Sex-differential non-specific effects of adjuvanted and non-adjuvanted rabies vaccines versus placebo on all-cause mortality in dogs (NERVE-Dog study): a study protocol for a randomized controlled trial with a nested case-control study.

BACKGROUND: It has been proposed that childhood vaccines in high-mortality populations may have substantial impacts on mortality rates that are not explained by the prevention of targeted diseases, nor conversely by typical expected adverse reactions to the vaccines, and that these non-specific effects (NSEs) are generally more pronounced in females. The existence of these effects, and any implications for the development of vaccines and the design of vaccination programs to enhance safety, remain controversial. One area of controversy is the reported association of non-live vaccines with increased female mortality. In a previous randomized controlled trial (RCT), we observed that non-live alum-adjuvanted animal rabies vaccine (ARV) was associated with increased female but not male mortality in young, free-roaming dogs. Conversely, non-live non-adjuvanted human rabies vaccine (NRV) has been associated with beneficial non-specific effects in children. Alum adjuvant has been shown to suppress Th1 responses to pathogens, leading us to hypothesize that alum-adjuvanted rabies vaccine in young dogs has a detrimental effect on female survival by modulating the immune response to infectious and/or parasitic diseases. In this paper, we present the protocol of a 3-arm RCT comparing the effect of alum-adjuvanted rabies vaccine, non-adjuvanted rabies vaccine and placebo on all-cause mortality in an owned, free-roaming dog population, with causal mediation analysis of the RCT and a nested case-control study to test this hypothesis. METHODS: Randomised controlled trial with a nested case-control study. DISCUSSION: We expect that, among the placebo group, males will have higher mortality caused by higher pathogen loads and more severe disease, as determined by haematological parameters and inflammatory biomarkers. Among females, we expect that there will be no difference in mortality between the NRV and placebo groups, but that the ARV group will have higher mortality, again mediated by higher pathogen loads and more severe disease. We anticipate that these changes are preceded by shifts in key serum cytokine concentrations towards an anti-inflammatory immune response in females. If confirmed, these results will provide a rational basis for mitigation of detrimental NSEs of non-live vaccines in high-mortality populations.

Reference intervals for hematological and biochemical analytes in a single herd of clinically healthy gelding donkeys in Saint Kitts.

There is a large population of donkeys in Saint Kitts; however, hematological and biochemical reference intervals (RIs) are lacking. This study addressed this deficiency by following the American Society for Veterinary Clinical Pathology RI guidelines. Sixty-six healthy, gelding standard donkeys with a median and interquartile range age of 5 years (3.5 - 8 years) and a mean ± standard deviation body weighed of 156 ± 16.7 kg were used to produce a five-part differential complete blood count using an impedance-based analyzer. Clinical chemistry analytes were quantified using a photometric-based analyzer utilizing two reagent rotors that determined 14 and 11 analytes, respectively. An electrochemical-based analyzer quantified chloride, sodium and potassium. Reference intervals were computed using Reference Value Advisor. Results of analytes determined using different rotors/analyzers were assessed using Passing-Bablok regression and Bland-Altman plot analyses. Reference intervals for 43 hematological and biochemical analytes were generated. Reference intervals for hematocrit, red blood cells, white blood cells, total protein, glucose, blood urea nitrogen, and creatinine were 23.67% - 38.08%, 4.08 - 6.42 1012/L, 4.7 - 12.34 109/L, 5.84 - 6.93 g/dL, 64.7 - 130.9 mg/dL, 11.1 - 13.4 mg/dL, and 0.67 - 1.36 mg/dL, respectively. There was good agreement between detection system for albumin, aspartate aminotransferase, gamma glutamyl transferase, total protein, globulin, and potassium, but not for blood urea nitrogen, calcium, creatinine kinase, and sodium. This study is the first to establish hematological and biochemical RIs in donkeys in Saint Kitts. These values will be useful for clinical decision-making.

Serologic and Molecular Diagnosis of Anaplasma platys and Ehrlichia canis Infection in Dogs in an Endemic Region.

