Experimental transmission of Hepatozoon americanum to rodents

Laboratory-raised cotton rats (Sigmodon hispidus), outbred white mice (Mus musculus), and C57BL/6J-Lystbg-J/J mice (M. musculus) that were administered approximately 50 sporulated oocysts of Hepatozoon americanum (AF176836) by gavage developed inflammatory lesions containing parasitic cystozoites in cardiac and skeletal muscle, kidney, and lung. Sprague-Dawley rats (Rattus norvegicus) similarly exposed showed no evidence of infection. Cystozoites were first detected by histopathologic examination four weeks after exposure to oocysts. Globular, PAS-positive material accumulated around the cystozoites as the duration of infection lengthened. Nested PCR analysis of tissues collected 16 weeks post-exposure was positive for the 18S rRNA Hepatozoon sp. gene and the DNA sequence of the fragment amplified was 99.6% and 99.8% identical to H. americanum sequences previously reported from naturally-infected dogs (AF176836 and AY864676, respectively). Merogonous and gamontogonous stages of the parasite were not detected in any of the cystozoite-infected rodents.     

Analysis of the 18S rRNA gene sequence of a Hepatozoon detected in two Japanese dogs

Partial sequences of the 18S rRNA gene (625 bp) from a Hepatozoon detected in two canine hepatozoonosis cases, one clinical and one subclinical, in Japan were analyzed. Both sequences were identical to each other and they were closely related to the Hepatozoon canis strain found in Israel with 99% (617/625) nucleotide identity. Both Hepatozoon americanum and Hepatozoon catasbianae were distantly related to the Japanese Hepatozoon with 94% (586/625) and 91% (566/625) identities, respectively. In a phylogenetic tree, the Japanese Hepatozoon was most closely related to H. canis from Israel but was significantly different than H. americanum and H. catasbianae. These results suggest that the Hepatozoon detected in the Japanese dogs might be a strain variant of H. canis, but is apparently a different species than H. americanum.     

Molecular characterization of Hepatozoon sp. from Brazilian dogs and its phylogenetic relationship with other Hepatozoon spp

To characterize phylogenetically the species which causes canine hepatozoonosis at two rural areas of Rio de Janeiro State, Brazil, we used universal or Hepatozoon spp. primer sets for the 18S SSU rRNA coding region. DNA extracts were obtained from blood samples of thirteen dogs naturally infected, from four experimentally infected, and from five puppies infected by vertical transmission from a dam, that was experimentally infected. DNA of sporozoites of Hepatozoon americanum was used as positive control. The amplification of DNA extracts from blood of dogs infected with sporozoites of Hepatozoon spp. was observed in the presence of primers to 18S SSU rRNA gene of Hepatozoon spp., whereas DNA of H. americanum sporozoites was amplified in the presence of either universal or Hepatozoon spp.-specific primer sets; the amplified products were approximately 600bp in size. Cloned PCR products obtained from DNA extracts of blood from two dogs experimentally infected with Hepatozoon sp. were sequenced. The consensus sequence, derived from six sequence data sets, were blasted against sequences of 18S SSU rRNA of Hepatozoon spp. available at GenBank and aligned to homologous sequences to perform the phylogenetic analysis. This analysis clearly showed that our sequence clustered, independently of H. americanum sequences, within a group comprising other Hepatozoon canis sequences. Our results confirmed the hypothesis that the agent causing hepatozoonosis in the areas studied in Brazil is H. canis, supporting previous reports that were based on morphological and morphometric analyses.     

