Effects of CD28 blockade on subsets of naïve T cells in cats

OBJECTIVE: To determine whether human CTLA4-Ig ([hu]CTLA4-Ig) inhibits costimulation-dependent lymphocyte proliferation in vitro, compare the effects of (hu)CTLA4-Ig with cyclosporine and steroids on CD4+ and CD8+ T-cell lymphocyte proliferation, and determine whether memory T-cell function remains intact in the presence of (hu)CTLA4-Ig. ANIMALS: 29 cats. PROCEDURE: Peripheral blood mononuclear cells (PBMCs) were stimulated with concanavalin A (costimulation-dependent mitogen) or phorbol 12-myristate 13-acetate and ionomycin (costimulation independent mitogens) alone or in the presence of (hu)CTLA4-Ig, cyclosporine, or dexamethasone; effects of these treatments on lymphocyte proliferation were assessed by incorporation of thymidine labeled with tritium or flow cytometry. Antigen-specific proliferation was determined by stimulating PBMCs from 2 healthy cats seropositive for Toxoplasma gondii with soluble Toxoplasma antigen alone or in the presence of (hu)CTLA4-Ig or cyclosporine. RESULTS: (hu)CTLA4-Ig inhibited costimulation-dependent lymphocyte proliferation in vitro but had no effect on costimulation-independent lymphocyte proliferation. Compared with mitogen alone, (hu)CTLA4-Ig caused a significant decrease in responder frequency and proliferative capacity of CD4+ T cells; however, the effect on CD8+ T cells was not significant. Cyclosporine alone or with dexamethasone had a significantly greater suppressive effect on responder frequency and proliferative capacity of CD4+ and CD8+ T cells, compared with (hu)CTLA4-Ig. Compared with cyclosporine, (hu)CTLA4-Ig appeared to have a sparing effect on antigen-specific proliferation of memory CD4+ and CD8+ T cells. CONCLUSIONS AND CLINICAL RELEVANCE: (hu)CTLA4-Ig selectively inhibited costimulation-dependent proliferation of lymphocytes in vitro and had a sparing effect on antigen-specific proliferation of memory cells. The specificity of its mechanism of action suggests that (hu)CTLA4-Ig may prevent allograft rejection but leave memory responses to previously encountered antigens intact.     

Survey of the large-animal diplomates of the American College of Veterinary Internal Medicine regarding knowledge and clinical use of polymerase chain reaction: implications for veterinary education

A questionnaire was developed to document the knowledge base of large-animal diplomates of the American College of Veterinary Internal Medicine (ACVIM) regarding polymerase chain reaction (PCR) technology and to identify the common use of this technology in equine practice. Ninety-three of the 278 mailed questionnaires were returned, for an overall response rate of 33.4%. Ninety respondents (99%) reported being familiar with the general principles of nucleic acid probe technology; however, only 52 (57%) knew the difference between conventional (traditional) and real-time (second-generation) PCR. The majority of the respondents (88%) emphasized the need for continuing education on molecular diagnostics. Eighty-four (92%) of the respondents regularly use PCR (conventional and/or real-time) for the detection of equine pathogens, and 80 (88%) commonly submit their samples to university/state veterinary laboratories. Blood, nasal swabs, and feces are the three equine specimens most commonly submitted for PCR analysis of Streptococcus equi, Lawsonia intracellularis, Neorickettsia risticii, equine herpesvirus 1/4, Rhodococcus equi, Sarcocystis neurona, and equine influenza virus. Diplomates reported costs associated with molecular diagnostics and unreliability of PCR as the most common limitations of PCR. Didactic training in veterinary curricula and during continuing-education opportunities continues to be necessary to produce veterinarians who have an understanding of the clinical applications of molecular diagnostics.     

Phylogeny, diversity, and species delimitation in some species of the Xiphinema americanum-group complex (Nematoda: Longidoridae), as inferred from nuclear and mitochondrial DNA sequences and morphology

During nematode surveys in southern Spain and Italy 14 populations of Xiphinema species tentatively identified as Xiphinema americanum-group were detected. Morphological and morphometrical studies identified three new species and six known Xiphinema americanum-group species, viz.: Xiphinema parabrevicolle n. sp., Xiphinema parapachydermum n. sp., Xiphinema paratenuicutis n. sp., Xiphinema duriense, Xiphinema incertum, Xiphinema opisthohysterum, Xiphinema pachtaicum, Xiphinema rivesi, and Xiphinema santos. The Xiphinema americanum-group is the most difficult Xiphinema species group for diagnosis since the morphology is very conservative and morphometric characters often overlap. This group includes vectors of several important plant pathogenic viruses that cause significant damage to a wide range of agricultural crops. Molecular characterisation of these species using D2-D3 expansion regions of 28S rRNA, 18S rRNA, ITS1-rRNA and the protein-coding mitochondrial gene, cytochrome oxidase c subunit 1 was carried out and maximum likelihood and Bayesian inference analysis were used to reconstruct phylogenetic relationships among these species and with other Xiphinema americanum-group species.     

