Prevalence of rat virus infection in progeny of acutely or persistently infected pregnant rats

Infant rats are susceptible to persistent rat virus (RV) infection, but risk of persistent infection after prenatal exposure to virus is unclear. We examined this aspect of RV infection in the progeny of dams inoculated with virus during or prior to pregnancy. Sprague-Dawley (SD) dams were infected during pregnancy (gestation day 9) by oronasal inoculation with 10(5) TCID50 of the UMass strain of RV. SD rats were infected prior to pregnancy by oronasal inoculation of two-day-old females with 10(2) TCID50 of RV-UMass, which induced persistent infection. They were mated to non-immune males after reaching sexual maturity. Rats were assessed for RV infection by virus isolation, in situ hybridization, contact transmission, or serologic testing. The progeny of dams inoculated with virus during gestation had high prevalence of infection through postpartum week 9 (9 of 12 rats were virus positive at week 3, and 7 of 10 were virus positive at week 9). Additionally, 2 of 10 rats were virus positive at least through postpartum week 15. The progeny from persistently infected, seropositive dams had no evidence of infection and did not transmit infection to contact sentinels. However, 12 dams were virus positive at necropsy and 9 had transmitted infection to their breeding partners. These results indicate that prenatal infection in non-immune dams can lead to RV persistence in their progeny. By contrast, the progeny of persistently infected dams are protected from infection, presumably by maternal antibody, although their dams can transmit infection to their breeding partners.     

Persistent rat virus infection in smooth muscle of euthymic and athymic rats

Rat virus (RV) infection can cause disease or disrupt responses that rely on cell proliferation. Therefore, persistent infection has the potential to amplify RV interference with research. As a step toward determining underlying mechanisms of persistence, we compared acute and persistent RV infections in infant euthymic and athymic rats inoculated oronasally with the University of Massachusetts strain of RV. Rats were assessed by virus isolation, in situ hybridization, and serology. Selected tissues also were analyzed by Southern blotting or immunohistochemistry. Virus was widely disseminated during acute infection in rats of both phenotypes, whereas vascular smooth muscle cells (SMC) were the primary targets during persistent infection. The prevalence of virus-positive cells remained moderate to high in athymic rats through 8 weeks but decreased in euthymic rats by 2 weeks, coincident with seroconversion and perivascular infiltration of mononuclear cells. Virus-positive pneumocytes and renal tubular epithelial cells also were detected through 8 weeks, implying that kidney and lung excrete virus during persistent infection. Viral mRNA was detected in SMC of both phenotypes through 8 weeks, indicating that persistent infection includes virus replication. However, only half of the SMC containing viral mRNA at 4 weeks stained for proliferating cell nuclear antigen, a protein expressed in cycling cells. The results demonstrate that vasculotropism is a significant feature of persistent infection, that virus replication continues during persistent infection, and that host immunity reduces, but does not eliminate, infection.     

Protective effect of colostrum in Mycoplasma pneumoniae infection induced in infant mice

The immunological responses and mechanism of maternal immunity in Mycoplasma pneumoniae infection of mice were investigated. ICR female mice, 4 weeks old, and infant mice, 2 to 4 days old, were infected with M. pneumoniae. Anti-M. pneumoniae antibodies in serum and colostrum were determined by enzyme-linked immunosorbent assay. The specific IgG antibody production persisted for 9 months or longer in both the young and infant mice. These infected mice were protected from rechallenge with M. pneumoniae. In addition, the infected dams conferred passive immunity on their offspring. The infant mice born to uninfected normal dams were protected from the challenge with M. pneumoniae when fed by infected foster dams. Conversely, the infant mice born to infected dams were not protected from the challenge with M. pneumoniae when the infants were fed by uninfected dams. The specific IgG antibody appeared in serum of infant mice inoculated orally with M. pneumoniae-infected mouse serum and the infants were protected from challenge with M. pneumoniae, while the infants given protein A-absorbed serum were not protected from the challenge. These results suggest that one of the factors involved in the resistance of infant mice to M. pneumoniae infection is the specific IgG antibody present in the colostrum rather than the result of transplacental transfer.     

