Indirect Fluorescent Antibody Staining Research Articles

Infection with agents of human granulocytic ehrlichiosis, lyme disease, and babesiosis in wild white-footed mice (Peromyscus leucopus) in Connecticut.

White-footed mice, Peromyscus leucopus, were captured in southern Connecticut during 1997 and 1998 to determine the prevalence of infections caused by granulocytic Ehrlichia sp., Borrelia burgdorferi, and Babesia microti. Of the 50 mice captured and recaptured, 25 of 47 (53.2%) and 23 of 48 (47.9%) contained antibodies to the BDS or NCH-1 Ehrlichia strains, respectively, as determined by indirect fluorescent antibody (IFA) staining methods. The majority (83.3%) of 48 mice also contained antibodies to B. burgdorferi, as determined by enzyme-linked immunosorbent assay. Moreover, 20 of 26 (76.9%) contained antibodies to B. microti by IFA staining methods. In nested PCR tests using the 16S rRNA gene, the DNA of the human granulocytic ehrlichiosis (HGE) agent was detected in 17 of 47 mice (36.2%), but only 4 (23.5%) of these 17 mice were PCR positive at each capture. Antibody-positive reactions to granulocytic Ehrlichia sp. organisms were detected in 17 of 23 (73. 9%) of the PCR-positive mice. The sequences from PCR products from nine positive blood samples were identical to the HGE agent. Ehrlichia spp. were cultured from three of five mice captured in April 1998, including one that was PCR positive in April 1997. In addition, 2 of 14 larval Ixodes scapularis pools, which were attached to two PCR-positive mice, contained DNA of the HGE agent. A high percentage of white-footed mice are infected or have been infected naturally by the HGE agent with low-level persistent infection or frequent reinfection in some individual mice. However, the changes noted in the presence of DNA and antibodies in repeated blood and serum samples from individual mice over several months of field collection suggests that infection with granulocytic Ehrlichia is transient in most wild P. leucopus.

Serologic testing of horses for granulocytic ehrlichiosis, using indirect fluorescent antibody staining and immunoblot analysis

Abstract Objective To diagnose granulocytic ehrlichiosis in horses, compare results of indirect fluorescent antibody (IFA) staining procedures with those of immunoblot analysis, and compare serologic test findings with polymerase chain reaction (PCR) results. Animals 69 horses with high rectal temperatures (≥ 39 C) and lethargy, anorexia, or limb edema. Procedure 43 convalescent serum samples obtained from 38 horses 2 to 18 weeks after onset of illness were analyzed by use of immunoblot procedures and IFA staining methods, using the NCH-1 or BDS ehrlichial strains. Blood samples from 69 acutely ill horses were tested by PCR to detect ehrlichial DNA. Results Antibodies to Erlichial equi were detected in serum samples obtained during all seasons; seropositivity rates ranged from 50 to 93%. In IFA assays using the BDS or NCH-1 strain, seropositivity rates were 70 and 79%, respectively, whereas in immunoblot analyses using the NCH-1 strain, a seropositivity value of 79% was recorded. By immunoblot analysis, all serum samples of all seropositive horses were reactive to a protein having molecular mass of about 44 kd. Blood samples from 29 of 69 (42%) acutely ill horses contained ehrlichial DNA. Conclusion and Clinical Relevance Results of the various serologic testing procedures were in close agreement with each other. All serologic testing methods are suitable for laboratory diagnosis of equine granulocytic ehrlichiosis. (Am J Vet Res 1999; 60:631–635)

Infections of granulocytic ehrlichiae and Borrelia burgdorferi in white-tailed deer in Connecticut.

