Oral vaccination of dogs with a monovalent live-avirulent vaccine confers 1 year of immunity against Bordetella bronchiseptica challenge.

This experimental challenge study assessed immune protection 1 year after a single dose of live-attenuated oral Bordetella bronchiseptica (Bb) vaccine in dogs. Forty Bb-seronegative 7-9-week-old puppies were randomly assigned at Day 0 to receive a single oral dose of either Bb vaccine (n = 20; vaccinated group) or sterile water (n = 20; control group). Groups were housed separately until comingling 1 day pre-challenge (Day 365). Challenge with virulent aerosolized Bb occurred at Day 366. Clinical scores were obtained at Days 1-7, and 366-380. Bb microagglutination test (MAT) titers were obtained at Days -7, 0, monthly post-vaccination, and Days 358, 365, and 380. Nasal swabs were collected for microbiological assessment at Days -7, 0, 365, and 367-380. Oral Bb vaccination was not associated with side effects. Pre-challenge, vaccinated dogs developed persistent Bb MAT titers and control dogs remained seronegative. Post-challenge, duration of cough was longer in control dogs (least square means [LSM], 8.6 days) than vaccinated dogs (LSM, 1.5 days; P < 0.0001), with more control dogs having cough on 2 or more consecutive days (control group, n = 17/19, 89.5%; vaccinated group, n = 3/19, 15.8%; P = 0.0011). Post-challenge, Bb shedding occurred in all control dogs and 5/19 (26%) vaccinated dogs. Average duration of Bb shedding was longer in the control group (11.9 days vs. 0.6 days; P < 0.0001) and nasal Bb loads were higher in the control group (P < 0.00001). Orally administered Bb vaccine stimulated immunity that was still protective against virulent Bb challenge after 1 year.

Evaluation of 3 Serological Tests for Early Detection Of Leptospira-specific Antibodies in Experimentally Infected Dogs.

BACKGROUND: Leptospirosis in dogs is a disease of global importance. Early detection and appropriate therapeutic intervention are necessary to resolve infection and prevent zoonotic transmission. However, its diagnosis is hindered by nonspecific clinical signs and lack of rapid diagnostic tests of early infection. Recently, 2 rapid point-of-care tests (WITNESS Lepto [WITNESS Lepto, Zoetis LLC, Kalamazoo, MI, USA] and SNAP Lepto [SNAP Lepto, IDEXX Laboratories, Westbrook, ME, USA]) for detection of Leptospira-specific antibodies in canine sera were developed. HYPOTHESIS: Immunoglobulin M-based WITNESS Lepto containing multiple detection antigens can detect Leptospira-specific antibodies to common leptospiral serovars earlier in the course of infection as compared to microscopic agglutination test (MAT) and SNAP Lepto. ANIMALS: Four groups of 8 6- to 8-month-old male Beagle dogs were used. METHODS: Thirty-two healthy seronegative dogs were inoculated experimentally with serovars Canicola, Grippotyphosa, Icterohaemorrhagiae, and Pomona (8 dogs/serovar). Acute-phase sera were collected at regular intervals and monitored for Leptospira-specific antibodies by WITNESS Lepto, MAT, and SNAP Lepto. RESULTS: Seroconversion was detected in all dogs by day 10 by WITNESS Lepto and in 30 of 32 dogs by day 14 by MAT. The SNAP Lepto test detected seroconversion in 3 dogs during the 2 weeks postchallenge. CONCLUSIONS: Immunoglobulin M-based WITNESS Lepto detected immune responses specific to multiple leptospiral serovars early in the course of infection and identified seroconversion in all animals earlier than did the gold standard MAT. The SNAP Lepto test displayed considerably lower and inconsistent performance during the study period. At the point-of-care, WITNESS Lepto should be the test of choice for rapid and reliable screening of acutely ill dogs suspected to have leptospirosis.

Evaluation of a rapid IgM detection test for diagnosis of acute leptospirosis in dogs.

Recently, a lateral flow assay (LFA) for detection of Leptospira-specific IgM in canine sera became commercially available in Europe. The present study aims to evaluate the diagnostic performance of this assay using canine sera from a collection of diagnostic accessions. Diagnostic sensitivity was assessed by testing 37 acute-phase and 9 corresponding convalescent-phase sera from dogs with a confirmed diagnosis of leptospirosis. Specificity was determined by testing sera from sick dogs with non-leptospiral infections (n=15) and healthy dogs with incomplete history of vaccination (n=45). During acute phase of illness, LFA scored positive for 28/37 sera with a sensitivity of 75.7 per cent while only 9/37 (24.3 per cent) samples were positive on microscopic agglutination test. The specificity of the LFA was 98.3 per cent (59/60). This test showed 89.7 and 100 per cent overall agreements with clinical diagnosis for acute-phase and convalescent-phase sera, respectively. The impact of vaccination on the LFA was also determined and vaccine-stimulated IgM responses were negative in 19/25 (76 per cent) dogs at 12 weeks post vaccination. In conclusion, the LFA is a rapid and reliable test for early detection of Leptospira-specific IgM during acute phase of canine leptospirosis. However, interpretation of a positive result must be made in the context of clinical signs and vaccination history.

