Salmonella Survival on Pecans as Influenced by Processing and Storage Conditions

Survival of Salmonella senftenberg 775W, S. anatum, and S. typhimurium during exposure to currently practiced, as well as abusive, pecan processing and storage conditions was studied. Thermal treatments normally carried out during the processing of pecans are inadequate to consistently destory salmonellae in highly contaminated inshell nuts. Pecan nut packing tissue was toxic to salmonellae, thus affording some protection against high initial contamination and subsequent survival of the organisms. Examinations of inoculated inshell pecans stored at -18, -7, 5, and 21 C for up to 32 weeks revealed that the extent of survival was inversely correlated to the storage temperature. S. senftenberg 775W and S. anatum were not detectable on inshell nuts after 16 weeks of storage at 21 C. Little decrease in viable population of the three species was noted on inoculated pecan halves stored at -18, -7, and 5 C for 32 weeks. Due to organoleptic quality deterioration in pecan nutmeats at elevated temperatures, sterilization methods other than thermal treatment appear to be required for the elimination of viable salmonellae from pecan nuts.     

Preparation of conjugates of 19s and 7s globulins of antitularemic sera for the determination of specific fluorescence ofPasteurella tularensis

Pasteurella tularensis was detected by means of conjugates of 19S and 7S globulins from antitularemic serum with fluorescein isothiocyanate (FITC). The serum was obtained by short-term immunization of rabbits. The capacity of both fractions to produce the immunofluorescence reaction with pure culturesof Pasteurella tularensis and the agglutinin titre of these fractions are compared here. It was found that the 19S globulin fraction which contained most agglutinating antibodies showed only weak immunofluorescence. The 7S fraction in which the agglutinin content was very low as compared with the 19S globulin fraction gave more intense fluorescence than the 19S fraction. On the basis of this finding the opinion is advanced that the agglutinating antibodies need not be the actual carrier of fluorescence due to fluorescent antibodies (FA) in the immunofluorescence reaction ofPasteurella tularensis. The different degrees of labelling with FITC of the individual globulin fractions as observed during simultaneous conjugation are also discussed. The 19S globulin fraction is regularly conjugated to a higher degree than the 7S component. The view is presented that the avirulent strains lack a certain part of the antigen structure as compared with virulent or vaccine strains (with residual virulence), the part being responsible for the positive reaction of immunofluorescence with preparations of fluorescent antibodies.     

Inoculation of Scytalidium thermophilum in Button Mushroom Compost and Its Effect on Yield

Scytalidium thermophilum isolates in culture, as well as the endogenous strain(s) in mushroom compost, were inactivated at 70°C. This temperature was used to pasteurize composts for experiments. Of nine thermophilic fungal species, only S. thermophilum and Myriococcum thermophilum grew well on pasteurized compost in test tubes. The effect of both species on the crop yield of Agaricus bisporus mushrooms was studied. In solid-state fermentation rooms called tunnels, compost was pasteurized and inoculated. After incubation, the inoculated organisms were reisolated and counted, showing their successful colonization. The yield of mushrooms on inoculated composts was almost twice that on the pasteurized control. This result demonstrates the effectiveness of S. thermophilum in compost preparation. Inoculation is not necessary for traditional compost preparation. Naturally occurring strains of S. thermophilum, present in ingredients, readily colonize compost during preparation. Inoculation may be vital if compost is pretreated at a high temperature in tunnels. This finding is of relevance for the environmentally controlled production of high-yielding compost.     

Properties of natural 19S antibodies in normal pig serum against the ΦX 174 and T2 phages

The physicochemical properties of natural phage-neutralizing antibodies were studied. Natural neutralizing antibodies against phages ΦX 174 and T 2 were found in the 19 S fraction isolated from normal pig serum by gel filtration on a Bio-Gel P-300 column. The 7 S fraction of normal pig serum possessed no neutralizing activity. The neutralizing activity of the 19 S fraction against phage ΦX 174 was not modified either by inactivation or by the addition of neutralizing cofactor; its activity against phage T 2 was lost by inactivation, but was restored by the addition of cofactor. The neutralizing activity of the 19 S fraction of normal pig serum was completely destroyed by 2-mercaptoethanol and was not restored by the subsequent addition of antibody cofactor. The results of attempts to release phage ΦX 174 from the neutralization complex with normal porcine serum 19 S macroglobulin antibody by the dilution method were the same as those of attempts to dissociate phage from the complex with 7 S type hyperimmune antibody. The virus particle was firmly and irreversibly adsorbed to both types of antibody and was not released by dilution. It is concluded from the results that neutralization of phage ΦX 174 by 19 S macroglobulin molecule of antibody is a simple, irreversible process, for which the thermolabile nonspecific serum components are not required.     

