Enhancing casein micelle dissociation in diluted skim milk systems using combined processing techniques.

The present work aims to evaluate the dissociation of casein micelles in diluted skim milk (SM) systems after undergoing solvent- or emulsifying salt-based dissociation coupled with ultra-high-pressure homogenization (UHPH). Specifically, part I evaluated dilute SM solutions combined with varying ethanol concentrations (0%-60%) at varying temperatures (5-65°C) in combination with UHPH (100-300 MPa), and part II evaluated dilute SM solutions combined with varying concentrations (0-100 mM) of either sodium hexametaphosphate (SHMP) or sodium citrate (SC) in combination with UHPH (100-300 MPa). In part I, high concentrations of ethanol (40%-60% vol/vol) at elevated temperatures (45-65°C) achieved extensive dissociation of casein micelles, especially in combination with UHPH at ≥200 MPa, as shown by a reduction in sample absorbance and in casein particle size compared with the control (dilute SM, 65°C) under optimum conditions (dilute SM, 60% ethanol, 65°C, ≥200 MPa). In part II, the level of casein micelle dissociation using emulsifying salts (ES) was dependent on the ES type and concentration. Considerable casein micelle dissociation in dilute SM systems was achieved with SHMP concentrations ≥1 mM and SC concentrations ≥10 mM, resulting in decreased sample absorbance, bimodal casein size distributions, and increased hydrophobicity (∼2-fold increase in intrinsic fluorescence) compared with the control (dilute SM). This dissociation was further enhanced with UHPH (≥200 MPa). These results indicate that both solvent- and ES-based casein dissociation techniques can be optimized when used in combination with UHPH. Together, these processing techniques can be used to extensively dissociate casein micelles with the potential to use these altered systems for value-added applications such as functional ingredients or encapsulation agents.

Effect of direct-fed fibrolytic enzymes on the lactational performance of dairy cows.

In trial 1, 30 midlactation (213 d in milk) Holstein cows were randomly assigned to a control or enzyme treatment in a two-period crossover design and were fed a total mixed ration based on alfalfa hay and silage. Cows on the enzyme treatment received an enzyme solution containing cellulases and xylanases, which was sprayed on the forage component of the ration at a rate of 1.65 ml/kg of forage dry matter (DM) between 8 and 24 h prior to feeding. Cows consuming the forage treated with enzyme produced more milk (27.2 vs. 25.9 kg/d) and digested more DM per day than did cows fed the control forage. In trial 2, 40 early lactation Holstein cows were assigned to one of four treatments for 16 wk. Following a 2-wk covariate period, cows were assigned to 1) no enzyme treatment, 2) a low (1.25 ml/kg of forage DM) enzyme treatment, 3) a medium (2.5 ml/kg of forage DM) enzyme treatment, or 4) a high (5.0 ml/kg of forage DM) enzyme treatment. Enzymes were a 2:1 combination of cellulase and xylanase diluted in water and sprayed on a combination of alfalfa hay and silage and whole cottonseed immediately before mixing with a concentrate based on barley. Dry matter intakes were similar for cows on treatments 2, 3, and 4 and were greater than those for cows on treatment 1. Production of milk, 3.5% fat-corrected milk, and energy-corrected milk was greater for cows on treatment 3 than for cows on treatment 1. Fibrolytic enzymes applied to the forage portion of the rations prior to feeding improved lactational performance of early and midlactation cows.

Effect of direct-fed fibrolytic enzymes on the digestive characteristics of a forage-based diet fed to beef steers.

