Widespread detection of antibodies to Leptospira in feral swine in the United States.

As feral swine continue to expand their geographical range and distribution across the United States, their involvement in crop damage, livestock predation, and pathogen transmission is likely to increase. Despite the relatively recent discovery of feral swine involvement in the aetiology of a variety of pathogens, their propensity to transmit and carry a wide variety of pathogens is disconcerting. We examined sera from 2055 feral swine for antibody presence to six serovars of Leptospira that can also infect humans, livestock or domestic animals. About 13% of all samples tested positive for at least one serovar, suggesting that Leptospira infection is common in feral swine. Further studies to identify the proportion of actively infected animals are needed to more fully understand the risk they pose.

Histology of AlphaCor skirts: evaluation of biointegration.

PURPOSE: To report histologic findings in 14 AlphaCor artificial corneas implanted during clinical trials and subsequently explanted from human subjects following complications, so as to evaluate biointegration within the device skirt. METHODS: Explants were fixed and sectioned in paraffin. Histologic findings related to the device skirt were compared with earlier histologic results from animal studies and correlated with clinical histories. RESULTS: Two devices had been removed due to complications related to the optic alone, 11 following stromal melting overlying the biointegratable sponge skirt and 1 due to a retroprosthetic membrane. All devices demonstrated normal skirt porosity. Biointegration was similar to that found in animal studies but qualitatively appeared reduced in the affected areas in patients with overlying stromal melting prior to explantation. Patients with a longer history of melting prior to explantation demonstrated presence of inflammatory cells around the device. CONCLUSIONS: Histologic findings of the AlphaCor skirt in humans are consistent with earlier animal studies. This study confirms that biointegration by host fibroblastic cells, with collagen deposition occurs after AlphaCor implantation in humans. In cases in which stromal melting had occurred, biointegration is seen to be reduced. On correlating preoperative clinical factors with biointegration observed histologically, preoperative vascularization appears not to be required for AlphaCor biointegration.

Cell viability and inflammatory response in hydrogel sponges implanted in the rabbit cornea.

In the quest for the development of a functional keratoprosthesis, the biocompatibility of the porous skirt material in the Chirila keratoprosthesis (KPro) was investigated. The population of live and dead cells within, and the inflammatory response to, a tissue-integrating poly(2-hydroxyethyl methacrylate) (PHEMA) sponge were studied. Samples of the hydrogel sponge were implanted in rabbit corneas and explanted at predetermined time points up to 12 weeks. The explanted sponges were subjected to cell viability assay using two types of fluoroprobes, 5-chloromethylfluorescein diacetate and ethidium homodimer-1. A semiquantitative analysis was performed to assess the number of dead cells within the sponge and in the area of corneal stroma proximal to the sponge. Five rabbits were used for each end point (2, 4 and 12 weeks). To investigate the inflammatory response to the sponge, immunocytochemistry, using specific antibodies to rabbit macrophages, enzyme histochemistry of chloroacetate esterase (to detect neutrophils) and transmission electron microscopy (TEM) were also employed at 24 h, 2, 4 and 12 weeks after implantation. Four weeks after implantation, fewer viable cells were observed in the sponge when compared to the 2-week implant. However, the proportion of viable cells increased dramatically by 12 weeks. The proportion of nonviable cells decreased gradually with time; central sponge contained 34+/-11 % dead cells after 2 weeks, and 15+/-4.3% after 12 weeks. The staining of inflammatory cells demonstrated the presence of macrophages and neutrophils up to 12 weeks after implantation. TEM confirmed the presence of these cell types and others. including eosinophils and myofibroblasts, as well as blood capillaries. The presence of a significant number of viable cells at each time point and the uniform reduction of the nonviable cell proportion with time suggests that the sponge is a conducive environment supporting a prolific, viable cellular colonization. Dead cells observed in the first instance indicate a normal injury pattern. However, the presence of a small but significant proportion of invading inflammatory cells 12 weeks after implantation confirms a characteristic pattern of wound healing within the sponges.

Keratoprostheses: advancing toward a true artificial cornea.