Diagnosis of canine Hepatozoon spp. infection by quantitative PCR

Hepatozoon (H.) americanum and H. canis are the etiological agents of canine hepatozoonosis, a disease that is found worldwide and is also prevalent in the southeastern United States. Current laboratory diagnosis of canine hepatozoonosis caused by H. americanum is usually dependent on visual identification of Hepatozoon "onion skin cysts" in muscle biopsies, an approach that requires invasive sampling and can result in false negatives. We have developed a diagnostic method for detection of Hepatozoon spp. DNA that integrates nucleic acid extraction with extensive agitation to maximize DNA extraction efficiency. The DNA extracted from canine EDTA-whole blood is subjected to real-time PCR, and fluorescence resonance energy transfer (FRET) probes detect a signature polymorphism in the amplified DNA. This PCR method amplifies a fragment of the Hepatozoon 18S rDNA gene, detects as few as 7 genomic copies of Hepatozoon spp. per ml of blood with high specificity, and differentiates between H. americanum and H. canis amplicons. A surprising 300-fold increase of H. americanum 18S rDNA targets occurred during 3-0 days of storage of positive blood specimens. Examination of 614 EDTA-blood samples submitted mostly from the southeastern Unites States from dogs with suspected hepatozoonosis identified H. americanum in 167 samples (27.2%). An additional 14 samples (2.3%) were positive for H. canis, and 14 samples (2.3%) were positive for both H. americanum and H. canis. These results suggest that the Hepatozoon spp. 18S rDNA quantitative PCR may be a valuable tool that can improve diagnosis and therapy of canine hepatozoonosis.     

Infection with a Hepatozoon sp. closely related to Hepatozoon felis in a wild Pampas gray fox (Lycalopex -Pseudalopex -gymnocercus) co-infected with canine distemper virus

A species of Hepatozoon closely related to Hepatozoon felis found in the skeletal and cardiac muscle of a wild Pampas gray fox (Lycalopex gymnocercus) is described. The fox was euthanized after showing severe incoordination. On necropsy and histopathology there was bilateral, diffuse, severe, sub-acute, necrotizing bronchointerstitial pneumonia, with intracytoplasmic and intranuclear eosinophilic inclusion bodies. Canine distemper virus was detected by immunohistochemistry in the bronchiolar epithelium, syncytial cells, alveolar macrophages and pneumocytes. The skeletal muscle and myocardium contained multiple round to oval protozoan cysts ranging from 64 μm × 75 μm to 98 μm × 122 μm, with a central eosinophilic meront-like core surrounded by concentric rings of mucinous material resembling Hepatozoon americanum cysts but smaller in size. Macrophages within rare pyogranulomas and monocytes/macrophages in adjacent sinusoidal blood vessels in the skeletal muscle contained intracytoplasmic round protozoa consistent with merozoites or developing gamonts of Hepatozoon. Hepatozoon sp. infection was confirmed by PCR of skeletal muscle and the sequenced 18S rRNA PCR product was found to be 99% identical to H. felis by BLAST analysis and deposited in GenBank as accession number HQ020489. It clustered together in the phylogenetic analysis with published H. felis sequences and separately from H. canis, H. americanum and other Hepatozoon species. However, the close relatedness of the fox Hepatozoon to H. felis does not rule out infection with a different and possibly unknown Hepatozoon species.     

Experimental evidence of host specificity of Bartonella infection in rodents

A large number of Bartonella species and genetic variants were compared for their ability to cause bacteremia in different rodent species: the cotton rat (Sigmodon hispidus), white-footed mouse (Peromyscus leucopus), BALB/c mouse and Wistar rat. Experimental data supported field observations that host specificity can occur among certain Bartonella species and rodent species. Bacteremia could only be readily produced in cotton rats or white-footed mice if the strains used for inoculation were originally obtained from the same species or from a phylogenetically close species. A few Bartonella colonies could be observed in the blood of some BALB/c mice by 7 days after inoculation, but no evidence of the persistence of the infection was found. Host specificity suggests the possibility of a long co-speciation of Bartonella species with their rodent hosts. Host–parasite relationships measured by the duration and level of bacteremia and the minimal infectious dose may serve as additional criteria for classification of Bartonella isolates obtained from natural environments.     

