Genetic diversity of Hepatozoon spp. in coyotes from the south-central United States.

To better define the strains and species of Hepatozoon that infect coyotes in the south-central United States, whole blood and muscle samples were collected from 44 coyotes from 6 locations in Oklahoma and Texas. Samples were evaluated by a nested polymerase chain reaction (PCR) using primers amplifying a variable region of the apicomplexan 18S rRNA gene as well as histopathology (muscle only) for presence of tissue cysts. Hepatozoon spp. infections were identified in 79.5% (35/44) of coyotes tested including 27 of 44 (61.4%) whole blood samples and 17 of 44 (38.6%) muscle samples tested by PCR and 23 of 44 (52.3%) muscle samples evaluated by histological examination. Analysis revealed 19 distinct sequences comprising 3 major clusters of Hepatozoon spp., i.e., 1 most closely related to Hepatozoon americanum, another most closely related to Hepatozoon canis , and the third an intermediate between the 2 groups. The diversity of Hepatozoon spp. in wild canids appears greater than previously recognized and warrants further investigation.

Hyperphosphatasemia and concurrent adrenal gland dysfunction in apparently healthy Scottish Terriers.

OBJECTIVE: To determine causes of hyperphosphatasemia (high serum alkaline phosphatase [ALP] activity) in apparently healthy Scottish Terriers. DESIGN: Prospective case-controlled study. ANIMALS: 34 apparently healthy adult Scottish Terriers (17 with and 17 without hyperphosphatasemia). PROCEDURES: Serum activities for 3 isoforms (bone, liver, and corticosteroid) of ALP were measured. Concentrations of cortisol, progesterone, 17-hydroxyprogesterone, androstenedione, estradiol, and aldosterone were measured before and after cosyntropin administration (ie, ACTH; 5 microg/kg [2.27 microg/lb], IM). Liver biopsy specimens from 16 dogs (11 with and 5 without hyperphosphatasemia) were evaluated histologically. RESULTS: In dogs with hyperphosphatasemia, the corticosteroid ALP isoform comprised a significantly higher percentage of total ALP activity, compared with the percentage in dogs without hyperphosphatasemia (mean +/- SE, 69 +/- 5.0% and 17 +/- 3.8%, respectively). In 6 dogs with hyperphosphatasemia, but none without, serum cortisol concentrations exceeded reference intervals after ACTH stimulation. Six dogs with and 15 without hyperphosphatasemia had increased concentrations of >or = 1 noncortisol steroid hormone after ACTH stimulation. Serum ALP activity was correlated with cortisol and androstenedione concentrations (r = 0.337 and 0.496, respectively) measured after ACTH stimulation. All dogs with and most without hyperphosphatasemia had abnormal hepatocellular reticulation typical of vacuolar hepatopathy. Subjectively, hepatocellular reticulation was more severe and widespread in hyperphosphatasemic dogs, compared with that in nonhyperphosphatasemic dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Hyperphosphatasemia in apparently healthy Scottish Terriers was most likely attributable to hyperadrenocorticism on the basis of exaggerated serum biochemical responses to ACTH administration and histologic hepatic changes, but none of the dogs had clinical signs of hyperadrenocorticism.

Pathogenesis and pathology of bovine pneumonia.

Pneumonia is a major cause of death and economic losses to the cattle industry. Recognizing the patterns of pneumonic lesions and understanding the pathogenesis of the various types of pneumonia are important for correct diagnosis and interpretation of the lesions. Bacterial pneumonias consist of bronchopneumonia, fibrinous pneumonia, and pleuropneumonia as well as caseonecrotic, aspiration, and tuberculous pneumonias. Two major patterns of interstitial pneumonia are recognized in cattle, and verminous pneumonia is associated with Dictyocaulus viviparus infection.

Parasitological and molecular features of the Hepatozoon species in the myocardium of Japanese Martens (Martes melampus melampus).

The Hepatozoon species in the myocardium of Japanese martens (Martes melampus melampus) was characterized by histological and molecular methods. Histologically, granulomatous nodules with Hepatozoon sp. merozoites/gametocytes, or schizonts, or both were observed in the hearts of Japanese martens. The most frequently observed forms were merozoites/gametocytes within phagocytes; each host cell included a zoite, which was not microscopically identifiable as a merozoite or gametocyte. Schizonts were oval in shape and 36.9 ± 5.7 x 28.9 ± 3.4 µm in size; each schizont had approximately 20-60 nuclei. The size of the merozoites could not be measured because no mature schizonts were observed. In the analyses of the partial 18S rRNA gene sequence, it was strongly suggested that the Hepatozoon sp. in Japanese marten and the Hepatozoon sp. in pine marten (Martes martes) in Scotland were the same species.

Experimental transmission of Hepatozoon americanum to New Zealand White rabbits (Oryctolagus cuniculus) and infectivity of cystozoites for a dog.

