An optimized assay for detecting Encephalitozoon intestinalis and Enterocytozoon bieneusi in dairy calf feces using polymerase chain reaction technology.

The purpose of this study was to optimize primary and nested polymerase chain reaction (PCR) assays for detecting the microsporidia Encephalitozoon intestinalis and Enterocytozoon bieneusi in fecal samples from dairy calves. PCR for these microsporidia were compared to immunofluorescence assays (IFA) based on commercially available monoclonal antibodies specific for outer wall proteins of Enc. intestinalis or Ent. bieneusi. Fecal samples were collected from 15 dairy calves and processed by molecular sieving followed by salt floatation to recover Enc. intestinalis and Ent. bieneusi spores. An aliquot of the final supernatant was applied to glass slides for IFA testing; another aliquot was extracted for total DNA using a QIAamp Stool Mini-Kit for primary and nested Enc. intestinalis- and Ent. bieneusi-specific PCR analysis. Internal standards were generated for both Enc. intestinalis and Ent. bieneusi PCR assays to control for false negative reactions due to the presence of inhibitors commonly found in fecal samples. Using the commercial MicrosporIFA (Waterborne, Inc.) as the gold standard, the optimized Enc. intestinalis PCR method provided 85.7% sensitivity and 100% specificity with a kappa value = 0.865. Likewise, using the commercial BienusiGlo IFA (Waterborne, Inc.) as the gold standard, the optimized Ent. bieneusi PCR method provided 83.3% sensitivity and 100% specificity with a kappa value = 0.857. Sequencing of amplicons from both PCR assays confirmed the presence of Enc. intestinalis or Ent. bieneusi. In conclusion, our optimized assays for recovering and detecting Enc. intestinalis or Ent. bieneusi in feces from dairy calves provides a valuable alternative to traditional IFA methods that require expertise to identify extremely small microsporidia spores (~ 2.0 µm). Our assays also improve upon existing molecular detection techniques for these microsporidia by incorporating an internal standard to control for false negative reactions.

Viable Eimeria oocysts in poultry house litter at the time of chick placement.

The purpose of this study was to determine if Eimeria oocysts recovered from litter at the time of chick placement in commercial broiler houses contained oocysts that were infectious for chickens. Over 100 litter samples were collected from 30 poultry farms representing a total of 60 different broiler houses with 9 houses sampled more than once over 1.5 yr. The samples were collected just before the placement of newly hatched chicks and after an anticoccidial drug (ACD) or Eimeria vaccine (VAC) program, and processed for counting oocysts followed by Eimeria species determination using ITS1 PCR. Broiler chicks were inoculated with recovered Eimeria oocysts to determine if the litter oocysts were viable and capable of causing patent infection. At placement, E. maxima (Emax) oocysts were detected in 70 of 75 houses after ACD program and 46 of 47 houses after VAC program. Eimeria acervulina, E. praecox, and/or E. tenella (Eapt) were detected in 75 of 75 houses after ACD program and 47 of 47 houses after VAC program. Viability testing revealed that 33.0% of broiler houses contained viable Emax oocysts, while 46.9% contained viable Eapt oocysts. During VAC programs, the concentration of Emax oocysts at placement and the total number of Emax oocysts shed by chickens in viability studies showed a very strong correlation (r = 0.83). Likewise, during ACD programs, the concentration of Eapt oocysts at placement and the total number of Eapt oocysts shed by chickens in the viability study showed a strong correlation (r = 0.62). In general, Eimeria oocyst levels at placement and number of viable oocysts shed by chickens in the viability study were similar among houses on the same farm. However, the number of Eimeria oocysts shed in the viability studies was considerably less than expected based on the number of oocysts given. These data suggest that nearly 100% of all poultry houses contain Emax and Eapt oocysts at placement with 30 to 50% of the houses containing viable Eimeria oocysts, thus possibly representing a source of the protozoa to newly hatched chicks.

Changes in the levels of Cryspovirus during in vitro development of Cryptosporidium parvum.