Molecular prevalence and characterization of Hepatozoon ursi infection in Indian sloth bears (Melursus ursinus)

Hepatozoon species are parasites that infect a wide variety of domestic and wild animals. The objective of the study was to detect the occurrence of Hepatozoon ursi in Indian sloth bears and to characterize the parasite based on phylogenetic analysis of the partial 18S rRNA gene sequence. Hepatozoon infection could be detected in 38 (70%) out of fifty-four blood samples of Indian sloth bears (captive and wild), suggestive of high prevalence of Hepatozoon infection in Indian sloth bears. Sequencing of partial 18S rRNA gene of the positive samples and BLAST analysis indicated that the nearest phylogenetic neighbour was H. ursi with which they exhibited 99-100% similarity. Additionally, Hepatozoon sp. isolated from wild sloth bears of India were identical to those in captive sloth bears and phylogenetically related to H. ursi reported from Japanese black bears from Japan. To our knowledge, this is the first report on the molecular characterization of H. ursi infection in Indian sloth bears.     

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.     

Disease patterns in field and bank vole populations during a cyclic decline in central Finland

Declining field vole (Microtus agrestis) and bank vole (Clethrionomys glareolus) populations were sampled (117 field voles and 34 bank voles) in south-central Finland during the winter of 1988-89. The last surviving field voles were caught in April and bank voles in February. A subsample (16) of the April field voles were taken live to the laboratory for immunosuppression. The histopathology of the main internal organs and the presence of aerobic bacteria and certain parasites were studied. In the lungs, an increase in lymphoid tissue, probably caused by infections, was the most common finding (52% of all individuals). The prevalences in the voles, in the whole material, of Chrysosporium sp. and Pneumocystis carinii in lungs were 13 and 10% in field voles, and 9 and 0% in bank voles, respectively. Cysts of Taenia mustelae (9 and 27%) were the most common pathological changes in the liver. Enteritis was also rather common (14 and 34%). In field voles the prevalences of Frenkelia sp. in the brain and Sarcocystis sp. in leg muscles were low (both 6%). Bordetella bronchiseptica was commonly (31%) isolated from field vole lungs and Listeria monocytogenes from the intestines (34%). Salmonella spp. could not be found. The dynamics and abundance of inflammations in the lungs and intestines, as well as B. bronchiseptica isolations from the lungs, indicate that obvious epidemics took place in declining vole populations. Of the Luhanka subsample of 16 field voles brought to the laboratory in April, one died of listeriosis, two of Bordetella, and five died for unknown reasons. Even if small mustelids are the driving force in microtine cycles, it is possible that diseases also contribute to the decline.     

Further studies on leptospirosis in small rodents and shrews in Finland. A report of investigation made in 1963-64

In previous investigations on small mammals in Finland (Rislakki & al 1954, Rislakki & Salminen 1955, Salminen 1956), the Leptospira icterohaemorrhagiae-frequency in rats (Rattus norvegicus) was rather high (43.1 %). Leptospira-positive cases were also found in house mice (L. sejroe 23.3 %), harvest mice (L. hataviae 9.0 %), yellow necked field mice (L. poi 12.5 %), common voles (L. sejroe and L. bataviae together 12.1 %), field voles (L. sejroe and L. bataviae together 10.7 %) and in common shrews (L. poi 1.2 %). Specimens of other species sent in for investigation (Norway rat, common red backed vole, large tooth backed vole, northern red backed vole, root vole, water vole, wood lemming, Laxmann''s shrew, lesser shrew and water shrew) gave negative results.     

An indirect enzyme-linked immunosorbent assay for diagnosis of American canine hepatozoonosis.

American canine hepatozoonosis (ACH), caused by Hepatozoon americanum, is an emerging tick-borne disease of dogs. An indirect enzyme-linked immunosorbent assay (ELISA) that should facilitate diagnosis of infection and study of the epidemiology of ACH has been developed using H. americanum sporozoites as antigen. Efficacy of the new test as a diagnostic tool was compared with that of skeletal muscle biopsy, the current gold standard for confirming H. americanum infection. Results show that the test is sensitive (93%) and specific (96%) and that it is as reliable as histopathologic examination of skeletal muscle for detecting infection. The ELISA would be suitable as a routine laboratory test for diagnosis of ACH.     

