Fluorescent Antibody Examination Research Articles

A survey of camel (Camelus dromedarius) diseases in Jordan.

Information on incidences of camel (Camelus dromedarius) diseases in Jordan is scarce. In this survey, 369 live and 156 slaughtered camels were examined in four Jordanian geographic regions and the proportion of diseased camels was calculated. Intestinal parasite ova were detected in 98% of camels; one or more species of external parasites were found on the skin of all camels; 33% had nasal myiasis; and hydatid cysts were identified in 44% of the slaughtered animals. Sarcoptic mange (Sarcoptes scabiei var. cameli) and trypanasomiasis, two diseases of economic importance, were also diagnosed in 83% and 33% of the 32 and 257 examined camels, respectively. Rabies virus was detected in eight camels by use of fluorescent antibody examination of brain tissues. Foreign-body accumulation within the first and second stomach compartments was the predominant gastrointestinal disease of slaughtered adult camels (22%). Ten percent of slaughtered camels had bacterial pneumonia, with Pasteurella hemolytica most often isolated (56% of pneumonic lungs). Further investigation into the relationship between parasite burden and health in camels is required to assess the significance of the high prevalence of parasites.

Porcine reproductive and respiratory syndrome virus infection in neonatal pigs characterised by marked neurovirulence

Neonatal pigs from three herds of pigs were somnolent and inappetent and had microscopic lesions characterised by severe meningoencephalitis, necrotic interstitial pneumonia and gastric muscular inflammation. Porcine reproductive and respiratory...

Immunohistochemical investigations of parvalbumin localization in the skeletal muscle fibers of rats.

Parvalbumin (PA) is a water soluble, low-molecular weight, calcium-binding protein which has been thought to be involved in the relaxation of skeletal muscle fibers. Although it is well known that PA concentrations are higher in fast twitch fiber than slow twitch fiber, the localization of PA within the cytoplasm of single muscle fibers is still unknown. The present study, therefore, was undertaken to clarify the PA localization by immunohistochemical methods using the confocal laser scanning microscope (CLSM) and transmission electron microscope (TEM). Wistar strain male rats were fixed by vascular perfusion with 4% paraformaldehyde solution, and the extensor digitorm longus muscle was dissected out. For fluorescent antibody examination, these specimens were quickly frozen in melting isopentan and sections were cut using a cryostat at -25 degrees C. These sections were incubated in anti-PA and anti-Troponin (TR) respectively, and then exposed to Texas-Red- and FITC-labelled secondary antibodies. For TEM study, the pre-embedding method was used. Fluorescent immunohistochemical study has clearly shown that both PA and TR are located intimately in the I-band of the skeletal muscle fibers. The finding by the immunofluorescent study correlated well with those which have been seen at the ultrastructural level. The fact that PA is located in close proximity to TR is considered to be very reasonable when we consider it in terms of the muscular contraction-relaxation cycle.

Antigenic variant of swine influenza virus causing proliferative and necrotizing pneumonia in pigs.

A new antigenic variant of swine influenza virus was isolated from the lungs of pigs experiencing respiratory problems in 7 different swine herds in Quebec. Pigs of different ages were affected, and the main clinical signs were fever, dyspnea, and abdominal respiration. Coughing was not a constant finding of the syndrome. At necropsy, macroscopic lesions included the overall appearance of pale animals, general lymphadenopathy, hepatic congestion, and consolidation of the lungs. Histopathologic findings were mainly proliferative pneumonia with a significant macrophage invasion, necrotic inflammatory cells in the alveoli and the airways, a marked proliferation of type II pneumocytes, and thickening of the alveolar septae. Fluorescent antibody examination of lungs of sick piglets did not demonstrate porcine parvovirus, transmissible gastroenteritis virus, or encephalomyocarditis virus. However, evidence of the presence of an influenza type A infection was demonstrated by indirect immunofluorescence (IIF) staining using monoclonal antibody directed to nucleocapsid protein (NP) of human type A influenza virus. The virus was isolated either by intra-allantoic inoculation of specific-pathogen-free embryonating hens' eggs or propagation in canine kidney (MDCK) cells in the presence of trypsin. By hemagglutination inhibition tests, no cross-reactivity was demonstrated with human influenza H1N1, H2N2, and H3N2 strains, and infected MDCK cells did not react by IIF with monoclonal antibodies to NP protein of type B influenza virus. The hemagglutination activity of plaque-purified isolates was only partly inhibited by hyperimmune serum produced to subtypes A/Wisconsin/76/H1N1 and A/New Jersey/76/H1N1 of swine influenza virus. Gnotobiotic piglets that were infected intranasally with egg-adapted isolates of this new antigenic variant of swine influenza virus developed the very same type of lesions observed in field cases.

