An ELISA to Detect Serum Antibodies to the Salivary Gland Toxin of Ixodes holocyclus Neumann in Dogs and Rodents.

The Ixodes holocyclus tick causes paralysis in up to 10,000 companion and domestic animals each year in Australia. Treatment requires the removal of the parasite and the administration of a commercial tick antiserum that is prepared from hyperimmune dogs. Each batch of this serum is initially tested for toxin-neutralising potency in a mouse bioassay that is expensive, time consuming, and subjective. With the aim of developing a rapid in vitro assay to replace the bioassay, we used a partially purified antigen prepared from I. holocyclus salivary glands to develop an ELISA to detect toxin-reactive antibodies in hyperimmune dog sera. The optimised ELISA reliably detected antibodies reactive to I. holocyclus salivary gland antigens. Parallel testing of sera with a negative control antigen prepared from the salivary glands of the nontoxic tick Rhipicephalus (Boophilus) microplus provided further evidence that we were detecting toxin-specific antibodies in the assay. Using the ELISA, we could also detect antibodies induced in rats after experimental infestation with I. holocyclus. This assay shows promise as an alternative means of assessing the potency of batches of hyperimmune dog serum and to screen for toxin-reactive monoclonal antibodies produced from immunised rodents.

Tick paralysis in Australia caused by Ixodes holocyclus Neumann.

Ticks are obligate haematophagous ectoparasites of various animals, including humans, and are abundant in temperate and tropical zones around the world. They are the most important vectors for the pathogens causing disease in livestock and second only to mosquitoes as vectors of pathogens causing human disease. Ticks are formidable arachnids, capable of not only transmitting the pathogens involved in some infectious diseases but also of inducing allergies and causing toxicoses and paralysis, with possible fatal outcomes for the host. This review focuses on tick paralysis, the role of the Australian paralysis tick Ixodes holocyclus, and the role of toxin molecules from this species in causing paralysis in the host.

Auscultation and echocardiographic findings in Bull Terriers with and without polycystic kidney disease.

OBJECTIVE: To investigate a possible association between Bull Terrier polycystic kidney disease (BTPKD) and cardiac disease, to determine the prevalence of mitral valve disease (MVD) and left ventricular outflow tract obstruction (LVOTO) in the Australian Bull Terrier population, and to compare auscultation and echocardiography in detection of cardiac disease in Bull Terriers. DESIGN: Ninety-nine Bull Terriers, ranging in age from 8 weeks to 13 years and 11 months were auscultated and examined using renal ultrasonography; 86 were also examined using echocardiography. The prevalence and severity of heart defects in dogs with BTPKD was compared with that in dogs without BTPKD. RESULTS: Nineteen of these 99 dogs were diagnosed with BTPKD. Forty-two percent of Bull Terriers with BTPKD and 28% of those without BTPKD had murmurs characteristic of mitral regurgitation or LVOTO. How recently an animal was descended from an ancestor with BTPKD was associated with presence (P = 0.008) and loudness of a murmur (P = 0.009). Overall, echocardiography detected MVD in 39% of Bull Terriers, with increased prevalence in older animals (P = 0.003). Mitral stenosis was found in eight cases. Fifty-three percent of dogs in this study had evidence of LVOTO, with obstruction consisting of a complex of lesions including dynamic or fixed subvalvular LVOTO, significantly narrowed left ventricular outflow tract or valvular aortic stenosis. Dogs with BTPKD, or those descended from dogs with BTPKD, were more likely to have MVD (P = 0.006), and while LVOTO was not more common in these dogs, if they did have LVOTO, they were more likely to have severe obstruction than dogs with no ancestors with BTPKD (analysed in three ways P = 0.028 to 0.001). In this study, 46% of Bull Terriers without a murmur or arrhythmia had cardiac disease detected on echocardiographic examination. CONCLUSION: Cardiac disease, especially MVD and LVOTO, was common in Bull Terriers in this study, and those with BTPKD had an increased risk of cardiac abnormalities. Auscultation did not detect a significant number of Bull Terriers with cardiac disease.

No disease-associated mutations found in the coding sequence of the canine polycystic kidney disease gene 1 in Bull Terriers with polycystic kidney disease.

