Cell mediated immunity and specific IgG1 and IgG2 antibody response in natural and experimental canine leishmaniosis

In the present study, we have followed up Leishmania infantum infection in dogs: (1) naturally infected; (2) experimentally infected with amastigotes; and (3) experimentally infected with culture promastigotes. The main objective was to evaluate the differences of the humoral and cellular immune responses of each group. Sera from 12 beagle dogs were analysed for total anti-leishmanial antibodies and IgG1 and IgG2 subclasses by enzyme-linked immunosorbent assay (ELISA). Lymphoproliferation to L. infantum antigen was also performed. All naturally infected animals were symptomatic with a marked humoral response. Dogs inoculated with amastigotes were asymptomotic and presented lower antibody titres than naturally infected. Dogs inoculated with culture promastigotes were asymptomotic with no significant humoral response. Strong proliferative responses to Leishmania antigen was observed in dogs inoculated with promastigotes. In our experimental model, IgG1 antibody levels presented a similar pattern in all infected animals, and IgG2 reactivity was high in naturally infected dogs.     

Canine visceral leishmaniasis: relationships between clinical status, humoral immune response, haematology and Lutzomyia (Lutzomyia) longipalpis infectivity

The main source of Leishmania infantum infection in humans is a naturally infected dog. This study reports on the infectivity to phlebotomine sandflies (Lutzomyia longipalpis) of serologically positive mongrel dogs that differed in clinical status, haematology and humoral responses to immunoglobulin (Ig) G(T) (total anti-Leishmania IgG), IgG(1) and IgG(2) subclasses of antibody to crude antigen of L. infantum. Forty-five female L. longipalpis were allowed to feed directly on the ears of dogs classified as asymptomatic, oligosymptomatic or symptomatic before being dissected five days later. Promastigotes were detected in 88% of the dissected sandflies. The highest rate of infectivity to sandflies was found in symptomatic dogs, followed by oligosymptomatic and asymptomatic animals. The results suggest that dogs naturally infected with L. infantum with higher total IgG and IgG(2) concentrations and lower haematocrit levels were able to infect the highest proportion of L. longipalpis. No correlation was observed between anaemia and the intensity of clinical signs. Symptomatic dogs presented the highest infection rate and intensity of infection.     

Clinical, Serologic, and Parasitologic Follow-Up after Long-Term Allopurinol Therapy of Dogs Naturally Infected with Leishmania infantum

Canine leishmaniasis usually is treated with antimony compounds, but frequent relapses, adverse effects, high costs, and development of resistance to long-term antimonial therapy emphasize the importance of searching for alternative antileishmanial drugs. Allopurinol was used at a dosage of 10 mg/kg/day PO to treat 10 dogs naturally infected with Leishmania infantum for a period of 2-24 months. Nine dogs recovered within 2-6 months of chemotherapy, and no relapses were observed during the treatment of up to 20 months. However, 3 of 4 dogs relapsed after treatment was discontinued. These dogs again recovered clinically when therapy was resumed. Parasite-specific immunoglobulin concentrations (IgG2) were high in all dogs before therapy and remained high even in clinically cured dogs during or after therapy. On the other hand, specific IgG1 reactions, which have been shown to be detectable in symptomatic animals, persisted in 7 dogs for long periods after clinical recovery. Three of these dogs relapsed within 2-4 weeks after interrupting therapy. However, 1 dog with no detectable specific IgG1 reaction at the end of therapy did not relapse in the following 4 months. Parasites could be detected in 8 of 9 dogs after clinical improvement by in vitro cultivation or polymerase chain reaction (PCR) testing of lymph node aspirates. In 4 of these dogs, parasites also were detected in blood samples by PCR. Hence, these clinically cured dogs must be regarded as reservoirs of Leishmania and allopurinol cannot be recommended in endemic areas.     

