Clinical and serologic evaluation of cats with cryptococcosis.

OBJECTIVE: To evaluate the long-term clinical outcomes and serologic changes in cryptococcal antigen and antibody titers in cats with confirmed Cryptococcus neoformans infection. DESIGN: Prospective case series. ANIMALS: 47 cats with cryptococcosis. PROCEDURE: Cats included in this study were determined to have cryptococcosis on the basis of identification of C neoformans on histologic or cytologic examination, isolation of C neoformans in culture, or positive serologic test results for cryptococcal antigens. Information concerning the signalment, history, physical examination findings, FeLV and feline immunodeficiency virus status, serologic testing, treatment, and outcome for each cat was requested on a survey form. Follow-up measurements of serum cryptococcal antigen and antibody titers were requested for all surviving cats. RESULTS: Signalment and clinical signs of cats with cryptococcosis reported here were consistent with previous reports. Treatment consisted primarily of azole antifungal drugs. All cats were seronegative for cryptococcal antibody titers, whether tested initially or at follow-up examination. All but 1 cat tested were seropositive for cryptococcal antigens when initially tested. Cats with and without clinical signs of C neoformans infection were seropositive for cryptococcal antigens months to years after initial diagnosis of cryptococcosis. CLINICAL IMPLICATIONS: The results of this study indicate that serum titers to cryptococcal antigens in cats can persist with or without clinical signs for months to years after an initial diagnosis of cryptococcosis is made. Repeated evaluation of serum cryptococcal antigen titers is advised during the treatment of cats to monitor progress, evaluate prognosis, and guide cessation of treatment.

Proficiency testing of selected antigen and antibody tests for use in dogs and cats.

OBJECTIVE: To determine the correlation of seroimmunologic test results between reference and nonreference laboratories. DESIGN: Retrospective data analysis. PROCEDURE: Serum samples obtained from naturally infected dogs and cats were distributed to reference and nonreference laboratories for seroimmunologic testing. Correlation of test results was evaluated by use of nonparametric analysis. RESULTS: Correlation coefficients were high between laboratory groups for samples tested for feline immunodeficiency virus antibodies, FeLV antigen, and toxoplasmosis antibodies in cats. Results for feline immunodeficiency virus antibody tests from reference laboratories were more likely to be positive than results from nonreference laboratories. Test results for feline infectious peritonitis antibodies, antinuclear antibodies, and Borrelia antibodies in cats were not significant. Coefficient correlations were significant for results of heart-worm antigen, Brucella antibodies, Toxoplasma antibodies, antinuclear antibodies, and rheumatoid factor in dogs. Results for Borrelia antibodies were not correlated between laboratory groups. CLINICAL IMPLICATIONS: Results were highly correlated between reference and nonreference laboratories for 8 of 14 seroimmunologic tests. Seroimmunologic tests for use in cats were less correlated as a group than those for use in dogs. Poor correlation of results between laboratories was attributed to variations in control agents, antigens, reagents, technical expertise, and cutoff values and end-point titers used for diagnosis.

Post-therapy antibody titers in dogs with ehrlichiosis: follow-up study on 68 patients treated primarily with tetracycline and/or doxycycline.

The clinical and serological responses to therapy were evaluated for at least 1 year in 68 dogs with antibody titers positive for Ehrlichia canis. Treatments were of variable periods with primarily tetracycline hydorchloride and/or doxycycline. Sixteen dogs had initial titers of 1:20 and, at the end of the year, were asymptomatic, no longer receiving medication, and had negative serology. The average length of treatment with tetracycline HCl and/or doxycycline was 85 days (range, 14 to 360 days). Of 39 dogs with initial titers of 1:2,560 or greater, 1 died, 25 were asymptomatic, and 13 were lost to follow-up at the end of the study. The average length of treatment was 210 days (range, 21 to 630 days). Twenty-seven dogs were seropositive at > or = 1:2,560 when the sera was last tested. Thirteen dogs had initial titers of 1:80 to 1:1,280. Of these 13 dogs, 2 died, 2 were lost to follow-up, and 9 were asymptomatic and had titers ranging from negative to > or = 1:2,560 at the end of the study. The persistence of antibodies, prolonged subclinical phase, and delayed relapses despite long-term medication, suggest inadequate chemotherapeutic agents or may be natural features of latency of ehrlichiosis in dogs.