Serum or whole blood samples, obtained from 141 white-tailed deer (Odocoileus virginianus) in Connecticut (USA) during 1980, 1991, and 1996, were analyzed to detect past or current infections of Ehrlichia phagocytophila genogroup organisms and Borrelia burgdorferi. When the BDS or NCH-1 strains of granulocytic ehrlichiae were used separately in indirect fluorescent antibody (IFA) staining methods, antibody positivity rates varied from 25 to 64% in 1991 and 1996, respectively. All 50 sera tested from 1980 collections were negative. Although percentages of sera with B. burgdorferi antibodies, as detected by an enzyme-linked immunosorbent assay, also differed (23 to 53%), there were coexisting antibodies to both bacteria in 20 (49%) of 41 sera. In tests on specificity, 19 deer sera with ehrlichial antibodies also were tested by IFA staining procedures for Anaplasma marginale antibodies; one serum with a titer of 1:5,120 to ehrlichial antigen reacted to A. marginale antigen at a serum dilution of 1:320. In parallel analyses of 69 sera, results of Western blot analyses for ehrlichial infections in deer were concordant (72% agreement) with those of IFA staining methods containing ehrlichial antigen. All positive immunoblots showed bands to peptides of the NCH-1 strain of the human granulocytic ehrlichiosis (HGE) agent having molecular masses of about 44, 105, or 110 kDa. In polymerase chain reaction (PCR) studies of blood samples from 63 deer, 11 (18%) specimens were positive for 16S ribosomal DNA of an Ehrlichia phagocytophila genogroup organism, whereas 23 (37%) samples were positive for the DNA of the 44 kDa gene of the HGE agent. White-tailed deer are exposed to different tick-borne bacteria in areas where Ixodes scapularis ticks are abundant and may, in some instances, have had concurrent infections.

Antibodies to granulocytic ehrlichiae in white-footed and cotton mice in eastern United States.

Serum samples, collected from Peromyscus leucopus (white-footed mouse) or Peromyscus gossypinus (cotton mouse) during 1987 through 1990 in Florida, Georgia, Maryland, Mississippi, and North Carolina (USA), and in 1997 in southern Connecticut were analyzed by indirect fluorescent antibody (IFA) staining methods or Western blot procedures for antibodies to granulocytic ehrlichiae. Of the 82 sera from white-footed mice in Connecticut tested by IFA methods with either the BDS or NCH-1 strain of the human granulocytic ehrlichiosis (HGE) agent, 45 (55%) and 42 (51%) of the samples contained antibodies to these strains, respectively, at concentrations ranging from 1:80 to 1:2560. One (2%) of 43 sera from P. leucopus captured in Assateague Island (Maryland) had a titer of 1:80, while three (20%) of 15 sera from P. gossypinus, captured in Sapelo Island (Georgia) and four (40%) of 10 sera from cotton mice caught in Amelia Island (Florida) had antibodies to the NCH-1 strain at titers of 1:160 to 1:1,280. Fifty-five sera from P. leucopus in Cape Hatteras (North Carolina) and 30 sera from P. gossypinus in Mississippi were negative. Western blot analyses confirmed seropositivity for 19 (95%) of 20 mouse sera positive by IFA staining methods, including samples from both mouse species captured in Connecticut, Maryland, or Florida. There were key banding patterns to proteins having molecular masses of about 44, 80, 105, 110, or 120 kDa. Both serologic assays can be used to determine if mice have been exposed to granulocytic ehrlichiae. These rodents also may be useful in surveillance programs to identify endemic sites for HGE and in performing laboratory studies on immune responses to the etiologic agent.

Development and duration of antibody response against Ehrlichia equi in horses

Objective To characterize antibody response in horses with clinical signs of Ehrlichia equi infection. Design Prospective study. Animals 13 horses with confirmed acute E equi infection. Procedure Sequential serum sampling was performed in Connecticut and New York during 1995 and 1996 to identify horses with naturally acquired equine granulocytic ehrlichiosis (EGE). Horses with clinical signs of EGE (ie, fever without respiratory involvement) were confirmed as having E equi infection by polymerase chain reaction detection of ehrlichial DNA and by a minimum fourfold increase in total antibody titer by indirect fluorescent antibody staining methods. Infection was corroborated by use of DNA sequencing. Results 11 of 13 horses did not have detectable antibody in serum samples obtained at onset of disease. Seroconversion was evident in samples obtained 19 to 81 days thereafter. Median time to peak antibody response was 46 days after onset and median titer was 1:320. For 11 of 13 horses, antibody titers were ≤ 1:40 by 215 days after onset. Clinical Implications E equi was found in horses in the northeastern United States and caused EGE. Concentration of antibodies to E equi increased within 19 to 81 days of disease onset and were low during early weeks of infection. Therefore, antibody detection may be of limited value for early serologic diagnosis. We suggest that disease may develop from a reinfection, and retrospective serologic studies to determine exposure to E equi may reflect a disproportionate number of negative reactions. (J Am Vet Med Assoc 1998;212:1910–1914)

Temporal correlations between tick abundance and prevalence of ticks infected with Borrelia burgdorferi and increasing incidence of Lyme disease.