Temporal IgG subclasses response in dogs following vaccination against Leishmania with Leishmune®

Canine Visceral Leishmaniasis (CVL) is a widespread zoonotic disease with mandatory euthanasia of infected dogs determined by the Brazilian Ministry of Health. Development of vaccines against CVL may provide a prophylactic barrier, but transitory peak of antibody response detected by standard diagnostic techniques in vaccinated dogs may be interpreted as natural infection. Accordingly, the aim of the present study was to sequentially evaluate total and IgG subclasses response between naturally Leishmania-infected and dogs vaccinated with Leishmune(?). A total of 172 mongrel dogs were divided in four groups: Group 1 (G1) with 45 clinically healthy dogs, Group 2 (G2) and Group 3 (G3) with 45 dogs naturally infected by Leishmania sp. each, symptomatic and asymptomatic respectively, and G4 (G4) with 37 healthy dogs submitted to a complete protocol of a commercially available vaccine against CVL, monitored and evaluated in 5 different chronological moments (M0-M4) up to 180 days after M0. Total IgG, IgG1 and IgG2 were unable to differentiate between infected (G2 and G3) and vaccinated (G4) dogs, demonstrating that polyclonal commercial antibodies do not distinguish these groups apart. Total and IgG subclasses antibodies were not detected until 21 days of the second vaccination dose; however, seroconversion was observed on 21 days and sustained positivity up to 6 months after the vaccination start. A peak of antibodies response was observed on 90 days (M3), when results for total IgG, IgG1, IgG2, IgG3 and IgG4 where highly significant when compared to M0 (P<0.0001). Neither total IgG nor IgG1 effectively differentiated between infected (G2 and G3) and vaccinated (G4) dogs. In conclusion, despite dogs may test serologically negative immediately after vaccination against CVL with Leishmune(?), subsequent seroconversion, antibody peak and positivity up to six months may lead vaccinated dogs to be mistakenly identified as naturally infected dogs during this period.     

Leishmania infantum-specific IgG, IgG1 and IgG2 antibody responses in healthy and ill dogs from endemic areas. Evolution in the course of infection and after treatment

The expression of IgG, IgG1 and IgG2 specific antibodies for Leishmania infantum was studied in five groups of dogs in Catalonia (Spain): I, 99 asymptomatic dogs (infected and uninfected) from a highly endemic area for leishmaniosis; II, 139 untreated dogs with clinically patent leishmaniosis; III, 11 naturally infected asymptomatic dogs monitored for up to 5 years since they were found seropositive to Leishmania antigen and without treatment; IV, 25 naturally infected dogs with clinically patent leishmaniosis and treated with either meglumine antimoniate and allopurinol or allopurinol alone and V, six experimentally infected dogs, treated with meglumine antimoniate and controlled for 5 years. The levels (ELISA units) of IgG, IgG1 and IgG2 in asymptomatic dogs (group I) were very variable (24+/-33, 32+/-31 and 26+/-31, respectively), and, as expected, lower than in ill dogs (group II) (168+/-34, 84+/-71 and 172+/-31, respectively). In both groups, the correlation between IgG and IgG2 levels (r=0.95, P<0.001 in group I and r=0.63, P<0.001 in group II) was higher than between IgG and IgG1 levels (r=0.01, P>0.05 in group I and r=0.31, P<0.001 in group II). In group III, IgG and IgG2 expression increased during infection, while IgG1 expression remained the same. In dogs of group IV, IgG levels after 1 year of treatment decreased more in responsive (mean values, 163+/-42 before treatment (b.t.) and 100+/-36 after treatment (a.t.)) than in unresponsive dogs (158+/-29 b.t. and 124+/-51 a.t.), especially for IgG1 (94+/-89 b.t. and 20+/-21 a.t. in responsive dogs and 35+/-25 b.t. and 22+/-13 a.t. in unresponsive dogs) rather than for IgG2 (156+/-16 b.t. and 114+/-45 a.t. in responsive and 151+/-11 b.t. and 125+/-36 a.t. in unresponsive dogs). Similar results were observed in the evolution of experimentally infected animals after consecutive and specific treatments. Overall results show the great variation in Leishmania-specific IgG1 expression in asymptomatic and symptomatic dogs, their lack of correlation with that of IgG2 and chemotherapy is more effective in dogs with initially high expression of IgG1.     

