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.     

Sporogonic development of Hepatozoon americanum (Apicomplexa) in its definitive host, Amblyomma maculatum (Acarina)

Light microscopic observations of the sporogonic development of Hepatozoon americanum are described in its acarine host, Amblyomma maculatum. Laboratory-reared nymphal ticks were fed on 2 dogs infected with H. americanum. Nymphal ticks were sampled daily, starting 3 days after being placed on a parasitemic dog, until 18 days after infestation (PI), and then every 3 or 4 days until replete nymphs molted. Ticks were examined as unstained wet mounts and hematoxylin-eosin-stained paraffin sections. Gametes were found within the gut cells of nymphs 4 and 6 days PI. Although differentiation of gamonts into gametes was not detected, syngamy and sporogony were observed. Sporogony appears to occur wholly within tick gut cells, followed by release of mature oocysts into the hemocoel. The earliest evidence of sporoblast formation was observed 23 days PI and of sporozoite formation, 10 days later. Mature oocysts were first found 42 days PI in newly molted adult ticks. Most adult ticks (>98%) that were dissected contained mature oocysts. Oocysts were multisporocystic, and sporocysts contained a variable number of sporozoites. Oocysts in various stages of development were often seen within the same tick, and the number of mature oocysts ranged from 4 to 573.     

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.     

Cultivation of Eimeria tenella in Japanese quail embryos (Coturnix coturnix japonica).

Complete development of Eimeria tenella in Japanese quail embryos was observed. Sporozoites were inoculated into the allantoic cavity of 7-day-old Japanese quail embryos (Coturnix coturnix japonica), after which the infected embryos were incubated at 41 C. In the chorioallantoic membrane mature first generation schizonts, mature second generation schizonts, and gametes were detected at 48 hr postinoculation of sporozoites (PI), 84 hr PI, and 126 hr PI, respectively. Mature gametes and zygotes were found at 132 hr PI, and oocysts were detected at 138 hr PI. Mortality of embryos increased with increment of inoculum size of sporozoites. LD50 was 1.7 x 10(2) sporozoites. Oocyst production was also dependent on inoculum size. Oocysts harvested from embryos sporulated. The oocysts were inoculated into 13-day-old chickens, and oocysts, capable of sporulating normally, were recovered from ceca 7 days after inoculation.     

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.     

Persistence of Hepatozoon americanum (Apicomplexa: Adeleorina) in a naturally infected dog

To determine the persistence of Hepatozoon americanum in a naturally infected dog, skeletal muscle biopsies were performed at approximately 6-mo intervals over a period of 5.5 yr, and the samples were examined for presence of lesions of American canine hepatozoonosis (ACH). Nymphal Amblyomma maculatum (Gulf Coast tick) were allowed to feed to repletion on the dog periodically over the 5.5-yr period, and adult ticks were dissected and examined for presence of H. americanum oocysts. With 3 exceptions, the biopsied muscle contained lesions characteristic of ACH; no evidence of infection was found at 36, 54, and 67 mo after the original diagnosis. In every instance, nymphal Gulf Coast ticks became infected, indicating that dogs naturally infected with H. americanum can remain infectious for Gulf Coast ticks for at least 5.5 yr. Skeletal muscle biopsy is a reasonably reliable method of determining whether dogs are infected with the parasite. Xenodiagnosis using nymphal Gulf Coast ticks is an even more sensitive method, but the procedure is practicable only experimentally. Design of prevention and control measures for ACH must take into account knowledge that the parasite can survive in dogs, and presumably other vertebrate host(s), for long periods. Preventing ingestion of Gulf Coast ticks is an effective control measure.     

Cryptosporidium canis n. sp. from domestic dogs.

