Indirect Fluorescent Antibody Titers Research Articles

Adjuvant activity of muramyl dipeptide derivatives to enhance immunogenicity of a hantavirus-inactivated vaccine

The adjuvant effect of two lipophilic derivatives of muramyl dipeptide (MDP), B30-MDP and MDP-Lys(L18), on the ability of an inactivated vaccine of B-1 virus (B-1 vaccine) to induce immune response against Hantavirus causing hemorrhagic fever with renal syndrome (HFRS) was examined. When mice were immunized subcutaneously (s.c.) twice at 2-week intervals with B-1 vaccine admixed with or without 100 μg mouse −1 of B30-MDP (B-1/B30-MDP) or MDP-Lys(L18) [B-1/MDP-Lys(L18)], mice immunized with B-1/B30-MDP as well as B-1/MDP-Lys(L18) showed significantly higher indirect fluorescent antibody (IFA) titers against HFRS virus than mice immunized with B-1 vaccine alone. Both mice treated with B-1/B30-MDP and B-1/MDP-Lys(L18) also exhibited significantly higher neutralizing antibody titers against HFRS virus than mice immunized with B-1 vaccine alone during 3–9 weeks after the primary immunization. The evaluation of antibody-producing cells by enzyme-linked immunospot (ELISPOT) assay on week 4 revealed that both MDP derivatives enhanced the number of HFRS virus-specific IgG1 and IgM antibody-producing cells. Furthermore, mice treated with B-1/B30-MDP as well as B-1/MDP-Lys(L18) showed a higher level of Th-2 type cytokines, IL-4 and IL-6, in sera than mice treated with B-1 alone. In an in-vitro analysis of T lymphocyte proliferation to baculovirus-expressed recombinant nucleocapsid protein (rNP) of Hantaan 76–118 strain, the splenocytes of mice treated with B-1/B30-MDP and B-1/MDP-Lys(L18) on week 4 showed a significantly higher proliferating activity than those treated with B-1 vaccine alone. In addition, when mice were immunized once with B-1 vaccine admixed with or without B30-MDP and MDP-Lys(L18) and followed by intrafootpad (i.f.) injection of B-1 vaccine on day 7, mice immunized with B-1/B3-MDP and B-1/MDP-Lys(L18) induced a higher delayed-type hypersensitivity (DTH) reaction than mice immunized with B-1 vaccine alone. These results suggest that B30-MDP and MDP-Lys(L18) are useful immunoadjuvants to enhance the ability of inactivated B-1 vaccine to induce a humoral and cellular response to HFRS virus.

Susceptibility to tropical theileriosis of calves born to dams immunized with Theileria annulata (Hisar) cell culture vaccine.

The susceptibility/immune status to tropical theileriosis of calves born of immunized dams was evaluated. Six cows were vaccinated with the Theileria annulata cell culture vaccine in the eighth month of pregnancy. Sera from the immunized dams exhibited very high post-vaccination antibody titres as determined by the indirect fluorescent antibody (IFA) test. The calves born to these dams did not show antibodies against T. annulata at the time of birth (IFA titres of < 1:20). The new-born calves were fed colostrum from their mothers and were challenged with T. annulata-infected ground tick supernate at 5-7 days of age. All the calves developed fever (from day 5-6 onwards) and parasitological reactions (from day 8-9 onwards) after challenge. There was a significant decrease in the haemoglobin and packed cell volume of the calves after challenge. All the calves showed signs of acute theileriosis by day 9-10 after challenge and had to be treated with buparvaquone in order to save their lives. The study indicated that detectable levels of anti-theilerial antibodies were not transferred from immune dams to their offspring. All the calves born to immunized dams were fully susceptible to theileriosis and thus themselves needed vaccination.

Distribution, diversity, and host specificity of Bartonella in rodents from the Southeastern United States.

