Detection of equine antiplatelet and antineutrophil antibodies by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (elisa) was standardised and applied for the detection of anti-platelet and antineutrophil antibodies using a heterologous system consisting of equine platelets or neutrophils and antisera raised in rabbits. The standardised technique consisted of using Immulon type 3 plate, I per cent gelatine as a blocking solution, poly-L-lysine buffer as a coating solution, unfixed antigen, 90 μl test serum, horseradish peroxidase conjugated antibody and o-phenylenediamine dihydrochloride as a substrate. The number of unfixed platelets or neutrophils required for optimum detection of antibodies was 250,000 per well. Unfixed cellular antigens were as good as their extracts and superior to paraformaldehyde-fixed antigens in detecting specific antibodies. Microtitre plates coated with platelet or neutrophil antigens could be stored at 4° and −70°C for four to five weeks without significant loss of antigenicity. The ELISA was very sensitive in that antiplatelet antibody was detected up to a titre of 1:204,800 and antineutrophil antibody to a titre of 1:51,200. Some cross-reactivity (1:1600) was detected in antiplatelet and antineutrophil sera for neutrophil and platelet antigens, respectively. Platelet-associated antibody was also detected in extracts from platelets pretreated with 1:2 and 1:8 dilutions of antiplatelet serum. Standardised elisa detected antiplatelet antibodies in nine and antineutrophil antibodies in three of 100 isologous equine blood typing sera.     

Mechanisms of platelet destruction in immune-mediated thrombocytopenia: in vitro studies with canine platelets exposed to heterologous and isologous antiplatelet antibodies

Normal canine platelets coated with heterologous or isologous antiplatelet antibody were interacted with viable canine neutrophils in vitro. Platelet phagocytosis was assessed by detecting nitroblue tetrazolium (NBT) reduction, measuring uptake of opsonised 51Cr-labelled platelets, and electron microscopy. The NBT reduction and uptake of 51Cr-labelled opsonised platelets were markedly increased. Electron microscopy revealed phagocytosis of antibody-coated platelets and their degradation intracellularly. Exposure of canine platelets to rabbit anti-canine platelet antibody in vitro produced morphological changes in platelets and caused serotonin release. Serotonin was not released in the absence of antiplatelet antibody or in the presence of normal rabbit gamma-globulin. Morphological changes in the platelets included disappearance of alpha and dense granules and exaggeration of the open canalicular system. These observations indicate that circulating platelets may be vulnerable to an antiplatelet antibody and that antibody-mediated phagocytosis of platelets is an important mechanism in the pathogenesis of immune-mediated thrombocytopenia.     

Canine distemper virus-induced thrombocytopenia

Effects of canine distemper virus (CDV) infection on circulating platelet values were studied in gnotobiotic dogs inoculated with R252-CDV. Thrombocytopenia (less than 200,000 platelets/microliter) was present on postinoculation day (PID) 5 and persisted through PID 15. Peak thrombocytopenia occurred on PID 10 (less than 85,000 platelets/microliter). Thrombocytopenia was accompanied by lymphopenia, neutropenia, and monocytopenia. Platelet membrane-bound CDV antigen and IgG were present from PID 7 onward; neither the third component of complement nor IgM was detected on platelets from CDV-inoculated dogs. The mean number of megakaryocytes per unit of bone marrow surface area was unchanged. Megakaryocyte infection was present in dogs euthanatized on PID 4 (0.33%), increased slightly in dogs euthanatized on PID 8 (3%), and increased sharply in dogs euthanatized on PID 9 and 10 to 17.8% and 8.3%, respectively. Phagocytosis of platelets by stellate reticuloendothelial (Kupffer's) cells in the liver was prominent in dogs euthanatized from PID 5 onward. Seemingly, CDV-induced thrombocytopenia was mediated by virus-antibody immune complexes on platelet membranes. Decreased platelet production after PID 8 resulting from direct viral infection of megakaryocytes was a likely contributing factor and occurred against a background of profound virus-induced dysfunctions of all hematopoietic cellular elements.     

