Determination of specific anti-leptospiral immunoglobulins M and G in sera of experimentally infected dogs by solid-phase enzyme-linked immunosorbent assay

The development and evaluation of an enzyme-linked immunosorbent assay (ELISA) to detect specific anti-leptospiral IgM and IgG in sera of dogs experimentally infected with Leptospira interrogans serotype canicola are reported. In all dogs specific anti-leptospiral IgM was detected from the second half of the first week after infection, the maximum being attained during the second week. Subsequently the IgM titre gradually decreased. Specific anti-leptospiral IgG was detected later and increased gradually to reach almost the same level as the IgM titre after two to three months. During the initial stage of the infection, when the microscopic agglutination titre was still negative or very low, a high IgM titre was accompanied by a negative or very low IgG titre in every case. After the initial stage a substantial IgG titre was also detectable. It is suggested that the test is suitable for serodiagnostic purposes, particularly for the diagnosis of a current infection in an individual.     

Rabies antibody titres in vaccinated dogs

In a field study, rabies virus neutralizing antibody titres were determined by the microtest modification of the rapid fluorescent focus inhibition test before and after primary vaccination in 30 puppies, and before and after booster vaccination in 59 previously vaccinated dogs. A commercial modified live virus vaccine was used. Three weeks after primary vaccination the mean antibody titre was 102 ± 90, but only 24 dogs presented for booster vaccination had detectable antibody levels (mean titre 12 ± 16). The antibody responses three weeks after booster vaccination (mean 380 ± 216) were significantly greater than the responses to primary vaccination. It was concluded that previously vaccinated dogs could have an anamnestic response to booster vaccination, even when antibodies were not detected in their sera before revaccination.     

Clinical, pathological and serological features of spontaneous canine leptospirosis. An evaluation of the IgM- and IgG-specific ELISA

The clinical, pathomorphological and serological features of acute canine leptospirosis are evaluated and the IgM- and IgG-specific ELISA for leptospirosis serology in dogs is assessed. The clinical syndrome of acute canine leptospirosis was characterized by depression, anorexia, vomiting and often haemorrhagic diarrhoea. In addition, jaundice, uraemia, elevated creatinine and alkaline phosphatase were observed in the majority of the dogs. In pups invagination of the intestines was a noteworthy finding. The clinical signs and the post-mortem findings were rather non-specific so that the clinical and post-mortem diagnosis had to be confirmed serologically. In acute clinical cases of canine leptospirosis a high anti-leptospiral IgM titre, ranging from 160 in pups to 10240 in adults, was always present, whereas the anti-leptospiral IgG titre and the agglutination titre usually were negative or low. Dogs died from leptospirosis in spite of a high anti-leptospiral IgM titre. Only two dogs having, at the first examination, a high IgM titre in conjunction with a high IgG titre survived an acute infection. The possible role of IgM and IgG in the pathogenesis of an acute leptospiral infection is discussed. Different serological patterns in reference dogs, which were not suffering from acute leptospirosis, are presented.     

Effect of age on immune parameters and the immune response of dogs to vaccines: a cross-sectional study

The evaluation of anti-aging intervention strategies in dogs would benefit from reliable quantitative biomarkers of aging. In the present study, the expression of various immune parameters was measured in young and old dogs to identify potential biomarkers of aging. The second goal of the study was to determine the effect of age on the immune response to vaccines. The immune function, including the antibody response to vaccines, was determined in 32 young adult (3.15+/-0.8 years of age) and 33 old dogs (12.1+/-1.3 years of age) of various breeds. Old dogs had a significantly lower lymphocyte proliferative response and a lower percentage of CD4+ T cells and CD45R+/CD4+ T cells, and a higher percentage of CD8+ T cells and a higher concentration of serum and salivary IgA. The most significant differences (P<0.001) occurred in the lymphocyte proliferative responses to ConA and PHA, the CD4:CD8 ratio, and the percentage of CD45R+/CD4+ T cells suggesting that these parameters are potential biomarkers of aging. There was no difference in the percentage of total T and B lymphocytes and the concentration of serum IgM and IgG. Both groups of dogs had protective titers against distemper virus, parvovirus and rabies virus before annual revaccination. The pre-vaccination titer against rabies virus was higher in the old dogs than in the young dogs, and there were no differences in post-vaccination titers against any of the viruses. This suggests that annual vaccination protocols provide adequate protection for old dogs.     