The aim of this study was to identify possible disease-associated mutations in the canine homologue of the polycystic kidney disease gene 1 (PKD1) in Bull Terriers with autosomal dominant polycystic kidney disease. Messenger RNA was obtained from the blood or renal tissue of five Bull Terriers with the disease and four close relatives without the disease. Reverse transcription, PCR and 3' rapid amplification of cDNA ends were used to amplify the coding and 3' untranslated regions of this transcript. Comparison of PKD1 sequence between the affected and unaffected Bull Terriers, revealed six polymorphisms, but no disease-associated mutations.

Effects of the paralysis tick, Ixodes holocyclus, on the electrocardiogram of the Spectacled Flying Fox, Pteropus conspicillatus.

OBJECTIVE: To evaluate cardiac electrical function in the Spectacled Flying Fox (bat) infested with Ixodes holocyclus. DESIGN: Prospective clinical investigation of bats treated for naturally occurring tick toxicity. PROCEDURE: ECGs were performed on bats with tick toxicity (n = 33), bats that recovered slowly (n = 5) and normally (n = 5) following treatment for tick toxicity, and on normal bats with no history of tick toxicity (n = 9). RESULTS: Bats with tick toxicity had significantly prolonged corrected QT intervals, bradycardia and rhythm disturbances which included sinus bradydysrhythmia, atrial standstill, ventricular premature complexes, and idioventricular bradydysrhythmia. CONCLUSIONS: The QT prolongation observed on ECG traces of bats with tick toxicity reflected delayed ventricular repolarisation and predisposed to polymorphic ventricular tachycardia and sudden cardiac death in response to sympathetic stimulation. The inability to document ventricular tachycardia in bats shortly before death from tick toxicity may be explained by a lack of sympathetic responsiveness attributable to the unique parasympathetic innervation of the bat heart, or hypothermia-induced catecholamine receptor down-regulation. Bradycardia and rhythm disturbances may be attributable to hypothermia.

Echocardiographic parameters in 14 healthy English Bull Terriers.

OBJECTIVE: To determine the range of various cardiac parameters using echocardiography in apparently normal, healthy English Bull Terriers. DESIGN: Fourteen English Bull Terriers were selected for study. Cardiac auscultation of the parents of these dogs was normal. Echocardiographic examination of one parent of each animal showed: no mitral or aortic valve abnormalities; no myocardial lesions; no two dimensional evidence of fixed or dynamic left ventricular outflow tract obstruction; and no systolic aortic or left ventricular outflow tract turbulence on colour flow Doppler examination. The 14 selected dogs did not have arrhythmias or murmurs, and on echocardiographic examination had similar findings to their parents. Systolic blood pressure was measured in all dogs and they had no clinical evidence of Bull Terrier polycystic kidney disease or Bull Terrier hereditary nephritis. PROCEDURE: All dogs were auscultated and subjected to a sequential global echocardiographic assessment of the heart, including two dimensional long and short axis, and colour flow Doppler interrogation of the mitral and aortic valves. Dimensional measurements, including those from the left atrium, aortic annulus and left ventricle, were taken from a right parasternal window, and derived values such as fractional shortening, stroke volume and left atrial to aortic annulus ratio were calculated. Peak systolic aortic velocity was measured from the left parasternal window using two dimensional-guided pulsed wave Doppler with angle correction. Systolic blood pressure was measured using a Doppler monitor. The absence of Bull Terrier polycystic kidney disease was determined using renal ultrasonography, and of Bull Terrier hereditary nephritis using urinary protein to creatinine ratio. RESULTS: These 14 dogs had greater left ventricular wall thickness and smaller aortic root diameters than those reported as normal for other breeds of comparable body size. Left atrial dimensions were also larger, however this may have been due to the "maximising" method of measurement. These apparently normal English Bull Terriers also had higher aortic velocities than those reported for other breeds, possibly due to a smaller aortic root diameter or other anatomic substrate of the left ventricular outflow tract, lower systemic vascular resistance, or breed-specific "normal" left ventricular hypertrophy. While these dogs were selected to be as close to normal as possible, the breed may have a particular anatomy that produces abnormal left ventricular echocardiographic parameters. CONCLUSION: These echocardiographic parameters may be used to diagnose left ventricular outflow tract obstruction and left ventricular hypertrophy, and inaccurate diagnoses may result if breed-specific values are not used.

