Overexpression of kermit/dGIPC is associated with lethality in Drosophila melanogaster

Insertional mutagenesis is an important tool for functional genomics in Drosophila melanogaster. The insertion site in the KG00562 mutant fly line has been mapped to the CG8709 (herein named DmLpin) locus and to the 3’ of kermit (also called dGIPC). This mutant line presents a high lethality rate resulting from a gain of function. To obtain some insight into the biological role of the mutated locus, we have characterized the mutation and its relation to the high mortality of the KG00562 fly line. In this mutant, we did not detect one of the DmLpin transcripts, namely DmLpinK, but we did detect an unusual 2.3-kb mRNA (LpinK-w). Further investigation revealed that the LpinK-w transcript results from an aberrant splicing between the untranslated first exon of DmLpinK and the mini-white marker gene. Lack of DmLpinK or LpinK-w expression does not contribute to lethality, since heterozygous KG00562/Def7860 animals presented lethality rates comparable to those of the wild type. In contrast, the overexpression of kermit was associated with lethality of the KG00562 fly line. Significantly higher levels of kermit were detected in the Malpighian tubules of KG00562/+ flies that presented higher lethality rates than wild-type or KG00562/Def7860 animals, in which the lethality was rescued. In agreement with a recently reported study, our data support the hypothesis that misexpression of kermit/dGIPC could interfere with Drosophila development, with further investigations being needed in this direction.

Overexpression of kermit/dGIPC is associated with lethality in Drosophila melanogaster.

Insertional mutagenesis is an important tool for functional genomics in Drosophila melanogaster. The insertion site in the KG00562 mutant fly line has been mapped to the CG8709 (herein named DmLpin) locus and to the 3' of kermit (also called dGIPC). This mutant line presents a high lethality rate resulting from a gain of function. To obtain some insight into the biological role of the mutated locus, we have characterized the mutation and its relation to the high mortality of the KG00562 fly line. In this mutant, we did not detect one of the DmLpin transcripts, namely DmLpinK, but we did detect an unusual 2.3-kb mRNA (LpinK-w). Further investigation revealed that the LpinK-w transcript results from an aberrant splicing between the untranslated first exon of DmLpinK and the mini-white marker gene. Lack of DmLpinK or LpinK-w expression does not contribute to lethality, since heterozygous KG00562/Def7860 animals presented lethality rates comparable to those of the wild type. In contrast, the overexpression of kermit was associated with lethality of the KG00562 fly line. Significantly higher levels of kermit were detected in the Malpighian tubules of KG00562/+ flies that presented higher lethality rates than wild-type or KG00562/Def7860 animals, in which the lethality was rescued. In agreement with a recently reported study, our data support the hypothesis that misexpression of kermit/dGIPC could interfere with Drosophila development, with further investigations being needed in this direction.

Neosporosis in Beagle dogs: clinical signs, diagnosis, treatment, isolation and genetic characterization of Neospora caninum.

Clinical neosporosis was diagnosed in a litter of five pups born to a Beagle bitch from Virginia, USA. Four of the pups developed limb weakness starting at 4 weeks of age. The dogs were suspected to have neosporosis based on clinical signs and empirically treated with Clindamycin (75 mg, oral, twice daily, total 150 mg) starting at 9 weeks of age and the dosage was doubled at 13 weeks of age. Antibodies to Neospora caninum were detected in sera of the dam and pups when first tested serologically at the age of 4 months. The owner donated the pup with the worst clinical signs and the dam for research; both dogs were euthanized. Viable N. caninum was isolated in gamma interferon gene knock out (KO) mice and in cell culture from the pup killed at 137 days of age. Tissue cysts, but no tachyzoites, were found in histological sections of brain and muscles. The isolate was also identified as N. caninum by PCR and sequence analysis and designated NC-9. N. caninum was neither isolated by bioassay in KO mice nor found in histological sections of tissues of the bitch. Clinical signs in the remaining three pups improved considerably after a 6-month treatment with Clindamycin; N. caninum antibody titers were still persistent in these pups at 23 months of age. Results indicate that medication with Clindamycin can improve clinical condition but not eliminate N. caninum infection.

