Effects of aerosolized dust in goats on lung clearance of Pasteurella and Mannheimia species

The objective was to determine whether the inhalation of large quantities of feedyard dust predisposed the animals to pulmonary bacterial proliferation. Two control groups, C1 and C2, did not receive dust treatments, and two principal groups (P1 and P2) received a total of 14 dust treatments each. The C1 and P1 groups of goats each received a transthoracic challenge of live Mannheimia haemolytica (4 x 10(6) colony forming units, CFU) The C2 and P2 groups of goats each received a transthoracic challenge of live Pasteurella multocida (1.0 x 10(6) CFU/goat). The results showed that dusted animals had fever when compared with non-dusted controls. In addition, dusted animals demonstrated a leukocytosis with neutrophilia after the first dust treatment that was not sustainable. Finally, dusted animals demonstrated pulmonary clearance of two potential bacterial pathogens that was not significantly different from that shown by control (not dusted) animals.     

Cross-Protection Studies with Three Serotypes of Pasteurella haemolytica in the Goat Model

Cross-protection studies employing three serotypes of Pasteurella haemolytica (Ph) were performed in goats, with challenge exposure by transthoracic injection. Indirect hemagglutination (IHA) serum titers showed that the herd had been naturally infected with Ph biovar A, serovar 2 (PhA2) prior to the study. Sixty-four weanling male Spanish goats were randomly allotted to 16 groups. Fifteen goats were given two transthoracic injections into the lungs 21 days apart with live Pasteurella haemolytica biovar A, serovar 1 (PhA1) in agar beads. Fifteen goats were given two transthoracic injections into the lungs 21 days apart with live PhA2 in agar beads. Sixteen goats were given two transthoracic injections into the lungs 21 days apart with live P. haemolytica biovar A, serovar 6 (PhA6) in agar beads. Eighteen control (CON) goats were given two transthoracic injections into the lungs 21 days apart with agar beads alone. Fourteen days after the second injection, goats were challenge-exposed to either live PhA1, PhA2, or PhA6 by transthoracic injection into the lung, and 4 days later, all goats were euthanatized and necropsied. Serum antibody to P. haemolytica antigens was measured throughout the experiment. Mean volumes of consolidated lung tissue for the CON goats challenged with PhA1, PhA2, and PhA6 were 28.29 cm³, 8.36 cm³, and 16.29 cm³, respectively. Mean volumes of consolidated lung tissue for the PhA1-immunized goats challenged with PhA1, PhA2, and PhA6 were 4.38 cm³, 0.25 cm³, and 1.90 cm³, respectively. Mean volumes of consolidated lung tissue for the PhA2-immunized goats challenged with PhA1, PhA2, and PhA6 were 9.68 cm³, 0.05 cm³, and 3.39 cm³, respectively. Mean volumes of consolidated lung tissue for the PhA6-immunized goats challenged with PhA1, PhA2, and PhA6 were 14.05 cm³, 1.27 cm³, and 4.53 cm³, respectively. These data demonstrate protection in immunized goats challenged with the homologous serotype of P. haemolytica. PhA1-immunized animals were protected against serotype 2 challenge as well as against serotype 6 challenge. PhA2-immunized animals were not protected against serotype 1 challenge, but were protected against transthoracic PhA6 challenge. PhA6-immunized animals were not protected against serotype 1 challenge, but were protected against transthoracic PhA2 challenge. There appears to be some cross-protection among the P. haemolytica serotypes, and this fact should be taken into consideration when developing vaccines against this organism. Received: 13 June 1997 / Accepted: 6 October 1997     

Pasteurella haemolytica Ultraviolet-Irradiated Vaccine by Parenteral and Aerosol Routes Compared in the Goat Model

