Pasteurella multocida toxin type D serological assay as an alternative to the toxin neutralisation lethality test in mice.

The objective of this work was to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Pasteurella multocida toxin type D, that correlated to a mouse lethality test. Currently, the mouse lethality test is one of several tests used world-wide to evaluate serological responses in animals immunised with vaccines containing toxoids. The mouse lethality test involves injecting mice with a mixture of toxin and test serum sample (from animals that have been vaccinated with a toxoid), and then determining antibody titre of the test serum from the number of mice that survive. Thus, the titre calculated is based on the neutralising activity of the test serum. The mouse lethality test requires large numbers of animals and causes severe distress to the animals. Organisations world-wide are working towards alternatives to animals in the development and control of biological products for human and veterinary use. Additionally, the mouse lethality test is labour-intensive, costly and lacks robustness and may be difficult to reproduce between different technicians. We have developed a double sandwich ELISA to measure anti- P. multocida toxoid type D antibodies in swine serum. Sera from swine immunised with vaccines containing type D toxoid showed good correlation to the mouse lethality assay (Spearman analysis=0.94 and Pearson analysis=0.84). When compared to the mouse lethality test, titres obtained using the ELISA format had higher correlation with protective immunity (i.e., lower turbinate atrophy) following challenge with virulent P. multocida. The ELISA assay is more robust, reproducible and costs less than the mouse lethality assay; and it complements efforts to reduce the use of animals in testing.

Mohs micrographic surgery for the treatment of spindle cell tumors of the skin.

BACKGROUND: Because of the uncommon nature of dermal spindle cell tumors, the effectiveness of various treatment modalities is difficult to assess. OBJECTIVE: Our purpose was to measure the effectiveness of treating dermatofibrosarcoma protuberans, atypical fibroxanthoma, malignant fibrous histiocytoma, and leiomyosarcoma by means of Mohs micrographic surgery (MMS). In addition, we attempted to determine whether MMS is useful in treating dermal spindle cell tumors when no definitive histopathologic diagnosis can be rendered. METHODS: In a retrospective chart review, demographic data, tumor data, treatment characteristics, recurrence, and follow-up data were tabulated. RESULTS: The recurrence rate for dermatofibrosarcoma protuberans treated by MMS was 3.0%, for atypical fibroxanthoma was 6.9%, for malignant fibrous histiocytoma was 43%, and for leiomyosarcoma was 14%. The recurrence rate for spindle cell tumors not otherwise specified was 0%. CONCLUSION: These data establish the effectiveness of MMS in the treatment of dermal spindle cell tumors, including those for which no definitive histopathologic diagnosis can be rendered.

Latex sensitivity in a macaque (Macaca mulatta).

UNLABELLED: BACKGROUND AND HISTORY: An adult Macaca mulatta was examined because of a history of multiple episodes of conjunctivitis and an acute, pruritic, dermatitic eruption that affected the axillary and inguinal regions, forearms, thorax, and neck. METHODS AND RESULTS: Results of corneal staining, examination of skin scrapings and feces, fungal culture, CBC, and a thyroid profile (thyroxine/triiodothyronine concentrations) were negative or normal, with the exception of eosinophilia (1,040/mm3). Examination of a punch biopsy specimen of the skin indicated chronic, nonsuppurative eosinophilic dermatitis. Skin patch testing against 25 contact allergens was negative for a delayed-type hypersensitivity reaction. Allergen-specific IgE testing, using six monkey chow additives, also yielded negative results, but testing against latex revealed a strong positive result (0.74 KU/L) consistent with a latex allergy. A skin prick test performed by use of a latex supernatant revealed significant inflammation at the latex site at 72 h and one week. Vinyl gloves were substituted for latex gloves, and that resulted in a marked decrease in erythema, pruritus, and lichenification with no flares of dermatitis for four years. Repeat skin biopsy fourteen weeks after the original biopsy revealed normal epidermis; however, mild chronic active nonsuppurative, perifolliculitis persisted. CONCLUSION: Latex can induce allergic dermatitis in nonhuman primates and should be included in the differen tial diagnosis for atopic dermatitis.

A single postoperative application of nitroglycerin ointment does not increase survival of cutaneous flaps and grafts.

BACKGROUND: Nitroglycerin is a vasodilator that has been reported to improve cutaneous flap and graft survival. It has not been tested in controlled studies. OBJECTIVE: We designed our study to test the effectiveness of a single postoperative application of nitroglycerin on flap and graft survival. METHODS: Eighty-eight surgical repairs received topical nitroglycerin and 85 received control ointment (polysporin). Treatment sites were evaluated on postoperative day 7 and assigned a percentage of surface area survival. RESULTS: There was no significant difference in the complication rate of flaps and grafts treated with nitroglycerin (12.5%) compared with those treated with control ointment (8.4%) (P = .244). Subset analysis of flaps as a group and grafts as a group were not meaningful because the complication rates were so low. CONCLUSION: There is no survival increase of flaps and grafts treated with a single application of nitroglycerin ointment.

