Franco Guscetti

Aberrant chlamydial developmental forms in the gastrointestinal tract of pigs spontaneously and experimentally infected with Chlamydia suis.

The phenomenon of persistence is well known from in vitro studies, where it is associated with the production of aberrant bodies, but its occurrence in vivo is less well documented. The objective of this study was to search for aberrant bodies in intestinal tissues from pigs, describe their ultrastructure, and investigate the suitability of immunohistochemical staining for chlamydial heat shock protein 60 (cHSP60) to detect such forms. Intestinal tissues derived from pigs naturally and experimentally infected with Chlamydia (C.) suis were examined by immunohistochemistry, transmission electron microscopy and immunogold electron microscopy. The chlamydial species involved in the natural infection were determined using an Array Tube Microarray to C. suis and Chlamydophila abortus. Ultrastructurally, aberrant bodies were detected in the gut of both naturally and experimentally infected pigs. Immunogold electron microscopy showed that the aberrant bodies were labeled less strongly than the normal forms by antibodies against LPS and cHSP60 respectively. It was concluded that aberrant bodies occur in vivo in pigs and that the gnotobiotic pig model might be suitable for the study of chlamydial persistence in vivo. The antibody against cHSP60 does not appear to be suitable to specifically detect such forms.

A retrospective analysis of radiation therapy for the treatment of feline vaccine-associated sarcoma.

We retrospectively evaluated predictive prognostic factors in 73 cats with vaccine-associated sarcoma given postsurgical curative (n = 46, most with clean margins) or coarse fractionated radiotherapy (n = 27, most with either macroscopic disease or dirty margins). The former animals displayed a median survival of 43 months and a median progression free interval (PFI) of 37 months, the latter reached a median survival of 24 months and a median PFI of 10 months. In cats undergoing coarse fractionated therapy, factors predictive of a better outcome included lack of visible mass (n = 10) as opposed to macroscopic disease (n = 17, survival: 30 versus 7 months, P = 0.025; PFI: 20 versus 4 months, P = 0.01), adjuvant chemotherapy for gross disease (n = 5/17, survival: 29 versus 5 months, P = 0.04) and a smaller number of surgeries preceding radiation therapy (coeff = 0.41, P = 0.03). The Ki67 index was not predictive for survival. We concluded that postsurgical curative and coarse fractionated radiotherapy are effective legitimate options for managing vaccine-associated sarcomas.

Intestinal Chlamydia in Finishing Pigs

Gut and blood samples from 119 finishing pigs derived from 11 farms were collected during routine slaughter at an abattoir. Sections of formalin-fixed, paraffin-embedded tissues were labeled immunohistochemically using genus-specific, mouse monoclonal antibody against chlamydial lipopolysaccharide; goat polyclonal antiserum against the major outer membrane protein of Chlamydia trachomatis; and mouse monoclonal antibody against the ovine abortion subtype of C. psittaci. Gut samples from 33 of 111 (29.7%) individual pigs stained positive with the genus-specific monoclonal antibody, and of these 30 of 32 (93.7%) also reacted with the C. trachomatis-specific antiserum. Labeled inclusions were restricted to mature enterocytes of the large intestine in 33 of 111 cases. Infection of small intestinal enterocytes was noted in only one of 82 ileal samples. The blood samples were tested for antichlamydial antibodies by enzyme-linked immunosorbent assay (ELISA) and complement fixation test (CFT). With ELISA, 95 of the 115 sera tested (82.6%) yielded positive antichlamydial reactions. With CFT, 34 of the 119 sera tested (28.6%) were unequivocally positive (≥ 1: 10, 100% binding), and 10 (7.6%) yielded doubtful positive reactions (1: 10, 50-75% binding). Positive ELISA and CFT titers showed poor agreement (K = 0.112), whereas the agreement between positive findings by immunohistochemical labeling and CFT was fair (K = 0.205).

CHLAMYDIAE IN PORCINE ABORTION

Formalin-fixed, paraffin-embedded fetal livers and lungs from 139 cases of swine abortion were investigated retrospectively for chlamydiae by means of immunohistochemistry. Using a genus-specific antibody, chlamydial antigen was found in eight livers obtained from five (3.6%) abortion cases from different herds. All lung sections were negative. Chlamydiae were also labeled in five of the eight positive livers using a monoclonal antibody against immunotype 1 of Chlamydia psittaci; the remaining three livers were negative. No reactivity was seen using an antibody specific for C. trachomatis. Chlamydiae should be considered a cause of abortion in sows in Switzerland. Porcine abortigenic strains identified in this study differed immunologically from intestinal strains (known to be mainly C. trachomatis) but shared similarities with abortigenic chlamydiae of ruminants.

