Neospora caninum and Toxoplasma gondii antibodies in dogs from Durango City, Mexico

Toxoplasma gondii and Neospora caninum are structurally similar parasites, with many hosts in common. The prevalence of antibodies to T. gondii and N. caninum was determined in sera from dogs from Durango City, Mexico. Using a modified agglutination test, antibodies to T. gondii were found in 52 (51.5%) of the 101 dogs with titers of 1:25 in 27, 1:50 in 11, 1:100 in 5, 1:200 in 4, 1:400 in 2, 1:800 in 2, and 1:3,200 or higher in 1. Antibodies to N. caninum were determined by the indirect immunofluorescent antibody test (IFAT) and the Neospora sp. agglutination test (NAT). Two of the 101 dogs had N. caninum antibodies; these dogs did not have T. gondii antibodies, supporting the specificity of the tests used. The N. caninum antibody titers of the 2 dogs were: 1:400 by IFAT and 1:200 by NAT in 1, and 1:25 by NAT and IFAT in the other. Results indicate that these 2 structurally similar protozoans are antigenically different.     

Prevalence of antibodies to Neospora caninum and Sarcocystis neurona in sera of domestic cats from Brazil

Parasite Biology, Epidemiology and Systematics Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, United States Department of Agriculture, Building 1001, Beltsville, Maryland 20705-2350 Antibodies to Neospora caninum and Sarcocystis neurona were determined in serum samples of 502 domestic cats from Brazil using direct agglutination tests with the respective antigens. Antibodies to S. neurona were not found in 1:50 dilution of any serum in the S. neurona agglutination test. suggesting that domestic cats from S?o Paulo city were not exposed to S. neurona sporocysts from opossums. Antibodies to N. caninum were found in 60 (11.9%) of 502 cats with titers of 1:40 in 36 cats, 1:80 in 18 cats, 1:160 in 5 cats, and 1:800 in 1 cat using the Neospora agglutination test (NAT). Antibodies to N. caninum were confirmed by Western blotting in the sera of 10 cats with NAT titers of 1:80 to 1:800; this finding suggests that at least 10 cats had N. caninum-specific antibodies confirmed by 2 tests. This is the first documentation of natural exposure of cats to N. caninum.     

Seroprevalence of Neospora caninum antibodies in cattle and water buffaloes in India

Neospora caninum is now recognized as a major cause of abortion in cattle worldwide, but there is no report of N. caninum infection in cattle in India. Serum samples from 427 dairy cattle and 32 dairy water buffaloes from 7 organized dairy farms located in Punjab, India, were tested for N. caninum antibodies using a commercial monoclonal antibody-based competitive enzyme-linked immunosorbent assay (ELISA). Antibodies to N. caninum were found in 35 of 427 cattle from 6 of the 7 farms; 9.6% of cows, 5.1% of heifers, and 5.0% of calves were seropositive, suggesting postnatal transmission of N. caninum on the farm. Antibodies to N. caninum were found in 16 of 32 buffaloes tested from 2 dairy farms. In total, 64 cattle and 16 buffalo sera already tested by ELISA were also evaluated by an indirect fluorescent antibody test (IFAT) to verify ELISA results. Of the 64 cattle samples, 29 sera were negative by both tests and of the 35 ELISA-positive sera, 12 had IFAT titers of 1:100 or higher (1 had IFAT titer of 100, 2 had IFAT titer of 200, and 9 had IFAT titers of 400 or higher). Of the 16 buffalo sera positive by ELISA, 1 had an IFAT titer of 1:400. Thus, antibodies to N. caninum were demonstrated in cattle sera by 2 serologic methods. To our knowledge this is the first report of N. caninum infection in cattle and buffaloes in India.     

Toxoplasma gondii and Neospora caninum antibodies in dogs from Grenada, West Indies

Toxoplasma gondii and Neospora caninum are structurally similar parasites with many common hosts. The prevalence of antibodies to T. gondii and N. caninum was determined in sera from dogs in Grenada, West Indies. Using a modified agglutination test, antibodies to T. gondii were found in 52 (48.5%) of the 107 dogs, with titers of 1:25 in 17, 1:50 in 19, 1:100 in 7, 1:1,600 in 5, and 1:3,200 or higher in 4. Seroprevalence increased with age from 2.2% in dogs <6 mo old to 18.9% in dogs older than 2 yr, indicating postnatal transmission of T. gondii in this population of canines. There was no correlation between the health of the dogs and the seroprevalence or magnitude of the T. gondii titer. Antibodies to N. caninum were determined by the indirect immunofluorescent antibody test (IFAT). Two of the 107 dogs had N. caninum antibodies (IFAT titers 1:100 and 1:400); these dogs had T. gondii titers of 1:1,600 and 1:50, respectively. Results indicate that these 2 structurally similar protozoa are antigenically different.     

