Objective—To determine whether immune function
can be accurately assessed in blood samples obtained
from horses and refrigerated overnight and whether a
nonradioactive lymphocyte proliferation assay can be
used to evaluate samples obtained from horses.
Sample Population—224 blood samples from 28
clinically normal adult horses.
Procedure—Heparinized blood samples were collected.
Each sample was divided into 2 equal aliquots.
One aliquot was refrigerated overnight to simulate
overnight shipping of blood samples, and the other
aliquot was evaluated on the day of blood collection.
Lymphocytes were isolated and enumerated by use
of a modified single-gradient procedure. Cell viability
and function were assessed by use of cytologic
examination, flow cytometry, and mitogen-induced
proliferation assays. Lymphocyte proliferation in
response to T- and B-cell mitogens was measured by
use of [3H]-thymidine incorporation and a nonradioactive
lymphocyte proliferation assay.
Results—Lymphocytes refrigerated for up to 24 hours
continued to be acceptable for use in immunologic
analysis on the basis that they maintained viability and
did not have significant alterations in lymphocyte subsets,
except for CD8, when compared with freshly isolated
lymphocytes. Furthermore, results for mitogeninduced
lymphocyte proliferation assays were also
comparable between fresh and refrigerated aliquots.
Conclusions and Clinical Relevance—The nonradioactive
lymphocyte proliferation assay is a reliable alternative
to [3H]-thymidine assay for assessing proliferation of
equine lymphocytes. Collectively, our results imply that
blood samples refrigerated and shipped overnight to a
laboratory can be used to perform cellular-immune
assays; results of those assays would enhance a clinician's
diagnostic abilities to monitor the efficacy of treatment.
(Am J Vet Res 2003;64:1003–1009)
Objective—To determine whether antibodies against
Sarcocystis neurona could be detected in CSF from
clinically normal neonatal (2 to 7 days old) and young
(2 to 3 months old) foals.
Animals—15 clinically normal neonatal Thoroughbred
Procedure—Serum and CSF samples were obtained
from foals at 2 to 7 days of age and tested for antibodies
against S neurona by means of western blotting.
Serum samples from the mares were also tested
for antibodies against S neurona. Additional CSF
and blood samples were obtained from 5 foals
between 13 and 41 days after birth and between 62
and 90 days after birth.
Results—Antibodies against S neurona were detected
in serum from 13 mares and their foals; antibodies
against S neurona were detected in CSF from 12 of
these 13 foals. Degree of immunoreactivity in serum
and CSF decreased over time, and antibodies against
S neurona were no longer detected in CSF from 2
foals 83 and 84 days after birth. However, antibodies
could still be detected in CSF from the other 3 foals
between 62 and 90 days after birth.
Conclusions and Clinical Relevance—Results indicate
that antibodies against S neurona can be detected
in CSF from clinically normal neonatal (2 to 7 days
old) foals born to seropositive mares. This suggests
that western blotting of CSF cannot be reliably used
to diagnose equine protozoal myeloencephalitis in
foals < 3 months of age born to seropositive mares.
(J Am Vet Med Assoc 2002;220:208–211)
Objective—To identify apoptosis in equine intestines
and determine whether apoptosis is associated with
gastrointestinal tract disease or a specific tissue layer
Animals—38 horses that underwent surgery or were
euthanatized for small or large intestine obstruction,
strangulation, or distension and 9 control horses
euthanatized for reasons other than gastrointestinal
tract disease or systemic disease.
Procedure—Specimens were collected at surgery
from intestine involved in the primary lesion and distant
to the primary lesion site or at necropsy from
several sites including the primary lesion site.
Histologic tissue sections were stained with H&E,
and apoptosis was detected by use of the terminal
deoxynucleotidyl transferase-mediated dUTP nick end
labeling technique. The number of apoptotic cells per
hpf was counted in the mucosa, circular muscle, longitudinal
muscle, and serosa.