Plasmodium berghei: vaccination of mice against malaria with heat inactivated parasitized blood

Heat inactivated Plasmodium berghei-infected blood acted as a vaccine against P. berghei infection in mice. The heat inactivated blood was noninfective. Intact or splenectomized vaccine-treated mice, as well as P. berghei susceptible mice inoculated with whole blood or homogenized spleens from vaccine-treated animals, did not become infected. A/J, DDS and Carworth CF₁ mice were all protected against P. berghei challenge after vaccination. A/J and DDS mice developed good immunity after a single vaccination injection. Similar levels of immunity were obtained in CF₁ mice after at least two vaccine injections. Immunized mice responded to P. berghei challenge with mild anemias and low level parasitemias. Resolution of infection occurred between the first and third weeks after challenge. Nonvaccinated mice developed progressive anemia and parasitemia during the same time period. The immunity appears to be caused by P. berghei antigens; it could not be induced by homologous or heterologous noninfected red blood cells, P. gallinaceum-infected blood or Freund's Complete Adjuvant.     

Schistosoma japonicum: A PCR assay for the early detection and evaluation of treatment in a rabbit model

A specific PCR assay for the detection of Schistosoma japonicum DNA in rabbit fecal and serum samples was developed by amplifying a 230-bp fragment from the sequence information of the clone G55A of the highly repetitive retrotransposon SjR2. The minimum amount of DNA detectable using the PCR assay was 0.8 pg, and the expected PCR product was amplified when DNA equivalent of 1.1 egg from feces was used as template. In the meantime, serum anti-worm IgG was examined by ELISA. ELISA gave positive results at 4-6 weeks post-infection depending on the cercarial doses. The parasite eggs were detected in feces at 7 weeks post-infection. In contrast, S. japonicum DNA was detected in sera at first week post-infection, and it became negative at 10 weeks post-treatment, whereas the anti-worm IgG was still at high levels at 23 weeks post-treatment. These data demonstrated that the PCR assay established provides a potential tool for the early diagnosis and therapy evaluation for S. japonicum infection in humans.     

Biologically active abietane and ent-kaurane diterpenoids and other constituents from Erythroxylum suberosum

Bioassay-guided fractionation of the ethanol extract from the branches of Erythroxylum suberosum, which was toxic to brine shrimp larvae, afforded five diterpenes bearing abietane and ent-kaurane-type skeletons from an active fraction. From these, four were new, 7-oxo-16-hydroxy-abiet-15(17)-en-19-al, 16-hydroxyabiet-15(17)-en-7-one, 7α,16-dihydroxy-abiet-15(17)-en-19-al and ent-12α-hydroxy-kaur-16-en-19-al, while methyl ent-7α,15β-dihydroxy-kaur-16-en-19-oate is reported for the first time as a natural product. This is also the first reported occurrence of abietane-type diterpenes in the genus Erythroxylum. The flavonoid ombuin-3-rutinoside was isolated from an inactive fraction, while rutin (quercetin-3-rutinoside) was obtained from the non-toxic ethanol extract of the leaves. The structures of the new and known compounds were established by analyses of 1D- and 2D-NMR and mass spectrometry data.     

