B bronchiseptica vaccine may be used to stimulate antibody responses in seropositive dogs. There was no apparent benefit to administering these vaccines simultaneously. Intranasal vaccines may not be effective for booster vaccination of dogs previously exposed to or immunized against B bronchiseptica. Dogs should be vaccinated at least 5 days prior to exposure to B bronchiseptica.     

Salivary and serum immunoglobulin levels in cats with chronic gingivostomatitis

The salivary and serum concentrations of immunoglobulins G, M and A (IgG, IgM and IgA), and the salivary concentrations of albumin were measured by ELISA in 30 cats with chronic gingivostomatitis and 32 healthy cats. The cats with chronic gingivostomatitis had significantly higher salivary concentrations of IgG, IgM and albumin, and higher serum concentrations of IgG, IgM and IgA, but significantly lower salivary concentrations of IgA than the healthy cats. The cats with chronic gingivostomatitis were treated with either methylprednisolone, sodium aurothiomalate, metronidazole and spiramycin, or oral hygiene products. After three months of treatment, the cats receiving methylprednisolone had a significant reduction in serum IgG levels compared to the cats treated with sodium aurothiomalate or metronidazole and spiramycin, but after six months of treatment there were no significant differences between the groups. Before the treatments, the levels of oral inflammation were not correlated significantly with any of the serum or salivary immunoglobulin levels. However, the changes in oral inflammation were correlated significantly with the changes in the salivary IgM concentration after three and six months of treatment, and with the change in the salivary IgA concentration after six months of treatment.     

Immunoglobulin class response to canine distemper virus in gnotobiotic dogs

Serial serum samples from 27 gnotobiotic dogs infected with R252-canine distemper virus (CDV) were tested for anti-viral IgG, IgM and IgA immunoglobulins using an enzyme-linked immunosorbent assay (ELISA). The results were compared retrospectively to clinicopathological course of disease and to previously reported patterns of complement-fixing and virus neutralizing antibody titers determined in these same sera. Virus-specific IgA was never detected in the sera. High levels of IgG correlated with recovery from disease, whereas the antiviral IgM levels were equivalent in both persistently infected animals and those animals which recovered from disease. The inability to sustain a significant antiviral antibody response in either IgM or IgG classes was characteristic of dogs with fatal encephalitis. The data suggests that IgG is the most important Ig class for recovery from disease.     

Detection of Leishmania infantum DNA by real-time PCR in canine oral and conjunctival swabs and comparison with other diagnostic techniques

The use of non invasive sampling, such as collection of conjunctival swabs, as a diagnostic tool for the detection of Leishmania DNA is of interest. The purpose of this study was to evaluate the diagnostic utility of detecting Leishmania infection with the use of conjunctival swab samples in dogs living in a highly endemic area for leishmaniosis and to investigate, for the first time, the presence of Leishmania DNA in oral swabs in the same population. One hundred sixty-three dogs living outdoor and recruited in various provinces of Sicily were studied. Leishmania infantum indirect fluorescent antibody test (IFAT), delayed-type hypersensitivity reaction to leishmanin (DTH) and real-time PCR of blood (BL), lymph node (LN), conjunctival (CS) and oral swab (OS) samples were performed. The positive PCR percentages in LN, CS, OS and BL samples were: 24.5%, 22.1%, 8.7% and 5.5%, respectively. Serological and DTH positive percentages were 27.0% and 73.8%, respectively. Seropositive and LN-PCR positive dogs had a high likelihood to be positive by CS-PCR. The similar positive PCR percentages found in CS and LN samples suggest the use of CS-PCR as non-invasive alternative technique to LN-PCR for the detection of Leishmania infection in dogs. In addition, this study demonstrated, for the first time, the presence of Leishmania DNA in oral swabs in dogs.     

Detection of anti-Leishmania infantum antibodies in sylvatic lagomorphs from an epidemic area of Madrid using the indirect immunofluorescence antibody test

An outbreak of human leishmaniosis was confirmed in the southwest of the province of Madrid, Spain, between July 2009 and December 2012. Incidence of Leishmania infection in dogs was unchanged in this period, prompting a search for alternative sylvatic infection reservoirs. We evaluated exposure to Leishmania in serum samples from animals in the area with an indirect immunofluorescence test (IFAT). Using promastigotes from six culture passages and a 1/25 threshold titer, we found anti-Leishmania infantum seroreactivity in 9.3% of cats (4 of 43), 45.7% of rabbits (16/35) and 74.1% of hares (63/85). Use of promastigotes from >10 in vitro passages resulted in a notably IFAT lower titer, suggesting antigenic changes during extended culture. Postmortem inspection of seropositive animals showed no clinical signs of infection. The results clearly suggest that asymptomatic hares were the main reservoir in the outbreak, and corroborate IFAT as a sensitive serological surveillance method to detect such cryptic Leishmania infections.     