The abundance of host-seeking Ixodes scapularis nymphs, the principal vector for the Lyme disease agent, Borrelia burgdorferi, in Old Lyme, Lyme, and East Haddam, Connecticut, was compared with the incidence of reported human Lyme disease in the 12-town area around the Connecticut River and the State of Connecticut for the period 1989 to 1996. Ticks were sampled from lawns and woodlands by dragging flannel over the vegetation and examined for the presence of B. burgdorferi by indirect fluorescent antibody staining. The infection rate of the nymphal ticks by B. burgdorferi during the 9-year period was 14.3% (of 3,866), ranging from 8.6% (1993) to 24.4% (1996). The incidence of Lyme disease was positively correlated with tick abundance in the 12 town area (r = 0.828) and the State of Connecticut (r = 0.741). An entomological risk index based upon the number of I. scapularis ticks infected by B. burgdorferi was highest in 1992, 1994, and 1996 and was highly correlated with the incidence of Lyme disease in Connecticut (r = 0.944). The number of Lyme disease cases has been influenced, in part, by annual changes in population densities of I. scapularis and, presumably, a corresponding change in the risk of contact with infected ticks. Based upon tick activity and spirochetal infection rates, epidemiologically based Lyme disease case reports on a regional scale appear to reflect real trends in disease.

Serologic survey for antibodies to Borrelia burgdorferi in white-tailed deer in Ontario.

Serum samples collected from 623 white-tailed deer (Odocoileus virginianus) in southern Ontario (Canada) from 1985 to 1989 were tested for antibodies to Borrelia burgdorferi using an indirect fluorescent antibody (IFA) staining method. Samples from 150 of the deer were also tested using an enzyme-linked immunosorbent assay (ELISA). At IFA titers of 1:64 and 1:128 deer with antibodies to B. burgdorferi appeared to be widespread throughout southern Ontario, with an apparent prevalence ranging from 3 to 47%. At IFA titres > or = 1:256 and ELISA titres > or = 1:160 deer with antibodies to B. burgdorferi were only present on Long Point which is the only known endemic focus of Ixodes scapularis, the primary vector for B. burgdorferi, in southern Ontario. At these titres the apparent prevalence of antibodies to B. burgdorferi on Long Point was only 5 to 7%, even though the mean intensity of infestation of adult I. scapularis on deer was > 180, and 60% of the adult ticks are infected with B. burgdorferi. Based on these results, white-tailed deer do not appear to be a good sentinel species for the distribution of B. burgdorferi.

Cryptosporidium parvum Development in the BS-C-1 Cell Line

Cryptosporidium parvum is a worldwide parasitic protozoon capable of causing life-threatening disease in immunocompromised patients. In vitro cultivation of C. parvum has been under investigation for development of a well-defined in vitro model for C. parvum infectivity assay. This is the first report of C. parvum completing its life cycle in BS-C-1, an African green monkey kidney cell line. Both sodium hypochlorite-stimulated oocysts and purified sporozoites were able to initiate infection that led to completion of the whole life cycle, although inoculating purified sporozoites was less efficient. Beside Giemsa staining and normal light microscopy, an indirect fluorescent antibody staining method was developed to facilitate the detection and interpretation of C. parvum life stages developed in vitro. A C. parvum-specific polymerase chain reaction was also applied to detect and confirm the presence of C. parvum life stages in liquid from oocyst-infected cell cultures. In addition to the 452-base-pair (bp) product that can be specifically amplified from C. parvum oocysts and sporozoites, a fragment near 280 bp was also obtained. Various applications of this in vitro culture system are envisioned.