Comparison of tissue stages of Hepatozoon americanum in the dog using immunohistochemical and routine histologic methods

American canine hepatozoonosis is caused by Hepatozoon americanum, a recently described species of apicomplexan protozoan parasite. An immunohistochemical procedure using a polyclonal antibody to sporozoites of H. americanum clearly identified asexual stages of H. americanum in canine striated muscle. The method also detects hepatozoa present in naturally infected coyotes and raccoons and reacts with certain other apicomplexans. Use of this immunohistochemical procedure confirms the canine intermediate host-parasite relationships that were presumptively established using conventional histopathologic methods.     

A PCR-based epidemiological survey of Hepatozoon canis in dogs in Nigeria

The prevalence of Hepatozoon canis infections in dogs in Nigeria was surveyed using molecular methods. DNA was extracted from blood samples obtained from 400 dogs. A primer set that amplified the Babesia canis 18S rRNA gene, which has high similarity to the H. canis 18S rRNA gene, was used for the PCR. As a result, samples from 81 dogs (20.3%) produced 757 bp bands, which differed from the 698 bp band that corresponded to B. canis infection. The sequence of the PCR products of 10 samples were determined, all of which corresponded with the H. canis sequence.     

山羊催乳素受体基因部分片段的克隆与序列分析

采用PCR技术扩增出济宁青山羊催乳素受体基因长892 bp的片段，该片段含有97 bp的部分外显子8序列（外显子8全长为100 bp）、683 bp的内含子8、70 bp的外显子9及42 bp的部分内含子9。将该片段克隆到pGEM-T Easy质粒中，重组质粒用PCR进行阳性克隆鉴定，然后测定核苷酸序列，并推导其氨基酸序列。该序列与绵羊、母牛、人、大鼠、小鼠的催乳素受体基因mRNA的对应序列的核苷酸同源性分别为99.4 %、97.01%、89.22%、89.22%、88.02%，氨基酸同源性分别为100%、94.55%、81.88%、81.82%、83.64%。     

Alternate Pathway of Infection with Hepatozoon americanum and the Epidemiologic Importance of Predation

Background: The range of American canine hepatozoonosis (ACH) is expanding from the southern USA northward. Transmission of Hepatozoon americanum occurs by ingestion of infected Gulf Coast ticks, Amblyomma maculatum. The source of the protozoan for the tick remains undetermined; infected dogs are unusual hosts for the tick. Objective: Compare possible sources of infection by field investigations of 2 multiple‐dog outbreaks of ACH. Animals: Twenty‐eight privately owned dogs (Canis familiaris), 1 coyote (Canis latrans), 31 wild‐trapped cotton rats (Sigmodon hispidus), 24 wild‐trapped field mice (Peromyscus leucopus), and 9 wild‐caught rabbits (Sylvilagus spp.) from sites in eastern Oklahoma were monitored for hepatozoonosis. Six laboratory‐raised cotton rats (S. hispidus), 6 Sprague‐Dawley rats (Rattus norvegicus), 6 C57BL/6J‐Lystbg‐J/J mice (Mus musculus), 6 outbred white mice (M. musculus), 6 New Zealand white rabbits (Oryctolagus cuniculus), and 2 dogs were acquired through commercial vendors for experimental transmission trials of H. americanum. Methods: Four of 15 dogs in a rural neighborhood and 5/12 hunting Beagles were confirmed to be infected by blood smear examination, muscle biopsy, and polymerase chain reaction assay of the 18S rRNA gene of Hepatozoon species. Histories and tick host preferences led to field collections of common prey of canids and experimental transmission trials of H. americanum to selected prey (M. musculus, S. hispidus, R. norvegicus, and O. cuniculus). Results: Dogs with ready access to prey (4/15 dogs) or that were fed prey retrieved from hunts (5/12 hunting Beagles) became infected, providing evidence that predation is an important epidemiologic component of ACH infection. Experimental transmission studies identified a quiescent, infectious stage (cystozoite) of the parasite that provides an alternate mode of transmission to canids through predation, demonstrating that cotton rats, mice, and rabbits but not brown rats may act as paratenic hosts of H. americanum. Conclusions and Clinical Importance: Predation of prey harboring infected A. maculatum or containing cystozoites of H. americanum in their tissues provide 2 modes of transmission of ACH to dogs, putting unconfined dogs at increased risk of infection in endemic areas.     