Postirradiation morphea of the breast presentation of two cases and review of the literature.

The advent of radiation therapy as a common modality in the treatment and palliation of breast cancer has led to the observation of morphea developing months to years after supervoltage radiation therapy, in and around the site of treatment. We report 2 new cases of morphea at the site of previous supervoltage radiation therapy for breast cancer. The time period between irradiation and onset of morphea in our 2 patients were an atypically long 6.5 years and 32 years, the latter being the longest reported such interval. With conservative treatment, the inflammatory component of the lesions resolved over an approximately 1-year period, leaving residual sclerosis. These patients are compared to those previously reported in the medical literature so as to summarize the range of clinical presentation and course. Recognition of postirradiation morphea is important in distinguishing it from infectious cellulitis, recurrent carcinoma, metastatic carcinoma or development of a second primary carcinoma.

Morphine alteration of histamine release in vivo.

Studies were performed to evaluate the effects of either acute or chronic morphine exposure on histamine release in vivo and supporting studies in vitro. In order to effectively assess histamine release in swine, studies were undertaken to evaluate the effectiveness of compound 48/80 as an intradermal skin test and determine its ability to release histamine in swine cells. Compound 48/80 skin testing was found to be a useful measure of histamine release in swine as evidenced by dose dependent wheal and flare reaction in vivo and histamine release from swine cells in vitro. Acute effects of morphine were determined on swine administered a single injection of morphine alkaloid. Skin tests using intradermal compound 48/80 and histamine, were performed using compound 48/80 both prior to, and 24 h following initiation of morphine treatment. Morphine tolerant swine were subjected to in vivo skin tests and the resulting wheal and flare responses measured. In select swine skin samples from the test sites were measured for mast cell numbers. Swine dermal mast cells were found to release histamine in a dose dependent manner upon compound 48/80 exposure. Both acute and chronic morphine-treated swine had significantly depressed responses to compound 48/80, however this difference was not due alteration in mast cell number or morphology, and skin responsiveness to histamine remained intact.

Comparison of serum responses in swine after vaccination and challenge exposure with Actinobacillus pleuropneumoniae serotype 1.

Clinical trials have shown that currently available commercial vaccines against porcine pleuropneumonia provide inconsistent, serotype-specific protection from the disease. Recovery from naturally acquired infection, however, provides solid, serotype cross-protective immunity. We examined various serum responses of pigs receiving 1 of 4 commercial vaccines or a cell extract, and compared the serologic responses of these pigs after challenge exposure with virulent Actinobacillus pleuropneumoniae serotype 1. Evaluation of serum included complement-mediated killing, opsonizing capacity, IgG titers to whole organisms, and cytotoxin neutralization titers. Pigs that received the cell extract had fewer clinical signs of pleuropneumonia than pigs in other vaccinated groups, and also were significantly (P < 0.05) better protected from development of lung lesions and death. Such vaccinates were the only pigs that developed significant (P < 0.05) serum antibody titers (ie, protective immune response) to whole-cell antigens and to cytotoxin.

The importance of secreted virulence factors in Actinobacillus pleuropneumoniae bacterin preparation: a comparison.

Current bacterins provide only partial protection against morbidity and mortality in swine following infection by Actinobacillus pleuropneumoniae. We compared the efficacy of a cell-free concentrate from mid-log phase growth cultures of Actinobacillus pleuropneumoniae (APP) serotype 1 to four commercial bacterins. This cell-free preparation contained carbohydrate, endotoxin, and protein, and had hemolytic and cytotoxic activity. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis analysis indicated the presence of one major 110,000-molecular-weight protein. This protein band also stained by the periodic acid Schiff method, indicating the presence of carbohydrate. Cell-free concentrates of APP serotypes 5 and 7 had identical profiles following electrophoresis and staining with either Coomassie blue for protein or Schiff reagent for carbohydrate. Lipopolysaccharide profiles for the cell-free concentrates of serotypes 1 and 5 were semi-rough while the LPS profile for serotype 7 was smooth. Five A. pleuropneumoniae-free SPF pigs per group were vaccinated on days 0 and 21 with cell-free concentrate of serotype 1 plus adjuvant, or one of four commercial bacterins according to the manufacturer's directions. Control pigs were vaccinated with PBS mixed with adjuvant. All pigs were challenged intranasally on day 35 with serotype 1 and necropsied on day 50. Protection was greatest in the cell-free concentrate group, as compared with all other groups, in that no deaths occurred, clinical scores were less severe, and percent lung affected was significantly reduced (P < 0.05). In addition, whole-cell ELISA titers were significantly increased (P < 0.05) postvaccination in the cell-free concentrate group, and postvaccination and postchallenge sera neutralized the hemolytic activity of the cell-free concentrate from serotypes 1 and 5 (P < 0.05), as compared with all other groups. No serum neutralization to the hemolysin of serotype 7 was observed. Immunoblot analysis using antisera derived from gnotobiotic pigs indicated that the cell-free vaccine generated a response that was identical to the response observed following live challenge. Similar, but not identical, responses were observed when antisera generated against the bacterins was used. This study indicates that an acellular vaccine containing multiple virulence factors can provide complete protection from mortality and significantly reduced morbidity to homologous challenge.