Application of a dot enzyme-linked immunosorbent assay for evaluation of the immune status to canine parvovirus and distemper virus in adult dogs before revaccination.

A growing body of literature has been published indicating that the current practice of annual vaccination of dogs may not be beneficial and in some cases may even be harmful. A number of publications have proposed assessing the immune status of dogs before annual revaccination. In this study the usefulness of a commercially available dot-ELISA kit was evaluated to determine the duration IgG antibody titers to canine parvovirus (CPV) and canine distemper virus (CDV) in 158 dogs vaccinated at least one year ago. Overall, the percentage of dogs with protective antibody titers to both CPV and CDV was 84%. The percentage of dogs with borderline antibody titers was 11% for CPV and 10% for CDV. Four percent of the dogs had no detectable antibody to CPV and 6% had no antibody to CDV. The results reported here are in good agreement with other studies measuring IgG antibody levels. It is concluded that the kit offers veterinarians the opportunity of determining antibody titers and revaccinating only those pets whose antibody titers to specific diseases have waned.     

Humoral immune response to avian influenza vaccination over a six-month period in different species of captive wild birds

In December 2005, the four major Swiss zoos carried out the vaccination of selected zoo birds with the adjuvant inactivated vaccine H5N2 Nobilis influenza. Pre- and post-vaccination antibody titers were determined either by hemagglutination inhibition (HI) test (non-Galliformes) or by enzyme linked immunosorbent assay (ELISA) (Galliformes) at Week 0, 5, 10, and 26 (Day 0-1, 35-36, 70-71, and 182 respectively) to determine the humoral immune response to H5 antigen. After the first vaccination, the overall geometric mean titer of non-Galliformes was 65 (n = 142), which increased to 187 (n = 139) after booster vaccination and dropped to 74 (n = 65) six months after first vaccination. For the Galliformes group, the mean titers were found to be 2.09 at Week 5 (n = 119), 3.24 at Week 10 (n = 113), and 1.20 at Week 26 (n = 39). Within the non-Galliformes, significant differences in geometric mean titers were found among different species representatives. In general, the flamingos (Phoenicopteriformes) showed a strong response to vaccination, reaching a geometric mean titer of 659 at Week 10, while the Sphenisciformes did not show high antibody titers even after booster vaccination, reaching a maximum geometric mean titer of only 65. Based on the antibody titer profiles of all investigated species, we recommend at least annual revaccination for the species that we investigated.     

Serological response to a booster foot-and-mouth disease vaccination with strains different from the primary vaccine

Young calves were vaccinated with Belgian foot-and-mouth disease (FMD) vaccine and revaccinated with either the same vaccine or with a foreign FMD vaccine.There was a significant serological response to the primary vaccine strains after the first vaccination which was greater following revaccination. At one and two months after revaccination there was no significant difference between the responses to revaccination with vaccine identical to the primary vaccine or with the foreign FMD vaccine.It was concluded that revaccination of young calves is effective even with an FMD vaccine different from the primary vaccine.     

Onset and duration of immunity against Babesia canis infection in dogs vaccinated with antigens from culture supernatants

It has previously been shown that dogs can be vaccinated against heterologous Babesia canis infection using a vaccine containing soluble parasite antigens (SPA) from in vitro cultures of B. canis and B. rossi that are adjuvanted with saponin. In the present study the onset and duration of immunity of vaccinated dogs were studied. Results showed that 3-26 weeks after initial vaccination, dogs effectively limit the level of SPA in plasma upon challenge infection, which was reflected in limited duration and extent of clinical manifestations. There was no statistically significant effect of vaccination on the parasite load in the circulation, which was determined from blood smears. It was further shown that the level of immunity of primary vaccinated dogs (priming and booster vaccination with a 6-week interval) and that of repeatedly vaccinated dogs (a single additional vaccination 6 months after primary vaccination) is comparable. From this study it is concluded that vaccination with this preparation induces protective immunity against clinical babesiosis from 3 weeks after booster vaccination onwards, and remains effective for a period of at least another 6 months. A single booster vaccination is sufficient to maintain immunity for at least another 6 months.     