Results—Apoptotic nuclei were seen in all layers of
intestine. An increased number of apoptotic cells was
found in the circular muscle of the intestine from horses
with simple obstruction, compared with strangulating
obstruction or healthy intestine. Intestine distant
from a primary strangulating lesion had higher numbers
of apoptotic cells than did intestine distant from
a simple obstructive lesion or intestine taken at the
site of a strangulating or simple obstructive lesion.
Conclusions and Clinical Relevance—Intestine
from horses with obstructing or strangulating lesions
in the small intestine and large colon had high numbers
of apoptotic cells possibly because of ischemic
cell injury and subsequent inflammation. Whether
substantial apoptosis affects intestinal function is not
yet known. (Am J Vet Res 2003;64:982–988)
Objective—To measure the ascorbic acid (AA) concentration in bronchoalveolar lavage fluid (BALF) and cellular glutathione peroxidase (cGPx) activity in RBCs and WBCs from peripherally obtained blood and in cells from BALF to determine whether differences existed between the 2 major redox systems in recurrent airway obstruction (RAO)-affected and -nonaffected (control) horses and between systemic and local pulmonary responses in the glutathione redox system.
Animals—16 adult horses in pairs: 8 healthy (control) and 8 RAO-affected horses.
Procedures—Physical examination data and biological samples were collected from horses before (remission), during, and after (recovery) environmental challenge with dusty straw and hay. At each stage, BALF cell AA concentration and RBC, WBC, and BALF cell cGPx activity were measured.
Results—Compared with control horses, RAO-affected horses had significantly higher cGPx activity in RBCs at all points and in WBCs during remission and challenge. The BALF cell cGPx activity was higher in RAO-affected horses during recovery than during remission The BALF cell AA concentration did not differ significantly in control horses at any point, but total and free AA concentrations were significantly lower in RAO-affected horses during the challenge period than during remission and recovery periods.
Conclusions and Clinical Relevance—High cGPx activity suggested this redox system was upregulated during exposure to dusty straw and hay to combat oxidative stress, as AA was depleted in RAO-affected horses. The relative delay and lack of comparative increase in cGPx activity within the local environment (represented by BALF cells), compared with that in RBCs and WBCs, might contribute to disease in RAO-affected horses.
Objective—To quantify peripheral blood neutrophil apoptosis in equine patients with acute abdominal disease (ie, colic) caused by strangulating or nonstrangulating intestinal lesions and compare these values with values for horses undergoing elective arthroscopic surgery.
Animals—20 client-owned adult horses.
Procedures—Peripheral blood was collected from horses immediately prior to and 24 hours after surgery for treatment of colic (n = 10) or elective arthroscopic surgery (10), and neutrophils were counted. Following isolation by means of a bilayer colloidal silica particle gradient and culture for 24 hours, the proportion of neutrophils in apoptosis was detected by flow cytometric evaluation of cells stained with annexin V and 7-aminoactinomycin D. Values were compared between the colic and arthroscopy groups; among horses with colic, values were further compared between horses with and without strangulating intestinal lesions.
Results—Percentage recovery of neutrophils was significantly smaller in preoperative samples (median, 32.5%) and in all samples combined (35.5%) for the colic group, compared with the arthroscopy group (median, 66.5% and 58.0%, respectively). No significant differences in the percentages of apoptotic neutrophils were detected between these groups. Among horses with colic, those with strangulating intestinal lesions had a significantly lower proportion of circulating apoptotic neutrophils in postoperative samples (median, 18.0%) than did those with nonstrangulating lesions (66.3%).
Conclusions and Clinical Relevance—The smaller proportion of apoptotic neutrophils in horses with intestinal strangulation suggested that the inflammatory response could be greater or prolonged, compared with that of horses with nonstrangulating intestinal lesions. Further investigations are needed to better understand the relationship between neutrophil apoptosis and inflammation during intestinal injury.
