Experimental Infection Of Dogs Research Articles

Recombinant Canine Coronaviruses Related to Transmissible Gastroenteritis Virus of Swine Are Circulating in Dogs

Four canine coronavirus type II (CCoV-II) strains were identified in the guts and internal organs of pups which had died of acute gastroenteritis. The CCoV-II strains were strictly related to porcine transmissible gastroenteritis virus (TGEV) in the N-terminal domain of the spike protein, whereas in the other parts of the genome, a higher genetic relatedness to recent CCoV-II isolates was observed. Experimental infection of dogs with a TGEV-like isolate induced mild gastroenteritis without any systemic involvement. By virus neutralization tests, antigenic differences between reference and TGEV-like CCoVs were found. Our data support the potential recombinant origin of the TGEV-like CCoVs.

Experimental infection of dogs with a Brazilian strain of Rickettsia rickettsii: clinical and laboratory findings

The bacterium Rickettsia rickettsii is the etiological agent of an acute, severe disease called Rocky Mountain spotted fever in the United States or Brazilian spotted fever (BSF) in Brazil. In addition to these two countries, the disease has also been reported to affect humans in Mexico, Costa Rica, Panama, Colombia and Argentina. Like humans, dogs are also susceptible to R. rickettsii infection. However, despite the wide distribution of R. rickettsii in the Western Hemisphere, reports of R. rickettsii-induced illness in dogs has been restricted to the United States. The present study evaluated the pathogenicity for dogs of a South American strain of R. rickettsii. Three groups of dogs were evaluated: group 1 (G1) was inoculated ip with R. rickettsii; group 2 (G2) was infested by R. rickettsii-infected ticks; and the control group (G3) was infested by uninfected ticks. During the study, no clinical abnormalities, Rickettsia DNA or R. rickettsii-reactive antibodies were detected in G3. In contrast, all G1 and G2 dogs developed signs of rickettsial infection, i.e., fever, lethargy, anorexia, ocular lesions, thrombocytopenia, anemia and detectable levels of Rickettsia DNA and R. rickettsii-reactive antibodies in their blood. Rickettsemia started 3-8 days after inoculation or tick infestation and lasted for 3-13 days. Our results indicate that a Brazilian strain of R. rickettsii is pathogenic for dogs, suggesting that canine clinical illness due to R. rickettsii has been unreported in Brazil and possibly in the other South American countries where BSF has been reported among humans.

Immunohistochemical Demonstration of the Putative Canine Distemper Virus Receptor CD150 in Dogs with and without Distemper

Signaling lymphocyte activation molecule (SLAM) or CD150 can function as a receptor for the canine distemper virus (CDV) in vitro. The expression of SLAM was studied using immunohistochemistry in order to evaluate the presence and distribution of the receptor in dogs in vivo. Additionally, receptor expression was assessed after experimental infection of dogs with CDV. In 7 control dogs without distemper virus, the receptor was found in various tissues, mostly on cells morphologically identified as lymphocytes and macrophages. In 7 dogs with early distemper lesions characterized by presence of the virus, higher numbers of SLAM-expressing cells were found in multiple tissues recognized as targets of CDV compared with those in control dogs. These findings suggest that SLAM, a putative distemper receptor, is expressed in dogs in vivo. Additionally, virus infection is associated with up-regulation of SLAM, potentially causing an amplification of virus in the host.

Experimental infection of dogs with a novel strain of canine coronavirus causing systemic disease and lymphopenia

A pantropic canine coronavirus (CCoV) strain (CB/05) has been recently associated to a fatal outbreak of systemic disease in young dogs. We report the clinical, virological and serological findings in dogs experimentally infected with strain CB/05. The dogs, three 2.5-month-old and two 6-month-old pups, were successfully infected, shedding viral RNA with their faeces for the entire observation period (21 days) and displaying systemic clinical signs resembling those observed during the course of natural infection. Leucopenia (acute lymphopenia) occurred in all infected dogs, with values dropping below 60% of the initial counts. Considering the severity of the CB/05-induced disease, two of the youngest pups were euthanized for ethical reasons at days 8–9 postinfection, whereas the other pups underwent a slow but progressive improvement of their clinical status with complete recovery. At postmortem examination, remarkable lesions were observed in the internal organs of the euthanized pups, that tested positive for CCoV by real-time RT-PCR and virus isolation on cell cultures. All pups seroconverted for CCoV, as shown by the high optical density values and antibody titres detected by ELISA and virusneutralisation tests, respectively. The present study confirms that strain CB/05 is highly pathogenic for dogs, being able to induce a severe disease (and in some cases the death) even in experimental conditions.

