New perspectives about Hemotrophic mycoplasma (formerly, Haemobartonella and Eperythrozoon species) infections in dogs and cats.

The new perspectives about hemotrophic mycoplasma infections in cats and dogs can be summarized as follows: Haemobartonella and Eperythrozoon species infecting the dog and cat have been reclassified as mycoplasmal parasites and given the names M haemofelis (Ohio or large form of H felis), M haemominutum (California or small form of H felis), and M haemocanis (H canis). The prevalence of hemotrophic mycoplasma infections in anemic cats in the United States is about 25% and usually involves M haemofelis. However, nonanemic cats may also be infected most commonly with M haemominutum. Chronic infections with hemotrophic mycoplasmas may promote myeloproliferative disorders in FeLV-infected cats. M haemocanis infection in dogs may be a widespread latent disease in kennel-raised dogs and is being investigated. The PCR assay is exquisitely sensitive for detection of M haemofelis and M haemominutum, and testing of blood donor cats and perhaps dogs should be done regularly. Fleas are involved in the transmission of M haemofelis to the cat, whereas R sanguines may be involved with transmission of M haemocanis to the dog. Treatment with doxycycline effectively controls acute infection in the cat and dog, and enrofloxacin may also be effective in the cat, but none of the antibiotics tested to date consistently clears the parasites.     

Use of a polymerase chain reaction assay to detect and differentiate two strains of Haemobartonella felis in naturally infected cats

OBJECTIVE: To develop a polymerase chain reaction (PCR) assay that detects and differentiates the Ohio strain of Haemobartonella felis (H. felis-OH) and the California strain of H. felis (H. felis-CA) and to apply the assay to blood samples from cats with and without suspected haemobartonellosis (suspect and control cats, respectively). SAMPLE POPULATION: 220 blood samples were examined; 82 were from suspect cats, and 138 were from control cats. PROCEDURE: A PCR assay was designed to detect and differentiate H. felis-OH and H. felis-CA. RESULTS: On the basis of PCR assay results, the overall prevalence of H. felis infection was 19.5% (43/220). Suspect cats (28.0%; 23/82) were significantly more likely than control cats (14.5%; 20/138) to be H. felis infected. Significantly greater numbers of suspect cats were H. felis-OH infected (12.2%, 9/82) or H. felis-OH and H. felis-CA infected (4.9%, 4/82) than control cats (0% [0/138] and 0.7% [1/138], respectively). Significantly more anemic cats were H. felis-OH infected (14.3%; 4/28) or H. felis-OH and H. felis-CA infected (7.1%; 2/28) than nonanemic cats (2.3% [3/128] and 0.8% [1/128], respectively). The PCR assay was more accurate than cytologic examination for detection of H. felis. CONCLUSIONS AND CLINICAL RELEVANCE: Haemobartonella felis infections are more common in cats than previously recognized. Haemobartonella felis-OH is apparently more pathogenic than H. felis-CA. The PCR assay is more accurate than cytologic examination for detection of H. felis infection and is an effective clinical tool for the detection and differentiation of both H. felis strains known to infect cats.     

Erythrocytic rickettsia and protozoa of the dog and cat

This article summarizes the biologic and clinical features of the rickettsia and protozoa of canine and feline erythrocytes that are significant in North America: Haemobartonella canis, Haemobartonella felis, Cytauxzoon felis, Babesia canis, and Babesia gibsoni.     

Haemobartonella felis: recent developments in diagnosis and treatment

Haemobartonella felis is a pleomorphic uncultivated wall-less haemotrophic bacterial parasite. Phylogenetic analysis of 16S rRNA gene sequences from a number of isolates of H felis has demonstrated that these bacteria are most closely related to species in the genus Mycoplasma, and Haemobartonella and related organisms are currently being reclassified as Mollicutes. Diagnosis by cytological examination of blood smears has been problematic, but recent molecular studies have led to the development of sensitive polymerase chain reaction (PCR) assays for diagnosis. Such studies have also resulted in the recognition of two distinct strains of H felis, which are divided into different groups based on phylogenetic analysis. This evolutionary divergence between strains is accompanied by differences in pathogenecity. This review discusses new developments in the diagnosis and treatment of H felis, focusing on the use of, and interpretation of, PCR assays.     