Titin and ryanodine receptor autoantibodies in dogs with thymoma and late-onset myasthenia gravis

Similar to human autoimmune myasthenia gravis (MG), canine MG occurs spontaneously and is associated with autoantibodies against the nicotinic acetylcholine receptor (AChR). In addition to AChR, human MG patients with thymoma or late-onset MG have antibodies against titin and ryanodine receptor (RyR). The objective of this study was to establish if dogs with confirmed MG (AChR antibody titer >0.6 nmol/l) also developed titin and RyR antibodies and identify possible associations with thymoma, late age of onset, or severity of clinical signs. Sera from dogs (n=430) with previously diagnosed autoimmune MG (N=415), other immune-mediated neuromuscular disorders including polymyositis (PM) and masticatory muscle myositis (N=5), and control dogs (N=10) were evaluated for the presence of titin antibodies in ELISA using MGT-30 as antigen, a peptide representing the main immunogenic region (MIR) for human titin antibodies. Titin antibody positive sera were further examined for RyR antibodies in Western blots using a RyR fusion protein (pc2-RyR) as antigen, which covers the MIR for human MG sera. Titin antibodies were found in sera of 80/430 dogs. Thymoma was present in 11/80 and age of onset was after 4 years in 66/80 titin positive dogs. Two of the titin positive dogs had PM. RyR antibodies were found in 13/80 sera (8/13 thymoma, 12/13 age of onset after 4 years, and 1/13 PM). Neither titin nor RyR antibodies were found in sera of healthy control dogs. Acute fulminating MG was described in five dogs with both titin and RyR antibodies. From these studies we conclude that titin and RyR antibodies in canine and human MG have a similar association with thymoma, late-onset MG, and possibly with more severe forms of MG.     

Immune responses in vaccinated dogs with autoclaved Leishmania major promastigotes.

A comparative study was undertaken on the immunogen power of autoclaved Leishmania major promastigotes (ALM) vaccines given simultaneously with either BCG or saponin against canine leishmaniasis. The humoral immune response was assessed by ELISA and western blotting. The cellular immune response was evaluated by the lymphocyte transformation test. Dogs vaccinated simultaneously with ALM and saponin showed high antibody titres to crude L. infantum antigens after the first vaccine booster and reacted with several antigens, with molecular weights from 26 to 108 kDa by western blotting. However, the lymphocyte proliferation of these dogs to the crude L. infantum antigen was not significantly different from the control group. In contrast, in dogs vaccinated simultaneously with ALM and BCG, the antibody titres to crude antigen were low. Their sera reacted with the same proteins recognised by sera from dogs vaccinated simultaneously with ALM and saponin by western blotting. However, the 85-kDa protein was only identified by sera taken from dogs vaccinated simultaneously with ALM and BCG. These latter exhibited specific lymphocyte proliferation to the L. infantum antigen. This cell proliferation was observed for approximately 9 months after the first dose of the vaccine. This study indicates that a combination of ALM as the vaccine and BCG as the adjuvant, in the dog model, was successful in inducing a cell immune response, which is implicated in protection of dogs against a Leishmania infection.     

Antibody and lymphoblastogenic responses of dogs experimentally infected with Trypanosoma cruzi isolates from North American mammals