Serum-Dependent Enhancement of Coxsackievirus B4-Induced Production of IFNα, IL-6 and TNFα by Peripheral Blood Mononuclear Cells

Only a few reports have been published on the interactions between Coxsackievirus B4 (CVB4) and human peripheral blood mononuclear cells (PBMC) but have not been extensively documented. Human serum containing non-neutralizing anti-CVB4 antibodies increased CVB4-induced synthesis of IFNα by PBMC. In this study, we determined if CVB4 and human serum have the ability to activate inflammatory cytokines in addition to IFNα in PBMC cultures. PBMC from healthy donors were inoculated with infectious, inactivated CVB4 or with CVB4 incubated with dilutions of human serum or polyvalent IgG with anti-CVB4 activity. Levels of IFNα, TNFα, IL-6, IL-12, IFNγ and IL-10 in the cell-free supernatants of PBMC cultures were measured using ELISA. Infection was assessed by real-time PCR. PBMC inoculated with CVB4 produced inflammatory cytokines but not IFNα. When CVB4 was incubated with serum or IgG, IFNα was detected in the culture supernatants, and high concentrations of TNFα and IL-6 were measured. The concentrations of TNFα and IL-6 were not reduced in cultures inoculated with inactivated CVB4, whereas the IgG-dependent enhancement of IFNα, IL-6 and TNFα production with inactivated virus was suppressed. The potentiation of IFNα production was associated with a high intracellular viral load. Infectious and non-infectious CVB4 can induce the production of inflammatory cytokines but not IFNα by PBMC. High levels of IFNα, in addition to TNFα and IL-6, in culture supernatants were obtained when infectious CVB4 was combined with immune serum or IgG, and they were associated with high amounts of intracellular viral RNA.     

The simultaneous production of two classes of cytoplasmic immunoglobulins by single cells in Xenopus laevis

The classes of cytoplasmic immunoglobulins of individual lymphoid cells from the spleen and peripheral blood of normal and immunized Xenopus laevis were investigated. Immunofluorescence microscopy and simultaneous double staining of the cytoplasm, using a mixture of class-specific TRITC-anti-19S Ig and FITC-anti-7S Ig conjugated antisera, showed that 70% of the immunoglobulin producing cells are “double producers” and contain the two classes of 19S and 7S immunoglobulins. The remaining 30% are composed of “single producers.” The proportion of splenic cells containing either 19S Ig or 7S Ig, respectively, was unequal, and reversed between two groups of animals, one examined at the primary immune response (29% 19S Ig and 4% 7S Ig) and the second group examined at the secondary immune response (8% 19S Ig and 30% 7S Ig). A spontaneous lymphoid tumor with an increased serum level of 19S Ig was examined in the same way. The tumor cells appear to produce 19S Ig exclusively.     

Detection of Amsacta moorei entomopoxvirus and vaccinia virus proteins in cell cultures restrictive for poxvirus multiplication

The titer of Amsacta entomopoxvirus (EPV) protein detected in murine L-929 cells by enzyme-linked immunosorbent assay (ELISA) decreased to within preimmune serum levels by 24 hr after inoculation of the virus which indicates that Amsacta EPV structural protein biosynthesis does not occur in the vertebrate cell line. A viral-induced protein of approximately 100,000 M_r was detected by [³⁵S]methionine incorporation 4 hr after inoculation of Tn-368 cells with Amsacta EPV. Biosynthesis of protein which reacted with vaccina antiserum was detected in Estigmene acrea (BTI-EAA) cells by ELISA 10 hr after inoculation with 10 PFU of virus per cell. The amount of putative vaccinia structural protein detected in BTI-EAA cells increased approximately twofold by 70 hr after virus inoculation. No increase in vaccinia structural protein biosynthesis was detected in BTI-EAA cells inoculated with vaccinia virus previously inactivated by heat and UV light.     