Oocysts of Cryptosporidium, from the feces of a naturally infected dog and from an HIV-infected human, were identified as the previously reported canine genotype of Cryptosporidium parvum, hereafter referred to as Cryptosporidium canis n. sp. Also among the oocysts from the dog, a trace amount of C. parvum bovine genotype was detected. Cryptosporidium canis oocysts from both the dog and human were infectious for calves. Oocysts excreted by calf 1 (dog source) were approximately 90% C. canis and 10% C. parvum, whereas those excreted by calf 3 (human source) were 100% C. canis. Oocysts from calf 1 infected calf 2 resulting in excretion by calf 2 of oocysts approximately 90% C. parvum and 10% C. canis. Oocysts of C. canis were not infectious for BALB/c neonatal mice or immunosuppressed C57 juvenile mice, although all control mice became infected with the C. parvum Beltsville isolate. Oocysts of C. canis from calf 1 and the human were structurally indistinguishable from oocysts of the C. parvum Beltsville isolate (bovine). However, C. canis oocysts differed markedly at the molecular level from all known species of Cryptosporidium based on sequence data for the 18S rDNA and the HSP 70 gene. The differences in genetics and host specificity clearly differentiate C. canis as a new species.     

Endogenous development of Eimeria intestinalis in rabbits (Oryctolagus cuniculus).

The endogenous life cycle of a pure strain of Eimeria intestinalis was studied by light and electron microscopy in coccidia-free rabbits. Four schizont generations could be observed: the first one, not previously described, was seen between 36 and 144 hr postinoculation (PI), the second one between 64 and 168 hr PI, the third one between 96 and 192 hr PI, and the fourth one between 168 and 240 hr PI. Gamogony apparently started as early as 144 hr PI. Thus, it was possible for oocysts to develop from third generation merozoites, later oocysts developing after the fourth schizont generation. Electron microscopic observation suggested that oocysts were derived mainly from merozoites of the fourth schizont generation. During the first stage of the life cycle, sporozoites were seen in intraepithelial lymphocytes. All asexual generations, except the fourth, were characterized by 2 schizont types: the first, regarded as female, contained mononuclear merozoites and the second, regarded as male, contained polynuclear merozoites.     

Endogenous stages of Eimeria tuskegeensis (Protozoa: Eimeriidae) in the cotton rat, Sigmodon hispidus

Endogenous stages of Eimeria tuskegeensis were studied in experimentally infected cotton rats, Sigmodon hispidus. Almost all parasites were located on the basilar side of the nucleus in epithelial cells on the sides and tips of villi of the small intestine. The endogenous cycle consisted of three generations of schizogony followed by gametogony. First-, second-, third-generation schizonts could be distinguished by time of appearance, size and shape of the schizont, and number, size, shape, and arrangement of merozoites. Immature gametogonous stages appeared to 84 hr postinoculation (PI) and developed into mature microgametocytes and macrogametes by 96 hr PI. Microgametocytes had a mono-centric type of development. Intermediate macrogametes had small, basophilic wall-forming bodies and mature macrogametes had large, eosinophilic wall-forming bodies. It was not possible to determine whether these were two distinct types of wall-forming bodies or whether they were different stages of a single type. Two nuclei were seen in the host's epithelial cells parasitized by schizonts, microgematocytes, macrogametes, and oocysts. This binucleate condition was apparently parasite-induced.     

Experimental life cycle of Lagochilascaris major leiper, 1910 (Nematoda: Ascarididae) in cats (Felis domesticus)

The life cycle of Lagochilascaris major was studied using eggs collected from a natural clinical case in a domestic cat. Twenty-seven white mice (Mus musculaus), 5 hamsters (Mesocricetus auratus), and 1 vesper mouse (Calomys callosus) were orally inoculated with 800-1,300 embryonated eggs. When examined from 73 to 246 days postinoculation (PI), encysted third-stage larvae were seen in skeletal muscles and less frequently in connective tissue, liver, and lungs. Twenty-two of the 23 cats orally inoculated with 40-430 encysted larvae from these rodents, and necropsied from 1 hr to 185 days PI, became infected. Third-stage larvae were located in the stomach, esophagus, and oropharynx from 1 to 24 hr PI. At 48 hr, larvae, from mainly the fourth stage, were only found, unilaterally or bilaterally, inside a "sac" in the region of the semilunar fold of the palatine tonsil at the base of the tongue. Adult worms were found in this location from 10 to 175 days PI. No fistulated abscess to the outside medium was found. Adult worms were also found in the middle ears of 2 cats showing purulent otitis. Eggs in the ear secretion were under different stages of development. Eggs in feces were first observed on days 14 and 15 PI, and 1 cat shed them until 178 days PI. Six infected cats were treated with fenbendazole at 50 mg/kg of body weight for 3 consecutive days, eliminating all the parasites present in the tonsils. The drug was not effective against the parasites present in the middle ear. No stage of the parasite was found in the tissues of 5 cats given 4,000-5,200 eggs orally and examined after 19 and 50 days PI. This indicates that the life cycle of L. major requires an obligate paratenic host and is characterized by heteroxenic cycle.     