Positive control 1 (PC1) (n = 9) goats were injected transthoracically into the left lung with live Pasteurella haemolytica biovar A, serovar 1 (PhA1) in polyacrylate (PA) beads on days 0 and 21. Positive control 2 (PC2) (n = 6) goats were nebulized with live PhA1 and PA beads on days 0 and 21. Negative control (NC) goats (n = 6) were each injected transthoracically into the left lung with PA beads alone on days 0 and 21. Four groups (n = 6) were administered PA beads mixed with ultraviolet (UV) killed PhA1 on days 0 and 21. The treatment doses of bacteria for these groups were principal group 1 (PR1) injected into the left lung (7.7 × 10¹⁰ cfu); PR2, 7.7 × 10¹⁰ UV-killed PhA1 injected subcutaneously (SC); PR3, 7.7 × 10¹⁰ UV-killed PhA1 injected SC only on day 21; PR4, nebulized with PA beads mixed with 5.6 × 10¹⁰ cfu of UV-killed PhA1; and PR5, nebulized with PA beads mixed with 5 × 10⁸ cfu of UV-killed PhA1. All goats were challenged transthoracically in the right lung with 1 × 10⁸ cfu of live PhA1 on day 42 and necropsied on day 46. The sizes of consolidated lung lesions at the challenge site were used as a measure of immunity. The data show that the introduction of live PhA1 into the lungs of goats, either by injection or aerosolization, offers excellent protection against a subsequent homologous challenge. The data also demonstrate that two transthoracic injections (21 days apart) of UV-killed PhA1 (PR1), and subcutaneous injection of UV-killed PhA1(PR2) also offer excellent protection against a subsequent homologous live PhA1 challenge. One SC injection of UV-killed PhA1 (PR3) appears to offer only partial protection against a subsequent homologous live PhA1 challenge. Inhalation of UV-killed PhA1 mixed with PA beads (PR4 and PR5) induced no protection in goats against a subsequent live PhA1 transthoracic challenge. Received: 3 February 1998 / Accepted: 18 March 1998     

In vivo production of neuraminidase by Pasteurella multocida A:3 in goats after transthoracic challenge

Six goats were injected transthoracically with live Pasteurella multocida A:3 to examine if an extracellular enzyme, neuraminidase, was produced in vivo during infection with this organism. The principal group of goats (n = 6) each received 1 ml of live 7.5 × 10⁴ cfu of P. multocida mixed with polyacrylate beads transthoracically in the left lung on day 0 and 1 ml of live P. multocida (2.2 × 10⁸ cfu) mixed with polyacrylate beads transthoracically in the left lung on day 22. Six goats were used as negative controls and received 0.3 g of polyacrylate beads subcutaneously in the right flank on days 0 and 22. Serum was obtained from all animals on days 0, 7, 14, 22, 29, and 36. Preimmune sera from all animals showed no detectable antibody to P. multocida A:3 neuraminidase in an enzyme neutralization assay. None of the sera from the negative control animals demonstrated a significant antibody titer against the P. multocida A:3 neuraminidase. On day 36, serum samples from the six infected animals possessed complete enzyme-neutralizing activity. Anti-neuraminidase antibody could be detected as early as day 14 in the infected animals. These data show that neuraminidase is produced in vivo during an active P. multocida A:3 lobar infection. Received: 16 March 1996 / Accepted: 19 April 1996     

Effect of Lactobacillus fermentum alone, and in combination with zinc(II) propionate on Salmonella enterica serovar Düsseldorf in Japanese quails

The influence of zinc(II) propionate on the efficacy of Lactobacillus fermentum against Salmonella enterica serovar Düsseldorf was tested in Japanese quails. Twenty one 3-day old Japanese quails were divided into 3 groups each consisting of 7 birds and inoculated orally: (i) group A (control) with Rogosa broth; (ii) group B with rifampicin-resistant L. fermentum (2.5 × 10⁷ CFU/animal); and (iii) group C with rifampicin-resistant L. fermentum cultured in Rogosa broth supplemented with zinc(II) propionate (1 g Zn²⁺ /L of broth; i.e. 2.5 × 10⁷ CFU/animal and 0.1 mg Zn²⁺ /animal). After 16 hours all birds were infected with a single dose of S. enterica serovar Düsseldorf (5.8 × 10⁷ CFU /animal). During the next 6 days the chicks received the same inoculations as they had received earlier through the medium of drinking water. The viable counts of Salmonella, rifampicin-resistant lactobacilli and total aerobes in the feces and cecal content, zinc concentration in the cecal content and growth performance were evaluated. L. fermentum alone, and in combination with Zn(II) propionate significantly reduced shedding of Salmonella in the feces and also the amount of salmonellae present in cecal content as compared to control. The numbers of Salmonella in group C in all collections were lower than in group B, but a significant difference (p < 0.05) was noted only 24 h after infection. The viable counts of rifampicin-resistant lactobacilli were similar in groups B and C and numbers of total aerobes were reduced in these groups compared with control. L. fermentum and its combination with zinc(II) propionate increased daily weight gains in the chicks in comparison with control. Zinc concentration in the control and C group was 34.9 ± 6.2 mg · kg⁻¹ and 676.3 ± 106.6 mg · kg⁻¹ of cecal content, respectively. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