Estuarine fish and turtles as intermediate and paratenic hosts of Gnathostoma binucleatum in Nayarit, Mexico

Human gnathostomosis is a severe public health problem in the State of Nayarit, Mexico. Between 1995 and 2005, the registration of human cases numbered 6,328, which makes it one of the largest focal points of the disease in the country. The present study determined the presence of natural hosts of Gnathostoma binucleatum larvae at the Laguna de Agua Brava in Nayarit, Mexico. A total of 5,450 fish and 247 turtles were sampled. Muscular tissue was ground and observed against the light using a 100-W lamp to identify advanced third-stage larvae. The estuarine species Cathorops fuerthii, Pomadasys macracanthus, Mugil curema, and Dormitator latifrons were found positive for presence of larvae, and annual prevalence was 4.8, 1.83, 2.16, and 4.0%, respectively. The species Oreochromys aureus and Chanos chanos were negative. The species of estuarine turtles Kinosternum integrum and Trachemys scripta were positive with annual prevalence of 79.1 and 52.5%, respectively. The criteria of identification of the Gnathostoma species were: mean number of nuclei in intestinal larval cells (2.3), larval morphometry with optic microscopy, larval morphometry with scanning electron microscopy, and number and sequence of ribosomal deoxyribonucleic acid of adult parasites obtained from experimental infection in dogs. The estuarine fish Pomadasys macracanthus and Mugil curema are reported as intermediate hosts for the first time and likewise the estuarine turtle Kinosternon integrum as a paratenic host.

Morphological and molecular identification of two Paragonimus spp., of which metacercariae concurrently found in a land crab, Potamiscus tannanti, collected in Yenbai Province, Vietnam

Paragonimosis is an important food-borne zoonosis especially in Asian countries. Among Paragonimus species, Paragonimus westermani followed by P. skrjabini complex are the major pathogens for human paragonimosis in Asia. In addition, P. heterotremus is an important pathogen in southern China and the Indochina Peninsula and is the only proven species to cause human paragonimosis in Vietnam. During a recent survey in Yenbai Province in northern Vietnam, we found small and large types of Paragonimus metacercariae often concurrently in mountainous crabs, Potamiscus tannanti. Adult worms from those small and large metacercariae were obtained separately by experimental infection in dogs and cats. Morphological and molecular phylogenetic study based on sequences of ITS2 and a part of CO1 genes were performed for the identification of small and large metacercariae and their adults. The results showed that small metacercariae and their adults are completely identical with P. heterotremus in morphology and molecular genetic profiles. In contrast, large metacercariae and their adults have some morphological similarities with P. skrjabini and P. harinasutai, but are unidentifiable from each other by morphology alone. Molecular phylogenetic tree analyses on ITS2 and CO1 genes revealed that large metacercariae and their adults were grouped in the same clade and different from any known Paragonimus species. Although they share the same ancestor with P. skrjabini complex, their genetic distance was considerably different from two other known subspecies, P. skrjabini skrjabini and P. skrjabini miyazakii. Our results provide a new insight on the phylogeny of the genus Paragonimus.

Experimental Infections in Dogs with Ehrlichia canis Strain Borgo 89

This study attemps to clarify the virulence and the pathogenicity of the Borgo 89 strain of Ehrlichia canis isolated from a sick dog in Corsica (France). Four unscathed beagles were intravenously injected with an inoculum of leukocytes infected with the Borgo 89 strain and the animals were examined daily for clinical signs of disease, and blood samples were drawn at frequent intervals for biochemical and hematologic assessment. Serologic (IFI) and PCR assays were also carried out. The results at autopsy are presented in this paper, leading to the conclusion that the Borgo 89 strain has a pathogenicity comparable to that of the known strains. However, the discovery of a case of completely unapparent infection raises the question of a possible individual immunization whose origin remains unexplained.