Long QT syndrome in dogs with tick toxicity (Ixodes holocyclus).

OBJECTIVE: To evaluate cardiac electrical function in dogs with tick toxicity. DESIGN: A prospective clinical investigation of 39 client-owned dogs treated for naturally occurring tick toxicity. PROCEDURE: An ECG was performed on each dog on several occasions; at admission to hospital with tick toxicity, 24 h later, at discharge from hospital when clinically normal and approximately 12 months later. RESULTS: The mean QT interval corrected for heart rate (QTc) was prolonged at admission, 24 h and at discharge compared to the QTc measured 12 months later. T wave morphology was altered in dogs at admission. All other parameters were within normal limits. CONCLUSIONS: The prolonged QTc interval and altered T wave morphology of dogs with tick toxicity reflects delayed cardiac repolarisation and is comparable with long QT syndrome (LQTS) in people who are predisposed to polymorphic ventricular tachycardia and sudden death. Resolution of ECG changes lagged behind clinical recovery.

Surgical extraction of transplanted adult Dirofilaria immitis in cats.

A jugular venotomy technique was developed to attempt extraction of Dirofilaria immitis in cats. Seven cats were first examined by cardiac ultrasound to establish the location of adult D. immitis that had been inserted by jugular venotomy. The efficiency of an extractor catheter in surgically removing filariae similarly inserted into 13 experimental cats was then assessed. A mean extraction success rate of 96% was achieved. This technique would seem to be worthy of clinical use provided the filariae are in the right atrial area.

Reactions to tick antitoxin serum and the role of atropine in treatment of dogs and cats with tick paralysis caused by Ixodes holocyclus: a pilot survey.

OBJECTIVE: To determine the incidence and nature of adverse reactions of dogs and cats to tick antitoxin serum and to re-evaluate the role of atropine in the treatment of tick paralysis. DESIGN: A retrospective questionnaire of veterinarians. PROCEDURE: Questionnaires were posted to 320 veterinarians in tick-endemic regions of Australia. Questions referred to dogs and cats treated for tick paralysis over a period of three years: the number treated, treatment protocols and adverse systemic reactions to tick antitoxin serum. Ninety completed questionnaires were returned and responses analysed. RESULTS: Veterinarians reported that approximately 3% of dogs exhibited adverse reactions immediately following treatment with tick antitoxin serum. Eighteen percent of these reactions were described as anaphylaxis, with the remaining 82% attributed to the Bezold-Jarisch reflex. Six percent of cats treated with tick antitoxin serum reacted adversely and the majority of reactions (63%) were ascribed to the Bezold-Jarisch reflex. Atropine was used routinely by 10% of responding veterinarians in the treatment of dogs and cats with tick paralysis. A similar number of veterinarians used atropine only in selected cases. Most veterinarians (76%) reported that they never used atropine in the treatment of tick paralysis in either dogs or cats. Within the survey population, premedication with atropine reduced the number of Bezold-Jarisch reactions following tick antitoxin administration approximately five-fold in dogs and four-fold in cats. CONCLUSIONS: Data from this pilot survey indicate that more cats than dogs have adverse systemic reactions to tick antitoxin serum and that the majority of these reactions in both dogs and cats could be related to the Bezold-Jarisch reflex. The number of reactions to tick antitoxin serum in dogs and cats could be significantly reduced by the routine use of atropine prior to administration of tick antitoxin serum.

Prospective survey of tick paralysis in dogs.