The humoral and cellular immune responses of dogs infected with either a non-pathogenic Trypanosoma cruzi isolated from a North American dog (Tc-D) or a pathogenic T. cruzi isolate from an opossum (Tc-O) were studied over a 240 day period. Antibody to T. cruzi epimastigote antigens prepared from Tc-O or Tc-D isolates were first detected by ELISA by Day 26 post infection (PI), peaked by day 175 PI and remained elevated throughout the experimental period in both Tc-O and Tc-D infected dogs. Differences in antibody levels between infected groups were not detected. Western blot analyses were performed using Tc-O and Tc-D epimastigote antigens probed with pooled sera and sera from individual Tc-O and Tc-D infected dogs prior to infection (Day 0), and during the acute (Day 16-35 PI), indeterminate (Day 50-135 PI) and chronic (Day 235 PI) stages of infection. Generally, the patterns, number of protein bands, and temporal appearance of the protein bands identified by pooled sera and sera from individual dogs within each antigen preparation were similar. However, similarities and differences were present in antibody responses between sera from Tc-O and Tc-D infected dogs. Blastogenic responses of peripheral blood mononuclear cells (PBMC) from Tc-O and Tc-D infected dogs to mitogens (concanavalin A, phytohemagglutinin and pokeweed) were not significantly different from controls at any time during the experimental period. The PBMC from both groups of dogs were unresponsive to epimastigote antigens during the acute stage of infection. Statistically significant differences (P less than 0.05) in PBMC responsiveness from controls were observed on Days 70 and 175 PI. Responses decreased to pre-infection levels by Day 240 PI. These studies demonstrate that although two North American T. cruzi isolates have markedly different virulence for dogs, some aspects of their cellular and humoral immune responses are similar while other responses, such as antibody recognition of specific T. cruzi antigens, vary.     

Establishment of central nervous system infection by canine distemper virus: breach of the blood-brain barrier and facilitation by antiviral antibody

Morphologic, immunologic and virologic data implicating antiviral antibody in promoting entry of canine distemper virus (CDV) into brain and reticuloendothelial tissues are reviewed. Infection of central nervous system (CNS) endothelium precedes invasion of virus-positive and -negative leukocytes into Virchow-Robin spaces and central nervous system (CNS) parenchyma by 1-3 days. Platelets are implicated in initiation of endothelial infection in that: CDV-infected dogs are thrombocytopenic; platelets from CDV-infected dogs contain IgG-virus complexes on their plasma membranes; platelet microthrombi were observed adjacent to foci of endothelial infection, and; CDV-susceptible ferrets rendered thrombocytopenic by antiplatelet antibody exhibit delayed viral entry into CNS tissues. Renal glomerular-bound IgG, IgM and occasionally CDV antigen were demonstrated in CDV-infected dogs by immunocytochemical techniques. Distemper-infected dogs with inherited C3 deficiency exhibited enhanced renal glomerular disease associated chiefly with deposition of IgM in mesengial regions vs. their homozygous normal CDV-infected littermates. Direct infusion of virus-positive leukocytes, plasma and platelets into the CNS capillary bed via the right carotid artery should establish the primacy of each in the initiation of CNS vascular endothelial infection by CDV.     

Antibodies against Helicobacter felis in sera of cats and dogs.

Serum samples from 61 dogs and 49 cats were screened for circulating antibodies against Helicobacter felis by an enzyme-linked immunosorbent assay (ELISA) using sonicated bacteria as an antigen. To improve the specificity of the ELISA, sera were absorbed with Campylobacter jejuni subsp. jejuni H. pylori as well as H. felis. Sera from 26 dogs (43%) and 19 cats (39%) revealed clear positive absorbance readings determined as an optical density (OD) that was statistically significant above the OD mean value [P < 0.025 (one-tailed); log10]. The absorbance pattern of ELISA-positive sera corresponded to results obtained with bovine and human reference sera. Furthermore, a correlation between the immune response and results from histopathological examination of gastric specimens from 22 dogs was demonstrated. It could be shown that antibodies against H. felis in sera of cats and dogs can easily be detected using an ELISA. The diagnostic value of this test must be evaluated in further investigations.     

