Isolation of a monoclonal antibody with specificity for commonly employed vaccine strains of Newcastle disease virus

A monoclonal antibody, AVS-I, was produced from a hybridization of murine myeloma cells and splenocytes from mice immunized with the La Sota strain of Newcastle disease virus (NDV). The hybridoma producing AVS-I, selected from 184 NDV-positive supernatants, is one of two supernatants that reacted exclusively with lentogenic strains in an indirect enzyme-linked immunosorbent assay. AVS-I can also be assayed by hemagglutination-inhibition (HI), which was used to test selected reference avian paramyxovirus (PMV) strains of types 1 to 3. NDV vaccines La Sota and B1 and field isolates from chickens, turkeys, pigeons, and cockatoos were also used as antigens. AVS-I had a high binding affinity for all La Sota and B1 strains, including vaccines. The antibody bound with a lower titer to the Australian Queensland V4 and Ulster strains, but it did not bind to the F strain, a lentogenic strain from England. AVS-I was HI-negative against the other PMV reference strains. AVS-I may be valuable for identifying field isolates antigenically similar to La Sota and B1 and rapidly differentiate those vaccine strains from more virulent viruses.     

An immunogold labelling method for the enumeration of canine T-lymphocytes

An immunogold procedure using the monoclonal antibody F3-20-7 to canine Thy-l has been used to label T-lymphocytes in peripheral blood samples taken from healthy Beagles. By this method, approximately 64% of peripheral blood lymphocytes were identified as T-lymphocytes.     

Chemiluminescence of canine peripheral blood lymphocytes stimulated by mitogens

Luminol-dependent chemiluminescence of peripheral blood lymphocytes from dogs stimulated with concanavalin A (Con A) or phytohemagglutinin P (PHA) was measured with a Pico-Lite luminometer. 10 microliter of luminol gave optimal quantum yield from 1 X 10(6) lymphocytes sensitized with either 80 micrograms Con A or 160 micrograms PHA. Addition of superoxide dismutase did not influence the course of chemiluminescence. Whereas catalase produced 41% increase in quantum yield, mannitol caused a 51% inhibition of chemiluminescence. Lymphocytes exposed to varying doses of short term x-irradiation or lymphocytes isolated from dogs kept under continuous exposure through a gamma irradiation source showed dose-related depression of chemiluminescence. Membrane factors may be involved in lymphocyte stimulation to chemiluminescence as pulse experiments with Con A and PHA revealed. It is proposed that chemiluminescence measurements may be useful in monitoring early events in lymphocyte stimulation by antigens and mitogens.     

Enzyme-linked immunosorbent assays of canine apolipoproteins B-100 and A-I

An enzyme-linked immunosorbent assay (ELISA) for canine blood apolipoprotein (apo) B-100 and A-I was developed. The working range for the assay was 1.8 to 28.7 ng/well for apoB-100 and it was 50 to 410 ng/well for apoA-I. The intra- and inter-assay coefficients of variation for the assay for apoB-100 were 5.4 and 6.9%, respectively, and for apoA-I they were 5.8 and 10.6%, respectively. The average concentrations of apoB-100 and A-I in 25 beagles (males, aged 3-4 years) were 0.084 +/- 0.028 (mean +/- SD) mg/ ml and 6.29 +/- 1.55 mg/ml, respectively. The ratios of canine (C) apoB-100 to CapoA-I were 1.41 +/- 0.58%. The respective concentrations in one case of hyperlipidemia with systemic atherosclerosis were 0.454 mg/ml and 11.28 mg/ml (a ratio of 4.03%). These values were larger than those of the controls. These results suggest that the measurements of CapoB-100 and A-I concentrations by this newly developed ELISA are helpful for diagnosis of lipidosis.     