A study was conducted to examine the method of delivery of a solution containing cellulases and xylanases on the digestion of a forage-based diet. Five ruminally cannulated beef steers (536 kg BW) were randomly assigned to a control (CON) or one of four enzyme treatments in a 5 x 5 Latin square experiment. Steers were fed a 70:30 (DM basis) grass hay:barley diet. Enzyme-treated rations contained a solution of fibrolytic enzymes at the rate of 1.65 mL/kg of forage DM. Enzyme application treatments were 1) enzyme to forage 24 h before feeding (F-24), 2) enzyme to forage 0 h before feeding (F-0), 3) enzyme to barley 0 h before feeding (B-0), and 4) enzyme infused ruminally 2 h after feeding (RI). Dry matter and NDF intakes were not different (P > .10) across treatments. Ruminal pH was lower and total VFA concentration at 16 h after-feeding was greater (P < .10) for steers fed enzyme treatments compared with CON. Rate of NDF disappearance was greater (P < .05) for enzyme-treated than for untreated grass substrate. Ruminal infusion of enzymes compared with F-24 and F-0 produced lower disappearance of DM at 8 and 32 h (P < .10), NDF at 32 h (P < .10), and DM and NDF at 96 h (P < .05). Rate of DM disappearance of enzyme-treated grass hay was greater (P < .10) for steers fed B-0 than for those fed F-24 and F-0 and for CON than for F-24 and F-0. Total tract digestibility of DM, NDF, and ADF was greater (P < .10) for F-24 and F-0 than for CON. Forage transit time was shorter (P < .10) for B-0 than for F-24 and F-0; however, all other contrasts for particulate passage did not differ (P > .10). Results from this study indicate that direct application of enzymes to forages is capable of improving forage digestion.

Pilot-Scale Selenium Bioremediation of San Joaquin Drainage Water with Thauera selenatis.

This report describes a simple method for the bioremediation of selenium from agricultural drainage water. A medium-packed pilot-scale biological reactor system, inoculated with the selenate-respiring bacterium Thauera selenatis, was constructed at the Panoche Water District, San Joaquin Valley, Calif. The reactor was used to treat drainage water (7.6 liters/min) containing both selenium and nitrate. Acetate (5 mM) was the carbon source-electron donor reactor feed. Selenium oxyanion concentrations (selenate plus selenite) in the drainage water were reduced by 98%, to an average of 12 (plusmn) 9 (mu)g/liter. Frequently (47% of the sampling days), reactor effluent concentrations of less than 5 (mu)g/liter were achieved. Denitrification was also observed in this system; nitrate and nitrite concentrations in the drainage water were reduced to 0.1 and 0.01 mM, respectively (98% reduction). Analysis of the reactor effluent showed that 91 to 96% of the total selenium recovered was elemental selenium; 97.9% of this elemental selenium could be removed with Nalmet 8072, a new, commercially available precipitant-coagulant. Widespread use of this system (in the Grasslands Water District) could reduce the amount of selenium deposited in the San Joaquin River from 7,000 to 140 lb (ca. 3,000 to 60 kg)/year.

Significance of circumsporozoite-specific antibody in the natural transmission of Plasmodium falciparum, Plasmodium vivax, and Plasmodium malariae in an aboriginal (Orang Asli) population of central peninsula Malaysia.

Two hundred and seventy-five Orang Asli volunteers living in nine villages in the Pos Legap Valley of Perak State, peninsular Malaysia, participated in a prospective study designed to characterize the epidemiological, parasitological, and entomological characteristics of Plasmodium falciparum, P. vivax, and P. malariae malaria transmission. Prevalence rates for the three plasmodial species at initiation of the study ranged from 56% in the 0-4-year-old age group to 0% in individuals over the age of 40. Entomological surveys were conducted, enabling us to determine mosquito salivary gland-positive rates and entomological inoculation rates of 1.2 infectious mosquito bites per person per month for P. falciparum, 2.4 for P. vivax, and 0.3 for P. malariae. Cumulative incidence rates over the 16 weeks of the study, following radical cure of all volunteers, were 22.5% for P. falciparum, 12.7% for P. vivax, and 1.5% for P. malariae. The median baseline antibody titer against the immunodominant repetitive B cell epitope of P. falciparum or P. vivax circumsporozoite protein was significantly higher for volunteers who did not become parasitemic. Volunteers were selected for further study if they had evidence of being challenged with P. falciparum sporozoites during the study, based on a two-fold or greater increase in antibody titer against the immunodominant repetitive B cell epitope of the circumsporozoite protein. Resistance to infection was seen in six of 10 individuals who had high (greater than 25 OD units) baseline ELISA titers, compared with only three of 24 individuals who had low baseline ELISA titers (chi 2 P less than 0.02). A similar analysis for P. vivax did not show a significant correlation.

