Henry S. Marr

Evaluation of Four DNA Extraction Methods for the Detection of Tritrichomonas Foetus in Feline Stool Specimens by Polymerase Chain Reaction

Feces are increasingly valued as practical samples for molecular diagnosis of infectious disease. However, extraction of polymerase chain reaction (PCR) quality DNA from fecal samples can be challenging because of coextraction of PCR inhibitors. Because the type and quantity of PCR inhibitors is influenced by diet, endogenous flora, and concurrent disease, it is unlikely that extraction method performance with human feces can be directly extrapolated to that of domestic cats. In the present study, 4 commercially available DNA extraction methods were examined for their influence on the sensitivity of PCR for the detection of Tritrichomonas foetus in feline stool. DNA was extracted from serially diluted feline-origin T. foetus trophozoites in the absence or presence of feline feces. The ZR Fecal DNA kit was identified as affording the greatest analytical sensitivity and reproducibility and was able to detect ≥10 T. foetus organisms per 100 mg feces in 100% of PCR reactions. Further, the identified extraction method could be completed in the shortest time of all kits tested.

Bartonella DNA in Loggerhead Sea Turtles

To the Editor: Bartonella are fastidious, aerobic, gram-negative, facultative, intracellular bacteria that infect erythrocytes, erythroblasts, endothelial cells, monocytes, and dendritic cells, and are transmitted by arthropod vectors or by animal scratches or bites (1–6). Currently, 20 species or subspecies of Bartonella have been characterized, of which 8 are known zoonotic pathogens (7). B. henselae has been recently identified from canine blood (8) and from harbor porpoises (9). Pathogenic bacteria are an important threat in terrestrial and marine environments, and in the case of B. henselae, reservoir hosts may be more diverse than currently recognized.

Re-emergence of Babesia conradae and effective treatment of infected dogs with atovaquone and azithromycin

Babesia conradae (B. conradae) causes hemolytic anemia in dogs. This organism has not been reported clinically since it was originally described in southern California in 1991. To date, no anti-protozoal therapies have been associated with clearance of B. conradae. This report describes the use of atovaquone and azithromycin for the treatment of dogs naturally infected with B. conradae and report the re-emergence of B. conradae in southern California. Twelve dogs naturally infected with B. conradae were identified by practicing veterinarians and public health officials in southern California. Treatments consisted of a 10 day course of atovaquone (13.3mg/kg PO q 8h) and azithromycin (10-12.5mg/kg PO q 24h). Four dogs were treated in a randomized blinded placebo-controlled fashion, four additional cases were treated in a non-random, non-blinded fashion and one dog received no treatment. All dogs were tested for B. conradae DNA by polymerase chain reaction (PCR) initially and then once or 3 times post treatment (60-210 days). B. conradae infected dogs that received treatment did not have any detectable Babesia DNA by PCR after treatment. In contrast, dogs receiving placebo had detectable Babesia DNA by PCR throughout the study period. Combination therapy with atovaquone and azithromycin appears to be effective for acute and chronic babesiosis caused by B. conradae.

Hypocalcemia and Hypovitaminosis D in Dogs with Induced Endotoxemia

BACKGROUND Hypocalcemia is a documented electrolyte disturbance in people and animals with sepsis, but its mechanism is poorly understood. OBJECTIVE To investigate mechanisms of hypocalcemia in dogs with experimentally induced endotoxemia. ANIMALS Six healthy mixed breed dogs were included in this nonrandomized, placebo-controlled, crossover study. METHODS Dogs initially were injected with placebo (0.9% NaCl; 1 mL, IV) and then lipopolysaccharide (LPS; 2 μg/kg, IV) after a 5-day washout period. Blood and urine samples were collected for measurement of serum total calcium (tCa), ionized calcium (iCa), total magnesium (tMg), ionized magnesium (iMg), parathyroid hormone (PTH), 25-hydroxyvitamin D (vitamin D), venous blood gases, and fractional excretion (FE) of calcium. RESULTS After LPS administration, body temperature increased and blood pressure decreased. Both iCa and tCa decreased (P < .01), but iMg was not significantly different between control and LPS treatments. PTH concentrations increased (P < .01) and vitamin D concentrations decreased (P < .01). Venous pH, bicarbonate, base excess, and blood glucose also decreased (P < .01). Urine tCa concentration was below the limit of detection for all dogs after LPS administration. CONCLUSIONS Hypocalcemia occurs during endotoxemia in dogs and is associated with hypovitaminosis D. Hypomagnesemia, hypoparathyroidism, alkalosis, and increased calciuresis are not associated with hypocalcemia in endotoxemic dogs.