Salivary testosterone measurements in growing pigs: validation of an automated chemiluminescent immunoassay and its possible use as an acute stress marker

This study aimed to validate the use of an automated chemiluminescent immunoassay analyser for salivary testosterone measurements in growing pigs and study how circadian pattern during daytime and stress can influence its values. The test method had intra- and inter-assay coefficient of variation lower than 10%. The method showed good linearity and recovery, and detection limits were low enough to detect salivary testosterone levels. No significant differences were observed in testosterone concentrations at different sampling time, and age and gender did not influence circadian pattern. In addition, this assay was used to quantify testosterone in two models of acute stress and, in both cases, significant increases (P < 0.01) in salivary testosterone were detected. Therefore, the automated assay system tested for porcine testosterone determinations would be suitable for its use in saliva samples and, furthermore, salivary testosterone levels could be used as a possible marker of acute stress in pigs.     

Change of antibody levels to ferritin in the sera of foals after birth: Possible passive transfer of maternal anti‐ferritin autoantibody via colostrum and age‐related anti‐ferritin autoantibody production

Antibody (immunoglobulin G (IgG), IgM or IgA) levels relative to ferritin in six foal sera (three male and three female) after birth (day 0 and 2, 6, 10, 20, 28, 36, 40, 52 and 56 weeks of age) were semi‐quantitatively measured with normalization with antibody activity to ferritin in one adult horse serum. After addition of horse spleen ferritin to the serum sample, the complex formed between antibodies to ferritin in the serum and ferritin was co‐immunoprecipitated using antibody to horse spleen ferritin. Antibody classes of the co‐immnoprecipitate were detected with antibodies specific for horse IgG, IgM or IgA heavy chain. Six adult horse serum samples were found to have ferritin‐binding activities in all immunoglobulin classes examined. Although ferritin antibody activities (IgG, IgM and IgA) were scant in the foal sera before sucking colostrum (day 0), their activities increased at 2 weeks of age. IgG antibodies showed a biphasic response and IgM antibody activity increased up to 40 weeks of age. Antibody (IgG, IgM and IgA) activities to ferritin in three colostrum samples were significantly higher than in adult horse serum samples. These results demonstrate that antibody to ferritin in foal serum is derived from colostrum after birth and is produced thereafter.     

Quantitative Changes Associated with Calving in the Levels of Bovine Immunoglobulins in Selected Body Fluids I. Changes in the Levels of IgA, IgG1 and Total Protein

Levels of bovine IgA, IgG1 and total protein (TP) were determined in serum, saliva, tears and individual quarter lacteal secretions of six Holstein-Friesian cows sampled from six weeks before to four weeks after parturition. Hierarchal analyses of variance indicated significant variations among weeks, cows and quarters of the udder. A precipitous but non proportional drop in the levels of IgA and IgA1 in lacteal secretions occurred at calving. There was a concomitant increase in IgG1, and decrease in IgA, in serum. Correlation studies supported the concept of selective transport of IgG1 from serum to lacteal secretions in regulated amounts independent of serum IgG1 levels. Changes in the IgG1/TP ratio of serum and lacteal secretions supported the idea of a decrease in the selective transport mechanism. Correlation studies and estimations of secretory IgA (SIgA) in serum suggest that serum IgA is derived from IgA synthesized in secretory tissues. Highly significant correlations between IgA and IgG1 levels in all secretions postpartum suggest that local IgA synthesis and either IgG1 transport or local IgG1 synthesis are initiated by the same stimuli. Although some of the variation in the level reported for IgA and IgG1 in secretions resulted from protein dilution, much of the variation represents physiological differences between individual animals and tissues in the same animal. An IgG2/IgG1 ratio approaching that of serum occurred in a mastitic quarter of one cow. IgA was the principal immunoglobulin in saliva and tears, comprised a greater proportion of the immunoglobulin in milk whey than in prepartum lacteal secretions and was a minor immunoglobulin in bovine serum.     