The purpose of this study was to develop and utilize semi-quantitative RT-PCR and PCR assays for measuring the level of Cryspovirus, the viral symbiont of Cryptosporidium parvum, during in vitro development of the protozoan. Cultures of human carcinoma cells (HCT-8) were inoculated with excysting C. parvum sporozoites, followed by harvest of cells and culture medium at 2-, 24-, 48-, and 72-h post-infection. Changes in viral RNA levels were detected by RT-PCR using primers specific for RNA encoding the 40-kDa capsid protein (CP) or RNA-dependent RNA polymerase (RdRp). Parasite or host DNA was quantified by PCR specific for C. parvum or human glyceraldehyde-3-phosphate dehydrogenase (HuGAPDH). An internal standard (competitor) was incorporated into all assays as a control for PCR inhibition. Intracellular levels of C. parvum DNA increased between 2- and 48-h post-infection, and then decreased at 72 h. Culture medium overlying these C. parvum-infected cells displayed a similar increase in CP and RdRp signal, reaching peak levels at 48 h. However, the CP and RdRp levels in cellular RNA displayed only a modest increase between 2 and 48 h, and exhibited no change (CP) or decreased (RdRp) at 72 h. These data suggest that during the first 48 h of C. parvum in vitro development, Cryspovirus is released into the media overlying cells but remains at fairly constant levels within infected cells.

A rapid method for determining salinomycin and monensin sensitivity in Eimeria tenella.

Standard methods of determining the ionophore sensitivity of Eimeria rely on infecting chickens with an isolate or a mixture of Eimeria spp. oocysts in the presence of different anti-coccidial drugs. The purpose of this study was to develop a rapid in vitro method for assessing salinomycin and monensin sensitivity in Eimeria tenella. Cultures of MDBK cells were grown to 85% confluency, and then inoculated with excysted E. tenella laboratory strain (APU-1) sporozoites in the presence of different concentrations of salinomycin or monensin. At various timepoints, the monolayers were fixed for counting intraceullar sporozoites, or were subjected to DNA extraction, followed by molecular analysis using quantitative (qPCR) or semi-quantitative PCR (sqPCR). Preliminary experiments showed that 24h was the optimum time for harvesting the E. tenella-infected cell cultures. The average number of E. tenella sporozoites relative to untreated controls displayed a linear decrease between 0.3 and 33.0 µg/ml salinomycin and between 0.3 and 3.3 µg/ml monensin. A similar pattern was observed in the relative amount of E. tenella DNA as measured by sqPCR. A linear decrease in the relative amount of E. tenella DNA was observed over the entire range of salinomycin and monensin concentrations as measured by qPCR possibly reflecting the greater sensitivity of this assay. Comparison of sporozoite counting, sqPCR, and qPCR signals using a criterion of 50% inhibition in sporozoite numbers or level of PCR amplification product showed good agreement between the three assays. E. tenella field isolates (FS-1 and FS-2) displaying resistance to salinomycin and monensin were evaluated in the in vitro assay using qPCR and sqPCR. Compared to E. tenella APU-1, the E. tenella FS-1 and FS-2 isolates showed higher levels of E. tenella DNA at 24h by both qPCR and sqPCR. This in vitro assay represents a significant advance in developing rapid, cost-effective methods for assessing ionophore sensitivity in E. tenella.

Analysis of giardin expression during encystation of Giardia lamblia.

The present study analyzed giardin transcription in trophozoites and cysts during encystation of Giardia lamblia . Encystment was induced using standard methods, and the numbers of trophozoites and cysts were counted at various time points during the process. At all time points, RNA from both stages were assayed for levels of alpha2-, beta-, and delta-giardin mRNA as well as for cyst wall protein 3 (CWP3) mRNA using quantitative RT-PCR. In encystation medium, the number of G. lamblia trophozoites decreased, while the number of cysts increased between 0 and 72 hr. In trophozoites, alpha2- and beta-giardin transcription decreased over time, while delta-giardin transcription remained unchanged during the same time period. CWP3 transcription exhibited a slight increase in trophozoites at 8 hr, followed by a decrease at subsequent time points. Expression of alpha2-giardin increased at 48 hr in cysts followed by decreased expression at 72 hr, while beta- and delta-giardin expression was unchanged during encystation. CWP3 transcription gradually decreased from 24-72 hr in cysts. Consistent with previous studies, giardin proteins appeared to be disassembled into amorphous structures inside cysts during encystation. These findings represent the first analysis of giardin transcription in separate populations of trophozoites and cysts during encystation and indicate differential regulation of giardin mRNA expression by these developmental stages.

