Short communication: Attempts to identify Clostridium botulinum toxin in milk from three experimentally intoxicated Holstein cows.

Three adult lactating Holstein cows were injected in the subcutaneous abdominal vein with 175 ng/kg of body weight of Clostridium botulinum type C toxin (451 cow median toxic doses) to determine if this botulinum toxin crosses the blood-milk barrier. Whole blood (in sodium heparin) and clotted blood serum samples were taken at 0 min, 10 min, and 3, 6, 9, and 12 h postinoculation. Milk samples were taken at 0 min and at 3, 6, 9 and 12 h postinoculation. All samples were tested for the presence of the toxin using the mouse bioassay and immunostick ELISA test. The immunostick ELISA identified the toxin in whole blood and the mouse bioassay identified the toxin in serum at all times examined in all 3 animals. Toxin was not identified by either detection method in milk samples collected from the 3 animals. From these results, it appears that Clostridium botulinum type C toxin does not cross from the blood to the milk in detectable concentrations.

The isolation of exotic Newcastle disease (END) virus from nonpoultry avian species associated with the epidemic of END in chickens in southern California: 2002-2003.

During the first 11 months of the 2002-2003 exotic Newcastle disease (END) epidemic in chickens in southern California, a total of 27,688 cloacal and tracheal (oropharyngeal) swab pools and/or tissue pools from 86 different avian species other than chickens and turkeys were submitted for Newcastle disease virus (NDV) isolation and characterization. Fifty-seven specimens (0.23%), representing 12 species of birds and 13 unspecified species, from a total of 24,409 accessions or submissions were positive for NDV. The NDV isolate was characterized as ENDV by real-time reverse transcription-polymerase chain reaction (RT-PCR). Of the 11,486 premises with other avian species, 1599 also had chickens. There were 1900 positive chicken samples from 164 premises, and 56 positive other avian species from 51 premises. Twelve premises had both positive chickens and positive other avian species. All positive other avian species were located on premises either on or within a 1 km radius of known infected premises. In this epidemic, premises with positive other avian species were significantly more likely to have chickens, and were significantly more likely to have positive chickens (OR = 3.7, P < 0.0001).

Interaction of Rhodococcus equi with phagocytic cells from R. equi-exposed and non-exposed foals.

The interaction of Rhodococcus equi with alveolar macrophages from adult horses, foals experimentally exposed to R. equi (sensitized foals) and non-exposed foals was studied using in vitro bactericidal assays, cytochemical staining and transmission electron microscopy. It was demonstrated that R. equi is a facultative intracellular parasite, able to survive and multiply within the alveolar macrophages of the host by interfering with phagosome-lysosome fusion. Opsonization of R. equi with antibody against capsular components was associated with increased phagosome-lysosome fusion and significantly enhanced (P less than 0.05) killing of the organism by alveolar macrophages from non-exposed foals. Macrophages from non-exposed foals were able to ingest the non-opsonized organism, but unable to kill greater than 65% of the infective dose by 6 h post-exposure. Alveolar macrophages from sensitized foals behaved as adult macrophages, able to kill greater than 95% of the infective dose by 6 h. Lymphocyte factors, derived by in vitro incubation of sensitized peripheral blood lymphocytes with R. equi surface antigens, enhanced macrophage bactericidal activity. Macrophages from non-exposed foals incubated in the presence of the lymphocyte factors had a 50% increase in killing of R. equi, while sensitized macrophages incubated with lymphocyte factors had a greater than 100% increase in killing capacity.

Enzyme-linked immunosorbent assay for the detection of antibodies to bovine viral diarrhea virus in bovine sera.

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was established for the detection of antibodies to bovine viral diarrhea virus (BVDV) in bovine sera. Polyethylene-glycol concentrated, equilibrium density gradient purified BVDV was used as test antigen at an optimal amount of 1 microgram/well, whereas the optimal concentration of conjugate was at 1/2000 dilution. The standardized test encountered no non-specific reaction with test sera at a starting dilution of 1/10. A total of 50 bovine serum samples was assayed for the presence of antibodies against BVDV by ELISA and serum neutralization test (SNT). A positive correlation between the 2 tests was found. However, ELISA could be as much as 500-fold more sensitive than SNT in detecting low levels of BVDV antibodies.