Development and validation of a SYBR green real-time PCR for the quantification of porcine circovirus type 2 in serum, buffy coat, feces, and multiple tissues

The emergence of multiple genotypes of PCV2, as demonstrated by phylogenetic analysis of whole genome or capsid sequences, makes it necessary to have quantitative diagnostic assays that perform equally well on all strains. The objectives of this study were to develop and validate a novel real-time polymerase chain reaction (PCR) assay targeting the highly conserved rep gene (ORF1) and investigate the effects of diagnostic specimen choice on its performance. The assay was tested in naturally infected conventional pigs, experimentally infected gnotobiotic pigs, and plasmid-spiked negative serum, lung tissue, and feces and found to have a linear detection range of 2.2x10(3) to 2.2x10(10) copies of PCV2 per mL. The assay successfully detected and quantified PCV2 DNA in serum, buffy coat, feces, and multiple lymphoid (bronchial, mesenteric, and superficial inguinal lymph nodes; thymus; tonsil; ileal Peyer's patches; and spleen), and non-lymphoid (myocardium; lung; kidney; liver; and gluteal muscle) tissues from naturally infected pigs. Across all tissues and sera of naturally infected pigs, the mean PCV2 concentration was 3.0logs higher in wasting versus non-wasting pigs. PCV2 concentration measured by tissue culture and immunohistochemical staining in homogenized liver samples of experimentally infected gnotobiotic pigs were compared to the concentrations estimated by quantitative PCR. Similar trends were noted with increasing PCV2 concentration detected in subclinically infected to severely PMWS-affected pigs across all assays. Our diagnostic assay was developed with a conserved target sequence, and performed efficiently in quantification of PCV2 in a variety of tissues from naturally and experimentally infected pigs.     

Dominance of Chlamydia psittaci-specific IgG2 subclass in the humoral immune responses of naturally and experimentally infected cattle

Indirect enzyme-linked immunosorbent assays were applied to differentiate Chlamydia (C.) psittaci-specific IgG1 and IgG2 levels in 143 individual serum samples from cattle with naturally occurring chlamydial infections and in 190 sequential serum samples from 26 experimentally infected pregnant cows, calves, and a bull. The mean IgG1:IgG2 ratio of naturally infected cattle was 1:4 indicating a significant (p less than 0.001) IgG2 dominance. Similar ratios were detected in the experimentally infected cattle. The dominance of IgG2 was independent of breed, sex, and age. Twenty-nine cattle had significant immunoglobulin levels to both C. psittaci and Coxiella (C.) burnetii simultaneously. The predominance of C. psittaci-specific IgG2, in contrast to the predominance of C. burnetti-specific IgG1 detected in these same individual serum samples under identical conditions, indicates that the ability to preferentially produce either IgG1 or IgG2 was not limited in these individual cattle. A transient yet significant IgG1 response was also developed in cows following chlamydia-induced abortions (immunotype 1) or in cattle infected with the polyarthritis-serositis-encephalomyelitis agents (immunotype 2). IgG1 levels decreased faster than IgG2 levels. These findings have diagnostic implications and identify the need for determining the immunoglobulin classes and subclasses of the humoral immune responses of animals and man to chlamydial infections.     

Development and evaluation of a flow cytometry microsphere assay to detect anti-histone antibody in dogs

Anti-nuclear antibody (ANA) is one of the diagnostic parameters that support a diagnosis of autoimmune disorders in humans, dogs, and horses, particularly the condition systemic lupus erythematosus (SLE). The most commonly used method for detecting ANA in canine serum is the indirect immunofluorescence antibody assay (IFA) that detects dog IgG with reactivity towards mammalian cell nuclei. Interpretation of the IFA results is very subjective and dependent on the source of tissue/cellular substrate. We have developed a flow cytometry based assay to detect canine serum antibodies specific to histones. Histones were chosen as the target antigen because these nuclear proteins are the most common nuclear substrate for ANA in dogs with SLE. Microsphere beads were coated with histones and incubated with canine sera. Bound anti-histone antibodies were detected by FITC-conjugated rabbit F(ab')2 anti-dog IgG. Sera from four groups of dogs (47 dogs total) were tested for anti-histone antibodies and compared with the traditional IFA assay. The groups included 15 healthy dogs, 15 dogs with noninflammatory diseases, 9 dogs with polyarthritis and positive ANA, and 8 German shepherds with perianal fistulas. The microsphere assay results indicated that only one dog in the noninflammatory group and four out of nine dogs in the polyarthritis group had mean fluorescent intensity values above our established cut-off (defined as 2 S.D. above the mean of healthy controls). There was moderate agreement between the anti-histone assay and the traditional ANA (kappa statistic=0.54). Absorption of ANA positive serum with total histones dramatically diminished the fluorescent signal detected by flow cytometry and the speckled nuclear pattern observed by IFA, whereas preabsorption did not change the diffuse nuclear staining pattern. These findings indicate that the anti-histone assay will not replace the ANA test and that other nuclear proteins, such as ribonucleoproteins may contribute to the diffuse ANA patterns.     