Biotypes and ScM types of isolates of Streptococcus canis from diseased and healthy cats.

Lancefield group G Streptococcus canis is a component of the normal urogenital and pharyngeal flora of the cat. It is also frequently implicated in epizootics of severe disease in closed cat colonies and animal shelters. Given the importance of S canis as a feline pathogen and relative lack of published information on characteristics potentially associated with virulence, the authors have compared isolates from healthy and diseased cats in New York and California using fermentation profiles (biotype) and ScM sequences. With few exceptions, isolates associated with disease were biotype 1. Four alleles of scm were identified of which type 1 dominated in diseased cats. Type 4 allelic variants were found only in healthy cats and all but one were biotype 2. Type 2 and 3 alleles showed extensive N-terminal variation suggesting a plasminogen-binding site as found on the type 1 allele was absent. Cat antisera to ScM were opsonobactericidal, and these potentially protective antibodies increased during convalescence.

Preliminary evaluation of a dual antigen ELISA to distinguish vaccinated from Leptospira infected horses.

Immunogenic proteins of Leptospira interrogans serovar Pomona type kennewicki (Lk) including Sph1, LigA, Hsp15 and LipL45 (Qlp42) are up-regulated in infected horses but are undetectable or expressed in trace amounts on cultured organisms. In contrast, LipL32 is abundant on cultured Lk and elicits infection antibody responses. The aim of this study was to develop an ELISA based on LipL32 or Lk sonicate and host-induced proteins to differentiate vaccine from infection serum antibody. IgG specific for recombinant Sph1, LigA, Lk90 (LigA; 379-1225 a.a), Hsp15, LipL45 and LipL32 of Lk were assayed in sera of horses infected naturally with Lk and before and after immunisation with serovar Pomona bacterin. Infection but not vaccine sera reacted strongly with Sph1, LigA and Lk90. LipL45 and Hsp15 reacted moderately with infection sera and weakly with vaccine sera. Lk sonicate and LipL32 reacted strongly with both infection and vaccine sera. As expected, culture-based vaccine failed to stimulate antibody to host-induced proteins. Therefore a dual antigen ELISA based on Lk sonicate or LipL32 combined with host-induced Sph1 and Lk90 will be valuable in differentiating infection from vaccine responses.

Multiple specificities of immunoglobulin M in equine fetuses infected with Leptospira interrogans indicate a competent immune response.

REASONS FOR PERFORMING STUDY: Foals of mares infected with Leptospira interrogans serovar Pomona type kennewicki (Lk) may be aborted/stillborn or delivered as healthy foals. Is fetal survival explained in part by the immune response of the fetus to Leptospira antigens? OBJECTIVES: To describe an outbreak of Leptospira abortion in which infected mares delivered dead/sick or normal foals and determine specificities of antibody in a collection of 54 fetuses from similar outbreaks. STUDY DESIGN: Outbreak investigation in combination with a case-control study of a larger set of samples from aborted fetuses. METHODS: Serology and polymerase chain reaction (PCR) on urine and amniotic fluids were used to diagnose infection during an outbreak of Leptospira abortion. Specificities of immunoglobulin (Ig)M, IgGa and IgGb for recombinant proteins of Lk in archived fluids of fetuses from similar outbreaks were compared by ELISA with those of fluids of fetuses not infected with Leptospira spp. RESULTS: Five fetuses of 11 infected mares in an outbreak survived in utero in the presence of persistent placental infection and were healthy at foaling. Fetuses of 6 mares in the outbreak were aborted or died soon after birth. Significantly greater (P<0.05) IgM reactivity with all recombinant proteins and with Lk sonicate was observed in 54 archived fluids from Leptospira infected fetuses than in fluids of 30 of non-Leptospira infected fetuses. Low levels of IgGa and IgGb specific for LipL32 and Lk sonicate and traces of LigA and Hsp15 specific IgGa were detected in a minority of archived fluids from Leptospira infected fetuses. CONCLUSION: Although mainly mediated by IgM, a high level of immune competence in aborted fetuses was evidenced by the multiplicity of Leptospira proteins targeted. This is likely to contribute to survival of foals in mares with evidence of placental infection at foaling as detailed in a typical outbreak.