Molecular characterization of Hepatozoon spp. infection in endangered Indian wild felids and canids

Hepatozoon species are parasites that infect a wide variety of domestic and wild animals. The objective of this study was to perform the molecular detection and characterization of Hepatozoon spp. in Asiatic lion, Indian tiger, Indian leopard, Indian wild dog, Indian domestic dog and cat based on partial 18S rRNA gene sequences from Hepatozoon spp. in the naturally infected animals. Hepatozoon spp. could be detected in blood samples of 5 out of 9 Asiatic lions, 2 out of 5 Indian tigers, 2 out of 4 Indian leopards and 2 out of 2 Indian wild dogs and, 2 out of 4 domestic cats and 2 out of 3 domestic dog samples by PCR. Sequencing of PCR amplicon and BLAST analysis of partial 18S rRNA gene sequences indicated that the Hepatozoon spp. in Asiatic lion, Bengal tiger, Indian leopard and domestic cat was Hepatozoon felis (98-99% similarity) and in the Indian wild and domestic dog the phylogenetic neighbour was Hepatozoon canis (97-100% similarity). Presence of H. felis and H. canis in both domestic and wild animals suggested that they are not host specific and the same parasite causes infection in domestic and wild felids and canids in India and from different parts of the world. To our knowledge, this is the first report on detection and molecular characterization of H. felis infection in Asiatic lions, Indian tigers, Indian leopards and H. canis in Indian wild dog. Hepatozoon spp. may be a potential pathogen and an opportunistic parasite in immuno-compromised animals and could thus represent a threat to endangered Indian wild felids and canids.     

Differentiation of Haemobartonella canis and Mycoplasma haemofelis on the basis of comparative analysis of gene sequences

OBJECTIVE: To determine whether Haemobartonella canis and Mycoplasma haemofelis (formerly known as H felis [large form]) can be differentiated by use of comparative analysis of gene sequences. SAMPLE POPULATION: Blood samples obtained from 3 dogs infected with H canis and 2 cats infected with M haemofelis. PROCEDURE: The partial 16S rDNA and ribonuclease P RNA (RNase P) genes were amplified, cloned, and sequenced in blood samples obtained from H canis-infected dogs and M haemofelis-infected cats. The DNA sequences were subjected to comparative analysis. RESULTS: The 16S rDNA sequences of H canis and M haemofelis were nearly identical (homology of 99.3 to 99.7%). In contrast, RNase P gene sequences had a lower degree of sequence homology between the 2 organisms (94.3 to 95.5%). CONCLUSIONS AND CLINICAL RELEVANCE: Haemobartonella canis and M haemofelis are not identical organisms. Molecular differentiation of H canis and M haemofelis is more clearly evident by use of comparative analysis of RNase P gene sequences than by comparative analysis of 16S rDNA gene sequences.     

华南虎源血巴尔通体的PCR检测

为了快速准确检测华南虎感染的血巴尔通体种类,我们根据已发表的猫血巴尔通体的16S rRNA基因序列,利用软件Primer Premier 5.0设计2对特异性引物,以华南虎源血巴尔通体基因组DNA进行PCR检测。2对引物分别扩增出1452 bp和170 bp的rDNA序列,1452 bp序列与GenBank~（TM）发表的3种猫血巴尔通体序列相似性分别为99.3%、91.7%和79.5%,而170 bp序列与序列AM748929的相似性为100%。实验结果表明华南虎感染的血巴尔通体为大型猫血巴尔通体,实验建立的PCR检测方法为华南虎血巴尔通体病的诊断及分子流行病学的调查提供了新的手段。     

Development and evaluation of a polymerase chain reaction assay using the 16S rRNA gene for detection of Eperythrozoon suis infection.