The use of copper and silver in carbon point-of-use filters for the suppression of Legionella throughput in domestic water systems

Aims: To evaluate throughput of seeded Legionella pneumophila bacteria in domestic point‐of‐use filters. Methods and Results: The filters were challenged with tap water seeded with Leg. pneumophila. After multiple challenge events (4·25 × 10¹¹ CFU per filter), the levels of Legionella were lower in the effluent from the filter containing both copper and silver (mean 4·48 × 10³ CFU ml^?1) than in the effluent from the filter containing copper only (1·26 × 10⁴ CFU ml^?1; P < 0·001). After a single challenge event of approx. 5 × 10⁹ CFU L. pneumophila per filter, there was no significant difference between the levels of Legionella in the effluents from a carbon filter containing copper and a carbon filter with no metals (mean 6·87 × 10² and 6·89 × 10² CFU ml^?1, respectively; P = 0·985). Conclusions: Legionella was detected in filter effluent up to 6 weeks after being challenged, indicating that while filters may reduce the levels during an initial contamination event, the exposure is extended as the accumulated bacteria slough off over time. Significance and Impact of the Study: This study has provided an understanding of the response of Legionella to the use of silver and copper in domestic point‐of‐use carbon filters.     

Protective Immunity to Sarcocystis capracanis-Induced Abortion in Dairy Goats1

ABSTRACT. Eleven female goats (Nos. 1 to 11) were each inoculated orally with 10⁴ sporocysts of Sarcocystis capracanis, and four female goats (Nos. 12 to 15) were not inoculated. Between 31 and 69 days after inoculation (DAI) goats were mated with a single buck; one goat (No. 5) did not breed. Eight inoculated goats were challenged with 10⁵or 10⁶ sporocysts, 135 DAI. Two of four goats challenged with 10⁶ sporocysts and one of three goats challenged with 10⁵ sporocysts aborted one month before the expected time of parturition. The three inoculated goats that were not challenged delivered healthy kids. All inoculated goats including the nonpregnant one (No. 5) were only mildly ill from the primary or challenge inoculations. Two of the four control goats challenged with 5 × 10⁴ or 10⁵ sporocysts aborted 21 days later, and both died of sarcocystosis 25 and 88 DAI. The two remaining control goats delivered normal kids. The results indicate that immunization prior to pregnancy protects some but not all goats from .Sarc0c.es/is-induced abortion.     

Utilization of oligo‐ and polysaccharides at microgram‐per‐litre levels in freshwater by Flavobacterium johnsoniae

Aims: To obtain a bacterial strain that can be used to quantify biodegradable polysaccharides at concentrations of a few micrograms per litre in freshwater. Methods and Results: Flavobacterium johnsoniae strain A3 was isolated from tap water supplemented with laminarin, pectin or amylopectin at 100 μg C l^?1 and river Rhine water. The organism utilized 14 of 23 oligo‐ and polysaccharides, and 1 of 9 monosaccharides, but none of the sugar acids, sugar alcohols, carboxylic acids or aromatic acids tested at 10 μg C l^?1. Amino acids promoted growth of strain A3, but not in coculture with assimilable organic carbon (AOC) test strain Pseudomonas fluorescens P17, which utilized these compounds more rapidly than strain A3. Compounds released by strain P17 and AOC test strain Spirillum sp. NOX grown on acetate promoted the growth of strain A3 at N_max values of ≥ 2 × 10⁵ CFU ml^?1 of strain P17 and ≥ 5 × 10⁵ CFU ml^?1 of strain NOX. Significant growth of strain A3 was observed in surface water and in tap water in the presence of strain P17 (N_max P17 < 2 × 10⁵ CFU ml^?1). Conclusions: Strain A3 utilizes oligo‐ and polysaccharides at microgram‐per‐litre levels. In surface water and in tap water, the organism was able to utilize compounds that were not utilized by strain P17. These compounds may include oligo‐ and/or polysaccharides. Significance and Impact of the Study: Phytoplanktonic and bacterial polysaccharides can constitute an important biodegradable fraction of natural organic matter in water and may promote growth of heterotrophic bacteria during water treatment and drinking water distribution. Strain A3 can be used to quantify a group of compounds that includes oligo‐ and polysaccharides at microgram‐per‐litre levels in freshwater.     