IgG subclass responses in a longitudinal study of canine visceral leishmaniasis

Visceral leishmaniasis (VL), caused by Leishmania infantum, is an important disease of domestic dogs. Here, we present data on the IgG subclass antibody response to crude L. infantum antigen in a cohort of naturally infected Brazilian dogs. Specific IgG1-IgG4 responses could be detected in 98, 58, 70 and 82%, respectively of 57 dogs that were seropositive for specific IgG. Levels of all IgG subclasses were strongly inter-correlated. Levels of all IgG subclasses increased at the time of seroconversion, before reaching a plateau after several months. Levels of all IgG subclasses were higher in sick dogs than healthy dogs, and levels of all except IgG2 were higher in parasite-positive (by PCR) than parasite-negative dogs. However, levels of IgG2 relative to IgG1 were lower in sick or parasite-positive dogs compared to healthy or parasite-negative infected dogs. In contrast to previous studies, the results suggest that canine VL is associated with upregulation of specific antibody of all IgG subclasses, particularly IgG1, IgG3 and IgG4.     

Antinuclear antibodies can be detected in dog sera reactive to Bartonella vinsonii subsp. berkhoffii, Ehrlichia canis, or Leishmania infantum antigens

The presence of antinuclear antibodies (ANAs) is used to support a clinical diagnosis of systemic lupus erythematosus (SLE) in dogs. However, clinicians must interpret the detection of ANAs with caution, particularly in light of increasing evidence that dogs with known bacterial and protozoal infections can have high ANA titers. Retrospectively, medical records were reviewed for all dogs that were concurrently tested for antinuclear antigens and Bartonella vinsonii (berkhoffii), Ehrlichia canis, or Rickettsia rickettsii antigens between 1990 and 2000. When analyzed on the basis of reactivity to a specific infectious agent, 75% of the B vinsonii (berkhoffii) seroreactors, 16.7% of the E canis seroreactors, and 0% of the R rickettsii seroreactors had concurrent ANAs. Subsequent prospective testing did not detect ANAs in convalescent sera from dogs experimentally infected with B vinsonii (berkhoffii), E canis, or R rickettsii. However, 10-20% B vinsonii (berkhoffii), E canis, or Leishmania infantum reactive sera from naturally infected dogs contained ANAs. In addition, 45% of sera from dogs that are reactive to multiple vectorborne organisms were more likely to contain ANAs when compared to sera from dogs reactive to only 1 test antigen. When interpreting the relevance of seroreactivity to nuclear antigens, clinicians should recognize that dogs with seroreactivity to B vinsonii (berkhoffii), E canis, or L infantum antigens (especially those with seroreactivity to more than one of these pathogens) may produce ANAs.     

Utility of diagnostic tests used in diagnosis of infection in dogs experimentally inoculated with a North American isolate of Leishmania infantum infantum

Eight female beagles were infected with 1 x 10(7) (low dose, LD) or 2 x 10(8) (high dose, HD) promastigotes of a North American isolate of Leishmania infantum infantum (LIVT-1 strain) isolated from naturally infected Virginia Foxhounds. Two female beagles served as negative controls and 2 male beagles chronically infected (> 3 years) with Leishmania infantum chagasi were positive controls. Bone marrow (BM) and lymph node (LN) aspirates were collected every 6-8 weeks for cytologic evaluation, parasite culture, and polymerase chain reaction (PCR). Serum samples were collected monthly for determination of serologic responses by indirect fluorescent antibody test (IFAT) and diagnostic rK39 antigen. Cultures of BM and LN aspirates and cytology evaluation were consistently positive in positive control dogs during the course of study. Negative control dogs were negative on BM and LN cultures and on cytologic evaluation of aspirates. Amastigotes were present on cytological examination of BM aspirates in 2 experimentally infected dogs. Cultures of LN aspirates were positive on 22 samples, whereas BM cultures were positive on 12 samples for all dogs. IFA titers ranged from 0 to 1 :400 in experimentally infected dogs during the course of the study. Recombinant K39 immunoassay tests were consistently positive in positive control dogs and in the HD dogs by approximately 8 weeks after infection. BM PCR products were identified more consistently in the HD dogs compared with the LD dogs. Kappa statistics indicated PCR correlated better with cultures and cytology than did IFAT or the rK39 immunoassay results in the experimentally infected dogs.     

