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.     

Experimental infection of dogs (Canis familiaris) with sporulated oocysts of Neospora caninum

Neospora caninum is widely distributed in the world and this parasite is one of the major causes of abortion in cattle. Dogs and coyotes are definitive hosts of N. caninum and several species of domestic and wild animals are intermediate hosts. Dogs can become infected by the ingestion of tissues containing cysts and then excrete oocysts. It is not yet known whether sporulated oocysts are able to induce a patent infection in dogs, i.e. a shedding of N. caninum oocysts in feces. The objective of this study was to experimentally examine the infection of dogs by sporulated oocysts. The oocysts used in the experiment were obtained by feeding dogs with brain of buffaloes (Bubalus bubalis) positive for anti-N. caninum antibodies by indirect fluorescent antibody test (IFAT ≥200). Oocysts shed by these dogs were confirmed to be N. caninum by molecular methods and by bioassay in gerbils, and sporulated N. caninum oocysts were used for the oral infection of four dogs. The dogs were 8 weeks old and negative for antibodies to N. caninum and Toxoplasma gondii. Dogs 1 and 4 received an inoculum of 10,000 sporulated oocysts each; dog 2 an inoculum of 5000 sporulated oocysts and dog 3 received 1000 sporulated oocysts of N. caninum. The total feces excreted by these dogs were collected and examined daily for a period of 30 days. No oocysts were found in their feces. The dogs were monitored monthly for a 6-month period to observe a possible seroconversion and when this occurred the animals were eliminated from the experiment. Dogs 1 and 4 seroconverted 1 month after the infection with titer, in the IFAT, of 1600 and 800, respectively; the other two dogs presented no seroconvertion during the 6-month period. Dogs 1 and 2 were euthanized 180 days after infection and were examined for the detection of N. caninum in tissues (brain, muscle, lymph node, liver, lung, heart and bone marrow) by immunohistochemistry and PCR with negative results in both techniques. Bioassay in gerbils with brain of these dogs was also performed and again the results were negative. In conclusion, dogs infected with sporulated oocysts of N. caninum were not able to shed oocysts in feces. However, a higher dose of infection stimulated the production of antibodies against N. caninum in the dogs.     

Identification of Hammondia heydorni oocysts by a heminested-PCR (hnPCR-AP10) based on the H. heydorni RAPD fragment AP10

Toxoplasma gondii, Hammondia hammondi, Neospora caninum, Neospora hughesi and Hammondia heydorni are members of the Toxoplasmatinae sub-family. They are closely related coccidians with similarly sized oocysts. Molecular diagnostic techniques, especially those based on polymerase chain reaction (PCR), can be successfully applied for the differentiation of Hammondia-like oocysts. In this paper, we describe a rapid and simple method for the identification of H. heydorni oocysts among other members of the Toxoplasmatinae sub-family, using a heminested-PCR (hnPCR-AP10) based on a H. heydorni RAPD fragment available in molecular database. DNA of oocysts of H. heydorni yielded a specific fragment of 289-290 bp in the heminested-PCR assay. No product was yielded when the primers were used for the amplification of DNA extracted from T. gondii, N. caninum, N. hughesi and H. hammondi, thus allowing the differentiation of H. heydorni among other members of the Toxoplasmatinae sub-family. The hnPCR-AP10 was capable of detecting H. heydorni genetic sequences from suspensions with at least 10 oocysts. In conclusion, the hnPCR-AP10 proved to be a reliable method to be used in the identification of H. heydorni oocysts from feces of dogs.     

Incidence of coccidia in dogs in Brno and the surrounding area

In the course of two years, 663 dogs from Brno and its vicinity were subjected to clinical and parasitological examination with a special reference to the occurrence of coccidia. A saturated sugar solution with the specific weight of 1150 was used as the flotation medium. Coccidia were found in 2.17% of the dogs examined. The oocysts of the groups Isospora ohioensis, I. burowsi and I. neorivolta were identified most frequently (2.11% of the dogs). Four dogs (0.60%), all alike, eliminated the oocysts of Hammondia heydorni and sporocysts of Sarcocystis species, and two dogs oocysts of Hammondia heydorni and sporocysts of Sarcocystis species, and two dogs (0.30%) excreted oocysts of I. canis. Out of the animals infected, 58% were younger than six months. Clinical signs were observed only at a medium to strong invasion with oocysts of the species I. ohioensis, I. burowsi and I. neorivolta. The digestive tract was affected most severely--the animals suffered from bloodless diarrhoeas lasting two to twelve days, from occasional vomiting and systemic dehydration. Invasion by I. canis, Hammondia heydorni and Sarcoystis sp. did not cause any clinical changes.     