Detection of Murine Typhus Infected Fleas with an Enzyme-Linked Immunosorbent Assay

An enzyme-linked immunosorbent assay (ELISA) for the detection of Rickettsia typhi antigen in homogenates of pooled or individual laboratory infected fleas is described. The assay uses a double sandwich technique, employing a pool of monoclonal antibodies to capture the antigen and a hyperimmune rabbit serum for antigen detection. Using pools of R. typhi infected Xenopsylla cheopis, Ctenocephalides felis, and Leptopsylla segnis, the sensitivity of the ELISA was compared with direct fluorescent antibody examination of individual fleas for rickettsiae and with rickettsial titers determined by plaque enumeration on primary chicken embryo fibroblasts (PFU). Pooled samples with less than 4 PFU of viable rickettsiae gave ELISA results which were not significantly above background. Both ELISA OD and ELISA titer (last dilution giving an OD that was 2 SD above the control) of a 1:10 dilution of homogenate (4 fleas/ml) were linearly related to rickettsial titer up to 10(6.8) PFU/sample. Multiple freeze-thaws of pools of infected fleas led to a rapid loss of ELISA sensitivity. ELISA assays on single fleas demonstrated large individual variability in rickettsial content. This was independent of the number of days postinfectious feeding or the mean number of PFU/flea (10(1.7-6.9) found for pooled fleas in the same cohort. The sensitivity and ease of performance of ELISA should make it usable under field conditions.

Diagnosis of Anaplasmosis in Formalin-Fixed Tissue Using the Wolbach's Giemsa Stain

Bovine anaplasmosis is an economically significant disease that is widespread throughout the world and enzootic in 40 of the 50 states of the USA. 2 The causative agent, Anaplasma marginale, can be transmitted from infected blood by arthropod and mechanical vectors. Cattle of all ages can be infected, but severe clinical signs and death are common only in mature cattle. Clinical disease is due to anemia resulting from the intracellular destruction of parasitized erythrocytes by phagocytes. In fatal cases severe anemia, icterus, and splenomegaly are typical. Histologically, centrilobular hypoxic hepatic necrosis and splenic hemosiderosis related to erythrocyte destruction are commonly present. Diagnosis is established by identification of anaplasma bodies in the margins of erythrocytes using Giemsa or Wright-Giemsa stains or fluorescent antibody examination of blood smears or tissue impressions.’ Field necropsy specimens submitted to the California Veterinary Diagnostic Laboratory occasionally include samples from cattle that have a clinical history and histologic lesions of hypoxic liver damage or splenic hemosiderosis suggestive

Antigen detection for the rapid diagnosis of mycoplasma and legionella pneumonia

Immunologic attempts to detect mycoplasma antigens in fluids of infected patients have been rare and largely unsuccessful. Nucleic acid hybridization procedures appear promising on the basis of successes in detecting mycoplasmal contamination of tissue culture cells; results of attempts to apply these techniques to human infections have not been reported. Antigens can be detected in the urine of about 80% of patients with serogroup 1 Legionella pneumophila pneumonia and of some patients with serogroup 4 Legionella pneumophila and Legionella dumoffii pneumonia. The specificity of these assays is greater than 99%. In a test population in which the prevalence of Legionella pneumophila was 4%, the posterior probabilities of positive and negative results of tests for antigen were 86.5% and 99.3%, respectively. Antigen is detectable within the first 3 days of illness approximately as often as at later periods, and antigen may remain detectable for a few days to 1 yr after successful therapy. Antigen is detectable in serum, but the concentrations are considerably lower than in urine. Combining urinary antigen detection with direct fluorescent antibody examination of secretions increases the rapid diagnostic yield by 10%-20%. Monoclonal antibody studies demonstrate that subgroup specificities are present among the serogroup 1 urinary antigens. Radiometric and enzyme immunoassays detect antigen in equal proportions of patients. Latex agglutination results are positive in about 80% of those cases positive by the other methods.

