Stephen C. Barr

Feline clinical parasitology.

Description: Although there are books available dealing with canine parasitology, there is at present no book detailing parasites that offers clinical information specific to felines. Cats differ significantly from dogs in their parasitic infections and infestations. Although dogs and cats do share a few parasites, the vast majority of the parasites of these pets are specific to either cats or dogs, not to both. This must–have reference offers an in–depth examination of feline parasites. Because of the immense worldwide popularity of cats and due to the amount of travel undertaken by cats and their owners, the authors have produced a book that is international in scope. Consequently, this exhaustive reference has strong appeal to practitioners and veterinary parasitologists in North America and around the world.

Immunogenicity and protective efficacy of recombinant Leptospira immunoglobulin-like protein B (rLigB) in a hamster challenge model

Leptospiral immunoglobulin-like protein (LigB) was truncated into conserved (LigBcon) and variable (varB1, varB2) fragments and expressed as GST/His-tag fusion proteins. Four-week-old hamsters were immunized with equal amounts of each fragment individually or combined in alum adjuvant at days 0 and 21 and subsequently challenged three weeks after the booster with 2.5 LD(50) live virulent Leptospira interrogans serovar Pomona. Our results demonstrate that immunization with LigB produced strong humoral immune responses as revealed by high titers against each fragment and significant enhancement in Th2 cytokines (IL-4, IL-10). A significant activation of CMI is revealed by enhanced proliferation of lymphocytes and up regulation of Th1 cytokines (IL-12p40, IFN-gamma) was also noted. Of the peptides studied, rLigBcon was able to impart maximum protection (71%), followed by rVarB1 (54%), whereas rVarB2 was not able to impart a significant level of protection (33%) against lethal infection as revealed by enhanced survival and reduced severity of histopathological lesions in vital organs (viz. kidney, liver, spleen) of the immunized animals. Moreover, concurrent administration of all three fragments significantly enhanced the protective efficacy of the vaccine (83%). Overall, our results clearly demonstrate that LigB has emerged as novel protective antigen that can be used in future subunit vaccines against leptospirosis.

Canine Leishmaniasis in North America: Emerging or Newly Recognized?

Canine leishmaniasis is a fatal zoonotic visceralizing disease usually associated with tropical areas. The etiologic agent is an obligate intracellular protozoan, Leishmania infantum. In 1999, an outbreak of a canine leishmaniasis was reported in a Foxhound kennel in New York, and since that report, several other outbreaks have occurred across the United States in additional Foxhound kennels. Because of the high mortality and transmissibility associated with these outbreaks, it is essential that clinicians be aware of this disease to permit its rapid recognition and institution of control measures. Cases with a travel history may suggest imported disease; these are mainly observed from Southern Europe (eg, south of France, Spain, and Italy). Breeds from these and other endemic areas may be at higher risk of infection with Leishmania because of vertical transmission. The purpose of this report is to discuss the clinical signs, epidemiology, diagnosis, control, and treatment of canine leishmaniasis with focus on the aspects of this disease within North America.

Influence of treatment with ultralow-dose aspirin on platelet aggregation as measured by whole blood impedance aggregometry and platelet P-selectin expression in clinically normal dogs.

OBJECTIVE To evaluate the influence of treatment with ultralow-dose aspirin (ULDAsp) on platelet aggregation, P-selectin (CD62P) expression, and formation of platelet-leukocyte aggregates in clinically normal dogs. ANIMALS 18 clinically normal dogs. PROCEDURES Studies were conducted before and 24 hours after ULDAsp administration (0.5 mg/kg, PO, q 24 h, for 2 days). Whole blood impedance aggregometry for the assessment of platelet function was performed with sodium citrate-anticoagulated blood and aggregation agonists (ADP at 20, 10, and 5 μmol/L; collagen at 10, 5, and 2 μg/mL). Onset, maximum response, and rate of platelet aggregation were recorded. Flow cytometric assays were configured to detect thrombin-induced CD62P expression and platelet-leukocyte aggregates in EDTA-anticoagulated whole blood. Externalized platelet CD62P and constitutive CD61 (GPIIIa) were labeled with antibodies conjugated to phycoerythrin (PE) and fluorescein isothiocyanate (FITC), respectively. Red blood cell-lysed paraformaldehyde-fixed EDTA-anticoagulated whole blood was dual labeled with CD61-FITC and a panleukocyte antibody (CD18-PE) to characterize platelet-leukocyte aggregates. RESULTS ULDAsp significantly delayed platelet aggregation onset with ADP at 20 μmol/L by 54% to 104%, attenuated maximum aggregation with various concentrations of ADP and collagen by ≥ 41%, and slowed aggregation rate with the highest ADP and collagen concentrations by ≥ 39%. Depending on the parameter tested, up to 30% of dogs failed to have an ULDAsp effect. Thrombin stimulation significantly increased CD62P expression in platelets and platelet-leukocyte aggregates, but ULDAsp did not alter basal or thrombin-stimulated CD62P expression. CONCLUSIONS AND CLINICAL RELEVANCE ULDAsp treatment of clinically normal dogs impaired platelet aggregation in most dogs, but did not influence CD62P platelet membrane expression.