A rapid method for producing highly purified Cryptosporidium parvum oocysts.

Most procedures that have been described for purifying Cryptosporidium parvum oocysts are designed to either identify the parasites in clinical specimens or isolate oocysts from a small volume of feces from infected animals. The present study describes a rapid method for purifying high numbers of C. parvum oocysts from feces of infected calves that contains minimal contaminating fecal material and bacteria. The isolation method is based on differential flotation of C. parvum oocysts in NaCl, followed by ether extraction to solubilize lipids in calf feces. This procedure regularly yields > 10(9) purified C. parvum oocysts within 1-2 days of feces collection.

Immunization with Staphylococcus aureus lysate incorporated into microspheres.

Antibiotics are of limited value against Staphylococcus aureus due to development of resistant strains, scar tissue formation, and blockage of ducts due to inflammation. Though macrophages are the predominant cell type in the mammary gland, they are primarily scavenger cells and are not effective against bacteria entering the gland. Neutrophil phagocytosis is the bovine's primary defense against S. aureus mastitis. Attempts to develop vaccines that enhance neutrophil phagocytosis by stimulating production of opsonizing antibodies to S. aureus have met with limited success because of the low immunogenicity of the exopolysaccharide capsule surrounding S. aureus. Staphylococcus aureus can also adhere to and penetrate epithelial tissue. This study was conducted to determine whether lysates of S. aureus encapsulated in biodegradable microspheres would increase the production of opsonizing antibodies to capsule and block adherence. Four groups of four cows each were injected with 1 ml of the respective treatment in the area of the supramammary lymph node and 1 ml in the hip muscle. The treatments were: lysate in NaCl, lysate in Freund's incomplete adjuvant (FICA), lysate in microspheres in NaCl, and lysate in microspheres in FICA. Antigen in microspheres produced a similar antibody response to antigen emulsified in FICA, but to a lesser magnitude. Antigen in microspheres produced antibodies that were more opsonic for neutrophils at 20 and 52 wk postimmunization and inhibited S. aureus adherence to mammary epithelium. Ability to control antigen release and presentation, and the benefit of a single injection for long-term immunity using microspheres warrants additional studies.

Production of antibodies to Staphylococcus aureus serotypes 5, 8, and 336 using poly(DL-lactide-co-glycolide) microspheres.

Staphylococcus aureus is responsible for a major portion of the economic losses due to mastitis. Attempts to produce a vaccine to prevent S. aureus mastitis have been hampered by the low immunogenicity of the polysaccharide, which forms on the surface of the organism when it enters the mammary gland. The polysaccharide inhibits phagocytosis and destruction of the organism by neutrophils. This study was conducted to determine if S. aureus polysaccharide serotypes 5, 8, and 336 conjugated to a protein and incorporated in poly(DL-lactide-co-glycolide) microspheres would enhance the production of opsonizing antibodies to the polysaccharide. Cows were immunized with either polysaccharide conjugates emulsified in Freund's incomplete adjuvant or polysaccharide conjugates encapsulated in poly (DL-lactide-co-glycolide) microspheres emulsified in Freund's incomplete adjuvant. All cows produced sustained antibody titers to the three polysaccharide serotypes. Cows immunized with microspheres had higher antibody titers. Cows in both groups produced increased concentrations of IgG1 and IgG2 antibodies; neither group produced an increase in IgM. Immune sera from cows immunized with conjugates alone increased phagocytosis, which decreased at the end of the study. Sera from cows immunized with conjugates in microspheres increased phagocytosis, which was sustained at the end of the study. Immune sera from both groups decreased bacterial adherence to bovine mammary epithelial cells. These data showed that a single injection of antigen in microspheres produced higher titers and more sustained enhancement of phagocytosis, which could aid in the defense of the cow against S. aureus infections.

Oregon's guidelines for physician-assisted suicide: a legal and ethical analysis.