Identification of envelope and nucleocapsid proteins of infectious bovine rhinotracheitis virus by SDS-polyacrylamide gel electrophoresis.

Infectious bovine rhinotracheitis (IBR) virus was purified by rate zonal and isopycnic centrifugation in potassium tartrate gradients. Viral nucleocapsids were isolated from purified virions by treatment with the nonionic detergent Triton X-100 followed by high speed centrifugation. This treatment was shown to produce a suspension of 74% completely de-enveloped nucleocapsids, 24% incompletely de-enveloped nucleocapsids, and 2% whole virions. The viral nucleocapsids contained DNA and banded at a density of 1.25 g/cm3. Analysis of the viral polypeptides by gradient SDS-polyacrylamide gel electrophoresis revealed that 33 virion proteins, ranging in molecular weight from 13,000 to 275,000 dalton, were present in the complete virus particle. Detergent treatment of the virus quantitatively removed two of the major proteins (vp8, 90,000 dalton, and vp13, 73,000 dalton) and partially removed eleven other proteins. Fifteen viral polypeptides appeared to remain firmly associated with the viral nucleocapsids.

Neutrophil phagocytic and serum opsonic response of the foal to Corynebacterium equi.

This study was undertaken to examine the neutrophil response to Corynebacterium (Rhodococcus) equi, and to assess the possibility of neutrophil immaturity or malfunction in predisposition to C. equi pneumonia in foals. Neutrophil phagocytosis of Corynebacterium (Rhodococcus) equi was studied in foals from birth to 6 months of age. Chemiluminescence (CL) and bactericidal assays were used to assay the phagocytic response of peripheral blood neutrophils to C. equi in vitro. Results of in vitro bactericidal and CL assays indicate that foal neutrophils are able to ingest and kill C. equi, however are significantly more efficient in the presence of opsonization with specific antibody, and less importantly complement. Neutrophil CL was significantly decreased (p greater than .05) or eliminated by antibody adsorption, heat-inactivation, or removal of serum from the assay. The ability of the neutrophil to kill C. equi, as measured by in vitro bactericidal assay, was greater than 90% killing by 6 hours, in the presence of C. equi antiserum. Bactericidal activity was reduced to less than 40% killing when C. equi adsorbed serum was used as the opsonin source. As CL results indicated complement involvement in the opsonization of C. equi, the temporal development of hemolytic and conglutinating complement was measured in normal and C. equi infected foals. Neither defects nor age-related suppression of neutrophil function or complement activity were detected in C. equi affected foals, suggesting that these are not pathogenic mechanisms involved in foal pneumonia.

The longevity of immunoglobulin preservation in canine skin utilizing Michel's fixative.

Skin biopsy specimens from 7 dogs with immune-mediated skin diseases diagnosed by routine histology and 5 dogs with other skin diseases were placed in Michel's transport medium for 4 to 9 years. Direct immunofluorescence yielded positive results in tissue samples from 3 dogs with pemphigus foliaceus and 2 dogs with discoid lupus erythematosus. Direct immunofluorescence was not seen in tissue samples from 1 dog with pemphigus foliaceus and 5 dogs with non immune-mediated skin diseases. Direct immunofluorescence was seen in skin biopsy specimens maintained in Michel's medium for 4 to 8 years.

Canine discoid lupus erythematosus.

Two dogs were found to have clinical, histopathological and immunofluorescent findings compatible with a diagnosis of canine discoid lupus erythematosus. The primary lesions included erythema and depigmentation of the nasal planum. Both dogs responded favorably to systemic corticosteroid therapy.

Experimental reproduction of bovine fetal Neospora infection and death with a bovine Neospora isolate.

