Presumptive neural microsporidiosis in a young adult German Shepherd dog from rural Australia.

CASE REPORT: A 1-year-old, neutered male German Shepherd was presented with a 5-month history of episodic lethargy, intermittent fever, weight loss and a hunched posture. The dog was diagnosed with presumptive microsporidian meningoencephalitis based on cytological findings on cerebrospinal fluid analysis and a positive PCR test. The dog initially responded favourably to a 4-week course of trimethoprim-sulfadiazine, pyrimethamine and fenbendazole, and remained well for 12 weeks following cessation of treatment. Disease then recurred, and despite an initial positive response to treatment, he deteriorated and was euthanased 11 weeks later, 7.5 months after definitive diagnosis and 13 months after clinical signs were first reported. CONCLUSION: To the authors knowledge, this is the first case of canine microsporidiosis in Australia.

A single exposure to the tremorgenic mycotoxin lolitrem B inhibits voluntary motor activity and spatial orientation but not spatial learning or memory in mice.

The indole diterpenoid toxin lolitrem B is a tremorgenic agent found in the common grass species, perennial ryegrass (Lolium perenne). The toxin is produced by a symbiotic fungus Epichloë festucae (var. lolii) and ingestion of infested grass with sufficient toxin levels causes a movement disorder in grazing herbivores known as 'ryegrass staggers'. Beside ataxia, lolitrem B intoxicated animals frequently show indicators of cognitive dysfunction or exhibition of erratic and unpredictable behaviours during handling. Evidence from field cases in livestock and controlled feeding studies in horses have indicated that intoxication with lolitrem B may affect higher cortical or subcortical functioning. In order to define the role of lolitrem B in voluntary motor control, spatial learning and memory under controlled conditions, mice were exposed to a known dose of purified lolitrem B toxin and tremor, coordination, voluntary motor activity and spatial learning and memory assessed. Motor activity, coordination and spatial memory were compared to tremor intensity using a novel quantitative piezo-electronic tremor analysis. Peak tremor was observed as frequencies between 15 and 25Hz compared to normal movement at approximately 1.4-10Hz. A single exposure to a known tremorgenic dose of lolitrem B (2 mg/kg IP) induced measureable tremor for up to 72 h in some animals. Initially, intoxication with lolitrem B significantly decreased voluntary movement. By 25 h post exposure a return to normal voluntary movement was observed in this group, despite continuing evidence of tremor. This effect was not observed in animals exposed to the short-acting tremorgenic toxin paxilline. Lolitrem B intoxicated mice demonstrated a random search pattern and delayed latency to escape a 3 h post intoxication, however by 27 h post exposure latency to escape matched controls and mice had returned to normal searching behavior indicating normal spatial learning and memory. Together these data indicate that the tremor exhibited by lolitrem B intoxicated mice does not directly impair spatial learning and memory but that exposure does reduce voluntary motor activity in intoxicated animals. Management of acutely affected livestock suffering toxicosis should be considered in the context of their ability to spatially orientate with severe toxicity.

Treatment with potassium bromide mitigates ataxia and reduces tremor in lambs with perennial ryegrass toxicosis.

