Naturally occurring and experimentally transmitted Hepatozoon americanum in coyotes from Oklahoma

Twenty free-ranging coyotes (Canis latrans) in Oklahoma (USA) were examined for the presence of naturally occurring infections with Hepatozoon americanum and to determine if bone lesions attributable to H. americanum were present. Although eight of the 20 free-ranging coyotes were found to be naturally infected with H. americanum, no bone lesions were detected. In addition, two coyote pups were exposed to H. americanum oocysts collected from experimentally infected ticks and the course of the resulting infection was followed. Both experimentally infected coyotes developed hepatozoonosis detectable by specific muscle lesions beginning 4 wk after exposure. Bone lesions were detected grossly and histologically at necropsy. Histologic evidence of periosteal bone proliferation ranged from segmental areas of plump hypercellularity and thickening of the periosteum, with minor degrees of osteogenesis, to extensive proliferation of woven bone and periosteal hypercellularity and thickening. Nymphal Amblyomma maculatum that fed on one of the experimentally infected coyote pups became infected and mature H. americanum oocysts were recovered when the ticks molted to adults. These results demonstrate that coyotes in some parts of Oklahoma are naturally infected with H. americanum, that experimentally infected coyotes can develop clinical disease, including characteristic bone lesions, and that A. maculatum nymphs can acquire infections by feeding on them.     

Morphological changes of Babesia gibsoni grown in canine red blood cell-substituted severe combined immune deficiency mice

Canine red blood cell-substituted severe combined immune deficiency (Ca-RBC-SCID) mice were prepared for canine Babesia gibsoni infection. The Ca-RBC-SCID mice infected with B. gibsoni developed a high level of parasitemia, and showed clinical symptoms such as anemia and hemoglobinuria, which are similar to those observed in dogs infected with B. gibsoni. The B. gibsoni parasites grown in Ca-RBC-SCID mice showed marked morphological changes, including a significantly larger size of parasites than those in dogs and abundant RBCs containing 4, 8, 16, and 32 parasites. The multiple infection may have resulted from 1 parasite because the posterior end of each parasite in a multiply infected cell was connected. The parasites grown in SCID mice retained their infectivity and virulence to dogs and their morphology was dramatically restored to the original state when they were returned to dogs.     

Salmon poisoning disease in juvenile coyotes: clinical evaluation and infectivity of metacercariae and rickettsiae

Clinical salmon poisoning disease (SPD), and survival of Neorickettsia helminthoeca and metacercariae of Nanophyetus salmincola in fish were evaluated experimentally in 12-wk-old coyotes (Canis latrans) to determine the potential of SPD for biological control of coyotes. Coyotes readily ate fish that contained metacercariae and rickettsiae. They developed diarrhea, anorexia and lethargy within 7 days after feeding. Infected coyotes lost 58% of their body weight when compared to uninfected controls. They died or became moribund and were euthanatized within 17 days after feeding. Rickettsiae were present in the macrophages of lymph nodes of all affected coyotes. Clinical disease occurred in coyotes fed fresh fish, but not in coyotes fed fish stored at 4 C for greater than or equal to 30 days or at -20 C for 14 days. Metacercariae in fish were viable after 60 days at 4 C. These trematodes developed in coyotes, but clinical SPD did not occur. This indicated survival of metacercariae, but not rickettsiae. Metacercariae were not viable after 14 days at -20 C.     

PCR detection of Anaplasma platys in blood and tissue of dogs during acute phase of experimental infection

Four dogs were experimentally infected with Anaplasma platys to determine changes in real-time TaqMan PCR detection in blood and tissue, microscopically detectable parasitemia, and platelet concentrations during the first 28 days of infection. Buffy-coat blood cells were PCR positive for A. platys DNA at 4 days after inoculation and remained positive in all dogs until day 14. Marked thrombocytopenia and low parasitemia occurred in dogs during that initial period. During 17 and 28 days post-inoculation, the PCR results on buffy-coat blood cells were intermittently negative in each dog with marked thrombocytopenia and no microscopic evidence of parasitemia. Bone marrow and splenic aspirates collected from the A. platys-infected dogs were tested by real-time TaqMan PCR. Two dogs were PCR positive in spleen and marrow at 28 days post-inoculation, when PCR results for buffy-coat blood cells were negative. Spleen and/or bone marrow samples should be considered as additional samples for PCR testing of dogs, particularly when blood samples are PCR negative during the acute phase of A. platys infection.     

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.     