A number of Bartonella isolates were obtained from seven species of rodents sampled from 12 geographic sites representing the major biotic communities of the southeastern United States. Bartonella were isolated from the blood of 42.2% of 279 tested rodents. The highest prevalence of infection typically occurred among the most commonly captured species in the rodent community. Four phylogenetic groups, uniting 14 genotypic variants of Bartonella, were identified by sequence analysis of the citrate synthase gene. The level of sequence homology between genotypic groups varied from 88.8% to 96.4%, and the degree of homology among variants within groups was > or = 97%. Cotton rats (Sigmodon hispidus) harbored up to three phylogenetic groups of Bartonella at a single site, and Bartonella of two phylogenetic groups were isolated from a single rodent. All the Bartonella isolated from three species of Peromyscus clustered in a single distinct phylogenetic group, suggesting some host specificity may occur. Mouse ascitic fluids produced in BALB/c mice inoculated with Bartonella of three phylogenetic groups demonstrated high indirect fluorescent antibody (IFA) titers to homologous antigens. However, use of eight Bartonella antigens in an IFA test with sera from 394 wild-caught rodents resulted in either little or extremely low titers of antibody.

Legionellosis linked with a hotel car park--how many were infected?

An outbreak of legionellosis associated with a hotel in Sydney, Australia, and the subsequent epidemiological and environmental investigations are described. Four cases of Legionnaires' disease were notified to the Public Health Unit. A cross-sectional study of 184 people who attended a seminar at the hotel was carried out. Serological and questionnaire data were obtained for 152 (83%) of these. Twenty-eight (18%) respondents reported symptoms compatible with legionellosis. Thirty-three subjects (22%) had indirect fluorescent antibody (IFA) titres to Legionella pneumophila serogroup 1 (Lp-1) of 128 or higher. The only site which those with symptoms of legionellosis and IFA titre > or = 128 were more likely to have visited than controls was the hotel car park (adjusted odds ratio [OR] 14.7, 95% confidence interval [CI]: 1.8-123.1). Those with symptoms compatible with legionellosis, but whose IFA titres were < 128 were also more likely to have visited the hotel car park (adjusted OR 4.4, 95% CI: 1.5-12.9). Seroprevalence of Lp-1 antibodies was higher in those who attended the seminar than in a population sample of similar age. Findings suggested that the 4 cases represented a small fraction of all those infected, and highlighted difficulties in defining illness caused by Lp-1 and in interpreting serology.

Screening for Antinuclear Antibodies by Enzyme Immunoassay

Indirect fluorescent antibody (IFA) ia most widely used method in clin clinical laboratories to screen for autoantibodies against a wide variety of nuclear antigens. Recently, a number of antinuclear antibody (ANA) enzyme immunoassay (EIA) screens have become commercially available and claim to be an alternative method to screen for ANAs. Given the subjectivity of technical interpretation of IFA and the high number of ANA negative samples, a suitable EIA method for ANA screening would be beneficial to clinical laboratories with large samples volumes. Five ANA EIA screens were compared (Elias, Helix, Sanofi, TheraTest and Zeus) to IFA using a human epithelial cell line (HEp-2). Sera from 601 patients submitted to our reference laboratory for autoimmune testing, and from 202 normal healthy blood donors, were included in this study. Samples with discordant results between IFA and EIA were further analyzed using single antigen EIAs for SSA, SSB, Sm, RNP, Scl-70, histones, dsDNA, and ssDNA. Analyses were based on clinically significant IFA titers of > or equal to 1:160 as positive and <1:40 as negative. When compared to IFA, agreement, sensitivity and specificity for each ANA EIA screen were as follows: Elias: 87.0%, 69.5% and 97.9%; Helix: 94.6%, 90.2%, and 97.3%; Sanofi: 95.0%, 93.7%, and 95.9%; TheraTest: 95.3%, 97.7%, and 93.5%; Zeus: 87.1%, 96.2%, and 81.4%, respectively. In conclusion, screening for ANAs by EIA using several commercial assays was both sensitive and specific when compared to IFA. Moreover, the EIA is objective and much less labor intensive when screening a large number of clinical specimens. None of the EIAs were 100% sensitive and, thus, may fail to detect a few of the nonspecific ANAs that demonstrate atypical as well as classical IFA patterns. The advantages of employing these nonsubjective assays to screen out the vast majority of ANA negative sera is clear. The authors still recommend confirming titers and patterns of sera with positive EIA screens using classical IFA methods employing HEp-2 cells.