Confirmation that the dog is a definitive host for Neospora caninum.

Two mixed-breed littermate dogs were fed mouse brains containing tissue cysts of the NC-beef isolate of Neospora caninum. Both dogs excreted N. caninum oocysts in their feces. Dog 1 which was given methylprednisolone acetate (MPA) prior to ingesting tissue cysts, excreted oocysts on days 5 to 10 inclusive and on day 17 after ingesting tissue cysts. Dog 1 had a serum antibody titer of 1:200 in the indirect fluorescent antibody test (IFAT) 35 days after it was fed tissue cysts. Dog 2, which was not treated with MPA, excreted oocysts on Day 6 and Day 9 after ingesting tissue cysts. Antibodies to N. caninum were not found in a 1:25 dilution of serum on any examination period for Dog 2 during the study. Neospora caninum was not found in the tissues of either dog by histological or immunohistochemical means following necropsy 42 days after being fed tissue cysts. The identity of the oocysts excreted in the feces of the dogs was confirmed by mouse inoculation studies.     

Coprodiagnosis of Hammondia heydorni in dogs by PCR based amplification of ITS 1 rRNA: differentiation from morphologically indistinguishable oocysts of Neospora caninum

Hammondia heydorni is thought to be a non-pathogenic coccidian parasite of dogs that is closely related to Neospora caninum, an important parasite of cattle and dogs. Oocysts of these two species are morphologically indistinguishable from each other. A population of 2240 dogs in the Czech Republic was screened for the presence of H. heydorni/N. caninum oocysts and five (0.22%), represented by five of 3135 faecal samples (0.16%), were positive. The internal transcribed spacer 1 region of the rRNA gene (ITS1) from two isolates were cloned and the DNA sequences were identical with those of the ITS1 of H. heydorni. Based on the rRNA sequences available for H. heydorni and related coccidia, the primer pair JS4-JS5 was designed to amplify the 3' end of the small subunit (SSU) rRNA gene and ITS1 of H. heydorni. When tested on DNA extracted from a variety of parasites, the primers amplified a specific 267 bp fragment in our isolates only. The presence of DNA equivalent to 10 oocysts was sufficient for the amplification of the ITS1. We present a PCR-based diagnostic method as the only fast and reliable method for the diagnosis of H. heydorni in dogs.     

Effect of broiler chicken age on susceptibility to experimentally induced Cryptosporidium baileyi infection

Clinical signs of respiratory tract disease were observed in chickens that were inoculated intratracheally with 1 x 10(6) oocysts of Cryptosporidium baileyi at 2 or 14 days of age (10 chickens/group), but not in chickens inoculated at 28 or 42 days of age (10 chickens/group). Orally inoculated chickens in all age groups (10 chickens/group) did not develop clinical signs of disease. Orally and intratracheally inoculated chickens in all age groups were infected, as determined by the finding of cryptosporidia in tissue sections of the trachea, bursa of Fabricius, and cloaca, and by the recovery of oocysts from their feces. Chickens inoculated at 2 and 14 days of age excreted oocysts for a longer period and had greater numbers of cryptosporidia in their tissues, compared with chickens inoculated at 28 and 42 days of age.     

Hammondia heydorni oocysts in the faeces of a greyhound in New Zealand

AIMS: To identify oocysts found in faecal material of a greyhound. METHODS: Polymerase chain reaction (PCR) and DNA sequencing were used to study genomic DNA isolated from oocysts purified from faeces of a greyhound. RESULTS: Database searches with the DNA sequences obtained showed they were derived from Hammondia heydorni. A species-specific PCR was developed to detect H. heydorni DNA. CONCLUSIONS: Light microscopy in conjunction with PCR and DNA sequencing definitively identified the presence of H. heydorni oocysts in faeces of a greyhound. CLINICAL RELEVANCE: This study confirms the presence of H. heydorni in New Zealand and indicates the need to correctly identify similar oocysts from dogs, rather than assume they are Neospora caninum.     