Oregon's Death with Dignity Act was first passed by a ballot initiative in 1994, but numerous judicial challenges delayed implementation of the Act. In November of 1997, following the United States Supreme Court decisions in Vacco v. Quill and Washington v. Glucksberg, which left the states' power to regulate physician-assisted suicide undisturbed, the Oregon voters upheld their law. Oregon remains the only state in the nation to authorize physician-assisted suicide. The Task Force to Improve the Care of Terminally Ill Oregonians published a Guidebook for health care providers on the Oregon Act, and the New England Journal of Medicine recently issued a special report on the first year's experience under the Act. This paper analyzes the legal context of the Oregon Death with Dignity Act, discusses the efficacy of the tenets in the Guidebook, and explores ethical issues underlying the guidelines, particularly those pertaining to the meaning of a patient's request for assisted suicide and processes supporting informed consent.

A bovine mammary endothelial/epithelial cell culture model of the blood/milk barrier.

The complex nature of the mammary gland has hampered in-depth studies of the relationship of the circulatory system to cells lining the teat ducts and alveoli of the gland. This study reports an in vitro model of endothelial and epithelial cells separated by a subcellular matrix that simulates the blood milk barrier of the bovine mammary gland. Dual chamber culture dishes with a porous membrane separating the upper and lower chamber were used. Endothelial and epithelial cells were cultured on opposite sides of the porous membrane. A collagen and fibroblast subcellular matrix, separating the 2 cell layers, simulated the in vivo interstitial tissue. Changes in surface binding of anti-bodies to polymorphonuclear neutrophils (PMN) following their migration from the upper to the lower chamber simulated the passage of PMN from blood to milk. Changes in the binding of antibodies to PMN agreed with results observed following the migration of PMN from blood to milk in vivo. This gives credence to the model's potential value for studies where more direct observation of the blood/milk barrier is required. The model will be further tested for its usefulness as an assay for determining: 1) antibiotic diffusion from milk to blood and from blood to milk, 2) cytotoxicity of prophylactic and therapeutic mammary infusion products, 3) factors affecting bacterial adhesion and penetration of mammary epithelial tissue, 4) effectiveness of antibodies present in lacteal secretions in preventing bacterial adhesion, and 5) the feasibility of gene constructs to induce synthesis and secretion of mastitis-preventing compounds and prophylactic and therapeutic compounds for treatment of human disorders.

Formulation of poly(DL-lactide-co-glycolide) microspheres and their ingestion by bovine leukocytes.

This study determined optimal parameters for producing controlled-release microspheres and examined their suitability for vaccines via ingestion by bovine leukocytes. Microspheres elicit an immune response when ingested by antigen-presenting cells and provide sustained exposure of antigen to sensitized cells. Ingestion of microspheres is determined by their size (< 10 microns), and antigen release is governed by composition. Poly(DL-lactide-co-glycolide) microspheres were prepared using different polymer concentrations, stir rates, emulsifier concentrations, and emulsifier molecular masses. Microspheres that were < 10 microns were prepared using a 6% 50:50 lactide to glycolide polymer solution emulsified at 12,000 rpm in a low molecular mass, 5% polyvinyl alcohol solution. Microspheres that were > 20 microns were prepared using a 10% 85:15 lactide to glycolide polymer solution emulsified at 1200 rpm in a low molecular mass, 3% polyvinyl alcohol solution. Small microspheres released 90% of the antigen after 7 d, and large microspheres released only 24% of the antigen after 56 d. Both monocytes and neutrophils selectively ingested small microspheres that were opsonized with normal bovine serum (heated and unheated). Ingestion of microspheres that had been opsonized with fetal bovine serum (heated and unheated) was minimal. Lymphocytes did not ingest microspheres. Ingestion of small microspheres by bovine monocytes and sustained release of antigen by large microspheres suggested that microspheres have the ability to produce a sustained immune response with a single injection.

Effect of antibodies to staphylococcal alpha and beta toxins and Staphylococcus aureus on the cytotoxicity for and adherence of the organism to bovine mammary epithelial cells.