Diagnostic use of cytologic examination of bone marrow from dogs with thrombocytopenia: 58 cases (1994-2004)

OBJECTIVE: To determine the diagnostic use of cytologic examination of bone marrow from dogs with thrombocytopenia. DESIGN: Retrospective case series. ANIMALS: 58 dogs with thrombocytopenia. PROCEDURES: Medical records were searched and reviewed for dogs with thrombocytopenia. Dogs that had thrombocytopenia and cytologic examination of bone marrow were included in the study. Dogs with other hematologic abnormalities, with a previous diagnosis of hematopoietic neoplasia, or that had previous treatment with cytotoxic drugs were excluded. Bone marrow cytologic findings were reviewed. Results were compared between dogs with severe thrombocytopenia (< 20,000 platelets/microL) and dogs with mild to moderate thrombocytopenia (20,000 to 200,000 platelets/microL). RESULTS: 58 dogs met the inclusion criteria. Of 55 dogs with diagnostic bone marrow aspirates, 36 had severe thrombocytopenia. Cytologic evaluation of bone marrow did not reveal substantial nonmegakaryocytic bone marrow abnormalities or result in a definitive diagnosis in any of these dogs. Nineteen dogs with mild to moderate thrombocytopenia had diagnostic bone marrow aspirates. Bone marrow cytologic findings revealed nonmegakaryocytic abnormalities in 4 of these dogs. Significantly fewer dogs with severe thrombocytopenia had abnormalities identified on cytologic examination of bone marrow, compared with dogs with mild to moderate thrombocytopenia. CONCLUSIONS AND CLINICAL RELEVANCE: Cytologic examination of bone marrow is unlikely to provide specific diagnostic or prognostic information in dogs with severe thrombocytopenia.     

Antibodies to Ehrlichia canis, Ehrlichia platys, and Spotted Fever Group Rickettsiae in Louisiana Dogs

Antibodies to Ehrlichia canis, Ehrlichia platys, and spotted fever group (SFG) rickettsiae were detected by indirect immunofluorescence in sera from 27 ill individually owned thrombocytopenic dogs (platelet concentrations less than 200,000 platelets/microliters) and 59 healthy kenneled dogs located in southern Louisiana. Platelet concentrations less than 100,000 platelets/microliters were detected in 63% of ill thrombocytopenic dogs and 6.8% of healthy kennel dogs. One ill thrombocytopenic dog had intracytoplasmic E platys morulae detected within platelets. The prevalence of increased serum antibody titers to E canis and E platys was 25.9% and 40.7% for the ill thrombocytopenic dogs and 20.3% and 54.2% for the healthy kennel dogs, respectively. All dogs with seropositivity to E canis had increased antibody titers of greater than or equal to 1:100 to E platys. Simultaneous examination of increased serum antibody titers (greater than or equal to 1:64) to four SFG rickettsiae indicate that Rickettsia rhipicephali and Rickettsia montana accounted for the majority of the antibodies detected in these dogs. Of 86 dogs tested, 44.2% were seronegative to E canis, E platys, and SFG rickettsiae.     

Detection of antibody to turkey coronavirus by antibody-capture enzyme-linked immunosorbent assay utilizing infectious bronchitis virus antigen

An antibody-capture enzyme-linked immunosorbent assay (ELISA) for detection of antibody to turkey coronavirus (TCV) utilizing infectious bronchitis virus (IBV) antigen was developed. Anti-TCV hyperimmune turkey serum and normal turkey serum were used as positive or negative control serum for optimization of the ELISA system. Goat anti-turkey immunoglobulin G (light plus heavy chains) conjugated with horseradish peroxidase was used as detector antibody. The performance of the ELISA system was evaluated with 45 normal turkey sera and 325 turkey sera from the field and the cutoff point was determined. Serum samples of turkeys experimentally infected with TCV collected sequentially from 1 to 63 days postinfection were applied to the established antibody-capture ELISA using IBV antigens. The optimum conditions for differentiation between anti-TCV hyperimmune serum and normal turkey serum were serum dilution at 1:40 and conjugate dilution at 1:1600. Of the 325 sera from the field, 175 were positive for TCV by immunofluorescent antibody (IFA) assay. The sensitivity and specificity of the ELISA relative to IFA test were 93.1% and 96.7%, respectively, based on the results of serum samples from the field turkey flocks using the optimum cutoff point of 0.18 as determined by the logistic regression method. The ELISA values of all 45 normal turkey sera were completely separated from that of IFA-positive sera. The ELISA results of serum samples collected from turkeys experimentally infected with TCV were comparable to that of the IFA assay. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the IBV antigens coated in the commercially available ELISA plates coated with IBV antigens could be utilized for detection of antibodies to TCV in antibody-capture ELISA.     