Conserved and variant epitopes of target antigens of transmission-blocking antibodies among isolates of Plasmodium falciparum from Malaysia.

Monoclonal antibodies (MAbs) directed against different epitope regions on three sexual stage-specific gamete surface proteins of Plasmodium falciparum, Pfs 25, Pfs 230, and Pfs 48/45, were used to study the genetic diversity of these epitopes among fresh isolates of P. falciparum from Malaysia, using immunofluorescence microscopy (IFA). Among 45 Malaysian isolates, one epitope of Pfs 25, designated region I, showed evidence of variable reactivity with MAbs among different isolates; the Pfs 25 epitope, region II, was universally recognized by MAbs in all isolates. Two apparently distinct epitope regions of Pfs 230 were defined by MAbs, one of which was universally recognized by MAbs among the 45 isolates; the other was conserved in all but three isolates. The epitope regions of gamete-surface protein Pfs 48/45, designated regions I, IIa, IIb, IIc, III, and IV, were examined for reactivity by IFA in 33 isolates. Epitope regions I, IIb, III, and IV were conserved in all isolates; regions IIa and IIc existed in variant forms.

Multi-laboratory evaluation of a scrub typhus diagnostic kit.

Scrub typhus is a major cause of febrile illness throughout the Asia-Pacific region. It is commonly undiagnosed, partly because of the lack of a simple, reliable diagnostic test which can be used in clinical laboratories. The indirect immunoperoxidase technique, configured into a test kit, was provided to technicians who were trained in its use. They used the kit during a 2 year field trial in their respective clinical hospital laboratories throughout Malaysia. In an evaluation using 1,722 consecutive sera tested in those laboratories, the kit was found to have a median sensitivity for IgG detection of 0.85 (range 0.33-0.95), a median specificity of 0.94 (range 0.88-1.00), reproducibility of 0.86, and efficiency of 0.92 when compared to the reference laboratory. In a proficiency survey in which 10 laboratories received 3 coded test samples, all but 2 laboratories had results within 1 dilution of the reference laboratory in quantitating specific IgG, whereas 7 laboratories were within 1 dilution in quantitating IgM. The shelf life of the kit was at least 1 year at 4 degrees C.

A rapid sporozoite ELISA using 3,3',5,5'-tetramethylbenzidine as the substrate chromogen.

A modified version of the standard 2-site sporozoite enzyme-linked immunosorbent assay (ELISA) using 3,3',5,5'-tetramethylbenzidine (TMB) as the substrate chromogen solution was adapted for rapid detection and identification of Plasmodium falciparum and P. vivax circumsporozoite (CS) proteins. The TMB-ELISA was evaluated using sporozoites from experimentally infected mosquitoes and laboratory colonized uninfected mosquitoes. Our data indicate comparable sensitivity levels between the TMB-ELISA and the standard ELISA, i.e., 50 P. falciparum or P. vivax sporozoites/50 microliters of test solution. Reactions inherent to the method were specific and background reactivity was minimal. The TMB-ELISA is rapid (1 hr), simple, uses a minimal amount of monoclonal antibodies, and is suitable for use in a wide range of laboratories.

Antimalarial drug susceptibility of Plasmodium falciparum isolates from forest fringe dwelling aborigines (Orang Asli) of Peninsular Malaysia.

The drug susceptibility of 70 isolates of Plasmodium falciparum to standard and experimental antimalarials was evaluated using a radioisotope microdilution method. All isolates were from forest fringe dwelling Orang Asli, the aborigines of Peninsular Malaysia. The geometric mean IC50 values were: chloroquine, 10 ng/ml; amodiaquine, 4.7 ng/ml; mefloquine, 2.8 ng/ml; quinine, 40.5 ng/ml; halofantrine, 1.5 ng/ml; enpiroline, 3 ng/ml; and pyrimethamine, 21 ng/ml. Four isolates exhibited decreased susceptibility to chloroquine (IC50 greater than 60 ng/ml), and one exhibited decreased susceptibility to quinine (IC50 = 161 ng/ml). Three isolates showed decreased susceptibility to mefloquine (IC50 = 10-11 ng/ml). The lack of drug pressure may account for the high prevalence of P. falciparum isolates susceptible to chloroquine.