Inflammatory lesions containing parasitic cystozoites developed in multiple organs and tissues of laboratory-raised Oryctolagus cuniculus that were administered approximately 100 sporulated oocysts of Hepatozoon americanum (Oklahoma isolate, GenBank accession AF176836) orally. The predominantly granulomatous inflammatory lesions were detected histologically 8 weeks after exposure to oocysts. Cystozoites, recognized by cresent-shaped, uninucleated bodies surrounded by an accumulation of globular, PAS-positive polysaccharide material, were evident within macrophages as monozoic and dizoic cysts. Neither meronts nor gamonts were detected in any of the laboratory-raised lagomorphs during the 24-week observation period. Nested PCR assay of rabbit tissues for a 488 bp fragment of the 18S rRNA Hepatozoon spp. gene was positive at 8 and 24 weeks post-exposure. The sequence was 97.1% similar with sequence from the H. americanum carrier used to infect ticks. A Hepatozoon-free dog fed tissues from the 24-week post-exposure rabbit principal developed American canine hepatozoonosis. Gamonts were first detected 5 weeks after the dog ingested the rabbit tissues containing cystozoites. PCR assay of blood from the dog was positive for the Hepatozoon spp. gene fragment. Sequencing confirmed that the parasite in the dog was H. americanum.

New developments in canine hepatozoonosis in North America: a review.

Canine hepatozoonosis is caused by Hepatozoon canis and Hepatozoon americanum, apicomplexan parasites transmitted to dogs by ingestion of infectious stages. Although the two agents are phylogenetically related, specific aspects, including characteristics of clinical disease and the natural history of the parasites themselves, differ between the two species. Until recently, H. canis infections had not been clearly documented in North America, and autochthonous infection with H. americanum has yet to be reported outside of the southern United States. However, recent reports demonstrate H. canis is present in areas of North America where its vector tick, Rhipicephalus sanguineus, has long been endemic, and that the range of H. americanum is likely expanding along with that of its vector tick, Amblyomma maculatum; co-infections with the two organisms have also been identified. Significant intraspecific variation has been reported in the 18S rRNA gene sequence of both Hepatozoon spp.-infecting dogs, suggesting that each species may represent a complex of related genogroups rather than well-defined species. Transmission of H. americanum to dogs via ingestion of cystozoites in muscle of infected vertebrates was recently demonstrated, supporting the concept of predation as a means of natural transmission. Although several exciting advances have occurred in recent years, much remains to be learned about patterns of infection and the nature of clinical disease caused by the agents of canine hepatozoonosis in North America.

Infectivity of Hepatozoon americanum cystozoites for a dog.

Hepatozoon americanum cystozoites from experimentally infected, laboratory-raised rodents were fed to a Hepatozoon-free dog. Gamonts were detected by examination of blood smear 42 and 56 days post-exposure. PCR analysis of blood was positive for the 18S rRNA Hepatozoon gene on days gamonts were demonstrated. Meronts were detected histologically in a skeletal muscle biopsy 90 days after ingestion of cystozoites. Sequencing confirmed that the parasite in the dog was H. americanum. Xenodiagnosis was conducted by replete feeding of Ambylomma maculatum larvae on the dog; 40 days after detachment, sporulated oocysts were recovered from recently molted nymphs.

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.

Hyperphosphatasemia in Scottish terriers: 7 cases.

Increased serum alkaline phosphatase (ALKP) activity in dogs is commonly encountered. In the study reported here, 7 Scottish Terriers were identified with hyperphosphatasemia, for which a cause could not be determined. The clinicopathologic findings of the syndrome are described and correlated with hepatic pathologic changes in biopsy specimens and in specimens obtained at postmortem examination. Five of the 7 dogs were related. The ALKP activity ranged from 1.7 to 17 times the reference value at the time of biopsy. Increased ALKP activity was present for >6 months in 2 dogs and >12 months in 5 dogs; activity was > 1,000 U/L for at least 1 measurement in 5 dogs. Results of liver function testing, adrenocortical function testing, and hepatic ultrasonography were reviewed. Results of histological examination were normal in 6 dogs. One dog had regional, chronic cholangitis without evidence of cholestasis. The lesion was judged unlikely to account for the degree of hyperphosphatasemia. This study provides evidence of possible benign hyperphosphatasemia in Scottish Terriers or of another familial disorder causing asymptomatical hyperphosphatasemia without corresponding histopathological abnormalities in the liver. Prospective studies of ALKP isoenzyme characterization, investigation of skeletal integrity, evaluation of additional related dogs to determine prevalence, and longer follow-up evaluation are necessary to better characterize this finding.

Experimental infection of adult and juvenile coyotes with domestic dog and wild coyote isolates of Hepatozoon americanum (Apicomplexa: Adeleorina).

Each of five adult and four juvenile coyotes (Canis latrans) was exposed to an oral dose of 50 Hepatozoon americanum oocysts recovered from Amblyomma maculatum ticks that previously fed on either naturally infected domestic dogs (Canis familiaris) or naturally infected wild coyotes. All coyotes exposed to H. americanum became infected, regardless of isolate source, and all exhibited mild to moderate clinical disease that simulated American canine hepatozoonosis in naturally infected dogs. At 100 days postexposure, parasitemia was greater in juvenile than adult coyotes (0.9% and 0.3%, respectively); radiographic imaging of femurs revealed moderate exostosis in all juveniles and mild to moderate new bone growth in four of five (80%) adult coyotes. Gross postmortem analysis of bone lesions demonstrated variation between age groups of coyotes but not between isolates of H. americanum. Microscopic evaluation of skeletal muscle revealed that parasite-induced lesions were significantly more numerous (t = 5.0, df = 7, P = 0.001) in juvenile than adult coyotes. Results of this study indicate that juvenile and adult coyotes are equally susceptible to experimental infection with H. americanum isolated from domestic dog and wild coyote sources. The age of coyotes at the time of exposure, and possibly the number of H. americanum oocysts ingested, might influence morbidity and mortality, but it appears that both adult and juvenile coyotes could be reservoirs of H. americanum.