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.     

The life cycle of Eimeria falciformis var. pragensis (Sporozoa: Coccidia) in the mouse, Mus musculus

The life cycle of Eimeria falciformis var. pragensis, established from a single oocyst, is described in experimentally infected mice (Mus musculus). The coccidium had a prepatent period of 7 days and a patent period of 10--16 days. Oocysts were spherical to ellipsoidal in shape and measured 21.2 x 18.3 micron. Sporulation time was 3 to 3.5 days. Sporocysts measured 12.2 x 7.2 micron and contained a circular to avoid granular sporocyst residuum measuring 5.5 X 5.0 micron. One, 2 or 3 circular to rectangular polar granules were observed within each sporulated oocyst. The endogenous stages developed primarily in the cecum and colon and only occasionally in the lower ileum. Four generations of schizonts were found. Mature 1st-generation schizonts, first observed 48 hr postinfection (PI), measured 17.8 x 12.3 micron and had 12 merozoites that measured 13.3 x 2.0 micron. Mature 2nd-generation schizonts appeared 78 hr PI. They measured 10.2 x 9.3 micron and had 8 merozoites measuring 5.0 x 1.6 micron. Mature 3rd-generation schizonts appeared first at 114 hr PI and measured 17.5 x 10.2 micron and had 10 merozoites that measured 12.4 x 1.8 micron. Mature 4th-generation schizonts appeared first at 144 hr PI. They measured 18.2 x 15.3 micron and had 18 merozoites. The merozoites of the 4th-generation schizont were 4.5 x 1.2 micron. Mature macrogamonts and microgamonts developed simultaneously appearing at 156 hr PI. Macrogamonts measured 16 x 14.5 micron and microgamonts were 18.2 x 15.3 micron. In experimentally infected rats (Rattus norvegicus), development of E. falciformis var. pragensis progressed only as far as mature 1st-generation schizonts.     

Besnoitia darlingi (Protozoa: Toxoplasmatinae): cyclic transmission by cats

Two of five opossums examined from the Kansas City area were found to be infected with Besnoitia darlingi. Inoculation of cysts resulted in acute lethal infections in mice and hamsters. Chronic infections with development of tissue cysts were obtained in mice by prophylaxis with sulfadiazine. Cysts were fed to cats and a dog which resulted in the shedding of isosporoid oocysts by cats. The prepatent period was 11 to 14 days. Unsporulated oocysts averaged 11.9 by 12.3 micrometer and sporulated in 48 to 72 hr. Mouse-to-mouse tramsmission was achieved by injecting triturated tissue containing cysts. This is the third species of Besonitia to be cyclically transmitted by cats.     

The life cycle and pathogenicity of coccicium Eimeria nocens (Kotlán, 1933) in domestic goslings