The 16S ribosomal RNA (rRNA) gene of Eperythrozoon suis was amplified using gene-specific primers developed from GenBank sequence accession U88565. The gene was subsequently cloned and sequenced. Based on these sequence data, 3 sets of E. suis-specific primers were designed. These primers selectively amplified 1394, 690, and 839 base-pair (bp) fragments of the 16S rRNA gene from DNA of E. suis extracted from the blood of an experimentally infected pig during a parasitemic episode. No polymerase chain reaction (PCR) products were amplified from purified DNA of Haemobartonella felis, Mycoplasma genitalium, or Bartonella bacilliformis using 2 of these primer sets. When the primer set amplifying the 690-bp fragment was used, faint bands were observed with H. felis as the target DNA. No PCR products were amplified from DNA that had been extracted from the blood of a noninfected pig or using PCR reagents without target DNA. The detection limits for E. suis by competitive quantitative PCR were estimated to range from 57 and 800 organisms/assay. This is the first report of the utility of PCR-facilitated diagnosis and quantitation of E. suis based on the 16S rRNA gene. The PCR method developed will be useful in monitoring the progression and significance of E. suis in the disease process in the pig.     

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.     

Neutropenia associated with osteomyelitis due to Hepatozoon canis infection in a dog

A 4-year-old, intact male Shiba dog was referred to Yamaguchi University Animal Medical Center, Yamaguchi, Japan, for the following complaints: anorexia, lethargy, intermittent fever, gingival bleeding and abdominal purpura. The dog presented with persistent neutropenia. Histopathological examination of a bone marrow sample revealed round to oval structures that resembled Hepatozoon micromerozoites and formed a "wheel-spoke" pattern. Furthermore, mature neutrophils were observed around these structures. PCR and sequencing using bone marrow aspirate confirmed Hepatozoon canis (H. canis) infection. These findings suggest that the neutropenia observed in this case was associated with osteomyelitis due to H. canis infection. This is the first report of neutropenia associated with H. canis infection. H. canis infection can be included in the differential diagnosis in canine cases of neutropenia in areas where the disease is endemic.     

Concurrent bartonellosis and babesiosis in a dog with persistent thrombocytopenia

A 12-year-old castrated male West Highland White Terrier was referred because of recurrent episodes of collapsing. The dog was mildly anemic and severely thrombocytopenic and had high serum alanine aminotransferase activity. Infection with Bartonella vinsonii (berkhoffii) was initially diagnosed on the basis of serologic testing. Despite treatment with a series of antimicrobials and prolonged use of immunosuppressive drugs, thrombocytopenia persisted. After 5 months of treatment, Babesia canis organisms were seen during examination of a direct blood smear. The dog was treated with imidocarb dipropionate for babesiosis, after which thrombocytopenia resolved, and administration of immunosuppressive drugs was discontinued. Retrospective review of blood smears failed to identify organisms; however, polymerase chain reaction (PCR) analysis of multiple stored blood samples obtained during the 5-month period of persistent thrombocytopenia identified DNA of B. canis vogeli. Babesiosis may cause persistent, unexplained thrombocytopenia in dogs that are not anemic. A PCR assay can facilitate a diagnosis of babesiosis when organisms are not evident or when serologic testing fails to detect Babesia-specific antibodies.     

Hypocellular marrow and extramedullary hematopoiesis in a dog: hematologic recovery after splenectomy

Nonregenerative anemia, poikilocytosis, thrombocytopenia, and splenomegaly were found in a 6-year-old dog. Marrow aspiration did not yield marrow particles, and microscopic examination of a marrow-core biopsy indicated hypocellular marrow. Marked extramedullary hematopoiesis was found in liver and spleen biopsy specimens. After splenectomy, the clinical and hematologic abnormalities resolved. The dog subsequently developed hemolytic anemia caused by Haemobartonella canis, which was resolved with oral tetracycline treatment. The dog remained healthy without recurrence of cytopenias or hypocellular marrow for 2 years after splenectomy. The clinical, hematologic, and microscopic findings in this dog were compatible with hypersplenism.     

A parasitological, molecular and serological survey of Hepatozoon canis infection in dogs around the Aegean coast of Turkey

Canine hepatozoonosis is caused by the tick-borne protozoon Hepatozoon spp. The prevalence of the infection in the Aegean coast of Turkey was investigated by examination of blood smear parasitology and polymerase chain reaction (PCR) using blood samples from 349 dogs collected from Central Aydin, Kusadasi, Selcuk, Central Manisa, Bodrum and Marmaris within the Aegean coast of Turkey. The indirect fluorescent antibody test (IFAT) for the detection of Hepatozoon canis antibodies was also used to detect the exposure rate to H. canis. PCR amplifying a 666bp fragment of 18S rRNA gene of Hepatozoon spp. was used in the epidemiological survey. The prevalence of Hepatozoon spp. infection was 10.6% by blood smear parasitology and 25.8% by PCR. IFAT revealed that 36.8% of serum samples were positive for antibodies reactive with Hepatozoon spp. The PCR products of 18S rRNA gene of Hepatozoon spp. isolated from six infected dogs, one isolate originating from each of the six different locations, were sequenced. The results of sequence analysis indicate that they are closely related to Indian and Japanese isolates of H. canis. This is the first epidemiological study on the prevalence of H. canis infection in the dog, in Turkey.     