Experimental enteric infection of gnotobiotic piglets with Chlamydia suis strain S45

Enteric chlamydial infections of pigs with Chlamydia (C.) suis are frequent and often subclinical. The enteric pathogenicity of C. suis strain S45 was investigated in gnotobiotic piglets. Piglets from three litters (n=31) were inoculated with egg-grown chlamydiae at 2-3 days of age (n=17) or used as controls (n=14). They were observed for clinical signs, killed and necropsied sequentially at 2-13 days postinoculation (DPI). Feces were collected daily and investigated with an ELISA for chlamydial antigen. At necropsy, specimens were collected for histopathology and for immunohistochemical, PCR-based, and serological (complement fixation test, ELISA) detection of chlamydiae. Chlamydial replication and associated symptoms and lesions were observed from 2 to 13 DPI and were particularly pronounced within the first week PI. Clinical symptoms consisted of moderate-to-severe diarrhea, slight and transient anorexia, weakness and body weight loss. Immunohistochemistry and ELISA revealed that chlamydial replication was particularly marked at 2-4 DPI and primarily located in the small intestinal villus enterocytes. Further sites of replication included large intestinal enterocytes, the lamina propria and Tunica submucosa, and the mesenteric lymphnodes. Histopathological changes included moderate-to-severe villus atrophy with flattened enterocytes and focal villus tip erosions, and moderate mucosal inflammatory cell infiltrates and lymphangitis in the small intestine. PCR of spleen tissue and blood was mostly negative for chlamydiae, indicating that they did not substantially disseminate into the host up to 13 DPI. All sera were negative for anti-chlamydial antibodies. In conclusion, C. suis strain S45 elicited significant enteric disease and lesions in gnotobiotic piglets indicating its pathogenic potential for swine.

Clinical, Histologic, and Immunohistochemical Analyses of Feline Squamous Cell Carcinoma In Situ

Actinic keratosis (AK) and Bowenoid in situ carcinoma (BISC) are two distinct forms of in situ squamous cell carcinoma in felines. They usually occur on different locations and present with specific clinical and histologic features. However, in some cases, these diseases cannot be distinguished either clinically or histopathologically. The aim of the present study was to determine the accuracy of diagnosis based on clinical or histologic criteria alone, and whether immunohistochemistry for papillomavirus or p53 can improve the accuracy of diagnosis. A series of in situ squamous cell carcinoma cases (n = 45) were selected according to their location and initial histologic classification and subsequently classified as AK (n = 22) or BISC (n = 23) according to the clinical criteria and were reevaluated histologically by 2 dermatopathologists. All BISC cases and most of the AK cases (n = 15) were confirmed histologically. In 7 cases clinically classified as AK, this diagnosis was not unanimously confirmed histologically because of the presence of overlapping features. P53 immunoreactivity was observed in 11/14 (79%) confirmed AK cases and in 4/22 (18%) BISC cases, while papillomavirus antigen was not detected in any confirmed AK case but was detected in 11/23 (48%) BISC cases. It was concluded that BISC can usually be reliably diagnosed histologically. The histologic diagnosis of lesions clinically suggestive of AK might sometimes be difficult. Results of immunohistochemistry for p53 and papillomavirus antigen were supportive for a role of sun exposure and papillomavirus in the pathogenesis of AK and BISC, respectively.

Functional characterization of human proapoptotic molecules in yeast S. cerevisiae

The presence of a complete (BH1‐3) proapoptotic molecule is necessary for the induction of the intrinsic apoptotic cascade in mammalian cells. It is unclear, however, what distinct roles the members of the large family of BH3‐only proapoptotic molecules play in apoptosis. Although biochemical analysis of these molecules can characterize binding efficiencies of BH3 family members, the biologic consequences of these interactions are difficult to predict. We have, therefore, established three functional categories of BH3‐only human proapoptotic proteins based on their toxicity after expression in budding yeast: directly killing (tBid), sensitizing in Bax/Bcl‐2 expressing cells (Bad or Puma), and non‐toxic (BNip3, BNip3L, and Noxa). The mechanism of killing by the proapoptotic molecules in yeast, however, is not due to activation of the recently described yeast metacaspase MCA1.

Subacute Endotoxemia Induces Adipose Inflammation and Changes in Lipid and Lipoprotein Metabolism in Cats