Diverse and atypical genotypes identified in Toxoplasma gondii from dogs in São Paulo, Brazil.

The prevalence of Toxoplasma gondii in 118 unwanted dogs from São Paulo City, São Paulo State, Brazil, was determined. Antibodies to T. gondii were assayed by the modified agglutination test and found in 42 (35.8%) dogs, with titers of 1:20 in 10, 1:40 in 6, 1:80 in 5, 1:160 in 5, 1:320 in 6, 1:640 in 7, and 1:1,280 or higher in 3. Hearts and brains of 36 seropositive dogs were bioassayed in mice, or cats, or both. Tissues from 20 seropositive dogs were fed to 20 T. gondii-free cats. Feces of cats were examined for oocysts. Toxoplasma gondii was isolated from 15 dogs by a bioassay in mice, from the brain alone of 1, from the heart alone of 4, and from both brains and hearts of 10. All infected mice from 5 of 15 isolates died of toxoplasmosis during primary infection. Four additional isolates were obtained by bioassay in cats. Genotyping of these 19 T. gondii isolates using polymorphisms at 10 nuclear markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and a new SAG2 (an apicoplast marker Apico) revealed 12 genotypes. One isolate had Type III alleles at all 11 loci, and the remaining 18 isolates contained a combination of different alleles and were divided into 11 genotypes. The absence of Type II in Brazil was confirmed. The result supports previous findings that T. gondii population genetics is highly diverse in Brazil.

Characterization of Toxoplasma gondii isolates in free-range chickens from Amazon, Brazil.

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 50 free-range chickens (Gallus domesticus) from Amazon, Brazil, was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT) and found in 33 (66%) chickens with titers of 1:5 in 3, 1:10 in 2, 1:20 in 1, 1:40 in 1, 1:80 in 2, 1:160 in 5, 1:200 in 9, 1:400 in 5, 1:800 in 2, 1:1,600 in 2, and 1:3,200 or higher in 1. Hearts and brains of 33 seropositive chickens were bioassayed individually in mice. Tissues from 17 seronegative chickens were pooled and fed to 2 T. gondii-free cats. Feces of cats were examined for oocysts, but none was found. Toxoplasma gondii was isolated from 24 chickens with MAT titers of 1:5 or higher. Genotyping of these 24 T. gondii isolates by polymorphisms at the SAG2 locus indicated that 14 were type I, and 10 were type III; the absence of type II strains from Brazil was confirmed. Fifty percent of the infected mice died of toxoplasmosis, irrespective of the genotype.

Characterization of Toxoplasma gondii isolates in free-range chickens from Portugal.

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 225 free-range chickens (Gallus domesticus) from Portugal was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT) and found in 61 chickens with titers of 1:5 in 8, 1:10 in 6, 1:20 in 3, 1:40 in 23, 1:80 in 5, 1:160 in 4, 1:320 in 8, and 1:640 or higher in 4. Hearts, leg muscles, and brains of 15 seropositive (MAT 1:10 or higher) chickens were bioassayed individually in mice. Tissue from 38 chickens with titers of 1:5 or less were pooled and fed to a T. gondii-free cat. Feces of the cat were examined for oocysts, but none was found. Toxoplasma gondii was isolated from 16 of 19 chickens with MAT titers of 1:10 or higher. Genotyping of 12 of these 16 isolates with polymorphisms at the SAG2 locus indicated that 4 were type III, and 8 were type II. None of the isolates was lethal for mice. Phenotypically, T. gondii isolates from chickens from Portugal were different from those of T. gondii isolates from chickens from Brazil.

Differential detection of Hammondia hammondi from Toxoplasma gondii using polymerase chain reaction.