OBJECTIVE: To obtain information on tick paralysis in dogs, including the nature of disease, host signalment, tick-host relationship, treatment, disease progression and recovery, and preventive measures. DESIGN: A prospective survey of 577 dogs affected by tick paralysis was conducted during 1998. Forty-two veterinary clinics along the eastern coast of Australia were instructed to complete survey forms for the first 15 dogs that presented with tick paralysis during September to November. RESULTS: Five percent of dogs died from tick paralysis. Younger dogs were more likely to survive. Long coat length was associated with a greater tick burden but not greater tick size, whereas coat thickness had no bearing on either. Dogs with mild disease recovered more quickly from tick paralysis. Respiratory and gait scores reflected disease severity and were good prognostic indicators. The size of the tick did not reflect the severity of the clinical condition it induced in the host. No method of tick removal or in situ treatment improved recovery time or reduced mortality. However, the time spent in hospital was significantly less for dogs from which the live tick was manually removed. Inspiratory stridor, evident in some dogs with tick paralysis, was not related to tick attachment on the neck. The use of acepromazine maleate or dexamethasone did not reduce recovery time or mortality. Increasing the dose of tick antitoxin serum (TAS) above 0.1 mL/kg had no effect on mortality or recovery time. Dogs with severe disease that received an additional dose of TAS were significantly less likely to survive. Subcutaneous use of TAS at the site of tick attachment was of no benefit in reducing mortality or time to initial clinical improvement. A registered preventative product had not been used on the majority of dogs. Clipping the coat to search for ticks did not reduce mortality. CONCLUSIONS: Therapy needs to address cardiopulmonary dysfunction that may be due directly to the effect of tick toxin and not just respiratory compromise caused by progressive respiratory muscle failure.

Levels of total alpha-macroglobulin and trypsin-like immunoreactivity are poor indicators of clinical severity in spontaneous canine acute pancreatitis.

Trypsin-like immunoreactivity and total alpha-macroglobulin levels were assessed in serum and plasma samples taken at presentation from 60 cases of spontaneous canine acute pancreatitis of varying clinical severity. Total alpha-macroglobulin was significantly decreased in all severity groups when compared to 119 healthy controls, however there were no significant differences between severity groups. Trypsin-like immunoreactivity was significantly elevated above assay reference range in all groups. Trypsin-like immunoreactivity was significantly elevated in dogs with severe disease when compared to those with mild disease. These results suggest that zymogen release and protease activation, while components of the pathology of spontaneous canine acute pancreatitis, are not directly associated with the onset of multiple organ failure seen in the most severe cases.

Detection and measurement of canine alpha-macroglobulins by enzyme immuno-assay.

The alpha-macroglobulins are broad-specificity protease inhibitors important in the regulation and clearance from circulation of biologically active proteases. Inappropriate protease activation may be a feature of canine acute pancreatitis and the ability of the animal to clear these proteases may be important in determining survival. An enzyme immunoassay for the detection and measurement of canine alpha-macroglobulins in plasma samples was developed. A reference range for the canine alpha-macroglobulins of 1.20-2.72 mg ml-1 was established from a panel of canine plasma samples, and the stability of the alpha-macroglobulins in plasma samples stored at 4 degrees C was investigated. Changes in the level of the alpha-macroglobulins during disease states involving increased endogenous protease activity can now be investigated using a rapid, repeatable and quantifiable assay.

Tumor necrosis factor-alpha at presentation in 60 cases of spontaneous canine acute pancreatitis.

Tumor necrosis factor-alpha (TNF) is a pleiotropic cytokine with profound and broad ranging effects on many cell types. There have been few publications investigating the role of TNF in spontaneous disease processes of dogs, particularly the role of this cytokine during endotoxaemia, shock and multiple organ dysfunction syndromes. Plasma samples taken at presentation from 60 dogs with spontaneous acute pancreatitis of varying severity levels (scored 0-4 in ascending severity) were assessed for TNF activity by bioassay and total TNF protein levels through a dot-blot immunoassay. TNF activity by bioassay was detected in 31% (4/13) of dogs presenting with severe disease (>50% expected mortality) as defined using a scoring system for organ compromise, and was not detectable in the remaining animals or healthy controls. TNF activity was detected in 66% (4/6) animals in the highest severity group (Score 4), these animals were showing severe multiple organ dysfunction. Total TNF protein levels, measured by dot-blot immunoassay, exhibited a wide range in all severity groups and healthy dogs. Dogs with detectable TNF activity were not distinguished from the other severity or healthy groups by immunoassay. The absence of detectable differences in total TNF protein levels between the various severity groups suggests that other factors may be crucial in determining the role of TNF in spontaneous canine acute pancreatitis and subsequent endotoxaemia and shock.

Unguided bronchoalveolar lavage techniques and residual effects in dogs.