Expression and function of triggering receptor expressed on myeloid cells-1 (TREM-1) on canine neutrophils

The dog is both a valued veterinary species and a widely used translational model for sepsis research. However, relatively little work has been performed evaluating potential biomarkers present during canine infection. Triggering receptor expressed on myeloid cells-1 (TREM-1) has shown promise as a biomarker for infection and pneumonia in humans. Here we describe, for the first time, the expression and function of the canine orthologue of TREM-1. Expression of TREM-1 on canine neutrophils is significantly up-regulated by stimulation with microbial agonists of TLR2/6, TLR1/2, and TLR4/MD2. Kinetics of TREM-1 protein up-regulation are rapid, with significant increases observed within 2 hr of neutrophil activation. Functionally, canine TREM-1 synergistically enhances LPS-induced production of IL-8, TNF-α and a canine orthologue of CXCL1. Collectively, these data suggest that TREM-1 expression in dogs, as it is in humans, is an amplifier of pro-inflammatory responses to microbial products. These results have direct application to veterinary diagnostics as well as the potential to enhance the utility of canine disease models in the assessment of potential therapeutics in the treatment of human sepsis.

Investigation of a commercial ELISA for the detection of canine procalcitonin.

Background Rapid identification of sepsis enables prompt administration of antibiotics and is essential to improve patient survival. Procalcitonin (PCT) is a biomarker used to diagnose sepsis in people. Commercial assays to measure canine PCT peptide have not been validated. Objective To investigate the validity of a commercially available enzyme‐linked immunosorbent assay (ELISA) marketed for the measurement of canine PCT. Animals Three dogs with sepsis, 1 healthy dog, 1 dog with thyroid carcinoma. Methods Experimental study. The ELISAs ability to detect recombinant and native canine PCT was investigated and intra‐assay and interassay coefficients of variability were calculated. Assay validation including mass spectrometry of the kit standard solution was performed. Results The ELISA did not consistently detect recombinant canine PCT. Thyroid lysate yielded a positive ELISA signal. Intra‐assay variability ranged from 18.9 to 77.4%, while interassay variability ranged from 56.1 to 79.5%. Mass spectrometry of the standard solution provided with the evaluated ELISA kit did not indicate presence of PCT. Conclusions and Clinical Importance The results of this investigation do not support the use of this ELISA for the detection of PCT in dogs.

Failure to identify an association between serologic or molecular evidence of Bartonella infection and idiopathic rhinitis in dogs

OBJECTIVE To determine whether infection with or exposure to Bartonella spp was associated with idiopathic rhinitis in dogs. DESIGN Case-control study. ANIMALS 44 dogs with idiopathic nasal discharge and 63 age- and weight-matched control dogs without nasal discharge and no clinical signs of bartonellosis. Procedures-Serum was tested for antibodies against Bartonella henselae and Bartonella vinsonii subsp berkhoffii with indirect fluorescent antibody assays. Blood was tested for Bartonella DNA with a PCR assay. RESULTS Results of the antibody and PCR assays were negative for all 44 dogs with idiopathic nasal discharge. One control dog had antibodies against B henselae; a second control dog had positive PCR assay results. We did not detect a significant association between assay results and group designation. CONCLUSIONS AND CLINICAL RELEVANCE The present study failed to confirm an association between idiopathic rhinitis and exposure to or infection with Bartonella spp in dogs. Findings do not rule out the possibility that Bartonella infection may cause nasal discharge in some dogs, but the failure to find any evidence of exposure to or infection with Bartonella spp in dogs with idiopathic nasal discharge suggested that Bartonella infection was not a common cause of the disease.