Interaction of Malaysian sera with Plasmodium vivax sporozoite antigen.

A seroepidemiologic survey of Plasmodium vivax and Plasmodium falciparum transmission was conducted in 94 Orang Asli children and adults. The prevalence of malaria was 46% in this population, and infections due to P. vivax and P. falciparum occurred with equal frequency. Multi-species infection was common, particularly in children less than 10 years of age. Circumsporozoite (CS) antibodies to P. vivax were detected by ELISA, using the recombinant protein NS181V20, in sera from 53-95% of all subjects in this study. The specificity of reactivity to NS181V20 was confirmed by immunofluorescence using air-dried sporozoites. CS antibodies to P. falciparum were present in less than 50% of the population less than 30 years of age. These data support further testing of this protein as a candidate vivax vaccine.

Evaluation of the whole blood lymphocyte transformation assay for scrub typhus exposure: adaptability to field sample collection.

The optimal conditions for the determination of exposure to scrub typhus by the whole blood lymphocyte transformation assay was 7 days culture of 10% blood in RPMI 1640 medium supplemented with 10% human AB-negative serum and L-glutamine with 50-200 micrograms protein/ml of Karp, Kato, or Gilliam strain membrane antigen. A simple exponentially decaying linear model shows the decrease in lymphocyte viability, the ability of sensitized cells to be stimulated with PHA mitogen, and the corresponding decrease in stimulation by scrub typhus antigens with increasing time of preincubation on ice. The lower limit of stimulation index for the detection of scrub typhus by whole blood lymphocyte transformation assay was 4.0 with a type I error of 1%.

Pathologic activity of Plasmodium berghei prevented but not reversed by dexamethasone.

Dexamethasone has recently been shown to block the production of cachectin (implicated in the pathogenesis of cerebral malaria) if administered prior to endotoxin induction of mouse macrophages. Using the hamster cheek pouch-cerebral malaria model, we tested the hypothesis that dexamethasone is effective as a therapeutic agent in severe malaria if given before some yet undefined trigger point in the disease. Infected hamsters were treated with dexamethasone (0.7 mg/kg) daily on days 7-12, 4-12, or 1-12 post-challenge. When treatment was started on day 1, whole body oxygen consumption (used as a measure of erythrocyte transport to sites of diffusion) on day 12 was greater than (P less than 0.05) that of infected control animals, though the degree of anemia was no different in treated and untreated groups. Furthermore, treatment produced a reduction in monocyte accumulation, capillary malfunction, and monocyte/red blood cell aggregate formation observable in the cheek pouch in vivo and a similar reduction in monocyte presence, capillary pathologic change, and multifocal hemorrhage in the brain on postmortem. These data suggest that mediator(s), whose production can be blocked by pretreatment with dexamethasone, are involved in the pathogenesis of disease leading to death of the Plasmodium berghei infected hamster.

Comparative evaluation of the indirect immunoperoxidase test for the serodiagnosis of rickettsial disease.

An indirect immunoperoxidase test was compared with an indirect fluorescent antibody test and the Weil-Felix OXK test for serodiagnosis of scrub typhus by measuring the rickettsial antigen specific activity of IgG, IgM, and whole globulin. Acute and convalescent sera from 50 Rickettsia tsutsugamushi isolate-positive scrub typhus patients and from 45 febrile patients diagnosed as having diseases other than scrub typhus were tested. The receiver operating characteristic for each test showed that the indirect immunoperoxidase and indirect fluorescent antibody tests were more sensitive and specific than the Weil-Felix test using convalescent and acute as well as paired sera. The indirect immunoperoxidase test showed no cross-reactivity when R. tsutsugamushi antigen was tested against sera collected from patients living outside the scrub typhus-endemic area with diseases other than scrub typhus. The indirect immunoperoxidase and indirect fluorescent antibody tests were comparable in measured response to R. tsutsugamushi, R. typhi, and TT-118 (spotted fever group) antigen. Thus the indirect immunoperoxidase test represents a sensitive, specific, reproducible, and practical semiquantitative test for rickettsial disease diagnosis.

