Generation of equine TSLP-specific antibodies and their use for detection of TSLP produced by equine keratinocytes and leukocytes

Allergic horses react to innocuous environmental substances by activation of Th2 cells and production of allergen-specific IgE antibodies. The mechanisms leading to Th2 differentiation are not well understood. In humans and mice, epithelial cell-derived thymic stromal lymphopoietin (TSLP) plays a central role in this process. Little is known about equine TSLP (eqTSLP) and its role in allergic diseases and our current knowledge is limited to the assessment of TSLP mRNA expression. In order to be able to study eqTSLP at the protein level, the aim of the present study was to produce recombinant eqTSLP protein and generate TSLP-specific antibodies. EqTSLP was cloned from a skin biopsy sample from a horse with chronic urticaria and eqTSLP protein was expressed in E.coli and in mammalian cells. Recombinant proteins were designed to include C-terminal Histag, which allowed subsequent purification and detection by Histag-specific Ab. Polyclonal and monoclonal eqTSLP-specific Ab were generated after immunization of mice with E.coli-expressed TSLP. Their specificity was tested by western blotting and ELISA. In addition, a commercially available polyclonal human TSLP-specific antibody was tested for cross-reactivity with eqTSLP. Expression of TSLP protein was confirmed by western blotting using Histag-specific Ab. E.coli-expressed TSLP appears as a band of ～13 kDa, whereas mammalian cell-expressed TSLP showed several bands at 20-25 kDa, probably representing several glycosylation forms. Polyclonal and monoclonal antibodies generated in this study, as well as commercially available human TSLP-specific Ab reacted with both E.coli- and mammalian cell-expressed TSLP in western blotting and ELISA. A capture ELISA was established to quantitate TSLP in cell supernatants and validated using supernatants from primary equine keratinocytes and peripheral blood leukocytes (PBL). Increased TSLP concentrations were found after stimulation of keratinocytes with PMA+ionomycine and with Culicoides extract. Similarly, increased TSLP concentrations were detected in PBL after stimulation with ConA, Culicoides extract, or IgE cross-linking. In conclusion, recombinant TSLP proteins and TSLP-specific antibodies produced in this study will allow further studies of the role of TSLP in equine allergic diseases.     

Characterization of the inflammatory infiltrate and cytokine expression in the skin of horses with recurrent urticaria

Background – Recurrent urticaria (RU) is a common skin disease of horses, but little is known about its pathogenesis. Hypothesis/Objective – The aim of this study was to characterize the inflammatory cell infiltrate and cytokine expression pattern in the skin of horses with RU. Animals – Biopsies of lesional and nonlesional skin of horses with RU (n = 8) and of skin from healthy control horses (n = 8) were evaluated. Methods – The inflammatory cell infiltrate was analysed by routine histology. Immunohistochemistry was used to identify T cells (CD3), B cells (CD79), macrophages (MAC387) and mast cells (tryptase). Expression of T‐helper 2 cytokines (interleukins IL‐4, IL‐5 and IL‐13), a T‐helper 1 cytokine (interferon‐γ), IL‐4 receptor α and thymic stromal lymphopoietin was assessed by quantitative RT‐PCR. Results – In subepidermal lesional skin of RU‐affected horses, increased numbers of eosinophils (P ≤ 0.01), CD79‐positive (P ≤ 0.01), MAC387‐positive (P ≤ 0.01) and tryptase‐positive cells (P ≤ 0.05) were found compared with healthy horses. Subepidermal lesional skin of RU‐affected horses contained more eosinophils (P ≤ 0.05) and tryptase‐positive cells (P ≤ 0.05) compared with nonlesional skin. There was no significant difference in infiltrating cells between nonlesional skin and skin of healthy horses. Expression of IL‐4 (P ≤ 0.01), IL‐13 (P ≤ 0.05), thymic stromal lymphopoietin (P ≤ 0.05) and IL‐4 receptor α (P ≤ 0.05) was increased in lesional skin of RU‐affected horses compared with control horses. Expression of IL‐4 was higher (P ≤ 0.05) in lesional compared with nonlesional RU skin. Conclusions and clinical importance – Analysis of cytokine expression and inflammatory infiltrate suggests that T‐helper 2 cytokines, eosinophils, mast cells and presumptive macrophages play a role in the pathogenesis of equine RU.     