Immunoglobulin isotype distribution of antinuclear antibodies in dogs with leishmaniasis

A modified indirect immunofluorescence method, using rat liver as substrate, was developed to determine the immunoglobulin isotypes forming antinuclear antibodies in sera from 12 antinuclear antibody-positive dogs out of 121 dogs with natural Leishmania infection. Immunoglobulin M was found to be the most frequent component of antinuclear antibodies (91·7 per cent), followed by IgG (41·7 per cent) and IgA (33·2 per cent). When these immunoglobulin isotypes were titrated, IgG antinuclear antibodies showed higher titres (1:200) than IgM and IgA antinuclear antibodies (1:50 and 1:20 respectively). Most of the antinuclear antibody-positive dogs simultaneously had two immunoglobulin isotypes, whereas none had all three immunoglobulin isotypes at the same time. Spearman rank correlation analysis showed no significant correlation between antinuclear antibody titres and circulating immune complexes or immunoglobulin levels. The low incidence of antinuclear antibodies and the absence of a clear relationship between isotype titres and clinical signs suggest a minor pathogenic role of antinuclear antibodies in canine leishmaniasis.     

Role of fimbriae F18 for actively acquired immunity against porcine enterotoxigenic Escherichia coli

Enterotoxigenic (ETEC) and enterotoxaemic (ETEEC) Escherichia (E.) coli that express F18 (F107) fimbriae colonize the small intestine and cause diarrhoea and/or oedema disease in weaned pigs. So far, two antigenic variants of F18 can be distinguished with a common antigenic factor designated ‘a’ and two specific factors called ‘b’ and ‘c’. In this study the existence of crosswise anti-colonization immunity between E. coli strains that express F18ab or F18ac fimbrial variants, respectively, was demonstrated. Weaned pigs of susceptible genotype with respect to susceptibility to adhesion of E. coli with fimbriae F18 were inoculated with E. coli strains 3064^STM (O157:K-:H-:F18ab; resistant to streptomycin) and 8199^RIF (O141ab:K-:H4:F18ac; resistant to rifampicin). The faecal shedding was compared subsequent to immunization and homologous or heterologous challenge. An enzyme-linked immunosorbent assay (ELISA) was applied to measure IgA, IgM and IgG antibodies against the F18ab and F18ac antigens in saliva, faeces, serum and intestinal wash samples. About 8 log CFU/g of the inoculated strains were found in faeces of all pigs following immunization as well as in non-immunized controls after challenge. Bacterial counts of the inoculated strains after challenge were between 2 and 5 log lower, without any difference between homologous and heterologous challenge. Intestinal colonization with fimbriated E. coli resulted in production of significantly increased levels of anti-fimbrial antibodies, especially IgA, in serum and intestinal wash samples. There were higher levels of homologous than of heterologous anti-fimbrial antibodies. Production of antibodies against F18a or against another common fimbrial antigen is probably responsible for crosswise anti-colonization immunity between E. coli strains with F18ab and F18ac fimbrial variants. Serum F18-specific IgA may be a useful indicator of a mucosal immune response directed against F18 fimbriae.     

Secretory patterns of growth hormone in dogs: circannual, circadian, and ultradian rhythms

The objective was to characterize the circannual, circadian, and ultradian secretory patterns of growth hormone (GH) in intact crossbred and purebred dogs. In all experiments, blood samples were collected with minimal stress by direct peripheral venipuncture and GH was measured in plasma by a homologous radioimmunoassay. For circannual studies, samples were collected monthly from 6 male dogs between 15:00 and 17:30 h over a 1-year time span. For circadian studies, blood samples were collected at 145-minute intervals from 09:00 to 06:45 h of the following day in 14 female dogs. In ultradian experiments, blood samples were collected at 15-minute intervals for 2.5 h (15:00 to 17:30 h) in 7 males and 7 females. Plasma GH in male dogs remained without change in summer, autumn, and winter but declined (P ≤ 0.01) in spring (LSM ± SEM; 6.9 ± 0.5; 6.0 ± 0.5; 6.3 ± 0.5; 4.3 ± 0.5 ng/mL, respectively). No plasma GH circadian rhythmicity was detected. Nor was any ultradian pattern evident in either males or females. No gender-related differences were observed in ultradian GH plasma profiles. It is concluded that, while basal GH levels show seasonal fluctuations in dogs, neither circadian nor ultradian GH secretory fluctuations were present in the dogs assessed.