Duffy phenotypes in Malaysian populations: correction of previous unusual findings.

Duffy phenotypes were determined for 314 Malaysian Orang Asli. The most common gene, Fya, was present in 313; there were no Duffy negative individuals. A previous study found evidence of Plasmodium vivax infection in 5 of 7 Orang Asli reported to be of the Duffy negative genotype. In this study, 5 of the 7 previously tested Orang Asli were retested in triplicate, and each of the 5 was found to be Duffy positive, having the Fya gene and a phenotype of Fy (a + b-).

Murine typhus among Khmers living at an evacuation site on the Thai-Kampuchean border.

An outbreak of febrile disease involving 170 Khmer adults at an evacuation site in Thailand occurred during the dry season of 1986, only 8 months after the camp was constructed. The illnesses were characterized by persistent fever, retro-orbital headache, myalgias, and clinical response to tetracycline within 2-3 days. The symptoms, effectiveness of tetracycline, and presence of a large rat population raised the suspicion of murine typhus. Fourteen (74%) of 19 patients had elevated or rising antibody titers against Rickettsia typhi, confirming the clinical diagnosis. Rats were caught, and they and their fleas were identified. In agreement with the known Thai host and vector, 80 (93%) of 86 rats were Rattus exulans, and all of 32 fleas were Xenopsylla cheopis. This first reported outbreak of murine typhus in Thailand is notable for its occurrence in a new human settlement only 8 months after construction.

Peripheral vascular pathophysiology of Plasmodium berghei infection: a comparative study in the cheek pouch and brain of the golden hamster.

Four- to six-week-old hamsters were infected with 1.5 X 10(7) Plasmodium berghei-parasitized hamster red blood cells by intraperitoneal injection. Cheek pouch circulation was observed microscopically in the anesthetized animal; the brain and contralateral pouch were collected for histopathologic examination on days 3-12 post-challenge. Cheek pouch vascular lesions, observed in vivo, appear to involve three phenomena; early (beginning 3-4 days) adhesion of pigment-laden mononuclear cells to endothelium within venous vessels and loss of function of the small capillaries supplying the skeletal muscle fibers and, later (6-9 days), the apparent attraction of erythrocytes to venular and venous endothelium and to adherent monocytes. The aggregation of formed elements on endothelial walls leads to progressive occlusion of venules and small veins and contributes to the observed disruption of flow through capillary networks. Histopathology of the brain and pouch shows vascular changes similar to those seen in vivo; in addition, multifocal hemorrhages are seen commonly in the brain and occasionally in the pouch on postmortem. In severe disease, evidence of cerebral edema is seen in the brain. The data suggest that failure of capillary flow and disruption of venous outflow tracts by cell aggregates are central to vascular failure in both the cheek pouch and brain of the P. berghei infected hamster. This hamster model of human cerebral malaria allows the in vivo observation, still and video photomicrography, and manipulation of the peripheral vascular pathogenesis of a disease process similar to that seen in humans.

A serological survey of scrub, tick, and endemic typhus in Sabah, East Malaysia.

A seroepidemiological survey of 837 people and 383 febrile patients was performed in rural areas of Sabah. We determined that the rickettsial diseases scrub typhus and endemic typhus were uncommon causes of febrile illness, as was tick typhus, except in forest dwelling peoples. The rate of occurrence of SFGR specific antibody was 16.5% among 412 forest dwellers, indicating that tick typhus may be a frequent cause of illness in this population.

A light and electron microscopic examination of Ehrlichia sennetsu in cultured human endothelial cells.

Umbilical cord-derived human endothelial cells were used for the propagation of Ehrlichia sennetsu. Scanning electron microscopic studies revealed numerous pleomorphic ehrlichiae extending from the surface of infected human endothelial cells. Transmission electron microscopic examination of the cells revealed E. sennetsu-induced cytopathic changes at the ultrastructural level. Ehrlichiae of variable size, shape and density were located individually and in clusters enclosed within membrane lined vacuoles. The cultured human endothelial cell provides an optimal environment for the growth of E. sennetsu and is a suitable in vitro model for the study of the cytopathic effects of this human pathogen.