Hantavirus infection among Rattus norvegicus in Japan.

Seroepizootiological study of hantavirus infection among 393 urban rats (Rattus norvegicus) captured in six regions in Japan during the period from 1990 to 1994 was carried out by the indirect fluorescent antibody (IFA) test and Western blot (WB). Fifteen out of 393 (3.8%) rat sera were antibody-positive by IFA, i.e., Tokyo Port (12.8%, 6/47), Shimizu Port (5.7%, 2/35), Otaru Port (1.5%, 1/65) and Nagoya City (3.6%, 6/167). In two other regions, i.e., Kasai Seaside Park and Haneda Airport, rat sera were antibody-negative. One serum with a lower IFA titer, 1:64, from Otaru Port was confirmed to be antibody-positive by WB, while two sera from Shimizu Port (IFA titer, 1:32 and 1:64) were not. In Nagoya City, one out of four sera (IFA titer, 1:32) and one of two sera (IFA titer, 1:64) were also confirmed to be antibody-positive by WB. Continuous hantavirus infection among rats in Tokyo Port, Shimizu Port and Nagoya City and the existence of hantavirus among rats in Otaru Port were demonstrated.

Histologically confirmed clinical toxoplasmosis in cats: 100 cases (1952–1990)

Summary Tissue sections from 119 cats that died or were euthanatized (1952-1990) because of toxoplasmosis-like illness were reexamined for Toxoplasma gondii by direct microscopy and immunohistochemical staining with anti-T gondii serum. Clinical and pathologic data from 100 of these cats with histologically verified toxoplasmosis were then analyzed. Of these 100 cats, 36 were considered to have generalized toxoplasmosis, 26 predominantly pulmonary lesions, 16 abdominal, 2 hepatic, 1 pancreatic, 1 cardiac, 2 cutaneous, 7 neurologic, and 9 had neonatal toxoplasmosis. In 14 cats, concurrent microbial infections or other maladies were seen. Cats were 2 weeks to 16 years old (median, 2 years; mean, 4 years). Sixty-five cats were males and 34 were females; sex was not recorded for 1 cat. Of 67 cats that had rectal temperatures recorded, 49 (73%) had fever (40.0 to 41.7 C). Dyspnea, polypnea, and signs of abdominal discomfort were frequently observed. Toxoplasmosis had been confirmed antemortem in 8 cats; 4 had a serum antibody titer to T gondii of ≥ 1:1,024; and T gondii had been found in cytologic evaluation of tracheal aspirates from 2 cats and pleural fluid from 1 cat, as well as in a biopsy specimen of a mesenteric lymph node from another. Of the 15 cats with T gondii serum-antibody titers determined by the Sabin-Feldman dye test, 6 had no antibody detected in 1:4 dilution of their serum. Indirect fluorescent antibody titers were found in 10 of 10 cats' sera tested. Forty-one eyes from 27 of the cats were examined microscopically. Twenty-two of the 27 cats (81.5%) had evidence of intraocular inflammation in one or both eyes. Multifocal iridocyclochoroiditis was the most common lesion and was seen in 18 (81.8%) of the cats with ophthalmitis. The ciliary body was the most often severely affected portion of the uvea. Of the 22 cats with ocular toxoplasmosis, T gondii was found in eyes of 10. Toxoplasma gondii was found in the retina of 5 cats, the choroid of 2, the optic nerve of 1, the iris of 3, and the ciliary body of 4. Toxoplasma gondii was identified in 80% of 55 brains, 70.0% of 90 livers, 76.7% of 86 lungs, 64.4% of 45 pancreata, 62.7% of 59 hearts, 45.8% of 72 spleens, 41.5% of 65 intestines, 17.7% of 61 kidneys, and 60.0% of 30 adrenal glands.

Seroprevalence of indirect fluorescent antibody to porcine reproductive and respiratory syndrome virus in selected swine herds.