Canine and bovine Neospora caninum control sera examined for cross-reactivity using Neospora caninum and Neospora hughesi indirect fluorescent antibody tests

Neospora caninum is a well known protozoan parasite of domestic and wild animals. Neospora hughesi is a closely related protozoan with an unknown life cycle, host range, and infection prevalence. Many serologic surveys of N. caninum have been performed without consideration of potential cross-reactions with N. hughesi, which could confound results. The aim of this study was to investigate whether postexposure sera from animals experimentally infected with N. caninum exhibit significant reactivity differences when tested using N. caninum and N. hughesi Immunofluorescent Antibody Tests (IFAT). Pre- and postinfection serum samples from 10 dogs, 20 calves, and 17 cows were tested by dual IFATs. All pre-exposure samples for N. caninum tested seronegative for both organisms. All postexposure samples that were seropositive for N. caninum were also positive for N. hughesi, although N. hughesi antibody titers were usually 1 dilution lower (P < 0.02). Serologic surveys for N. caninum may be confounded by cross-reacting titers with N. hughesi, but true positive N. caninum antibody titers are greater than, or equal to, cross-reacting N. hughesi antibody titers.     

Seroprevalence of Neospora caninum and Toxoplasma gondii antibodies in the South American opossum (Didelphis marsupialis) from the city of São Paulo, Brazil

Antibodies to Neospora caninum and Toxoplasma gondii were assayed in sera of 396 opossums (Didelphis marsupialis) from the city of S?o Paulo, Brazil. Antibodies to N. caninum were assayed using the indirect immunofluorescent antibody test (IFAT). Antibodies (IFAT, approximately 1:25) to N. caninum were found in 84 opossums (D. marsupialis) in titers of 1:25 in 46, 1:50 in 20, 1:100 in 17, and 1:400 in 1. Antibodies to T. gondii were assayed with the modified agglutination test (MAT) and the IFAT. Antibodies to T. gondii (MAT, approximately 1:25) were found in 82 (20.4%) of the 396 opossums, in titers of 1:25 in 24, 1:50 in 26, 1:100 in 18, 1:200 in 13, and 1:800 in 1. The IFAT antibodies to T. gondii were found in 148 of 396 opossums, in titers of 1:16 in 41, 1:32 in 23, 1:64 in 13, 1:128 in 6, 1:256 in 20, 1:512 in 17, 1:1,024 in 10, 1:2,048 in 10, 1:4,096 in 7, and 1:8,192 in 1. This is the first report of N. caninum and T. gondii infections in D. marsupialis.     

Neospora caninum-like oocysts observed in feces of free-ranging red foxes (Vulpes vulpes) and coyotes (Canis latrans)

The aim of this study was to examine the feces of free-ranging foxes and coyotes for the presence of Neospora caninum oocysts. Feces were collected from 271 foxes and 185 coyotes in the Canadian province of Prince Edward Island, processed by sucrose flotation, and examined by light microscopy for the presence of coccidian oocysts. In 2 fox and 2 coyote samples, oocysts morphologically and morphometrically similar to oocysts of N. caninum were observed. DNA was extracted from these samples and subjected to nested polymerase chain reaction (PCR) using primers to the N. caninum-specific Nc5 genomic sequence. Through DNA sequencing, alignment of the sequences of at least 3 clones from each isolate to sequences deposited in GenBank revealed 95-99% similarity to the Nc5 sequence of N. caninum. PCR using primers specific for Hammondia heydorni failed to yield an amplification product from these DNA samples.     

Seroprevalence of antibodies to Neospora caninum in dogs from Spain

The prevalence of Neospora caninum antibodies was determined in sera of 139 dogs from Catalonia (northeastern Spain) using the indirect immunofluorescence antibody test (IFAT). Antibodies in the IFAT were found in 17 of 139 dogs (12.2%) with titers ranging from 1:50 to 1: 1,600. Seroprevalence was higher in dogs over 1 yr old compared with dogs younger than 1 yr (P < 0.05). No statistical difference was observed when sex, breed, purpose, or modus vivendi was compared with seropositivity. Most dogs had low antibody titers, which indicated subclinical infection in the area studied. No neosporosis-related disease was reported from any dog, although a German shepherd with an antibody titer of 1:800 showed pododermatitis. All sera were also screened using a commercial direct agglutination test (DAT). The DAT showed a similar specificity but a lower sensitivity when compared with IFAT as a reference technique.     