Efficacy of a modified polymerase chain reaction assay for detection of Ehrlichia canis infection.

Detection of Ehrlichia canis in acutely infected and convalescent dogs is important for effective treatment and control. However, accurate detection has been difficult to achieve, in part because dogs that have been treated therapeutically often remain seropositive for extended periods. A new method, polymerase chain reaction (PCR) assay using biotinylated E. canis-specific primers (PCR-BP), was developed for detection of E. canis. Four dogs experimentally infected with E. canis by intravenous inoculation of whole blood from carrier dogs and 2 naturally infected convalescent carriers were used to compare the specificity and sensitivity of the new method with that of microscopy/blood smear evaluation, serologic test, and conventional PCR assay using E. canis-specific primers. In experimentally infected animals, infection was detected as early as 7 days post-exposure using PCR-BP. Although the 2 naturally infected dogs were positive by serologic test and PCR-BP, both were negative by conventional PCR. Results suggest that the new method is a sensitive assay for detection of E. canis infection. In addition, results were obtained more rapidly than with other PCR-based assays.     

Caprine herpesvirus-1-specific IgG subclasses in naturally and experimentally infected goats

Enzyme-linked immunosorbent assays (ELISAs) were developed to detect caprine herpervirus-1 (CpHV-1)-specific IgG1 and IgG2 in sera from 43 naturally infected goats. The analysis of the IgG subclasses showed a dual pattern of distribution in seropositive goats with a major group of animals (36 out of 43) exhibiting significantly higher levels of IgG2 over IgG1 and a minor group (7 out of 43) possessing equal levels of IgG1 and IgG2. Four goats were experimentally infected with a virulent CpHV-1 Ba.1 strain by the intranasal or the intravaginal route and the kinetics of appearance of CpHV-1-specific IgG, IgG1 and IgG2 in the serum were studied. Two weeks following infection, both IgG1 and IgG2 levels increased although convalescent sera (i.e., collected 5–8 weeks post-infection) showed a clear prevalence of the IgG2 subclass. To determine the contribution of the different IgG subclasses to herpesvirus immunity, serum neutralization (SN) assays were performed in both naturally and experimentally infected goats. The kinetics of SN showed that neutralization activity was mainly associated to the IgG1 subclass and this was also confirmed in naturally infected goats. The results are discussed from the standpoint that the profile of the IgG subclasses is instrumental to study immune responses to CpHV-1 and that vaccination strategies may benefit from this information.     

Molecular specificity of the antibody responses of cattle naturally and experimentally infected with cytopathic and noncytopathic bovine viral diarrhea virus biotypes

The specificity of the humoral IgG response of cattle naturally or experimentally infected with bovine viral diarrhea virus (BVDV) was studied by radioimmunoprecipitation. Serum samples were tested against radiolabeled lysates of cells infected with cytopathic and noncytopathic biotypes of BVDV. A biotype-specific serologic marker was not detected. The specificity of the IgG induced in cows naturally or experimentally infected with either BVDV biotype was essentially the same. A strong IgG response to the 2 glycoproteins (56 to 58 kilodaltons, [kD] and 48 kD) of both biotypes and to the major polypeptides was induced in infected cells: 118 kD and 80 kD by cytopathic BVDV and only 118 kD by noncytopathic BVDV. The most consistent difference among cattle was the presence of IgG specific for the 37-kD polypeptide. Sequential serum sample collection after spontaneous and induced infections with either BVDV biotype did not indicate specific IgG markers for determining infection history. Sera from cattle with a confirmed diagnosis of mucosal disease and lacking neutralizing antibodies to BVDV usually lacked (greater than 80%) nonneutralizing BVDV-specific IgG. One animal had substantial amounts of IgG to the 80-kD polypeptide. Other cattle had less readily detectable amounts of IgG specific for 80-kD or 37-kD viral polypeptides.     