Coccidioidomycosis in 48 cats: a retrospective study (1984-1993)

Coccidioidomycosis was diagnosed in 48 cats. Forty-one cases were identified within a period of 3 years. Coccidioides immitis was revealed by cytological or histopathological examinations, or culture in 70% of cats. The remaining 30% of cases were diagnosed by appropriate clinical signs, radiographic lesions, and serological test results. The average age of affected cats was 6.2 years with a median age of 5.0 years. Fifty-four percent (n = 26) were female and 46% (n = 22) were male. Domestic shorthaired and longhaired breeds comprised 89% (n = 41) of affected cats. Sixty-seven percent of cases were diagnosed during the 6-month period of December through May. Cats infected with C immitis were presented for evaluation of dermatologic (56%), respiratory (25%), musculoskeletal (19%), and neurological or ophthalmologic signs (19%). Fever, inappetence, and weight loss were present in 44% of the cats. Duration of clinical signs before diagnosis was less than 4 weeks in 85% (n = 42) of cats, with an average of 3.8 weeks and a median of 2 weeks. Agar gel immunodiffusion tests were positive in all 39 cats tested at sometime during the course of their disease. Hyperproteinemia (greater than 7.9 g/dL) was present in 52% (10/23) of cases. The majority of cats (n = 39) were negative for feline leukemia virus. Antibodies to feline immunodeficiency virus were absent in the 19 cats tested. Ketoconazole was the most common antifungal agent used to treat cats with coccidioidomycosis. Duration of treatment ranged from less than 1 week to 43 months. Thirty-two cats are currently asymptomatic, with or without treatment. Eleven cats died or were euthanized.(ABSTRACT TRUNCATED AT 250 WORDS)

Staphylococcus intermedius: current knowledge on a pathogen of veterinary importance.

It has been 16 years since the identification of S. intermedius as a new species. Numerous investigations using cell wall and DNA analytic methods have now clearly demonstrated significant differences to warrant the creation of this new species. However, studies investigating virulence factors associated with S. intermedius have not uncovered evidence that differentiates virulent from non-virulent isolates. Therefore, at the present time, it is difficult for veterinary clinicians and microbiologists to determine the clinical significance of many S. intermedius isolates. Host-bacterial interactions and the hosts' immune status appear to be the factors most crucial in determining the outcome of infections, not the virulence of the organism. Continued research in the virulence factor field will hopefully lead to a better understanding on how we can differentiate virulent from nonvirulent isolates of S. intermedius.

Isolation and characterization of immunoglobulin binding proteins from Staphylococcus intermedius and Staphylococcus hyicus.

125-I-IgG binding activities were observed with 15 (17%) of 90 S. intermedius isolates from dogs and 39 (95%) of 41 S. hyicus isolates from pigs. Binding activities were not detected with S. hyicus isolates from cows. The IgG binding proteins of 2 S. intermedius, 2 S. hyicus, and protein A from S. aureus Cowan I were isolated from their cell surfaces. The proteins precipitated with IgG preparations from human, rabbit, pig, dog and horse, but not with IgG from cow, mouse and chicken. This indicated that these IgG binding proteins could be classified as type I receptors. In addition, the isolated proteins from all 3 staphylococcal species precipitated with polyclonal chicken anti-protein A antiserum. SDS-PAGE, Western blotting and gel isoelectric focussing of the proteins revealed numerous bands in the 42,000 D range and acid isoelectric points. The isoelectric point of the isolated proteins from both S. intermedius cultures was slightly more acidic than those from S. hyicus and S. aureus. The present results indicate a close functional and antigenic similarity, if not identity, between IgG binding proteins of S. intermedius and S. hyicus, and protein A of S. aureus.

