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Biomedical subjects

L A Magnarelli

Publications and source records attributed to L A Magnarelli.

At least 19 recordsLinked to original sources

Epizootiology of Lyme disease and methods of cultivating Borrelia burgdorferi.

Lyme disease is prevalent over vast areas of land in the northern hemisphere. The etiologic agent is a spirochete, Borrelia burgdorferi, that is transmitted by Ixodes ticks. At least five species of anthropophilic ticks are competent vectors, and the causative agent or closely related species have been detected in 20 wild or domestic mammals and eight birds. The ability to isolate and culture B. burgdorferi from humans, ticks, and wildlife has significantly contributed to our understanding of the epizootiology of Lyme disease. Specific procedures for isolating borreliae from ticks and rodents in Barbour-Stoenner-Kelly medium are described. The authors have preserved in liquid nitrogen more than 500 isolates from ticks, wildlife, and a human.

Animals

Relationship between development of antibodies to Borrelia burgdorferi in dogs and the subsequent development of limb/joint borreliosis.

The relationship between antibody production and the subsequent development of limb/joint disorders of borreliosis was examined in dogs from south central Connecticut. Dogs without signs of illness, determined by physical examination, were selected from dogs being tested for Dirofilaria immitis. An ELISA was used to detect antibodies to Borrelia burgdorferi in 234 apparently healthy dogs during 1988. These dogs were monitored for 20 months after initial analyses to determine the prevalence of limb/joint disorder in seropositive and seronegative dogs. Of 234 dogs from which samples were initially obtained, 125 had antibodies to B burgdorferi and 109 were seronegative. The development of limb/joint disorder (eg, lameness, swelling, and signs of pain) accompanied by lethargy, fever, and inappetence in each group was nearly equal. Rates of 4.8% (6/125) and 4.6% (5/109) were recorded for seropositive and serosurvey of dogs, respectively. We conclude the serosurvey of apparently healthy dogs had no predictive value for the subsequent development of limb/joint disorder.

Animals

Comparison of whole-cell antibodies and an antigenic flagellar epitope of Borrelia burgdorferi in serologic tests for diagnosis of Lyme borreliosis.

A recombinant protein (p41-G) of an antigenic region of flagellin was used in a standard and amplified enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Borrelia burgdorferi, the causative agent of Lyme borreliosis. Comparable sensitivities (88 to 94%) were noted when sera from 17 persons who had erythema migrans and antibodies to whole-cell B. burgdorferi were tested against the p41-G antigen. In tests of a second study group of 36 persons who had erythema migrans but no detectable antibodies to whole-cell B. burgdorferi, 3 (8%) were positive when the p41-G antigen was used. Assay specificity likewise increased when the p41-G fragment was included in an ELISA with human sera containing treponemal antibodies. Recombinant flagellar proteins of B. burgdorferi, such as the p41-G antigen, can be used in an ELISA and may help confirm Lyme borreliosis during early stages of infection and improve specificity.

Antibodies, Bacterial

Antibodies to Borrelia burgdorferi in rodents in the eastern and southern United States.

Serologic studies were conducted to determine whether white-footed mice (Peromyscus leucopus) and cotton mice (Peromyscus gossypinus) contained serum antibodies to Borrelia burgdorferi, the causative agent of Lyme borreliosis. Enzyme-linked immunosorbent assays detected antibodies to this spirochete in 35.7 and 27.3% of 56 P. leucopus and 535 P. gossypinus serum samples, respectively, collected in Connecticut, North Carolina, South Carolina, Georgia, Florida, Alabama, and Mississippi. Antibody titers ranged from 1:160 to greater than or equal to 1:40,960. On the basis of adsorption tests, the antibodies detected appeared to be specific to Borrelia spirochetes. Seropositive rodents in the eastern and southern United States, areas where human cases of Lyme borreliosis have been reported, indicate a widespread geographic distribution of B. burgdorferi or a closely related spirochete.

Animals

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.

Adult

Rickettsiae and Borrelia burgdorferi in ixodid ticks.