RELATIVE ABUNDANCE OF TREE HOLE–BREEDING MOSQUITOES IN BOONE COUNTY, MISSOURI, USA, WITH EMPHASIS ON THE VECTOR POTENTIAL OF AEDES TRISERIATUS FOR CANINE HEARTWORM, DIROFILARIA IMMITIS (SPIRURIDA: FILARIIDAE)1

Aedes (Protomacleaya) triseriatus currently shares its habitat in the USA with the introduced species Aedes (Finlaya) japonicus and Aedes (Stegomyia) albopictus. In the late 1980s, before the introduction of these 2 species, Ae. triseriatus was the dominant tree hole- and artificial container-breeding mosquito in central Missouri. Aedes triseriatus represented 89% of the mosquito immatures collected from water-filled tree holes and artificial containers at 3 forested field sites in central Missouri, from May to October, 1986 to 1988. Laboratory-reared female Ae. triseriatus were able to support larval development of Dirofilaria immitis (canine heartworm) to the infective 3rd larval stage. A blood meal from a microfilaremic Collie-mix dog was sufficient to infect adult female mosquitoes, indicating that Ae. triseriatus is a possible vector of canine heartworm in central Missouri. Confirmation of the vector status of this species depends on the yet-to-be observed transmission of D. immitis by Ae. triseriatus in the field, possibly by experimental infection of dogs by wild-caught mosquitoes. Defining the role of this species in epizootic outbreaks could contribute toward accurate risk assessment as the abundance of Ae. triseriatus increases and decreases in response to the success of Ae. albopictus, Ae. japonicus, or other introduced container-breeding mosquitoes.

Experimental infection of dogs with Echinococcus granulosus (Batsch, 1986) from protoscoleces of donkey's hydatid cyst

This study was conducted, for the 1st time in Iraq, to infect 5 puppies, 1 - 5 month old, with protoscoleces of hydatid cyst isolated from the liver of donkey, slaughtered at zoo abattoir of Al-Zawra park in Baghdad, at a dose rate of 10000 protoscoieces i puppy (n=2) and 20000 protoscoleces / puppy (n=3) for 35 and 40 days post infection. The dogs were killed and the developed Echinococcus granulosus were isolated from the small intestine. Results of the study revealed 
 variation in the period of maturation of the collected cestodes, and the mean total length of the mature worms at 35 days PI was 4.53+ 0.24 mm, and for those at 40 days PI was 7+ 0.20 mm, whereas for the gravid ones at the same age of the latter was 8.57 + 0.25 mm. Results found that infection rate with E. granulosus, which their strobilae composed from 4 proglottids at 35 days PI, was 29.03%, and with those, which their strobilae composed from 5 proglottids at 40 days PI, the rate was 0.76%. It was found that the mean length of the gravid proglottid equal to or longer than half the total worm length with a ratio of 1.77 + 0.05 for those at 35 days of age, and 2.2 + 0.35 and 1.93 + 0.01 for mature and gravid cestodes at the age of 40 days.

The Echinococcus granulosus antigen EgA31: localization during development and immunogenic properties.

EgA31 is a fibrillar protein from Echinococcus granulosus that behaves as a potent antigen during infestation of dogs. The localization of this antigen during development of the parasite was investigated by immunohistochemistry in optical and electron microscopy. The protein is mostly abundant in the microtriches and subtegumental cells of the adult, whereas it is absent from protoscolex microtriches. Eggs, the periphery of calcareous corpuscles, and the germinal layer were other sites of accumulation. Immunogenicity of different domains of the protein was assessed during experimental infection of dogs. It was shown that the polypeptide encoded by the Pst I-Hind III fragment of the complete cDNA is the most antigenic during the infection. The uses of such polypeptide for infection diagnosis and as a candidate vaccine protein are discussed.