Efficacy and toxicity of estrogens commonly used to terminate canine pregnancy

Three groups of bitches were treated with diethylstilbestrol (75 micrograms/kg) orally for 7 days (n = 12), estradiol cypionate intramuscularly once (22 micrograms/kg; n = 12), or estradiol cypionate intramuscularly once (44 micrograms/kg; n = 12). Treatments commenced during late proestrus (n = 4/group), the fourth day of behavioral estrus (n = 4/group), or the second day of diestrus (n = 4/group). All bitches were bred on alternate days throughout estrus to stud dogs of known fertility. Ovariohysterectomies were performed on day 25 of diestrus to diagnose pregnancy and to assess any pathologic changes in the uterus. Eleven bitches treated with diethylstilbestrol, 6 bitches treated with the low dosage of estradiol cypionate, and 4 bitches receiving the high dosage of estradiol cypionate were pregnant at the time of surgery. Ten of the bitches treated with estrogens during proestrus, 6 treated during estrus, and 4 treated during diestrus were pregnant. The serum concentration of progesterone in 2 bitches treated with the high dosage of estradiol cypionate decreased to less than 2 ng/ml by day 25 of diestrus, suggesting premature luteal regression. Diethylstilbestrol appeared to have little efficacy in terminating pregnancy. Estradiol cypionate appeared to have greater efficacy when administered during estrus or early diestrus; however, pyometra developed in 2 bitches treated with this estrogen during diestrus.     

Antigen requirements and specificity of enzyme-linked immunosorbent assay for detection of canine IgG against canine distemper viral antigens

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of specific immunoglobulin G (IgG) against canine distemper virus (CDV) antigens. Sucrose gradient separation of viral and cellular proteins was required to produce coating antigens for the ELISA. The specificity of the ELISA was demonstrated by blocking CDV-positive canine sera with CDV-specific antisera produced in goats and rabbits and adsorption of positive sera with CDV antigens. A comparison of the ELISA with the serum-neutralization technique for the detection of CDV antibodies was conducted. Anti-CDV IgG was detected in conventional dogs as early as 6 days after inoculation with a commercial vaccine to CDV. Paired sera from the immunized dogs were evaluated by both techniques and a statistically (P less than 0.01) significant agreement between the ELISA and the serum-neutralization technique was shown (r = 0.6121, n = 75).     

Immunohistochemical detection of apolipoprotein A-I and B-100 in canine atherosclerotic lesions

We attempt to determine and compare the localization of apolipoproteins (apo) apoA-I and B-100 in atherosclerotic lesions of canine aortas, coronary arteries, and the peripheral arteries, using immunohistochemical techniques. Histopathologically, atherosclerotic lesions were characterized by deposition of lipids and infiltration of lipid-laden foamy cells in the tunica intima and tunica media, sometimes forming fibrofatty plaques containing abundant sudanophilic and mineralized material. Canine apoA (CapoA)-I and canine apoB (CapoB)-100 immunopositive signals were simultaneously observed in mild and severe atherosclerotic lesions of the aorta, coronary arteries, splenic arteries, and renal arteries in the double-immunolabeled sections. Both CapoA-I and CapoB-100 positive signals were seen in the cytoplasm of endothelial cells, smooth muscle cells, and macrophages. The subendothelial space and extracellular matrix in the tunica intima and media were also positive. Neither CapoA-I nor CapoB-100 positive signals were seen in normal arteries. These findings closely resemble those of the localization of apoA-I and apoB-100 in human atherosclerotic lesions.     

Lymphocyte responsiveness to mitogens and quantitation of T and B lymphocytes in canine malignant lymphoma.

Two canine malignant lymphoma cases were studied, one from the time of detection of enlarged palpable lymph nodes through the terminal stage and another at the terminal stage. Hematologic and histopathologic studies were confirmative of leukemia. The lymphocyte subpopulations, T and B cells, were quantitated as identified by the presence or absence of surface immunoglobulin and erythrocyte-antibody-complement-rosette formation. The average number of B cells in the peripheral blood lymphocytes throughout the study were approximately 80%. The B cells in the lymph node lymphocytes were 82%. There was considerable fluctuation in the number of blood lymphocytes, but the percentage of T and B lymphocytes remained nearly constant. There was marked impairment in the lymphocytic response to mitogens. The results of this study indicate that the canine malignant lymphoma is predominantly a B-lymphocyte type.     