Experimentally induced infection with autonomous parvoviruses, minute virus of mice and H-1, in the African multimammate mouse (Mastomys coucha)

To determine whether the multimammate mouse (Mastomys coucha) could be used to evaluate rodent parvovirus-based vectors, neonates were subcutaneously inoculated with minute virus of mice (prototype strain, MVMp) or rat parvovirus H-1. The course of infection with both viruses was similar. Seroconversion occurred within two weeks after virus inoculation, as detected by use of hemagglutination-inhibition assays, and antibody titers remained high for the entire observation period of 12 months. Viral DNA and infective virions were detected in several organs of inoculated animals prior to seroconversion, as measured by use of Southern blotting and plaque assays, respectively. Infective particles subsequently became undetectable, whereas viral DNA imprints persisted in distinct organs for at least nine months. Clinical signs of parvovirus infection appeared around six weeks after virus inoculation, and consisted of hemorrhages, stunted growth, and transient hair color changes. Sudden death occurred in a significant fraction of animals infected with MVMp, but not H-1 virus, at the time of weaning. Altogether, MVMp, which is innocuous to its natural host, the mouse, and H-1 virus, which is poorly pathogenic to the rat, appear to be pathogenic for Mastomys coucha.     

Hygienic monitoring of Mongolian gerbils: which mouse viruses should be included?

The Mongolian gerbil (Meriones unguiculatus) serves as an animal model for a wide range of diseases. A practical limitation in its use is the definition of the hygienic status, as not much is known about viruses that potentially infect gerbils and might be transmitted to other rodents. As successful re-derivation was recently described for gerbils, we now aimed at investigating which mouse viruses induce seroconversion in gerbils and might be transmitted to mice. Gerbils were inoculated with viral agents of mice and co-housed with mouse contact sentinels. Seroconversion in gerbils was observed after oronasal inoculation with Sendai virus (SeV), mammalian orthoreovirus serotype 3 (Reo-3) and rotavirus A (RV-A, EDIM), seroconversion to RV-A also in sentinel mice. Pneumonia virus of mice (PVM) was not detected by serology but by polymerase chain reaction in gerbils and respective sentinel mice. No seroconversion towards or transmission of murine hepatitis virus, murine norovirus, minute virus of mice or mouse cytomegalovirus was detected. Anti-gerbil IgG antibodies did not increase sensitivity of indirect immunofluorescence (IFA) compared with anti-mouse IgG. In conclusion, seroconversion to SeV, Reo-3 and RV-A as well as transmission of RV-A and PVM indicate that these agents should be included in health monitoring of gerbils. Furthermore, anti-mouse IgG is suitable as a secondary antibody for IFA with gerbil serum.     

Intrauterine transmission of human T-cell leukemia virus type I in rats.

To analyze intrauterine transmission, MT-2 cells, a human T-cell line producing human T-cell leukemia virus type I (HTLV-I), were injected into eight pregnant F344 rats, and cesarean section was performed at day 23 of pregnancy. HTLV-I provirus was detected by PCR in the liver and spleen taken from one of the eight fetuses. Moreover, 71 offspring were delivered by cesarean section from the remaining seven dams and fostered by seven normal rats. HTLV-I provirus was detected in peripheral blood mononuclear cells in 2 of the 71 offspring 4 weeks after cesarean section. These results indicate for the first time the intrauterine transmission of HTLV-I. To confirm the postnatal transmission, MT-2 cells were injected into a dam within 24 h after delivery, and six offspring were fostered by this dam. HTLV-I provirus was detected in peripheral blood mononuclear cells of all six offspring. This animal model may be useful for analysis and prevention of mother-to-child transmission of HTLV-I.     

Minute virus of mice: antibody response, viral shedding, and persistence of viral DNA in multiple strains of mice

Minute virus of mice (MVM) is a major concern for laboratory animal facilities because it remains with considerably high prevalence despite strict barrier systems. The aim of this study was to elucidate potential risks associated with MVM infection by investigating the role of the genetic background on antibody production and persistence as well as viral shedding. Mice of various strains and stocks were inoculated oronasally with the immunosuppressive strain MVMi; in addition, natural infection was modeled through contact exposure. As determined by serology, seroconversion and serum levels of IgG differed considerably among strains and stocks, especially in the contact-exposed group. For example, C57BL/6J mice responded well to exposure in contrast to FVB/N, NMRI, ICR, and C3H/HeN mice. Titration studies indicated that the viral dose necessary to induce seroconversion was strain-dependent. Experiments to dissect the role of the major histocompatibility complex haplotype in the response to MVMi gave inconclusive results. To detect viral persistence, spleens and feces were analyzed by PCR at 16 wk after exposure, and the infectivity of PCR-positive spleens was investigated by IP and oronasal inoculation of naive mice. Although DNA was detected in the spleens of some mice, feces remained negative, and naive mice were not infected by inoculation. In addition, viral shedding declined rapidly after day 20 postinoculation. In summary, the data show that seroconversion and antibody response to MVMi infection depend on the genetic background of mice, with the infective dose being a critical factor. The role of viral DNA in chronically infected mice will require further elucidation.     