Oral Administration of Live <Emphasis Type="Italic">Bifidobacterium</Emphasis> Substrains Isolated from Centenarians Enhances Intestinal Function in Mice

We studied the effects of two bifidobacteria strains isolated from healthy centenarians on intestinal function in mice. Bifidobacterium adolescentis BBMN23 and Bifidobacterium longum BBMN68 were orally administrated to specific pathogen-free BALB/c mice at different doses (2 × 10¹¹, 2 × 10⁹, or 2 × 10⁷ CFU/kg body weight) each day for 4 weeks. Villus height, crypt depth, villus width, and villus/crypt ratio (V/C) were determined. The content of duodenal secreted immunoglobulin A (sIgA) was also evaluated. There were clear increases in height and width of duodenal villi in both treated groups. Crypt depths were deeper in animals treated with BBMN23 than in controls, while depths were reduced in animals receiving BBMN68. The V/C ratio was increased after feeding with BBMN68, while BBMN23 had no significant effect. Both strains improved the sIgA content of the duodenum. These results suggest that BBMN23 and BBMN68 may improve intestinal digestion and ability and enhance immune barrier function in the intestine.     

Prevalence of fungi in carpeted floor environment: Analysis of dust samples from living-rooms,bedrooms, offices and school classrooms

The results of 100 carpet dust analyses from atopic individuals' environment were compared according to the sampling period or the location. Dust samples were collected with a standard domestic vacuum cleaner, in locations with carpeted floor: in residences (living-room and/or bedroom), in school classrooms and in offices. The quantities of fungi vary from 5000 CFU/g to 66 000 000 CFU/g of dust. More than 100 species were isolated by dilution plating. The main species found in carpet dust wereEurotium repens, Penicillium chrysogenum, Alternaria alternata, Aureobasidium pullulans andPhoma herbarum. Strict xerophilic species were rather rare and detected in small quantities. Differences in the distribution of the CFU concentrations were examined for the four different sampling locations and were statistically significant (P=0.0174). In this study, schools were open spaces, and offices, mostly with air conditioning systems, were locations in which air is not confined. This, added to frequent professional carpet cleaning, probably explains the lowest levels of fungal concentration found in these locations. The majority of the homes had the largest fungal concentration in the living-room (median: 2×10⁵ CFU/g) while some bedrooms (median: 7×10⁴ CFU/g) had the highest concentrations. It is suggested that, when fungi are suspected to be the origin of respiratory allergy or irritating symptoms, the mycoflora of the bedroom, principally, should be investigated first.     

Development of emulsion from rhizobial fermented starch industry wastewater for application as Medicago sativa seed coat

Starch industry wastewater was efficiently employed for the production of Sinorhizobium meliloti and the concentrated culture was used for the development of a biofertilizer formulation. Tween‐80 (0.02 g/L) acted as the best emulsifier for a Sinorhizobium–canola oil emulsion. The stability of the emulsion and survival of the organism was enhanced by supplementation of xanthan gum at pH 8. The refrigerated condition was most favorable for stability and survival of the microorganism. The survival of microorganism at 4±1°C was 2.78×10¹⁰ and 2.01×10¹⁰ CFU (colony forming unit)/mL on storage for 1 and 2 months, respectively. The values were higher than the prescribed cell count (×10³ CFU/mL) for field application. At 40°C, the survival of bacteria reduced from 3×10¹⁰ CFU/mL to 8.1×10⁹ and 8.8×10⁶ CFU/mL in 1 and 2 months, respectively. Emulsion‐coated seed was incubated at different temperatures and a cell count of 10⁵ CFU/seed was observed after 2 months of storage at 4°C, which was equal to the highest level of the described requirement (10³–10⁵ CFU/seed). Emulsion supplemented with xanthan gum improved the shelf‐life under optimized conditions (Sinorhizobium concentrate – canola oil (1:1) emulsion with 0.02 g/L Tween‐80; storage at pH 8 and temperature 4±1°C) and this emulsion with the required cell count and prolonged viability was used for the pre‐inoculation of seed or for in situ soil application.     