Hepatozoon species infection in wild red squirrels (Sciurus vulgaris) on the Isle of Wight

Postmortem examinations of 49 red squirrels (Sciurus vulgaris) found dead on the Isle of Wight revealed the presence of a Hepatozoon species in 18 of them (37 per cent). The prevalence of infection was highest in subadult animals and no juveniles were infected. The prevalence was higher in the squirrels dying from natural causes (nine of 12) than in squirrels killed in road accidents (seven of 27). The weight of infection varied, and there were heavy infections in squirrels dying from toxoplasmosis and bacterial pneumonia. A PCR-based assay was used to identify the presence of Hepatozoon species DNA in the lungs, and immunoperoxidase staining was used to confirm the identity of schizonts observed in histological sections. The nucleotide base sequence of the PCR products indicated that the organism was a novel species closely related to, but distinct from, Hepatozoon erhardovae of bank voles (Clethrionomys glareolus).     

Clinical and hematological signs associated with dogs naturally infected by Hepatozoon sp. and with other hematozoa: a retrospective study in Uberlândia, Minas Gerais, Brazil

An evaluation was made of the clinical and hematological aspects of 115 dogs infected naturally by Hepatozoon sp. and treated at the Hospital School of the Faculty of Veterinary Medicine in Uberlandia, MG, Brazil. Of the 115 dogs for whom peripheral blood films were evaluated, 89 (77.39%) presented parasitemia by Hepatozoon sp. solely, while 26 (22.61%) had combination of Hepatozoon sp., Babesia sp. and Ehrlichia sp. Young male dogs less than a year old, of undefined breed (UB), were the most commonly affected. Thirty-nine (33.92%) of the dogs were asymptomatic while 76 (66.08%) presented varied clinical symptoms, the most frequent being anorexia, pulmonary alterations, hyperthermia, pale mucosae, apathy and/or prostration, and diarrhea. The majority of hematological alterations were normochromic-normocytic anemia, leukocytosis, neutrophilia, and nuclear deviation of neutrophils to the left (NDNL). The findings of this study confirm that Hepatozoon sp. causes clinical and hematological alterations of varied intensity, which, albeit not specific to canine hepatozoonosis, reinforce the notion that the discovery of the agent in dogs, even with low parasitemia, should be taken into consideration.     

The first report of Hepatozoon sp. (Apicomplexa: Hepatozoidae) in neotropical felids from Brazil

In order to investigate the occurrence of Hepatozoon infection in Neotropical felids from Brazil, blood from the jugular or cephalic vein was taken from 29 non-domestic felids including ocelot (Leopardus pardalis), little spotted cat (Leopardus tigrinus), margay (Leopardus wiedii), and jaguarondi (Puma yagouaroundi) from the Northeast region of Brazil. Hepatozoon infection was confirmed by light microscopy and molecular techniques. The results showed five naturally infected felids. Partial sequences of the 18S rRNA gene of the Hepatozoon sp. from these felids were further analyzed. Sequences revealed that the isolates found are closely related to Hepatozoon sp. from domestic cats in Spain. Hepatozoon species from Neotropical felids were identified molecularly and characterized for the first time. This is also the first report of Hepatozoon infection in a little spotted cat.