Perinatal transmission of SHIV-SF162P3 in Macaca nemestrina

We developed a SHIV/macaque model of transmission from infected dams to their infants. Ten pregnant dams were infected intravenously with 100 MID(50) of macaque-titered SHIV-SF162P3 during the second trimester. Nine infants were born; the seven surviving beyond day of birth suckled for 6 months. Four of nine infants were infected (transmission rate = 44.4%), with one infection in utero, and three intrapartum and/or immediately post-birth via suckling. Varying levels of binding and neutralizing antibodies were transplacentally transferred to infants. Passive antibodies were detected in plasma on the day of birth and persisted for 5 weeks. Infants infected at or after birth controlled acute and post-acute viremia. Exposure to maternal SHIV-SF162P3 during birth and suckling in the presence of autologous maternal neutralizing antibodies may have affected transmission or pathogenesis in the infants. This transmission model can allow investigation of key parameters involved in perinatal transmission of HIV.     

Rabbit model of rotavirus infection.

A new small animal model was developed to study parameters of rotavirus infections, including the active immune response. Seronegative New Zealand White rabbits (neonatal to 4 months old) were inoculated orally with cultivatable rabbit rotavirus strains Ala, C11, and R2 and with the heterologous simian strain SA11. The course of infection was evaluated by clinical findings, virus isolation (plaque assay and enzyme-linked immunosorbent assay), and serologic response. All four strains of virus were capable of infecting rabbits as determined by isolation of infectious virus from intestinal contents or fecal samples, by seroconversion, or by a combination of these methods. The responses differed depending on the virus strain used for inoculation. Rabbits remained susceptible to primary infection to at least 16 weeks of age (upper limit examined). Virus excretion in intestinal contents was detected from 6 h to 7 days postinoculation. RNA electropherotypes of inocula and viruses isolated from rabbits were the same in all samples tested. Transmission of Ala virus and R2 virus but not SA11 virus from inoculated animals to uninoculated controls also occurred. In a challenge experiment with Ala virus, 74- and 90-day-old rabbits were rechallenged with Ala 5 weeks after a primary infection with Ala. Virus was excreted in feces from 2 to 8 days after the primary infection but was not excreted after challenge. These results indicate that the rabbit provides an ideal model to investigate both the primary and secondary active immune responses to rotavirus infections and to evaluate candidate vaccines.     

Experimental inoculation of male rats with Coxiella burnetii: successful infection but no transmission to cage mates

Beginning in 2007, the largest human Q fever outbreak ever described occurred in the Netherlands. Dairy goats from intensive farms were identified as the source, amplifying Coxiella burnetii during gestation and shedding large quantities during abortions. It has been postulated that wild rodents are reservoir hosts from which C. burnetii can be transmitted to domestic animals and humans. However, little is known about the infection dynamics of C. burnetii in wild rodents. The aim of this study was to investigate whether brown rats (Rattus norvegicus) can be experimentally infected with C. burnetii and whether transmission to a cage mates occurs. Fourteen male brown rats (wild type) were intratracheally or intranasally inoculated with a Dutch C. burnetii isolate obtained from a goat. At 3 days postinoculation, a contact rat was placed with each inoculated rat. The pairs were monitored using blood samples and rectal and throat swabs for 8 weeks, and after euthanasia the spleens were collected. Rats became infected by both inoculation routes, and detection of C. burnetii DNA in swabs suggests that excretion occurred. However, based on the negative spleens in PCR and the lack of seroconversion, none of the contact animals was considered infected; thus, no transmission was observed. The reproduction ratio R(0) was estimated to be 0 (95% confidence interval = 0 to 0.6), indicating that it is unlikely that rats act as reservoir host of C. burnetii through sustained transmission between male rats. Future research should focus on other transmission routes, such as vertical transmission or bacterial shedding during parturition.     

Vertical transmission of Neospora caninum in BALB/c mice determined by polymerase chain reaction detection.