Antibodies to Ehrlichia equi in dogs from the northeastern United States

Objective To determine whether dogs living in tick-infested areas of the northeastern United States had been exposed to Ehrlichia equi, an etiologic agent of granulocytic ehrlichiosis. Design Analyses of dog sera. Animals 106 ill dogs and 12 clinically normal dogs. Procedure Antibodies to E equi were detected by indirect fluorescent antibody staining methods and western blot analyses. Results 10 of 106 (9.4%) sera tested from ill, privately owned dogs living in tick-infested areas of Connecticut and New York state had antibodies to E equi, a member of the E phagocytophila genogroup. Titration end points ranged from 1:80 to 1:1,280. Immunoblots revealed antibodies to proteins of E equi having molecular masses of predominantly 29, 40, 44, 105, 120, and 160 kd. There was good agreement between results of serologic testing methods, but use of the human isolate (NCH-1 strain) in western blot analyses detected 2 additional seropositive dogs found to be negative by indirect fluorescent antibody staining methods with the MRK strain. Clinical Implications Dogs living in areas where Ixodes scapularis is abundant may be exposed to multiple pathogens, such as E equi or Borrelia burgdorferi. Although mild or subclinical infections with E equi may develop, dogs with marked leukopenia, thrombocytopenia, or anemia should be viewed as possibly having ehrlichiosis. Laboratory diagnosis should include examinations for morulae in granulocytes or monocytes in addition to serologic analyses. (J Am Vet Med Assoc 1997;211:1134–1137)

Pneumocystis carinii Infection of the Conjunctiva in a Patient with Acquired Immune Deficiency Syndrome

Background: Ocular disease, especially the development of choroidal lesions, is a known extrapulmonary manifestation of Pneumocystis carinii infection in the acquired immune deficiency syndrome (AIDS). To our knowledge, conjunctival involvement due to P. carinii has not been described previously.Methods: The authors describe a 33-year-old homosexual male with AIDS in whom a large placoid, white lesion developed involving the tarsal conjunctiva of the right upper lid. Conjunctival malignancy was suspected and biopsies and swabs were taken. At this time, the patient had been receiving monthly aerosolized pentamidine prophylaxis for 18 months, and there was neither a history of pneumonia nor any clinical signs of disseminated infection due to P. carinii.Results: Histopathologic examination results of the conjunctival biopsy specimen showed a necrotic, frothy tissue surrounded by activated fibroblasts. Within this material, Gomori methenamine silver stains showed numerous round and cup-like cysts of P. carinii, confirming the diagnosis that had already been obtained by an indirect fluorescentantibody stain of a conjunctival smear specimen.Conclusions: The presence of white placoid conjunctival lesions in a patient with AIDS may indicate an infection due to P. carinii. Conjunctival disease due to P. carinii widens the spectrum of AIDS-associated ophthalmic pneumocystosis.

Antibodies to multiple tick-borne pathogens of babesiosis, ehrlichiosis, and Lyme borreliosis in white-footed mice.

Serum samples from Peromyscus leucopus (white-footed mouse), collected in Connecticut (USA) in 1983, 1985, and during 1990 to 1993, were analyzed by an enzyme-linked immunosorbent assay (ELISA) or indirect fluorescent antibody (IFA) staining methods for antibodies to Borrelia burgdorferi (strain 2591), Babesia microti, Ehrlichia chaffeensis (Arkansas strain), and Ehrlichia equi (MRK strain). Of the 294 serum samples tested, 160 (54%) contained immunoglobulins to one or more of these pathogens. There were antibodies to two or more etiologic agents in 77 (48%) of the seropositive mice. Although it was uncommon to detect coexisting antibodies to all four pathogens (n = 5 positive mice), E. chaffeensis-reactive antibodies or immunoglobulins to E. equi were present along with those produced to B. burgdorferi and B. microti in 24 other mice. These rodents carry antibodies to several tick-borne pathogens at numerous sites in Connecticut and may play a role in the epizootiology of ehrlichiosis as well as babesiosis and Lyme borreliosis.

Toxoplasma gondii: In vivo and In vitro Cystogenesis of the Virulent RH Strain

Models of cystogenesis were developed in vivo and in vitro with the virulent RH Toxoplasma gondii strain. In vivo in a first assay, 7.5-wk-old Fischer rats were inoculated intraperitoneally with 10(7) tachyzoites from mouse peritoneal fluid. Between 9 and 17 wk after inoculation, 1-5 cysts of 40-60 microns, containing numerous bradyzoites, were isolated from the brain of 5 of the rats. In a second assay, 24 Wistar rats were inoculated intraperitoneally with 10(2), 10(4), and 10(8) tachyzoites at 7, 11, 24, and 46 days. An inoculum of 10(8) was lethal for the 6 rats inoculated before 46 days and there was no correlation between the inoculum and the number of cysts except in the rats inoculated at 46 days. Fibroblast cell cultures (MRC5), at pH 7.5, were destroyed in 72 hr by 10(5)/ml RH-strain tachyzoites. However, when pH culture was increased to pH 8, up to 140 cysts were observed with indirect fluorescent antibody staining. Electron microscopy showed a 15 microns cyst, containing bradyzoites and surrounded by a 0.5-1-micron irregular wall.