Studies were conducted to determine the pathogenic potential of the recently isolated bovine Neospora protozoa (BPA-1) for the bovine fetus. Cows chosen for study had Neospora titers < 160 using an indirect immunofluorescent antibody (IFA) test. Four experimental groups were studied. In group 1, 2 fetuses were inoculated in utero at 118 days gestation with culture-derived Neospora tachyzoites. A pregnant control cow was housed in the same pen, observed daily and screened serologically for evidence of exposure to Neospora. In group 2, 2 cows were infected with Neospora tachyzoites at 138 or 161 days gestation, and 1 control cow was given uninfected cell culture suspension simultaneously at 154 days gestation. Groups 3 (85 days gestation) and 4 (120 days gestation) each consisted of 2 cows infected with Neospora tachyzoites and 1 control cow given uninfected material at the same stage of gestation. Dead fetuses were surgically removed from the infected cows in group 1 on postinfection day (PID) 17. The histopathology was compatible with protozoal fetal infection, and protozoa were identified by immunohistochemistry. Viable fetuses were removed surgically from cows in group 2 on PID 28-30. The histopathology was compatible with protozoal fetal infection, protozoa were identified by immunoperoxidase techniques, and Neospora tachyzoites were reisolated in vitro from tissues of the 2 infected fetuses. In groups 3 and 4, the control fetus and 1 infected fetus were removed surgically between PID 26 and PID 33. The remaining infected cows were observed until fetal death or abortion occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonspecific reactions in an enzyme-linked immunosorbent assay caused by binding of immunoglobulins in situ to egg-propagated infectious bronchitis virus.

High levels of nonspecific background absorbance and increased variability were found in a previously optimized enzyme-linked immunosorbent assay (ELISA) for infectious bronchitis virus (IBV) antibody after changing to commercially available non-pathogen-free eggs for viral antigen production. An increase in bound viral antigen in the assay caused a proportionate increase in the nonspecific binding of the conjugate, independent of other variables, in the absence of serum. Virus was propagated in non-pathogen-free eggs, and individual viral proteins were separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose. Localization of chicken IgG-virus complexes were identified by immunoprecipitation with peroxidase-conjugated anti-chicken IgG. Specific staining at molecular weights corresponding to major proteins of IBV was demonstrated in these viral preparations. Virus grown in specific-pathogen-free eggs and treated in the same manner showed only slight amounts of staining. This evidence suggests that viral antigens grown in eggs from a non-pathogen-free flock bind with maternal chicken immunoglobulins present in the allantoic cavity of eggs. This IgG caused nonspecific reactions in our chicken ELISA system and gives cause for concern in any diagnostic system requiring the propagation of agents in fertile eggs.

Optimization of parameters for detecting antibodies against infectious bronchitis virus using an enzyme-linked immunosorbent assay: temporal response to vaccination and challenge with live virus.

Critical parameters affecting sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) for avian infectious bronchitis virus (IBV) were evaluated and optimized. The use of purified IBV as antigen at 50 ng protein/well and high-ionic-strength serum dilution buffer has resulted in a test with minimal nonspecific binding of chicken immunoglobulins and very high sensitivity. Optimum conditions for serum dilution, conjugate dilution, and substrate incubation were determined for minimizing background and nonspecific reactions. The use of this test in a controlled challenge study with chickens vaccinated with live IBV demonstrated its effectiveness in monitoring circulating antibody levels to infectious bronchitis. The IBV ELISA, which is rapid, inexpensive, highly sensitive, and capable of handling very large numbers of samples, should provide the poultry industry with a reliable means for IBV flock monitoring.

An association between complete and incomplete stress fractures of the humerus in racehorses.

Twenty-one horses had a complete unilateral humeral fracture during race training or racing at a California racetrack during the period 24 February 1990 to 10 July 1991. Fractures occurred approximately equally in left and right limbs, and in males and females. Most fractures occurred during training, and in 2- and 3-year-old horses. Only 5 of 16 Thoroughbred horses with known racing records had previously raced more than once, and their mean time between races was less than the time between their last race and fracture (P = 0.07). Ten of 13 humeri studied further had gross evidence of periosteal callus bridging one portion of the fracture line, indicative of a pre-existing stress fracture.

Detection of Corynebacterium equi-specific antibody in horses by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbant assay was developed to measure naturally occurring Corynebacterium equi specific antibody in horse serum. Antibody against C equi was demonstrated in normal adults and was passively transferred to foals. Adult levels of specific antibody were reached by 5 to 6 months of age in healthy foals. Decreased early antibody levels were demonstrated in a limited number of foals with confirmed C equi infection.

Application of the log-linear model in the prediction of the antinuclear antibody test in the dog.