Experimental infection of dogs with Borrelia burgdorferi sensu stricto using Ixodes scapularis ticks artificially infected by capillary feeding.

Specific pathogen-free dogs were experimentally infected with Borrelia burgdorferi sensu stricto using nymphal or adult female Ixodes scapularis ticks artificially infected with spirochetes by capillary feeding. The ticks were capillary fed B. burgdorferi isolate 610, previously isolated from a dog with Lyme disease and grown in BSK medium. This isolate induced clinical signs in the dogs similar to those for dogs infested with ticks naturally infected with B. burgdorferi. Adult ticks were more efficient than nymphs in transmitting spirochetes to the dogs. One of five dogs infested with nymphal ticks capillary fed B. burgdorferi was skin biopsy culture and serologically positive, and demonstrated lameness. In contrast, all five dogs infested with adult female ticks that had been capillary fed with B. burgdorferi were culture and serologically positive, with one dog developing lameness. The immunoblot profiles of dogs challenged with female ticks infected by capillary feeding (8 weeks post challenge) were similar to immunoblots (4 weeks post challenge) from dogs challenged with naturally infected females collected in the field. These studies demonstrated that B. burgdorferi cultured in BSK medium can be capillary fed to either nymphal or adult female ticks under laboratory controlled conditions for the purpose of transmitting the spirochete to dogs during the tick's blood meal. This tick infection system would be useful for a controlled and defined challenge of vaccinated and non-vaccinated dogs for proper evaluation of vaccine efficacy, which is difficult to achieve using field-collected ticks. Furthermore, this system may also be useful for investigation of the pathogenesis of Lyme disease, evaluation of the pathogenicity of new isolates of B. burgdorferi, or evaluation of antibiotic therapy.

Ocorrência de apoptose em leucócitos no esfregaço de sangue periférico e em sincícios na infecção in vivo pelo vírus da cinomose canina

The aim of this paper is to report the occurrence of apoptosis in peripheral blood leukocytes and in syncytia induced by experimental infection of dogs with canine distemper virus. Blood was collected from nine puppies that were inoculated with the Snyder Hill strain of CDV, and stained with May Grunwaldt-Giemsa. Dogs were necropsied and retropharyngeal lymph nodes were collected and processed for paraffin embedding. Five micrometers thick sections were stained with hematoxylin-eosin and methyl green pyronin. Other sections were submitted to TUNEL reaction for in situ detection of apoptosis, and immunohistochemistry to detect CDV antigen. Immunohistochemistry confirmed the presence of viral antigen. Syncytia were present in all retropharyngeal lymph nodes. Several syncytia contained apoptotic nuclei as detected by hematoxylin-eosin and methyl green pyronin stains. Apoptotic nuclei were labeled inside syncytia by the TUNEL reaction. Blood smear analyses indicated higher proportion of apoptotic leukocytes in the peripheral blood of infected dogs.

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.

Pig, donkey and buffalo meat as a source of some coccidian parasites infecting dogs

Experimental infection of dogs with meat samples (oesophagus, heart and diaphragm) from each of 105 pigs, 11 donkeys and 17 Egyptian water buffaloes indicated that they contained the infective stages of some coccidian parasites of dogs. The dogs which were fed pig meat shed in their faeces Isospora ohioensis, I. canis oocysts and Sarcocystis miescheriana sporocysts after prepatent periods of 3–5, 4–7 and 9–10 days, respectively. The dogs which were fed donkey meat excreted only I. ohioensis oocysts and Sarcocystis bertrami sporocysts after prepatent periods of 3 and 11 days, respectively. However, the dogs which were fed buffalo meat shed in their faeces I. ohioensis, I. canis and Hammondia heydorni oocysts with prepatent periods of 1, 1 and 7 days, respectively.