Hammondia hammondi and Toxoplasma gondii are two related coccidian parasites, with cats as definitive hosts and warm-blooded animals as intermediate hosts. It is difficult to differentiate them by morphological and serological parameters. In the present study, primers were designed to specifically amplify the ITS-1 region of H. hammondi to differentiate it from T. gondii. Attempts were made to detect the presence of H. hammondi DNA in the tissues of mice infected with H. hammondi alone, as well as from mixed infections with T. gondii, using the newly designed primers. The de novo primers effectively amplified the H. hammondi-specific target fragment from all samples containing H. hammondi, including those with concomitant T. gondii infection. Further, the primers did not amplify any fragment from the related parasites like T. gondii, Neospora caninum and Hammondia heydorni. The new primers provide simple and efficient means to differentially diagnose H. hammondi from T. gondii even in samples containing both parasites, thus obviating the need for other labourious techniques like mouse bioassay and in vitro cultivation.

Isolation, tissue distribution, and molecular characterization of Toxoplasma gondii from chickens in Grenada, West Indies.

The prevalence of Toxoplasma gondii in free-range chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 102 free-range chickens (Gallus domesticus) from Grenada was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT). Antibodies were found in 53 (52%) chickens with titers of 1:5 in 6, 1:10 in 4, 1:20 in 4, 1:40 in 4, 1:80 in 15, 1:160 in 9, 1: 320 in 5, 1:640 in 4, and 1:1,280 or greater in 2. Hearts, pectoral muscles, and brains of 43 seropositive chickens with MAT titers of 1:20 or greater were bioassayed individually in mice. Tissues of each of 10 chickens with titers of 1:5 and 1:10 were pooled and bioassayed in mice. Tissues from the remaining 49 seronegative chickens were pooled and fed to 4 T. gondii-free cats. Feces of cats were examined for oocysts; they did not shed oocysts. T. gondii was isolated from 35 of 43 chickens with MAT titers of 1:20 or greater; from the hearts, brains, and pectoral muscles of 2, hearts and brains of 20, from the hearts alone of 11, and brains alone of 2. T. gondii was isolated from 1 of 10 chickens with titers of 1:5 or 1:10. All 36 T. gondii isolates were avirulent for mice. Genotyping of these 36 isolates using polymorphisms at the SAG2 locus indicated that 29 were Type III, 5 were Type I, 1 was Type II, and 1 had both Type I and Type III. Genetically, the isolates from Grenada were different from those from the United States; Type II was the predominant type from the United States. Phenotypically, all isolates from Grenada were avirulent for mice, whereas those from Brazil were mouse-virulent. This is the first report of isolation of T. gondii from chickens from Grenada, West Indies.

Genetic and biologic characteristics of Toxoplasma gondii isolates in free-range chickens from Colombia, South America.

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 77 free-range chickens (Gallus domesticus) from Colombia, South America was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT), and found in 32 (44.4%) of 72 chickens with titers of 1:5 in 4, 1:10 in 3, 1:20 in 1, 1:40 in 1, 1:80 in 8, 1:160 in 8, 1:320 in 3, and 1:640 or higher in 4. Hearts and brains of 31 seropositive chickens were pooled and bioassayed in mice. Tissues from 32 (16+16) seronegative chickens were pooled and fed to two, T. gondii-free cats, and tissues from nine chickens without matching sera were fed to one T. gondii-free cat. Feces of cats were examined for oocysts. T. gondii oocysts were excreted by a cat that was fed tissues of 16 seronegative chickens. T. gondii was isolated by bioassay in mice from 23 chickens with MAT titers of 1:20 or higher. All infected mice from 16 of the 23 isolates died of toxoplasmosis. Overall, 82 (81.1%) of 101 mice that became infected after inoculation with chicken tissues died of toxoplasmosis. Genotyping of these 24 isolates using polymorphisms at the SAG2 locus indicated that seven T. gondii isolates were Type I, 17 were Type III, and none was Type II. Phenotypically, T. gondii isolates from chickens from Colombia were similar to isolates from Brazil but different from the isolates from North America; most isolates from chickens from Brazil and Colombia were lethal for mice whereas isolates from North America did not kill inoculated mice. Genetically, none of the T. gondii isolates from Colombia and Brazil was SAG2 Type II, whereas most isolates from chickens from North America were Type II. This is the first report of genetic characterization of T. gondii isolates from Colombia, South America.