Keratoprosthesis surgery is carried out in very few centers. Elaborate surgical techniques and high complication rates limit the application of currently available keratoprostheses (KPros). However, the clinical need for an alternative to donor tissue has sparked considerable research interest in the development of new KPros. This paper charts the evolution of KPros from the earliest devices to those currently used, describes their drawbacks and discusses the specifications of an ideal device. Recent research focuses upon the use of porous polymers as the skirt component of core-and-skirt KPros in order to obtain improved biological integration of the prosthetic material. Developments in biomaterials technology make a KPro analogous to a donor corneal button an increasingly realistic goal. However, two particular problems still need to be addressed. First, it must be demonstrated that secure long-term fixation that is able to withstand trauma is achievable in a full-thickness artificial cornea. Second, an ideal artificial cornea for a wet eye requires an epithelialized surface, and this has yet to be achieved.

Correlation of histological findings with gadolinium enhanced MRI scans during healing of a PHEMA orbital implant in rabbits.

BACKGROUND/AIMS: To investigate a poly(2-hydroxyethyl methacrylate) (PHEMA) orbital implant with a spongy anterior hemisphere and a smooth gel posterior hemisphere, by histology correlated with magnetic resonance images. METHODS: Following enucleation, eight rabbits received PHEMA implants to which the muscles were directly sutured, and underwent gadolinium enhanced magnetic resonance imaging (MRI) from 3 to 52 weeks. After the rabbits were killed, the implants were removed, cut in a plane corresponding to the scan, and processed for light and electron microscopy. RESULTS: All eight rabbits retained their implant to the end of the study period without complications. The scans demonstrated muscle attachment to the anterior half of the implant, and enhancement was seen on injection of gadolinium chelate. Histology confirmed muscle attachment, and cellular and vascular ingrowth. Over time, a transformation from reactive inflammatory to relatively non-vascular scar tissue was seen within the implant. Calcium deposits in one implant were detected by imaging and histology. CONCLUSION: The implants are readily visualised on MRI. Muscle attachment and fibrovascular ingrowth into the anterior hemisphere are seen, while encapsulation of the posterior hemisphere is minimal. Histological findings confirm the progress of the healing response, with initial inflammation and marked vascularisation, developing later into quiescent scar tissue predominantly of fibroblasts.

Clinical results of implantation of the Chirila keratoprosthesis in rabbits.

AIMS/BACKGROUND: An ideal keratoprosthesis (KPro) would closely resemble a donor corneal button in terms of its surgical handling, optics, and capacity to heal with host tissue in order to avoid many of the complications associated with the KPros which are currently in clinical use. This study was carried out to assess the long term clinical outcomes on implantation of the core and skirt poly(2-hydroxyethyl methacrylate) KPro in animals. METHODS: 20 KPros were made and implanted as full thickness corneal replacements into rabbits and followed for up to 21 months to date. RESULTS: 80% of the prostheses have been retained, with a low incidence of complications such as cataract, glaucoma, and retroprosthetic membrane formation which are frequently associated with KPro surgery. CONCLUSIONS: KPros of this type may offer promise in the treatment of patients for whom penetrating keratoplasty with donor material carries a poor prognosis. Refinement of the KPro and further animal trials, including implantation into abnormal corneas, are however mandatory before human implantation could be planned.

Histologic evaluation during healing of hydrogel core-and-skirt keratoprostheses in the rabbit eye.