Ivermectin treatment of naturally acquired and experimentally induced Strongyloides stercoralis infections in dogs.

Treatment of Strongyloides stercoralis infection was investigated in 2 dogs with naturally acquired, chronic-active infections, and in 3 dogs with corticosteroid-enhanced, experimentally induced hyperinfections. A single oral dose of ivermectin was given to naturally infected (200 micrograms/kg of body weight) and experimentally infected (800 micrograms/kg) dogs. Five dogs with experimental hyperinfections served as controls. Dogs with naturally acquired infections ceased to shed first-stage larvae in the feces 1 week after treatment, but 1 dog had recrudescence and required a second dose. Ivermectin was 100% effective in removing adult S stercoralis from the intestinal tract of the experimentally infected dogs, but it was not effective in removing third-stage larvae from parenteral sites. Ivermectin-treated dogs had few intestinal parasites of any stage, whereas at necropsy, 4 of 5 experimentally infected dogs not treated had massive infections (greater than 100,000 adults, greater than 92,000 larvae) in the intestinal tract, and 3 of 5 had larvae (greater than 2,500) in parenteral sites.     

Detection of platelet-bound antibodies in beagle dogs after artificial infection with Ehrlichia canis

Six dogs were infected with Ehrlichia canis by intravenous injection of heavily infected DH82 cells. All dogs developed typical signs of canine monocytic ehrlichiosis. Using flow cytometric technology, platelet-bound IgG (PBIgG) were detected in 5 of the 6 dogs after experimental infection with E. canis over a period of 3-10 days post infection (PI). The first detection of PBIgG was made as early as day 3 PI in 2 out of 6 dogs, and on day 5 PI in 1 dog. On day 7 PI, PBIgG was detected in 2 dogs, and on day 10 PI in 3 out of 6 dogs. This is the first report documenting the presence of PBIgG following E. canis infection in dogs. This finding further supports the theory that the thrombocytopenia seen in canine monocytic ehrlichiosis has an immunological component and that exposure to an infectious agent, in this case the rickettsia E. canis, can trigger autoimmune mechanisms. Due to the heterogeneous appearance of PBIgG among the infected dogs it was concluded that other non-immunological mechanisms are probably also involved in the pathogenesis of the thrombocytopenia seen in canine monocytic ehrlichiosis.     

Immune-mediated fever in the dog. Occurrence of antinuclear antibodies,rheumatoid factor,tumor necrosis factor and interleukin-6 in serum

Contents of antinuclear antibodies (ANA), rheumatoid factor (RF), tumor necrosis factor (TNF-alpha) and interleukin-6 (IL-6) were measured in serum from 20 dogs with immune-mediated fever. Seven out of 20 patients were ANA positive, 1 out of 20 was positive to antibodies against extractable nuclear antigens (ENA), 1 out of 20 was positive to antibodies against deoxynucleoproteins (DNP), 2 out of 13 were RF positive and none out of 20 patients had antibodies against native DNA in the serum. TNF-alpha was not detected in any serum of 15 dogs with immune-mediated fever, while 10 out of 13 presented with elevated IL-6. The results varied between patients, but the IL-6 level was high in most of them. This indicate a role for IL-6 in the pathogenesis of immune-mediated fever in most cases.     

Concentrations of cartilage oligomeric matrix protein in dogs with naturally developing and experimentally induced arthropathy

