Biomedical subjects
A F Azad
Publications and source records attributed to A F Azad.
Quantitation of cat immunoglobulins in the hemolymph of cat fleas (Siphonaptera: Pulicidae) after feeding on blood.
Passage of ingested cat immunoglobulin G (IgG) into the hemocoel of cat fleas, Ctenocephalides felis (Bouché), was examined using antibody capture enzyme-linked immunosorbent assays (ELISA) and Western blotting. Fleas were fed heparinized cat blood via membrane feeders. Cat IgG was present in the hemolymph of engorged female fleas 1 h after ingestion at an estimated quantity of 35 +/- 14 micrograms/ml. The prevalence of fleas with demonstrable cat IgG in their hemolymph 1 h after feeding was 100% for both female and male fleas. Following a single blood meal, cat IgG was present in the hemolymph of all 15 fleas tested 1 h after ingestion but dissipated below detectable levels in 10 of 20 fleas examined 3 h after ingestion, and was detectable in only 1 of 10 fleas examined 18 h after ingestion. However, when fleas were provided with continual access to blood over a 72-h period, IgG content in hemolymph, as measured in excised, triturated legs of individual fleas, remained fairly constant (3-16 pg IgG per sample). Flea feeding studies using specific antisera indicated that IgG in flea hemolymph retained its binding activity, and that at least a portion of the IgG was intact. Passage of ingested host antibody from gut into hemocoel is a prerequisite for the possible development of antiflea vaccines that target antigens outside of the flea midgut lumen (e.g., key components of the flea endocrine system controlling oogenesis).
Molecular identification of Rickettsia typhi and R. felis in co-infected Ctenocephalides felis (Siphonaptera: Pulicidae).
Rickettsia typhi and R. felis, 2 closely related rickettsial species, often have been identified in cat fleas, Ctenocephalides felis (Bouché) from the same geographical location. However, no fleas have been found to be naturally infected concurrently with both rickettsial species. To examine whether one rickettsial species can develop simultaneously with another species in the same flea host, cat fleas, naturally infected with R. felis, were allowed to feed on blood containing R. typhi (Ethiopian strain, 10(7) PFU/ml). Experimental controls consisted of uninfected cat fleas, fleas infected with only R. typhi; and fleas naturally infected with only R. felis. After 9 d at 28 degrees C, the fleas were examined by PCR amplification and subsequent restriction digest analysis and dot blot hybridization of PCR products. Results from these studies demonstrated that R. felis and R. typhi are capable of co-existing in the same flea host. Subsequent isolation of R. typhi from dually infected fleas by tissue culture indicated that R. typhi was viable and capable of being maintained in fleas naturally infected with R. felis. As more studies confirm the presence of R. felis and R. typhi in domestic pets and peridomestic vertebrates in urban areas, the ability of the individual cat fleas, which live on these animals, to support both rickettsial species could be an epidemiologically important consideration.
Detection of point mutations in rpoB gene of rifampin-resistant Rickettsia typhi.
The rpoB gene of rifampin-resistant Rickettsia typhi (Rif mutant) and wild-type R. typhi were sequenced and compared. The Rif mutant rpoB had three nucleotide substitutions, which resulted in amino acid changes at residues 151, 201, and 271 and may be the basis for the rifampin resistance.
Reverse transcriptase PCR amplification of Rickettsia typhi from infected mammalian cells and insect vectors.
We developed a reverse transcriptase PCR assay to detect expression of 120- and 17-kDa antigen genes in Rickettsia typhi. Infected Vero cell and flea RNAs were reverse transcribed by using random hexamers. The cDNA was amplified by using high concentrations of primer and template in an inexpensive, nonradioactive assay.
Inhibition of nitric oxide interrupts the accumulation of CD8+ T cells surrounding Plasmodium berghei-infected hepatocytes.
The elimination of liver-stage malaria parasites by nitric oxide (NO)-producing hepatocytes is regulated by T cells. Both CD8+ and CD4+ T cells, which surround infected hepatocytes, are evident by 24 h after sporozoite challenge in Brown Norway rats previously immunized with irradiated Plasmodium berghei sporozoites. While the number of CD4+ T cells remained the same beyond 24 h postchallenge, the number of CD8+ T cells increased three- and sixfold by 31 and 44 h, respectively. This increase in the number of CD8+ T cells correlated with a decrease in the number of intrahepatic parasites. In immunized rats, intrahepatic parasites were reduced in number by 31 h after sporozoite challenge and cleared from the liver by 44 h, as visualized by P. berghei-specific DNA in situ hybridization. If immunized rats were treated with aminoguanidine, a substrate inhibitor of NO synthase, at the time of challenge, liver-stage protection was blocked, as shown by the increase in parasite liver burden. Further histological examination of infected livers from immunized animals treated with aminoguanidine revealed fewer and smaller cellular infiltrates surrounding the infected hepatocytes, and the number of CD8+ T cells that normally accumulate within the infiltrates was drastically reduced. Consequently, the infected hepatocytes were not cleared from the liver. We hypothesize that the early production of NO may promote the influx and/or enhance local proliferation of malaria parasite-specific CD8+ T cells or a CD8+ T-cell subset which is required for parasite clearance.
