Infected Puppies Research Articles

Acute Canine Adenovirus 2 Infection Increases Histamine Airway Reactivity in Beagle Puppies

Acute infection with canine adenovirus was studied in 23 specific pathogen-free outbred beagle puppies (median age = 78 days, range = 67 to 86 days) to determine its effects on pulmonary function and airway responsiveness to aerosolized histamine. The following groups were studied: uninoculated (n = 6, Control); inoculated with canine adenovirus type 2 (CAV2) (n = 11, Infected); and subclinical spontaneous infection with CAV2 (n = 6, Subclinical). While anesthetized with chloralose and mechanically ventilated, lung function and responsiveness to aerosolized histamine were measured 3 days before inoculation (Day -3), the day of inoculation (Day 0), and 3 to 4 (Day 3-4), 6 (Day 6), 8 to 10 (Day 8-10), and 12 to 14 (Day 12-14) days after inoculation. Histamine responsiveness was assessed by calculating the provocation concentration of histamine diphosphate to increase lung resistance (RL) to 150% (PC 150% RL), or decrease dynamic lung compliance (Cdyn) to 75% (PC 75% Cdyn) of the response to saline [RL(sal) and Cdyn(sal), respectively]. Arterial blood gases, functional residual capacity (FRC), specific static lung compliance (spCst), RL, Cdyn, and histamine responsiveness were not significantly different on Day 0 among the groups (p greater than 0.05). Control and Subclinical puppies remained healthy, had a mean weight gain of 0.7 kg, and did not change their histamine responsiveness during the study period. Infected puppies developed moderate to severe clinical illnesses, had poor weight gain, and were histamine hyperresponsive on Days 3-4 and 6. One infected puppy died on Day 3-4, and two died on Day 6 of their illness.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth of a Japanese isolate ofTaenia crassiceps in intermediate and definitive hosts

Higher infection rates were observed in gerbils and voles than in ICR mice after oral inoculation with eggs of a Japanese isolate of Taenia crassiceps. Asexual reproduction of T. crassiceps cysticerci was observed in all gerbils and voles infected i.p. with the cysticerci. However, ICR mice and Wistar rats were not suitable for the asexual proliferation of T. crassiceps. The hooks of cysticerci from mice were smaller than those from gerbils. In experimentally infected puppies, parasite development was noted as follows: strobilation and initial differentiation of the genital primordia on day 7 postinoculation (p.i.), appearance of the testes on day 9, observation of the ovaries on day 10, and development of the lateral branches of the uterus on day 15. The prepatent period was 27-31 days. After day 15 p.i., most of the worms were recovered from the middle third of the small intestine. The number of proglottids shed per day by each strobila was about 1. The number of eggs contained in a gravid segment was about 13,000.

Metabolic studies of parasitic helminths using n.m.r. spectroscopy

To.vocuru c.uni.s is a n intestinal nematode which infects dogs; it is of zoonotic significance as ingestion of its eggs can cause visceral larva migrans in man (Jacobs, 1985), whereas D i c t j ~ o c m h s vivipurus is a nematode which infects the respiratory tract of cattle giving rise to parasitic bronchitis. To date, very little research has been performed regarding the metabolic pathways and metabolites present in these nematodes. Although n.m.r. spectroscopy is an established technique for the study of metabolism in lower organisms (Ogino ct d.. 1980) and mammalian tissues (Behar cJt al., 1985). its application to helminths has, so far, been limited (Matthews et a/., 1985). The aim ofour present research is to obtain a more detailed understanding of the carbohydrate metabolism occurring in both nematodes, and possibly to identify metabolic pathways which may be used as potential targets for drug design. N.m.r. spectra were recorded on a Varian XL300 spectrometer operating at 299.9 and 75.4MHz for ' H and C studies, respectively. Typical I H spectral parameters included a spectral width of 3000 Hz, 90deg. pulse angle and a re-cycle time of 3-7 s; for C parameters see Table 1. T. cnnis (l'rom naturally infected greyhound puppies) and D. vivipurus (from artificially infected Jersey calves) were maintained under sterile conditions in Eagle's minimum essential medium at 37°C and Earle's balanced salt solution at 2°C. respectively. Equal numbers of male and female nematodes (approx. 300 mg wet wt.) were fed with [ 1 -13C]glucose (20 mg/lO ml). 1 ml aliquots of medium were removed as a function of time, and replaced with 1 ml of substrate-free medium. Samples were frozen and stored a t 14°C before n.m.r. analysis. I3C n.m.r. spectroscopic studies revealed that the C label from [ I -C]glucose was incorporated into [2-C]succinate, [3-C]lactate and [?-C]acetate in D. vivipurus maintained under aerobic conditions. Maintenance of T . cmnis under aerobic and anaerobic conditions roduced differing end-

Control of Toxocara canis in puppies: a comparison of screening techniques and evaluation of a dosing programme.