Aims: To assess the use of potassium bromide (KBr) as a therapeutic intervention for perennial ryegrass toxicosis (PRGT) in lambs fed ryegrass seed containing lolitrem B. Methods: Male lambs aged 10-12 months (n = 43) were assigned to receive ryegrass seed containing lolitrem B, at a dose of 0.16 mg/kg/day (Groups 2, 3 and 4), or lucerne chaff and molasses (Groups 1 and 5). Lambs in Groups 2 and 3 were observed for clinical signs and gait changes until defined signs of PGRT were observed, when they were transferred, with lambs in Group 1, to the Testing phase of the trial. Lambs in Group 3 were then treated with a single oral dose of 300 mg/kg bromide. Lambs in Groups 4 and 5 received KBr daily from the start of the trial (540 mg/kg bromide over 3 days then 20 mg/kg daily) and were transferred to the Testing phase after 18 days. Clinical examination and gait assessment, and surface electromyography of the triceps muscle, measuring root-mean-square (RMS) voltages, were carried out on Days 0, 1 and 2 of the Testing phase followed by necropsy, histopathology, measurement of concentrations of bromide in serum and CSF and faecal cortisol metabolites (FCM). Results: In Group 3 lambs, mean composite gait scores decreased between Testing phase Day 0 and Days 1 and 2 (p < 0.001), but increased in lambs in Group 2 between Day 0 and Day 2 (p = 0.015). Scores for lambs in Group 3 on Day 2 were lower than for lambs in Group 2 (p < 0.001). Mean RMS voltages on Day 2 were higher in lambs in Group 2 than Group 3 (p = 0.045). Mean concentrations of bromide in serum were >800 µg/mL in lambs in Groups 3 and 4 on Day 2. Concentrations of FCM were higher in lambs from Group 2 than in Groups 1 or 5, but were similar in Groups 2, 3 and 4. Histopathological findings in the cerebellum of lambs from Groups 2, 3 and 4 were similar, showing pyknosis of neurons within the granular layer of the cerebellum and Purkinje neuron proximal axonal spheroid formation. Conclusions and clinical relevance: A single oral dose of 300 mg/kg bromide in lambs with neurological signs of PRGT resulted in reduced composite gait scores and reduced RMS voltages, indicating a significant improvement in clinical signs of ataxia, movement disorder and muscle tremor associated with the neurotoxic effects of lolitrem B.

Development of a model for investigation of perennial ryegrass toxicosis in sheep.

AIMS To develop a clinical model of perennial ryegrass toxicosis (PRGT) based on feeding a known dose of lolitrem B and ergotamine, and to produce a consistent clinical presentation for assessment of disease pathophysiology, neurological changes and neurohistopathology. METHODS Male lambs, aged between 10-12 months, were randomly assigned to either Treatment (n=9) or Control (n=9) groups. Lambs in the Treatment group received feed containing a novel endophyte-infested perennial ryegrass seed, commencing on Day 0 of the Feeding phase with a low induction dose, then increasing after 3 days to provide 0.16 mg/kg live bodywight (LBW)/day of lolitrem B and 0.054 mg/kg LBW/day ergotamine. Lambs were examined daily and when defined signs of PRGT were observed they were transferred to the Testing phase. Neurological examinations, assessment of gait, surface electromyography (EMG) and mechanosensory nociceptive threshold testing were carried out and blood samples collected during both phases of the trial, with a full necropsy, histopathological examination and measurement of faecal cortisol metabolites (FCM) performed on Day 2 of the Testing phase. RESULTS Typical clinical signs of PRGT, including ataxia of vestibulocerebellar origin leading to stumbling, were observed in all Treatment lambs. The median interval from the start of the Feeding phase to entry into the Testing phase was 21 (min 18, max 34) days. Histopathological characterisation of neurological lesions included the presence of Purkinje cell vacuolation, pyknotic granular layer neurons and proximal axonal Purkinje cell spheroids. Lesions were most apparent within the vestibulocerebellum. Mean root-mean-square voltages from triceps EMG increased in Treatment lambs between Feeding phase Day 0 and Testing phase Day 2 (p<0.001). Daily water intake during the Testing phase for the Treatment group was less than in Control group lambs (p=0.002), and concentrations of FCM at necropsy were higher in Treatment compared to Control lambs (p=0.02). CONCLUSIONS AND CLINICAL RELEVANCE Lolitrem B and ergotamine dosing in feed on a live weight basis combined with neurological/gait assessment provides an effective model for investigation of PRGT and potential therapeutics. Assessment of gait changes using defined criteria and RMS voltages from EMG appear to be useful tools for the assessment of the severity of neurological changes.

Spatial variation in the parasite communities and genomic structure of urban rats in New York City.