Neospora caninum infections in bovine foetuses and dairy cows with abortions in Argentina

Antibodies to Neospora caninum were measured in bovine foetuses, dairy cows and beef cows in Argentina using the IFAT, the N. caninum agglutination test, and the recombinant NCDG1 and NCDG2 ELISA. Serum antibodies (IFAT titre 1:80) were found in 20 of 82 (24.4%) dairy cow foetuses and one of 22 (4.5%) beef cow foetuses. Microscopic lesions suggestive of neosporosis were seen in brains of seven of eight foetuses with IFAT titres of 1:80. Antibodies (IFAT) were found in 122 of 189 (64.5%) dairy cows that aborted. Serum antibody titres (IFAT) of 189 dairy cows that aborted were: < 1:25 (67 cows), 1:25 (four cows), 1:50 (16 cows), 1:200 (seven cows), 1:> or = 800 (95 cows). Of the 87 sera with IFAT titres of < or = 1:50, 57 had no antibodies in 1:40 dilution and 30 had titres of 1:40 in the N. caninum agglutination test. Thus, sera from at least 56 dairy cows which had aborted were seronegative both in the N. caninum agglutination test and the IFAT. The distribution of positive and negative sera was similar when measured by ELISA, except that, depending on cut-off titre, the ELISA indicated a greater number of seropositive cows that were negative by the IFAT and N. caninum agglutination test. These results suggest that transplacental transmission of N. caninum in dairy cows in Argentina is frequent.     

Prevalence of Neospora caninum and Toxoplasma gondii antibodies in wild ruminants from the countryside or captivity in the Czech Republic

In the Czech Republic, sera from 720 wild ruminants were examined for antibodies to Neospora caninum by screening competitive-inhibition enzyme-linked immunosorbent assay and confirmed by indirect fluorescence antibody test (IFAT); the same sera were also examined for antibodies to Toxoplasma gondii by IFAT. Neospora caninum antibodies were found in 14% (11 positive/79 tested) roe deer (Capreolus capreolus), 14% (2/14) sika deer (Cervus nippon), 6% (24/ 377) red deer (Cervus elaphus), 1% (2/143) fallow deer (Dama dama), 3% (3/105) mouflon (Ovis musimon), and none of 2 reindeer (Rangifer tarandus). Toxoplasma gondii antibodies were found in 50% (7/14) sika deer, 45% (169/377) red deer, 24% (19/79) roe deer, 17% (24/143) fallow deer, 9% (9/105) mouflon, and 1 of 2 reindeer. In 42 samples of wild ruminants that tested positive for N. caninum antibodies, 28 (67% of the positive N. caninum samples) reacted solely to N. caninum. This is the first evidence of N. caninum infection in mouflon, the first N. caninum seroprevalence study in farmed red deer, and the first survey of N. caninum in wild ruminants from the Czech Republic.     

Seroprevalence of antibodies to Neospora caninum in dogs and raccoon dogs in Korea

Neospora caninum is an important cause of abortion in cattle, and dogs are its only known definitive host. Its seroprevalence among domestic urban and rural dogs and feral raccoon dogs (Nyctereutes procyonoides koreensis) in Korea was studied by indirect fluorescent antibody test (IFAT) and by the neospora agglutination test (NAT), respectively. Antibodies to N. caninum were found in 8.3% of urban dogs and in 21.6% of dogs at dairy farms. Antibody titers ranged from 1:50 to 1:400. Antibodies to N. caninum were found in six (23%) of 26 raccoon dogs. However, the potential role of raccoon dogs as a source of horizontal transmission of bovine neosporosis needs further investigation. The results of this study suggest that there is a close relationship between N. caninum infection among dairy farm dogs and cattle in Korea. This study reports for the first time upon the seroprevalence of N. caninum infection in raccoon dogs in Korea.     