Coexistence of antibodies to tick-borne pathogens of babesiosis, ehrlichiosis, and Lyme borreliosis in human sera

Serum specimens from persons with or without Lyme borreliosis were analyzed by indirect fluorescent antibody staining methods for total immunoglobulins to Babesia microti, Ehrlichia chaffeensis (Arkansas strain), and Ehrlichia equi (MRK strain). There was serologic evidence of human exposure to multiple tick-borne agents in 15 (6.6%) of 227 serum samples obtained in Connecticut and Minnesota. Of these, 10 serum samples were from Connecticut patients who had erythema migrans and antibodies to Borrelia burgdorferi (range, 1:160 to 1:40, 960). A maximal antibody titer of 1:640 was noted for a B. microti infection, whereas titration end points of 1:640 and 1:1,280 were recorded for E. chaffeensis and E. equi seropositives, respectively. In specificity tests, there was no cross-reactivity among the antisera and antigens tested for the four tick-borne pathogens. On the basis of serologic testing, a small group of persons who had Lyme borreliosis had been exposed to one or more other tick-borne agents, but there was no clinical diagnosis of babesiosis or ehrlichiosis. Therefore, if the clinical picture is unclear or multiple tick-associated illnesses are suspected, more extensive laboratory testing is suggested.

Telomerase RNA localized in the replication band and spherical subnuclear organelles in hypotrichous ciliates.

The intranuclear distribution of telomere DNA-binding protein and telomerase RNA in hypotrichous ciliates was revealed by indirect fluorescent antibody staining and in situ hybridization. The Oxytricha telomere protein colocalized with DNA, both being dispersed throughout the macronucleus except for numerous spherical foci that contained neither DNA nor the protein. Surprisingly, the telomerase RNA was concentrated in these foci; therefore, much of telomerase does not colocalize with telomeres. These foci persist through the cell cycle. They may represent sites of assembly, transport or stockpiling of telomerase and other ribonucleoproteins. During S phase, the macronuclear DNA replication machinery is organized into a disc-shaped structure called the replication band. Telomerase RNA is enriched in the replication band as judged by fluorescence intensity. We conclude that the localization of a subfraction of telomerase is coordinated with semiconservative DNA replication.

Hemocytic rickettsia-like organisms inIxodes scapularis: transovarial and transstadial transmission

Unengorged, host-seeking female Ixodes scapularis were collected from the northeastern United States (Rhode Island and Massachusetts) in April 1992. Hemolymph preparations from F1males and females were tested to determine if hemocytic rickettsia-like organisms, having shared antigens with ehrlichiae, are passed transovarially and transstadially. Fluorescein-labeled dog anti-Ehrlichia canis antiserum was used to detect infected hemocytes by means of direct fluorescent antibody staining methods. In duplicate tests of F1ticks, hemocytes from 25 males and 25 females were tested by indirect fluorescent antibody staining methods with antisera from two persons who had Rocky Mountain spotted fever. These hemolymph preparations were nonreactive, but those from cohorts of ticks reared from five infected females and tested with anti-E. canis antiserum contained rickettsia-like organisms in the cytoplasm of hemocytes. Thirty-four (54.8%) of 62 males and 71 (63.4%) of 112 females were positive. Prevalence of positive males among the five cohorts ranged from 18.8 to 80%, while results for females varied between 21.2 and 91.7%. The unidentified rickettsia-like hemocytic organisms, which have shared antigens with Ehrlichia species, are transmitted transovarially and transstadially in I. scapularis.