Immunological analysis of agglutination in Dirofilaria immitis microfllariae

The mechanism of agglutination phenomenon of Dirofilaria immitis microfilariae was analyzed. Circulating microfilariae were collected from a D. immitis-infected microfilaremic dog and cultured in the several kinds of sera from dogs and animals. The agglutination of D. immitis microfilariae is a specific phenomenon due to some immune complexes formed with the anti-microfilarial antibody, heat-instable factor(s) and excretory-secretory products of microfilariae. Only live microfilariae were agglutinated and the agglutinated microfilariae remained alive as long as 27 days in culture in vitro.     

Presence of anti-platelet IgM and IgG antibodies in dogs naturally infected by Leishmania infantum

Thirty-three dogs, naturally infected by Leishmania infantum, were enrolled in the study and were classified as oligo-symptomatic (n. 15) and symptomatic or markedly symptomatic (n. 18). A control group was 10 healthy dogs. A haematological profile was obtained and the dogs serum was employed to assess the presence of platelet binding IgM and IgG antibodies (PBIgM, PBIgG) using flow cytometry. FITC labelled goat anti-dog IgM or IgG were used to detect PBIgM and PBIgG. Samples with a mean fluorescence intensity (MFI) that was 100 channels higher on a log scale for more than 30% of the platelets than seen in negative control platelets from a healthy dog were considered positive for the presence of anti-platelet antibodies (PBIg). Twenty-one (63.3%) dogs revealed the presence of PBIg. Six of them were oligo-symptomatic while 15 showed moderate or severe clinical signs of illness. All the dogs with PBIg showed the presence of PBIgM, with nine animals showing both PBIgM and PBIgG. Nine of 18 symptomatic or markedly symptomatic dogs showed thrombocytopenia, while normal platelet counts were observed in all oligo-symptomatic animals. Eight of 9 thrombocytopenic animals showed the presence of PBIgM, while six of them showed PBIgG. One thrombocytopenic dog was negative for PBIg. This study is the first report documenting the presence of PBIg in natural canine leishmaniasis implying a pathogenic association between thrombocytopenia and the presence of antibody against platelet membrane.     

Virion polypeptide specificity of immune complexes and antibodies in cats inoculated with feline infectious peritonitis virus

Immune complexes purified from sera and ascitic fluids of cats after inoculation with feline infectious peritonitis (FIP) virus contained proteins and proteolytic fragments of the peplomer, nucleocapsid, and envelope polypeptides; in addition, host proteins were demonstrated in the immune complexes. Free (uncomplexed) antibodies against the 3 classes of virion polypeptides were detected and quantitated; the weakest and latest response was directed against the peplomer protein. Immunofluorescence titers showed the best correlation with the antibody response directed against the envelope polypeptides. Differences in reactivity were not found between sera and ascitic fluids from the same animals and between seropositive healthy cats and cats which had died of FIP. Humoral antibody and hypergammaglobulinemia showed a linear correlation, but the wide variation in antiviral titers at a given concentration of gamma-globulin indicated that additional (autoimmune) reactions occur during the pathogenesis of FIP.     

