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PubMed1998

Development and evaluation of a PCR-based assay for detection of Haemobartonella felis in cats and differentiation of H. felis from related bacteria by restriction fragment length polymorphism analysis.

The 16S rRNA gene of Haemobartonella felis was amplified by using universal eubacterial primers and was subsequently cloned and sequenced. Based on this sequence data, we designed a set of H. felis-specific primers. These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of H. felis from each of four experimentally infected cats at peak parasitemia. No PCR product was amplified from purified DNA of Eperythrozoon suis, Mycoplasma genitalium, and Bartonella bacilliformis. Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment. The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats. By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data. In this PCR assay, the minimum detectable number of H. felis organisms was determined to be between 50 and 704. The potential usefulness of restriction enzymes DdeI and MnlI for distinguishing H. felis from closely related bacteria was examined. This is the first report of the utility of PCR-facilitated diagnosis and discrimination of H. felis infection in cats.

Anaplasmataceae↗
PubMed1999

Western immunoblot analysis of the antigens of Haemobartonella felis with sera from experimentally infected cats.

Cats were experimentally infected with a Florida isolate of Haemobartonella felis in order to collect organisms and evaluate the immune response to H. felis. Cryopreserved organisms were thawed and injected intravenously into nonsplenectomized and splenectomized cats. Splenectomized animals were given 10 mg of methylprednisolone per ml at the time of inoculation. Blood films were evaluated daily for 1 week prior to infection and for up to 60 days postinfection (p. i.). Blood for H. felis purification was repeatedly collected from splenectomized animals at periods of peak parasitemias. Organisms were purified from infected blood by differential centrifugation, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes for immunoblot analysis. Serum was collected from nonsplenectomized animals prior to and for up to 60 days p.i. and was used on immunoblots to identify antigens. The combination of splenectomy and corticosteroid treatment resulted in marked, cyclic parasitemias without concurrent severe anemia, providing an opportunity to harvest organisms in a manner that was not lethal to the animals. Several antigens (150, 52, 47, 45, and 14 kDa) were identified. An antigen with a molecular mass of approximately 14 kDa appeared to be one of the most immunodominant and was consistently recognized by immune sera collected at various times during the course of infection. These data suggest that one or more of these antigens might be useful for the serologic diagnosis of H. felis infections in cats.

Anaplasmataceae↗
PubMed1986

Haemobartonella felis infection in cats from the Glasgow area.

A survey of 155 cats for Haemobartonella felis infection in the Glasgow area revealed a prevalence of 23.2 per cent. The infection occurred in all age groups and there was no significant difference between sex or breed type. Infection with H felis was more prevalent among cats with fleas and those with feline leukaemia virus infection than among the other cats in the sample. H felis organisms identified were mainly coccoid with diameters between 0.63 micron and 1.73 micron. Rod forms, found in two cats, had lengths ranging from 0.79 micron to 1.1 micron and diameters between 0.17 micron and 0.24 micron.

Age Factors↗
PubMed2002

Characteristics of the 16S-23S rRNA intergenic spacer region of Mycoplasma haemomuris, previously classified as 'Haemobartonella muris'.

The intergenic spacer region between the 16S and 23S rRNA genes of Mycoplasma haemomuris, previously classified as 'Haemobartonella muris', was amplified by PCR and sequenced for analysis of the primary and secondary structures of the RNA transcript. The spacer region consisted of 219 base-pairs and lacked the spacer tRNA gene. A hypothetical secondary structure predicted in the RNA transcript of the spacer region was tentatively assigned box A and box B loci peculiar to the members of the Mycoplasma. Mycoplasma haemomuris and the other species of the genus Mycoplasma are consistent with these characteristics of the spacer region.

Base Sequence↗
PubMed2003

Molecular detection and characterization of Haemobartonella felis in domestic cats in Japan employing sequence-specific polymerase chain reaction (SS-PCR).