OBJECTIVE: To determine the effect of antibodies to staphylococcal alpha and beta toxins and Staphylococcus aureus on the toxicity for and adherence of S aureus to bovine mammary epithelial cells. SAMPLE POPULATION: Cultured bovine mammary epithelial cells and Staphylococcus aureus obtained from a cow with mastitis. PROCEDURE: Cultured bovine epithelial cells were incubated with antisera to alpha and beta toxins of S aureus and culture supernatant; cell damage and S aureus adherence to cells were measured. RESULTS: Antisera to alpha, beta, and alpha + beta toxins inhibited cytotoxicity of S aureus culture supernatant. Antiserum to alpha + beta toxin was the most effective inhibitor of cytotoxicity and antiserum to beta toxin was the least effective. All 3 antisera decreased the percentage of S aureus adhered to the mammary epithelial cell monolayers and numbers of organisms per cluster of adhered bacteria. In this study, antisera to alpha and alpha + beta toxins decreased the number of S aureus clusters per dish, but antiserum to beta toxin had no significant effect. Antiserum to alpha + beta toxin decreased the percentage of epithelial cells with adhered S aureus, but neither antiserum to alpha nor beta toxin had significant effect. Antiserum to S aureus decreased the percentage of S aureus adhered, number of clusters perdish, number of organisms per cluster, and percentage of epithelial cells with S aureus adhered. CONCLUSIONS: Antibodies to staphylococcal alpha and beta toxins inhibit adherence to and cytotoxicity of S aureus for bovine mammary epithelial cells, and antibodies to S aureus inhibit adherence of S aureus to bovine mammary epithelial cells.

A method for measuring specific antibodies in bovine lacteal secretions during the nonlactating period.

A large portion of new IMI in dairy cattle occurs during the nonlactating period. Because antibiotic infusions at the beginning of the nonlactating period are only partially effective, attempts have been made to stimulate the production of protective antibodies in lacteal secretions during this period. However, measurement of antibodies in mammary secretions during the nonlactating period has been hampered by the complex, viscous nature of these secretions. This report describes the use of caprylic acid to clarify secretions from the bovine mammary gland during the nonlactating period to provide a more accurate measurement of specific antibody. Six healthy Jersey cows were injected in the area of the supramammary lymph node with an encapsulated strain of Staphylococcus aureus in dextran sulfate at the beginning of the nonlactating period and 15 and 30 d later. Seven healthy unimmunized Jersey cows served as controls. Lacteal secretions taken at the beginning of the nonlactating period; at 15, 30, and 45 d into the nonlactating period; and at calving were treated with caprylic acid prior to assay for specific antibodies using ELISA. Purified S. aureus capsule was used as the antigen in the ELISA. Caprylic acid lowered non-specific binding of IgG1 and IgM in secretions during the dry period from unimmunized control cows and lowered IgM from immunized cows. The most pronounced effect of caprylic acid was an increase in IgG2 binding in secretions from immunized cows. Treatment with caprylic acid more accurately measured specific activity of Ig in mammary secretions during the nonlactating period.

Effect of alpha-toxin and capsular exopolysaccharide on the adherence of Staphylococcus aureus to cultured teat, ductal and secretory mammary epithelial cells.

Cultures of teat, ductal and secretory epithelial cells were used to study the role of alpha-toxin and the capsular exopolysaccharide on the adherence of Staphylococcus aureus to mammary epithelium. The adherence of S aureus to the cells and their susceptibility to damage by alpha-toxin increased from teat to ductal to secretory cells. Alpha-toxin increased the susceptibility of epithelial cell monolayers to adherence by S aureus, and the extent of the adherence increased with the time of exposure to alpha-toxin. The exopolysaccharide capsule deterred the adherence of S aureus to mammary epithelial cells and to collagen. Organisms with a rigid capsule adhered to a smaller extent than those with a flaccid capsule. Both encapsulated and unencapsulated S aureus adhered more readily to collagen than to either healthy monolayers of epithelial cells or monolayers of cells damaged by alpha-toxin.

Effect of whole Staphylococcus aureus and mode of immunization on bovine opsonizing antibodies to capsule.

Exopolysaccharide capsule is a major virulence factor of Staphylococcus aureus because it inhibits neutrophil recognition of antibodies to highly antigenic S. aureus cell wall. To circumvent this inhibition, two modes of immunization were tested for ability to induce anticapsular opsonins. Cows were immunized at drying off and boosted on d 14 and 28 by injection of Smith diffuse S. aureus plus dextran sulfate in the area of the supramammary lymph node or intramammarily. In cows immunized in the area of the supramammary lymph node, IgG1 and IgG2 sera antibody titers to capsule increased and remained elevated to the end of the study, 120 d postcalving. The IgM titers increased during the dry period but declined to preimmunization levels at calving. Response of serum IgG1 and IgM to intramammary immunization was similar to that with supramammary lymph node immunization, but more delayed and lower in magnitude. Antibodies of all four isotypes, IgG1, IgG2, IgA, and IgM, increased in dry secretions following immunization via lymph node. In cows immunized in the lymph node, IgG1 antibodies remained elevated throughout the study, but IgG2 antibodies dropped to baseline 15 d postcalving. In cows immunized intramammarily, only IgA antibodies increased significantly in lacteal secretions and remained elevated throughout the study. Immunization of cows in the lymph node during the dry period enhanced the ability of dry secretions and colostrum to support phagocytosis.