Anaplasma platys and Ehrlichia canis are obligate intracellular, tick-borne rickettsial pathogens of dogs that may cause life-threatening diseases. In this study, we assessed the usefulness of PCR and a widely used commercial antibody-based point-of-care (POC) test to diagnose A. platys and E. canis infection and updated the prevalence of these pathogens in dogs inhabiting the Caribbean island of Saint Kitts. We detected A. platys in 62/227 (27%), E. canis in 84/227 (37%), and the presence of both in 43/227 (19%) of the dogs using PCR. POC testing was positive for A. platys in 53/187 (28%), E. canis in 112/187 (60%), and for both in 42/187 (22%) of the samples tested. There was only a slight agreement between A. platys PCR and POC test results and a fair agreement for E. canis PCR and POC test results. Our study suggests that PCR testing may be particularly useful in the early stage of infection when antibody levels are low or undetectable, whereas, POC test is useful when false-negative PCR results occur due to low bacteremia. A combination of PCR and POC tests may increase the ability to diagnose A. platys and E. canis infection and consequently will improve patient management.

First report of Aelurostrongylus abstrusus in St. Kitts.

A 4-month-old intact male domestic shorthair kitten living in St Kitts, West Indies presented with respiratory distress, cachexia, and mucopurulent nasal discharge. Thoracic radiographs revealed a diaphragmatic hernia. The diaphragmatic hernia as well as subpleural pulmonary nodules suspicious for verminous pneumonia were identified during a postmortem examination. Histology showed multifocal to coalescing pyogranulomatous and eosinophilic pneumonia centered on larvae and morulated eggs. The lesion and nematode morphology were consistent with Aelurostrongylus abstrusus. Although Aelurostrongylus abstrusus has been reported worldwide, this is the first report of a metastrongyloid lungworm in cats in St. Kitts and for the West Indies. This case report should increase the awareness of A. abstrusus pneumonia in cats from St. Kitts and other locations in the eastern Caribbean.

Hereditary ß-mannosidosis in a dog: Clinicopathological and molecular genetic characterization.

Hereditary ß-mannosidosis causing progressive lysosomal neuropathy and other clinical signs, has been previously described in humans, Nubian goats, and Salers cattle. Here we report the clinicopathological, metabolic, and molecular genetic features of canine beta-mannosidase (MANBA, EC 3.2.1.25) deficiency. A 1-year-old male mix-breed dog from St. Kitts was presented with progressive stumbling, weakness, and regurgitation. Vacuolated lymphocytes were observed on the blood film. Postmortem findings included marked enlargement of nerves, megaesophagus, and internal hydrocephalus. Vacuolated macrophages, neurons, and secretory epithelial cells suggested an oligosaccharide storage disease. Plasma concentration of the ß-mannosidosis specific oligosaccharide was approximately 75 fold that of controls. The plasma beta-mannosidase activity was severely reduced to ~5% of controls; five other lysosomal acid hydrolase activities were increased or within their normal reference interval. Genomic sequencing of this dog's MANBA gene identified a homozygous exonic five bp tandem duplication in the penultimate exon of the MANBA gene (c.2377_2381dupTATCA) which results in a reading frame shift, altering the subsequent amino acid sequence and creating a premature stop codon. The truncated beta-mannosidase enzyme is expected to be dysfunctional. This enzyme deficiency causes the accumulation of un-degraded oligosaccharides in cells, which affect the myelination of the peripheral and central nervous systems. This insertion was not encountered in 121 and 80-screened samples from dogs on St. Kitts (all were homozygous for wild-type) and Philadelphia region (wild-type), respectively. In conclusion, canine ß-mannosidosis has similar clinicopathological features with some human patients, but milder signs than in ruminants and more severe than in knockout mice. Hence, dogs with ß-mannosidosis could become a valuable disease model for the human disease.

Hemolytic anemia, spherocytosis, and thrombocytopenia associated with honey bee envenomation in a dog.