Effect of Spatial Separation of Pigs on Spread of Streptococcus suis Serotype 9

The spread of an infectious agent in a population can be reduced by interfering in the infectiousness or susceptibility of individuals, and/or in their contact structure. The aim of this study was to quantify the effect of prevention of direct contact between infectious and susceptible pigs on the transmission of Streptococcus suis (S. suis). In three replicate experiments, S. suis-free pigs were housed in boxes either in pairs (25 pairs) or alone (15 pigs). The distance between the boxes was ±1 m. At 7 weeks of age, one pig of each pair was inoculated intranasally with S. suis serotype 9; the other pigs were exposed to S. suis by either direct (pairs) or indirect contact (individually housed pigs). Tonsillar brush and saliva swab samples from all pigs were collected regularly for 4 weeks post inoculation to monitor colonization with S. suis. All inoculated pigs became infected, and their pen mates became colonized within 2 days. Thirteen indirectly exposed pigs became positive within 7–25 days after exposure. The rate of direct transmission β_dir was estimated to be 3.58 per pig per day (95% CI: 2.29–5.60). The rate of indirect transmission increased in time, depending on the cumulative number of days pigs tested positive for the presence of S. suis. The estimate β’_ind was 0.001 (95% CI: 0.0006–0.0017) new infections per pig per day for each day that an infected pig was tested positive for S. suis. We conclude that prevention of direct contact reduces the rate at which susceptible pigs become colonized. Simulation studies using these parameters showed, however, that such intervention measure would not limit S. suis serotype 9 spread in a commercial pig farm to a relevant extent, implying that spatial separation of groups op pigs within a compartment would not be effective on a farm.     

Influenza C Virus Infection in Rats

Four-week-old rats (WKA/Hkm strain) were infected intranasally with the Ann Arbor/1/50 strain of influenza C virus and examined for clinical symptoms, virus replication, and serum antibody response. Although the animals showed no definite signs of illness, the virus replicated in the nose, and the hemagglutination-inhibiting (HI) and neutralizing antibodies were produced in their sera. When the inoculum sizes of 10^6.2 and 10^3.2 PFU were used, virus was recovered from nasal homogenates between days 1 and 10, and serum HI antibody became detectable by 10 days after infection. The rats infected with 10^1.2 PFU of the virus continued to shed virus until as late as day 20 without producing serum HI antibody. The amount of virus recovered from the nose was not affected significantly by either sex. age, or strain of the rat except that a slower virus growth was seen in the LE strain. It was also observed that the rats, previously inoculated with 10^3.2 PFU of the virus, showed no virus shedding when reinfected 7 weeks later but produced virus though in low titers when reinfected 50 to 55 weeks later. Virus was also recovered from rats once inoculated with 10^1.2 PFU of the virus when challenged 7 weeks later. Thus repeated infections characteristic of human influenza C can be produced in rats under the restricted conditions.     

Viricidal Effects of Lactobacillus and Yeast Fermentation

The survival of selected viruses in Lactobacillus- and yeast-fermented edible waste material was studied to determine the feasibility of using this material as a livestock feed ingredient. Five viruses, including Newcastle disease virus, infectious canine hepatitis virus, a porcine picornavirus, frog virus 3, and bovine virus diarrhea, were inoculated into a mixture of ground food waste (collected from a school lunch program) containing Lactobacillus acidophilus. Mixtures were incubated at 20, 30, and 40°C for 216 h. In a second trial, four viruses, including Newcastle disease virus, infectious canine hepatitis virus, frog virus 3, and a porcine picornavirus, were inoculated into similar edible waste material containing Saccharomyces cerevisiae. Mixtures were incubated at 20 and 30°C for 216 h. Samples were obtained daily for quantitative (trial 1) and qualitative (trial 2) virus isolation. Temperature, pH, and redox potential were monitored. Controlled pH and temperature studies were also done and compared with the inactivation rates in the fermentation processes. In trial 1 (Lactobacillus fermentation), infectious canine hepatitis virus survived the entire test period in the fermentation process but was inactivated below pH 4.5 in the controlled studies. Newcastle disease virus was inactivated by day 8 in the fermentation process and appeared to be primarily heat sensitive and secondarily pH sensitive in the controlled studies. The porcine picornavirus survived the fermentation process for 8 days at 20°C but was inactivated more rapidly at 30 and 40°C. The controlled studies verified these findings. Frog virus 3 was inactivated by day 3 in the fermentation process and appeared to be sensitive to low pH in the controlled studies. Bovine virus diarrhea was rapidly inactivated in the fermentation process (less than 2 h) and was pH and temperature sensitive. In trial 2 (yeast fermentation), infectious hepatitis virus survived the entire test period in the fermentation process. Newcastle disease virus was inactivated by day 7 at 20°C and day 6 at 30°C. The porcine picornavirus was inactivated by day 7 at 30°C but survived the entire test period at 20°C. Frog virus 3 was inactivated by day 3 at 20°C and day 2 at 30°C.     