Rapid identification of material colonization with group B streptococci by use of fluorescent antibody.

To identify women colonized with group B streptococci during parturition, we used pooled type-specific fluorescent antibody to examine vaginal swabs enriched by preincubation in selective broth medium. In preliminary experiments, group B streptococcus strain III-Bell was reliably detectable with fluorescent antibody at concentrations of greater than 10(5) colony-forming units per ml, achieved after 6 h of incubation of small inocula (18 to 26 colony-forming units). Of the vaginal swabs from 924 parturient women examined prospectively by both fluorescent antibody and selective bacteriology techniques, group B streptococci were isolated in 154. The sensitivity of the fluorescent antibody technique increased with increasing incubation time and ranged from 49% (3 to 6 h) to 81% (7 to 12 h) to 83% (13 to 18 h) to 93% (greater than 18 h). Colonized mothers identified within 6 h by the fluorescent antibody technique had higher rates of vertical transmission to their newborn infants (61%) than colonized mothers whose fluorescent antibody examinations were negative within this time interval (32%; P = 0.027). However, because of the timing of their admissions, none of the colonized mothers of the four infants who developed early-onset group B streptococcal sepsis were identified with fluorescent antibody until after delivery. Although its sensitivity approaches selective culture methods after 6 h of incubation, fluorescent antibody examination of vaginal swabs does not appear to offer a practical approach to identifying colonized parturient women for intrapartum antibiotic prophylaxis of group B streptococcal infection.

Distribution of Rickettsia tsutsugamushi in organs of Leptotrombidium (Leptotrombidium) fletcheri (Prostigmata: Trombiculidae).

Microdissection techniques and direct fluorescent antibody staining were used to demonstrate scrub typhus rickettsiae in all stages of infected Leptotrombidium (Leptotrombidium ) fletcheri (=Malaysian L. akamushi ). Rickettsiae were detected in some gut and hemolymph samples from each stage of mite development. Unfed larvae had the highest and protonymphs the lowest numbers of samples positive for rickettsiae. The high incidence of rickettsiae in the salivary glands of unengorgcd larvae along with the low incidence in engorged larvae suggest that rickettsiae in the salivary fluid are the source of infection for the mammalian host. Fluorescent antibody examination of eggs and unfed larvae appears to be a practical method of screening field-collected mites for scrub typhus rickettsiae.

Demonstration of Neisseria gonorrhoeae with fluorescent antibody in patients with disseminated gonococcal infection.

Among patients with disseminated gonococcal infection and pustular skin lesions, Neisseria gonorrhoeae was identified in exudates from the skin lesions by the direct fluorescent antibody test in 57%, by gram stain in 11%, and by culture in only 9%. However, gonococci were cultured from four of seven pustular biopsies of skin lesions. Gonococci were also identified by fluorescent antibody examination in four of eight buffy coat smears from patients with negative blood cultures, but in only one of eight culture-negative synovial fluids. Among patients without disseminated gonococcal infection, smears of synovial fluid, buffy coat, and exudate from skin pustules were uniformly negative by the fluorescent antibody test. This test has contributed to studies of the pathogenesis of disseminated gonococcal infection and is frequently useful in the diagnosis of this condition.

Role of Bursa of Fabricius in Chicken Resistance to Salmonella typhimurium

SUMMARY Surgically bursectomized and control birds were inoculated intraperitoneally with Salmonella typhimurium and necropsied 3 to 48 hours later. Salmonella counts in artificially induced abdominal fluid at necropsy at 6 hours were significantly greater in bursectomized than in unbursectomized birds, indicating an active role for the bursa of Fabricius in the immune response to parenterally inoculated salmonellae. S. typhimurium was inoculated via the rectum into the ceca of bursectomized and control birds. The groups did not differ in salmonella counts in cecal contents at 24 and 48 hours, but fluorescent antibody examination of frozen sections of cecal walls showed tissue invasion to be at a statistically significant higher rate in bursectomized than in unbursectomized birds. Groups of bursectomized, thymectomized, and control birds were inoculated orally with either 10 or 108 organisms of S. typhimurium and inoculated intraperitoneally with 108 organisms 48 days later. Rectal swabs at intervals thereafter further indicated a significant role for the bursa of Fabricius in the immune response. No difference was noted between the thymectomized and control birds.