Experimental infection of dogs with the nasal mite Pneumonyssoides caninum

A successful experimental transmission of the canine nasal mite, Pneumonyssoides caninum, is described. Some 11 weeks after repeated systemic ivermectin treatment, four Beagles were inoculated via the right nostril with 20 P. caninum mites of different sexes and life stages, obtained at the necropsy of an infected dog. The inoculated dogs and a matching uninoculated control were observed for clinical signs for 14 weeks and then euthanised. Vague upper respiratory signs and a transient minor increase in the number of eosinophils in peripheral blood were recorded in the inoculated dogs. At necropsy 4–12 P. caninum mites were found in the nasal cavities and sinuses of the inoculated dogs, but none in the control. In three out of the four infected dogs mites were found in both the right and left nasal cavities and sinuses of the skull. Since in no case more mites than the number used for inoculation were detected it is not clear if the mites managed to reproduce in the dogs. Inflammatory lesions were seen most consistently in the olfactory mucosa, respiratory mucosa and tonsils, and growth of opportunistic bacteria was observed in the tonsils of the infected dogs. The inflammatory lesions seen in the olfactory mucosa may explain why dogs infected with P. caninum sometimes appear to suffer from impaired scenting ability.

Experimental infection of dogs with Borrelia burgdorferi

Four beagle dogs were inoculated subcutaneously with the BITs1 Italian strain of Borrelia burgdorferi. Only one dog became infected and B burgdorferi was isolated from its blood and urine three and four weeks after infection. B burgdorferi antibodies were detected by immunofluorescence from four to 11 weeks after infection. An uninoculated dog kept in the same run as the infected dog, developed a positive serological response, but none of the five dogs showed clinical signs.

Detection of Neospora caninum in tissue sections using a murine monoclonal antibody.

A murine monoclonal antibody (MAb 6G7), isotype IgG2a, produced against tachyzoites of Neospora caninum (isolate NC-1) reacted specifically with tachyzoites of N. caninum in an indirect immunofluorescent antibody test. MAb 6G7 did not react with tachyzoites of Toxoplasma gondii, sporozoites of Isospora suis, Eimeria bovis, or E. tenella, or merozoites of E. bovis in the indirect immunofluorescent antibody test. MAb 6G7 reacted positively with both tachyzoites and bradyzoites of N. caninum in an avidin-biotin peroxidase complex immunohistochemical test on formalin-fixed paraffin-embedded tissues. No reaction was observed with the following: tachyzoites and bradyzoites of T. gondii, T. gondii-like parasites, or Hammondia hammondi; bradyzoites of Frenkelia microti; schizonts and merozoites of Sarcocystis-like organisms; schizonts, sarcocysts, and oocysts/sporocysts of S. cruzi; schizonts and merozoites of S. canis; schizonts of S. hirsuta, S. tenella, and S. capracanis; merozoites of S. neurona and S. neurona-like organisms, E. bovis, or Haemoproteus sp.; bradyzoites and merozoites of S. montanaensis; bradyzoites of S. odocoileocanis, S. cruzi, and S. tenella; meronts, sexual stages, and caryocysts of Caryospora sp. and C. bigenetica; micromerozoites, macromerozoites, and schizonts of Hepatozoon canis; sporozoites, sexual stages, and oocysts of Cryptosporidium parvum and C. baileyi; trophozoites of Monocystis lumbrici, Tritrichomonas foetus, and Balantidium coli; tissue cysts and bradyzoites of Besnoitia sp. and B. jellisoni; amastigotes of Leishmania sp.; and trophic theronts of Ichthyopthirius multifilis. MAb 6G7 reacted with tachyzoites and bradyzoites of N. caninum in natural and experimental infections in dogs, cattle, mice, rats, sheep, and goats, indicating that host origin of the tissues did not affect the performance of the test.

Camel ( Camelus dromedarius) and sheep ( Ovis aries) meat as a source of dog infection with some coccidian parasites

Experimental infection of dogs with camel ( Camelus dromedarius) meat resulted in infection of the dogs with Isospora canis, Hammondia heydorni and Sarcocystis cameli. The dogs fed sheep ( Ovis aries) meat passed oocysts of Isospora canis, Isospora ohioensis and sporocyts of Sarcocystis spp. Extraintestinal stages were detected in the intestinal lymph node of a rabbit killed 4 days following inoculation with Isospora ohioensis oocysts. Dogs fed the rabbit (killed 4 days after inoculation with I. ohioensis) passed I. ohioensis oocysts in their faeces 8 days post-infection.