Use of a multiplex polymerase chain reaction to rapidly differentiate Neospora caninum from Toxoplasma gondii in an adult dog with necrotizing myocarditis and myocardial infarct

This report describes a 3-year-old male castrated Mastiff dog that died unexpectedly with locally extensive, acute, necrotizing myocarditis and myocardial infarction. Intralesional protozoal tachyzoites in the affected myocardium were confirmed to be Neospora caninum by a novel multiplex polymerase chain reaction (PCR) and immunohistochemistry. Protozoal organisms were not identified in other tissues by histology, immunohistochemistry, or PCR. The multiplex PCR assay was used to quickly provide preliminary results on fresh myocardium to differentiate N. caninum and Toxoplasma gondii. Neosporosis is an uncommon cause of myocarditis in adult dogs and differential diagnoses for myocarditis in this population of dogs are reviewed.

Upregulation of cardiac cell plasma membrane calcium pump in a canine model of Chagas disease.

We have previously demonstrated that cardiac myocytes isolated from the hearts of adult dogs develop rapid repetitive cytosolic Ca2+ transients, membrane depolarization, and cell contraction by mobilization of sarcoplasmic reticulum Ca2+ stores when exposed to a soluble factor from the trypomastigotes of Trypanosoma cruzi. These findings led us to investigate the regulatory mechanisms of cytosolic Ca2+ in cardiac tissues from dogs chronically infected with T. cruzi. Expression of the plasma membrane calcium pump (PMCA) RNA and protein was determined by Northern and Western blotting, respectively, followed by densitometric analyses. A 642-bp PMCA 1b complementary DNA probe derived from canine epicardial tissue hybridized to 2 major transcripts (7.3 and 5.3 kb) in canine epicardium. Expression of the dominant transcript (7.3 kb) was 77% greater in cardiac tissues obtained from dogs with chronic T. cruzi infection (140 days after inoculation) in comparison with constitutive expression levels in normal dogs. Monoclonal antibody 5F10, known to recognize all isoforms of the PMCA, was used to detect expression of the PMCA protein in epicardial tissue. Expression of a 142-kDa protein was increased by 58% in the cardiac tissues of infected dogs when compared with those from uninfected dogs. To establish a link between the upregulation of PMCA in dogs chronically infected with Chagas disease and the ventricular-based arrhythmias and myocardial failure that occur during this stage of disease both in dogs and humans, further study will be required.

Prevalence of Antibodies to Trypanosoma cruzi, Toxoplasma gondii, Encephalitozoon cuniculi, Sarcocystis neurona, Besnoitia darlingi, and Neospora caninum in North American Opossums, Didelphis virginiana, from Southern Louisiana