Molecular and biological characterization of Hammondia heydorni-like oocysts from a dog fed hearts from naturally infected white-tailed deer (Odocoileus Virginianus)

Neospora caninum and Hammondia heydorni are morphologically and phylogenetically related coccidians that are found in dogs. Although there is serological evidence of N. caninum infection in the white-tailed deer (Odocoileus virginianus), the parasite has not been yet isolated from the tissues of this host. In an attempt to isolate N. caninum from deer, hearts from 4 deer with antibodies to N. caninum were fed to 2 dogs. One of these dogs shed unsporulated oocysts 12-14 microm in diameter. Sporulated oocysts were not infective to Mongolian gerbils (Meriones ungulatus), and DNA isolated from these oocysts was not amplified using N. caninum-specific primers. However, positive amplification with the H. heydorni-specific first internal transcribed spacer (ITS-1) primers and common toxoplasmatiid ITS-1 primers confirmed the presence of H. heydorni DNA in the samples. The oocysts were considered to be H. heydorni on the basis of their morphology, biology, and molecular characteristics. This is the first record of a H. heydorni-like parasite in the white-tailed deer.     

Prevalence of Neospora caninum infection in dogs from beef-cattle farms, dairy farms, and from urban areas of Argentina

Prevalence of anti-Neospora caninum antibodies was determined in sera of 320 dogs from Argentina using the indirect fluorescent antibody test (IFAT). Antibodies to N. caninum were found in 121 of 320 (37.8%) sera with titers of 1:50 (21 dogs), 1:100 (23 dogs), 1:200 (23 dogs), 1:400 (17 dogs), 1:800 (23 dogs), and > or = 1:1.600 (14 dogs). The seropositivity (IFAT, > or = 1:50) was higher in dogs from dairy (48% of 125) and beef (54.2% of 35) farms than in dogs from urban areas (26.2% of 160). Prevalence of anti-N. caninum antibodies was higher in dogs more than 12 mo of age (47.7%, 105 of 222) versus in 12-mo-old or younger dogs (12.7% of 86), suggesting postnatal exposure of N. caninum infection in dogs.     

Prevalence of Neospora caninum antibodies in dogs from dairy cattle farms in Parana,Brazil

Serum samples from 134 dogs from 22 cattle dairy farms in the northern region of Parana State, Brazil, were tested for antibodies to Neospora caninum in an indirect fluorescent antibody test. Antibodies (> or = 1:50) to N. caninum were found in 29 (21.6%) of the 134 dogs, and seropositive dogs were found on 14 (63.6%) of the 22 dairy cattle farrms. The antibody titers of dogs were 1:50 (3 dogs), 1:100 (7 dogs), 1:200 (7 dogs), 1:400 (6 dogs), and > or = 1:800 (6 dogs). The low prevalence (9%) in < 1-yr-old dogs compared with the 2- to 3-fold higher prevalence in older dogs (17-29%) suggests postnatal exposure to N. caninum infection.     

Transplacental Neospora caninum infection in cats

Transplacental transmission of Neospora caninum was studied in 2 pregnant cats (queens). Queen 1 was inoculated subcutaneously with 2 x 10(6) cell culture-derived N. caninum tachyzoites on day 47 of gestation. She gave birth to a full-term kitten on the 17th day after inoculation. The kitten died the second day after birth due to generalized N. caninum infection. The mother cat was killed on the third day after parturition and was found to have a macerated kitten in the uterus. Severe placentitis, metritis, hepatitis, and nephritis due to N. caninum were seen in tissues from the queen. Queen 2 was fed N. caninum tissue cysts and mated 111 days later. She gave birth to 3 healthy full-term kittens. The kittens were necropsied at 2, 22, and 30 days of age. Neospora caninum was recovered from the organs and was seen in histologic sections in 1 of the 3 kittens. Results indicate that N. caninum can be transplacentally transmitted in cats during acute and chronic stages of infection. Neospora caninum-specific IgG antibodies were demonstrated in the sera of inoculated cats and nursing kittens.     

Prevalence of agglutinating antibodies to Neospora caninum in raccoons, Procyon lotor.

Neospora caninum is an apicomplexan parasite that causes neonatal neuromuscular disease in dogs and abortions in cattle. Dogs are the only proven definitive host. Little is known about the prevalence of antibodies to this parasite in wildlife. Sera from 99 raccoons (Procyon lotor) were examined for agglutinating antibodies to N. caninum using the modified agglutination test employing formalin-fixed tachyzoites as antigen. Raccoons originated in Florida (n = 24, collected in 1996), New Jersey (n = 25, collected in 1993), Pennsylvania (n = 25, collected in 1999), and Massachusetts (n = 25, collected in 1993 and 1994). Ten (10%) had antibodies to N. caninum; 9 had titers of 1:50, and 1 (1%) had a titer of 1:100. The present study indicates that raccoons have minimal exposure to N. caninum. The sera were also tested for agglutinating antibodies to Toxoplasma gondii and 46 (46%) were positive; 16 had titers of 1:50, 8 had titers of 1:100, and 22 had titers of > or = 1:500.     

