Bartonella quintana: A species of gram-negative bacteria in which man is the primary host and the human body louse, Pediculus humanus, the principal vector. It is the etiological agent of TRENCH FEVER.Trench Fever: An intermittent fever characterized by intervals of chills, fever, and splenomegaly each of which may last as long as 40 hours. It is caused by BARTONELLA QUINTANA and transmitted by the human louse.Bartonella: A genus of gram-negative bacteria characteristically appearing in chains of several segmenting organisms. It occurs in man and arthropod vectors and is found only in the Andes region of South America. This genus is the etiologic agent of human bartonellosis. The genus Rochalimaea, once considered a separate genus, has recently been combined with the genus Bartonella as a result of high levels of relatedness in 16S rRNA sequence data and DNA hybridization data.Bartonella Infections: Infections by the genus BARTONELLA. Bartonella bacilliformis can cause acute febrile anemia, designated Oroya fever, and a benign skin eruption, called verruga peruana. BARTONELLA QUINTANA causes TRENCH FEVER, while BARTONELLA HENSELAE is the etiologic agent of bacillary angiomatosis (ANGIOMATOSIS, BACILLARY) and is also one of the causes of CAT-SCRATCH DISEASE in immunocompetent patients.Bartonella henselae: A species of gram-negative bacteria that is the etiologic agent of bacillary angiomatosis (ANGIOMATOSIS, BACILLARY). This organism can also be a cause of CAT-SCRATCH DISEASE in immunocompetent patients.Pediculus: Lice of the genus Pediculus, family Pediculidae. Pediculus humanus corporus is the human body louse and Pediculus humanus capitis is the human head louse.Angiomatosis, Bacillary: A reactive vascular proliferation that is characterized by the multiple tumor-like lesions in skin, bone, brain, and other organs. Bacillary angiomatosis is caused by infection with gram-negative Bartonella bacilli (such as BARTONELLA HENSELAE), and is often seen in AIDS patients and other IMMUNOCOMPROMISED HOSTS.Phthiraptera: An order of small, wingless parasitic insects, commonly known as lice. The suborders include ANOPLURA (sucking lice); AMBLYCERA; ISCHNOCERA; and Rhynchophthirina (elephant and warthog lice).Cat-Scratch Disease: A self-limiting bacterial infection of the regional lymph nodes caused by AFIPIA felis, a gram-negative bacterium recently identified by the Centers for Disease Control and Prevention and by BARTONELLA HENSELAE. It usually arises one or more weeks following a feline scratch, with raised inflammatory nodules at the site of the scratch being the primary symptom.Homeless Persons: Persons who have no permanent residence. The concept excludes nomadic peoples.Typhus, Epidemic Louse-Borne: The classic form of typhus, caused by RICKETTSIA PROWAZEKII, which is transmitted from man to man by the louse Pediculus humanus corporis. This disease is characterized by the sudden onset of intense headache, malaise, and generalized myalgia followed by the formation of a macular skin eruption and vascular and neurologic disturbances.Arthropod Vectors: Arthropods, other than insects and arachnids, which transmit infective organisms from one host to another or from an inanimate reservoir to an animate host.Bartonellaceae: A family of small gram-negative bacteria whose organisms are parasites of erythrocytes in man and other vertebrates and the etiologic agents of several diseases.Bartonella bacilliformis: The type species of the genus BARTONELLA, a gram-negative bacteria found in humans. It is found in the mountain valleys of Peru, Ecuador, and Southwest Columbia where the sandfly (see PHLEBOTOMUS) vector is present. It causes OROYA FEVER and VERRUGA PERUANA.Lice Infestations: Parasitic attack or subsistence on the skin by members of the order Phthiraptera, especially on humans by Pediculus humanus of the family Pediculidae. The hair of the head, eyelashes, and pubis is a frequent site of infestation. (From Dorland, 28th ed; Stedman, 26th ed)Rickettsia felis: A species of gram-negative bacteria transmitted by the flea Ctenocephalides felis, and known to infect CATS, oppossums, and humans.Endocarditis, Bacterial: Inflammation of the ENDOCARDIUM caused by BACTERIA that entered the bloodstream. The strains of bacteria vary with predisposing factors, such as CONGENITAL HEART DEFECTS; HEART VALVE DISEASES; HEART VALVE PROSTHESIS IMPLANTATION; or intravenous drug use.Rickettsiaceae: A family of small, gram-negative organisms, often parasitic in humans and other animals, causing diseases that may be transmitted by invertebrate vectors.Bacteremia: The presence of viable bacteria circulating in the blood. Fever, chills, tachycardia, and tachypnea are common acute manifestations of bacteremia. The majority of cases are seen in already hospitalized patients, most of whom have underlying diseases or procedures which render their bloodstreams susceptible to invasion.DNA, Ribosomal Spacer: The intergenic DNA segments that are between the ribosomal RNA genes (internal transcribed spacers) and between the tandemly repeated units of rDNA (external transcribed spacers and nontranscribed spacers).DNA, Bacterial: Deoxyribonucleic acid that makes up the genetic material of bacteria.Heart Valves: Flaps of tissue that prevent regurgitation of BLOOD from the HEART VENTRICLES to the HEART ATRIA or from the PULMONARY ARTERIES or AORTA to the ventricles.Coxiella burnetii: A species of gram-negative bacteria that grows preferentially in the vacuoles of the host cell. It is the etiological agent of Q FEVER.Hemin: Chloro(7,12-diethenyl-3,8,13,17-tetramethyl-21H,23H-porphine-2,18-dipropanoato(4-)-N(21),N(22),N(23),N(24)) ferrate(2-) dihydrogen.Antibodies, Bacterial: Immunoglobulins produced in a response to BACTERIAL ANTIGENS.Siphonaptera: An order of parasitic, blood-sucking, wingless INSECTS with the common name of fleas.Cats: The domestic cat, Felis catus, of the carnivore family FELIDAE, comprising over 30 different breeds. The domestic cat is descended primarily from the wild cat of Africa and extreme southwestern Asia. Though probably present in towns in Palestine as long ago as 7000 years, actual domestication occurred in Egypt about 4000 years ago. (From Walker's Mammals of the World, 6th ed, p801)Polymerase Chain Reaction: In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships.Cat Diseases: Diseases of the domestic cat (Felis catus or F. domesticus). This term does not include diseases of the so-called big cats such as CHEETAHS; LIONS; tigers, cougars, panthers, leopards, and other Felidae for which the heading CARNIVORA is used.RNA, Ribosomal, 16S: Constituent of 30S subunit prokaryotic ribosomes containing 1600 nucleotides and 21 proteins. 16S rRNA is involved in initiation of polypeptide synthesis.Angiomatosis: A condition with multiple tumor-like lesions caused either by congenital or developmental malformations of BLOOD VESSELS, or reactive vascular proliferations, such as in bacillary angiomatosis. Angiomatosis is considered non-neoplastic.World War I: Global conflict primarily fought on European continent, that occurred between 1914 and 1918.Immunoglobulin M: A class of immunoglobulin bearing mu chains (IMMUNOGLOBULIN MU-CHAINS). IgM can fix COMPLEMENT. The name comes from its high molecular weight and originally being called a macroglobulin.Rickettsia: A genus of gram-negative, aerobic, rod-shaped bacteria often surrounded by a protein microcapsular layer and slime layer. The natural cycle of its organisms generally involves a vertebrate and an invertebrate host. Species of the genus are the etiological agents of human diseases, such as typhus.Rickettsia Infections: Infections by the genus RICKETTSIA.Rickettsia prowazekii: A species of gram-negative, aerobic bacteria that is the etiologic agent of epidemic typhus fever acquired through contact with lice (TYPHUS, EPIDEMIC LOUSE-BORNE) as well as Brill's disease.Rickettsia rickettsii: A species of gram-negative, aerobic bacteria that is the etiologic agent of ROCKY MOUNTAIN SPOTTED FEVER. Its cells are slightly smaller and more uniform in size than those of RICKETTSIA PROWAZEKII.Dictionaries, MedicalDictionaries as Topic: Lists of words, usually in alphabetical order, giving information about form, pronunciation, etymology, grammar, and meaning.