Serodiagnosis of canine Babesia gibsoni infection by enzyme-linked immunosorbent assay with recombinant P50 expressed in Escherichia coli

The entire P50 gene encoding a surface protein of Babesia gibsoni was cloned into the bacteria expression vector pGEX-4T-3 and subsequently expressed in Escherichia coli as a glutathione S-transferase fusion protein. The purified recombinant P50 was evaluated in an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of B. gibsoni infection in dogs. ELISA was able to differentiate clearly among B. gibsoni-infected, Babesia canis-infected, and uninfected dog sera. The antibody response against the recombinant P50 was maintained at a high level until the chronic stage of infection in dogs experimentally infected with B. gibsoni. When serum samples collected from domestic dogs in Japan were examined for the diagnosis of B. gibsoni infection by the ELISA, 3 of 209 samples (1.4%) were positive for the antibody to B. gibsoni. This result was completely identical to those of Western blot analysis and the indirect fluorescent antibody test. These results indicate that the recombinant P50 expressed in E. coil is a useful diagnostic antigen for practical use in the diagnosis of B. gibsoni infection in dogs.     

Babesia gibsoni: preferential multiplication in reticulocytes is related to the presence of mitochondria and a high concentration of adenosine 5'-triphosphate in the cells

To determine the cause of the predilection of Babesia gibsoni for reticulocytes, the parasites were cultivated with various types of reconstituted erythrocyte ghosts, which were prepared by resealing erythrocyte ghosts together with variously treated erythrocyte lysate, in vitro. The level of parasitemia in the culture with reconstituted reticulocyte ghosts containing untreated reticulocyte lysate was significantly higher than that in the culture with reconstituted normocyte (mature erythrocyte) ghosts containing untreated normocyte lysate. The removal of mitochondria from reconstituted reticulocyte ghosts by filtration or centrifugation resulted in decreased of parasitemia in those cultures. In contrast, when mitochondria from reticulocytes were loaded into reconstituted normocyte ghosts, the parasitemia in the ghosts loaded mitochondria was increased to the same level as that in reconstituted reticulocyte ghosts. Furthermore, the parsitemia in the culture with reconstituted normocyte ghosts was proportional to the concentration of adenosine 5'-triphosphate in the ghosts. These results suggested that mitochondria of reticulocytes might enhance the multiplication of B. gibsoni through the generation of adenosine 5'-triphosphate within the cells.     

Babesia gibsoni: Serodiagnosis of infection in dogs by an enzyme-linked immunosorbent assay with recombinant BgTRAP

The thrombospondin-related adhesive protein of Babesia gibsoni (BgTRAP) is known as an immunodominant antigen and is, therefore, considered as a candidate for the development of a diagnostic reagent for canine babesiosis. The recombinant BgTRAP (rBgTRAP) expressed in Escherichia coli was tested in an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to B. gibsoni in dogs. The ELISA with rBgTRAP clearly differentiated between B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. The sera collected from dogs experimentally infected with closely related parasites, B. canis canis, B. canis vogeli, B. canis rossi, and Neospora caninum, showed no cross-reactivity by the ELISA with rBgTRAP. A total of 107 blood samples collected from dogs that had been diagnosed as having babesiosis at veterinary hospitals in Japan were examined for the diagnosis of B. gibsoni infection by the ELISA and PCR. Ninety-six (89.7%) and 89 (83.2%) of the tested samples were positive by the ELISA and PCR, respectively, while 11 (10.3%) and 4 (3.7%) were ELISA+/PCR- and ELISA-/PCR+, respectively. In addition, the sensitivity of the ELISA with rBgTRAP was much higher than that of previously established ELISAs with rBgP50, rBgSA1, and rBgP32. These results indicate that the rBgTRAP is the most promising diagnostic antigen for the detection of an antibody to B. gibsoni in dogs and that the combined ELISA/PCR approach could provide the most reliable diagnosis for clinical sites.     

The parasitemia of cloned Trypanoplasma borreli Laveran and Mesnil, 1901, in laboratory-infected common carp (Cyprinus carpio L.)