This case report describes a massive honey bee envenomation in a 14-month-old male Belgian Malinois dog from St. Kitts, West Indies. Acute and delayed onsets of hemolytic anemia, echinocytosis, spherocytosis, thrombocytopenia, hemoglobinemia, and hemoglobinuria developed following envenomation. The dog recovered after treatment with glucocorticoids and supportive therapy. Spherocytosis, hemolysis, and thrombocytopenia in patients with massive bee envenomation are likely due to the direct toxic effects of the primary components of bee venom, melittin and phospholipase A2 (PLA2 ). Mellitin causes hemolysis by forming large pores in erythrocytes resulting in leakage of hemoglobin and also causes spectrin stiffening and resultant echinocyte and spherocyte formation. Melittin also stimulates PLA2 , a hydrolase that causes echinocytosis and spherocytosis, in vivo and in vitro, and mitochondrial breakdown in platelets. However, delayed manifestations could be attributed to immune-mediated mechanisms from the generation of antibodies against damaged erythrocytes and platelet membrane proteins.

Thoracic and paraspinal extramedullary hematopoiesis in a cat with chronic non-regenerative anemia.

CASE SUMMARY: A 6-year-old neutered male domestic shorthair cat presented with non-regenerative macrocytic anemia of 2 years' duration and minimally ambulatory paraparesis. Neurologic examination suggested an upper motor neuron paresis or T3-L3 myelopathy. The cat was positive for feline immunodeficiency virus (FIV), neutropenic, had polyclonal gammopathy and was euthanized following a hemolytic crisis. At autopsy, multifocal bilateral dark red masses were observed subpleurally around the costochondral junctions, extradurally and paraspinally in the spinal canal, and paravertebrally, on the lateral and ventral subpleural surfaces of the T4-11 vertebrae. Histologic examination of the masses revealed extramedullary hematopoietic tissue composed primarily of erythroid precursors and megakaryocytes, with occasional myeloid precursors and blood-filled sinuses. Bone marrow findings supported ineffective granulopoiesis, and decreased erythropoiesis and megakaryopoiesis, with probable myelodysplasia as the underlying cause of the hematologic abnormalities. RELEVANCE AND NOVEL INFORMATION: Thoracic, paraspinal and paravertebral extramedullary hematopoietis presenting as masses has not been described previously in cats with chronic anemia. This is a unique case of a thoracic-spinal-epidural extramedullary hematopoietic masses resulting in possible spinal cord compression and paraparesis in a cat.

Safety and efficacy of high-dose fomepizole compared with ethanol as therapy for ethylene glycol intoxication in cats.

OBJECTIVE: To determine the safety and efficacy of high-dose fomepizole compared with ethanol (EtOH) in cats with ethylene glycol (EG) toxicosis. DESIGN: Prospective study. SETTING: University veterinary research laboratory. ANIMALS: Thirteen cats. INTERVENTIONS: Two cats received injections of high-dose fomepizole (Study 1). Three cats received lethal doses of EG and fomepizole treatment was initiated 1, 2, or 3 hours later (Study 2). Eight cats received a lethal dose of EG and were treated with fomepizole or EtOH (Study 3). Cats treated with fomepizole received 125 mg/kg IV initially, then 31.25 mg/kg at 12, 24, and 36 hours. Cats treated with EtOH received 5 mL of 20% EtOH/kg IV initially, then every 6 hours for 5 treatments, then every 8 hours for 4 treatments. Cats also received fluids and supportive therapy as needed. MEASUREMENTS AND MAIN RESULTS: Clinical signs were monitored and serial blood analyses performed. Cats receiving fomepizole experienced mild sedation but no biochemical evidence of toxicity. Cats receiving fomepizole for EG intoxication survived if therapy was initiated within 3 hours of EG ingestion. One of the 6 developed acute renal failure (ARF) but survived. Only 1 of the 3 cats treated with EtOH 3 hours following EG ingestion survived; 2 developed ARF and were euthanized. Cats treated 4 hours following EG ingestion developed ARF, whether treated with EtOH or fomepizole. CONCLUSIONS: Fomepizole is safe when administered to cats in high doses, prevents EG-induced fatal ARF when therapy is instituted within 3 hours of EG ingestion, and is more effective than treatment with EtOH.