PURPOSE: We developed two models that are modifications of our original poly(2-hydroxyethyl methacrylate) (PHEMA) core-and-skirt keratoprosthesis. In these keratoprostheses, the mechanical strength of the skirt has been considerably increased with divinyl glycol (DVG) as a cross-linking agent during polymerization. In one (KPro I), methyl methacrylate (MMA) was added as comonomer to increase cell adhesion, and in the other (KPro II), HEMA was polymerized with DVG without comonomer. The aim of this study was to evaluate the process of healing and biocolonization and to ascertain whether KPro I demonstrates better ingrowth than the mechanically stronger KPro II, after implantation in rabbit eyes. METHODS: Ten rabbits were used for each model and studied at five predetermined end points up to 26 weeks. The device was implanted as a full-thickness keratoprosthesis covered with a conjunctival flap. RESULTS: Neither prosthesis demonstrated extrusion or retroprosthetic membrane formation. There was no significant difference between the two types of prosthesis with respect to tissue ingrowth and surrounding tissue melting. Histologically, inflammation was not severe, but calcification was seen in most specimens. Evidence of biodegradation of the prosthesis also was seen. CONCLUSION: In our original keratoprosthesis, fibrovascular invasion had occurred into the prosthetic skirt, but wound dehiscence and low mechanical strength resulted in an unfavorable outcome. In this series, the mechanical properties were improved, and KPro II was stronger than KPro I. Therefore KPro II would be the preferred polymer combination for surgical manipulation. However, biodegradation and calcification require further investigation into the degree and significance of these adverse reactions.

Assessment of anticollagenase treatments after insertion of a keratoprosthetic material in the rabbit cornea.

PURPOSE: This study was performed to evaluate the enzyme production in response to implantation of the hydrogel material used in the experimental Chirila keratoprosthesis (KPro) and to assess the effects of five topical drugs on enzyme production and activity. KPros may be extruded from the cornea as a result of tissue melting, a process that involves excessive enzyme activity. To reduce the possibility of implant loss for the hydrogel Chirila KPro, a number of antiinflammatory drugs that have been used to treat other corneal melting conditions were investigated for their effect on initial collagenase activity after the implantation of KPro material into the rabbit cornea. METHODS: Poly(2-hydroxyethyl methacrylate) sponge pieces were implanted into rabbit corneas. Prednisolone, tetracycline, medroxyprogesterone, acetylcysteine, and sodium citrate were assessed for effects on gelatinolytic activity and stromal collagenase [matrix metalloprotease-1 (MMP-1)] production in vivo and in vitro by using zymography and Western blotting techniques. RESULTS: Whereas all five anticollagenase drugs were effective in reducing gelatinolytic activity in vitro, many were ineffective in vivo. However, medroxyprogesterone caused a reduction of gelatinolytic activity in vivo. The amount of MMP-1, as measured by immunoblotting, also was reduced by medroxyprogesterone treatment when compared with untreated controls. An increase in the apparent molecular weight of MMP-1 in operated corneas appears to be the result of the association of MMP-1 with collagen fragments resulting from the surgical trauma. CONCLUSION: This study indicates that topical medroxyprogesterone may be a useful adjunctive therapy after prosthokeratoplasty.

Implantation of PHEMA keratoprostheses after alkali burns in rabbit eyes.

PURPOSE: We have previously examined histologically the healing of a PHEMA core-and-skirt keratoprosthesis (the Chirila KPro) as a full-thickness implant in healthy animal corneas. The present study was carried out to determine whether a diseased cornea could also generate biocolonization of the skirt region of a KPro. METHODS: Ten KPros were placed as full-thickness corneal implants under conjunctival flaps in 10 alkali-burned rabbit corneas. Histological findings at intervals from 2 weeks to 6 months postoperatively were compared with earlier findings in 10 rabbits that had received identical KPros without prior alkali injury. RESULTS: Despite severe corneal injury and the reduced keratocyte population present, there were no clinically detected complications in 60%. Histological findings established that, compared with healthy host tissue, skirt biocolonization and KPro-cornea healing after an alkali burn were impaired, with evidence of epithelial downgrowth in 40%. One animal required euthanasia earlier than the planned end point, but no KPro extrusions occurred. CONCLUSION: Biocolonization of a KPro skirt is reduced but not prevented in an alkali-induced corneal inflammation model. Although no extrusions occurred, close follow-up and anticollagenolytic medication would be required to minimize the complication rate.

Comparison of microbiologic culture, an enzyme-linked immunosorbent assay, and determination of somatic cell count for diagnosing Staphylococcus aureus mastitis in dairy cows.