Adherence of Staphylococcus aureus to cultured bovine mammary epithelial cells.

Bovine mammary secretory cells, isolated at necropsy, were cultured in vitro and used as a model to study the mode of adherence of Staphylococcus aureus to mammary epithelium. Cultured cells were characterized by their morphology and physiology as secretory epithelial cells. Cells showed characteristic growth patterns when grown on polystyrene, fibronectin, laminin, collagen, and reconstituted basement membrane from the Engelbreth-Holm-Swarm murine sarcoma. Cells cultured on collagen formed confluent monolayers and were the most suitable for bacterial adherence studies. Cultured cells stained intensely for cytokeratin and for specific milk proteins, i.e., alpha-casein, beta-casein, alpha-lactalbumin, beta-lactoglobulin, and lactoferrin. The effect of frozen storage for 10 mo on cell viability or presence of milk proteins was minimal. Staphylococcus aureus showed large affinity for extracellular matrix components, i.e., fibronectin, laminin, and collagen. Adherence to confluent cell monolayers was minimal. In preconfluent cell monolayers, most S. aureus adhered more readily to the exposed matrix than to the epithelial cells. Overnight exposure to staphylococcal alpha-toxin greatly increased adherence of S. aureus to confluent monolayers. However, whether bacteria adhered to alpha-toxin damaged cells or to exposed matrix is not clear. Unencapsulated S. aureus adhered in larger numbers than did encapsulated S. aureus.

Bovine mammary teat and ductal epithelial cell cultures.

Bovine mammary epithelial cells from teat and ductal tissue were isolated at necropsy and were grown in culture. Cells were characterized by the presence of cytokeratin filaments, cell morphologic features, synthesis of milk proteins, esterase activity, DNA content, and growth patterns on polystyrene, fibronectin, laminin, collagen, and reconstituted basement membrane from the Engelbreth-Holm-Swarm murine sarcoma. Cultured teat and ductal cells stained intensely for cytokeratin and had similar morphologic features. Both cell types synthesized alpha-casein, beta-casein, alpha-lactalbumin, beta-lactoglobulin, and lactoferrin to variable degrees. Cell type and culture conditions did not affect the DNA content of the cells, as indicated by similar amounts of DNA in G0G1 and G2M phases of the mitotic cycle in cultured cells and in cells from freshly isolated mammary explants. Cells cultured on polystyrene, fibronectin, laminin, and collagen formed pavement-like cell monolayers suitable for cytotoxicity and bacterial adherence studies. Cells cultured on the reconstituted basement membrane from the Engelbreth-Holm-Swarm murine sarcoma formed three-dimensional structures closely resembling lactiferous ducts and alveoli, which could be used for studying lactogenesis and galactopoiesis. Freshly isolated cells and cultured cells were stored at -70 C or in liquid nitrogen. The latter storage method affected the cells less than did freezing at -70 C.

A century of progress: nursing quality improvement at the Mount Sinai Medical Center.

Since The Mount Sinai Hospital in New York City began delivering care in the mid 1800s, the medical services and, shortly thereafter, the nursing service have consistently given priority to improving patient care. Through the decades of the late 19th and 20th centuries, the Department of Nursing has remained current with the evolving concept of quality improvement. A historical overview of key elements in the evolution of quality improvement in nursing is presented.

Quality improvement in action: a falls prevention and management program.

A departmental program for falls prevention and management, initiated in 1987, and arising out of the awareness of the economic, physical, and psychological effects of falls in hospitalized patients, has been described. Given the results outlined, there is no doubt that the efforts applied in the area of falls prevention have been extremely worthwhile in affecting major quality improvements in patient care. The benefits of the falls prevention program include a reduction in the incidence of falls as well as an increased awareness by staff, patients, and families of the importance of monitoring patients at risk for falls.