Culture of Bartonella quintana and Bartonella henselae from human samples: a 5-year experience (1993 to 1998). (1/97)

Bartonella quintana and Bartonella henselae are fastidious gram-negative bacteria responsible for bacillary angiomatosis, trench fever, cat scratch disease, and endocarditis. During a 5-year period, we received 2,043 samples for culture of Bartonella sp. We found Bartonella sp. to be the etiologic agent in 38 cases of endocarditis, 78 cases of cat scratch disease, 16 cases of bacteremia in homeless people, and 7 cases of bacillary angiomatosis. We correlated the results of positive cultures with the clinical form of the disease, type of sample, culture procedure, PCR-based genomic detection, and antibody determination. Seventy-two isolates of B. quintana and nine isolates of B. henselae from 43 patients were obtained. Sixty-three of the B. quintana isolates and two of the B. henselae isolates, obtained from patients with no prior antibiotic therapy, were stably subcultured. The sensitivity of culture was low when compared with that of PCR-based detection methods in valves of patients with endocarditis (44 and 81%, respectively), skin biopsy samples of patients with bacillary angiomatosis (43 and 100%, respectively), and lymph nodes of cat scratch disease (13 and 30%, respectively). Serological diagnosis was also more sensitive in cases of endocarditis (97%) and cat scratch disease (90%). Among endocarditis patients, the sensitivity of the shell vial culture assay was 28% when inoculated with blood samples and 44% when inoculated with valvular biopsy samples, and the sensitivity of both was significantly higher than that of culture on agar (5% for blood [P = 0.045] and 4% for valve biopsy samples [P < 0.0005]). The most efficient culture procedure was the subculture of blood culture broth into shell vials (sensitivity, 71%). For patients with endocarditis, previous antibiotic therapy significantly affected results of blood culture; no patient who had been administered antibiotics yielded a positive blood culture, whereas 80% of patients with no previous antibiotic therapy yielded positive blood cultures (P = 0.0006). Previous antibiotic therapy did not, however, prevent isolation of Bartonella sp. from cardiac valves but did prevent the establishment of strains, as none of the 15 isolates from treated patients could be successfully subcultured. For the diagnosis of B. quintana bacteremia in homeless people, the efficiency of systematic subculture of blood culture broth onto agar was higher than that of direct blood plating (respective sensitivities, 98 and 10% [P < 10(-7)]). Nevertheless, both procedures are complementary, since when used together their sensitivity reached 100%. All homeless people with positive blood cultures had negative serology. The isolation rate of B. henselae from PCR-positive lymph nodes, in patients with cat scratch disease, was significantly lower than that from valves of endocarditis patients and skin biopsy samples from bacillary angiomatosis patients (13 and 33%, respectively [P = 0.084]). In cases of bacillary angiomatosis for which an agent was identified to species level, the isolation rate of B. henselae was lower than the isolation rate of B. quintana (28 and 64%, respectively [P = 0.003]). If culture is to be considered an efficient tool for the diagnosis of several Bartonella-related diseases, methodologies need to be improved, notably for the recovery of B. henselae from lymph nodes of patients with cat scratch disease.  (+info)

Seroprevalence of Bartonella henselae in cats in Germany. (2/97)

Bartonella henselae and B. quintana infections in man are associated with various clinical manifestations including cat-scratch disease, bacillary angiomatosis and bacteraemia. While cats are the natural reservoir for B. henselae, the source of B. quintana is unclear. In this study, the sera of 713 cats from Germany were examined for the presence of antibodies against B. henselae, B. quintana or Afipia felis by an indirect immunofluorescence assay (IFA). Bartonella-specific antibody titres of > or =50 were found in 15.0% of the cats. There was substantial cross-reactivity among the various Bartonella antigens, although single sera showed high titres against B. henselae but not against B. quintana and vice versa. Antibodies against A. felis were not detected in any of these cats. Statistical analysis indicated that there is no correlation between Bartonella infections and the sex, age or breed of the cat or its hunting behavior. There was also no correlation between bartonella and toxoplasma infections in cats. However, whereas 16.8% of cats from northern Germany had B. quintana-specific antibodies, only 8.0% of cats from southern Germany were seropositive for B. quintana. No statistically significant difference was found for B. henselae. IFA-positive and IFA-negative sera were used for immunoblot analysis including B. henselae and B. quintana. Marked reactivity was observed with protein bands at 80, 76, 73, 65, 37, 33 and 15 kDa. The results of this study suggest that B. henselae, and possibly a B. quintana-related pathogen, but not A. felis, are common in cats in Germany, and that there are differences in the geographic distribution of bartonella infections in cats.  (+info)