OBJECTIVES: To investigate the use of unguided bronchoalveolar lavage techniques in dogs without fibreoptic bronchoscopy, using an adapted single vascular catheter and a double-lumen catheter made from two single vascular catheters. ANIMALS: Sixty-nine dogs were examined with the single-catheter technique and 110 dogs with the double-catheter technique. DESIGN: A prospective study. PROCEDURE: Sixty-nine and 220 samples, collected with the single catheter and the double catheter respectively, were examined cytologically. Lungs of 69 dogs were examined grossly and histologically. Radiographic examination was performed on 11 dogs. RESULTS: The double-catheter technique produced samples with significantly higher cellularity (P < 0.01) and fewer red blood cells (P < 0.01) than the single-catheter technique. Repeat samples collected with a double catheter were not significantly different (P > 0.01) in any value. A reference range for nucleated cell counts of 62 to 1210 x 10(6)/L was calculated from 57 clinically and histologically normal dogs. The major residual effects of the technique were localised pulmonary oedema, and alveolar distension with collapse and congestion of distant parenchyma. Thoracic radiographs revealed increased lung opacity for at least up to 7 h after the procedure. CONCLUSIONS: The cellularity of the bronchoalveolar lavage fluid obtained was adequate and sufficient fluid was retrieved when the single catheter was located in a proper position. However, the double catheter obtained better samples more quickly and easily, with less damage to the respiratory tract.

A severity score for spontaneous canine acute pancreatitis.

OBJECTIVE: To derive a severity score for spontaneous canine acute pancreatitis applicable to general practice. DESIGN: Cohort study of canine pancreatitis cases. PROCEDURE: Cases (n = 68) of spontaneous canine acute pancreatitis presented to general practitioners were identified among accessions to Veterinary Pathology Services Brisbane. The primary veterinarian was surveyed by telephone to ascertain the outcome of each case. Scores were assigned for extent of hyperamylasaemia, hyperlipasaemia and number of organ systems other than the pancreas compromised. The probability of mortality with each score of each analyte was calculated. The strength of interaction between scores for each analyte and mortality rate was assessed by chi-square analysis where appropriate. Relationships between the organ system score, other physiological variables and likelihood of euthanasia were analysed. RESULTS: Scores derived mathematically from analysis of enzyme activities had poor abilities to predict mortality. The score based upon the number of organ systems compromised showed good ability to predict mortality and the interaction between the organ system score and mortality rate was significant by chi-square analysis (P < 0.01). Distribution of data within the amylase and lipase scores was not compatible with chi-square analysis. CONCLUSION: Assessment of severity of spontaneous canine acute pancreatitis using pancreatic enzyme activities is potentially inaccurate. The use of a severity score based upon organ system compromise was more accurate in determining the likelihood of mortality in spontaneous canine acute pancreatitis. This is compatible with the hypothesis that severe canine acute pancreatitis is a multiple organ failure syndrome.

Variation in protein concentrations in acute and chronic ascites in Dirofilaria immitis-infected dogs.

Microfilaremic dogs, developing ascites acutely following a reaction to diethylcarbamazine therapy, had similar protein concentrations in their ascitic fluid and plasma. In contrast, in dogs chronically infected with Dirofilaria immitis, the protein concentrations of ascitic fluid were found to be significantly lower than plasma protein concentrations. The acute development of ascites in such dogs is associated with high ascitic protein levels.

Echocardiographic values in the greyhound.

Serial B- and M-mode echocardiography was performed on Greyhounds to determine normal cardiac values for this breed. These were generally of greater magnitude than predicted from previous echocardiographic research on other breeds and cross-breeds. In particular, left ventricular posterior wall thickness, measured at both systole and diastole, was consistently greater.

The effect of prostaglandin inhibition on the development of pulmonary pathology associated with dead Dirofilaria immitis.

Flunixin meglumine was used to examine the effect of prostaglandin inhibition on the pathogenesis of Dirofilaria immitis in the pulmonary arteries of dogs. Immunopathological reactions to dead filariae were monitored by light and transmission electron microscopy and serology. Lung lesions in prostaglandin-inhibited dogs exposed to dead filariae were enhanced compared to control dogs. This was associated with the persistence of parasitic antigen in lung tissue and in the blood. Serology demonstrated that after insertion of D. immitis in treated dogs, antibody levels did not change, while immune complex and antigen levels increased. These results indicate that prostaglandin may have a protective effect on the way the lung reacts to dead D. immitis, and that altered dynamics of the antigen processing may well contribute to the associated lung lesions.