Conventional chemoprophylaxis of canine toxocariasis is based upon the treatment of young puppies. It is proposed that physiological considerations and the complexity of the host-parasite relationship in the case of Toxocara canis demand that anthelmintic evaluation should properly be conducted in such animals rather than in adolescent or adult dogs. A screening model using the critical trial technique in early weaned pups gave variable results even with compounds known to be of high efficacy in older dogs. Circumstantial evidence suggested that this inconsistency may be related to the rate of passage of ingesta through such young animals. More satisfactory results were obtained by pairing unweaned naturally infected puppies by weight and sex and treating one of each pair with the test compound. Natural in utero and transmammary infection was found to give fairly consistent worm-burdens within each litter and a good indication of efficacy could therefore be estimated by comparing post mortem worm-counts. On this basis, the apparent efficacies of pyrantel pamoate (5 mg kg-1) and piperazine (100 mg kg-1) were found to be 83.5 and 82.5%, respectively. This method was extended to evaluate a prophylactic programme in which anthelmintic therapy was given to pups when aged 10, 20 and 30 days. Intestinal T. canis populations were reduced, at 35 days of age, by between 94.2% and 98.0%. This programme increased overall weight gains between 10 and 30 days of age by 18% (P less than 0.05) although there was great variation between litters. The biggest response was observed in male pups between 20 and 30 days old.

Prenatal and transmammary infections of Toxocara canis in dogs: effect of benzimidazole‐carbamate anthelmintics on various developmental stages of the parasite

ABSTRACTFenbendazole and albendazole, given at a dose rate of 150 mg/kg for 3 days, produced a 90 per cent reduction in the numbers of second stage larvae of Toxocara canis present in the tissues of dogs although no reduction in the number of larvae found in the brains of infected dogs occurred with this treatment. The results suggest that a course of 3 day therapy with these anthelmintics should prevent prenatal infections in puppies. However, if infection is acquired by bitches during late pregnancy or early lactation, the transmammary route of infection becomes important. Therefore, anthelmintic treatment of the bitch prior to pregnancy will not prevent transmission of infection to her puppies should the bitch acquire a new infection of T. canis during pregnancy or early lactation.Alternatively, infection with T. canis can be controlled through the treatment of neonatal puppies for migrating larvae of T. canis. Treatment of newborn puppies with fenbendazole, albendazole or oxfendazole at a dose of 100 mg/kg for 2–3 days produced a 91–99 per cent reduction in the number of adult parasites found. In addition, a single dose of fenbendazole, given at a dose rate of 40 mg/kg, eliminated 93–96 per cent of adult T. canis from the intestines of 4–5‐week‐old puppies. These latter treatments would need to be repeated to eliminate completely the infection from puppies.

Investigation of antibodies against toxocara canis in naturally infected puppies

A investigation of antibodies against Toxocara canis was carried out in 15 naturally infected puppies by enzyme-linked immunosorbent assay. For this, four different antigens --eggs containing second stage larvae, larvae, adult worms and excretory-secretory products of larvae--were used. At the same time, autopsy and fecal examinations were carried out in these puppies. Of the samples tested, 11 had adult worms and no eggs in the faeces were found in three puppies. The ELISA values against four different antigens obviously enlarged with the age. The correlation between age and ELISA values was significant in the positive direction. While the relationship between the number of worms and the ELISA values was also significant in the negative direction. The ELISA values in the puppies having unfertilized eggs were greater than those having fertilized eggs. These findings submit in the naturally infected puppies that there are intensive relationships among the antibody levels, age, the number of worms and the condition of egg fertilization.

Isolation of antigenic variants of leptospiras from puppies and pigs experimentally infected with Leptospira interrogans serovars canicola and pomona.