Antimicrobial susceptibilities of canine Clostridium difficile and Clostridium perfringens isolates to commonly utilized antimicrobial drugs

Clostridium difficile and Clostridium perfringens are anaerobic, Gram-positive bacilli that are common causes of enteritis and enterotoxemias in both domestic animals and humans. Both organisms have been associated with acute and chronic large and small bowel diarrhea, and acute hemorrhagic diarrheal syndrome in the dog. The objective of this study was to determine the in vitro antimicrobial susceptibilities of canine C. difficile and C. perfringens isolates in an effort to optimize antimicrobial therapy for dogs with clostridial-associated diarrhea. The minimum inhibitory concentrations (MIC) of antibiotics recommended for treating C. difficile (metronidazole, vancomycin) and C. perfringens-associated diarrhea in the dog (ampicillin, erythromycin, metronidazole, tetracycline, tylosin) were determined for 70 canine fecal C. difficile isolates and 131 C. perfringens isolates. All C. difficile isolates tested had an MIC of or=256 microg/ml for both erythromycin and tylosin. A third C. perfringens isolate had an MIC of 32 microg/ml for metronidazole. Based on the results of this study, ampicillin, erythromycin, metronidazole, and tylosin appear to be effective antibiotics for the treatment of C. perfringens-associated diarrhea, although resistant strains do exist. However, because there is limited information regarding breakpoints for veterinary anaerobes, and because intestinal concentrations are not known, in vitro results should be interpreted with caution.     

Sensitivity and specificity of canine serum total amylase and isoamylase activity determinations.

Serum isoamylases were determined prospectively in dogs with pancreatic and extrapancreatic diseases. Mean serum isoamylase determinations were significantly different (p less than 0.05) between normal dogs and dogs with pancreatitis and exocrine pancreatic insufficiency. The sensitivity of serum isoamylase determination exceeded that of total amylase activity for the diagnosis of pancreatitis. Serum isoamylase determinations were less influenced by extrapancreatic diseases compared to total amylase activity when used in the diagnosis of pancreatic disease. Neither serum isoamylase determination nor total amylase activity had adequate sensitivity to support their use in the diagnosis of exocrine pancreatic insufficiency. There were significant (p less than 0.05) linear correlations between isoamylase determinations, total amylase activity, and trypsin-like immunoreactivity concentration.     

Sensitivity and specificity of radioimmunoassay of serum trypsin-like immunoreactivity for the diagnosis of canine exocrine pancreatic insufficiency

Concentrations of serum trypsin-like immunoreactivity (TLI) measured by radioimmunoassay were low (less than 1.9 micrograms/L) in 25 dogs with exocrine pancreatic insufficiency (EPI), compared with 100 clinically normal (control) dogs (5.2 to 34.0 micrograms/L; P less than 0.001; sensitivity, 100%). Serum TLI concentrations (5.5 to 35.0 micrograms/L) in a group of 50 dogs with small intestinal disease (SID) were not significantly different from those of control dogs, values being greater than the lower limit of the control range in all cases (specificity, 100%). Results of bentiromide (N-benzoyl-L-tyrosyl-p-aminobenzoic acid [BT-PABA]) tests and fecal proteolytic activity (determined by use of an azocasein substrate) were abnormal in 21 of 22 dogs with EPI (sensitivity, 95%). Bentiromide test results were subnormal in 13 of 35 dogs with SID (specificity, 63%), whereas fecal proteolytic activity was subnormal in 7 of 34 dogs with SID (specificity, 79%). It was concluded that assay of serum TLI is a highly sensitive and specific test for the identification of dogs with EPI.     

Sensitivity and specificity of the indirect-fluorescent antibody test and two enzyme-linked immunosorbent assays in canine dirofilariasis

Ninety dogs naturally infected with Dirofilaria immitis and 103 noninfected dogs, as determined by necropsy, were used to compare the sensitivity and specificity of the cuticular and somatic reactions of the indirect fluorescent antibody test (IFA-C and IFA-S, respectively) and 2 enzyme-linked immunosorbent assays (ELISA). In microfilaremic and amicrofilaremic heartworm-infected dogs, negative results were common for all serotests. In dogs without adult heartworms at necropsy, 32% to 49% were positive, using 1 ELISA, 27% to 29% were positive with the other ELISA, 15% to 36% were positive with the IFA-S, and 0% to 1% were positive using the IFA-C, depending on the classification of borderline reactions. The prevalence of false positive serotests was probably not due to the detection of precardial stages of D immitis in dogs obtained from areas of low endemicity. Until the causes of the false-positive tests are resolved, the use of currently available serotests for routine diagnostic screening or as criteria for instituting treatment is not recommended.     