A novel PCR assay was developed in order to examine the prevalence of Haemobartonella felis (H. felis) in Japanese domestic cats and which was able to differentiate of the Ohio strain and the California strain of H. felis. Blood samples from a total of 21 cats suspected of having haemobartonellosis were examined employing a novel PCR assay and demonstrated positive results in 18 cats which was confirmed by cytological examination of blood smears. Four out of 18 positive cats (22%) were infected with the California strain, whilst the other 12 cats (67%) were infected with the Ohio strain and two animals (11%) were infected with both strains. As most of the cats with moderate to severe anemia were infected with the Ohio strain, it is suggested that the most prevalent strain of H. felis in Japanese domestic cats might be the Ohio strain. In the present study, it was thought that molecular detection and characterization of H. felis may provide valuable information regarding the severity and prognosis of this illness.

Animals↗
PubMed2000

Specific in situ hybridization of Haemobartonella felis with a DNA probe and tyramide signal amplification.

Haemobartonella felis is an epierythrocytic bacterium suspected to be the causative agent of feline infectious anemia. Previous studies with a polymerase chain reaction assay have identified a mycoplasmal 16S rRNA gene sequence that coincides with clinical disease and the presence of organisms in the blood. Tissues from a cat experimentally infected with H. felis were used for in situ hybridization studies to physically link this 16S rRNA gene to the organisms on the red cells. A biotin-labeled probe was used in conjunction with tyramide signal amplification to visualize the hybridization signal. This study clearly demonstrates a specific hybridization signal on the red cells in the tissues of the H. felis-infected cat. This in situ hybridization study is the final step in fulfilling the molecular guidelines for disease causation and proves that H. felis, a mycoplasmal organism, is the causative agent of feline infectious anemia.

Anaplasmataceae↗
PubMed2003

Identification of Haemobartonella felis (Mycoplasma haemofelis) in captive nondomestic cats.

This study was undertaken to determine whether Haemobartonella felis (Mycoplasma haemofelis), the causative bacterial agent of feline infectious anemia, infects nondomestic cats. Routine complete blood count and polymerase chain reaction (PCR) were performed to detect the gene for 16S ribosomal RNA for the organism. Sixty-four blood samples were collected from 54 nondomestic cats, including tigers (Panthera tigris), cheetahs (Acinonyx jubatus), lions (P. leo), mountain lions (Felis concolor), snow leopards (P. unica), and a jaguar (P. onca). Some cats were sampled on two or three different dates. Two tigers were positive for H. felis by PCR analysis. As previously described in domestic cats, the parasitemia appears to be intermittent in nondomestic cats.

Anemia↗
PubMed2002

Effect of preexisting FeLV infection or FeLV and feline immunodeficiency virus coinfection on pathogenicity of the small variant of Haemobartonella felis in cats.

OBJECTIVE: To investigate the effects of preexisting FeLV infection or FeLV and feline immunodeficiency (FIV) coinfection on the pathogenicity of the small variant of Haemobartonella felis (Hfsm, California variant) in cats. ANIMALS: 20 FeLV infected, 5 FeLV-FIV coinfected, and 19 retrovirus-free cats. PROCEDURES: A client-owned cat, coinfected with FeLV and Hfsm, was the source for Hfsm. Inoculum 1 (FeLV free) was obtained by passage of source Hfsm through 4 FeLV-resistant cats. Inoculum 2 was obtained by further passage of Hfsm (inoculum 1) through 2 specific pathogen-free cats. RESULTS: A mild-to-moderate anemia started 21 days after inoculation, with its nadir occurring at 35 to 42 days after inoculation. Infection with Hfsm induced greater decrease in hemoglobin concentration in FeLV infected cats, compared with retrovirus free cats. Reticulocytosis, macrocytosis, and polychromasia of erythrocytes developed in anemic cats regardless of retrovirus infection status. Mean neutrophil counts decreased during the hemolytic episode. For most cats, the anemia was transient. Four FeLV infected cats, 1 of which was also FIV infected, developed fatal FeLV-associated myeloproliferative diseases. Of the surviving cats, 8 died over the next 24 months from other FeLV-related diseases. Hemolysis did not recur after the initial episode. Inoculum 1 induced more severe anemia than inoculum 2. CONCLUSIONS AND CLINICAL RELEVANCE: Our results support the clinical observation that cats coinfected with FeLV and H felis develop more severe anemia than cats infected with H felis alone. Infection with Hfsm may induce myeloproliferative disease in FeLV infected cats. The small variant of H felis may lose pathogenicity by passage through FeLV-free cats.