For the purpose of isolating antigenic variants of leptospiras from domestic animals, Leptospira interrogans serovars canicola and pomona were inoculated into puppies and spesific-pathogen-free (SPF) pigs respectively, and the blood and kidneys of those animals were inoculated onto the solid medium each containing homologous immune serum. Colonies developed on the medium were divided into large, medium and small colonies. Some of these colonies picked at random were antigenically compared with the parent by a screening test using the precipitin-absorption test in gel. In the case of canicola, the antigenic variants were isolated from the blood of 7 of the 10 infected puppies. The antigenic variants were found in a large number of the large colonies from the blood and kidneys, and in a small number of the medium and small colonies from the blood, and none of the medium and small colonies from the kidneys. The antigenic variants were also isolated from the inocula, but they were isolated more frequently from the blood of the puppies than from the inocula. In the case of pomona, the antigenic variants were isolated only from the small colonies from the blood of 2 of the 5 infected pigs. Large colonies were never developed from the specimens of the pigs. The antigenic variants were isolated more frequently from the blood of the pigs than from the inocula. The variants were confirmed to be antigenically diffetent from their parent. The variants originated from the same parent were antigenically similar to one another. These results indicate that canicola Moulton and pomona MLS contained a small percentage of the antigenic variants and that the percentage of the variants was raised significantly, 3 to 7 days after the infection, in the blood of the puppies and pigs.

An immunofluorescence technique for the detection of Toxocara canis antibodies

An immunofluorescent (IF) test for the serodiagnosis of Toxocara canis infections in puppies is described. Frozen sections of male adult T. canis worms were used as antigen. A group of seven puppies, 6 weeks of age, was infected orally with 10 000 embryonated T. canis eggs each. In the sera of all animals IF antibodies could be detected from approximately 4 weeks after infection onwards. Titers were detectable until the end of the observation period (22 weeks). Two puppies of the same age were infected with 30 000 or 50 000 embryonated T. canis eggs respectively. Positive IF results were also obtained in the sera of these pups from week 4 post infection (p.i.) onwards. No correlation between titer and initial number of egges administered was observed. Furthermore, no correlation was noticed between titer and number of adult worms recovered from the dogs. For comparison all sera were tested with the complement fixation (CF) test, using cuticle material of adult worms as antigen. Complement fixing antibodies could be detected in none of the serum samples.

Structure of the Oocyst and Sporocyst Walls and Excystation of Sporozoites of Isospora canis

Excystation of sporozoites from oocysts of Isospora canis occurred 50 to 100 min after these were treated at about 22 C with trypsin and a bile salt (sodium taurocholate). Treatment with the bile salt alone resulted in excystation in 60 to 120 min, but no excystation was observed in oocysts treated with trypsin alone. Excystation occurred in 10 to 35 min when the oocysts were pretreated with Clorox before exposure to trypsin and the bile salt. Usually, the oocyst wall became flattened or indented at one or both ends; the sporocyst wall ruptured suddenly, and broke into several pieces, allowing the sporozoites to escape. The sporocyst wall consisted of 2 layers. The inner layer, about 4 times as thick as the outer, was made up of 4 separate plates. During excystation, the wall evidently breaks at the sites of apposition between the plates. Thus, the mechanism of excystation in I. canis differs considerably from that previously reported for Eimeria species. Excystation of various coccidian oocysts has been described previously (Doran and Farr, 1962; Hammond, Ernst, and Chobotar, 1970; Roberts, Speer, and Hammond, 1970). Bunch and Nyberg (1970) reported that Isospora canis differed from various Eimeria species in that excystation occurred readily without a CO2 stimulus in the former. Our findings with the electron microscope as to the structure of the oocyst and sporocyst walls of Isospora canis are described herein, as well as observations with the light microscope of excystation of I. canis sporozoites. MATERIALS AND METHODS Oocysts of Isospora canis obtained from experimentally infected beagle puppies were cleaned of fecal debris, sporulated (Nyberg and Hammond, 1964), and then stored in 2.5% K2CR207 at 5 C for 8 weeks. For light microscope studies, some oocysts were concentrated by centrifugation and resuspended in distilled water or saline A, whereas others were exposed to 5.25% sodium hypochlorite solution (Clorox) for 20 min, washed twice in saline A, concentrated, and then resuspended in saline A. One drop of the suspension of Cloroxtreated oocysts was placed on each of 5 slides, and to this drop was added a drop of 0.5% trypsin (1 to 300 Nutritional Biochemical) in saline A, Received for publication 29 August 1972. * This investigation was supported by PHS Research Grant No. AI-07488 to Utah State University from the NIAID. Published as Journal Paper No. 1301, Utah Agricultural Experiment Station. t Present address: Department of Histology, Dental Branch, University of Texas, Texas Medical Center, Houston 77025. t On leave from Department of Zoology, University of Alberta, Edmonton, Canada. 1.5% sodium taurocholate (Nutritional Biochemical) in saline A, 0.5% trypsin and 1.5% sodium taurocholate in saline A, saline A, and distilled water, respectively. In order to prevent drying, a cover slip was placed on each of the above preparations and sealed with Vaseline. The slides were then examined at room temperature (about 22 C) with bright-field and Zeiss-Nomarski interferencecontrast microscopy for observation of the excystation process. On each slide, 10 to 20 oocysts were observed to determine the timing of each event occurring during the excystation process. Oocysts not treated with Clorox were studied similarly. For electron microscope studies, sporocysts from oocysts which had been ground in a tissue grinder to break the walls were suspended for 10 min in a 0.25% trypsin and 0.75% sodium taurocholate excysting medium and then added to a cell suspension which had been harvested with a trypsin-versene solution from cell monolayers in two 2-ounce Brockway culture flasks. The suspension was centrifuged at 750 g for 5 min. The pellet was fixed in Karnovsky's fixative (Karnovsky, 1965) with cacodylate buffer for 2 hr at 22 C. The pellet was then dehydrated in 35 and 50% ethanol for 5 min each, and prestained with 1% uranyl acetate and 1% phosphotungstic acid in 70% ethanol at 4 C for 15 hr. It was further dehydrated in ethanol and 2 changes of propylene oxide, embedded in Dow epoxy resin (Lockwood, 1964), sectioned, placed on 200-mesh grids, stained with lead citrate, and examined with a Zeiss EM 9S electron microscope.