The course of parasitemia of cloned Trypanoplasma borreli in laboratory-infected common carp was investigated. In 25-42-g carp kept at 20 C, the prepatent period was 8 days; after a phase of exponential growth, the parasitemia peaked at day 39 postinjection (PI) at a level of about 10(3) T. borreli/microliters blood. This maximum was followed by a chronic phase of about 6 wk with large numbers of T. borreli. At 20 wk PI, T. borreli was absent in infected carp. In 2.2-g carp kept at 20 C, the prepatent period was 4 days only, and the parasitemia peaked at day 23 PI. At 30 C, T. borreli was present in the blood only for 12 wk, and the number of T. borreli did not exceed 162 trypanoplasms/microliters blood. Carp kept at 8 and 15 C showed retarded development of parasitemia. The prepatent period lasted longer and the generation time was increased, but the level of parasitemia was not affected. Carp, inoculated at 8 C and then warmed to 20 C on days 27 and 55 PI, developed a parasitemia of 10(4) flagellates/microliters blood and showed high mortalities. During the prepatent period, T. borreli was found in the muscle tissue of the inoculation area but in no other tissue. In the kidney, T. borreli was found 27 hr PI, whereas in the circulating blood it was manifest at day 3 PI. At the same time it was manifest in the liver and spleen.     

Serum hemolytic activity in dogs infected with Babesia gibsoni

The hemolytic activity in serum of Babesia gibsoni-infected dogs was examined. When the activity was assayed in a reaction system consisting of similar concentrations of the serum and canine red blood cells to those in blood, significant hemolysis was observed. The activity of the serum of B. gibsoni-infected dogs, either naturally or experimentally, was always higher than that of uninfected animals. Moreover, in the experimental infection with B. gibsoni, the change in serum hemolytic activity was parallel to those of anemia and parasitemia, whereas it was inversely parallel to that of the hematocrit value. The present study revealed the presence of a hemolytic factor(s) in the serum of B. gibsoni-infected dogs, suggesting that the progressive anemia was due to hemolysis by the factor(s).     

Studies on immunity to Babesia gibsoni in dogs immunostimulation by Bordetella bronchiseptica

Four species of bacteria, Corynebacterium anaerobium 578, Actinobacillus pleuropneumoniae G-4, Mycobacterium bovis BCG, and Bordetella bronchiseptica A-2, were injected intravenously into mice (5 weeks old, ICR-SPF). The clearance of carbon from the blood stream and the weights of the spleen and liver were determined as indicators of RES stimulation. Mouse footpad reaction was assessed as an indicator of delayed-type hypersensitivity to each species of bacteria. The immuno-stimulative activity of each species of bacteria against bovine serum albumin was monitored by passive hemagglutination assay and the macrophage migration-inhibition test in guinea pigs. Based on the results of the experiments described above, B. bronchiseptica was selected as an immunostimulator (Ims) for immunization trials of the hemo-protozoan parasite, Babesia gibsoni, with inactivated merozoites of B. gibsoni (BgK). Twelve dogs, pointers about 6 months old, were divided into four groups of three dogs each. Group 1 dogs were initially injected with Ims, and later injected with BgK and Ims (BgK+Ims) after a 3-week interval. Group 2 and Group 3 dogs were injected twice, at a 3-week interval, with BgK+Ims and BgK, respectively, and Group 4 served as a control. As the results, the serum antibody titres of Group 1 and 2 were several times higher than that of Group 3, and the cell-mediated immunity to parasites was noticeably stimulated by immunization with BgK+Ims. The peak level of parasitemia following the challenge were over 10% for Group 4 and 4.5% for Group 3, while levels for Group 1 and 2 were 2.5% and less than 1%, respectively. No such major clinical signs of babesiosis as jaundice and anemia were observed in Group 1 or 2.     

Safety of Brucella abortus strain RB51 vaccine in non-target ungulates and coyotes

Brucellosis is endemic in free-ranging elk (Cervus elaphus) and bison (Bison bison) in the Greater Yellowstone Area (GYA; USA). It is possible that an oral brucellosis vaccine could be developed and disseminated in the GYA to reduce disease transmission. Should this occur, non-target species other than elk and bison may come in contact with the vaccine resulting in morbidity or mortality. To assess biosafety, bighorn sheep (Ovis canadensis; n = 10), pronghorn (Antilocapra americana; n = 9), mule deer (Odocoileus hemionus; n = 11), moose (Alces alces shirasi; n = 10), and coyotes (Canis latrans; n = 24) were given a single oral dose of at least 1.0 x 10(10) colony-forming units of Brucella abortus strain RB51 vaccine (RB51). Animals were randomly divided into vaccinated and control groups. Ungulates were captured, blood sampled, and swabs taken from the nares, rectum, and vagina for bacterial culture on day 0, 42, and 84 post-inoculation (PI). On day 42, the vaccinated group became a control group and vice versa in a crossover design. Blood and swab samples were taken from coyotes on days 0, 14, 28, and 42 PI. There was no crossover for the coyote study. Two coyotes from each group were also euthanized and cultured for RB51 on days 42, 84, 168, and 336 PI. Blood samples were analyzed for hematologic changes and antibodies to RB51 using a modified dot-blot assay. No morbidity or mortality as a result of vaccination was observed in any animal. There were no differences in hematologic parameters at any time for ungulate species; vaccinated coyotes had higher hematocrit, hemoglobin, and eosinophil counts (P < or = 0.006). All individuals, except some moose, seroconverted to RB51. Strain RB51 was cultured from oropharyngeal lymph nodes from one coyote 42 days PI and from a moose 117 days PI. This study suggested that a single oral dose of RB51 was safe in these species.     