Porcine reproductive and respiratory syndrome (PRRS) virus has recently been identified in the USA as a cause for reproductive failure in pregnant sows and respiratory disease in young pigs. 1-4,6 The syndrome has been commonly referred to as swine infertility and respiratory syndrome in this country but there was a general agreement to adopt the name PRRS at an international PRRS symposium (St. Paul, MN, May 1992). A virus designated as Lelystad virus has also been reported to cause a similar syndrome in several European countries. 1,5 Both viruses can cause significant economic problems for swine producers because of acute reproductive losses and piglet mortality. Chronic production losses involving increased mortality and reduced growth rate in pigs at postweaning age have also been reported on some infected farms. 1 In our recent study, an indirect fluorescent antibody (IFA) test was developed to detect and quantitate PRRS virus antibody in swine sera. 7 This IFA test for swine sera collected between 1981 and 1991 revealed evidence of PRRS virus infection in this country as early as April 1986. The purpose of the present study was to report the recent Seroprevalence status of IFA to PRRS virus by testing sera collected from selected swine herds during the first 6 months of 1992. Swine sera submitted January-June 1992 for serodiagnosis to the Oxford Veterinary Laboratory, Worthington, Minnesota, were used. Since October 1988, the laboratory has provided a swine antibody profile service to practicing veterinarians for different pathogens. The service has included serology for Actinonobacillus pleuropneumonia serotypes 1, 5, and 7, encephalomyocarditis virus, porcine parvovirus, Leptospira spp., and group D Streptococcus. The IFA test for PRRS virus was included starting in January 1992. Between January and June 1992, a total of 2,787 sera from 263 swine farms (an average of 10.6 serum samples per farm), were submitted from 13 different states. Veterinarians provided a brief farm history and requested serology for specific agents. Of the 263 farms, 225 requested PRRS virus serology. For the PRRS virus IFA test, aliquots of each sample were sent to the swine virology laboratory, University of Minnesota. ethanol was used to fix the cell monolayers in each well. Test sera, diluted 4-fold serially from 1:4 to 1:1,024, were added to test plates. The plates were incubated, washed, and incubated again with an optimal dilution of rabbit anti-swine IgG conjugated with fluorescein isothiocyanate. The plates were then washed and examined under a fluorescent microscope. Negative and positive reference sera and an uninfected SAM control for each serum were included in each test. The IFA titers were recorded as the highest serum dilutions with specific cytoplasmic fluorescence, and titers of ≤ 1:4 and ≥ 1: 16 were considered negative and positive, respectively. Of 2,787 sera from 263 swine herds tested, 979 sera (35.1%) had PRRS IFA titers of 21: 16 and 148 farms (56.3%) had 1 or more pigs that were PRRS virus IFA test positive (Table 1). Of 1,726 sera from 148 farms with PRRS virus seropositive pigs, 747 sera (43.3%) had IFA titers of ≤ 1:4 and 979 sera (56.7%) had the titers of ≥ 1:16. Of 979 seropositive samples, 222 (22.7%) 314 (32.1%), 283 (28.9%), and 160 (16.3%) samples had IFA titers of 1:16, 1:64, 1:256, and ≥ 1: 1,024, respectively. Of 225 farms requesting PRRS serology, 129 (57.3%) had seropositive pigs, whereas 19 of 38 farms (50.0%) that did not request PRRS virus serology were positive. Results of the farms with seropositive pigs were 1 1/16 (68.8%), 16/41 (39.0%) 35/65 (53.8%) 21/35 (60.0%), 37/61 (60.7%), and 28/45 (62.2%) farms for January, February, March, April, May, and June, respectively. No significant difference for seropositive farms was found between the months by chisquare analysis (P = 0.190). The positive farms were located in 11 different states.

Immunogenicity of the Plasmodium falciparum asexual blood-stage synthetic peptide vaccine SPf66.