Marking of peripheral T-lymphocytes by retroviral transduction and transplantation of CD34+ cells in a canine X-linked severe combined immunodeficiency model

A retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) was used to mark and dynamically follow vector-expressing cells in the peripheral blood of bone marrow transplanted X-linked severe combined immunodeficient dogs. CD34(+) cells isolated from young normal dogs were transduced, using a 2 day protocol, with an amphotropic retroviral vector that expressed enhanced green fluorescent protein (EGFP) and the canine common gamma chain (gammac) cDNAs. Following transplantation of the transduced cells, normal donor peripheral blood lymphocytes (PBL) appeared by 1 month post-bone marrow transplant (BMT) and rescued three of five treated dogs from their lethal immunodeficiency. PCR and flow cytometric analysis of post-BMT PBL documented the peripheral EGFP expressing cells as CD3(+) T cells, which varied from 0% to 28%. Sorting of EGFP(+) and EGFP(-) peripheral blood T cells from two dogs, followed by vector PCR analysis, showed no evidence of vector shutdown. EGFP expression in B cells or monocytes was not detected. These marking experiments demonstrate that the transduction protocol did not abolish the lymphoid engraftment capability of ex vivo transduced canine CD34(+) cells and supports the potential utility of the MSCV retroviral vector for gene transfer to XSCID affected canine hematopoietic progenitor cells (HPC).     

Activation of the alternative complement pathway by lymphoblasts isolated from canine thymic lymphomas

The ability of lymphocytes isolated from cases of canine thymic lymphomas to activate the alternative complement pathway has been studied. In a series of 14 thymic lymphomas 5 were found to activate the alternative pathway. This ability to activate was not abrograted by trypsinisation of the cells. It was found that normal canine thymic lymphocytes did not activate the alternative pathway.     

Species specificity in the adherence of staphylococci to canine and human corneocytes: a preliminary study

Staphylococcal pyoderma is rarely contagious between dogs and humans, or humans and dogs. This study investigated the hypothesis that there are species differences in adherence of Staphylococcus intermedius (the most common isolate from dogs) and Staphylococcus aureus (the most common isolate from humans) to canine and human corneocytes. Sheets of corneocytes were collected from the ventral abdomen of 10 dogs and the medial forearm of 10 humans (all healthy and without any history or physical signs of skin disease) using double-sided tape. Staphylococcus intermedius from a case of canine bacterial pyoderma and a human strain of S. aureus were prepared in phosphate buffered saline (PBS) and applied in duplicate, respectively, to the canine and human corneocyte-covered tapes using PBS as negative control. After incubation, rinsing, and staining with crystal violet, quantification of the adherent bacteria was carried out blindly by computerized image analysis. Staphylococcus intermedius was found to adhere significantly more to canine corneocytes than S. aureus (P = 0.0006), whereas S. aureus showed greater adherence to human corneocytes than S. intermedius (P < 0.0001). In addition, the pattern of adherence differed between the two organisms, with S. intermedius adhering to the entire surface and S. aureus adhering mainly to the periphery of both canine and human corneocytes. Preference for adherence to these two hosts may explain, in part, why S. intermedius and S. aureus are uncommonly isolated from human and canine skin infections, respectively.     

Establishment of canine and feline cells expressing canine signaling lymphocyte activation molecule for canine distemper virus study

Antimicrobial therapy is a useful tool to control bovine mastitis caused by Staphylococcus aureus, as consequence an increase in staphylococci resistant cases has been registered. Alternative strategies are desirable and bacteriocins represent attractive control agents to prevent bovine mastitis. The aim of this work was to evaluate the activity of five bacteriocins synthesized by Bacillus thuringiensis against S. aureus isolates associated to bovine mastitis. Fifty S. aureus isolates were recovered from milk composite samples of 26 Holstein lactating cows from one herd during September 2007 to February 2008 in México and susceptibility of those isolates to 12 antibiotics and 5 bacteriocins from B. thuringiensis was evaluated. S. aureus isolates were mainly resistant to penicillin (92%), dicloxacillin (86%), ampicillin (74%) and erythromycin (74%); whereas susceptibility to gentamicin, trimethoprim and tetracycline was detected at, respectively, 92%, 88%, and 72%. All S. aureus isolates showed susceptibility to the five bacteriocins synthesized by B. thuringiensis, mainly to morricin 269 and kurstacin 287 followed by kenyacin 404, entomocin 420 and tolworthcin 524. Our results showed that S. aureus isolates had differences in the antimicrobial resistance patterns and were susceptible to bacteriocins produced by B. thuringiensis, which could be useful as an alternative method to control bovine mastitis.