Immune‐mediated hemolytic anemia and severe thrombocytopenia in dogs: 12 cases (2001–2008)

Objective – To identify and characterize the syndrome of immune‐mediated hemolytic anemia (IMHA) with concurrent severe thrombocytopenia (≤15.0 × 10⁹ platelets/L; [15.0 × 10³ platelets/μL]), and to evaluate prognostic factors, clinicopathologic findings, complications, treatment, outcome, and survival of dogs with this hematologic disorder. Design – Retrospective, observational study. Setting – Veterinary teaching hospital. Animals – Twelve client‐owned dogs with IMHA and severe thrombocytopenia (≤15.0 × 10⁹ platelets/L; [15.0 × 10³ platelets/μL]), without evidence of overt disseminated intravascular coagulation. Interventions – The following data were recorded and analyzed from the electronic medical record: signalment, history, concurrent diseases, clinical signs at presentation, clinicopathologic data, diagnostic testing, radiographic findings, treatment modalities, length of hospitalization, complications, and clinical outcome. All dogs were treated with immunosuppressive doses of corticosteroids. Measurements and Main Results – Twelve dogs were identified with the diagnosis of IMHA and severe thrombocytopenia; of these, 9 (75%) survived, 3 (25%) were euthanized, and none died. Dogs that survived were significantly younger than nonsurvivors (P=0.03). There were no specific clinical signs or therapies associated with survival. Conclusions – Dogs in this study had a mortality rate similar to reported rates for dogs with either disease alone. Overall, younger dogs were more likely to survive. No association between different treatment modalities and overall survival was identified.     

Detection of Antiplatelet Antibody With a Platelet Immunofluorescence Assay

An indirect platelet immunofluorescence assay (PIFA) was developed for detection of circulating antiplatelet antibody in dogs with suspected immune-mediated thrombocytopenia (ITP). The PIFA was performed on 10 healthy dogs with normal platelet counts; 76 thrombocytopenic dogs, 20 of which were suspected of having ITP; and 18 dogs with other diseases and normal platelet counts. All normal dogs had negative test results. Fourteen (70%) of 20 dogs suspected of having ITP had positive test results. Fifteen of the remaining 56 thrombocytopenic dogs had positive test results, 9 had cancer and 6 had other immune-mediated diseases including systemic lupus erythematosus (SLE). In this study, the PIFA assay seemed to be more sensitive (70%) than the megakaryocyte immunofluorescence assay (41 %) in the diagnosis of ITP. Of the 9 PIFA-positive dogs with neoplasia, 6 had lymphoproliferative disorders. The PI FA was positive in 5 of 18 diseased dogs with normal platelet counts. There was an inverse relationship between the platelet count and the intensity of fluorescence in the PIFA-positive dogs. We conclude that the PIFA is a sensitive screening method for detecting circulating antiplatelet antibody.     

Enzyme-linked immunosorbent assay for detection of antibody to avian encephalomyelitis virus in chickens

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to avian encephalomyelitis virus (AEV) has been developed for determining whether existing AEV control programs adequately protect breeder hens. A partially purified AEV antigen was bound to microcuvettes for reaction with specific primary antibody. A second antibody, rabbit anti-chicken immunoglobulin G (IgG) conjugated with horseradish peroxidase, was employed to react with bound primary IgG. The relative amount of bound primary IgG was detected using ortho-phenylenediamine as a substrate for enzymatic production of a chromogen by horseradish peroxidase. Intensity of absorbance of the chromogen at 490 nm was related to the bound primary antibody by the titration method. Negative antisera were surveyed to establish an appropriate positive/negative cutoff level at twice the mean absorbance of negative sera at a 1:100 dilution. The test reagents for the ELISA were optimized by reagent titrations utilizing known positive and negative antisera for discrimination. The optimized ELISA had a coefficient of variation of from 1.2 to 3.3 for within-assay titer and of 2.4 for between-assay mean titer. Even though the ELISA detected only specific IgG, it was as accurate as the virus-neutralization test for evaluating the immune status of hens to AEV. Moreover, the ELISA was more economical in the use of reagents, time, and personnel and was free from dependence on susceptible embryos. Since ELISAs can be standardized and measured with manual or automated instruments, the derived ELISA can be easily and economically used to evaluate the immune status of breeder hens in commercial poultry operations.     