Influence of the urban matrix on space use of coyotes in the Chicago metropolitan area

Expansion of the coyote’s (Canis latrans) distribution in North America has included most urban areas. Concerns for human safety have resulted in the need to understand the spatial relationship between humans and coyotes in urban landscapes. We examined the space use of coyotes with varying degrees of urban development in the Chicago metropolitan area, IL, USA, between March 2000 and December 2002. We compared home-range size, land use, and habitat use of 41 radio-collared coyotes (5 coyotes residing in developed areas, 29 in less-developed areas, and 7 in a matrix of developed and less-developed areas). The partitioning of coyotes into groups based on their level of exposure to urban development allowed us to examine if differences in use of land types by coyotes was evident in our study area. Coyotes in developed areas had home ranges twice the size of animals in less-developed areas. Nonurban habitats were used by all coyotes in the study area, while urban land was avoided. Coyotes in developed areas had large home ranges and high amounts of urban land in their range, but preferred nonurban habitat. This required the coyotes to travel through a matrix of urban land, thus encountering human activity and possibly increasing the risk of conflict with humans. However, coyotes in developed areas avoided crepuscular times when human activity was highest, suggesting that coyotes in developed areas may reduce conflicts with humans by traveling through the matrix of urban land late at night when the risk of contact with humans is lowest. Coyotes in less-developed areas were less affected by human activity at night and likely posed less risk to humans.     

Trichinella murrelli in scavenging mammals from south-central Wisconsin, USA

Tissues and serum from 59 raccoons (Procyon lotor), 42 coyotes (Canis latrans), and seven Striped Skunks (Mephitis mephitis) collected in Dane and Iowa Counties, Wisconsin, USA, between October 2005 and March 2006 were microscopically and serologically examined for the presence of Trichinella spp. Encapsulated larvae were found on compression slides prepared from tongue tissues from a few animals. Complete tissue digestion of tongues revealed that 19% of the raccoons, 26% of the coyotes, and none of the seven skunks tested were infected with Trichinella spp. Cats were subsequently experimentally infected by feeding them the raccoon tissues containing muscle larvae, and muscle larvae isolated from the collected tongues were experimentally transmitted to mice. Multiplex polymerase chain reaction analysis of the isolated muscle larvae demonstrated two distinct bands migrating at 127 base pairs (bp) and 316 bp in all samples, which together are diagnostic for Trichinella murrelli; the isolates were assigned Istituto Superiore di Sanita (ISS) codes ISS1656 through ISS1667, and ISS1708 through ISS1710 by the International Trichinella Reference Centre. These findings extend the geographic range of T. murrelli into Wisconsin, USA.     

Two species of canine Babesia in Australia: detection and characterization by PCR

The haemoprotozoan Babesia canis has been recognized in Australia for many years, and a second, smaller species has recently been discovered. Amplification and sequencing of a partial region of the 18S small subunit ribosomal RNA (rRNA) gene enabled detection and characterization of the large and small canine babesiae of Australia for the first time. Isolates from northern Australia were genetically characterized to be 99% homologous to Babesia canis vogeli, confirming previous speculation about the subspecies of B. canis endemic to Australia. The partial 18S rRNA gene sequence amplified from isolates obtained in southeastern Australia was genetically identical to Babesia gibsoni, a species not previously known in Australia. The polymerase chain reaction (PCR) used was shown to be specific to Babesia and had a high sensitivity, detecting DNA at a parasitemia of approximately 0.0000027%. This study also reports the first known detection and characterization of B. canis DNA in Rhipicephalus sanguineus ticks using PCR.     

Cryptosporidial infections in SCID mice reconstituted with human or murine lymphocytes.