Nymphs and adults of hard-bodied ticks were collected in Connecticut and tested by direct and indirect immunofluorescence staining methods for rickettsiae and Borrelia burgdorferi. Of the 609 Ixodes dammini ticks examined, 59 (9.7%) harbored rickettsialike microorganisms in hemocytes (blood cells). These bacteria reacted with fluorescein-conjugated antiserum to Ehrlichia canis, the etiologic agent of with fluorescein-conjugated antiserum to Ehrlichia canis, the etiologic agent of canine ehrlichiosis. Prevalence of infection ranged from 6.8 to 12.7% for males and females, respectively. Although the specific identities of the hemocytic rickettsialike organisms are unknown, they share antigens with ehrlichiae. Electron microscopy revealed rickettsiae in ovarian tissues of I. dammini that also had infected hemocytes. Rickettsialike organisms were also observed in the hemocytes of 5 (6.9%) of 73 Dermacentor variabilis ticks. In analyses for B. burgdorferi, 146 (23.7%) of 617 I. dammini ticks harbored these spirochetes in midguts. Hemocytic rickettsialike microorganisms coexisted with B. burgdorferi in 36 (6.7%) of the 537 nymphs and adults of I. dammini examined. I. dammini, with its broad host range, has the potential to acquire multiple microorganisms.

Animals

Spotted fever group rickettsiae or Borrelia burgdorferi in Ixodes cookei (Ixodidae) in Connecticut.

Immatures and females of Ixodes cookei, a hard-bodied tick, were collected from woodchucks and other mammals in the northeastern United States and examined for spotted fever group rickettsiae and Borrelia burgdorferi. Of the 93 nymphs analyzed by a hemolymph test, 4 (4.3%) harbored rickettsiae. Six (15%) of 40 females were also infected. All infected ticks were collected from woodchucks in Connecticut. Indirect fluorescent antibody staining of midgut tissues from 128 nymphs revealed B. burgdorferi in two (1.6%) ticks, whereas larval and female ticks were negative. Further consideration should be given to I. cookei as a possible vector of spotted fever group rickettsiae or spirochetes that cause Lyme borreliosis.

Animals

Adsorption and biotin-streptavidin amplification in serologic tests for diagnosis of Lyme borreliosis.

Serum samples from persons with Lyme borreliosis, periodontitis, or acute necrotizing ulcerative gingivitis were analyzed by an enzyme-linked immunosorbent assay (ELISA) with and without adsorption and amplification procedures. When biotin and streptavidin reagents were used as an amplification procedure in ELISA without the use of commercially prepared sorbent (Treponema phagedenis biotype Reiter), sensitivity increased. Of the 85 serum samples collected from persons with erythema migrans but no detectable antibodies to Borrelia burgdorferi by standard ELISA, 17 (20%) were reactive after amplification. Adsorption of serum samples with a 1:10 dilution of T. phagedenis biotype Reiter sorbent used in conjunction with amplified ELISA also improved the sensitivity of this method. However, cross-reactivity could not be completely eliminated. An adsorbed-amplified ELISA may be helpful in the diagnosis of Lyme borreliosis in the laboratory, particularly during early weeks of infection, when antibodies to B. burgdorferi can be present at a low concentration.

Antibodies, Bacterial

Antibodies to Borrelia burgdorferi in deer and raccoons.

An enzyme-linked immunosorbent assay (ELISA) was developed to detect serum antibodies to Borrelia burgdorferi, the causative agent of Lyme borreliosis, in deer (Odocoileus virginianus) and raccoons (Procyon lotor). Blood samples were collected from these mammals in Connecticut, Maryland, North Carolina, Georgia and Florida. Seropositivity for deer was highest in Connecticut (56% of 353 sera) and Maryland (51% of 35 sera). Raccoons in Connecticut, Maryland, North Carolina, and Florida also had antibodies to B. burgdorferi, but prevalence of positive sera was highest in Maryland (79% of 14 samples). Based on adsorption tests, the immunoglobulins detected in these mammals were probably specific to B. burgdorferi. The ELISA was more sensitive than an indirect fluorescent antibody staining method and was more suitable for analyzing large numbers of serum samples.

Animals

Tick parasitism and antibodies to Borrelia burgdorferi in cats.