Brown rats (Rattus norvegicus) are a globally distributed pest. Urban habitats can support large infestations of rats, posing a potential risk to public health from the parasites and pathogens they carry. Despite the potential influence of rodent-borne zoonotic diseases on human health, it is unclear how urban habitats affect the structure and transmission dynamics of ectoparasite and microbial communities (all referred to as "parasites" hereafter) among rat colonies. In this study, we use ecological data on parasites and genomic sequencing of their rat hosts to examine associations between spatial proximity, genetic relatedness and the parasite communities associated with 133 rats at five sites in sections of New York City with persistent rat infestations. We build on previous work showing that rats in New York carry a wide variety of parasites and report that these communities differ significantly among sites, even across small geographical distances. Ectoparasite community similarity was positively associated with geographical proximity; however, there was no general association between distance and microbial communities of rats. Sites with greater overall parasite diversity also had rats with greater infection levels and parasite species richness. Parasite community similarity among sites was not linked to genetic relatedness of rats, suggesting that these communities are not associated with genetic similarity among host individuals or host dispersal among sites. Discriminant analysis identified site-specific associations of several parasite species, suggesting that the presence of some species within parasite communities may allow researchers to determine the sites of origin for newly sampled rats. The results of our study help clarify the roles that colony structure and geographical proximity play in determining the ecology of R. norvegicus as a significant urban reservoir of zoonotic diseases. Our study also highlights the spatial variation present in urban rat parasite communities, indicating that rats across New York City are not reservoirs for a homogenous set of parasites and pathogens. As a result, the epidemiological risks may be similarly heterogeneous for people in urban habitats.

Admixture on the northern front: population genomics of range expansion in the white-footed mouse (Peromyscus leucopus) and secondary contact with the deer mouse (Peromyscus maniculatus).

Range expansion has genetic consequences expected to result in differentiated wave-front populations with low genetic variation and potentially introgression from a local species. The northern expansion of Peromyscus leucopus in southern Quebec provides an opportunity to test these predictions using population genomic tools. Our results show evidence of recent and post-glacial expansion. Genome-wide variation in P. leucopus indicates two post-glacial lineages are separated by the St. Lawrence River, with a more recent divergence of populations isolated by the Richelieu River. In two of three transects we documented northern populations with low diversity in at least one genetic measure, although most relationships were not significant. Consistent with bottlenecks and allele surfing during northward expansion, we document a northern-most population with low nucleotide diversity, divergent allele frequencies and the most private alleles, and observed heterozygosity indicates outcrossing. Ancestry proportions revealed putative hybrids of P. leucopus and P. maniculatus. A formal test for gene flow confirmed secondary contact, showing that a reticulate population phylogeny between P. maniculatus and P. leucopus was a better fit to the data than a bifurcating model without gene flow. Thus, we provide the first genomic evidence of gene flow between this pair of species in natural populations. Understanding the evolutionary consequences of secondary contact is an important conservation concern as climate-induced range expansions are expected to result in new hybrid zones between closely related species.

Epidemiological investigation of grass seed foreign body-related disease in dogs of the Riverina District of rural Australia.

OBJECTIVE: To characterise disease presentations caused by grass seed foreign body-related disease (GSFBD) in dogs, identify predisposing risk factors and suggest effective prevention strategies. METHODS: A retrospective, case-control, telephone survey was conducted to obtain information on GSFBD in dogs from the Riverina district of New South Wales (NSW). Additionally, retrospective case records were obtained from Wagga Wagga Veterinary Hospital and Charles Sturt University Veterinary Teaching Hospital over the period July 2006 to October 2011. Signalment, history, investigative strategies, location and severity of lesion(s), cost of therapy, length of hospitalisation, recurrence rate and microbiology data were recorded. RESULTS: Cases (473 dogs) meeting stringent inclusion criteria were identified. GSFBD comprised 2.0% of hospital presentations. Breeds with high-density coats were overrepresented. Otitis externa was the most common manifestation of GSFBD in the general practice (47%). In the referral practice pyothorax was the most common entity (34%). In both practices the range of clinical manifestations was broad, including a small number of dogs with catastrophic intracranial disease. Univariable and multivariable logistic regression analyses demonstrated dogs with GSFBD had 3-fold greater odds of having medium coats than short coats and 5-fold less odds of being groomed. CONCLUSIONS AND CLINICAL RELEVANCE: Grass seeds are a major cause of disease in the dogs of south-west rural NSW, with presentations ranging from mild lameness to severe neurological disease. Some protection from GSFBD was achieved with frequent grooming. Clipping or coat searching without grooming was ineffective as a prevention strategy.