Canine Adenovirus Pneumonia

SUMMARY Clinical pneumonia characterized by tachypnoea and coughing was induced in experimental puppies exposed to aerosolized virus of infectious canine hepatitis. Susceptible animals kept in separate pens, but having a common air space with infected puppies also developed the injection. The virus produced a necrotizing bronchiolitis in which many inclusion bodies were present; immunofluorescence studies showed large amounts of virus antigen within the bronchiolar lesions and in the surrounding alveoli. In the later stages of the disease, the main feature was bronchiolar epithelial hyperplasia. Virus was re-isolated from infected lungs, tonsils and turbinates up to 12 days after aerosolization. None of the infected dogs developed hepatitis.

The Pathology of Experimental Infectious Canine Hepatitis in Neonatal Puppies

Summary Day-old puppies were found to be highly susceptible to infectious canine hepatitis virus and intraperitoneal inoculation of the virus induced a severe acute, and often lethal, infection. Infected puppies did not survive beyond the fifth day after administration of the virus. Although many intranuclear inclusion bodies were present in the liver, hepatic necrosis was minimal or absent.

Immunological response of the hosts to Toxocara canis (Werner, 1782) infection. I. Effect of superinfection on naturally infected puppies.

Superinfection of naturally infected puppies with large numbers of Toxocara canis eggs induced a self-cure reaction. There was a sharp fall in Toxocara egg output following superinfection in most of the puppies. In most, but not all the puppies, the existing worm burden was eliminated. Eliminated worms were full of fertile eggs, but, in the puppies in which egg production fell sharply without expulsion of the worm burden, egg production by the naturally acquired worms was suppressed only transiently.Antibody production, measured by the complement-fixation test, was directly related to resistance to superinfection but not to self-cure.Acquired resistance reduced the pathogenic effects of superinfection, and prevented development of the parasite beyond the second stage.It is a pleasure to thank Professor P. Seneviratna, University of Ceylon, for his constructive criticisms of the manuscript and the facilities provided.The writer thanks Mr W. G. Senaratne for technical assistance and typing of the manuscript.This work was carried out with a special research grant from the University of Ceylon.

Observations on the development of Toxocara canis (Werner, 1782) in the dog.