Severe combined immunodeficient (SCID) mice were experimentally infected with Cryptosporidium parvum. Adoptive transfer of BALB/c thymocytes, spleen and bone marrow cells resulted in functional immunologic reconstitution followed by complete eradication of the cryptosporidial infection. Additional SCID mice were injected with human blood peripheral blood lymphocytes and were subsequently infected with C. parvum. The latter mice (SCID-hu-PBL) were at least partially reconstituted with human lymphoid tissues, as evidenced by flow cytometric identification of human cell populations in the SCID mouse spleens and the response of these cells to the T-cell mitogen phytohemagglutinin. The SCID-hu-PBL mice did not resolve the cryptosporidial infections, although a transient reduction in parasitemia was noted 4-6 wk post-reconstitution.     

Serologic survey for canine infectious diseases among sympatric swift foxes (Vulpes velox) and coyotes (Canis latrans) in southeastern Colorado

Swift foxes (Vulpes velox) and coyotes (Canis latrans) are sympatric canids distributed throughout many regions of the Great Plains of North America. The prevalence of canid diseases among these two species where they occur sympatrically is presently unknown. From January 1997 to January 2001, we collected blood samples from 89 swift foxes and 122 coyotes on the US Army Pi?on Canyon Maneuver Site, Las Animas County, SE Colorado (USA). Seroprevalence of antibodies against canine parvovirus (CPV) was 71% for adult (> 9 mo old) and 38% for juvenile (< or = 9 mo old) swift foxes. Adult (<1 yr old) and juvenile (<1 yr old) coyotes had a seroprevalence for CPV of 96% and 78%, respectively. Presence of antibodies against canine distemper virus (CDV) was 5% for adult foxes and 0% for juvenile foxes. Seroprevalence of CDV was 46% for adult coyotes and 18% for juvenile coyotes. No swift foxes had canine adenovirus (CAV) antibodies, whereas 81% and 63% of adult and juvenile coyotes, respectively, had antibodies for CAV. Seroprevalence of antibodies against Yersinia pestis was 68% among adult foxes and 34% among juvenile swift foxes. Seroprevalence of Y. pestis antibodies was 90% and 70% for adult and juvenile coyotes, respectively. No swift foxes had antibodies against Francisella tularensis, whereas seroprevalence was 4% among both adult and juvenile coyotes. Antibodies against CPV and plague were common in both species, whereas antibodies against CDV and CAV were more prevalent in coyotes compared to swift foxes.     

Protection against malaria due to innate immunity enhanced by low-protein diet

Mice were fed ad libitum with a normal diet (25% protein) or low-protein diets (0-12.5% protein) for a wk and then infected with a nonlethal or lethal strain of Plasmodium yoelii, that is, blood stage infection. The same diet was continued until recovery. Mice fed with a normal diet showed severe parasitemia during nonlethal infection, but survived the infection. They died within 2 wk in the case of lethal infection. However, all mice fed with low-protein diets survived without apparent parasitemia (there were small peaks of parasitemia) in cases of both nonlethal and lethal strains. These surviving mice were found to have acquired potent innate immunity, showing the expansion of NK1.1 -TCRint cells and the production of autoantibodies during malarial infection. Severe combined immunodeficiency (scid) mice, which lack TCRint cells as well as TCRhigh cells, did not survive after malarial infection of lethal strain of P. yoelii, even when low-protein diets were given. These results suggest that low-protein diets enhanced innate immunity and inversely decreased conventional immunity, and that these immunological deviations rendered mice resistant against malaria. The present outcome also reminds us of our experience in the field study of malaria, in which some inhabitants eventually avoided contracting malaria even after apparent malarial infection.     

Serological responses of coyotes to two commercial rabies vaccines.

Between August 1993 and September 1994 we documented serological responses of coyotes (Canis latrans) vaccinated with two commercial rabies vaccines licensed for use in domestic dogs. Serologic responses were documented by testing for rabies virus neutralizing antibodies with the rapid fluorescent focus inhibition test (RFFIT) at 30, 90, 180, 270, and 365 days post-vaccination. All coyotes vaccinated with Imrab 3 (Rhone-Merieux, Inc.), and 75% of those vaccinated with Dura-Rab 3 (Immunovet, Inc.) seroconverted, as evidenced by the presence of antirabies antibody titers > or = 1:5 in one or more of the five post-vaccination samples. The percent of coyotes showing a titer > or = 1:5 was generally greater and titer levels appeared higher and more persistent among animals vaccinated with Imrab 3 than Dura-Rab 3. Presence of titers via RFFIT tests demonstrates the antibodies produced in coyotes by these rabies vaccines functionally bind and neutralize rabies virus in vitro, but these results do not constitute a demonstration of protection required for licensure for use in coyotes.