Prevalence of antibodies to Neospora caninum, Sarcocystis neurona, and Toxoplasma gondii in wild horses from central Wyoming

Sarcocystis neurona, Neospora caninum, N. hughesi, and Toxoplasma gondii are 4 related coccidians considered to be associated with encephalomyelitis in horses. The source of infection for N. hughesi is unknown, whereas opossums, dogs, and cats are the definitive hosts for S. neurona, N. caninum, and T. gondii, respectively. Seroprevalence of these coccidians in 276 wild horses from central Wyoming outside the known range of the opossum (Didelphis virginiana) was determined. Antibodies to T. gondii were found only in 1 of 276 horses tested with the modified agglutination test using 1:25, 1:50, and 1:500 dilutions. Antibodies to N. caninum were found in 86 (31.1%) of the 276 horses tested with the Neospora agglutination test--the titers were 1:25 in 38 horses, 1:50 in 15, 1:100 in 9, 1:200 in 8, 1:400 in 4, 1:800 in 2, 1:1,600 in 2, 1:3,200 in 2, and 1:12,800 in 1. Antibodies to S. neurona were assessed with the serum immunoblot; of 276 horses tested, 18 had antibodies considered specific for S. neurona. Antibodies to S. neurona also were assessed with the S. neurona direct agglutination test (SAT). Thirty-nine of 265 horses tested had SAT antibodies--in titers of 1:50 in 26 horses and 1:100 in 13. The presence of S. neurona antibodies in horses in central Wyoming suggests that either there is cross-reactivity between S. neurona and some other infection or a definitive host other than opossum is the source of infection. In a retrospective study, S. neurona antibodies were not found by immunoblot in the sera of 243 horses from western Canada outside the range of D. virginiana.     

Prevalence of antibodies to Neospora caninum in white-tailed deer,Odocoileus virginianus,from the southeastern United States

Serum samples from 305 white-tailed deer (Odocoileus virginianus) from 14 states in the southeastern United States were examined for antibodies to Neospora caninum using a direct agglutination test. Positive agglutination titers were found in 145 (48%) of the white-tailed deer examined: 21 (7%) had titers of 1:25, 92 (30%) had titers of 1:50, and 32 (10%) had titers of > or = 1:500. These findings that antibodies to N. caninum are common in white-tailed deer support the concept that a sylvatic cycle might exist for this economically important parasite of domestic cattle.     

Isolation of Neospora caninum from the brain of a pregnant sheep.

Neospora caninum was isolated from the brain of a naturally infected pregnant sheep by inoculation of immunodeficient mice with a homogenate of the brain tissue. The ewe showed no clinical signs. Tachyzoites were observed in the tissues of the nu/nu mice injected with the brain tissue homogenate and the diagnosis was confirmed by immunohistochemical staining with anti-N. caninum antibodies and by detecting N. caninum-specific DNA by polymerase chain reaction.     

Characterization of the Oregon isolate of Neospora hughesi from a horse

Neospora hughesi was isolated in cell cultures inoculated with homogenate of spinal cord from a horse in Oregon. Tachyzoites of this Oregon isolate of N. hughesi were maintained continuously by cell culture passage and tachyzoites were infective to immunosuppressed mice. Gamma interferon gene knockout (KO) mice injected with tachyzoites developed fatal myocarditis and numerous tachyzoites were seen in lesions. Gerbils (Meriones unguiculatus) inoculated with tachyzoites developed antibodies (> or = 1:500) as indicated by the Neospora caninum agglutination test but did not develop clinical signs, and Neospora organisms were not demonstrable in their tissues. Tissue cysts were not found in gerbils, nude mice, KO mice, immunosuppressed outbred Swiss Webster mice, or BALB/c mice injected with the Oregon isolate of N. hughesi. Ultrastructurally, tachyzoites of the Oregon isolate from the myocardium of infected KO mice and from cell culture were similar to N. caninum tachyzoites. Western blot analysis using NcSAG1 and NcSRS2 polyclonal and monoclonal antibodies and characterization of the internal transcribed spacer 1 sequences from the equine isolates and different isolates of N. caninum from dogs and cattle indicated that the Oregon isolate of N. hughesi is distinct from N. caninum isolates from cattle and dogs.