Emerging bacterial zoonotic and vector-borne diseases. Ecological and epidemiological factors.
Among the etiologic agents of emerging infectious diseases are several bacterial organisms that naturally reside in animal and arthropod hosts. The most compelling emerging bacterial zoonotic and vector-borne diseases in the United States are Lyme disease; a Southern erythema migrans-like illness; human monocytic ehrlichiosis; human granulocytic ehrlichiosis; a novel cat flea-associated typhus group rickettsiosis; bartonelloses of immunocompetent and immunocompromised persons, particularly with AIDS; and sylvatic plague. Some of these antimicrobial-treatable infections are life threatening. During the acute stage of illness when antimicrobial agents are most effective, the flulike clinical signs and symptoms and available laboratory tests frequently do not point to a particular diagnosis. Epidemiological factors determined by the ecology of the bacteria are often the most useful diagnostic clues. The recognition of these evolving problems emphasizes the need for development of better laboratory diagnostic methods, for surveillance for and tracking of disease, and for continued research into factors contributing to transmission of the organisms. The continual appearance of previously unidentified bacterial infections requires prospective national strategies for timely recognition of the syndrome, identification of the agent, establishment of criteria and methods for diagnosis, optimization of the treatment regimen, and determination of successful approaches to prevention and control.
Presence of calreticulin in vector fleas (Siphonaptera).
Calreticulin has been defined in the cat flea, Ctenophalides felis (Bouché), and oriental rat flea, Xenopsylla cheopis (Rothschild). Calreticulin, a major endoplasmic reticulum protein, was previously identified as a component of ixodid tick saliva. Using a riboprobe generated from tick calreticulin complementary DNA (cDNA), we distinguished 2 transcripts for calreticulin in cat fleas by Northern blot analysis. Increased expression of calreticulin was not evident in fed versus unfed adult fleas. We were able to amplify a calreticulin flea product from fed female messenger RNa (mRNA) using primers designed from the tick calreticulin gene. One of these products hybridized to the tick riboprobe. Localization of specific antibody to cat flea tissues showed calreticulin in the midgut with no detection in the salivary glands. We also observed specific labeling of calreticulin with antibody in the ovaries of fed females. Several cat flea polypeptides appear to crossreact with anticalreticulin antibody in Western blots. We did not detect a calreticulin using antibody to the tick-secreted protein in cat flea salivary glands. This antibody did recognize a protein in the rate flea salivary glands. Our results show that fleas have calreticulin and, possibly, several isoforms. It appears that the salivary glands of the cat and oriental rat flea differ in detectable levels of calreticulin. The specific antibody labeling of the ovaries is interesting and remains to be understood. Calreticulin's appearance in the midgut suggests a possible source of calreticulin as a flea secretion. Further studies are in progress to complete the sequencing of the flea polymerase chain reaction (PCR) product to compare to tick-secreted calreticulin. Comparisons to other blood-feeding arthropods at the protein and gene level are also being done. We hope to define further the expression of calreticulin in fleas, and in general, blood-feeding arthropods, with respect to its role in feeding and pathogen transmission.
Acquisition of the cat scratch disease agent Bartonella henselae by cat fleas (Siphonaptera:Pulicidae).
We assayed the ability of cat fleas to become infected with Bartonella henselae, using an artificial feeding device. Fleas fed a concentration of 1 x 10(5) cfu/ml in blood were examined using immunofluorescent antibody assay and polymerase chain reaction. Bacteria were present in the gut at 3 h, and persisted up to 9 d after infection. Qualitatively, the density of B. henselae was greater in the flea gut at 9 d, indicating that replication was occurring in the gut. B. henselae also was detected in the feces of infected fleas 9 d after infection, and produced viable colonies upon inoculation onto heart infusion agar/rabbit blood plates. Our results indicate that fleas can maintain infection with B. henselae, and may play a role in the transmission of this bacterium from infected cats to humans.
Rickettsia felis: a new species of pathogenic rickettsia isolated from cat fleas.