Survival of Salmonella montevideo on Wheat Stored at Constant Relative Humidity

Eleven samples of Ottawa variety hard red winter wheat were inoculated with a standardized suspension of Salmonella montevideo. The contaminated wheat samples were placed in constant relative humidity (RH) chambers held at 25 C. Relative humidities were 7, 11, 22, 33, 43, 53, 62, 75, 84, 92, and 98%. Constant RH at 25 C was maintained with different saturated salt solutions in the sealed chambers. Periodic counts of viable S. montevideo cells per gram of wheat were made over a 28-week sampling period. Viable counts of S. montevideo on wheat held at 7, 11, and 22% RH decreased from an initial 10⁶ cells/g of wheat to a final count of 10⁴ cells/g in each sample. Samples stored at 33, 43, 53, and 62% RH decreased from 10⁶ viable cells/g to 3.6 × 10³, 10³, 10², and 20 viable cells/g, respectively. No viable S. montevideo cells were detected in the samples held at 75, 84, 92, and 98% RH after 22, 16, 26, and 16 weeks, respectively.     

Demonstration of human pancreatic anionic trypsinogen in normal serum by radioimmunoassay

A specific radioimmunoassay for human pancreatic anionic trypsin has been developed. The trypsin employed as radioiodinated tracer in the assay was inactivated with tosyl-L-lysine chloromethyl ketone in order to prevent binding of the tracer to the serum inhibitors α₁-antitrypsin and α₂-macroglobulin. A normal serum level of immunoreactive anionic trypsin of 5.45 ng/ml was determined. The results of experiments in which serum was fractionated by Sephadex G-200 gel filtration suggest that essentially all of the immunoreactive material in normal human serum is trypsinogen. This finding implies that a small fraction of the zymogens synthesized in the pancreas are released directly into the circulation.     

Trypanosoma equiperdum: Movement from the dermis

Twelve dogs were injected intradermally with 352,770 to 14,391,660 Trypanosoma equiperdum and afferent and efferent lymph, lymph nodes, and blood examined by mouse inoculation at minute, hourly, and daily intervals following inoculation. The log dosage of trypanosomes given each dog was closely related to their body weight (P < 0.01). Afferent lymph contained trypanosomes as soon as 5 and 27 min after inoculation. Lymph nodes on the side of injection became positive within 5 min of injection, while those on the contralateral side remained negative for at least 120 min after injection. Blood contained trypanosomes as soon as 5 min after injection, although the average time for all dogs, before trypanosomes were demonstrated in the blood, was 40 min postinjection. Efferent lymph did not contain organisms until 25–76 hr after inoculation. We consider this sequence to indicate that T. equiperdum can leave the dermis in afferent lymphatics, reach the local lymph node, invade the blood stream from this site, and only after a day or longer do they leave the node via the efferent lymphatics.     

Passive protection of mice with the 19 S and 7 S fractions of anti-pertussis sera in experiment.

Protective activity of anti-persussis rabbit and mouse sera and 19 S and 7 S fractions obtained from these sera was investigated in the test of passive protection of mice on a model of pertussis meningoencephalitis. The method of simultaneous intracerebral administration of the serum or fraction with live culture of a virulent B. pertussis strain was used. Hyperimmune rabbit serum containing mercaptoethanol-resistant agglutinins in a high titre was found to have the most pronounced protective effect. Serum of mice, collected 14 days after single immunization of the animals, did not show any protective properties. A small amount of protective activity was observed in the serum collected on the 30th day after a single administration of the vaccine. A sharp increase in the protective activity of the serum was observed after double immunization of mice. Correlation was found between the increase in the titre of agglutinins (in particular of 7 S antibodies) and the protective activity of the serum. Protective properties of 19 S and 7 S fractions isolated from immune rabbit and mouse sera by the method of gel filtration were investigated. Both fractions were found to possess protective properties, but fraction 7 S was more active than fraction 19 S.     