The immunogenicity of the cocktail vaccine SPf66 against Plasmodium falciparum was investigated in rabbits, monkeys, and human volunteers. This polymerized peptide vaccine incorporates a portion of each of the 35-kD, 55-kD, and 83-kD blood-stage proteins, linked by an amino acid sequence reproducing one repeat from the circumsporozoite protein of P. falciparum. Results from this study show that vaccination with this vaccine molecule elicited high titers of antibodies to SPf66 and its constituents, but these antibodies did not correlate with regular or sensitive indirect fluorescent antibody (IFA) titers to blood-stage malaria parasites. However, rabbits injected with individual peptides produced antibodies with low affinity to indefinite parasite structures by immunoelectron microscopy, and rabbits injected with SPf66 had high antibody titers against the peptide (NANP)40. Consequently, these anti-SPf66 serum samples recognized P. falciparum sporozoites in the IFA. Such reactivity was not observed in monkeys or human volunteers vaccinated with SPf66. In addition, SPf66 components were recognized by antibodies induced by natural infection in humans and by laboratory-monitored infections in Aotus monkeys. These results suggest that this vaccine candidate merits further developmental work to better define immune response elicited by the copolymer to the parasite.

Outbreak of fatal illness among captive macaques in the Philippines caused by an Ebola-related filovirus.

Following the detection of an Ebola-like virus in cynomolgus macaques recently imported into the United States from The Philippines, studies were initiated to document transmission at export facilities located in the latter country. At one export facility, 52.8% of 161 monkeys that died over a 2.5-month period were shown to be infected with this virus using an enzyme-linked immunosorbent assay to detect antigen in liver homogenates. A case fatality rate of 82.4% was documented for the infected monkeys. The initial anti-viral antibody prevalence among the captive macaques at this facility was 25.9% (indirect fluorescent antibody titer greater than or equal to 1:16). Followup documented infection of 24.4% of initially seronegative animals and 8.7% of initially seropositive monkeys. Being held in a gang cage versus a single cage was found to be a significant risk factor for subsequent virus infection, and the presence of IFA antibody was shown to predict protection. This study documents unequivocally for the first time the presence of an Ebola-related filovirus in Asia.

Immunisation of cattle against theileriosis in Nakuru District of Kenya by infection and treatment and the introduction of unconventional tick control

One hundred and one cross European-Boran cattle (50 cows and 51 calves), on a farm in Nakuru District, Kenya, were immunised against theileriosis using Theileria parva lawrencei and Theileria parva parva stocks from another district of Kenya. The stabilates used were T.p.lawrencei (Mara III) used at 10 −1.7 dilution and T.p.parva (Kilae) used at 10 −1.0 dilution. The stabilates were combined and inoculated simultaneously with a short-acting formulation of oxytetracycline hydrochloride given intramuscularly at 10 mg kg −1 body weight and was repeated on Day 4 after inoculation of the stabilate. Most of the theileriosis challenge on the farm was thought to be derived directly from the African buffalo ( Syncerus caffer). Nine percent of the cattle had significant indirect fluorescent antibody (IFA) titres before the immunisation and 99% after immunisation. The immunised cattle were exposed to tick-borne disease challenge on the farm by withdrawal of acaricide cover. The immunised cattle were divided into five groups plus two susceptible control cows and two calves for each group. Cattle in four of the groups had acaricidal ear tags, each group having a different type, applied to both ears and the fifth group remained untagged. The animals remained without conventional acaricide application for 134 days. Ten out of 20 (50%) non-immunised control cattle became T.p.lawrencei reactors while only one out of 97 (1%) of the immunised cattle reacted. A frequent complication noted was mild infections due to unidentified Theileria sp. which required expert differentiation from T.parva infections. An additional group of ten steers whose tick load was removed by hand at weekly intervals was introduced 79 days after exposure; these had no tick control and four became T.p.lawrencei reactors. Of 12 calves born during the exposure period and without tick control, four became theilerial reactors and one died. The application of acaricidal tags however, reduced tick infestation levels considerably compared with untagged controls but did not prevent transmission of theileriosis with the possible exception of tags on Group 4. A number of transient low grade fevers were noted and attributed to Theileria sp., Ehrlichia bovis, Ehrlichia (Cytoecetes) ondiri and Borrelia theileri infections, none of which were fatal. One immunised animal died of acute dual infection of Babesia bigemina and Borrelia theileri after acaricide control by spraying was re-introduced but no Anaplasma infections were detected. An analysis of the economic effects of immunisation was made.