A novel canine model of immune thrombocytopenia: has immune thrombocytopenia (ITP) gone to the dogs?

Canine immune thrombocytopenia (ITP) is analogous to human ITP, with similar platelet counts and heterogeneity in bleeding phenotype among affected individuals. With a goal of ultimately investigating this bleeding heterogeneity, a canine model of antibody‐mediated ITP was developed. Infusion of healthy dogs with 2F9, a murine IgG2a monoclonal antibody to the canine platelet glycoprotein GPIIb (a common target of autoantibodies in ITP) resulted in profound, dose‐dependent thrombocytopenia. Model dogs developed variable bleeding phenotypes, e.g. petechiae and haematuria, despite similar degrees of thrombocytopenia. 2F9 infusion was not associated with systemic inflammation, consumptive coagulopathy, or impairment of platelet function. Unexpectedly however, evaluation of cytokine profiles led to the identification of platelets as a potential source of serum interleukin‐8 (IL8) in dogs. This finding was confirmed in humans with ITP, suggesting that platelet IL8 may be a previously unrecognized modulator of platelet‐neutrophil crosstalk. The utility of this model will allow future study of bleeding phenotypic heterogeneity including the role of neutrophils and endothelial cells in ITP.

Diminazene Diaceturate for Treatment of Chronic Cytauxzoon felis Parasitemia in Naturally Infected Cats

BACKGROUND Cytauxzoon felis is a hemoprotozoal parasite that causes substantial morbidity and mortality during the acute phase of infection in cats. However, cats that survive the acute illness remain persistently infected and may serve as a reservoir for the tick-transmitted pathogen. OBJECTIVE We investigated the ability of the antiprotozoal compound diminazene diaceturate to eliminate the pathogen from naturally infected C. felis carriers. ANIMALS Seven healthy, chronically infected domestic cats housed in a research setting. METHODS Prospective clinical trial. Cats were treated in a masked fashion with diminazene diaceturate (3 mg/kg) or placebo IM in a series of 2 injections 7 days apart. Clearance of the organism was assessed by light microscopy and real-time polymerase chain reaction (PCR) at 0, 3, 6, and 10 weeks. In addition, cats were monitored for behavioral changes or for changes on physical examination, CBC, plasma biochemical profile, and urinalysis periodically. Cats that remained parasitemic at the end of 10 weeks were switched to the alternative treatment and similarly monitored for an additional 10 weeks. RESULTS Adverse events associated with treatment were limited to self-resolving hypersalivation and injection site soreness; the former was ameliorated by premedication with atropine. Parasite burden, as assayed by both light microscopy and real-time PCR, was similar between diminazene- and placebo-treated cats. CONCLUSIONS AND CLINICAL RELEVANCE Diminazene diaceturate was unable to eliminate the pathogen or decrease parasite burden in healthy, chronically infected cats.

Prevalence of selected vector-borne organisms and identification of Bartonella species DNA in North American river otters (Lontra canadensis).

Trapper-killed North American river otters (Lontra canadensis) in North Carolina, USA, were screened for multiple vector-borne bacteria known to be pathogenic to mammals. Blood was collected from 30 carcasses in 2006, from 35 in 2007, and from one live otter in 2008. Samples were screened using conventional polymerase chain reaction (PCR) tests for DNA from Bartonella spp., Ehrlichia spp., and spotted fever group Rickettsia spp. All samples were negative for Rickettsia spp. Twelve of 30 samples from 2006 produced amplicons using the assay designed to detect Ehrlichia spp., but sequencing revealed that the amplified DNA fragment was from a novel Wolbachia sp., thought to be an endosymbiote of a Dirofilaria sp. Between 2006 and 2007, DNA from a novel Bartonella sp. was detected in 19 of 65 animals (29%). Blood from one live otter captured in 2008 was found positive for this Bartonella sp. by both PCR and culture. The pathogenicity of this Bartonella species in river otters or other mammals is unknown.