Ticks were removed from naturally infested cats, and serum samples from these cats were tested for antibodies to Borrelia burgdorferi. Twenty-two of 93 cats (23.7%) had one or more motile stages of Ixodes dammini attached. Of 2 larvae and 20 nymphs removed from cats, 1 larva and 2 nymphs were infected with B burgdorferi. Spirochetes were not found in tissues of 13 female and 4 male ticks. Ten of 71 serum samples analyzed by indirect fluorescent antibody staining or ELISA contained antibodies to this spirochete. Maximal antibody titers were 1:256 and 1:2,560, respectively. At titers greater than or equal to 1:160 in ELISA, seropositivity ranged from 8.8% (n = 34 sera tested from 34 cats) in May through July to 33.3% (n = 12 cats tested) during February through April. In clinical studies of 30 cats, there were nearly equal percentages of seropositive cats with limb or joint disorders not accompanied by fever, anorexia, or fatigue (5 of 21 cats) and cats with these signs of illness but lacking lameness (2 of 9 cats.)

Animals

Serologic testing for Lyme disease.

Serologic testing for antibodies is, at present, the only practical means of diagnosing Borrelia burgdorferi infection. However, specific IgM spirochetal antibodies may not be at detectable concentrations in the early stage of Lyme disease (within 6 weeks of onset). Western blot analysis may be used to confirm indirect fluorescent antibody staining or enzyme-linked immunosorbent assay results, but it is laborious and expensive. False-positive reactions caused by cross-reactivity do occur. In patients who have had subclinical Lyme disease and contract another illness that causes similar symptoms, interpretation of serologic test results may be confused by residual antibodies. Therefore, assay results should always be interpreted in conjunction with clinical and epidemiologic findings.

Antibodies, Bacterial

Persistence of antibodies to Borrelia burgdorferi in dogs of New York and Connecticut.

Multiple blood samples were obtained from privately owned dogs living in tick-infested areas of New York (Westchester County) and Connecticut, where Lyme disease in human beings has been reported. Of the 175 dogs examined, 127 (72.6%) had limb/joint disorder, whereas the remaining 48 dogs were considered healthy. Results of analysis of 419 serum samples revealed IgM antibody to Borrelia burgdorferi in healthy and lame dogs during all seasons. Prevalence of seropositivity was significantly (P less than 0.01) greater, using a polyvalent ELISA (89.5%) than using a class-specific ELISA for IGM antibody (57.8%). Mean antibody titers obtained by use of polyvalent ELISA were likewise higher than IgM titers. Analysis of paired serum samples from dogs with limb/joint disorder indicated that 118 (92.9%) remained positive for IgM or IgG antibodies when retested weeks or months after initial testing. In 48 dogs without history of joint involvement or other signs of disease, 43 (89.6%) had antibody to B burgdorferi 2 or more times. Serotest results also revealed little or no change in antibody titer for lame dogs given antibiotics or for healthy dogs 2 or more months after initial sample collection.

Animals

Isolation of Borrelia burgdorferi from the blood of seven patients with Lyme disease.

PURPOSE: Borrelia burgdorferi, the etiologic agent of Lyme disease, has rarely been successfully cultured from blood. We report on seven patients from Westchester County, New York, with B. burgdorferi bacteremia diagnosed between April 1987 and August 1987. PATIENTS AND METHODS: One hundred thirty-two attempts to isolate spirochetes were made on blood specimens obtained from 104 patients. Twenty-two of these specimens were obtained from nine patients who had recently been bitten by Ixodes ticks but who were asymptomatic. Heparinized blood or serum specimens (0.2 to 0.4 mL) were inoculated onto 6 mL of modified Barbour-Stoenner-Kelly medium. Lyme serology was performed by enzyme-linked immunosorbent polyvalent, IgM, and IgG assays, fluorescent immunoassay, and microhemagglutination. RESULTS: Four of the seven patients had erythema migrans, two had facial nerve palsy, and one had a flu-like syndrome without rash. These patients represented 21% (four of 19) of all patients with the characteristic skin lesion who had blood cultures for B. burgdorferi, and 40% (two of five) of all those with facial nerve palsy. Serologic testing was frequently nonreactive; two patients had no detectable antibody on multiple sera by five different assays. All patients improved with antibiotic treatment, and had negative subsequent blood cultures, but five of seven had persistent complaints after completion of therapy. CONCLUSION: Culturing blood for B. burgdorferi may be useful in confirming the diagnosis of Lyme disease in selected patients. Use of spirochete blood cultures may facilitate a better understanding of the pathogenesis and natural history of Lyme disease.