Acute inflammation in humans is associated with transient insulin resistance (IR) and dyslipidemia. Chronic low-grade inflammation is a pathogenic component of IR and adipose tissue dysfunction in obesity-induced type 2 diabetes. Because feline diabetes closely resembles human type 2 diabetes, we studied whether lipopolysaccharide (LPS)-induced subacute inflammation, in the absence of obesity, is the potential primary cause of IR and metabolic disorders. Cats received increasing iv doses (10-1000 ng/kg(-1) · h(-1)) of LPS (n = 5) or saline (n = 5) for 10 d. Body temperature, proinflammatory and metabolic markers, and insulin sensitivity were measured daily. Tissue mRNA and protein expression were quantified on d 10. LPS infusion increased circulating and tissue markers of inflammation. Based on the homeostasis model assessment, endotoxemia induced transient IR and β-cell dysfunction. At the whole-body level, IR reverted after the 10-d treatment; however, tissue-specific indications of IR were observed, such as down-regulation of adipose glucose transporter 4, hepatic peroxisome proliferative activated receptor-γ1 and -2, and muscle insulin receptor substrate-1. In adipose tissue, increased hormone-sensitive lipase activity led to reduced adipocyte size, concomitant with increased plasma and hepatic triglyceride content and decreased total and high-density lipoprotein cholesterol levels. Prolonged LPS-induced inflammation caused acute IR, followed by long-lasting tissue-specific dysfunctions of lipid-, glucose-, and insulin metabolism-related targets; this ultimately resulted in dyslipidemia but not whole-body IR. Endotoxemia in cats may provide a promising model to study the cross talk between metabolic and inflammatory responses in the development of adipose tissue dysfunction and IR.

Luteal and placental function in the bitch: spatio-temporal changes in prolactin receptor (PRLr) expression at dioestrus, pregnancy and normal and induced parturition

BackgroundEndocrine mechanisms governing canine reproductive function remain still obscure. Progesterone (P4) of luteal origin is required for maintenance of pregnancy. Corpora lutea (CL) are gonadotrop-independent during the first third of dioestrus; afterwards prolactin (PRL) is the primary luteotropic factor. Interestingly, the increasing PRL levels are accompanied by decreasing P4 concentrations, thus luteal regression/luteolysis occurs in spite of an increased availability of gonadotropic support. PRL acts through its receptor (PRLr), the expression of which has not yet been thoroughly investigated at the molecular and cellular level in the dog.MethodsThe expression of PRLr was assessed in CL of non-pregnant dogs during the course of dioestrus (days 5, 15, 25, 35, 45, 65 post ovulation; p.o.) as well as in CL, the utero/placental compartments (Ut/Pl) and interplacental free polar zones (interplacental sites) from pregnant dogs during the pre-implantation, post-implantation and mid-gestation period of pregnancy and during the normal and antigestagen-induced luteolysis. Expression of PRLr was tested by Real Time PCR, immunohistochemistry and in situ hybridization.ResultsIn non-pregnant CL the PRLr expression was significantly upregulated at day 15 p.o. and decreased significantly afterwards, towards the end of dioestrus. CL of pregnancy showed elevated PRLr expression until mid gestation while prepartal downregulation was observed. Interestingly, placental but not interplacental expression of PRLr was strongly time-related; a significant upregulation was observed towards mid-gestation. Within the CL PRLr was localized to the luteal cells; in the Ut/Pl it was localized to the fetal trophoblast and epithelial cells of glandular chambers. Moreover, in mid-pregnant animals treated with an antigestagen, both the luteal and placental, but not the uterine PRLr were significantly downregulated.ConclusionsThe data presented suggest that the luteal provision of P4 in both pregnant and non-pregnant dogs may be regulated at the PRLr level. Furthermore, a role of PRL not only in maintaining the canine CL function but also in regulating the placental function is strongly suggested. A possible functional interrelationship between luteal P4 and placental and luteal PRLr expression also with respect to the prepartal luteolysis is implied.

THORACIC COMPUTED TOMOGRAPHY, ANGIOGRAPHIC COMPUTED TOMOGRAPHY, AND PATHOLOGY FINDINGS IN SIX CATS EXPERIMENTALLY INFECTED WITH AELUROSTRONGYLUS ABSTRUSUS

Aelurostrongylus abstrusus infection is common in endemic areas and may cause severe respiratory clinical signs. Computed tomography (CT) is an important tool to diagnose pulmonary disease, because it allows detection of small lesions and discrimination of superimposed structures. The purpose of this study was to characterize by CT and angiographic CT the pulmonary lesions in six cats before, and 48 and 81 days after inoculation with 100 or 800 A. abstrusus infective larvae. Histological examination of the accessory lung lobe was performed to determine the microscopic, pathomorphologic correlate of the CT findings. The predominant CT lesion consisted of multiple nodules of varying size distributed throughout the lungs, severity depending on infectious dose. The histological correlate of the nodular lesions was multifocal dense granulomatous to mixed inflammatory cell infiltrates, including eosinophils distributed in the parenchyma and obliterating the alveoli. Marked, multifocal, dose-dependent thickening of the bronchi and adjacent interstitial changes blurred the margins of the outer serosal surface of the bronchi and vessels. Histologically, this was due to peribronchial mixed cell inflammation. During the course of infection some of the nodular and peribronchial changes were replaced by areas of ground-glass opacity. In addition to providing detailed depiction of pulmonary lesions resulting from an infectious cause and clearly defining lesions with respect to time and severity of infection, CT allowed quantitative assessment of bronchial thickness and lymph node size during the course of disease. Findings indicated that CT characteristics of this disease are consistent with pathologic findings.