Photodynamic action inhibits compound 48/80-induced exocytosis in rat peritoneal mast cells.

Photostimulation of sulfonated aluminum phthalocyanine (SALPC)-loaded mast cells (20,000 lux, 2 min) itself caused neither exocytosis nor [Ca2+]i increase in isolated rat peritoneal mast cells. This result is incompatible with that reported in other cell types such as pancreatic acinar cells. Stimulation with 50 micrograms/ml compound 48/80, a direct G-protein activator, induced massive exocytosis which was easily detectable under conventional microscope. The fluorescent granules stained with sulforhodamine B were found to be numerous on the perimetry of mast cells, confirming occurrence of exocytosis. The stimulation also increased [Ca2+]i and cell volume before initiation of exocytosis. Pretreatment of the cells with photodynamic action with 5 microM SALPC inhibited the compound 48/80-induced exocytosis, but the [Ca2+]i increase and the increase of cell volume were unaffected. NaN3 at 0.5 mM could relieve the photodynamic action-induced inhibition of exocytosis. These results indicate that, unlikely to other secretory or contractile cells, photodynamic action with SALPC does not directly affect exocytotic machinery but modulates some functional proteins involved in signal transduction process which may be posterior to G-protein activation in mast cells. Singlet oxygen may be involved in the photodynamic action-induced modulation. A possible target protein can be a protein in the cell membrane which binds with a protein of a granular membrane during the course of exocytosis.

Evaluation of nasotracheal aspiration as a diagnostic tool for Rhodococcus equi pneumonia in foals.

The reliability of preparing bacteriological cultures from nasotracheal aspirates of foals routinely in order to diagnose R. equi pneumonia in foals was studied by isolating Rhodococcus equi from specimens obtained from 96 foals by nasotracheal aspiration with a silicon catheter. Results were compared with specimens obtained from 21 foals by transtracheal aspiration (percutaneous tracheal puncture). These 117 foals showed clinical signs of respiratory tract infection at sampling. R. equi was isolated from 14 of 21 (66.7%) specimens by transtracheal aspiration and from 59 of 96 (61.4%) specimens by nasotracheal aspiration, 649 of 655 isolates (99.1%) from the 73 positive specimens were virulent R. equi, and the culture-positive foals were diagnosed as having R. equi pneumonia. To assess the contamination of aspirates by organisms from the nasopharynx, the results of R. equi isolation from nasal swabs obtained from 56 of the 96 foals were compared to those obtained by nasotracheal aspiration from the same foals. R. equi was isolated from 2 of the 56 nasal swabs: one from a tracheal aspirate was positive, and the other was not. These results suggest that the nasotracheal aspiration technique, which is noninvasive and not associated with complications, could be used as an alternative to the transtracheal aspiration method, especially for the diagnosis of R. equi pneumonia in foals.

Effect of prophylactic administration of hyperimmune plasma to prevent Rhodococcus equi infection on foals from endemically affected farms.

The effect on foals of prophylactic administration of hyperimmune plasma to prevent R. equi infection was investigated on three farms at which R. equi infection was endemic. Sixteen foals between 10 and 39 days of age were intravenously given 1-21 of hyperimmune plasma. ELISA antibody titres against R. equi were significantly increased and maintained at high levels for over 30 days in most of the recipient foals. The prevalence of R. equi infection was 6.3% (1/16) in the foals that received the immune plasma, and 26.3% (5/19) in the control foals not given the immune plasma on the three farms. For 2 years before and after this field trial on the three farms, 18 of 64 foals (28.1%) showed clinical signs of respiratory tract infection and four of them died of R. equi pneumonia. Heavy contamination of horses and their environment with virulent R. equi was detected by colony blotting, and plasmid profiles also suggested that foals on the three farms were constantly exposed to virulent R. equi. The results of this field trial support previous observations by some researchers that the administration of hyperimmune plasma to foals in the early days of life promotes prevention of R. equi infection on endemic farms; however, the mechanism of hyperimmune plasma protection remains unclear.

Detection of virulent Rhodococcus equi in tracheal aspirate samples by polymerase chain reaction for rapid diagnosis of R. equi pneumonia in foals.

Polymerase chain reaction (PCR)-based assays were developed to detect virulent Rhodococcus equi in transtracheal aspirate samples from sick foals showing respiratory signs. An oligonucleotide primer pair from the sequence of the virulence-associated 15- to 17-kDa antigen gene of the virulence plasmid in virulent R. equi was used to amplify a 564 bp region by PCR, and the result was confirmed by Southern blot hybridization. No positive reaction was seen in DNA from 13 different microorganisms typically found in the respiratory tract. In tracheal aspirates seeded with virulent R. equi, a visible band could detect 10 to 10(2) bacteria per PCR assay (10(3) to 10(4)/ml of the aspirate). Virulent R. equi was demonstrated in 31 of 42 transtracheal aspirates by culture and colony blot analysis, whereas a positive PCR result was observed in only 12 of the 31 culture positive samples. To prevent false-negative results, two methods were developed: a nested PCR and a PCR in combination with enrichment cultures of aspirates in the selective medium to increase the number of bacteria to 10(4)/ml or more. All of the PCR-negative and culture-positive samples were positive by the two methods. These results indicated that PCR-based assays provide a specific and sensitive means to detect virulent R. equi in tracheal aspirates of foals, and they are more rapid than the routine culture procedures for the diagnosis of R. equi pneumonia in foals.