Experimental Coxiella burnetii infection in pregnant goats: excretion routes

Q fever is a widespread zoonosis caused by Coxiella burnetii. Infected animals, shedding bacteria by different routes, constitute contamination sources for humans and the environment. To study Coxiella excretion, pregnant goats were inoculated by the subcutaneous route in a site localized just in front of the shoulder at 90 days of gestation with 3 doses of bacteria (10(8), 10(6) or 10(4) i.d.). All the goats aborted whatever the dose used. Coxiella were found by PCR and immunofluorescence tests in all placentas and in several organs of at least one fetus per goat. At abortion, all the goats excreted bacteria in vaginal discharges up to 14 days and in milk samples up to 52 days. A few goats excreted Coxiella in their feces before abortion, and all goats, excreted bacteria in their feces after abortion. Antibody titers against Coxiella increased from 21 days post inoculation to the end of the experiment. For a Q fever diagnostic, detection by PCR and immunofluorescence tests of Coxiella in parturition products and vaginal secretions at abortion should be preferred to serological tests.     

Effects of lasalocid on coccidial infection and growth in young dairy calves.

Effects of lasalocid on coccidial infection and on calf growth were examined in 16 Holstein bull calves. Calves were assigned randomly to a 2 x 2 factorial arrangement of starter ration containing 0 or 40 mg of lasalocid/kg of starter, beginning when calves were 3 days old (SE = 0.046), and single oral inoculation with 0 or 30,000 sporulated oocysts (Eimeria bovis) at 28 days. Pelleted calf starter was fed ad libitum from day 1; milk replacer was fed at a rate of 3.6 kg/d until day 28. Mean daily gain, dry-matter intake, and body weight were increased in calves fed lasalocid and decreased in those inoculated with coccidia. Addition of lasalocid to the feed improved gains by 8% in uninoculated calves and by 50% in inoculated calves. Fecal oocyst numbers were reduced when lasalocid was fed to inoculated calves. Feces were more abnormal in calves inoculated with coccidia. Respiration rates, rectal temperatures, PCV, and serum sodium and potassium concentrations were unaffected by treatment. On the basis of findings in this study, lasalocid minimized effects of coccidial challenge inoculation and increased growth of calves.     

Coyote as a final host for Sarcocystis species of goats, sheep, cattle, elk, bison, and moose in Montana

Tissues (1 kg) from sheep, goats, cattle, moose, bison, or elk naturally infected with Sarcocystis species were fed to one to four Sarcocystis-free coyotes and the number of sporocysts in feces and intestines were counted. All 12 coyotes fed naturally infected tissues shed Sarcocystis in feces, with a prepatent period of 9 to 15 days. The four coyotes fed infected beef had 15, 25, 113, and 201 million sporocysts in their feces and intestines. The coyotes fed elk, moose, or bison had 2.5, 15, and 2.5 million sporocysts in their intestines, respectively. Sporocysts in feces of coyotes fed musculature of cattle, sheep, goats, and elk were structurally similar to those described previously from the feces of dogs. This is evidently the first report of the completion of life cycle of Sarcocystis species in moose and bison. Cross-transmission experiments indicated that one species of goat Sarcocystis completes its life cycle in both dog and coyote and that the ovine Sarcocystis is not transmissible to goats.     

Dogs as carriers of the emerging pathogen Arcobacter

Dogs and cats living in a household have previously been identified as a risk factor for human infection with Campylobacter and Helicobacter. In this study, carried out between July 2006 to September 2007, feces and oral swabs from 267 dogs and 61 cats were examined for the presence of the emerging pathogen Arcobacter. Isolates, obtained by an Arcobacter selective isolation procedure, were identified with an Arcobacter species-specific multiplex-PCR and characterized by modified enterobacterial repetitive intergenic concensus PCR. No arcobacters were isolated from cats. Five dogs excreted arcobacters in the feces and two other dogs carried arcobacters in the mouth. In the follow-up, one dog excreted the same Arcobacter butzleri strain for at least 1 week. Six dogs carried each an unique A. cryaerophilus strain although three of them lived in the same family. Therefore, beside the consumption of food and water, contact with dogs is another potential source of Arcobacter infection.     