Antibody response in bovine pharyngeal fluid following foot-and-mouth disease vaccination and, or, exposure to live virus

Samples of pharyngeal fluid and serum were collected from cattle after exposure to live foot-and-mouth disease (FMD) virus (with or without prior vaccination) or after subcutaneous vaccination with inactivated virus. The pharyngeal fluid samples were examined for FMD neutralising activity and specific anti-FMD IgG, IgM and IgA antibodies. The neutralising activity of the serum was also monitored. A peak of neutralising activity which occurred in the pharyngeal fluid of unvaccinated cattle seven days after virus exposure corresponded to a rise in specific IgM and IgA antibodies. This peak appeared to be due to serum and tissue fluid escaping from the damaged mucosa during the acute inflammatory phase of infection. At later stages (20 to 60 days after virus exposure) the pharyngeal fluid neutralising activity corresponded to a rise in specific IgA antibodies, suggesting that active local antibody production was taking place. The pharyngeal fluid neutralising activity detected after revaccination with oil emulsion or aqueous vaccines, without exposure to live virus, corresponded to a rise in specific IgG and IgM antibody levels and this may have been due to serum transudation.     

Diffusion in gel-enzyme linked immunosorbent assay (DIG-ELISA) to record the immunoglobulin response of calves vaccinated with Salmonella

A diffusion in gel-enzyme linked immunosorbent assay (DIG-ELISA) was used to record immunoglobulin development of colostrum-fed calves vaccinated with aromatic dependent (aro-) Salmonella and challenged with either the homologous as a heterologous serotype. IgG was detected by using a peroxidase conjugated rabbit antibovine IgG, whereas IgM and IgA were measured using a double sandwich technique. Although IgG levels to Salmonella endotoxins increased after exposure to Salmonella, they were found to be high in many calves prior to vaccination. However, IgM antibody levels were consistently low prior to vaccination, and their increase was a more reliable indicator of the vaccination and immune status of the calves. IgA levels were generally low and of less predictive value.     

Walking the dog and moving the cat: rabies serology in the context of international pet travel schemes

Data of 13'469 blood samples from 10'999 dogs and 2'470 cats tested for rabies neutralizing antibodies within the framework of pet travel schemes were analysed for single and combined factors influencing antibody titres and failures. The time span between vaccination and drawing the blood sample was confirmed as a major source of failure in dogs with a proportion of 23 % at 4 months after primary vaccination (single dose). Failures in dogs and cats (titre < 0.5 IU) were significantly reduced after double primary vaccination (2 doses within 7 - 10 days), although failures reached comparable levels in dogs as early as 6 months after vaccination. In contrast, failure after vaccination was generally below 5 % in dogs and absent in cats after a booster applied at earliest 12 months after single primary vaccination. Statistically significant differences between the failures of the vaccine brands ?Rabisin? (1.5 %), ?Defensor? (6.7 %), ?Nobivac Rabies? (11.0 %) and ?Rabdomun? (18.2 %) were found in dogs but also between the titres induced in cats. Significant differences were found between different dog breeds with some small breeds showing a significantly higher responsiveness. Taken together, a new regimen for rabies vaccination consisting of double primary vaccination with a short interval of 7 - 10 days and a one-year booster appears to be highly recommended for dogs and cats.     

Chromatographic Studies of Sera from Calves Vaccinated with Brucella Abortus, Strain 19

The presence of antibody was detected by agglutination tests in the serum of calves four days after vaccination with Brucella abortus strain 19. Titres had reached a maximum by seven to ten days post-vaccination. Sucrose density-gradient ultracentrifugation demonstrated that the earliest antibodies were macroglobulins, IgM (19Sγ; γM)-globulins. Lighter antibodies, IgG (7Sγ₂; γG)-globulins, appeared a few days later. With time, antibody titres fell, IgM declining somewhat more quickly than IgG. After revaccination some seven months later, there was a rapid rise in both IgM and IgG.