Pharmacokinetics of bromide in adult sheep following oral and intravenous administration.

OBJECTIVE: To determine the pharmacokinetics of bromide in sheep after single intravenous (IV) and oral (PO) doses. PROCEDURE: Sixteen Merino sheep were randomly assigned to two treatment groups and given 120 mg/kg bromide, as sodium bromide IV or potassium bromide PO. Serum bromide concentrations were determined by colorimetric spectrophotometry. RESULTS: After IV administration the maximum concentration (Cmax ) was 822.11 ± 93.61 mg/L, volume of distribution (Vd ) was 0.286 ± 0.031 L/kg and the clearance (Cl) was 0.836 ± 0.255 mL/h/kg. After PO administration the Cmax was 453.86 ± 43.37 mg/L and the time of maximum concentration (Tmax ) was 108 ± 125 h. The terminal half-life (t½ ) of bromide after IV and PO administration was 387.93 ± 115.35 h and 346.72 ± 94.05 h, respectively. The oral bioavailability (F) of bromide was 92%. No adverse reactions were noted in either treatment group during this study. The concentration versus time profiles exhibited secondary peaks, suggestive of gastrointestinal cyclic redistribution of the drug. CONCLUSIONS AND CLINICAL RELEVANCE: When administered PO, bromide in sheep has a long half-life (t½ ) of approximately 14 days, with good bioavailability. Potassium bromide is a readily available, affordable salt with a long history of medical use as an anxiolytic, sedative and antiseizure therapy in other species. There are a number of husbandry activities and flock level neurological conditions, including perennial ryegrass toxicosis, in which bromide may have therapeutic or prophylactic application.

Evidence of dehydration and electrolyte disturbances in cases of perennial ryegrass toxicosis in Australian sheep.

CASE REPORT: Perennial ryegrass toxicosis (PRGT) is a common disease entity in Australia, presenting as an association of clinical signs including alterations in normal behavioural, ataxia ('staggers'), ill thrift and gastrointestinal dysfunction ('scours'). Clinical signs can range in severity from mild (gait abnormalities and failure to thrive) to severe (seizures, lateral recumbency and death). Presentation across the flock is usually highly variable. PRGT is caused by toxins produced by the endophytic fungus Neotyphodium lolii, a symbiont of perennial ryegrass that is present in pastures across the temperate regions of Australia and Tasmania. A particular feature of PRGT in Australia is the occasional occurrence of large-scale sheep losses, suggesting other factors are influencing mortality rates compared with other PRGT risk zones such as North America and New Zealand. During 2011, producers in the state of Victoria experienced a mild outbreak of PRGT that affected large numbers of animals but with limited mortalities. Clinical samples taken from affected sheep showed a high incidence of dehydration and electrolyte abnormalities. CONCLUSION: We speculate that changes in hydration status may be a contributory aetiological factor in those years in which high numbers of deaths are associated with PRGT outbreaks in Australia.

Selecting an acceptable and safe antibody detection test can present a dilemma.

The Transfusion Service at Duke University Hospital has changed antibody detection methods from the use of albumin in indirect antiglobulin tests to low-ionic-strength solution (LISS), and from LISS to polyethylene glycol (PEG) in an effort to enhance the rapid detection of clinically significant antibodies. In 1996, staffing issues required the consideration of automation. Although previous studies indicated that the gel test was not as sensitive as PEG for detection of clinically significant antibodies, we chose to implement the gel test to be used with the Tecan MegaFlex-ID. We performed a retrospective analysis of identified antibodies and transfusion reactions to compare the outcomes of one year's experience with gel and PEG. We found comparable detection of potentially clinically significant antibodies by both methods and significantly fewer unwanted or clinically insignificant antibodies detected with the use of gel. Fewer delayed serologic transfusion reactions and no transfusion-associated hemolytic events occurred in the year that gel was used. Although we initially found the selection of the gel test to be a dilemma, our ultimate decision appears to have successfully protected patient safety and balanced sensitivity with specificity.

An investigation into the efficacy of intravenous diclofenac in post-operative dental pain.