Studies on the role of the red fox (Vulpes vulpes) as a potential definitive host of Neospora caninum

Neospora (N.) caninum is a protozoan parasite which is regarded as a major cause of abortion in cattle. Dogs and coyotes are definitive hosts of N. caninum which may shed environmentally resistant stages, oocysts, in their feces. Epidemiological studies in Germany showed that the presence of dogs increased the risk of a bovine herd to be N. caninum-positive in a bulk-milk ELISA test. However, there were also N. caninum-positive herds where dogs were not kept together with cattle.This leads to the question whether canids other than dogs, e.g., foxes, might be involved in the horizontal transmission of N. caninum. Therefore, the aim of our examinations in wild animals was to find out whether there are indications for a sylvatic cycle with foxes as definitive hosts and deer, roe deer and wild mice samples contained structures which resembled those of coccidian oocysts. In 13 of these 65 samples coccidian DNA was detected using a 18S rRNA gene based polymerase chain reaction (PCR).The examination of the 65 samples in a N. caninum-specific PCR revealed no positive result. Hammondia (H.) heydorni-DNA was detected in two samples. In addition, brain samples from 528 foxes, 224 wild mice, 16 deer and roe deer as well as from 1 wild boar were examined for the presence of N. caninum DNA by real time PCR. All samples tested negative by PCR. In conclusion, our study yielded no evidence indicating that the examined animals were part of a sylvatic cycle for N. caninum.     

Sarcocystis capracanis and Toxoplasma gondii infections in range goats from Texas

Specimens of tongues, esophagi, diaphragms, or abdominal muscles of 115 range goats from San Angelo, Tex, were examined for Sarcocystis and Toxoplasma gondii infections. Sarcocystis spp zoites were found microscopically in pepsin digests of muscles of 60.8% goats and sarcocysts of S capracanis were found in histologic sections of 27.8% goats. Sarcocysts were more common in sections of tongue (19.1%) than in those of other muscles (9.9% to 10.7%). A dog fed Sarcocystis-infected tissues shed sporocysts in feces, whereas 2 cats fed the same tissues did not shed sporocysts. Toxoplasma gondii was neither seen in histologic sections of goat tissues nor found by bioassays in mice or cats. Mice inoculated with pepsin digests of muscles did not develop T gondii infection and 2 cats fed goat tissues did not shed oocysts. Also, antibody to T gondii was not found in serum samples from goats. The low prevalence of T gondii infection in range goats may be because of the relative absence of domestic cats on Texas ranges.     

Effect of antibiotics in milk replacer on fecal shedding of Escherichia coli O157:H7 in calves

The objective of this study was to compare the concentration and duration of fecal shedding of Escherichia coli O157:H7 between calves fed milk replacer with or without antibiotic (oxytetracycline and neomycin) supplementation. Eighteen 1-wk-old Holstein calves were orally inoculated with a strain of E. coli O157:H7 (3.6 x 10(8) cfu/calf) made resistant to nalidixic acid (NA). Rectal samples were obtained three times weekly for 8 wk following oral inoculation. Fecal shedding of NA-resistant E. coli O157:H7 was quantified by direct plating or detected by selective enrichment procedure. Eight weeks after inoculation, calves were killed, necropsied, and tissues (tonsils, retropharyngeal and mesenteric lymph nodes, and Peyer's patches) and gut contents (rumen, omasum, abomasum, ileum, cecum, colon, and rectum) were sampled to quantify or detect NA-resistant E. coli O157:H7. The percentage of calves shedding NA-resistant E. coli O157:H7 in the feces in the antibiotic-fed group was higher (P < 0.001) early in the study period (d 6 and 10) compared with the control group fed no antibiotics. There was no difference between treatment and control groups in the concentration of E. coli O157 in feces that were positive at quantifiable concentrations. A comparison of the duration of fecal shedding between treated and untreated calves showed no significant difference between groups. At necropsy, E. coli O157:H7 was recovered from the rumen and omasum of one calf in the control group and from retropharyngeal lymph node and Peyer's patch of two calves in the antibiotic group. Supplementation of milk replacer with antibiotics may increase the probability of E. coli O157:H7 shedding in dairy calves, but the effect seems to be of low magnitude and short duration.     