Vertical transmission of Neospora caninum was evaluated in BALB/c mice using an N. caninum-specific polymerase chain reaction (PCR) assay as a means of detecting parasite transmission to offspring. BALB/c mice were infected with the NC-1 isolate of N. caninum during pregnancy (days 8-15 gestation). Transmission of parasite, detected by PCR, was determined in 2- to 23-day-old offspring. When dams were infected on days 13-15 of gestation, transfer of parasites was detected in only a proportion of the litter. Infection between days 8 and 12 of gestation resulted in a high frequency of parasite transmission; every offspring from all litters was infected. The tissue locations of parasites in pups of different ages were determined. In young pups (2- to 4-days-old), the predominant sites of infection were the lungs and the brain. In older pups (7- and 23-days-old) the predominant site of infection was the brain. This study shows that PCR may be useful for evaluation of candidate vaccines against horizontal N. caninum infection, vertical transmission, or both.     

Diversity of the human immunodeficiency virus type 1 (HIV-1) env sequence after vertical transmission in mother-child pairs infected with HIV-1 subtype A

Although several virologic and immunologic factors associated with an increased risk of perinatal human immunodeficiency virus type 1 (HIV-1) transmission have been described, the mechanism of mother-to-child transmission is still unclear. More specifically, the question of whether selective pressures influence the transmission remains unanswered. The aim of this study was to assess the genetic diversity of the transmitted virus after in utero transmission and after peripartum transmission and to compare the viral heterogeneity in the child with the viral heterogeneity in the mother. To allow a very accurate characterization of the viral heterogeneity in a single sample, limiting-dilution sequencing of a 1016-bp fragment of the env gene was performed. Thirteen children were tested, including 6 with in utero infections and 7 with peripartum infections. Samples were taken the day after birth and at the ages of 6 and 14 weeks. A homogeneous virus population was seen in six (46.2%) infants, of whom two were infected in utero and four were infected peripartum. A more heterogeneous virus population was detected in seven infants (53.8%), four infected in utero and three infected peripartum. The phylogenetic trees of the mother-child pairs presented a whole range of different tree topologies and showed infection of the child by one or more maternal variants. In conclusion, after HIV-1 transmission from mother to child a heterogeneous virus population was detected in approximately one-half of the children examined. Heterogeneous virus populations were found after peripartum infection as well as after in utero infection. Phylogenetic tree topologies argue against selection processes as the major mechanism driving mother-to-child transmission but support the hypothesis that virus variability is mainly driven by the inoculum level and/or exposure time.     

Maternal transmission and dental caries induction in Sprague-Dawley rats infected with Streptococcus mutans

Thirty-four female rats (18 days old) were infected with Streptococcus mutans MT8148R (serotype c) or 6715 (g). Diets containing different proportions of sucrose were used to prepare the dams which harbored various levels of S. mutans in their oral cavity. Around 66 days of age, the female rats were bred and 34 dams subsequently bore 322 offspring. The dams were killed upon weaning (20 days of age) of their respective litters. There were positive correlations between the recovery of inoculated S. mutans and the caries incidence in the dams. Transmission of S. mutans from a dam to her offspring was studied in 10-, 15-, 20-, 27-, 34-, 41-, 48-, and 55-day-old rats by evaluating the recover of S. mutans from the offspring. Positive correlation between the magnitudes of recovered S. mutans MT8148R from dams and their offspring was found in all ages of young rats examined. Furthermore, caries incidence in young rats was found to be positively correlated with the recovery of both strains of S. mutans as well as with incidence of caries in their respective dams.     

Transmission dynamics of simian T-lymphotropic virus type 1 (STLV1) in a baboon breeding colony: predominance of female-to-female transmission

We investigated the prevalence, distribution, and transmission of simian T-lymphotropic virus type 1 (STLV1) in a baboon breeding colony over a 4-y period. We used polymerase chain reaction amplification of the proviral tax gene to assess the infection status of 272 animals housed in 4 separate corrals. Sequencing the proviral envelope gene from individual baboons detected several molecular subtypes (genotypes) of STLV1. At the start of the study, 31% (54 of 176) of all baboons were infected; the majority of infections (91%) were in mature females, with only 3 of 12 mature males and 2 of 48 infants and juveniles being infected. Over the next 4 years, 41 new infections were diagnosed. Of these, 83% occurred in sexually mature female baboons (at least 3 y of age), 17% in infants and juveniles (younger than 3 y), and 0% in mature males. The 7 infections in juveniles were probably derived from mother-to-infant transmission because mother-infant pairs consistently were infected with the same viral genotype. Of the 34 new infections in sexually mature female baboons, the genotyping data showed that 25 (73%) originated from other infected females as opposed to males. Male-to-female sexual transmission may have accounted for the remaining 9 new infections. There was no evidence of female-to-male sexual transmission. The high percentage of female-to-female transmission of STLV1 in our baboons was unexpected; we speculate that transmission may have occurred due to blood contamination from biting during aggressive behavior between females in establishing hierarchical dominance.     