Effect of Cytopathic Bovine Viral Diarrhea Virus (BVDV) Superinfection on Viral Antigen Association with Platelets, Viremia, and Specific Antibody Levels in Two Heifers Persistently Infected with Noncytopathic BVDV

Two female Holstein heifers (Nos. 132,138) obtained from the same farm and persistently infected with noncytopathic (NCP) bovine viral diarrhea virus (BVDV) were inoculated intravenously with a heterologous cytopathic (CP) BVDV isolate (Burns). Each received an inoculum containing 103 CCID50 of CP BVDV. 7 Heifer No. 132 was inoculated at 302 days of age with a virus-neutralizing monoclonal antibody (MAb) escape variant of BVDV-Burns, and heifer No. 138 received at 636 days of age parent BVDV-Burns. The CP BVDV-Burns variant used in heifer No. 132 was recovered by in vitro selection for MAb neutralization resistance. Agar containing MAb 12G4a.a was poured over primary bovine testes cell monolayers infected with parent BVDV-Burns isolate. After 7 days of incubation, placques in the agar were harvested for viral expansion. Such a neutralization-escape variant of BVDV-Burns was selected based on its resistance to neutralization by MAb 12G4a.a, differing from BVDVBums parent in its neutralization reaction at only this one gp53-specific epitope. The BVDV-Burns parent and variant were differentiated from the persistently infecting NCP BVDV by reactivity with monoclonal antibody 39.a.7 in an indirect fluorescent antibody staining assay. Clinical responses to superinfection, as well as virus isolation using mononuclear cells (MNC) and necropsy tissues, were recorded. Heifer No. 132 was clinically normal when euthanized at 57 days postsuperinfection (DPSI), and heifer No. 138 was euthanized when moribund, displaying classic signs of mucosal disease at 16 DPSI. Several parameters monitored after inoculation with BVDV-Burns included platelet count, association of NCP BVDV antigen(s) with platelets, viral concentration in serum, and specific virusneutralizing antibody levels in serum. Rectal temperatures were obtained for each heifer twice daily prior to and after super-infection. Both heifers experienced pyrexia (greater than 39.2 C) 2-12 DPSI, with peak fever in No. 132 reaching 41.6 C and peak temperature in No. 138 attaining 42.1 C. Neither heifer showed hemorrhage at any time antemortem. In addition, heifer No. 138 developed other clinical signs of disease, including diarrhea, anorexia, and depression. Gross lesions observed in heifer No. 138 at necropsy were consistent with those previously described for experimentally induced mucosal disease. Tissue specimens harvested from each heifer at necropsy for virus isolation included spleen, mesenteric lymph node, Peyer’s patch, bone marrow, brain,

Current status of laboratory diagnosis for Lyme disease

Laboratory tests have been used extensively to help diagnose Borrelia burgdorferi infections. In many cases, results of indirect fluorescent antibody (IFA) staining methods or an enzyme-linked immunosorbent assay (ELISA), combined or separate from findings of Western blot analyses, have confirmed clinical diagnoses of Lyme disease. Alternative assays, such as culturing or DNA detection by polymerase chain reaction (PCR) methods, can provide more definitive evidence of B. burgdorferi infection than can antibody assays. However, aside from being more expensive, culturing B. burgdorferi from human tissues and fluids gives us a low yield, while results of PCR analyses can be as misleading as those obtained by performing IFA staining methods or an ELISA if there are false-negative or false-positive reactions. With increased knowledge of human immune responses to key proteins of B. burgdorferi, such as those with molecular masses of 21, 31, 34, 39, 41, and 93 kilodaltons, Western blot analyses are being used more frequently to confirm B. burgdorferi infections. These methods have been particularly helpful in identifying false-positive reactions in an ELISA. Until highly sensitive and specific assays have been adequately standardized, diagnosis of Lyme disease should be based primarily on clinical and epidemiologic evidence.