Perspectives and advances in in-clinic laboratory diagnostic capabilities: hematology and clinical chemistry.

The typical technologies used in veterinary hematology and biochemical analyzers are reviewed, along with associated advantages and disadvantages. Guidelines for implementing a successful in-clinic laboratory are provided, including criteria for system evaluation and expectations for comparative performance evaluations. The more common problems and limitations associated with in-clinic laboratory diagnostics and how to best prevent them are also discussed.

Quality control recommendations and procedures for in-clinic laboratories.

The design and use of quality control materials and rationale for implementation of a quality monitoring program are discussed. A simplified approach to a quality monitoring program suitable for in-clinic laboratories is presented. Use of blood films and the mean cell hemoglobin concentration value as adjuncts to quality monitoring in hematology is described. Over time, it is hoped that the profession more widely embraces, if not demands, implementation of quality monitoring for in-clinic laboratory diagnostics.

Transtracheal wash from a puppy with respiratory disease.

A 4-month-old, intact male Boxer puppy was presented to the Animal Emergency and Critical Care Services of South Florida because of nasal discharge, dehydration, dyspnea, and coughing. The dog had been diagnosed with intestinal parasites and kennel cough approximately 10 days before presentation. Lateral and ventrodorsal radiographs of the thorax revealed an increased bronchointerstitial pattern throughout the lungs. A transtracheal wash was performed. On cytologic examination of direct, Wright-Giemsa-stained smears, small basophilic coccoid structures (0.3-0.9 microm in diameter) were observed in low to moderate numbers within neutrophils and adherent to epithelial cells. The small size of the organisms raised suspicion for Mycoplasma. Culture of the transtracheal wash fluid resulted in growth of a Mycoplasma sp. The patient was treated with enrofloxacin and amoxicillin/clavulanate and made a full recovery. Recognizing Mycoplasma in transtracheal washes could aid in recommending the appropriate culture media or immunologic techniques, which could result in an accurate diagnosis of mycoplasmosis.

Heinz body anemia in a dog that had been sprayed with skunk musk.

A 5-year-old pit bull-type dog was examined because of lethargy and black feces, and a presumptive diagnosis of methemoglobinemia was made on the basis of grossly visible brown discoloration of the mucous membranes and urine. Heinz body formation was seen on examination of blood smears, and severe anemia developed 3 days later. The dog did not have any history of exposure to toxins or drugs that have been associated with Heinz body formation. The only remarkable historical finding was that the dog had been wandering loose the day prior to initial examination and returned home smelling strongly of skunk spray. Skunk spray contains thiols and other compounds that are strong oxidizing agents. It was speculated that the methemoglobinemia and Heinz body anemia were a result of exposure to these compounds. Skunk interactions with dogs usually have malodorous but otherwise harmless results. However, findings in this dog suggest that more serious consequences may develop in isolated cases.

Mutation analysis of feline Niemann-Pick C1 disease.

Niemann-Pick C (NPC) disease is an autosomal recessive neurovisceral lysosomal storage disorder that results in defective intracellular transport of cholesterol. The major form of human NPC (NPC1) has been mapped to chromosome 18, the NPC1 gene (NPC1) has been sequenced and several mutations have been identified in NPC1 patients. A feline model of NPC has been characterized and is phenotypically, morphologically, and biochemically similar to human NPC1. Complementation studies using cultured fibroblasts from NPC affected cats and NPC1 affected humans support that the gene responsible for the NPC phenotype in this colony of cats is orthologous to human NPC1. Using human-based PCR primers, initial fragments of the feline NPC cDNA were amplified and sequenced. From these sequences, feline-specific PCR primers were generated and designed to amplify six overlapping bands that span the entire feline NPC1 open reading frame. A single base substitution (2864G-C) was identified in NPC1 affected cats. Obligate carriers are heterozygous at the same allele and a PCR-based assay was developed to identify the geneotype of all cats in the colony. The mutation results in an amino acid change from cysteine to serine (C955S). Several of the mutations identified in people occur in the same region. Marked similarity exists between the human and feline NPC1 cDNA sequences, and is greater than that between the human and murine NPC1 sequences. The human cDNA sequence predicts a 1278aa protein with a lysosomal targeting sequence, several trans-membrane domains and extensive homology with other known mediators of cholesterol homeostasis.