OBJECTIVE To evaluate the lipidomic profile of surfactant obtained from horses with asthma at various clinical stages and to compare results with findings for healthy horses exposed to the same conditions.
SAMPLE Surfactant samples obtained from 6 horses with severe asthma and 7 healthy horses.
PROCEDURES Clinical evaluation of horses and surfactant analysis were performed. Samples obtained from horses with severe asthma and healthy horses before (baseline), during, and after exposure to hay were analyzed. Crude surfactant pellets were dried prior to dissolution in a solution of isopropanol:methanol:chloroform (4:2:1) containing 7.5mM ammonium acetate. Shotgun lipidomics were performed by use of high-resolution data acquisition on an ion-trap mass spectrometer. Findings were analyzed by use of an ANOVA with a Tukey-Kramer post hoc test.
RESULTS Results of lipidomic analysis were evaluated to detect significant differences between groups of horses and among exposure statuses within groups of horses. Significantly increased amounts of cyclic phosphatidic acid (cPA) and diacylglycerol (DAG) were detected in surfactant from severely asthmatic horses during exposure to hay, compared with baseline and postexposure concentrations. Concentrations of cPA and DAG did not change significantly in healthy horses regardless of exposure status.
CONCLUSIONS AND CLINICAL RELEVANCE cPA 16:0 and DAG 36:2 were 2 novel lipid mediators identified in surfactant obtained from asthmatic horses with clinical disease. These molecules were likely biomarkers of sustained inflammation. Further studies are needed to evaluate a possible correlation with disease severity and potential alterations in the plasma lipidomic profile of horses with asthma.
Objective—To evaluate the precision of intradermal
testing (IDT) in horses.
Animals—12 healthy adult horses.
Procedure—IDT was performed on the neck of each
horse by use of 2 positive control substances (histamine
and phytohemagglutinin [PHA]) and a negative
control substance. An equal volume (0.1 mL) for each
injection was prepared to yield a total of 20 syringes
([4 concentrations of each positive control substance
plus 1 negative control substance] times 2 positive
control substances times 2 duplicative tests) for each
side of the neck. Both sides of the neck were used for
IDT; therefore, 40 syringes were prepared for each
horse. Hair was clipped on both sides of the neck, and
ID injections were performed. Diameter of the skin
wheals was recorded 0.5, 4, and 24 hours after ID
Results—Intra- and interhorse skin reactions to ID
injection of histamine and PHA resulted in wheals of
uniform size at 0.5 and 4 hours, respectively.
Significant intra- and interhorse variation was detected
in wheals caused by PHA at 24 hours.
Conclusions and Clinical Relevance—ID injection of
histamine and PHA caused repeatable and precise
results at 0.5 and 4 hours, respectively.
Concentrations of 0.005 mg of histamine/mL and
0.1 mg of PHA/mL are recommended for use as positive
control substances for IDT in horses. This information
suggests that consistent wheal size is evident
for ID injection of control substances, and variation in
wheals in response to ID injection of test antigens
results from a horse's immune response to specific
antigens. (Am J Vet Res 2005;66:1341–1347)
Objective—To evaluate differences in response to ID
injection of histamine, phytohemagglutinin (PHA), and
Aspergillus organisms between clinically normal horses
and horses with recurrent airway obstruction
Animals—5 healthy adult horses and 5 adult horses
Procedure—Intradermal testing (IDT) was performed
on the neck with 2 positive control substances (histamine
and PHA) and a mixture comprising 5
Aspergillus species. Four concentrations of each test
substance plus a negative control substance were
used. Equal volumes (0.1 mL) of each test substance
were prepared to yield 15 syringes ([4 concentrations
of each test substance plus 1 negative control substance]
times 3 test substances) for each side of each
horse (ie, 30 syringes/horse). Intradermal injections
were administered; diameter of wheals was recorded
0.5, 4, and 24 hours after injection.