Construction of Otherwise Isogenic Serotype 6B, 7F, 14, and 19F Capsular Variants of Streptococcus pneumoniae Strain TIGR4

The polysaccharide capsule is the primary virulence factor in Streptococcus pneumoniae. There are at least 90 serotypes of S. pneumoniae, identified based on the immunogenicity of different capsular sugars. The aim of this study was to construct pneumococcal strains that are isogenic except for capsular type. Serotype 4 strain TIGR4 was rendered unencapsulated by recombinational replacement of the capsular polysaccharide synthesis (cps) locus with the bicistronic Janus cassette (C. K. Sung, J. P. Claverys, and D. A. Morrison, Appl. Environ. Microbiol. 67:5190-5196, 2001). In subsequent transformation with chromosomal DNA, the cassette was replaced by the cps locus derived from a strain of a different serotype, either 6B, 7F, 14, or 19F. To minimize the risk of uncontrolled recombinational replacements in loci other than cps, the TIGRcps::Janus strain was “backcross” transformed three times with chromosomal DNA of subsequently constructed capsular type transformants. Capsular serotypes were confirmed in all new capsule variants by the Quellung reaction. Restriction fragment length polymorphism (RFLP) analysis of the cps locus confirmed the integrity of the cps region transformed into the TIGR strain, and RFLP of the flanking regions confirmed their identities with the corresponding regions of the recipient. Transformants had in vitro growth rates greater than or equal to that of TIGR4. All four strains were able to colonize C57BL/6 mice (female, 6 weeks old) for at least 7 days when mice were intranasally inoculated with 6 × 10⁶ to 8 × 10⁶ CFU. The constructed capsular variants of TIGR4 are suitable for use in studies on the role of S. pneumoniae capsular polysaccharide in immunity, colonization, and pathogenesis.     

Promotion of Infection Thread Formation by Substances from Rhizobium

An extrinsic substance (ES-6000) was isolated from the periplasmic space of Rhizobium trifolii (strain 4S) cells by osmotic shock, using a high-density sucrose solution. This substance promoted infection thread formation in root hairs of white clover when inoculated together with the infectious strain (4S). However, ES-6000 obtained from another rhizobial species and from strain A1, which is a noninfectious mutant strain obtained from strain 4S, did not have this effect. The promoter in the ES-6000 from strain 4S is a relatively small molecule since it passed through a hollow-fiber membrane (molecular weight, 6,000). This substance was also recognized as an R_f 0.1 fraction by paper chromatography. Sucrose was effective in promoting nodulation and root elongation.     

Experimental Neospora caninum infection in chickens (Gallus gallus domesticus) with oocysts and tachyzoites of two recent isolates reveals resistance to infection

The importance of birds in the biological cycle of Neospora caninum is not clear. We report unsuccessful Neospora infection in chickens (Gallus gallus domesticus) using two isolates of N. caninum. In experiment #1, 30 White Leghorn chickens were orally inoculated with viable N. caninum oocysts (NC-SP1 isolate, 200 oocysts per bird) via the crop at 21 days of age. Groups of three birds were euthanised at intervals of 7 days (a total of 9 weeks) and one group was challenged with the same oocyst dose at 37 days p.i. and observed for 11 weeks. Blood samples were collected weekly, and sera were tested using IFAT. Chicken tissues were collected for PCR, quantitative PCR and immunohistochemistry. Two dogs approximately 45 days of age were fed with tissues from chickens euthanised at 138 and 159 days p.i. The results indicated that the chickens were resistant to neosporosis as revealed by failure to seroconvert, to detect parasite DNA or N. caninum antigen by immunohistochemistry in inoculated bird tissues, and by no oocyst excretion by the dogs fed avian tissues. Similar results were obtained in experiment #2, in which 34 1-week-old chickens were each s.c. inoculated with 100,000 tachyzoites of the NcWTDMn1 isolate of N. caninum. The chickens were euthanised on days 7, 15, 22, 28, 36 and 60 p.i. At necropsy, all tissues and serum from each bird were collected. All chickens remained asymptomatic, and N. caninum antigen was not detected by immunohistochemistry. Seven chickens euthanised at day 60 p.i. demonstrated low (1:25 dilution) levels of antibodies by using the Neospora agglutination test. Two 12-week-old dogs fed tissues pooled from 10 inoculated chickens euthanised at day 60 p.i. did not excrete N. caninum oocysts. This investigation indicates that chickens are resistant to experimental infection by N. caninum.