Semiquantitative species-specific detection of Bartonella henselae and Bartonella quintana by PCR-enzyme immunoassay. (3/97)

Bartonella henselae is the main causative agent of cat-scratch disease, and both B. henselae and Bartonella quintana cause angioproliferative disorders such as bacillary angiomatosis. To increase the sensitivity of Bartonella detection by PCR and to improve the species differentiation, we developed a semiquantitative, species-specific PCR-based enzyme immunoassay (EIA). The 16S rRNA gene was selected as the target sequence. Internal nucleotide sequences derived from the amplified 16S rRNA region were used to develop species-specific oligonucleotide probes for B. henselae and B. quintana. Biotin-labeled PCR products were immobilized on streptavidin-coated microtiter plates, hybridized to a digoxigenin-labeled probe, and detected with antidigoxigenin peroxidase conjugate. No cross-hybridization with other Bartonella or non-Bartonella species was observed. This EIA was as sensitive as dot blot hybridization and was 10 times more sensitive than visualization of PCR products on agarose gels. Serial dilutions of B. henselae and B. quintana suspensions demonstrated that an optical density (OD) of approximately 0.200 was equivalent to 5 CFU in the reaction mixture. By comparing the OD of the bacterial dilutions with that obtained from clinical specimens we could determine that the number of CFU in clinical samples ranged from 10(3) to 10(6) CFU/ml. The PCR-EIA developed in the present study is a rapid, sensitive, and simple method for the diagnosis of B. henselae and B. quintana infections.  (+info)

Species-specific monoclonal antibodies for rapid identification of Bartonella quintana. (4/97)

Seven species-specific monoclonal antibodies (MAbs) to Bartonella quintana were produced and characterized. The MAbs were of the immunoglobulin G class and reacted only with 13 B. quintana strains in indirect microimmunofluorescence and Western immunoblotting assays. They did not react with eight other Bartonella spp., including Bartonella henselae, the most closely related species, and a selected MAb did also not react with nine other strains of gram-negative bacteria. The MAbs reacted mainly with a 34-kDa protein epitope of B. quintana which was shown to be species specific by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four of five body lice experimentally infected with B. quintana were found to be positive for the organism in microimmunofluorescence assays with one MAb. These MAbs may provide a specific, simple, rapid, and low-cost tool for the identification of B. quintana and the diagnosis of infections due to the microorganism.  (+info)

In vitro activities of telithromycin (HMR 3647) against Rickettsia rickettsii, Rickettsia conorii, Rickettsia africae, Rickettsia typhi, Rickettsia prowazekii, Coxiella burnetii, Bartonella henselae, Bartonella quintana, Bartonella bacilliformis, and Ehrlichia chaffeensis. (5/97)

In vitro activities of telithromycin compared to those of erythromycin against Rickettsia spp., Bartonella spp., Coxiella burnetii, and Ehrlichia chaffeensis were determined. Telithromycin was more active than erythromycin against Rickettsia, Bartonella, and Coxiella burnetii, with MICs of 0.5 microg/ml, 0.003 to 0.015 microg/ml, and 1 microg/ml, respectively, but was inactive against Ehrlichia chaffeensis.  (+info)

Bartonella quintana and urban trench fever. (6/97)

Contemporary Bartonella quintana infections have emerged in diverse regions of the world, predominantly involving socially disadvantaged persons. Available data suggest that the human body louse Pediculus humanus is the vector for transmission of B. quintana. Descriptions of the clinical manifestations associated with contemporary B. quintana infections have varied considerably and include asymptomatic infection, a relapsing febrile illness, headache, leg pain, "culture-negative" endocarditis, and, in human immunodeficiency virus-infected persons, bacillary angiomatosis. Laboratory diagnosis is most convincing when B. quintana is isolated in blood culture, but growth often takes 20-40 days; problems exist with both sensitivity and specificity of serological assays. On the basis of available information, use of doxycycline, erythromycin, or azithromycin to treat B. quintana infections is recommended. Treatment of uncomplicated B. quintana bacteremia for 4-6 weeks and treatment of B. quintana endocarditis (in a person who does not undergo valve surgery) for 4-6 months are recommended, with the addition of a bactericidal agent (such as a third-generation cephalosporin or an aminoglycoside) during the initial 2-3 weeks of therapy for endocarditis.  (+info)