Twenty-four 10-day-old, artificially reared, coccidia-free goslings (Anser cygnoides var. domestica) were inoculated orally with 1.0x10(5)-1.0x10(6) sporulated oocysts of Eimeria nocens, and killed at intervals from 30 to 336 hr postinoculation (PI). Parts of the visceral organs, including intestines, kidney, liver, gallbladder, and spleen from inoculated goslings, were fixed and sectioned. The life cycle of E. nocens and histologic changes during infection were examined microscopically. The results showed that at least 3 generations of meronts developed in the endogenous stage of the life cycle of E. nocens. Two types of meronts were found. The first completed maturation at 54 to 78 hr PI. These meronts were the first generation, with each forming about 12 merozoites. The second completed maturation at 102 to 240 hr PI. These meronts were the second or third generations, with each meront forming about 24 merozoites. Development of gamonts began at about 198 hr after infection. The prepatent period was 9 days and discharge of oocysts continued for 4 days. Sporulation of oocysts occurred in 60-72 hr at 25 C. Eimeria nocens invaded the posterior jejunum, ileum, caecum, rectum, and cloaca. Developmental stages were localized within the epithelial cells of villi and crypts, and in lamina propria. Marked histological changes, including desquamation and necrosis of intestinal epithelium, submucosal edema, hemorrhages, infiltration of inflammatory cells, and villous atrophy, were seen during the periods of late merogony, gamogony, and oocyst shedding. They were most pronounced in the ileum and the regions nearby. The infected goslings showed severe diarrhea, bloody feces, anorexia, emaciation, and even death, suggesting that E. nocens is highly pathogenic for goslings.     

Detection of Echinococcus granulosus coproantigens in Australian canids with natural or experimental infection

Coproparasitological and purging methods for diagnosing canids infected with the intestinal helminth Echinococcus granulosus, an important zoonotic parasite, are unreliable. Detection of coproantigens in feces of infected dogs by enzyme-linked immunosorbent assay (ELISA) is suitable for detecting patent and prepatent infections with a high degree of sensitivity and specificity. In the present study, natural and experimental infections in domestic and wild Australian canids were investigated using a coproantigen capture ELISA. Experimental infection of dogs with E. granulosus was detected at between 14 and 22 days postinfection (PI), and optical density (OD) values remained high until termination of experiments 35 days PI. After chemotherapy, coproantigen levels in infected dogs dropped rapidly, becoming negative 2-4 days after treatment. In experimentally infected red foxes (Vulpes vulpes), the coproantigen excretion profile was different, with ELISA OD levels peaking 15-17 days PI, then falling to low or undetectable levels by 30 days PI. Coproantigens were detected in the feces of naturally infected Australian wild dogs (dingoes, dingo/domestic dog hybrids) with infection levels ranging between 2 worms and 42,600. Preliminary data on the stability of coproantigen in dog feces exposed to environmental conditions indicated that there was no change in antigenicity over 6 days. The results suggest the coproantigen ELISA could be successfully used to monitor E. granulosus prevalence rates in Australian domestic dogs, foxes, and wild dogs.     

Sarcocystis Idahoensis Sp. N. In Deer Mice Peromyscus Maniculatus (Wagner) and Geopher Snakes Pituophis Melanoleucus (Daudin)*,†