OBJECTIVE: To evaluate the efficacy of single doses of intravenous diclofenac sodium (25, 50 and 75 mg) in patients with post-operative pain after third-molar surgery in a randomised, placebo-controlled study. METHODS: Two hundred and sixty-nine patients (168 females) who required the removal of their impacted third molars participated in the study, which had received prior ethical approval. Surgery was completed under general anaesthesia and, during the early post-operative period, patients received either a single intravenous dose of diclofenac (25, 50 or 75 mg) or matched placebo in random, double-blind order. Pain intensity was assessed on 10-cm visual analogue scales at fixed time points throughout a 4-h investigation period. Other efficacy variables obtained included time until rescue medication and overall assessment at 4, 6, 12 and 24 h after dosing. RESULTS: Throughout the 4-h investigation period, patients treated with diclofenac reported significantly less pain than those treated with placebo (P < 0.001). No differences were observed among the three doses of diclofenac (P = 0.22). Similar results were observed at 6, 12 and 24 h after dosing. Significant differences were also noted between the placebo group and all the diclofenac treatment groups with respect to time until rescue medication (P < 0.001) and the proportion of patients taking such medication. CONCLUSION: Single doses of i.v. diclofenac (25, 50 and 75 mg) provide significant pain relief after third-molar surgery. The efficacy of this preparation does not appear to be dose related.

Large-scale use of red blood cell units containing alloantibodies.

Many transfusion services are reluctant to accept red blood cell (RBC) units containing antibodies. We evaluated the impact of accepting routine shipments of our region's inventory of alloantibody- positive RBC units over a 4-month period. All patients' samples received up to 30 days after transfusion of such units were evaluated for the presence of passively acquired antibody, and labor and reagent costs were determined. During the study period, we received 259 alloantibody-containing RBC units, and 253 of these were transfused to 187 patients. Follow-up samples were received on 99 of these 187 patients, and 10 of these patients had detectable passive antibody in posttransfusion antibody screening tests. Two patients had anti-C and -D and eight patients had anti-D. Due to our negotiation of a small discount for antibody-containing units and the use of 20 units based on labeled phenotype rather than antigen typing in our laboratory, we experienced a net savings of $3814 over the 4-month period. This savings was achieved despite some additional costs incurred, including costs of data entry and additional testing on patients' samples. We concluded that large-scale use of RBC units from donors with alloantibodies is safe and likely to have a minimal impact on a busy transfusion service's workload and costs. Furthermore, nationwide use of such units would help alleviate projected blood shortages.

Induction of anchorage independent growth by heparin-binding EGF-like growth factor (HB-EGF).

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is initially synthesized as a membrane bound protein that is subsequently processed to yield an approximately 74 amino acid secreted product. To investigate the biological activities of HB-EGF and its role(s) in tumor formation, the full-length HB-EGF cDNA was cloned under the regulation of the mouse metallothionein promoter and stably expressed in HB-EGF deficient mouse L cells. HB-EGF immunoreactive proteins of 21 and 24 kDa were observed from transfected MLC lysates, and these lysates exhibited the ability to bind to the EGF receptor, stimulate 3H-thymidine uptake in BALB/c-3T3 cells, and induce anchorage independent growth (AIG) of normal rat kidney (NRK) cells. Furthermore, NRK cells treated with either E. coli-derived or vaccinia virus-derived HB-EGF, as well as NRK cells directly transfected with the HB-EGF construct, demonstrated AIG. We conclude that HB-EGF is a potent growth factor capable of stimulating altered cell growth and anchorage independence.

Erythrocyte autoantibodies in paediatric patients with sickle cell disease receiving transfusion therapy: frequency, characteristics and significance.

The formation of erythrocyte autoantibodies following transfusion therapy has been described in case reports and small series. To determine the frequency, serological characteristics, and clinical significance of this phenomenon in paediatric patients with sickle cell disease, we analysed the patient database at the Duke University Pediatric Hematology Clinic. We identified children who received multiple erythrocyte transfusions, then reviewed clinical records to identify children who developed erythrocyte autoantibodies in association with transfusions. Among 184 paediatric patients who received multiple erythrocyte transfusions, 14 children (7.6%) developed warm (IgG) erythrocyte autoantibodies. Median transfusion exposure at the time of autoantibody formation was 24 erythrocyte units, range 3-341 units. The autoantibody reacted as a panagglutinin in 11 cases but had anti-e specificity in three patients. Surface complement also was detected in five patients. Clinically significant haemolysis was documented in four patients, each of whom had both surface IgG and C3 detected. The development of erythrocyte autoantibodies was associated with the presence of erythrocyte alloantibodies. Formation of warm erythrocyte autoantibodies in association with transfusions is not rare in paediatric patients with sickle cell disease. Clinicians should be aware of this complication and recognize that the presence of surface C3 is often associated with significant haemolysis.