A flea-borne rickettsia, previously referred to as ELB, has been implicated as a cause of human illness. Using sequence data obtained from a fragment of the citrate synthase gene, we compared ELB, Rickettsia australis, R. rickettsii, and R. akari with the louse-borne R. prowazekii. We tallied 24 base pair differences between ELB and R. prowazekii and 25 between R. rickettsii and R. prowazekii; there were 30 base pair differences between R. australis and R. prowazekii and 29 between R. akari and R. prowazekii. We observed 32 differences between Rickettsia typhi and ELB. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses of ELB, with typing sera against R. typhi indicate that ELB surface antigens are more closely related to the flea-borne R. typhi than to the mite-borne R. akari. On the basis of the results of citrate synthase gene sequence comparisons, as well as previous comparisons with 16S rRNA and 17-kDa-protein gene segments, we found that ELB is sufficiently genetically distinct from other rickettsiae to be designated a new species, Rickettsia felis.
Maintenance of protective immunity against malaria by persistent hepatic parasites derived from irradiated sporozoites.
Immunization of rodents and humans with irradiation-attenuated malaria sporozoites confers preerythrocytic stage-specific protective immunity to challenge infection. This immunity is directed against intrahepatic parasites and involves T cells and interferon gamma, which prevent development of exoerythrocytic stages and subsequent blood infection. The present study was undertaken to determine how protective immunity is achieved after immunization of rodent hosts with irradiated Plasmodium berghei sporozoites. We present evidence that irradiated parasites persist in hepatocytes of rats and mice for up to 6 months after immunization. A relationship between the persistence of parasites and the maintenance of protective immunity was observed. Protective immunity was abrogated in irradiated-sporozoite-immunized rats following the application of chemotherapy to remove preexisting liver parasites. Additionally, protective immunity against sporozoite challenge was established in rats vaccinated with early and late hepatic stages of irradiated parasites. These results show that irradiation-attenuated sporozoites produce persistent intrahepatic stages in vivo necessary for the induction and maintenance of protective immunity.
Co-localization of inducible-nitric oxide synthase and Plasmodium berghei in hepatocytes from rats immunized with irradiated sporozoites.
Both CD8+ T cells and IFN-gamma (IFN-gamma) are important components in the regulation of inducible-nitric oxide synthase (iNOS) which contribute to liver stage anti-malarial activity in rodents immunized with irradiated sporozoites. IFN-gamma, provided by malaria-specific CD8+ T cells, stimulates liver cells to produce nitric oxide (NO) for the destruction of infected hepatocytes or the parasite within these cells. To identify the cell source of iNOS in livers from Brown Norway rats challenged with Plasmodium berghei sporozoites, we probed tissue sections with antisera that recognize iNOS and the malarial exoerythrocytic stage parasite. Immunofluorescence analysis of parasitized livers demonstrate that 1) iNOS was found in infected hepatocytes, not Kupffer or endothelial cells; and 2) a higher proportion of infected hepatocytes express iNOS in immunized rats compared with naive animals after challenge. There was no immunoreactivity to the iNOS antisera in liver sections of immunized rats 15 h after sporozoite challenge, however, iNOS activity was present in 18% of the infected hepatocytes by 24 h and reached 81% by 31 h. In contrast, < 10% of the infected hepatocytes displayed iNOS activity in naive or immune animals 48 h after challenge. We also found a significant decrease in the ability of the immunized animals to express iNOS in response to sporozoite challenge by accelerating the removal of pre-existing irradiated-attenuated parasites from hepatocytes with the antimalarial drug, primaquine. Therefore, induction and maintenance of iNOS activity were dependent on intrahepatic persistence of the irradiated-attenuated parasite. These results suggest that liver-iNOS expression following sporozoite challenge is restricted to the infected hepatocyte and dependent on the presence of the irradiated-attenuated parasite in immune animals.
Use of polymerase chain reaction to detect bacteria in arthropods: a review.
Detection of bacteria in arthropod vectors traditionally has been pursued using serological and cell culture methodologies. The advent of the polymerase chain reaction (PCR) has made possible accurate, timely, and reproducible identification of bacteria in these vectors, particularly those microbes that are difficult to culture in vitro. We have reviewed the literature for PCR primers used to amplify gene segments of pathogenic bacteria from insect, tick, and mite vectors and provide the sequences of these primers, as well as notes on preparation of arthropod samples and direct sequencing of PCR products.
In vitro and in vivo antibiotic susceptibilities of ELB rickettsiae.
The activities of doxycycline, rifampin, chloramphenicol, and erythromycin against ELB rickettsiae (Rickettsia azadi) were determined by dye uptake and plaque assays. Plaque formation in Vero cells was inhibited by 0.12 microgram of doxycycline per ml. The data presented demonstrate the susceptibility of ELB rickettsiae to commonly used antibiotics for the treatment of rickettsial diseases.