Coproantigen detection for immunodiagnosis of echinococcosis and taeniasis in dogs and humans.

Three ELISA assays, based on hyperimmune rabbit serum raised against adult cestode somatic antigen, were applied in this study for the detection of Taenia- and Echinococcus-specific antigens in host faeces. The first assay, using an antiserum against Taenia pisiformis antigen extract, was used in a time-course of T. pisiformis experimental infection in dogs. The assay was shown to be considerably more sensitive than microscopical detection of eggs in faeces. Antigen was present in faeces before patency and antigen levels were independent of T. pisiformis egg output. The second assay, involving a test for human taeniasis based on antibodies against T. solium, was applied in two field studies carried out in China and Guatemala. The test was highly specific, no false positive reactions occurred with human faecal samples and the test was capable of diagnosing individuals who would not have been detected by coproscopy or treatment to recover the tapeworm. A third assay was designed for E. granulosus and demonstrated 87.5% sensitivity and 96.5% specificity with samples from naturally and experimentally infected dogs with Echinococcus or Taenia infections. In both the human Taenia and canine Echinococcus studies antigen could be detected in faecal samples from infected hosts stored in 5% formalin for 6 months. Further refinements to these tests for field application are discussed.

Experimental Infection of Dogs with Sarcoptes scabiei Derived from Naturally Infected Wild Red Foxes (Vulpes vulpes): Clinical Observations

Abstract— Sarcoptes scabiei, derived from wild red foxes (Vulpes vulpes) gave rise to self‐limiting sarcoptic mange (pseudoscabies) in 12 experimentally infected beagle dogs. The experimentally induced Sarcoptes scabiei infections caused skin lesions typical of sarcoptic mange in dogs on recognized predilection sites. The incubation periods varied from 6 to 13 days depending on the mode of infection. When six dogs were re‐infected two months after complete clinical recovery from a primary infection, the incubation period was three days.Résumé— Des sarcoptes, Sarcoptes scabiei, provenant de renards roux sauvages (Vulpes vulpes) ont provoqué une gale sarcoptique limitée (pseudogale) chez 12 Beagles infectés expérimentallement. Les infections par Sarcoptes scabiei ont entrainé l'apparition de lésions typiques de la gale sarcoptique sur les zones de prédilection habituelles. La période d'incubation était de 6 à 123 Jours selon le mode d'infection. Cette période n'était plus que de trois Jours chez 6 chiens reinfectés 2 mois après la guérison complète de la première infection. Zusamenfassung—Sarcoptes scabiei, isoliert aus wilden Rotfüchsen (Vulpes vulpes), verursachte bei 12 experimentell infizierten Beagle‐Hunden eine sich selbst begrenzende Sarkoptesräude (Pseeudoskabies). Die experimentell herbeigeführten Sarcoptes scabiei‐Infektionen führten zu für Sarkoptesräude des Hundes typischen Hautläsionen an den bekannten Prädilektionsstellen. Die Inkubationszeit vanierte, abhängig vom Infektionsmodus, zwischen 6 und 13 Tagen. Als sechs Hunde zwei Monate nach vollständiger klinischer Abheilung der Primärinfektion reinfiziert wurden, betrug die Inkubationszeit 3 Tage.Resumen En 12 perros beagle infectados experimentalmente con el ácaro Sarcoptes scabiei (pseudoscabies), procedente de zorros rojos salvajes (Vulpes vulpes), dió lugar a una sarna sarcóptica de carácter autolimitante. La infección inducida de Sarcoptes scabiei, provocó lesiones típicas de sarna sarcóptica, en las zonas de predileccion reconocidas como tales. Dependiendo de la forma de infección, el periodo de incubación osciló entre 6 y 13 días. Se observó que en seis perros, dos meses después de una recuperación clínica completa, el periodo de incubación fué de dos días.