To find possible associations between antinuclear antibody (ANA) pattern, ANA titer, and certain clinical changes and clinical laboratory test results in dogs, the veterinary medical records of 111 ANA-positive and 126 ANA-negative dogs were examined. Variables could not be found that had significant associations with ANA pattern (unlike the results in persons), because of the predominance of 2 patterns. A log-linear model for ANA titer adequately fit the observed frequency and included 2-way interactions between titer and polyarthritis, titer and hematologic disorders, and polyarthritis and lymphadenopathy.

Application of the log-linear and logistic regression models in the prediction of systemic lupus erythematosus in the dog.

This study sought to mathematically define canine systemic lupus erythematosus (SLE) by unifying diagnostic criteria proposed by others. Thirty-one cases of canine SLE were selected for modeling when 4 different published schemes agreed on the diagnosis, and 122 controls were selected when a patient's status met no scheme's criteria. The log-linear method showed an association between SLE and polyarthritis, hematologic abnormalities, renal damage, dermatologic disorders, and antinuclear antibody test response (positive). Logistic regression was then used to derive a predictive algorithm that could identify cases and controls with which all published criteria would be in accordance. The final equation correctly classified 93.5% of the affected dogs and 98.4% of the controls. It was concluded that the log-linear and logistic regression models are useful for the diagnosis of clinically similar, but distinguishable, disease states.

Postmortem evaluation of homotypic variation in shoe characteristics of 201 thoroughbred racehorses.

OBJECTIVES: To develop a standard technique for evaluation of racehorse shoes, to assess homotypic variation (interlimb variation) in shoe characteristics, and to determine whether shoe characteristics varied with age and sex. DESIGN: Cross-sectional study. ANIMALS: Thoroughbred racehorses (n = 201) that died or were euthanatized at California racetracks between August 1992 and July 1994. PROCEDURE: Shoe characteristics were measured on horses examined after death. Percentage of agreement was used to compare shoe characteristics between limbs (homotypic variation). Using chi 2 analysis, shoe characteristics were compared between horses grouped by age and sex. RESULTS: Toe grabs were present on 90.5% of horses, and rim shoes were present on 15.9% of horses. Heel traction devices were less frequent on front (2.5%) than rear (6%) hooves. Pads were present on 24.9% of horses, with bonded rim pads most common. Special types of shoes were present cn 5% of horses. Percentage of agreement between left and right front hooves and between left and right rear hooves was high (20/25 variables; % agreement > or = 99). In contrast, percentage of agreement between left front and left rear hooves and between right front and right rear hooves was low (2/25 variables; % agreement > or = 99). Presence of a pad was significantly (P < 0.05) associated with age, and several shoe variable (size, presence of a special shoe, overall wear matched) were significantly (P < 0.05) associated with sex. CLINICAL RELEVANCE: Except for variables related to special shoes, wear, and weight, 1 shoe for the respective fore- or hind limbs could be used as an indicator for the contralateral shoe worn by Thoroughbred racehorses without substantial loss of information. However, 1 shoe could not be used as an indicator for shoe characteristics of all 4 limbs. Some shoe characteristics are associated with age and sex, and these variables should be considered possible confounders in studies of shoe characteristics.

Horseshoe characteristics as possible risk factors for fatal musculoskeletal injury of thoroughbred racehorses.

OBJECTIVE: To evaluate selected shoe characteristics as risk factors for fatal musculoskeletal injury (FMI) and specifically for suspensory apparatus failure (SAF) and cannon bone condylar fracture (CDY) of Thoroughbred racehorses in California. DESIGN: Case-control study. ANIMALS: Thoroughbred racehorses (n = 201) that died of were euthanatized at California racetracks between August 1992 and July 1994. PROCEDURE: Shoe characteristics were compared between case horses affected by FMI (155), SAF (79), and CDY (41) and control horses that died for reasons unrelated to the appendicular musculoskeletal system (non-FMI; 46). Multivariable logistic regression was used to estimate odds ratios for FMI, SAF, and CDY. RESULTS: Toe grabs were identified as possible risk factors for FMI, SAF, and CDY. The odds of FMI, SAF, and CDY were 1.8, 6.5, and 7.0, respectively, times greater for horses shod with low toe grabs than for horses shod without toe grabs on front shoes. Horses shod with regular toe grabs on front shoes had odds 3.5, 15.6, and 17.1 times greater (P < 0.05) for FMI, SAF, and CDY, respectively, compared with horses shod without toe grabs. The odds of horses shod with rim shoes were a third (P < 0.05) of those shod without rim shoes for either FMI or SAF. The apparent association between toe grab type and CDY may, in part, be attributable to concurrent SAF and CDY injuries in many horses. CLINICAL RELEVANCE: Avoiding the use of toe grabs should decrease the incidence of FMI, especially SAF, in Thoroughbred racehorses. The use of rim shoes that are more consistent with natural hoof shape may decrease injury risk.