Isolation, cultivation, and partial characterization of the ELB agent associated with cat fleas.
ELB rickettsiae from cat flea homogenates were recovered in tissue culture cells following sequential passage through laboratory rats and the yolk sacs of embryonated chicken eggs. Seven days after inoculation of ELB from the infected yolk sacs, Vero cells and L929 cells were observed to contain intracellular bacteria as demonstrated by Diff Quik and indirect immunofluorescence assay staining. The rickettsial and ELB identity of the cultured agent was confirmed by PCR detection of the 16S rRNA and citrate synthase genes and PCR-restriction fragment length polymorphism analysis of the 17-kDa conserved rickettsial antigen gene. The ELB rickettsiae induced plaques in Vero cells on day 11 postinfection. Rat anti-ELB serum reacted at 1:4,096 to cultured ELB and had lower reactivity to Rickettsia typhi Wilmington (1:1,024), Rickettsia akari Kaplan (1:512), and Rickettsia australis JC (1:64). Spotted fever group polyclonal sera also exhibited lower reactivity to ELB than to the homologous antigen. Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of the ELB isolate and two R. typhi strains were identical.
Plasmodium berghei: production and quantitation of hepatic stages derived from irradiated sporozoites in rats and mice.
Immunization with irradiated-attenuated malaria sporozoites has been shown to protect both rodents and humans against a homologous sporozoite challenge. Irradiated-attenuated sporozoites retain their capacity to invade hepatocytes and transform into trophozoites without undergoing complete schizogony. As a result, the minute size of these trophozoites (4-8 microns) makes their detection by conventional microscopy difficult. An additional problem lies in obtaining sufficient quantities of exoerythrocytic stages of attenuated parasites in vivo to study their antigenic repertoire and the sequence of events that occur after immunization of hosts. We have used a previously described method of inoculating Plasmodium berghei sporozoites directly into specific liver lobes (HPBI = hepatic portal branch inoculation) to improve parasite yields. Comparing HPBI with tail vein inoculation of sporozoites in Brown Norway rats and C57BL/6 mice revealed up to a 6-fold increase in hepatic parasite yields by HPBI method. The inoculation of 3 x 10(6) irradiated sporozoites via HPBI yielded 139 +/- 2 and 69 +/- 2 exoerythrocytic parasites per cm2 of liver in Brown Norway rats and C57BL/6 mice, respectively. The HPBI method therefore not only facilitates visualization of a large number of irradiated hepatic stage parasites within the defined lobes of the liver but also provides ample numbers of parasites for immunization and for immunological analysis.
Murine typhus: updated roles of multiple urban components and a second typhuslike rickettsia.
Studies using serologic and polymerase chain reaction-(PCR) facilitated analysis of field samples from southern Texas indicate the presence of Rickettsia typhi and ELB agent infected cat fleas, Ctenocephalides felis (Bouché), and the first observation of ELB infected vertebrates (opossums). The ELB agent is a recently described typhus-like rickettsia that is not distinguished from R. typhi or R. prowazekii by currently available serologic reagents. Restriction digests of PCR products from 399 fleas revealed an ELB agent infection rate of 3.8% and a R. typhi infection rate of 0.8%. Three of nine tested opossums (Didelphis virginiana) were shown to harbor ELB agent infections. No R. typhi infected rats, Rattus norvegicus, or rat-fleas, Xenopsylla cheopis Rothschild, were detected among surveyed samples. The persistence of this murine typhus disease focus appears to be better accounted for by the presence of infected cat fleas, opossums, and other non-rat hosts found in close association with human populations. Involvement of the ELB agent in the biology of murine typhus is suggested by its prevalence among suspected vectors and reservoir hosts.
Molecular identification of rickettsia-like microorganisms associated with colonized cat fleas (Ctenocephalides felis).
Cat fleas (Ctenocephalides felis) from eight commercial flea colonies from various regions of the USA were examined by selective PCR amplification, and subsequent restriction digest analysis and Southern hybridization of PCR products, for the presence of a rickettsia-like organism (ELB agent). These flea colonies were either started with fleas from one supplier (EL Labs), in which ELB agent was first identified, or were started with fleas from stray cats and dogs and later came into contact with ELB-infected fleas. Infection rates in the colonies ranged from 43% to 93%. The successful propagation of ELB agent in these colonies may be due to efficient trans-stadial and transovarial transmission. While ELB agent has recently been identified in blood from human murine typhus cases, attempts to infect mammalian cells and SCID mice with flea isolates were unsuccessful.