HPLC/atmospheric pressure chemical ionization-mass spectroscopy of eight regulated sulfonamides.

Reversed phase high performance liquid chromatography coupled with on-line atmospheric pressure chemical ionization mass spectrometry, HPLC,APCI-MS, has been applied to a mixture of eight sulfonamides. In full scan mode, extracted ion chromatograms produced minimum detectable quantities (MDQ) of 0.8 ng on column, for six of the eight regulated sulfonamides investigated. Selected ion monitoring yielded a 50 pg MDQ for sulfamerazine, sulfadiazine and sulfamethazine, while, the other compounds presented higher values. Analysis of supercritical fluid extracts of chicken liver containing sulfadimethoxine were found to be easily detected by HPLC/APCI-MS. In extracts of chicken liver spiked with 25 microg/kg(-1) (25 ppb) of sulfadimethoxine this compound could be detected in selected ion mode, while 100 pg/microl(-1) was detectable in either full scan or single ion modes. The analysis method for extracted sulfadimethoxine also demonstrated good linearity and reproducibility in both single ion and scan mode.

Relationship between patient variables and plasma clozapine concentrations: a dosing nomogram.

BACKGROUND: Previous work has suggested factors such as gender, smoking behavior, dose, and age affect the amount of drug a patient requires to achieve a desired plasma concentration of clozapine. Plasma clozapine concentrations ranging from 350 to 504 ng/mL in treatment-refractory schizophrenics and schizoaffective patients produce response rates ranging approximately 55-80%. Without the aid of clozapine plasma concentration monitoring, 3-6 months are recommended for a therapeutic clozapine trial. Data suggest that the lag time to response can be reduced by administering a dose that produces a therapeutic clozapine concentration. METHODS: To generate a clozapine dosing nomogram to predict clozapine steady-state plasma concentrations, a cohort of 71 patients was collected via retrospective chart review and/or patient interview. Clozapine steady-state plasma concentrations and demographic variables were obtained. Multiple-linear regression was utilized to examine the relationship between the plasma clozapine concentration and the independent variables. RESULTS: The dosing model that optimally predicted steady-state clozapine plasma concentrations included the variables dose (mg/day), smoking (yes = 0 and no = 1), gender, and a dose-gender interaction variable. The model explained 47% of the variance in the clozapine concentrations (F = 14.42, p < .001, r2 = .47). Two equations, one for male subjects, i.e., clozapine (ng/mL) = 111 (smoke) + 0.464 (dose) + 145, and one for female subjects, i.e., clozapine (ng/mL) = 111 (smoke) + 1.590 (dose)-149, were derived to predict clozapine steady-state plasma concentrations to serve as a clozapine dosing guide for clinicians. CONCLUSIONS: A clozapine dosing nomogram was constructed as a clinical aid to facilitate clozapine dosing.

N-desmethylclozapine, an insensitive marker of clozapine-induced agranulocytosis and granulocytopenia.

We reviewed the charts of 58 patients with treatment-refractory schizophrenia who were receiving clozapine, to determine if the drug's active metabolite, N-desmethylclozapine, is a biologic marker for impending clozapine-induced granulocytopenia and agranulocytosis. No significant correlation between granulocyte counts and patient demographic variables of clozapine and N-desmethylclozapine steady-state plasma concentrations, clozapine:N-desmethylclozapine ratio, age, gender, clozapine dosage, smoking status, and race were found. We believe N-desmethylclozapine is not a clinically useful marker for monitoring the effect of clozapine on granulocyte integrity. On the contrary, its plasma concentrations correlated positively with granulocyte counts.