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.     

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.     

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.     

A survey of cats and dogs for fleas: with particular reference to their role as intermediate hosts of Dipylidium caninum

Three species of fleas, viz. Ctenocephalides felis, Ctenocephalides canis, and Pulex irritans were found in an examination of 81 cats and 48 dogs in Wellington. C. felis was the most prevalent flea in cats, and C. canis predominated in dogs. It is speculated that C. felis, and its primary host the cat, may assume greater medical and veterinary significance than C. canis. In an examination of 1578 fleas for cysticercoids of the dog tapeworm, Dipylidium caninum, all were found negative. These results do not necessarily reflect the availability of cysticercoids in the environment, and the reasons for this are discussed.     

Direct detection of Ehrlichia canis by PCR in the conjunctiva of a dog with bilateral anterior uveitis

The following report describes the direct detection of Ehrlichia canis by real-time PCR in the conjunctiva of a 1-year-old female Maltese dog. After being imported from Brazil, the dog was presented because of anorexia, dehydration, fever, and palpable mandibular lymph nodes. A few days later, the dog developed bilateral blepharospasm, photophobia and anterior uveitis. Monocytic ehrlichia was diagnosed by a positive PCR result and the detection of IgM and IgG antibodies. Because of the massive uveitis a conjunctival sample was taken with a cytobrush, which also tested positive for Ehrlichia canis DNA by real-time PCR. Only one week after starting treatment with systemic doxycycline and local anti-inflammatory and cyclopalgic therapy the dog recovered from systemic and eye diseases. After therapy the follow-up examination revealed a full remission of clinical and hematological parameters and negative PCR result.     

Recombinant DNA probe detecting Eperythrozoon suis in swine blood

A genomic library to Eperythrozoon suis DNA was constructed in lambda gt11, and from this library, E suis clone KSU-2 was identified as a potential diagnostic probe. In hybridization experiments that used 100-microliters samples of blood collected in chaotropic salt solutions, the KSU-2 probe hybridized strongly with purified E suis organisms and blood samples from splenectomized swine that were parasitized with E suis. However, the probe under stringent conditions did not give radiographic indications of hybridizing with equine blood DNA, bovine blood DNA infected with Anaplasma marginale, canine blood DNA infected with Ehrlichia canis, feline blood DNA infected with Haemobartonella felis, or uninfected swine blood DNA.     

Mixed Ehrlichia canis, Hepatozoon canis, and presumptive Anaplasma phagocytophilum infection in a dog

A 5-month-old, female, mongrel dog was admitted to the Clinic of Companion Animal Medicine, Aristotle University of Thessaloniki, Greece, with depression, anorexia, fever, peripheral lymphadenopathy, splenomegaly, oculonasal discharge, nonregenerative anemia, and mild thrombocytopenia. Cytology of Giemsa-stained buffy coat, bone marrow, and lymph node aspiration smears revealed numerous morulae in mononuclear leukocytes and in neutrophils, and Hepatozoon canis gamonts in neutrophils. The dog was seropositive to Ehrlichia canis (immunofluorescence assay [IFA]) and Hepatozoon canis (ELISA) but not to Anaplasma phagocytophilum (IFA). A nested polymerase chain reaction performed on bone marrow aspirates was positive for E canis. This method was not applied for the detection of A phagocytophilum. Treatment with doxycycline and imidocarb dipropionate resulted in both clinical and parasitologic cure. This is the first reported case of a mixed infection with E canis, H canis, and presumptive A phagocytophilum. The findings emphasize the value of cytology in offering a quick and inexpensive diagnosis in mixed tick-borne infections of dogs.