Anion-exchange column chromatography (DEAE-cellulose) and gel filtration (Sephadex G-200) were applied in separating the two forms of antibody. The former method, in which a gradient buffer system was used, proved to be the more efficient; the IgG antibodies apeared in early eluates at pH 7.8 to 8.0 and low ionic strength, 0.03M, whereas IgM was eluted late when the pH had fallen below 6.0 and the molarity had increased to beyond 0.2. DEAE cellulose chromatography detected IgG as well as IgM sera collected as early as five days after vaccination.

     

Immunoglobulins in cattle vaccinated with leptospiral bacterins.

The growth-inhibition test was used to detect specific antibodies against Leptospira interrogans serotype hardjo in isolated immunoglobulin (IgG and IgM) fractions of serums from cattle vaccinated with leptospiral bacterins. The growth-inhibiting antibodies were detected mainly in the IgG class. Agglutinated clumps also occurred with the IgM fraction. The serums collected from cattle 4 months after vaccination were negative in the microscopic agglutination test.     

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.     

Immune responses of Asian elephants (Elephas maximus) to commercial tetanus toxoid vaccine

Although captive elephants are commonly vaccinated annually against tetanus using commercially available tetanus toxoid vaccines marketed for use in horses and livestock, no data exists to prove that tetanus toxoid vaccination produces measurable antibody titers in elephants. An ELISA test was created to measure antibody responses to tetanus toxoid vaccinations in 22 Asian elephants ranging in age from 24 to 56 years (mean age 39 years) over a 7-month period. All animals had been previously vaccinated with tetanus toxoid vaccine, with the last booster administered 4 years before the start of the study. The great majority of elephants had titers prior to booster vaccination, and following revaccination all elephants demonstrated anamnestic increases in titers, indicating that this species does respond to tetanus vaccination. Surprisingly older animals mounted a significantly higher response to revaccination than did younger animals.     

The Response of Ponies to Myxovirus influenzae A-equi 2 I. Serum and Antibody Titres Following Exposure

The antibody response in serum and nasal secretions of groups of ponies vaccinated or infected with Myxovirus influenzae A-equi 2 was examined. Following infection by aerosol with live virus, a weak antibody response was recorded in both serum and secretions. Antibody levels were undetectable in secretions at 31 days after infection.

After primary intramuscular vaccination with killed virus, using sodium alginate as an adjuvant, antibody was detected only in the serum. However, following revaccination, a pronounced antibody response was demonstrated in both serum and secretions. Antibody was still detectable in all four ponies when tested 135 days later.

Only a serum antibody response was detected in ponies after primary intramuscular vaccination with a commercial vaccine. Upon revaccination nasal antibody occurred in all ponies but this only persisted for about 30 days.

Neither serum nor nasal antibody response occurred following intranasal vaccination and revaccination with a killed virus vaccine.

     

Immunoglobulin deficiency in Cavalier King Charles Spaniels with Pneumocystis pneumonia

Serum concentrations of immunoglobulin G (IgG), IgM, and IgA were measured in 9 Cavalier King Charles Spaniels with pneumonia caused by Pneumocystis sp that were examined at 4 veterinary surgeries in the United Kingdom (UK) between September 2001 and November 2002. Pneumocystis pneumonia was confirmed in all dogs by visualization of the organism in bronchoalveolar lavage fluid or a transthoracic lung aspirate. Two dogs had a history of demodicosis. Immunoglobulin concentrations also were measured in breed-and age-matched dogs sampled over the same period. IgG concentrations were significantly (P = .000) lower in the affected dogs (median 3.2 mg/mL) than in the control dogs (median 8.5 mg/mL). IgM concentrations were significantly (P = .002) higher in the affected dogs (median 1.95 mg/mL) than in the control dogs (median 1.12 mg/mL). One affected dog had no change in IgG concentration more than 3 months after resolution of infection or vaccination, but did have reduction in IgM concentration after resolution of infection and vaccination. Control dogs had low serum IgG and IgM concentrations, compared with the reference interval for all dogs. Lymphocyte count in blood was normal or high in 7 of 8 affected dogs. The results of this study suggest that there is a defect in immunity in Cavalier King Charles Spaniels that underlies the susceptibility of these dogs to pneumocystosis. Further studies are indicated to elucidate the mechanisms behind the defect, the prevalence within the breed, and the potential mode of inheritance of the problem.     