An indirect fluorescent antibody test for the detection of antibody to swine infertility and respiratory syndrome virus in swine sera.

An indirect fluorescent antibody (IFA) test was developed and standardized to detect and quantitate antibody for swine infertility and respiratory syndrome (SIRS) virus in swine sera. Test results were evaluated using sera of pigs infected both experimentally and naturally with SIRS virus. The IFA test used swine alveolar macrophage (SAM) monolayers prepared in 96-well microplates and infected with SIRS virus. The monolayers were incubated with test sera, washed, and stained with fluorescein isothiocyanate-labeled rabbit anti-swine IgG. After another wash step, the monolayers were examined under a fluorescent microscope. A noninfected SAM control well was included for each sample. The antibody titers for each serum sample were recorded as the highest serum dilutions with specific cytoplasmic fluorescence but no fluorescence in the control wells. To evaluate the test, sera of 4 6-week-old pigs that had been infected with SIRS virus, 2 contact pigs, and 13 experimentally infected sows were used. In the experimentally infected pigs, antibody was first detected at 7 days postexposure (PE) and peaked (1:256-1,024) between 11 and 21 days PE. All 13 sow sera were negative at time of infection but were positive (1:64- greater than or equal to 1:1,024) at 14-26 days PE. Seven hundred twenty sera collected from 25 different swine farms with or without a history of SIRS were also tested. Of 344 sera from 15 swine farms with a clinical history of SIRS, 257 (74.7%) sera had IFA titers greater than or equal to 1:4, whereas 371 (98.7%) of 376 sera from herds with no history of SIRS were negative.(ABSTRACT TRUNCATED AT 250 WORDS)

Seroprevalence of Lyme disease in gray wolves from Minnesota and Wisconsin.

To determine the seroprevalence of Lyme disease in gray wolves (Canis lupus) from various counties of Minnesota and Wisconsin (USA), 589 serum samples were collected from 528 wolves from 1972 to 1989. An indirect fluorescent antibody (IFA) test was used to detect the presence of antibodies against Borrelia burgdorferi. Titers of greater than or equal to 1:100 were considered positive. Results were confirmed by testing a few selected sera by Western blotting. Of the 589 sera tested, 15 (3%) had IFA titers of greater than or equal to 1:100. Three of the positive samples were collected from Douglas County in Wisconsin and twelve were from Minnesota counties. This study indicates that wolves are exposed to B. burgdorferi and are susceptible to Lyme disease.

Babesia microti, human babesiosis, and Borrelia burgdorferi in Connecticut

Babesia microti was isolated from a white-footed mouse (Peromyscus leucopus) that was captured in southeastern Connecticut in 1988, when the first human case of babesiosis acquired in Connecticut was recognized. To date, 13 cases of babesiosis have been reported in Connecticut, the largest number of human cases reported on the mainland United States. Two of nine patients quiried remembered a prior tick bite. Since Babesia parasites are known to be vectored only by ticks, we surmise that 12 of these infections were acquired via tick bites; 1 was obtained by blood transfusion (the patient was 46 years of age) from an endemically infected donor. The ages of the patients with tick-acquired babesiosis ranged from 61 to 95 years. Two patients died with active infections, and one patient died from chronic obstructive pulmonary disease soon after treatment with clindamycin and quinine. Indirect fluorescent-antibody titers of blood samples drawn at the time of hospitalization for 11 patients and at the time of active infection for 1 asymptomatic person ranged from 1:1,024 to 1:4,096. Five of eight patients with babesiosis also had significant immunoglobulin G or immunoglobulin M titers (1:640 to 1:5,120) to Borrelia burgdorferi. B. microti was isolated in Syrian hamsters inoculated with blood from 7 of 12 patients tested and was also isolated from mice captured in six towns. The peridomestic nature of the disease was demonstrated by isolating the parasite from white-footed mice captured in or near the yards of eight different patients. Of 59 mice tested, 27 were positive and 25 were coinfected with B. burgdorferi. The isolation of B. microti from a white-footed mouse captured in north-central Connecticut (West Hartford), away from the focus of human infections in southeastern Connecticut, suggests that this pathogen may spread into other areas where Ixodes dammini, the tick vector, becomes established.