Experimental Pathogenicity of a Clinical Isolate of <Emphasis Type="Italic">Trichosporon dermatis</Emphasis> in a Murine Model

The pathogenicity of Trichosporon dermatis isolated from skin lesions of a patient has been examined in mice. Balb/c mice were treated with two intraperitoneal injections of 100 mg/kg cyclophosphamide on days 4 and 1 and one subcutaneous injection of 10 mg/kg dexamethasone on day 1 pre-inoculation, and then challenged with 0.2 ml T. dermatis inoculum (1 × 10⁸ CFU/ml) by topical application on an abrasive wound in the dermabrasive group and by hypodermic injection in the subcutaneous group. In the intravenous group, 0.2 ml of high (1 × 10⁸ CFU/ml) or low (1 × 10⁷ CFU/ml) inoculum was injected into the tail vein. Histopathology and inverse fungal culture were performed on the skin lesion and viscera, and renal fungal burden was also determined. Inoculated sites developed localized infections after dermabrasive and subcutaneous challenge in all mice, but the maximum area of skin lesions, and number of positive cultures from the lesions, were higher for immunocompromised mice. In the intravenous group, all immunocompetent animals survived during the four-week period, whereas 100 and 70% of immunocompromised animals died by 3 and 5 days in the high and low-inoculum groups, respectively. The incidence of disseminated infection and the renal fungal burden of immunocompromised mice were higher than those of immunocompetent mice. Our results demonstrate that subcutaneous and intravenous injection of T. dermatis can successfully establish cutaneous and systemic infection models in immunocompromised mice, with the kidney and lung being most susceptible.     

Intranasal Immunization with Yersinia enterocolitica O:8 Cellular Extract Protects against Local Challenge Infection

Yersinia enterocolitica is enteropathogenic for humans and rodents. Immune protection from oral and respiratory pathogens may be most effectively elicited following intranasal (i.n.) vaccination. An experimental murine intranasal challenge model was used to evaluate the immunogenicity of a Y. enterocolitica O:8 cellular extract (CE) in mucosa. This antigenic preparation has demonstrated to induce protection by subcutaneous immunization. Mice were immunized intranasally with two doses of CE. Immunized and nonimmunized animals were challenged with 5×10⁶ colony-forming units (CFU) by nasal infection. Antibodies in serum and bronchoalveolar lavage (b.a.l.) fluid were assessed before and 48 hr after challenge. The CFU were determined by analysis of lung homogenate samples. The CE immunization induced significant b.a.l.-specific IgA and IgG, and serum-specific IgG, IgA and IgM. Histopathological studies 24 and 48 hr postchallenge demonstrated that immunization protected against progressive lesions resulting from Y. enterocolitica invasion of the pulmonary mucosa. The CFU in the lungs showed that CE immunization led to significant clearance as compared to the bacterial level in nonimmunized controls. From the results obtained, it can be concluded that CE can induce local and systemic immunity and protect against nasal infection.     

Immune Response to Nocardia brasiliensis Extracellular Antigens in Patients with Mycetoma