Hoof size, shape, and balance as possible risk factors for catastrophic musculoskeletal injury of Thoroughbred racehorses.

OBJECTIVE: To evaluate hoof size, shape, and balance as risk factors for catastrophic musculoskeletal injuries (CMI), including suspensory apparatus failure (SAF) and cannon bone condylar fracture (CDY) in Thoroughbred racehorses. ANIMALS: 95 Thoroughbred racehorses that died between 1994 and 1996. PROCEDURE: 38 quantitative measures of hoof size, shape, and balance were obtained from orthogonal digital images of the hoof and were compared between case horses with forelimb CMI (70), SAF (43), and CDY (10) injuries and control horses whose death was unrelated to the musculoskeletal system (non-CMI, 25). Comparison of group means between cases and controls was done using ANOVA, and multivariable logistic regression was used to estimate odds ratios. RESULTS: Odds of CMI were 0.62 times lower for a 5mm increase in ground surface width difference and 0.49 times lower for a 100-mm2 increase in sole area difference. Odds of SAF were 6.75 times greater with a 10 degrees increase in toe-heel angle difference and 0.58 times lower with a 100-mm2 increase in sole area difference. Odds of CDY were 0.26 times lower with a 3 degrees increase in toe angle, 0.15 times lower with a 5-mm increase in lateral ground surface width, and 0.35 times lower with a 100-mm2 increase in sole area difference. CLINICAL RELEVANCE: Decreasing the difference between toe and heel angles should decrease risk of SAF for Thoroughbred racehorses and should be considered in addition to increasing toe angle alone to help prevent catastrophic injury. Trimming the hoof to perfect mediolateral symmetry may not be a sound approach to avoiding injury.

Measurement of neonatal equine immunoglobulins for assessment of colostral immunoglobulin transfer: comparison of single radial immunodiffusion with the zinc sulfate turbidity test, serum electrophoresis, refractometry for total serum protein, and the sodium sulfite precipitation test.

Four procedures for assessment of adequacy of colostral immunoglobulin (Ig) transfer in foals were evaluated. Results of zinc sulfate turbidity test, serum electrophoresis, total serum protein refractometry, and sodium sulfite precipitation test were compared with immunoglobulin G content determined by single radial immunodiffusion. The zinc sulfate turbidity test gave acceptable results for IgG, except that hemolyzed serum samples gave higher than expected values. A correction factor for hemolyzed serum was found to be useful. Serum electrophoresis was a satisfactory method of estimating IgG content. Total serum protein values may not be a valid basis for estimating IgG content, inasmuch as postsuckling total protein values were found to decrease in some foals in which passive transfer of IgG had been adequate. Sodium sulfite precipitation reactions were too unpredictable to be of value for determination of neonatal IgG concentration.

Avian pox in blue-fronted Amazon parrots.

During a 1-month period at a quarantine station, an epornitic of avian pox occurred in blue-fronted Amazon parrots (Amazona aestiva). Clinical signs included conjunctivitis, blepharitis, and varying degrees of anorexia and respiratory distress. Lesions included periocular ulcerations and scabs and necrotic plaques in the oral cavity. Histologically, the lesions consisted of epithelial hyperplasia, secondary inflammatory changes, and eosinophilic inclusions which, by electron microscopy, were shown to contain poxvirus. When chicken embryos were inoculated with material from eyelid scabs and pharyngeal plaques, lesions of avian pox developed on the chorioallantoic membrane. The death rate of infected birds was high because of secondary bacterial and fungal infections, but uncomplicated cases were usually self-limiting. Periocular lesions also developed in 2 other species of psittacine birds housed in the same facility.