Results—Hypersensitive responses to ID injection of
histamine were detected 0.5 hours after injection, and
a delay in wheal formation after ID injection of
Aspergillus mixture 24 hours after injection was detected
in RAO-affected horses but was not observed in clinically
normal horses. No differences were detected
between the 2 groups after ID injection of PHA.
Conclusions and Clinical Relevance—RAO-affected
horses are hypersensitive to histamine, suggesting that
RAO is associated with a heightened vascular response
to histamine. Higher concentrations of Aspergillus mixture
may be needed to detect horses that are sensitive
to this group of antigens. Wheal reactions to Aspergillus
may be a delayed response, suggesting that IDT results
should be evaluated 0.5, 4, and 24 hours after ID injection.
(Am J Vet Res2005;66:1348–1355)
Objective—To identify ventilatory protocols that yielded good image quality for thoracic CT and hemodynamic stability in cats.
Animals—7 healthy cats.
Procedures—Cats were anesthetized and ventilated via 4 randomized protocols (hyperventilation, 20 seconds [protocol 1]; single deep inspiration, positive inspiratory pressure of 15 cm H2O [protocol 2]; recruitment maneuver [protocol 3]; and hyperventilation, 20 seconds with a positive end-expiratory pressure of 5 cm H2O [protocol 4]). Thoracic CT was performed for each protocol; images were acquired during apnea for protocols 1 and 3 and during positive airway pressure for protocols 2 and 4. Heart rate; systolic, mean, and diastolic arterial blood pressures; blood gas values; end-tidal isoflurane concentration; rectal temperature; and measures of atelectasis, total lung volume (TLV), and lung density were determined before and after each protocol.
Results—None of the protocols eliminated atelectasis; the number of lung lobes with atelectasis was significantly greater during protocol 1 than during the other protocols. Lung density and TLV differed significantly among protocols, except between protocols 1 and 3. Protocol 2 TLV exceeded reference values. Arterial blood pressure after each protocol was lower than before the protocols. Mean and diastolic arterial blood pressure were higher after protocol 3 and diastolic arterial blood pressure was higher after protocol 4 than after protocol 2.
Conclusions and Clinical Relevance—Standardization of ventilatory protocols may minimize effects on thoracic CT images and hemodynamic variables. Although atelectasis was still present, ventilatory protocols 3 and 4 provided the best compromise between image quality and hemodynamic stability.
Objective—To evaluate the phospholipid composition and function of surfactant in horses with recurrent airway obstruction (RAO) at various clinical stages and compare these properties with findings in horses without RAO.
Animals—7 horses with confirmed RAO and 7 without RAO (non-RAO horses).
Procedures—Pairs of RAO-affected and non-RAO horses were evaluated before, during, and after exposure to hay. Evaluations included clinical scoring, lung function testing, airway endoscopy, and bronchoalveolar lavage fluid (BALF) absolute and differential cell counts. Cell-free BALF was separated into crude surfactant pellet and supernatant by ultracentrifugation, and phospholipid and protein concentrations were determined. Phospholipid composition of crude surfactant pellets and surface tension were evaluated with high-performance liquid chromatography and a pulsating bubble surfactometer, respectively. Findings were compared statistically via mixed-effects, repeated-measures ANOVA.
Results—Total phospholipid concentration in BALF was lower in RAO-affected versus non-RAO horses at all sample collection times. In the RAO-affected group, total phospholipid concentration was lower during exposure to hay than before or after exposure. There were no significant differences in BALF protein concentration, percentages of phospholipid classes, or surface tension between or within groups of horses.
Conclusions and Clinical Relevance—All clinical stages of RAO-affected horses were characterized by low surfactant concentration in BALF. Exacerbation of RAO led to an additional decrease in surfactant concentration. Causes for low surfactant concentration in RAO-affected horses remain to be determined. Low phospholipid concentration may render RAO-affected horses more susceptible than unaffected horses to surfactant alterations and contribute to clinical disease status and progression.