SYNOPSIS Deer mice Peromyscus maniculatus (Wagner) were trapped near Hammett, Idaho, as a possible source of Besnoitia jellisoni Frenkel and species of Sarcocystis to be used for life cycle studies. Forty-nine deer mice were necropsied; 20 (40.8%) were positive for sarcocysts structurally identical with those of Sarcocystis idahoensis sp. N. the source of S. idahoensis used for life cycle studies was a Great Basin gopher snake Pituophis melanoleucus deserticola Stejneger killed near Hammett, Idaho; 20 sporulated sporocysts measured 11.1 × 13.4 (11-12 × 13-14) μm. Structurally identical sporocysts were found in 7 of 14 Pacific gopher snakes P. m. catenifer (Blainville), and in 6 of 10 San Diego gopher snakes, P. m. annectens Baird & Girard. Totals of 148 deer mice and 17 gopher snakes were necropsied in the course of life cycle studies. Development of the first generation meronts took place within the hepatocytes of deer mice 2-10 days post-inoculation (PI) with sporulated sporocysts. Rosette-shaped meronts (6-8 days PI) contained tachyzoites attached by their posterior poles to a residual body. After release from the residual body, tachyzoites were initially retained in a meront wall and later released from the hot cells Within muscle cells a single tachyzoite-shaped structure was found 11 days PI and PAS-negative metrocyte-containing sarcocysts (2nd generation meronts) 13-34 days PI. PAS-positive material was first seen in sarcocysts 34 days II at which time bradyzoite formation became apparent. At 160 days PI, 10 sarcocysts measured 0.4 × 5.8 (0.2-0.9 × 1.8-9.9) μ and appeared to be mature and structurally identical with those from naturally infected deer mice. After ingestion of S. idahoensis-infected deer mice by gopher snakes, bradyzoites developed directly into microgamonts and macrogametes. These stages were first seen 5 days PI. Microgamonts were generally located above and macrogametes below the epithelial host cell nucleus. Seven to 11 days PI microgamonts were seen with mature microgametes, and oocysts which had not yet begun sporogony were found with oocyst walls. Clinical signs of illness were generally not observed in infected gopher snakes; however, one snake developed anorexia and cachexia, and became moribund after repeated ingestion of heavily infected deer mice. Acute hepatitis associated with developing meronts often was noted in deer mice given over 15,000 sporocysts each. Five to 6 days PI anorexia, weakness, ataxia, and dyspnea were observed: these clinical signs increased in severity until 6-8 days PI, when mice became recumbent and died, or were killed while moribund. Hepatosplenomegaly, petechial hemorrhage o the serosal and cut surfaces of the liver, and icterus were common. Diffuse coagulative necrosis with cellular infiltration (primarily neutrophils) was noted on microscopic examination.     

First blood meal of Ctenocephalides canis (Siphonaptera: Pulicidae) on dogs: time to initiation of feeding and duration

Two experiments were conducted on dogs to evaluate interval to initiation and duration of the first blood meal of Ctenocephalides canis (Curtis). Percentage of fed male and female fleas was calculated for fleas held on dogs for 5, 15, 30, 60 min, 6, and 24 hr. Duration of first blood meal was also measured for individual fleas confined on dogs. When fleas were free in the hair coat, 21.2% had begun blood feeding within 5 min. After 1 hr, 72.5% of fleas had fed. After 6 hr, 95.2% of males and 100% of females had taken a blood meal, and 24 hr after deposition all fleas had fed. There was no significant difference between the 2 sexes. The mean delay between deposition and biting for fleas that began feeding within 15 min was 2 min 52 sec +/- 3 min 2 sec for female fleas and 3 min 8 sec +/- 2 min 45 sec for males. The mean duration of female and male meals was 5 min 3 sec +/- 3 min 41 sec and 6 min 9 sec +/- 6 min 8 sec, respectively. There was no significant difference between the 2 sexes. The dog flea took its blood meal on dogs more slowly than the cat flea did on cats; this meal was significantly longer for Ctenocephalides felis felis (Bouche) than for C. canis.     

Biology of Amblyomma aureolatum (Pallas, 1772) (Acari: Ixodidae) on some laboratory hosts in Brazil

The ixodid Amblyomma aureolatum is suspected to play a role in the epidemiology of wild life-cycle hemoparasites, which frequently infect dogs in rural and hunting areas in Brazil. Little is known about its bionomics. The objective of the present study was to evaluate some bionomic aspects of A. aureolatum ticks in Brazil. One engorged female, collected from a dog (Canis familiaris) in S?o Sebasti?o das Aguas Claras, State of Minas Gerais, was used to establish a colony in the laboratory. Subsequently its parasitic stage progeny were fed on domestic dogs and laboratory animals. The free-living stages were incubated at 27 degrees C +/- 2 degrees C and minimum 70% relative humidity in a BOD incubator. The egg incubation period ranged from 31 to 34 days; the parasitic period of larvae ranged from 4 to 6 days and ecdysis to nymphs occurred from day 19 up to day 22. The parasitic period of nymphs ranged from 5 to 8 days and the period of ecdysis to adults from 31 to 33 days. The parasitic period of adults ranged from 11 to 15 days, the pre-oviposition period from 6 to 12 days, and the oviposition period from 9 to 38 days. The total duration of the life cycle ranged from 116 to 168 days.