Serological testing—An alternative to boosters?

The issue of the duration of immunity, particularly for the modified live viral components of veterinary vaccines, has been a significant part of the recent vaccination debate. One manufacturer has increased the recommended booster interval for these components to 3 years give name and another now states 'up to 4 years' immunity. There remain many unanswered questions regarding this duration of immunity (DOI). Studies suitable for data sheet claims are time consuming and costly and can only be performed in laboratory dogs under tightly controlled conditions. Evidence from rabies serology testing in the UK shows that the response of individual animals to routine vaccination is highly variable. Much of the published field evidence on the persistence of antibody titres originates from North America, where vaccination strategies and reservoir species differ from Europe. Quantifying the effect of exposure to field virus on the maintenance of immunity in these studies is impossible, and little is known of the circulation of virus in unvaccinated dogs and wild mammals throughout Europe. If owners or vets are concerned about revaccination one option is to assess the need for each booster by performing a blood test. There is some published evidence of the relationship between antibody titres and protective immunity, and tests are available to measure responses to individual viral components of the routine canine and feline vaccines. It must be remembered that most commercial tests to assess immunity only measure antibodies, which are only one aspect of the immune response to vaccination. It is therefore possible that animals without or with low antibody titres are in fact protected. Serological tests are an option if owners are unwilling to have their animal boostered without evidence that it is needed. However, the cost of these tests is likely to exceed that of booster vaccination for the foreseeable future.     

Results of vaccination of Asian elephants (Elephas maximus) with monovalent inactivated rabies vaccine

OBJECTIVE: To evaluate the humoral immune response of Asian elephants to a primary IM vaccination with either 1 or 2 doses of a commercially available inactivated rabies virus vaccine and evaluate the anamnestic response to a 1-dose booster vaccination. ANIMALS: 16 captive Asian elephants. PROCEDURES: Elephants with no known prior rabies vaccinations were assigned into 2 treatment groups of 8 elephants; 1 group received 1 dose of vaccine, and the other group received 2 doses of vaccine 9 days apart. All elephants received one or two 4-mL IM injections of a monovalent inactivated rabies virus vaccine. Blood was collected prior to vaccination (day 0) and on days 9, 35, 112, and 344. All elephants received 1 booster dose of vaccine on day 344, and a final blood sample was taken 40 days later (day 384). Serum was tested for rabies virus-neutralizing antibodies by use of the rapid fluorescent focus inhibition test. RESULTS: All elephants were seronegative prior to vaccination. There were significant differences in the rabies geometric mean titers between the 2 elephant groups at days 35, 112, and 202. Both groups had a strong anamnestic response 40 days after the booster given at day 344. CONCLUSIONS AND CLINICAL RELEVANCE: Results confirmed the ability of Asian elephants to develop a humoral immune response after vaccination with a commercially available monovalent inactivated rabies virus vaccine and the feasibility of instituting a rabies virus vaccination program for elephants that are in frequent contact with humans. A 2-dose series of rabies virus vaccine should provide an adequate antibody response in elephants, and annual boosters should maintain the antibody response in this species.     

Homotypic and heterotypic serum and milk antibody to rotavirus in normal, infected and vaccinated horses.

The homotypic and heterotypic antibody response to rotavirus was determined in three pony mares and their foals. The normal concentrations of anti-rotavirus antibodies in mares' milk and mares' and foals' serum over the first 10 weeks post-partum were measured using IgA, IgG and rotavirus serotype-specific enzyme linked immunosorbent assays. Experimental infection of the foals with serotype 3 equine rotavirus produced a rapid, serotype-specific response which peaked 10 days after infection and a slower heterotypic response which peaked 32 days later. In contrast, vaccination of the mares with an inactivated, adjuvanted serotype 6 bovine rotavirus produced a heterotypic response similar to that of the homotypic response in both serum and milk, although the predominant response in serum was IgG, while in milk it was IgA. These results suggest that non serotype-restricted passive protection of foals against rotavirus may be achieved by parenteral vaccination of mares.