Results of using microbiologic culture of a single milk sample, determination of somatic cell count (SCC), an ELISA, and a combination of determination of SCC and ELISA to diagnose Staphylococcus aureus mastitis in dairy cattle were compared. Cows were considered to have S aureus intramammary infections if microbiologic culture of at least 2 of 3 consecutive sets of milk samples yielded growth of the organism. Data were analyzed from milk samples collected over a 4-month period from 185 cows in 5 herds. Sensitivity, specificity, and likelihood ratio of a positive test result for microbiologic culture of a single milk sample were 93%, 99%, and 93.0, respectively. Sensitivity, specificity, and likelihood ratio of a positive test result for ELISA were 69%, 61%, and 1.8, respectively, and for determination of SCC, they were 79%, 72%, and 2.9, respectively. Combination of determination of SCC and ELISA had sensitivity, specificity, and likelihood ratio of a positive test result of 80%, 62%, and 3.4, respectively. Results from microbiologic culture of consecutive milk samples were more consistent than results of ELISA performed on consecutive samples. These data suggest that microbiologic culture of a single milk sample is the best of the 3 tests studied for diagnosing S aureus intramammary infection.

Pseudomonas mastitis: difficulties in detection and elimination from contaminated wash-water systems.

Histories of 4 dairy herds with increased incidence of Pseudomonas mastitis associated with contaminated wash hoses in milking parlors are described. Problems of detection and elimination of the organism from contaminated water sources and the inadequacy of iodide germicides in eliminating Pseudomonas are discussed. In problem herds, greater numbers of organisms often are found in the water left standing in the wash hoses between milkings. Thus, flushing of hoses before their use in the milking process may be beneficial in reducing exposure of the cows to the organism.

The origins of the artificial cornea: Pellier de Quengsy and his contribution to the modern concept of keratoprosthesis.

There has been little recognition of the French ophthalmologist Guillaume Pellier de Quengsy and his contribution to the problem of artificial cornea (keratoprosthesis). This fact that he was the first to propose such a device was seldom acknowledged, and usually as a secondary reference. Based on the examination of original texts (1789), this study demonstrates that Pellier not only proposed an essentially correct keratoprosthesis, but also suggested a porous prosthetic skirt, a revolutionary concept which is currently fundamental to artificial cornea research.

Effects of neurokinins on ion transport and sulfated macromolecule release in the isolated ferret trachea.

The secretory actions of substance P (SP) were studied by examining its effects on ion transport and sulfated macromolecule release in the isolated ferret trachea. Responses to serosally administered SP were unmasked by thiorphan, suggesting that SP administered by this route was degraded by neutral endopeptidase. Therefore, the serosal actions of neurokinins and neurokinin analogues were studied in tissues pretreated with thiorphan. In thiorphan-treated tissues, SP dose-dependently increased the short-circuit current (Isc) with a maximum increase of 0.50 +/- 0.07 mueq/cm2.h above baseline. The SP-induced increase in Isc was inhibited by bumetanide but not by amiloride, suggesting the involvement of electrogenic Cl- secretion. The direct measurement of ion fluxes, however, failed to detect a significant unilateral movement of Cl- from the serosa to mucosa, probably due to a relatively small and transient increase in Isc produced by SP. Instead, SP (10(-6) M) induced large, electroneutral secretion of Na+ and Cl-. In addition, SP stimulated sulfated macromolecule output. The rank order of potency in increasing both Isc and sulfated macromolecule release among neurokinins and their analogues was SP = SP-methyl ester greater than neurokinin A greater than neurokinin B greater than senktide. These findings indicate that the secretory responses to SP are probably mediated by NK-1 receptors.

Altered interleukin-2 production by lymphocyte populations from bovine leukemia virus-infected cattle.