Adolescent

Infectious but nonpathogenic isolate of Borrelia burgdorferi.

We document for the first time an infectious but nonarthritogenic variant of Borrelia burgdorferi. Strain 25015, previously isolated from an Ixodes dammini larva collected in upstate New York, was infectious but failed to produce arthritis or carditis in laboratory rats and mice. By contrast, pathogenic strain N40 invariably caused arthritis. This nonarthritogenic variant, with proteins with molecular weights different from those of the standard B31 strain, was frequently isolated from normal joint tissues of experimentally infected rats. Outer surface proteins A and B of strain 25015 have molecular weights of about 32,500 and 35,500, respectively, in contrast to molecular weights of approximately 31,000 and 34,000, respectively, for outer surface proteins A and B of strains B31 and N40. A prominent low-molecular-weight protein of about 23,500 also characterizes strain 25015. Test animals infected for 30 to 60 days had relatively high antibody titers (greater than or equal to 1:1,280). The nonarthritogenic variant will be useful, along with pathogenic strains, in providing comparative insight into the pathogenesis of Lyme borreliosis. Homologous immunoblotting of sera from rats and mice inoculated with both the arthritogenic and nonarthritogenic strains revealed antibody reactivities to proteins of B. burgdorferi different from those revealed in the heterologous tests.

Animals

Canine ehrlichiosis in Connecticut.

The first case of canine ehrlichiosis in Connecticut is reported. A female Brittany spaniel from Milford presented with lethargy, anorexia, fever, petechiae, splenomegaly, thrombocytopenia, anemia, elevated serum alkaline phosphatase, lymphopenia, and hypoalbuminemia. Serologic analysis revealed antibodies to Ehrlichia canis (titer, 1:2,560). This documents a more northern geographic distribution in the United States for this infectious agent than had previously been suspected.

Animals

Serologic analyses of cottontail rabbits for antibodies to Borrelia burgdorferi.

An enzyme-linked immunosorbent assay was developed to detect antibodies to Borrelia burgdorferi in cottontail rabbits captured in Millbrook, N.Y., and New York, N.Y. Five antigenically variable strains of B. burgdorferi were analyzed to determine the variability of serologic test results. In analyses of 79 serum samples, seropositivity ranged from 56% for a strain cultured from kidney tissues of a cottontail rabbit to 68% for a strain isolated from a larva of Ixodes dentatus, a tick that parasitized a cottontail rabbit. There were false-positive results when reference rabbit antisera to B. hermsii and Treponema pallidum were screened against B. burgdorferi. Cross-reactivity with antisera to Leptospira interrogans serovars was less pronounced. Western blot (immunoblot) analyses revealed reactivities of test sera to two or more surface or subsurface proteins of B. burgdorferi with approximate molecular masses of 18, 25 to 27, 34, 36, 41, and 59 kilodaltons. Cottontail rabbits respond immunologically to B. burgdorferi, but the observed variations in serologic test results should not be a limitation in field and laboratory investigations of Lyme borreliosis.

Animals

Cross-reactivity of nonspecific treponemal antibody in serologic tests for Lyme disease.

Serum samples obtained from 59 persons who had acute necrotizing ulcerative gingivitis, periodontitis, syphilis, or Lyme disease were tested against Treponema phagedenis biotype Reiter, Treponema denticola, Treponema vincentii, and Treponema scoliodontum by indirect fluorescent-antibody staining methods. Although there were positive reactions for sera representing each of these study groups and for 20 (13%) of 156 samples collected from the general population (premarital screening for syphilis), titration endpoints were relatively low (less than or equal to 1:256). Serum samples from 18 persons who had gingivitis or periodontitis but no history of Lyme borreliosis were tested by enzyme-linked immunosorbent assay for antibodies to Borrelia burgdorferi. Of these, five (28%) had immunoglobulin M antibody and four (22%) contained immunoglobulin G antibodies to this spirochete. Adsorption with either sorbent commercially prepared from T. phagedenis biotype Reiter or with washed, whole cells of T. phagedenis biotype Reiter reduced cross-reactivity in the enzyme-linked immunosorbent assay for Lyme borreliosis.

Antibodies, Bacterial