Assessment of an optimized dog-culling program in the dynamics of canine Leishmania transmission

In Brazil, zoonotic visceral leishmaniasis (ZVL) control programs based on the mass elimination of seropositive dogs have failed to reduce the number of leishmaniasis cases. However, these programs have been done under sub-optimal conditions. We studied a cohort of dogs in an urban area in Brazil to determine, whether a dog-culling program optimized with: (i) replacement of a relatively low-sensitivity indirect immune-fluorescent test on blood eluate by a more sensitive enzyme-linked immunosorbent assay on serum blood samples; (ii) shortening of the time interval from serodiagnosis to removal of dogs; (iii) screening a high proportion of the dog population could reduce the incidence of canine Leishmania infection (CLI). The study ran from December 1997 to July 2000, with four follow-up assessments performed at approximately 8-month intervals. All dogs seropositive for anti-Leishmania antibodies were promptly eliminated. A large number of new dogs immigrated to the study area throughout the study period. They comprised 43.8-49.8% of the cohort at each follow-up assessment, and upto 15% of them already had Leishmania infection. Overall, 42 news cases of CLI were identified, for a crude incidence rate of 11.8 cases per 100 dog-years (95% CI 8.6-15.6). In the first, second, third and fourth follow-up assessments the incidence rates were 8.2 (95% CI 3.0-17.9), 12.2 (95% CI 6.3-21.2), 16.4 (95% CI 8.5-28.6) and 13.6 (95% CI 7.1-23.8), respectively. There was no statistically significant change in these rates throughout the study period. Our results suggest that dog-culling programs do not reduce the incidence of CLI, even with an optimized intervention. Possible reasons for this failure include: currently available serologic methods lack sufficient sensitivity and/or specificity to accurately identify all infected dogs warranting removal in order to prevent Leishmania transmission; destroyed dogs are immediately replaced by susceptible puppies, and quite often, by already infected dogs; and other reservoirs may be involved in maintaining canine infection. Further efforts on ZVL control should be directed to developing new strategies or to testing control methods already in place with properly designed trials.     

Variations in acaricidal effect of wettable sulfur on Tetranychus urticae (Acari:Tetranychidae): effect of temperature,humidity and life stage

Under laboratory conditions, the acaricidal effect of wettable sulfur is influenced by climatic conditions and the stage of development of Tetranychus urticae. Its ovicidal effect results from the combined action of temperature and relative humidity (RH). Wettable sulfur becomes effective at 27.5 degrees C and 75% RH. Beyond this threshold, the acaricidal effect increases with rising temperature or humidity, to become complete at a temperature of 35 degrees C and 90% RH. Within the range of temperatures and humidities 20 degrees C/30% RH and 35 degrees C/90% RH the mortality of immatures (from protonymphs to teleiochrysalis) was total. Under similar experimental conditions, females usually died before the end of the experiment. Temperature and relative humidity increased the adulticidal potential of wettable sulfur. The fecundity of the sulfur-treated females and the viability of their progeny was found to depend on temperature and RH. According to the same climatic conditions, eggs were less susceptible than females, which were in turn less susceptible than juvenile stages.     

Advances in equine immunology: Havemeyer workshop reports from Santa Fe,New Mexico,and Hortobagy,Hungary

The horse has been human kind's most important partner throughout history. Similarly, in the field of immunology, many critical scientific advances have depended on the horse. Equine immunology today is an active and important field of study, with a focus on control of many common infectious diseases and immunopathologic conditions of broad comparative interest. In 2001 two major equine immunology workshops were held, in Santa Fe, USA, and in Hortobagy, Hungary, with major sponsorship from the Havemeyer Foundation. This report summarizes the scientific themes and foci of those meetings.     

Canine parvovirus 2c infection in central Portugal

Canine parvovirus (CPV) has been evolving, generating new genetic and antigenic variants throughout the world. This study was conducted to determine the types of CPV circulating in dogs in Figueira da Foz, Portugal. Thirty fecal samples, collected between 2006 and 2007 from dogs with clinical signs of CPV infection, were tested for CPV by a rapid, in-clinic, enzyme-linked immunosorbent assay (ELISA)/immunomigration test, by conventional real-time polymerase chain reaction (PCR), and by minor-groove binding TaqMan PCR. Of the 29 PCR-positive samples, 15 were identified as CPV-2b and 14 as CPV-2c. No CPV-2a was detected. The sensitivity of the ELISA test was 82.76% compared with the PCR assays. No significant associations were found between CPV type, clinical outcome, breed, vaccination status, or age.     

In vitro analysis of expression vectors for DNA vaccination of horses: the effect of a Kozak sequence

One of the prerequisite for developing DNA vaccines for horses are vectors that are efficiently expressed in horse cells.We have analysed the ectopic expression of the human serum albumin gene in primary horse cells from different tissues. The vectors used are of pcDNA and pUC origin and include the cytomegalovirus (CMV) promoter. The pUC vectors contain CMV intron A whereas the pcDNA vectors do not.Insertion of intron A diminished the expression from the pcDNA vectors whereas insertion of a Kozak sequence upstream of the gene in two types of pUC vectors increased significantly the in vitro expression in primary horse cells derived from skin, lung, duodenum and kidney.We report for the first time the significance of full consensus Kozak sequences for protein expression in horse cells in vitro.