The effects of bovine leukosis virus (BLV) on the phenotypic and functional characteristics of peripheral blood mononuclear cells were investigated. Whole blood differentials showed that persistently lymphocytotic (BLV+PL) dairy cattle had more lymphocytes and fewer neutrophils than the aleukemic seropositive (BLV+AL) or seronegative (BLV-) animals. Flow cytometric analyses of peripheral blood mononuclear cells indicated that the BLV+PL animals had more B lymphocytes, with a concomitant decrease in CD2 positive cells when compared with the BLV- group. Mononuclear cells from the BLV+AL animals also had fewer CD2 positive cells, but no differences in B lymphocytes were observed when compared with BLV- cattle. Peripheral blood mononuclear cells were used in blastogenesis assays to assess the functional ability of lymphocytes. Lymphocytes from BLV+PL animals had lower proliferative responses to concanavalin A and pokeweed mitogen when compared with cells from the BLV- or BLV+AL groups. The level of spontaneous blastogenesis in the absence of mitogenic stimulation was high for lymphocytes obtained from BLV+AL cattle. Cultures of lymphocytes obtained from BLV+PL animals produced greater amounts of interleukin-2 (IL-2) than BLV+AL and BLV- groups, although no differences were observed in the expression of IL-2 receptors. The development of uncontrolled lymphocytosis in BLV-infected cattle may result from an altered responsiveness to IL-2-regulated B-lymphocyte proliferation.

Interactions between Pseudomonas aeruginosa and iodophor germicides in milking parlor udder wash water systems.

In a field study of 29 dairy farms, Pseudomonas aeruginosa was isolated more frequently (P = 0.05) from milking parlor udder wash water systems containing iodophor germicides than from those with no germicide. Most available iodine (AI2) concentrations were below the recommended level of 25 ppm (25 microgram/ml). Rubber and polyvinyl chloride hoses caused rapid decreases in the AI2 concentrations of 25 ppm iodophor solutions. AI2 dropped from 25 ppm to 6 ppm or less in 240 min for solutions contained in either polyvinyl chloride or rubber, compared with solutions in glass, which were unchanged in 240 min. Addition of inactivated iodophor solution to aqueous cultures resulted in significantly higher (P less than 0.05) numbers of P. aeruginosa at 10 and 24 h postinoculation. P. aeruginosa was grown in polyvinyl chloride tubing and exposed twice daily to 0, 10, or 25 ppm of AI2. None of the exposure concentrations eliminated the bacteria from the hoses, and bacterial numbers were not significantly different in hoses exposed to 0 and 10 ppm by the eighth treatment day. Bacteria taken from the water in these hoses were exposed to different concentrations of iodophor solution. Iodophor concentrations which will kill 50% of P. aeruginosa cultures previously exposed to 0, 10, and 25 ppm of AI2 were predicted to be 3.0, 11.8, and 20.8 ppm, respectively.

Effects of selenium status on bovine mononuclear cell function.

The effects of selenium (Se) status on the distribution and functional capabilities of bovine peripheral blood mononuclear cells (PBMC) were examined. No differences in proliferative responses to mitogens, interleukin (IL)-2 production, IL-2 receptor expression, or lymphocyte trafficking patterns were observed between PBMC from dairy cows maintained on Se adequate or Se deficient diets. In contrast, Se deficiency significantly reduced the numbers of circulating monocytes when compared to cows fed a Se adequate diet. Differences in the number of circulating monocytes had no apparent effect on the accessary functions of these cells with respect to antigen presentation and IL-2 production. These results suggest that the effect of Se-modified diets on bovine defense mechanisms is not mediated by the ability of peripheral blood lymphocytes to respond to antigen and mature into immunocompetent cells.

Development of a poly(2-hydroxyethyl methacrylate) orbital implant allowing direct muscle attachment and tissue ingrowth.

PURPOSE: To develop a poly(2-hydroxyethyl methacrylate) orbital implant that allows tissue ingrowth and direct muscle attachment to minimize the risk of extrusion and to enhance cosmesis. METHODS: Assessment of clinical outcomes and histologic findings after implantation of 18 prototype prostheses into rabbits. The implants were not wrapped with other tissues or materials. RESULTS: One case of infection was observed but there were no extrusions, with up to 21 months follow-up. Biocolonization was confirmed histologically. Good movement was observed when a cosmetic shell was fitted. CONCLUSIONS: The prototype prosthesis appears promising, with particular advantages being the direct attachment of extraocular muscles, good cosmesis and movement, and a low complication rate in this pilot study.