Abstract We examined the prevalence of antibodies to zoonotic protozoan parasites (Trypanosoma cruzi, Toxoplasma gondii, and Encephalitozoon cuniculi) and protozoans of veterinary importance (Neospora caninum, Sarcocystis neurona, and Besnoitia darlingi) in a population of North American opossums (Didelphis virginiana) from Louisiana. Samples from 30 opossums were collected as part of a survey for T. cruzi in Louisiana. Frozen sera from these 30 opossums were examined using an indirect immunofluorescent antibody test (IFAT) against in vitro-produced antigenic stages of these protozoans. Additionally, 24 of the 30 samples were examined using hemoculture, and all 30 were examined in the modified direct agglutination test (MAT) for antibodies to To. gondii. The prevalences of reactive IFAT samples were as follows: 60% for T. cruzi, 27% for To. gondii, 23% for E. cuniculi, 17% for S. neurona, 47% for B. darlingi, and 0% for N. caninum. Hemoculture revealed that 16 (67%) of 24 samples were positive for T. cruzi, compared to 18 of 30 (60%) by IFAT. The sensitivity and specificity for the IFAT compared to hemoculture was 100% for each. The modified direct agglutination test revealed that 9 (30%) of the 30 samples from opossums had antibodies to To. gondii, compared to 8 (27%) using the IFAT. The sensitivity and specificity of the IFAT compared to the MAT was 100% and 72%, respectively.

Trypanosoma manulis n. sp. from the Russian Pallas cat Felis manul

ABSTRACT. The morphology of Trypanosoma manulis n. sp. is described from living and stained specimens obtained from the blood of a Pallas cat, Felis manul, from Kazakhstan. the cat was also infected with a Hepatozoon sp. and feline immunodeficiency virus. the morphology of the trypanosome most closely resembles that of Trypanosoma mpapuense Reichenow and Trypanosoma heybergi Rodhain found in bats. Trypanosoma manulis does not grow well in conventional media, but co‐culture with African green monkey kidney cells in Eagles Minimum Essential Medium supplemented with 10% fetal calf serum at approximately 27° C resulted in luxuriant growth of trypanosomes. Under these growth conditions, epimastigotes adhered to the surface of the culture flask and to African green monkey kidney cells, as well as forming large rosettes. At 37° C, although growth was poor, transformation of the epimastigotes into the bloodstream forms occurred. This represents the first report of a trypanosome of the subgenus Megatrypanum in a felid.

Eurytrema procyonis and pancreatitis in a cat

A young adult male domestic shorthair cat was presented for physical examination, routine vaccinations, and a fecal examination. Physical examination revealed no significant abnormalities. Eggs of the raccoon pancreatic fluke Eurytrema procyonis were detected by fecal flotation. Results of a complete blood count and serum biochemistry panel were normal. Abdominal sonography revealed an enlarged hypoechoic pancreas with a hyperechoic rim, and a distended and thickened pancreatic duct. Serum pancreatic lipase immunoreactivity (PLI) was increased. These findings supported the possibility of fluke-associated pancreatitis. Treatment with praziquantel/pyrantel/febantel was associated with resolution of sonographic abnormalities and normalization of PLI.

Characterization of a serine protease activity in Sarcocystis neurona merozoites

Sarcocystis neurona merozoites were examined for their ability to invade and divide in bovine turbinate (BT) cell cultures after treatment with cysteine (iodoacetamide), aspartic (pepstatin A), metallo- (1,10-phenanthroline and ethylene glycol-bis(aminoethylether)-tetraacetic acid [EGTA]), or serine (4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride [AEBSF], phenylmethane sulphonyl fluoride [PMSF], and tosyl lysyl chloramethyl ketone [TLCK]) protease inhibitors. Significant (P < 0.01) inhibition of serine protease activity by PMSF and TLCK led to a reduction of 86 and 78% in merozoites produced in BT cell cultures, respectively, whereas AEBSF (1 mM) led to a 68% reduction in merozoites produced in BT cell cultures and a reduction of 84 and 92% at higher AEBSF concentrations (2 and 3 mM, respectively). Pepstatin A and iodoacetamide failed to cause any inhibition in merozoite production, whereas 1,10-phenanthroline and EGTA caused slight, but not significant, inhibition at 6 and 17%, respectively. In zymograms, 2 bands of protease activity between 65- and 70-kDa molecular weight were seen. The protease activity was inhibited by AEBSF but not by E-64 (cysteine protease inhibitor), EGTA, iodoacetamide, or pepstatin A. In native zymograms, the protease activity was highest between a pH range of 8 and 10. These data suggest that merozoites of S. neurona have serine protease activity with a relative molecular weight range between 65 and 70 kDa and optimal pH range between 8 and 10, which is essential for host cell entry at least in vitro. The protease activity described here could be a potential target for chemotherapy development.