Persistence of Sendai virus in a mouse breeder colony and possibility to re-establish the virus free colonies.

An epizootic of Sendai virus infection occurred in a mouse breeder colony with respiratory signs, mortality, retarded growth in young and prolonged gestation period in adults. Thereafter, the infection persisted in this colony without any clinical signs and with 90 to 100% antibody positivity in adults. Weanlings had maternal antibodies and no pneumonic lesions. Antibodies were hardly detected in 6-week-old mice with high incidence of red hepatization or congestion in their lungs, but mice over 8 weeks of age had antibody without the lesions. After isolating weanlings and pregnant mice with antibodies from the infected colony, the isolated weanlings and offspring from the isolated dams became negative for Sendai virus antibodies.     

Transmission of avian influenza A viruses among species in an artificial barnyard

Waterfowl and shorebirds harbor and shed all hemagglutinin and neuraminidase subtypes of influenza A viruses and interact in nature with a broad range of other avian and mammalian species to which they might transmit such viruses. Estimating the efficiency and importance of such cross-species transmission using epidemiological approaches is difficult. We therefore addressed this question by studying transmission of low pathogenic H5 and H7 viruses from infected ducks to other common animals in a quasi-natural laboratory environment designed to mimic a common barnyard. Mallards (Anas platyrhynchos) recently infected with H5N2 or H7N3 viruses were introduced into a room housing other mallards plus chickens, blackbirds, rats and pigeons, and transmission was assessed by monitoring virus shedding (ducks) or seroconversion (other species) over the following 4 weeks. Additional animals of each species were directly inoculated with virus to characterize the effect of a known exposure. In both barnyard experiments, virus accumulated to high titers in the shared water pool. The H5N2 virus was transmitted from infected ducks to other ducks and chickens in the room either directly or through environmental contamination, but not to rats or blackbirds. Ducks infected with the H7N2 virus transmitted directly or indirectly to all other species present. Chickens and blackbirds directly inoculated with these viruses shed significant amounts of virus and seroconverted; rats and pigeons developed antiviral antibodies, but, except for one pigeon, failed to shed virus.     

Comparative transmission of multiple herpesviruses and simian virus 40 in a baboon breeding colony

Little is known about the natural history of herpesviruses indigenous in baboons. Here, we describe the development of ELISAs for five herpesviruses. These assays were used to test more than 950 serum samples collected from approximately 210 infant/juvenile and 130 adult baboons in a captive breeding colony over a period of seven years. Results indicated that baboon cytomegalovirus, lymphocryptovirus, and rhadinovirus are transmitted efficiently within the colony and are acquired at an early age. Baboon alpha-herpesvirus HVP2 and polyomavirus simian virus 40 (SV40) were acquired later and by fewer juveniles than were the other three herpesviruses. More than 60% of baboons acquired HVP2 before reaching sexual maturity, indicating that oral infection of infants and juveniles, rather than sexual transmission between adults, is the predominant mode of transmission for this virus. Antibody to simian varicella virus (SVV) was found in about 40% of baboons. SVV was acquired principally by infants and juveniles; few adults seroconverted despite seronegative adults being in constant contact with infants and juveniles undergoing primary infection. Time of seroconversion was not statistically correlated to specific individual herpesviruses, suggesting that each virus is acquired as an independent infection event rather than multiple viruses being acquired at the same time. Several baboons that were delivered by cesarean section and were housed separate from, but in close proximity to, other baboons remained free of many or all viruses for several years, suggesting that, similar to human herpesviruses, baboon herpesviruses and SV40 are transmitted principally by direct contact.     