Hemin-binding surface protein from Bartonella quintana. (7/97)

Bartonella quintana, the agent of trench fever and a cause of endocarditis and bacillary angiomatosis in humans, has the highest reported in vitro hemin requirement for any bacterium. We determined that eight membrane-associated proteins from B. quintana bind hemin and that a approximately 25-kDa protein (HbpA) was the dominant hemin-binding protein. Like many outer membrane proteins, HbpA partitions to the detergent phase of a Triton X-114 extract of the cell and is heat modifiable, displaying an apparent molecular mass shift from approximately 25 to 30 kDa when solubilized at 100 degrees C. Immunoblots of purified outer and inner membranes and immunoelectron microscopy with whole cells show that HbpA is strictly located in the outer membrane and surface exposed, respectively. The N-terminal sequence of mature HbpA was determined and used to clone the HbpA-encoding gene (hbpA) from a lambda genomic library. The hbpA gene is 816 bp in length, encoding a predicted immature protein of approximately 29.3 kDa and a mature protein of 27.1 kDa. A Fur box homolog with 53% identity to the Escherichia coli Fur consensus is located upstream of hbpA and may be involved in regulating expression. BLAST searches indicate that the closest homologs to HbpA include the Bartonella henselae phage-associated membrane protein, Pap31 (58.4% identity), and the OMP31 porin from Brucella melitensis (31.7% identity). High-stringency Southern blots indicate that all five pathogenic Bartonella spp. possess hbpA homologs. Recombinant HbpA can bind hemin in vitro; however, it does not confer a hemin-binding phenotype upon E. coli. Intact B. quintana treated with purified anti-HbpA Fab fragments show a significant (P < 0.004) dose-dependent decrease in hemin binding relative to controls, suggesting that HbpA plays an active role in hemin acquisition and therefore pathogenesis. HbpA is the first potential virulence determinant characterized from B. quintana.  (+info)

Use of rpoB gene analysis for detection and identification of Bartonella species. (8/97)

Identification of Bartonella species is of increasing importance as the number of infections in which these bacteria are involved increases. To date, these gram-negative bacilli have been identified by various serological, biochemical, and genotypic methods. However, the development of alternative tools is required, principally to circumvent a major risk of contamination during sample manipulation. The aim of our study was to investigate the possible identification of various Bartonella species by comparison of RNA polymerase beta-subunit gene (rpoB) sequences. This approach has previously been shown to be useful for the identification of members of the family Enterobacteriaceae (C. M. Mollet, M. Drancourt, and D. Raoult, Mol. Microbiol. 26:1005-1011, 1997). Following PCR amplification with specific oligonucleotides, a 825-bp region of the rpoB gene was sequenced from 13 distinct Bartonella strains. Analysis of these sequences allowed selection of three restriction enzymes (ApoI, AluI, and AflIII) useful for discerning the different strains by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. To confirm the potential value of such an approach for identification of Bartonella, the rpoB PCR was then applied to 94 clinical samples, and the results obtained were identical to those obtained by our reference PCR method. Twenty-four isolates were also adequately identified by PCR-RFLP analysis. In all cases, our results were in accordance with those of the reference method. Moreover, conserved regions of DNA were chosen as suitable primer targets for PCR amplification of a 439-bp fragment which can be easily sequenced.  (+info)