Prevention of coccidiosis in domestic dogs and captive coyotes (Canis latrans) with sulfadimethoxine-ormetoprim combination

Sulfadimethoxine-ormetoprim combination was evaluated as a coccidiostat against experimentally induced coccidiosis in young dogs and coyotes (Canis latrans). The animals were experimentally inoculated with 50,000 or 100,000 sporulated oocysts of Isospora ohiohensis (98%) and Isospora canis (2%). In experiment 1, daily treatment for 13 to 23 days with a combination of 27.5 mg of sulfadimethoxine/kg of body weight (BW) and 5.5 mg of ormetoprim/kg of BW admixed to the feed resulted in no significant (P greater than 0.05) difference in fecal oocyst counts between treated and nontreated groups of dogs or coyotes. In experiment 2, treatment with a combination of 55 mg of sulfadimethoxine/kg of BW and 11 mg of ormetoprim/kg of BW for 23 days was 99.8% effective against Isospora spp infections in dogs. Significantly (P less than 0.05) fewer oocysts were present in feces of treated dogs than were present in feces of nontreated dogs from first passage of oocysts at day 4 to the end of the patent period at days 19 to 21. After the 2nd week of treatment, BW of treated dogs were significantly greater (P less than 0.05) than BW of nontreated dogs. Evidence of drug toxicity was not observed clinically or by serum chemical analyses.     

Response of goats to vaccination with temperature-sensitive mutants of Chlamydia psittaci obtained by nitrosoguanidine mutagenesis

Two temperature-sensitive strains of ovine Chlamydia psittaci, 1B and 1H, obtained by mutagenesis were used as live-organism vaccines; 31 goats were given 4 X 10(6) plaque-forming units (PFU) of strain 1B, and 31 were given 5 X 10(6) PFU of strain 1H 2 months before they were bred. The consequences of the vaccination of the goats were studied during pregnancy by recording complement-fixation antibody titer, chlamydial excretion, and kidding performances and were compared with those of goats inoculated under the same conditions with 100 X smaller dose of virulent caprine abortive strain AC1. The vaccination did not disturb pregnancy, and none of the vaccinated goats excreted chlamydiae. In contrast, 2 of 28 goats inoculated with AC1 aborted and shed chlamydiae. One year after the goats were vaccinated, they were challenge exposed by intradermal inoculation of 10(6) PFU of the caprine strain AC1 at 79 to 98 days of pregnancy. Although 13 of the 14 control nonvaccinated goats aborted and excreted chlamydiae, none of the pregnant goats vaccinated with 1H and only 1 of the pregnant goats vaccinated with 1B aborted and excreted chlamydiae.     

Prevention and control of coccidiosis in goats with decoquinate

Decoquinate was evaluated as a coccidiostat in domestic goats. Fifty goats less than 4 months of age were assigned to 5 groups (pens) of 10 goats each and were treated for 87 days with 0 (control), 0.3, 0.5, 1.0, or 4.0 mg of decoquinate in feed/kg of body weight. Goats were inoculated orally weight. Goats were inoculated orally with 30,000 oocysts, mainly Eimeria christenseni (74%) and E ninakohlyakimovae (20%) on day 19. Nontreated goats developed profuse watery diarrhea and tenesmus and gained weight poorly; 2 died. Treated goats did not develop clinical coccidiosis and gained significantly more weight (P less than 0.05), regardless of the dose used. Treated goats also had significantly fewer (P less than 0.05) oocysts in feces than did nontreated controls. Oocyst numbers were inversely related to dose; a more rapid decrease in oocyst numbers occurred as the dose was increased. At the doses used, decoquinate was safe in goats and was an effective drug for the prevention of clinical coccidiosis.     

Dog shedding oocysts of Neospora caninum: PCR diagnosis and molecular phylogenetic approach

Results of molecular determination of a dog isolate of Neospora caninum in the Czech Republic are presented. Colorless bisporocystic oocysts measuring 10-13 micro m x 10-11 micro m were recovered from feces and used for DNA isolation. A diagnostic PCR procedure using previously described molecular methods was performed to determine the species. The N. caninum species-specific primers based on the Nc 5 region produced a positive result, while primers specific for Hammondia heydorni rDNA internal transcribed spacer 1 (ITS1) was negative. Sequencing and phylogenetic comparison of ITS1 rDNA and the D2 domain of the large subunit rDNA (D2 LSU) determined our isolate to be N. caninum. Phylogenetic analysis of closely related genera Toxoplasma, Neospora and Hammondia based on ITS1 and D2 LSU robustly distinguished three clades: (i). Toxoplasma gondii + Hammondia hammondi, (ii). N. caninum + Neospora hughesi, and (iii). H. heydorni. Based on phylogenetic relationships we propose three acceptable suggestions to solve the problem of taxonomy of these genera.