Small antibiotic resistance plasmids in Staphylococcus intermedius.

Antibiograms and plasmid profiles were evaluated for 116 Staphylococcus intermedius isolates collected from dogs in Germany and in the USA. Of the 26 S. intermedius isolates from Germany, 9 (34.6%) carried plasmids, while 20 (22.2%) of the 90 S. intermedius isolates from the USA were found to be plasmid-positive. Eight small resistance plasmids were identified and characterized using protoplast transformations and restriction endonuclease analyses. Five plasmids (3.8 and 3.9 kb) encoded for chloramphenicol resistance, 2 plasmids (each 2.5 kb) carried determinants for macrolide-lincosamide resistance, and one plasmid (4.5 kb) conferred resistance to tetracycline. Detailed restriction maps of these plasmids were constructed and served for structural comparisons with other small resistance plasmids found in staphylococci. These comparisons implied marked structural homologies with those prototype plasmids initially characterized in S. aureus of human origin.

Surface hydrophobicity of Staphylococcus intermedius and Staphylococcus hyicus.

Surface hydrophobicity of 90 Staphylococcus intermedius and 55 S hyicus isolates was evaluated using the hexadecane adherence assay and the ammonium sulphate salt aggregation test. A strongly positive hydrocarbon adherence in the hexadecane adherence assay was demonstrated in 11 per cent of the S intermedius isolates and 7 per cent of the S hyicus isolates. Bacterial aggregation in 1.6 M, or less, ammonium sulphate was observed in 28 per cent of the S intermedius isolates and 37 per cent of the S hyicus isolates. There was no statistical correlation between the two assays. The adherence of both bacterial species to hexadecane was eliminated when the cells were first treated with pronase and trypsin, while it was mildly enhanced by prior heat treatment (60 degrees C and 95 degrees C for up to three hours). In contrast, aggregation of S intermedius in ammonium sulphate was not influenced by trypsin pretreatment, and aggregation of both bacterial species was diminished, or eliminated, with pronase or prior 95 degrees C heat treatment. Surface hydrophobicity, as measured in both assays, appeared to have no relationship with growth patterns in serum soft agar or production of slime. Similarly, the presence or absence of substantial surface receptor activity to fibrinogen, fibronectin or IgG did not appear to be related to surface hydrophobicity.

Phage typing of Staphylococcus intermedius.

Staphylococcus intermedius, a coagulase-positive staphylococcal species, is a common canine pathogen and a rare human wound pathogen. A total of 145 strains of S. intermedius (ATCC 29663, 4 reference strains, 4 human isolates, 44 canine infection isolates, and 92 isolates from canine gingiva) were screened for lysogenic phage by a modified Fisk method. Nineteen phage preparations were prepared for preliminary typing experiments. Lytic activity was observed on 93 of 145 (64.1%) isolates, yielding 44 lytic patterns with individual strains susceptible to one or more phages. Five phages lysed only a single strain, but lytic patterns varied from 1 to 11 lytic phages per isolate. A distinct lytic pattern did not separate canine or human wound isolates from canine gingival isolates. All human wound isolates fell into the two most common canine gingival or wound patterns; the single human nasopharyngeal isolate was not lysed by any phage. Twenty-two of 44 (55%) canine wound isolates and 65 of 92 (71%) gingival isolates yielded lytic patterns. Lysogenic phages are common in S. intermedius. This preliminary study suggests that phage typing may be a useful tool in distinguishing epidemiologically related strains.

Interlaboratory comparison of titers of antibody to Borrelia burgdorferi and evaluation of a commercial assay using canine sera.