Monospecific antiserum is suitable for the selective detection of Xylella fastidiosa

Monospecific antibodies were successfully obtained by using unique protein bands of the quarantine bacterium Xylella fastidiosa, the causal agent of Pierce's disease of grapevine, and by applying gel electrophoresis and electroblotting. The monospecific antibodies directed against the two best suited protein bands (20.7 kD, 19.8 kD) did not show any cross-reaction on a Western blot, a solid phase ELISA (Enzyme-Linked Immunosorbent Assay) or an IFAS (Indirect Fluorescent Antibody Staining). A monospecific antiserum was generated in rabbits directed against the 20.7 kD and 19.8 kD protein bands, and was applied in an ELISA. Formerly cross-reacting bacteria isolated from grapevine cuttings did not show any antibody reaction with this antiserum. When grapevine cuttings were pre-infiltrated with a suspension of 4.15 × 10 7 bacteria the detection limit was found to be 10 6 −10 5 cells in the sap extract using ELISA. Titer used in all assays of the monospecific antiserum was 1:100. Using pure cultures of Xylella fastidiosa the detection limit of the monospecific antiserum in an indirect ELISA was 8.30 × 10 5 cells/ml.

Membrane-binding domains and cytopathogenesis of the matrix protein of vesicular stomatitis virus

The membrane-binding affinity of the matrix (M) protein of vesicular stomatitis virus (VSV) was examined by comparing the cellular distribution of wild-type (wt) virus M protein with that of temperature-sensitive (ts) and deletion mutants probed by indirect fluorescent-antibody staining and fractionation of infected or plasmid-transfected CV1 cells. The M-gene mutant tsO23 caused cytopathic rounding of cells infected at permissive temperature but not of cells at the nonpermissive temperature; wt VSV also causes rounding, which prohibits study of M protein distribution by fluorescent-antibody staining. Little or no M protein can be detected in the plasma membrane of cells infected with tsO23 at the nonpermissive temperature, whereas approximately 20% of the M protein colocalized with the membrane fraction of cells infected with tsO23 at the permissive temperature. Cells transfected with a plasmid expressing intact 229-amino-acid wt M protein (M1-229) exhibited cytopathic cell rounding and actin filament dissolution, whereas cells retained normal polygonal morphology and actin filaments when transfected with plasmids expressing M proteins truncated to the first 74 N-terminal amino acids (M1-74) or deleted of the first 50 amino acids (M51-229) or amino acids 1 to 50 and 75 to 106 (M51-74/107-229). Truncated proteins M1-74 and M51-229 were readily detectable in the plasma membrane and cytosol of transfected cells as determined by both fluorescent-antibody staining and cell fractionation, as was the plasmid-expressed intact wt M protein. However, the expressed doubly deleted protein M51-74/107-229 could not be detected in plasma membrane by fluorescent-antibody staining or by cell fractionation, suggesting the presence of two membrane-binding sites spanning the region of amino acids 1 to 50 and amino acids 75 to 106 of the VSV M protein. These in vivo data were confirmed by an in vitro binding assay in which intact M protein and its deletion mutants were reconstituted in high- or low-ionic-strength buffers with synthetic membranes in the form of sonicated unilammelar vesicles. The results of these experiments appear to confirm the presence of two membrane-binding sites on the VSV M protein, one binding peripherally by electrostatic forces at the highly charged NH2 terminus and the other stably binding membrane integration of hydrophobic amino acids and located by a hydropathy plot between amino acids 88 and 119.

Colonization of professional divers by toxigenicVibrio choleraeO1 andV. choleraenon-O1 at dive sites in the United States, Ukraine and Russia

Vibrio cholerae, recognized as the causative agent of epidemic cholera, was isolated from healthy professional divers and from water samples collected at dive sites in the United States, Ukraine and Russia. Swabs of nose, ear and throat of divers and their tank regulators, i.e. the divers and their diving gear, were taken before and after routine dives. Blood samples were collected before and 30-60 days after each dive to measure IgG and IgA titers against the whole cell antigen of V. cholerae O1. Nine strains of V. cholerae O1 and nine strains of V. cholerae non-O1 were isolated during this study. These isolates were identified by conventional biochemical tests and indirect fluorescent antibody staining methods, using fluorescein isothiocyanate-labeled monoclonal antibody, COLTA, prepared against the 'A' antigenic factor of the lipopolysaccharide of V. cholerae O1, and serotyped by slide agglutination. Seven of the nine strains of V. cholerae O1 isolated and successfully cultured during the studies, were toxigenic by enzyme-linked immunosorbent assay and polymerase chain reaction. Analyses of IgG and IgA antibodies of the divers showed that most of the divers had prior exposure to V. cholerae O1. V. cholerae serotype non-O1 strains isolated during the study were found to be non-toxigenic.