Anaplasmataceae↗
PubMed2002

Use of enrofloxacin for treatment of large-form Haemobartonella felis in experimentally infected cats.

OBJECTIVE: To compare treatment with enrofloxacin and doxycycline with no treatment in cats experimentally infected with Haemobartonella felis. DESIGN: Prospective case-control study. ANIMALS: 16 cats. PROCEDURE: Cats were inoculated with large-form H. felis from a chronically infected donor. Cats were assigned to 1 of 4 treatment groups: doxycycline (5 mg/kg [2.3 mg/lb], p.o., q 12 h), low-dose enrofloxacin (5 mg/kg, p.o., q 24 h), high-dose enrofloxacin (10 mg/kg [4.5 mg/lb], p.o., q 24 h), and an untreated control group. Clinical signs, Hct, blood smears, and a polymerase chain reaction (PCR) assay were used to monitor progression of the infection. RESULTS: All cats were confirmed to be infected with H. felis via blood smear evaluations and PCR assay results. Treatment had no effect on Hct during the intratreatment period, but Hct values were significantly greater in the low-dose enrofloxacin group, compared with the control group, during the posttreatment period. During the intratreatment period, H. felis organism counts per 1,000 RBC in the doxycycline treatment and the high-dose enrofloxacin treatment groups decreased at a significantly faster rate than those in the control group. In the posttreatment period, organism counts in the doxycycline treatment group and the low- and high-dose enrofloxacin groups decreased at significantly faster rates than counts in the control group. There was no significant effect of treatment on the number of positive PCR assay results. Two cats treated with enrofloxacin and 1 cat treated with doxycycline completely cleared the H. fe is organism despite presumed immunosuppression caused by glucocorticoids. CONCLUSIONS AND CLINICAL RELEVANCE: Results support the hypothesis that enrofloxacin has anti-H. felis effects.

Anaplasmataceae↗
PubMed1975

Haemobartonella canis infection in research dogs.

During 1970-1972 haemobartonellosis occurred in research canines at 2 widely separated institutions. Clinical anemia occurred in a splenectomized dog at a Maryland facility, and subsequent screening disclosed an infection rate of 65% in a group of 20 splenectomized subjects. Treatment was successful, and the animals were used in research. A research institution in Texas encountered a number of dogs with fever (to 106 degrees F) and eosinophilia (to 42%) following minor surgery. Blood from affected animals was injected iv into splenectomized dogs, and 3 of 6 recipients developed haemobartonellosis. Further study was conducted, with some success, to establish a relationship between fever and eosinophilia and Haemobartonella canis infection in nonsplenectomized subjects. Our experiences suggest that haemobartonellosis is a widespread, latent disease of dogs and that significant potential exists for the infection to adversely affect research results.

Animals↗
PubMed1978

Ultrastructure of erythrocytes parasitized by Haemobartonella felis.

Experimental Haemobartonella felis infections were studied in 3 mature, intact cats by examining peripheral blood, lung, and spleen by electron microscopy. Coccoid, rod, or ring forms of the organism were found on or close to the erythrocytic membrane, and adjacent parasitized erythrocytes often were attached. Intracytoplasmic crystalloid inclusions occupying most of erythrocytic cytoplasm were seen in the 3 infected cats. The cat with the highest parasitemia had inclusions in about 10% of the erythrocytes. Less than 0.01% of the erythrocytes of a control cat contained inclusions. Parasitized erythrocytes, with and without inclusions, were seen in capillaries of the lung and spleen of infected cats. Macrophages in the lung and spleen of infected cats contained parasitized erythrocytes, either with or without inclusions. Some macrophages contained erythrocyte-free organisms in phagocytic vacuoles.

Anaplasmataceae↗
PubMed1990

Risk factors for Haemobartonella felis infection in cats.

A seroepidemiologic survey for Haemobartonella felis infection in cats of Wake County, NC was undertaken. To help assess risk factors, cat owners completed a 10-item questionnaire. Additionally, blood samples were obtained for determination of H felis presence, FeLV infection, and anemia. Prevalence rates for H felis presence were as follows: all cats, 4.9% (6/123); healthy cats, 3.6% (3/83); and ill cats, 7.5% (3/40). The estimated relative risk for haemobartonellosis was also increased in cats with any of the following: anemia, FeLV-positive status, lack of vaccinations, history of catbite abscesses and/or anemia, age less than or equal to 3 years, or outdoor-roaming status. The sex, breed, number of cats in the household, or presence of fleas were not significant factors, although ill male cats had a greater estimated relative risk for haemobartonellosis.