OBJECTIVE: To assay concentrations of cartilage oligomeric matrix protein (COMP) in canine sera and synovial fluid (SF), to compare COMP concentrations in clinically normal dogs and dogs with joint disease, and to analyze changes in COMP concentrations in dogs with experimentally induced acute synovitis. ANIMALS: 69 control dogs without joint disease, 23 dogs with naturally occurring aseptic arthropathy, and 6 dogs with experimentally induced synovitis. PROCEDURE: Serum (n = 69) and SF (36) were obtained from control dogs. Samples of serum (n = 23) and SF (13) were obtained from dogs with naturally occurring aseptic arthropathy with or without radiographic features of osteoarthritis (OA). Serum and SF were obtained before and 1, 2, 3, and 7 days after induction of synovitis. The COMP concentrations were determined by use of an inhibition ELISA that had canine cartilage COMP and monoclonal antibody against human COMP. RESULTS: Concentrations of COMP in serum and SF of control dogs were 31.3+/-15.3 and 298.7+/-124.7 microg/ml, respectively. In naturally occurring OA, COMP concentrations in serum (44.9+/-177 microg/ml) and SF (401.7+/-74.3 microg/ml) were significantly higher than corresponding concentrations in control dogs. The COMP concentration in SF peaked 24 and 48 hours after induction of synovitis, whereas concentration in serum peaked on day 3. CONCLUSIONS AND CLINICAL RELEVANCE: These results supported the hypothesis that COMP concentration in serum and SF of dogs may be altered after cartilage degradation or synovitis. Measurement of COMP concentrations can be useful when differentiating arthropathies in dogs.     

Serological diagnosis of Echinococcus granulosus infection in sheep using cyst fluid antigen processed by antibody affinity chromatography

Serum antibody responses in sheep naturally or experimentally infected with Echinococcus granulosus and/or other larval cestodes were examined using an enzyme-linked immunosorbent assay (ELISA) with various antigens prepared from sheep hydatid cyst fluid ( SHCF ). Serum donors included: sheep experimentally infected with E. granulosus and their age-matched non-infected controls; sheep experimentally infected with other helminth parasites; sheep naturally infected with E. granulosus both from Tasmania and the Australian mainland; sheep from Tasmania naturally infected with larval cestodes other than E. granulosus; and naturally reared sheep completely free from infection with larval cestodes. Attempts were made to eliminate serological reactions which were not specific for E. granulosus by using a series of antibody affinity chromatography steps to deplete crude SHCF antigen; these included adsorption with a monoclonal antibody, 3EgH 29-2, removal of host IgG using rabbit anti-sheep IgG antibody, and removal of antigens which bound non-specifically to normal sheep immunoglobulin. The final affinity-depleted antigen product was designated AD SHCF . Specific serological reactivity in infected sheep was very low. Affinity depletion of SHCF using 3EgH 29-2 did not appear to increase the specificity of serological diagnosis of E. granulosus infection when experimentally infected sheep were compared with their non-infected controls provided the latter were age-matched with experimental animals. The other affinity adsorption steps significantly reduced non-specific background binding to antigen by normal sheep serum. Despite this reduction in background in the ELISA, only low levels of antibody could be detected in naturally-infected sheep.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clinical and hematologic effects of experimental infection of dogs with recently identified Babesia gibsoni-like isolates from Oklahoma

OBJECTIVE: To characterize clinical and hematologic responses in dogs following experimental inoculation with Babesia gibsoni-like isolates from infected dogs in Oklahoma. DESIGN: Prospective study. ANIMALS: 6 mixed-breed dogs. PROCEDURE: 2 dogs were inoculated with organisms from a naturally infected dog, and 3 were inoculated with organisms from a second naturally infected dog (1 of these 3 dogs was splenectomized 1 week prior to inoculation). One dog was not inoculated. Complete blood counts were performed weekly. RESULTS: In the 5 dogs inoculated with organisms, parasites were initially detected 1 to 5 weeks after inoculation, and severity of parasitemia peaked with 1.9 to 6.0% of RBC infected by 4 to 6 weeks after inoculation. Parasitemia was easily detectable (> 0.1% of RBC infected) for 3 to 4 weeks. Clinical abnormalities included lethargy, fever, and pale mucous membranes but were mild to nearly inapparent in 2 dogs. All dogs developed regenerative anemia and marked thrombocytopenia. Thrombocytopenia developed before and lasted longer than the parasitemia. Profound but transient neutropenia was detected in some dogs. The splenectomized dog developed more severe parasitemia and anemia and more pronounced clinical abnormalities. Three dogs with intact spleens recovered without treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that 2 or more genotypically distinct, but morphologically identical, small Babesia parasites can infect dogs in the United States. Compared with infection with small Babesia parasites from California, infection with these isolates resulted in less severe parasitemia and clinical abnormalities. Parasitemia was transient, indicating that identification of organisms in blood smears may be difficult in some dogs.