Synthesis, physical characterization, and biological performance of sequential homointerpenetrating polymer network sponges based on poly(2-hydroxyethyl methacrylate).

A limitation in the use of hydrophilic poly(2-hydroxyethyl methacrylate) (PHEMA) sponges as implantable devices is their inherently poor mechanical strength. This precludes proper surgical manipulation, especially in the eye where the size of the implant is usually small. In this study a new method was developed to produce mechanically stronger PHEMA sponges. Sequential homointerpenetrating polymer network (homo-IPN) sponges were made by using HEMA as the precursor for generating both the first network and the successive interpenetrated networks. Following the formation of network I, the sponge was squeezed to remove the interstitial water, soaked in the second monomer (also HEMA), and squeezed again to remove the excess monomer from the pores before being subjected to the second polymerization leading to the formation of network II. Two two-component IPN sponges (K2 and K4) with increasing HEMA content in the network II and a three-component IPN sponge (K3) were produced, and their properties were compared to those of a homopolymer PHEMA sponge (control). Apart from elongation, the tensile properties were all significantly enhanced in the IPN sponges; the water content was the same as in the control sponge, except for sponge K4, which was lower. Light microscopy revealed similar pore morphologies of the control and IPN sponges K2 and K3, and the majority of the pores were around 25 microm. Sponge K4 displayed smaller pores of around 10 microm. Cellular invasion into the sponges was examined in vitro (incubation with 3T3 fibroblasts) and in vivo (implantation in rabbit corneas). Although the in vitro assay detected a change in the cell behavior in the early stage of invasion, which was probably due to the formation of IPNs, such changes were not reflected in the longer term in vivo experiment. There was a proper integration of sponges K2 and K3 with the corneal stroma, but much less cellular invasion and no neovascularization in sponge K4. We concluded that IPN formation is a valid method to enhance the strength of PHEMA sponges, provided that the content of HEMA in the successive networks is not too high.

Calcification of poly(2-hydroxyethyl methacrylate) hydrogel sponges implanted in the rabbit cornea: a 3-month study.

Poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels have been used in the past as ocular implants. In a recent development, PHEMA sponges have shown suitable properties as materials for the peripheral component of an artificial cornea (keratoprosthesis). However, the propensity of PHEMA to calcify could threaten the long-term stability of the implanted devices. In an attempt to improve the understanding of the calcification mechanism, the dynamics, extent, and nature of calcified deposits within PHEMA sponges implanted in the cornea were investigated in this study, and the possible correlation between necrosis of cells and calcification was critically examined. Samples of a PHEMA sponge were implanted in rabbit corneas and explanted at predetermined time points (2, 4, and 12 weeks). The samples were examined by microscopy (light, transmission, scanning) and energy dispersive analysis of X-rays. Histological assessment and semiquantitative analysis of the amount of calcium deposited was performed using image analysis. An in vitro experiment was also performed by incubating sponge samples for 2 weeks in a solution of calcium and phosphate ions at a ratio similar to that in hydroxyapatite, in the absence of cells. Calcification was not seen in the 2- and 4-week explants, however, small deposits were detected in two of the 12-week explants, both within and on the sponge's constituent polymer particles. The deposit volumes represented 0.094% and 0.21%, respectively, of the total sponge volumes. Calcium deposits were present in large amounts both within the constituent polymer particles and on the surface of the sponges incubated in the abiotic calcifying solution. Cooperative mechanisms are suggested for the calcification of PHEMA sponges in vivo. The initial event may occur at a molecular level, when plasma proteins are adsorbed onto the polymer surface and bound through chelation to the calcium ions present in the medium. After their natural degradation, these structures may act as nucleation sites for calcium phosphate crystallization. Concurrently, the calcium ions can diffuse into the hydrogel particles and then the spontaneous precipitation of calcium phosphate may be caused by supersaturation due to the lower content of water in polymer, an effect which is likely predominant in vitro. The second event is the recruitment of phagocytic cells to clear calcium debris. Degeneration of these cells may then form nucleation sites for secondary calcification.