Anaplasmataceae↗
PubMed1979

Sequestration and phagocytosis of Haemobartonella felis in the spleen.

Spleens of two cats infected with Haemobartonella felis were examined by electron microscopy to determine the means by which the organism was sequestered in this organ. The principal means of sequestration occurred when H felis, located on the erythrocytes was removed by phagocytosis by a cordal macrophage, apparently preceded by the adhesion of extended processes of the macrophage to H felis. The second and least frequent means of removal of H felis was by pitting, a process that did not cause destruction of the host erythrocyte. The H felis was pitted from the parasitized erythrocyte when H felis passed through gaps between reticular cells or when the parasitized erythrocyte passed among the cytoplasmic processes of the reticular cells in the splenic cords. Some H felis were closely associated with the plasmalemma of cordal reticular cells and also were located in intracytoplasmic vacuoles of the cells without being influenced by the phagocytic process.

Anaplasmataceae↗
PubMed1978

Cold-agglutinin hemolytic anemia and Haemobartonella canis infection in a dog.

Cold-agglutinin hemolytic anemia in a dog was manifested by weakness, progressive hemolytic anemia, hematuria, and hemoglobinuria. Corticosteroid therapy failed to alleviate the anemia. The condition became complicated by Haemobartonella canis infection berminally. Prominent postmortem findings included disseminated thrombosis and infarction, glomerulonephritis, and thickened alveolar membranes.

Agglutinins↗
PubMed1981

Haemolytic anaemia in the Gunn rat following transplantation of isolated hepatocytes injected with Haemobartonella muris.

Extensively purified preparations of isolated rat hepatocytes derived from Wistar rats infected with Haemobartonella muris induced haemolytic anaemia when injected into congenitally jaundiced Gunn rats. In the absence of overt parasitaemia and marked falls in haematocrit, elevation of total plasma bilirubin levels was a valuable indicator of red-cell destruction. Evidence is presented that the parasite is removed by the spleen from the surface of infected red cells which then remain in the circulation.

Anaplasmataceae Infections↗
PubMed1981

Immunoconglutinin and antibody against fibrinogen products in hemolytic anemia and nephritis resulting from infection with a Haemobartonella-like agent.

An agent morphologically similar to Haemobartonella muris was isolated from the blood of rats infected with a strain of Trypanosoma lewisi kept at this Department. It caused acute hemolytic anemia, splenomegaly, glomerulonephritis, and death within 5 to 8 days in mature Sprague-Dawley rats. The disease was less severe in weanling rats which usually recovered within 3 to 4 wk. The anemia was accompanied by phagocytosis of erythrocytes by monocytes of the spleen and bone marrow, by high titers of cold-active hemagglutinin, high titers of antibody to the third component of fixed complement (immunoconglutinin), and antibody to fibrinogen/fibrin related products. Filtrates of blood from anemic rats passing a 0.20-micron filter did not produce disease or signs of infections, but filtrate from a 0.45-micron filter was infective. Attempts to grow the agent on rat embryo fibroblast cultures and in embryonated chicken eggs were successful. Tests for bacteria, mycoplasma, and spirochetes gave negative results. Blood of infected rats did not produce signs of infections when inoculated into laboratory mice, and normal rats housed in cages with acutely infected rats did not develop signs of infection or disease. Morphological similarity did not allow differentiation of the agent from H. muris. However, its virulence for mature rats differs markedly from that usually seen in H. muris infection.

Agglutinins↗
PubMed1977

Metabolism of [14C]glucose in Haemobartonella-like infected erythrocytes in splenectomized calves.

A quantitative relationship was established between glucose utilization by Embden-Meyerhof glycolytic pathway and hexose monophosphate shunt in erythrocytes from splenectomized calves infected with a Haemobartonella bovis-like organism. Parasitized erythrocytes metabolized glucose 3.69-fold above normal. Embden-Meyerhof glycolytic pathway accounted for 99.4% of glucose utilization, and the remainder, by hexose monophosphate shunt.

Anaplasmataceae↗