Cloning, sequencing and regulation of an mRNA encoding porcine interleukin-1 beta.

Interleukin 1 (IL-1) is a cytokine that mediates a variety of immunological responses. We report here the isolation of a porcine IL-1 beta cDNA from an alveolar macrophage library. The complete cDNA sequence is 1458 bp in length and has an open reading frame of 803 bp, encoding a 267-amino-acid (aa) protein with an approximate molecular size of 31 kDa. The porcine IL-1 beta cDNA sequence shows homologies of 82.0, 81.0, 74.0, and 74.0% with the bovine, sheep, human, and murine sequences in the protein coding regions, respectively. Northern blot analysis reveals a single 1.7-kb mRNA species that is differentially regulated by lipopolysaccharide, phorbol myristate acetate, and tumor necrosis factor but is not regulated by human recombinant IL-1. Porcine IL-1 beta is down-regulated by dexamethasone, polymyxin B, and IL-4 in a fashion kinetically similar to the other reported species.

Actinobacillus pleuropneumoniae-induced thymic lesions in mice and pigs.

Actinobacillus pleuropneumoniae produces several hemolysins/cytotoxins that may be important in the pathogenesis of acute lesions. Little is known, however, about the role of these virulence factors in chronic disease or the carrier state. We investigated the effects of live bacterial infection and transthoracic injection of a sterile culture supernatant on primary lymphoid organs and lymphocyte populations. Transthoracic inoculation of mice or intranasal inoculation of pigs with virulent A. pleuropneumoniae serotypes 1 and 7 induced thymic cortical lymphoid necrosis. These lesions were reproduced in mice by transthoracic injection of a concentrated sterile culture supernatant. The cytotoxic effect of this culture supernatant was also demonstrated in vitro by using a tetrazolium dye reduction assay. Both porcine and murine thymic lymphocytes as well as splenic T lymphocytes were susceptible to the toxin. Porcine convalescent serum, but not preimmune serum, prevented thymic lesions and neutralized the in vitro cytotoxic effect of the culture supernatant on murine thymic lymphocytes. Thymic lesions also were reproduced in mice by using purified lipopolysaccharide (LPS) from Escherichia coli O111:B4; however, LPS had no in vitro cytotoxic effect on either porcine or murine thymic lymphocytes. These results suggest that secreted A. pleuropneumoniae toxin(s) is capable of affecting host T-lymphocyte populations and may affect host immune function.

Efficacy of a cell extract from Actinobacillus (Haemophilus) pleuropneumoniae serotype 1 against disease in swine.

We partially characterized a cell extract (CE) from Actinobacillus pleuropneumoniae serotype 1 and used the CE to test the efficacy of secreted proteins against disease. Secreted products from 4-h culture supernatants were precipitated with 20% polyethylene glycol. Analysis of the CE indicated the presence of protein, endotoxin, and carbohydrate. Hemolytic activity to bovine erythrocytes and cytotoxic activity to porcine mononuclear leukocytes was also demonstrated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the CE from a 4-h culture showed a major band at 110 kilodaltons (kDa), while a CE of a 26-h culture indicated the presence of a number of additional proteins, including the 110-kDa protein. The 110-kDa protein was also identified as a glycoprotein by periodic acid-Schiff and silver staining. A single band precipitated against convalescent-phase pig antiserum when the polyethylene glycol precipitate was used in an Ouchterlony plate. Vaccination with CE conferred greater protection against challenge with the homologous serotype than either a commercial bacterin or an outer membrane protein vaccine. Hemolysin-neutralizing titers were higher both pre- and postchallenge in the group vaccinated with the CE compared with in all other groups. We believe that this demonstrates the importance of secreted factors in protection against disease and suggests that the 110-kDa protein is an important immunogen.

Temperature-Sensitive, Photosynthesis-Deficient Mutants of Chlamydomonas reinhardtii.

Mutants of the unicellular, green alga Chlamydomonas reinhardtii were recovered by screening for the absence of photoautotrophic growth at 35 degrees C. Whereas nonconditional mutants required acetate for growth at both 25 and 35 degrees C, the conditional mutants have normal photoautotrophic growth at 25 degrees C. The conditional mutants consisted of two classes: (a) Temperature-sensitive mutants died under all growth conditions at 35 degrees C, but (b) temperature-sensitive, acetate-requiring mutants were capable of heterotrophic growth at 35 degrees C when supplied with acetate in the dark. The majority of mutants within the latter of these two classes had defects in photosynthetic functions. These defects included altered pigmentation, reduced whole-chain electron-transport activity, reduced ribulosebis-phosphate carboxylase activity, or pleiotropic alterations in a number of these photosynthetic components. Both nuclear and chloroplast mutants were identified, and a correlation between light-sensitive and photosynthesis-deficient phenotypes was observed.