The Niemann-Pick C1 protein in feline fibroblasts.

Niemann-Pick type C (NPC) disease is a rare inherited metabolic disorder characterized by hepatosplenomegaly, progressive neurodegeneration, and storage of lipids such as cholesterol and glycosphingolipids in most tissues. The current study was conducted to characterize the Niemann-Pick C1 (NPC1) protein in feline fibroblasts. This was accomplished by generating rabbit polyclonal antibodies against a peptide corresponding to amino acids 1256-1275 of the feline NPC1 protein. The results obtained using immunoblot analysis identified two major proteins that migrated at approximately 140 and 180 kDa. These two proteins were absent when immunoblots were incubated in the presence of feline NPC1 antibody and immunizing peptide, or preimmune serum. Fluorescence microscopy of feline fibroblasts incubated with the feline NPC1 antibody revealed granular staining within the perinuclear region of the cell. This granular staining was diminished when feline fibroblasts were incubated in the presence of feline NPC1 antibody and immunizing peptide, or was completely absent when feline fibroblasts were incubated in the presence of preimmune serum. Additional studies using double-labeled fluorescence microscopy indicated that feline NPC1 partially colocalized with markers for late endosomes/lysosomes, endoplasmic reticulum, and microtubules, but not the trans-Golgi network. In summary, the results presented in this report demonstrate that the NPC1 protein in feline fibroblasts has a similar distribution as that previously described for human and murine fibroblasts.

Blood precipitate associated with intra-abdominal carboxymethylcellulose administration.

A precipitate was observed on the blood films of horses (15 of 16) and one cow given a peritoneal infusion of 1 % sodium carboxymethylcellulose (SCMC) solution to prevent abdominal adhesions. The intensity of the precipitate seen 2 to 3 days post-infusion strongly correlated with the administered dose of SCMC (range 0.96 to 11.7 ml/kg). The dose given was inversely correlated with bodyweight and the most prominent precipitates were seen in foals. The precipitate was observed as early as 24 hours and persisted for as long as 9 days after SCMC administration. Fibrinogen was the only hematological or biochemical parameter consistently abnormal in horses receiving SCMC, mild increases (500 to 700 mg/dl) were noted in 11/16 cases but did not correlate with SCMC administration.

Cytology of canine malignant histiocytosis.

Cytologic features of bone marrow, tissue, and abdominal fluid in seven cases of malignant histiocytosis in dogs are described, and histopathology, hematology, and serum biochemistry of the cases are reviewed. Diagnosis of malignant histiocytosis was confirmed by tissue morphology and immunohistochemistry; neoplastic cells in all cases had positive immunoreactivity to lysozyme. This stain can be used to definitively establish the diagnosis of malignant histiocytosis on cytology specimens as well as tissue sections. Cytologic findings included numerous pleomorphic, large, discrete mononuclear cells with abundant, lightly basophilic, vacuolated, granular cytoplasm. Nuclei were round to oval to reniform with marked anisocytosis and anisokaryosis; nucleoli were prominent. Mitotic figures, often bizarre, were occasionally seen. Multinucleated giant cells and phagocytosis of erythrocytes and leukocytes were prominent features in cytologic preparations in four cases. Four dogs were anemic, five dogs were thrombocytopenic, and three dogs were hypercalcemic. Breeds affected included Doberman Pinscher (1), Golden Retriever (2), Flat Coated Retriever (3), and mixed-breed dog (1).