  • However, over the past 3 years we have identified one case of culture-positive and two cases of culture-negative endocarditis due to B. quintana . (
  • The 1984 edition of Bergey ' s Manual of Systematic Bacteriology lists only one species of Bartonella ( B. bacilliformis ) ( 13 ) and two species of Rochalimaea ( R. quintana and R. vinsonii ) ( 18 ), which were later included in the Bartonella genus. (
  • Lice have been demonstrated, as of recently, to be the key component in transmitting B. quintana. (
  • This has been attributed to living in unsanitary conditions and crowded areas, where the risk of coming into contact with other individuals carrying B. quintana and ectoparasites (body lice) is increased. (
  • Body lice are known to be the vectors of B. quintana. (
  • Body lice were collected and analyzed by the polymerase chain reaction and sequencing of a portion of the citrate synthase gene of B. quintana. (
  • Tests of lice from 3 of the 15 patients from whom they were collected were positive for B. quintana. (
  • Detection of Bartonella quintana from body lice ( Anoplura: Pediculidae ) infesting homeless people in Tokyo by molecular technique. (
  • To determine the presence of Bartonella quintana in head and body lice from persons in different locations in Ethiopia, we used molecular methods. (
  • Samples of blood and body lice were collected for culture for B. quintana and for serological testing. (
  • Examination for ectoparasites at the last day of reservoir hosts for B. quintana and that Pedicinus obtusus observation (day 36) revealed that all 10 monkeys were lice might act as efficient vectors. (
  • However, in spite of B. quintana 's emerging status, little is known about the pathogen's current reservoir and vector of transmission, although exposure to lice appears to correlate with incidence of disease ( 30 ). (
  • In 2015, we investigated Bartonella quintana and typhus group rickettsiae in body lice from homeless persons in Bogotá, Colombia. (
  • Comparison of the proliferation and excretion of Bartonella quintana between body and head lice following oral challenge. (
  • The mechanisms by which body lice became a vector for B. quintana, however, are poorly understood. (
  • Using this approach, we were able to confirm the presence of R. prowazekii in lice collected from refugees in Burundi, among whom typhus was epidemic, and the presence of B. quintana in lice collected from all locations except the Congo. (
  • To avoid exposure to B. quintana , HIV-infected patients should avoid body lice and, if infected, treat the infestation. (
  • Clinical diagnosis relies on a combination of epidemiological and clinical features such as male sex, homelessness, chronic alcoholism, contact with body lice and the absence of a previously known valvulopathy for B. quintana , and contact with cats or cat fleas and the presence of a previously known valvulopathy for B. henselae ( 10 ). (
  • Bartonella henselae-specific DNA was also detected in live deer ticks obtained from the households of 2 of these patients. (
  • A total of 1253 ixodid ticks (254 tick pools) collected between the end of 1995 and the spring of 1997 from six California counties (El Dorado, Los Angeles, Orange, Santa Cruz, Shasta and Sonoma) were examined for the presence of Bartonella DNA by PCR of the citrate synthase gene. (
  • Bartonella PCR-positive ticks were identified in five counties but none of the ticks from Los Angeles County was positive. (
  • Among the 54 Dermacentor occidentalis grouped in 12 pools from Orange County, one pool (8.3%) was PCR positive for Bartonella and similarly one pool (14.3%) was positive among the 30 Dermacentor variabilis ticks grouped in seven pools. (
  • Following our previous findings of Bartonella PCR-positive adult I. pacificus ticks in central coastal California, this is the first preliminary report of the presence of Bartonella DNA in I. pacificus nymphs and in Dermacentor sp. (
  • Distribution of Bartonella among ixodid ticks appears widespread in California. (
  • Parola P , Shpynov S , Montoya M , Lopez M , Houpikian P , Zeaiter Z , First molecular evidence of new Bartonella spp. (
  • Molecular detection of B. quintana using standard PCR targeting the 16S-23S intergenic spacer region [ 3 ] was positive for the cutaneous biopsy and had 100% homology with B. quintana strainFuller (Genbank accession number L35100). (
  • While the disorder is treatable and curable, it may be life threatening if untreated… Lesions can also occur in the oral mucosa , tongue, oropharynx, nose, penis, and anus. (
  • Bartonella quintana and Typhus Group Rickettsiae Exposure among Homeless Persons, Bogotá, Colombia. (
  • Four rhesus macaques confirmed to be Bartonella -negative by morphologic examination, blood test by nested PCR and serum test by indirect immunofluorescence assay were selected (all tests repeated after a week's interval), deloused, and held in a clean room for 7 days before the inoculation. (