Sixty canine serum samples were sent to 10 different diagnostic laboratories for anti-Borrelia burgdorferi antibody analysis. All laboratories knew of the study prior to receiving the samples. Agreement among all laboratories for all interpretations was 91% (546 of 600 samples). There was complete agreement among all the laboratories for only 32 (53%) of the samples. Most of the disagreements were due to differences reported by either one (15 samples) or two (7 samples) laboratories per sample. When discrepancies in interpretations existed, the interpretation reported by the majority of the laboratories was considered the standard for comparison. One laboratory had no discrepant interpretations from this standard, while the laboratory with the most discrepancies had 16. The median number of discrepancies per laboratory was five. By using pairwise comparisons between each laboratory and the majority standard, eight of the laboratories showed strong agreement and the remaining two showed fair to good agreement. The type of test used (enzyme-linked immunosorbent assay versus indirect immunofluorescence assay) did not appear to influence the number of discrepant interpretations reported. Sera considered to be positive by the majority of the laboratories usually reacted to more than five antigens in immunoblots, with at least three or more of those being intense reactions. For positive samples, reactivity was consistently present in the 60-, 41-, 31-, and 22- or 24-kDa regions. Samples considered negative usually reacted to fewer than three bands, with reactivity usually being faint. A commercially available, rapid dot blot assay showed strong agreement with the majority standard.

Comparison of an enzyme-linked immunosorbent assay to an indirect immunofluorescence assay for the detection of antibodies to Borrelia burgdorferi in the dog.

An enzyme-linked immunosorbent assay (ELISA) was compared to an indirect immunofluorescence assay (IFA) for detection of IgG antibodies to Borrelia burgdorferi in dog sera. The concordance of the two tests was 93.5% for sera from dogs from Maryland (n = 93), 98.0% for sera from dogs from North Carolina (n = 446), and 97.2% for the combined sample groups (n = 539). Twenty-five of the 27 samples with discordant or low positive results were tested, and showed immunoblot reactions to 1 to 10 different bands. Reaction patterns and intensity of the bands were quite variable, and did not explain a reason for the discordance.

Canine Lyme borreliosis.

Lyme borreliosis is now the most commonly reported tick-transmitted disease in humans and is an important disease in dogs. Case reports of canine Lyme borreliosis have appeared in the literature during the last 6 years, but a complete description of the disease still is not available. Until an accurate diagnostic scheme is developed, the disease will remain incompletely understood. A nonlocalizing polyarthropathy is the most commonly described clinical manifestation of canine Lyme borreliosis, but other syndromes probably also exist. The difficulty in making a diagnosis is a result of the fact that dogs do not develop a characteristic skin lesion to mark the beginning of their disease, and many dogs become seropositive but never develop clinical manifestations. Also, Borrelia burgdorferi has been isolated from the blood of healthy dogs, which suggests that detecting a spirochetemia may not have diagnostic significance. Newer diagnostic tests are being evaluated, but at present the diagnosis of canine Lyme borreliosis should be one of exclusion. After other common illnesses are ruled out, serology and response to antibiotic therapy help suggest a diagnosis. Once the disease is accurately diagnosed, efficient therapeutic schemes will be developed based on randomized therapeutic trials. In addition, vaccines are being developed. Currently, without the ability to diagnose the disease accurately, their efficiency can not be demonstrated adequately. Future findings surely will change our understanding of this disease.

Evaluation of four commercial anaerobic systems for identification of Eubacterium suis.

Four commercial anaerobic systems (CASs) were evaluated for usefulness in identification of Eubacterium suis. Twelve strains were evaluated in each system in triplicate, and results were interpreted independently by 5 individuals. Statistically significant differences (P less than 0.01) due to strain variation and reader interpretation accounted for discrepancies encountered. The reactivity, repeatability, and unique profiles generated made both CAS-1 and CAS-2 suitable adjuncts for identification of E. suis when colony morphology and Gram reaction were considered. Limited reactivity in CAS-3 limited its use as an aid in identification. Variability in test observations and the large number of numerical profiles generated precluded use of CAS-4.

An update on the serodiagnosis of canine Lyme borreliosis.