Antibodies Against Helicobacter felis in Sera of Cats and Dogs

Serum samples from 61 dogs and 49 cats were screened for circulating antibodies against Helicobacter felis by an enzyme-linked immunosorbent assay (ELISA) using sonicated bacteria as an antigen. To improve the specificity of the ELISA, sera were absorbed with Campylobacter jejuni subsp. jejuni H. pylori as well as H. felis. Sera from 26 dogs (43%) and 19 cats (39%) revealed clear positive absorbance readings determined as an optical density (OD) that was statistically significant above the OD mean value [P < 0.025 (one-tailed); log₁₀]. The absorbance pattern of ELISA-positive sera corresponded to results obtained with bovine and human reference sera. Furthermore, a correlation between the immune response and results from histopathological examination of gastric specimens from 22 dogs was demonstrated. It could be shown that antibodies against H. felis in sera of cats and dogs can easily be detected using an ELISA. The diagnostic value of this test must be evaluated in further investigations.     

Idiopathic asymptomatic thrombocytopenia in Cavalier King Charles Spaniels is an autosomal recessive trait

Cavalier King Charles Spaniels (CKCS) often have idiopathic asymptomatic thrombocytopenia. In affected dogs, the thrombocytes often are large, and it has been speculated that the condition could be an inherited macrothrombocytopenia. The aim of this study was to examine the inheritance of idiopathic, asymptomatic thrombocytopenia in CKCS. Sixteen families (both parents and > or = 3 offspring) of privately owned CKCS were included. There were 105 clinically healthy dogs (50 from Denmark and 55 from Sweden): 81 offspring and 26 parents (2 dogs had both roles). Because autoanalyzers have difficulty counting large platelets, the platelets were counted manually, with a counting chamber. Platelet counts were not influenced by age, gender, or heart murmur status. Thrombocytopenia (< or = 100,000 platelets/microL) was found in 46% of the parents. The pedigrees indicated that thrombocytopenia segregated as an autosomal recessive trait and that 100,000 platelets/microL was appropriate as a lower limit of normal. Affected offspring were found in all families, showing that all of the included parents were at least carriers. Therefore, the expected segregation ratios (which were in good accordance with the observed ones) were 1:0, 1:1, and 1:3 for the 3 crosses: affected x affected, normal x affected, and normal x normal. Within a given cross, the mean parental platelet count had no influence on the platelet counts of the offspring. We conclude that idiopathic, asymptomatic thrombocytopenia in CKCS is inherited in an autosomal recessive manner. The condition most likely constitutes an inherited macrothrombocytopenia in dogs.     

Fasciola gigantica excretory-secretory products for immunodiagnosis and prevention of sheep fasciolosis

Excretory-secretory products (ESP) products of ex vivo Fasciola gigantica adult worms were used for immunodiagnosis of sheep experimental infection with F. gigantica and natural infection with Fasciola spp. by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Specific IgG antibody binding to native or denatured ESP was detected as early as 2 weeks after experimental sheep infection with 100 or 200 metacercariae. No specific IgG antibody binding was displayed by sera obtained from 192 sheep considered to be Fasciola- and other parasite-free by microscopic examination of bile and feces. Additionally, sera from 200 apparently Fasciola-free sheep, yet infected with other parasites, were all negative. The data, thus, indicated that ESP-based ELISA reached nearly 100% sensitivity and specificity in immunodiagnosis of sheep fasciolosis. As expected, the ESP molecules were immunogenic in sheep eliciting interleukin-12p40 mRNA response and considerable amounts of antibodies, which were able to bind to the surface of newly excysted juvenile worms as judged by membrane indirect immunofluorescence, and mediate their attrition via antibody-dependent cell-mediated cytotoxicity. The ESP-induced cellular and humoral immune responses were associated with a modest reduction in worm count, yet with a highly significant (P<0.0001) decrease in size of recovered worms, thus suggesting that ESP immunization might be a safe and cost-effective strategy for reducing transmission of the infection.