Physical and serologic examinations of foals at 30 and 45 days of age for early diagnosis of Rhodococcus equi infection on endemically infected farms.

OBJECTIVE: To evaluate results of physical and serologic examinations of foals at 30 and 45 days of age on 3 types of farms with various prevalences of clinical disease (endemic, sporadic, none) caused by Rhodococcus equi and to determine whether evaluations were helpful in early diagnosis and control of the disease. DESIGN: Prospective cohort study. ANIMALS: 144 foals at 30 and 45 days of age. PROCEDURE: During a 2-year period, 36 foals on farms at which R equi infection was endemic, 71 foals on farms at which the disease was sporadically detected, and 37 foals on farms without the disease were examined by means of auscultation of lungs, serum biochemical and hematologic analyses, and determination of antibody titers against R equi, using ELISA. Transtracheal aspirates were obtained from 14 of 32 foals that had clinical signs of disease and 7 of 41 seropositive foals that did not have clinical signs of disease. RESULTS: Prevalences of respiratory tract disease and seropositive conversion rates for 45-day-old foals on endemically and sporadically infected farms were significantly higher than on farms without the disease. Rhodococcus equi was isolated from tracheal aspirates of seropositive foals, even when clinical signs were not evident. CLINICAL IMPLICATIONS: Physical and serologic examinations of foals at 30 and 45 days of age were useful for early diagnosis of R equi infection, especially for foals on farms at which the disease was endemic.

Clinical evaluation of the serodiagnostic value of enzyme-linked immunosorbent assay for Rhodococcus equi infection in foals.

An enzyme-linked immunosorbent assay (ELISA) for detection of serum IgG antibodies against Tween 20-extracted antigen of strain ATCC 6939 was applied in Hidaka, Japan to a total of 752 sick foals showing a variety of signs of infectious disease. An optical density (OD) value of more than 0.3 was tentatively fixed to be positive on the basis of readings made of healthy horse sera in previous studies. During a 2 year study, 138 of the 752 sick foals showed an OD value of 0.3 or higher and were designated as 'suspected of R. equi infection'. Age distribution during the initial medical examination of the 138 seropositive foals was significant in that most (64%) foals were age 31-60 days, with a sharp decrease in subjects beyond that age. Of the 138 foals suspected of having R. equi infection, 34 foals (25%) showed OD values of over 0.9 at the initial medical examination, in addition to high blood leucocyte counts and serum fibrinogen and alpha-globulin values. The infectious foals had been treated with antibiotics just before and after serodiagnosis and 126 foals (91%) recovered from the disease. However, no clinical improvement was observed in 12 foals (9%). At necropsy, these foals revealed suppurative pneumonia and lymphadenitis of gut associated lymph nodes accompanied by abdominal abscesses. All isolates from the pulmonary and abdominal abscesses revealed R. equi. These results suggest that OD readings in the high range are associated with severe disseminated infection with R. equi.

Isolation of virulent Rhodococcus equi from transtracheal aspirates of foals serodiagnosed by enzyme-linked immunosorbent assay.

Although isolation of Rhodococcus equi from tracheobronchial aspirates is thought to be a definitive diagnosis of R. equi pneumonia in foals, virulence of isolates from the aspirates of infected foals remains obscure. In the present study, transtracheal aspirates were collected from thirty-one 1- to 6-month-old foals, which showed clinical signs of respiratory tract infection, and R. equi isolates were analyzed for the presence of virulence plasmids and virulence-associated antigens. Moreover, this method was compared with a serodiagnosis by an enzyme-linked immunosorbent assay (ELISA) to evaluate the sensitivity of the ELISA. Of the 31 foals, 21 revealed positive cultures for R. equi. Of the 21 foals, 20 (95%) had an ELISA OD value of 0.3 (positive limit of this test) or higher at the initial medical examination. All of the isolates from the aspirates were virulent R. equi, which contained virulence plasmids and expressed virulence-associated antigens. In the remaining 10 foals showing a negative culture for R. equi, 3 foals had positive ELISA titers. Six foals died during the treatment, and necropsy revealed that 5 of the 6 foals had R. equi infection characterized by large abscesses in the lungs, and 3 of the 5 foals also had intestinal lesions. All clinical isolates from the lesions of the foals were virulent R. equi. These results support the assumption that isolates from the transtracheal aspirates of infected foals are virulent R. equi and the sensitivity of ELISA might demonstrate a serodiagnostic value for early diagnosis of R. equi infection in foals.