Capsular hyaluronic acid of equine isolates of Streptococcus zooepidemicus is upregulated at temperatures below 35°C.

REASONS FOR PERFORMING STUDY: Streptococcus zooepidemicus causes opportunist respiratory and other infections in the horse. Capsule expression is highly variable and known to affect resistance to phagocytosis. Most clinical isolates producing small, dry colonies at 37°C produce mucoid colonies at temperatures below 35°C. OBJECTIVES: The aim was to understand the molecular basis of increased capsule expression by equine isolates of S. zooepidemicus at temperatures lower than 35°C. STUDY DESIGN: Cross-sectional observational study. METHODS: Capsule production by groups of equine S. zooepidemicus strains was determined at 23, 30, 35 and 37°C. Hyaluronidase (HylC) at 23 and 37°C was measured by quantitative enzyme-linked immunosorbent assay. Expressions of hasA and hylC at 23 and 37°C were measured by quantitative reverse transcriptase-PCR. The covRS genes in representative S. zooepidemicus were sequenced and checked for mutations. RESULTS: Colonies of randomly selected S. zooepidemicus strains became mucoid or showed marked increase in colony mucoidy following the change in temperature to 23°C. Expression of hasA at 23°C was 45- to 700-fold greater than at 37°C. Transcription of hylC at 23°C was 2.5- to 200-fold greater than at 37°C, yet enzyme concentrations in cultures were significantly higher at 37°C (P<0.05), suggesting that production of HylC is regulated post transcriptionally. The covRS gene in S. zooepidemicus was not mutated as seen in isolates of Streptococcus pyogenes with increased capsule production at 25°C. CONCLUSIONS: Sensitivity of capsule expression to temperature above 35°C but not HylC by the general population of equine S. zooepidemicus indicates that capsule is not required for extended colonisation nor for opportunist invasion. Instead, capsule production at lower than body temperature may reflect adaptation to life on skin and mucosal surfaces, where hyaluronic acid contributes to adhesion and resistance to desiccation. Pathogenicity of S. zooepidemicus following opportunist invasion is possibly dependent on factors other than capsule that may be co-regulated with HylC. The Summary is available in Chinese - see Supporting information.

Surface-associated Hsp60 chaperonin of Leptospira interrogans serovar Autumnalis N2 strain as an immunoreactive protein.

The present study has been formulated in order to detect an immunoreactive protein whose identification can play a major role in the early diagnosis of disease. The identified protein will be produced by recombinant methods and used for the recombinant protein based ELISA. A comparison was made between the developed method and the gold standard MAT test to evaluate the serodiagnosis potential of the protein. The protein profile, immunoblot and MALDI-TOF analysis was carried out to identify the immunoreactive protein. The immunoreactive protein identified was used to develop ELISA for the diagnosis of leptospirosis using patients' sera with various clinical manifestations. The immunoreactive protein was identified as Leptospira GroEL chaperonin of molecular weight 60 kDa. The theoretical/experimental molecular weights, pI were found to be 58.5/60 kDa and 5.41/6, respectively. The overall results of the recombinant GroEL-IgM ELISAs showed cumulative sensitivity, specificity, positive predictive value, and negative predictive values of 90.6%, 94.9%, 94.6%, and 91.0%, respectively. The performance of such ELISA appeared better than that of any other serological tests previously evaluated for the diagnosis of leptospirosis in India. Thus, a highly conserved and immunogenic outer exposed GroEL protein during infection clearly merits further use in the serodiagnosis of leptospirosis.

Serological evaluation of leptospirosis in Hyderabad Andhra Pradesh: a retrospective hospital-based study.

PURPOSE: Leptospirosis is a zoonotic disease with humans getting the infection either from rodent hosts or from domestic animals. Urine contaminated environment is the common source of infection. This is an under-reported disease in Andhra Pradesh. We report a retrospective hospital-based study on 55 patients with suspected leptospirosis. METHODS: A total of 55 serum samples were collected from patients with suspected leptospirosis and subjected to serological testing by LeptoTek Dri-dot, microscopic agglutination test (MAT) and IgM enzyme-linked immunosorbent assay (ELISA). Identification of the predominant infecting serotype was done using a panel of 12 serovars. RESULTS: MAT analysis of all the 55 samples identified all cases to be positive. The predominant serogroup was Icterohaemorrhagiae (68%) followed by Australis (22%), Autumnalis (8%) and Javanica (2%). LeptoTek Dri-dot showed a sensitivity of 96% as compared to MAT. IgM ELISA done on 32 samples showed a sensitivity of 86.7% compared to MAT. CONCLUSIONS: MAT helped to identify Icterohemorrhagiae as the predominant serovar in this study. Despite the small number of samples analyzed, the data obtained establishes a need for a prospective study in this region.