Identification and functional expression of the mitochondrial pyruvate carrier

The transport of pyruvate, the end product of glycolysis, into mitochondria is an essential process that provides the organelle with a major oxidative fuel. Although the existence of a specific mitochondrial pyruvate carrier (MPC) has been anticipated, its molecular identity remained unknown. We report that MPC is a heterocomplex formed by two members of a family of previously uncharacterized membrane proteins that are conserved from yeast to mammals. Members of the MPC family were found in the inner mitochondrial membrane, and yeast mutants lacking MPC proteins showed severe defects in mitochondrial pyruvate uptake. Coexpression of mouse MPC1 and MPC2 in Lactococcus lactis promoted transport of pyruvate across the membrane. These observations firmly establish these proteins as essential components of the MPC.     

Attenuation of virulence of Lawsonia intracellularis after in vitro passages and its effects on the experimental reproduction of porcine proliferative enteropathy

Non-pathogenic Lawsonia intracellularis variants have been obtained through multiple passages in cell culture but there is no information regarding the number of passages necessary to attenuate a pathogenic isolate. The present study evaluated the susceptibility of pigs to L. intracellularis after 10, 20 and 40 passages in vitro. Three groups (six animals/group) were inoculated with pure culture of L. intracellularis on passage 10, 20 or 40 and one group with placebo. The animals were monitored for clinical signs, fecal shedding and serological IgG response during 28 days post-inoculation. Gross and histologic lesions and the level of infection based on the amount of L. intracellularis-specific antigen in the intestinal mucosa identified by immunohistochemistry were evaluated in two animals from each group on days 14, 21 and 28. Animals inoculated with passages 10 and 20 demonstrated proliferative lesions typical of porcine proliferative enteropathy associated with the presence of Lawsonia-specific antigen in the intestinal mucosa. Passage 40-inoculated pigs did not show proliferative lesions or presence of Lawsonia antigen at any time point throughout the study. Similar patterns of the fecal shedding were observed in passage 10 and 20-infected pigs but those infected with passage 40 shed for a short period. Serological IgG responses in passage 10 and 20-inoculated pigs were detected from day 14 post-infection but not at all in passage 40-inoculated animals. These results demonstrate attenuation of the virulence properties of L. intracellularis between 20 and 40 cell passages in vitro. This information will be valuable for design of future experimental models and for studying the mechanisms involved in the attenuation of L. intracellularis virulence.     

An update on canine coronaviruses: viral evolution and pathobiology

The emergence of human severe acute respiratory syndrome incited renewed interest in animal coronaviruses (CoVs) as potential agents of direct and indirect zoonoses. The reinforced epidemiological surveillance on CoVs has led to the identification of new viruses, genotypes, pathotypes and host variants in animals and humans. In dogs, a CoV associated with mild enteritis, canine coronavirus (CCoV), has been known since 1970s. CoV strains with different biological and genetic properties with respect to classical CCoV strains have been identified in dogs in the last few years, leading to a full reconsideration of the CoV-induced canine diseases. The genetic evolution of dog CoVs is paradigmatic of how CoVs evolve through accumulation of point mutations, insertions or deletions in the viral genome, that led to the emergence of new genotypes (CCoV type I), biotypes (pantropic CCoV) and host variants (canine respiratory coronavirus). This paper is a review of the current literature on the recent genetic evolution of CCoV and emergence of new CoVs in the dog. The significances of the newly acquired information for the canine health status and prophylaxis programmes are also discussed.     

Use of dried blood smears for detection of feline hemoplasmas using real-time polymerase chain reaction

The objective of the current study was to determine the sensitivity and specificity of real-time polymerase chain reaction (real-time PCR) for feline hemoplasmas when applied to DNA extracted from dried whole-blood smears in comparison to that for DNA extracted from liquid whole blood. Blood samples were collected into ethylenediamine tetra-acetic acid tubes from 305 cats with possible or suspected hemoplasmosis, and dried blood smears from each sample were prepared. DNA was extracted from blood smears and a 160-microl aliquot of each liquid blood sample by using a robotic extractor and was subjected to real-time PCR for feline glyceraldehyde-3-phosphate dehydrogenase (liquid blood), 18S ribosomal RNA (dried blood), and "Candidatus Mycoplasma haemominutum", Mycoplasma haemofelis, and "Candidatus Mycoplasma turicensis" DNA. When using the results for liquid whole blood as the gold standard, the sensitivity of each assay for "Ca. M. haemominutum", M. haemofelis, and "Ca. M. turicensis" was 49 of 66 (74%), 11 of 13 (85%), and 11 of 20 (55%), respectively. The specificity of each assay was 224 of 234 (96%), 287 of 287 (100%), and 280 of 280 (100%), respectively. When possible, liquid blood samples should be submitted for detection of feline hemoplasmas by using real-time PCR. The improved sensitivity of real-time PCR on blood smears for M. haemofelis compared with that of the other hemoplasma species may reflect the higher organism burdens associated with infection with this species.