The ability of culture-filtrate proteins to induce a cellular immune response in infected mice and humans was investigated. A crude extract culture filtrate of Nocardia brasiliensis (CFA) and five semi-purified CFA fractions (P1, P2, P3, P4, P5) were used to stimulate BALB/c mice spleen-cell cultures. The animals were divided into three groups: the first group was infected with 1 × 10⁷ CFU of N. brasiliensis in the footpad, the second group was immunized with heat-killed bacteria, and the third was injected with sterile saline. IFN-γ, IL-1α, and IL-4 concentrations were determined in culture supernatants. Protein fractions eliciting IFN-γ production in mice, as well as the CFA, were used to stimulate IFN-γ production and in vitro cell proliferation assays with peripheral blood mononuclear cells of patients with actinomycetoma by N. brasiliensis, individuals with pulmonary tuberculosis, and healthy controls. In mice, CFA and three of the protein fractions (P3, P4 and P5) induced significant IFN-γ production in the infected group. In humans, only the CFA-induced IFN-γ production and cell proliferation in the group of patients with actinomycetoma. There was no stimulation in tuberculosis patients nor healthy controls. These results suggest that some culture-filtrate antigens are recognized by patients with active actinomycetoma and do not cross-react with M. tuberculosis antigens, being therefore potential candidates to develop a diagnostic test.     

Airborne Penicillium in the grain shops of Nagpur (India)

The airborne Penicillium spp. and total airborne fungal spore concentration was investigated in the grain shops of Nagpur city, India, using a volumetric Hi‐Air sampler system Mark II (Hi Media Laboratories Ltd., India). The mycotoxins were analysed from the Penicillium isolates obtained from the seeds by thin layer chromatography.

The mean concentration of the total fungi isolated from different grain shops ranged from 7.8×10² to 1.1×10³ CFU/m³. The mean concentration of Penicillium isolated from the air of grain shops ranged from 8.6×10¹ CFU/m³ (10.8%) to 1.7×10² CFU/m³ (19.9%). Among the 13 species of Penicillium which were isolated, P. citrinum Thom was the most prevalent species (24.2%), followed by P. oxalicum Currie & Thom (16.5), P. digitatum Saccardo (8.9%), P. janthinellum Biourge (8.7%), P. funiculosum Thom (8.3%), P. chrysogenum Thom (6.4%), P. purpurogenum Stoll (6.2%), P. brevicompactum Dierckx (4.8%), P. frequentans Westling (4.2%), P. italicum Wehmer (3.8%), P. rubrum Stoll (3.4%), P. expansum Link (2.9%) and P. cyclopium Westling (1.6%).

Penicillium species were also isolated from seeds such as wheat, maize, soybean, and groundnut. The mycotoxins roquefortin C, citrinin, rubratoxin B, cyclopiazonic acid, verrucosidin, mitorubrinic acid and two unknown metabolites were isolated from Penicillium isolates.     

Activation of trehalase by heat shock in yeast ascospores: Correlation with total cellular cyclic-AMP content

An animal model resembling the human disease caused byCampylobacter jejuni has been developed. Characteristic illness followed intragastric challenge of neonatal mice with strains ofC. jejuni enhanced for virulence by serial intraperitoneal passage in weanling mice of organisms suspended in either mucin or iron dextran. Such passage lowered the LD₅₀ in weanlings from 2×10¹¹ colony-forming units (CFU) to 2×10⁵ CFU per mouse. Neonatal mice chellenged by intragastric intubation with 2×10⁹ CFU of the virulence-enhanced organisms suspended in mucin or iron dextran showed signs of infection by day 5, including severe diarrhea, increased musus discharge occasionally with blood, and reduced weight gain. Diarrhea contionued for eight days, after which most animals recovered. This mouse infection model provides a means for assessing the determinants of virulence among strains ofC. jejuni.     

A DNA vaccine candidate for <Emphasis Type="Italic">B. anthracis</Emphasis> immunization,pcDNA3.1+PA plasmid,induce Th1/Th2 mixed responses and protection in mice

The protective antigen (PA) of Bacillus anthracis (B. anthracis) is a potent immunogen and a candidate subunit vaccine. To address the question whether antibodies raised against PA following injection of pcDNA3.1+PA plasmid, encoding PA, can protect against virulent B. anthracis two different regimens of PA based vaccines (DNA and live spore) were used. The groups of BALB/c mice that received live spores of the Sterne strain, naked pcDNA3.1 and naked pcDNA3.1+PA were compared to control groups. All groups were injected three times with 30-day intervals. Two weeks after the last immunization, all mice were subjected to challenge with a pathogenic strain of B. anthracis (C2). Blood samples were taken before each injection and challenge. Evaluation of the sera by ELISA method showed that DNA immunization using pcDNA3.1+PA plasmid resulted in an antibody profile representative of a mixed Th1 and Th2 response, with a skewing to a Th1 response. The group which received the naked pcDNA3.1+PA had a survival rate of >80%. This challenge assay revealed that antibodies raised following DNA vaccination against PA can confer strong protection, and resistance against virulent species of B. anthracis.     