アメーバ症の血清学的診断法に関する基礎的研究

Sera of 375 blood donors which were seropositive for syphilis were examined for antibodies against Entamoeba histolytica. Antibody prevalences against E. histolytica using complement fixation (CF) test were 12.0%, and enzyme-linked immunosorbent assay (ELISA) were 2.7%. The positive rates of antibodies in the CF test were significantly higher in sera showing positive by the Venereal Disease Research Laboratory (VDRL) test than in the VDRL-negative sera, and not related to the results of Treponema pallidum hemagglutination (TPHA) test. Only one sample in the VDRL-negative sera was positive by CF test. On the other hand, the positive rates or mean absorbance in ELISA were not correlated to the results of VDRL or TPHA test. The percent positivity in CF test became higher with the level of antibodies in VDRL test, but not that in ELISA. The level of antibodies in CF and VDRL test were weakly correlated. These results suggested that the results of CF test for E. histolytica antibodies were most likely false-positive in relation to the results of VDRL test. 10 (2.7%) of sera were positive by ELISA, but all ELISA-positive samples showed low ELISA titers and absorbance. 9 of the ELISA-positive samples showed indirect fluorescent antibody (IFA) titers of over 1:50, and the level of antibodies in the IFA test correlated to that in ELISA, so ELISA-positive persons seemed to have been previous infected or were asymptomatic cyst carriers.

In Vitro Growth Inhibition of Plasmodium falciparum by Sera from Different Regions of the Philippines

Sera from different malaria endemic regions of the Republic of the Philippines were compared for their ability to inhibit growth of Plasmodium falciparum in vitro. Dialyzed serum was added to synchronous cultures containing schizonts for either the total 48 hr test period or only the last 24 hr in order to analyze the effects on erythrocytic invasion and intraerythrocytic growth, respectively. Reduction in 3H-hypoxanthine uptake was used to determine the percent of inhibition compared to nonimmune serum. One hundred seventy sera from Mindanao and Palawan in the South, the centrally located island of Mindoro, and Luzon in the North, were tested against 4 P. falciparum strains from the Philippines and 1 from Africa. Indirect fluorescent antibody titers were not predictive of inhibition. Inhibition of merozoite invasion rather than intraerythrocytic parasite growth is suggested by this study. Generally, sera were more inhibitory to parasite strains from the same geographical area than to those from more remote areas.

Diagnosis of Human Ehrlichiosis with the Indirect Fluorescent Antibody Test: Kinetics and Specificity

Human ehrlichiosis, an acute febrile illness caused by Ehrlichia canis or a closely related rickettsial organism, was first identified in 1986. From 1986 through 1988, sera from 85 patients demonstrated a fourfold rise or fall in antibody titer to E. canis. Seven (22%) of 32 patients initially tested during the first week after onset of illness. 17 (68%) of 25 tested during the second week, and all 18 tested during the third week had titers that exceeded the minimum positive titer of greater than or equal to 80. Of the 85 confirmed ehrlichiosis patients, 31 (36.5%) also had indirect fluorescent antibody titers considered diagnostic of infection with Rickettsia rickettsii, Rickettsia typhi, or Coxiella burnetti, but in most these diagnoses were not supported by epidemiologic, clinical, or serologic evidence. These results emphasize that patients suspected of having a tick-borne infection should be tested for antibodies to E. canis as well as for those to other rickettsiae.