Systematic pathogenesis and replication of avian hepatitis E virus in specific-pathogen-free adult chickens

Hepatitis E virus (HEV) is an important human pathogen. Due to the lack of a cell culture system and a practical animal model for HEV, little is known about its pathogenesis and replication. The discovery of a strain of HEV in chickens, designated avian HEV, prompted us to evaluate chickens as a model for the study of HEV. Eighty-five 60-week-old specific-pathogen-free chickens were randomly divided into three groups. Group 1 chickens (n=28) were each inoculated with 5 x 10(4.5) 50% chicken infectious doses of avian HEV by the oronasal route, group 2 chickens (n=29) were each inoculated with the same dose by the intravenous (i.v.) route, and group 3 chickens (n=28) were not inoculated and were used as controls. Two chickens from each group were necropsied at 1, 3, 5, 7, 10, 13, 16, 20, 24, 28, 35, and 42 days postinoculation (dpi), and the remaining chickens were necropsied at 56 dpi. Serum, fecal, and various tissue samples, including liver and spleen samples, were collected at each necropsy for pathological and virological testing. By 21 dpi, all oronasally and i.v. inoculated chickens had seroconverted. Fecal virus shedding was detected variably from 1 to 20 dpi for the i.v. group and from 10 to 56 dpi for the oronasal group. Avian HEV RNA was detected in serum, bile, and liver samples from both i.v. and oronasally inoculated chickens. Gross liver lesions, characterized by subcapsular hemorrhages or enlargement of the right intermediate lobe, were observed in 7 of 28 oronasally and 7 of 29 i.v. inoculated chickens. Microscopic liver lesions were mainly lymphocytic periphlebitis and phlebitis. The lesion scores were higher for oronasal (P=0.0008) and i.v. (P=0.0029) group birds than for control birds. Slight elevations of the plasma liver enzyme lactate dehydrogenase were observed in infected chickens. The results indicated that chickens are a useful model for studying HEV replication and pathogenesis. This is the first report of HEV transmission via its natural route in a homologous animal model.     

Investigation of the pathogenesis of transplacental transmission of Aleutian mink disease parvovirus in experimentally infected mink.

The transplacental transmission of Aleutian mink disease parvovirus (ADV) was studied in experimental infection of 1-year-old female non-Aleutian mink. The ADV-seronegative female mink were inoculated with ADV prior to mating or after the expected implantation of the embryos during pregnancy. A group of uninfected females served as a control group. Animals from each group were killed prior to or shortly after parturition. The in situ hybridization technique with radiolabeled strand-specific RNA probes was used to determine target cells of virus infection and virus replication. In both infected groups, ADV crossed the endotheliochorial placental barrier, although animals infected before mating already had high antibody titers against ADV at the time of implantation. The percentage of dead and resorbed fetuses was much higher in dams infected before mating. In the placentae of these mink, virus DNA and viral mRNA were detected in cells in the mesenchymal stroma of the placental labyrinth and hematoma but only occasionally in the cytotrophoblast of the placental hematoma. Placentae of animals infected during pregnancy showed in addition very high levels of virus and also viral replication in a large number of cytotrophoblast cells in the placental hematoma, which exhibited distinct inclusion bodies. In both groups, neither virus nor virus replication could be detected in maternal endothelial cells or fetal syncytiotrophoblast of the placental labyrinth. Fetuses were positive for virus and viral replication at high levels in a wide range of tissues. Possible routes of transplacental transmission of ADV and the role of trophoblast cells as targets for viral replication are discussed.     

Transmission of hepatozoon canis to dogs by naturally-fed or percutaneously-injected Rhipicephalus sanguineus ticks.

Hepatozoon canis is an apicomplexan protozoan parasite of dogs, prevalent in Asia, Africa, and southern Europe. Experimental transmission of H. canis to dogs was performed with laboratory-reared Rhipicephalus sanguineus nymphs that fed on a naturally infected dog or were percutaneously injected with canine blood containing H. canis gamonts. Dogs were inoculated by oral ingestion of adult ticks containing H. canis oocysts. Transstadial transmission of H. canis was recorded, whereas transovarial transmission could not be demonstrated. Oocysts were detected in 85% of the adult ticks that had engorged as nymphs on an infected dog and in 61% of the adult ticks resulting from nymphs injected percutaneously with blood from the same dog. Nine of 12 dogs (75%) inoculated with naturally fed or percutaneously injected ticks became parasitologically positive, and all showed seroconversion. Meronts were initially detected in the bone marrow 13 days postinoculation and gamonts 28 days after infection. The variation in the time of initial detection of parasitemia among infected dogs and the rapid appearance of gamonts in dogs immunosuppressed with corticosteroids suggest that immune mechanisms play an important role in controlling H. canis parasitism. The artificial acquisition of Hepatozoon parasites by percutaneous injection of ticks, demonstrated here for the first time, may serve as a useful tool for studies on transmission, vector-host relationships, and the immunology of infection with Hepatozoon species.