Definition and Application of a Histopathological Scoring Scheme for an Animal Model of Acute Mycoplasma pneumoniae Pulmonary Infection

A histopathological scoring system was developed to assess the pathology of acute Mycoplasma pneumoniae pulmonary infection in a hamster model. A final score per animal (ranging 0–26) is obtained by averaging scores from each lung which have been accumulated by the addition of subscores from the assessments of quantity and quality of peribronchiolar and peribronchial infiltrates, luminal exudates, perivascular infiltrates, and parenchymal pneumonia. The scoring scheme was then applied to test the ability of a heat-killed inoculum to induce pulmonary pathology and to the trial of a 43 kDa protein-associated antigen as a vaccine immunogen. A heat-killed inoculum delivered by both intratracheal and intranasal routes did not induce pulmonary pathology compared to a live inoculum (respective mean scores 0.1, 6.7; P<0.01). Animals prevaccinated with the 43 kDa antigen developed an accentuated pathological response after live challenge compared to those unvaccinated (respective mean scores 16.8, 5.8; P=0.00007). Hypersensitization to growth medium components may, however, have contributed to the accentuated disease since the lungs of vaccinated animals challenged with culture-negative media also were affected (mean score 5.4). Reproducibility of the scoring system was measured by duplicate reading of histology slides which were randomized to the observer upon the second reading (r=0.93; P=0.000009). The scoring system has the ability to differentiate disease severity in small groups of animals.     

Protective immunity by oral immunization with heat‐killed Shigella strains in a guinea pig colitis model

The protective efficacy of and immune response to heat‐killed cells of monovalent and hexavalent mixtures of six serogroups/serotypes of Shigella strains (Shigella dysenteriae 1, Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, Shigella boydii 4, and Shigella sonnei) were examined in a guinea pig colitis model. A monovalent or hexavalent mixture containing 1 × 10⁷ of each serogroup/serotype of heat‐killed Shigella cells was administered orally on Days 0, 7, 14 and 21. On Day 28, the immunized animals were challenged rectally with 1 × 10⁹ live virulent cells of each of the six Shigella serogroups/serotypes. In all immunized groups, significant levels of protection were observed after these challenges. The serum titers of IgG and IgA against the lipopolysaccharide of each of the six Shigella serogroups/serotypes increased exponential during the course of immunization. High IgA titers against the lipopolysaccharide of each of the six Shigella serogroups/serotypes were also observed in intestinal lavage fluid from all immunized animals. These data indicate that a hexavalent mixture of heat‐killed cells of the six Shigella serogroups/serotypes studied would be a possible broad‐spectrum candidate vaccine against shigellosis.     

Murine Model of Disseminated Fusariosis: Evaluation of the Fungal Burden by Traditional CFU and Quantitative PCR

Systemic disease is the most severe clinical form of fusariosis, and the treatment involves a challenge due to the refractory response to antifungals. Treatment for murine Fusarium solani infection has been described in models that employ CFU quantitation in organs as a parameter of therapeutic efficacy. However, CFU counts do not precisely reproduce the amount of cells for filamentous fungi such as F. solani. In this study, we developed a murine model of disseminated fusariosis and compared the fungal burden with two methods: CFU and quantitative PCR. ICR and BALB/c mice received an intravenous injection of 1 × 10⁷ conidia of F. solani per mouse. On days 2, 5, 7, and 9, mice from each mice strain were killed. The spleen and kidneys of each animal were removed and evaluated by qPCR and CFU determinations. Results from CFU assay indicated that the spleen and kidneys had almost the same fungal burden in both BALB/c and ICR mice during the days of the evaluation. In the qPCR assay, the spleen and kidney of each mouse strain had increased fungal burden in each determination throughout the entire experiment. The fungal load determined by the qPCR assay was significantly greater than that determined from CFU measurements of tissue. qPCR could be considered as a tool for quantitative evaluation of fungal burden in experimental disseminated F. solani infection.