1. Evidence from previous literature shows that puppies are commonly infected with larvae ofToxocara canisat birth and that prenatal infection can be produced by feeding embryonated eggs to pregnant bitches.2. Observations on fifty-eight dogs in Brisbane showed that all of twenty-nine puppies 1–6 months old were infected, while only three out of twenty-nine dogs over 6 months old were infected.3. In naturally infected puppies, 1–3 weeks old, it was found that at birth third-stage larvae were present in the lungs. Third-stage larvae continued to appear in the lungs for the first week of life; their length was 0·6–1·3 mm.4. Third-stage larvae were found in the stomach on the day after birth; at 3 days after birth fourth-stage larvae were found in the intestine.5. It appeared likely that the second moult occurs in puppies before birth, and that the third moult takes place at a length of 1·0–1·3 mm. in the lungs and stomach within the first week of life.6. By the beginning of the second week, fourth-stage larvae were fully grown and had commenced the fourth moult at a length of 5–7 mm.7. Throughout the second and third weeks, adults grew rapidly, reaching a length of about 67 mm. by the end of the third week, but no eggs had appeared in the faeces at this time.8. Experimental infection of mice with eggs showed that the larvae were distributed to the somatic tissues, few reaching the alimentary tract. The larvae did not progress beyond the second stage in the tissues of mice.9. Experimental infection of dogs with eggs showed that in dogs over 5 weeks old the larvae were distributed to the somatic tissues and did not reach the alimentary tract. The larvae did not progress beyond the second stage, though some of them showed signs of commencing the second moult. In contrast, 1 to 3-week-old puppies infected in the same way were found to harbour larvae in the alimentary tract. Though some of these larvae were probably derived from a naturally acquired prenatal infection, it was evident from the progress of development that the experimental infection resulted in the presence of second-stage larvae in the liver and lungs and that these larvae underwent the second moult, commenced development as third-stage larvae in the lungs, migrated into the stomach and developed to the adult stage in the intestine.10. Experimental infection of dogs and foxes with mice harbouring second-stage larvae in the tissues showed that, in some instances, development of larvae proceeded in the alimentary tract. No evidence of somatic migration was found in dogs infected in this way, but in foxes second-stage larvae were found in the lungs.11. The structure and development of the second, third and fourth stage is described in detail and found to resemble closely the development ofT. catilarvae (Sprent, 1956). Second-stage larvae from dog tissues had a length of 0·34–0·44 mm. and the second moult occurred at a length of 0·37–0·44 mm. Third-stage larvae varied in length from 0·46 to 1·36 mm., and the third moult occurred at a length of 0·98–1·3 mm. Fourth-stage larvae measured 1·2–6·3 mm., though moulting fourth-stage larvae were observed up to a length of 7·4 mm. Sexual differentiation occurred during the fourth stage and was evident at a length of about 1·5 mm.12. The migratory behaviour of the larvae ofT. canisandT. catiis compared and discussed in relation to their wide range of hosts. It is concluded that differences in migratory behaviour are adaptations to prevailing modes of nutrition and it is suggested that the somatic migration occurring in dogs is an adaptation to the non-predatory habits of this particular host.

Observations on experimental Chagas' disease in dogs.

Summary 1. Experimental Chagas' disease has been produced in adult dogs by inoculation with culture forms of “A” and “B” strains of Trypanosoma cruzi and with blood forms of “W” strain. 2. Occasional spontaneous recoveries occurred in adult dogs but untreated infections in puppies were uniformly fatal. 3. The prepatent periods for “W”, and “A”, and “B” strains were: 5 to 10 days, 9 to 13 days, and 10 to 42 days, respectively. 4. The prepatent period was a little shorter in animals infected subcutaneously than in those infected intraperitoneally. 5. The prepatent periods and the survival times did not differ between the sexes but the parasitemias in males were higher than in females. 6. In unmedicated puppies death from acute myocarditis ordinarily ensued between 8 and 39 days following the appearance of trypanosomes in the blood. 7. The median survival time in untreated infections in puppies was 34 days from the time of inoculation; the shortest was 27, the longest 49 days. 8. Paraplegia was occasionally observed in adult dogs infected with “B” and “W” and occurred once in a puppy which survived the acute myocarditic stage as a result of medication with Bayer 7602. 9. Agglutination tests, using live culture as antigen, did not give consistent results during any of the periods of the disease. 10. The intravenous administration of Bayer 7602 did not usually alter the course of Chagas' disease in puppies; isopentaquine was usually not curative in tolerated doses. 11. Pentaquine, given orally, produced cures in puppies as evidenced by suppression of symptoms, eradication of parasites from the blood, survival, and absence of leishmania forms in the tissues at autopsy. 12. Although isopentaquine is more toxic than pentaquine in animals, the opposite is apparently the case in man; either compound might prove to be useful in clinical Chagas' disease.

Prenatal Infection with the Dog Hookworm, Ancylostoma caninum

SUMMARY All the puppies of two litters born to mothers infected with hookworm larvae during pregnancy acquired prenatal infestations of the dog hookworm, Ancylostoma caninum. Development of the larvae in the prenatally infected puppies did not take place until after birth. The fact that approximately ten and twenty per cent of the larvae given the mothers during gestation were recovered from the puppies as mature worms, was taken as indicating a wide-spread migration of hookworm larvae after oral infection, a phenomenon which it is suggested may be associated with the resistance of the host. The marked susceptibility of the puppies born to very resistant mothers was pointed out to be difficult to reconcile with a serological explanation of this type of resistance. Since all of the prenatally infected puppies died within twenty-five days, the suggestion is made that there may be a relation between human hookworm infection during pregnancy and infant mor-