The first report of canine Lyme borreliosis was published in 1984, and since that time there have been numerous reports. Clinical manifestations have varied, and serology is often used to make the diagnosis. This review is designed to inform veterinary clinicians about the current serologic tests and their limitations. In endemic areas, the fact that up to 50% of the canine population can be asymptomatic, yet seropositive, suggests that positive antibody titers do not necessarily indicate the presence of disease. Instead, the presence of a positive antibody titer should be considered evidence of exposure to the agent. Many dogs apparently are exposed to the agent of Lyme borreliosis but do not come down with the disease. Immunoblots are not able to distinguish between infection versus exposure. Newer diagnostic tests, such as antigen assays, are being evaluated. However, the fact that spirochetes have been isolated from 8% of asymptomatic dogs living in an endemic area is evidence that these tests will also need to be cautiously interpreted. Our inability to characterize the relationship between clinical signs and serology has hampered our understanding of canine Lyme disease. Presently, the diagnosis should be one of exclusion. A diagnosis of Lyme borreliosis should be considered confirmed only when other diseases are completely ruled out.

Shared flagellar epitopes of Borrelia burgdorferi and Borrelia anserina.

Antigenic cross-reactivity between Borrelia burgdorferi and Borrelia anserina was studied using mouse immune sera and monoclonal antibodies. With immune sera, significant cross-reactivity between B. burgdorferi and B. anserina was demonstrated by indirect immunofluorescent assay. In immunoblots, most of the cross-reactivity was shown to be associated with the periplasmic flagella. Using monoclonal antibodies in immunoblots, it was shown that B. burgdorferi and B. anserina shared at least two flagellar epitopes, one of which was not shared with Borrelia hermsii or Borrelia coriaceae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of whole cell lysates and the use of a species-specific monoclonal antibody (H5332) which reacts with a major outer surface protein (Osp A) of B. burgdorferi readily differentiated the two species at the molecular level.

Heterogeneity in immunoblot patterns obtained by using four strains of Borrelia burgdorferi and sera from naturally exposed dogs.

This study evaluated the differences in immunoblot patterns when serum specimens from seropositive dogs were reacted against four strains of Borrelia burgdorferi. Intense bands were consistently detected for the 83-, 66-, 61- to 60-, 41-, and 31-kilodalton regions for all four strains. Most variations were observed in the regions of 45 to 34 and 26 to 15 kilodaltons. Adsorption studies suggested that one reason for the observed variability was a difference in proteins among the organisms, rather than a difference in migration of proteins. Therefore, knowledge and consistency of the test antigen are essential when evaluating and comparing canine immunoblot patterns to B. burgdorferi, but for diagnostic purposes all of the serum samples would have been considered positive regardless of the strain used.

Clinical and serologic evaluations of induced Borrelia burgdorferi infection in dogs.

Adult Beagles were used to evaluate clinical signs and serologic response after inoculation with, or exposure to, Borrelia burgdorferi. An indirect immunofluorescent assay (IFA) and 2 ELISA were used to monitor the serologic response to B burgdorferi. Feeding infected ticks on 4 dogs (group 1) failed to cause seroconversion, and SC inoculation with 500 organisms caused minimal seroconversion in 2 of 4 dogs (group 2). At 56 days, approximately 3.01 X 10(8) B burgdorferi organisms were injected IV into group-1 dogs, and intraperitoneally into group-2 dogs. A control group of 4 dogs (group 3) had noninfected ticks feed on them, and then were given IV injection of physiologic saline solution. Increases in immunoglobulin M (IgM) titers were detected in 2 of 4 group-2 dogs approximately 7 days after the initial exposure. These titers returned to negligible values 20 days later. Immunoglobulin G titers increased approximately 10 days after the initial exposure and were mildly increased 56 days later, when dogs were exposed a second time. Both the IV and intraperitoneal injections (second exposures) resulted in increased IgM titers, which in both groups eventually returned to preexposure values after approximately 2 months. Immunoglobulin G titers increased within a week after the second exposure, and in 3 dogs monitored for 8 months, returned to negligible values after the 8-month period. One control dog had a slightly increased IgG titer 24 days after the second inoculation. The possibility of urine transmission is suggested. Clinical status, hemograms, serum biochemical profiles, ECG and results of urinalyses remained normal throughout the study.(ABSTRACT TRUNCATED AT 250 WORDS)