Prevalence and implications of feline coronavirus infections of captive and free-ranging cheetahs (Acinonyx jubatus)

The extent and progression of exposure to feline infectious peritonitis (FIP) virus in the cheetah, Acinonyx jubatus, was monitored by a world-wide serological survey with indirect fluorescent antibody titers to coronavirus. The indirect fluorescent antibody assay was validated by Western blots, which showed that all indirect fluorescent antibody-positive cheetah sera detected both domestic cat and cheetah coronavirus structural proteins. There was a poor correlation between indirect fluorescent antibody results and the presence of coronaviruslike particles in cheetah feces, suggesting that electron microscopic detection of shed particles may not be an easily interpreted diagnostic parameter for FIP disease. Low, but verifiable (by Western blots [immunoblots]) antibody titers against coronavirus were detected in eight free-ranging cheetahs from east Africa as well as from captive cheetahs throughout the world. Of 20 North American cheetah facilities screened, 9 had cheetahs with measurable antibodies to feline coronavirus. Five facilities showed patterns of an ongoing epizootic. Retrospective FIP virus titers of an FIP outbreak in a cheetah-breeding facility in Oregon were monitored over a 5-year period and are interpreted here in terms of clinical disease progression. During that outbreak the morbidity was over 90% and the mortality was 60%, far greater than any previously reported epizootic of FIP in any cat species. Age of infection was a significant risk factor in this epizootic, with infants (less than 3 months old) displaying significantly higher risk for mortality than subadults or adults. Based upon these observations, empirical generalizations are drawn which address epidemiologic concerns for cheetahs in the context of this lethal infectious agent.

Indirect fluorescent antibody test versus enzyme-linked immunosorbent assay and agglutination tests in the serodiagnosis of patients with brucellosis

Anti- Brucella IgG, IgM and IgA in sera from patients with blood culture positive for B. melitensis and controls were measured by indirect fluorescent antibody (IFA) test and the findings compared with those of enzyme-linked immunosorbent assay (ELISA) and microagglutination test (MAT). Brucella melitensis and B. abortus antigens from three vendors (BioMerieux, Wellcome and Oxoid) and from reference strains (Ames, Iowa) were used in IFA and MAT while a whole cell heat-killed B. melitensis antigen was used in ELISA. Statistical analysis showed comparable results when using B. melitensis or B. abortus antigen, in IFA, from the same manufacturer but there were subtle differences among antigens from different manufacturers. Correlation between IFA and ELISA titers was poor, due to differences in the levels of these titers. However, the percentage of sensitivity, specificity, predictive positive, and predictive negative at different titers indicated the most reliable discriminative titers to be as follows: ELISA IgG 1 : 800 (100% for all), IgM 1 : 400 (100%, 93%, 100%, 100%, respectively) and IgA 1 : 200 (95%, 100%, 100%, 94%, respectively); IFA IgG 1 : 320 (95%, 93%, 95%, 93%, respectively) and IgM 1 : 80 (95%, 100%, 100%, 94%, respectively). IFA IgA showed either poor sensitivity or specificity at all titers. These findings and the subjective reading of IFA limit its value in Brucella diagnosis while the MAT showed high false negatives (5%–40%). Thus, ELISA proves to be the most reliable test for the diagnosis of patients with brucellosis.

Effects of bovine serum albumin on the ability of Barbour-Stoenner-Kelly medium to detect Borrelia burgdorferi

The ability of decreasing inocula of Borrelia burgdorferi to grow in otherwise identical Barbour-Stoenner-Kelly (BSK) media containing different lots of bovine serum albumin (fraction V) was determined. These media differed significantly in ability to detect B. burgdorferi. Some BSK media required inocula of 2 x 10(5) organisms per ml for detection, while other media could stimulate growth after inoculation with less than 2 organisms per ml. In addition, organisms from the less sensitive BSK media were thinner, longer, and less tightly coiled. The endpoint dilutions of indirect fluorescent-antibody titers, especially immunoglobulin M, exhibited up to 16-fold decreases, and both immunoglobulin G and M titers were more difficult to interpret with diagnostic slides prepared from some longer, thinner B. burgdorferi. These results demonstrate that, when performing laboratory investigations which rely on B. burgdorferi, it is essential that the quality of the BSK medium be determined.