Genotyping and detection of multiple infections of Toxoplasma gondii using Pyrosequencing.

A Pyrosequencing assay, based on SAG2 gene polymorphisms, was designed for genotyping and detection of multiple infections of Toxoplasma gondii. The assay was tested on samples spiked with DNA from single and multiple genotypes of T. gondii and also on a DNA sample from the brain of a rat with multiple infections. To evaluate the comparative efficacy of the assay, identical samples were also analysed by PCR-restriction fragment length polymorphism (RFLP) and dideoxy sequencing. The Pyrosequencing assay was found to be superior to the two conventional techniques. Genotyping and detection of multiple alleles were possible after a single PCR assay in duplex format, from both the spiked and direct samples. The simplex PCR assay enabled accurate quantification of the different alleles in the mix. In comparison, PCR-RFLP and dideoxy sequencing were neither able to unequivocally detect multiple genotype infections, nor quantify the relative concentrations of the alleles. We conclude that Pyrosequencing offers a simple, rapid and efficient means for diagnosis and genotyping of T. gondii, as well as detection and quantification of multiple genotype infections of T. gondii.

Genetic and biologic characteristics of Toxoplasma gondii infections in free-range chickens from Austria.

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in free-range chickens (Gallus domesticus) from 11 Bio-farms in Austria was determined. Antibodies to T. gondii assayed by the modified agglutination test (MAT) were found in 302 of 830 (36.3%) chickens with titers of 1:10 in 50, 1:20 in 69, 1:40 in 53, 1:80 in 40, 1:160 or higher in 90. Hearts of 218 chickens with MAT titers of 10 or higher were bioassayed individually in mice. Tissues from 1183 chickens were pooled and fed to 15, T. gondii-free cats. Feces of the cats were examined for oocysts; 11 cats shed T. gondii oocysts. T. gondii was isolated from 56 chickens by bioassay in mice. Thus, there were 67 isolates of T. gondii from these chickens. Genotyping of these 67 isolates using the SAG2 locus indicated that all 33 were Type II. Phenotypically and genetically these isolates were different from T. gondii isolates from Brazil. None of the isolates was virulent for mice. This is the first report of isolation of T. gondii from chickens from Austria.

Redescription of Besnoitia bennetti (Protozoa: Apicomplexa) from the donkey (Equus asinus).

Besnoitia bennetti tissue cysts were found in four naturally-infected donkeys (Equus asinus) from the USA. Infectivity of its bradyzoites and tachyzoites to animals and cell culture was studied. The bradyzoites were not infectious to out-bred Swiss Webster mice, rabbits or gerbils. When fed tissue cysts, cats did not excrete oocysts. However, the parasite was infectious to interferon-gamma gene knock out mice. The parasite from tissues of two donkeys was grown successfully in bovine monocyte monolayers for the first time. Non-dividing, uninucleate tachyzoites were approximately 6 x 1.5 microm in size. Longitudinally-cut bradyzoites in tissue sections measured 8.7 x 1.9 microm. Ultrastructurally, tachyzoites and bradyzoites were similar to those in other Besnoitia species, and in particular to parasites described from cattle (Besnoitia besnoiti) and reindeer (Besnoitia tarandi), in that their bradyzoites lacked enigmatic bodies. Based on comparative analysis of three portions of nuclear ribosomal DNA (the small and large subunits and the first internal transcribed spacer) B. bennetti was found to be more closely related to the other congeners described from ungulates. The parasite was formally redescribed and specimens deposited in the US National Parasite Collections.

Isolation of Neospora caninum from naturally infected white-tailed deer (Odocoileus virginianus).

Attempts were made to isolate Neospora caninum from naturally infected white-tailed deer (Odocoileus virginianus). A total of 110 deer killed during the 2003 hunting season in Virginia region were used for the isolation of N. caninum. Of these, brains from 28 deer that had NAT titer of 1:200 were inoculated into interferon-gamma gene knock out (KO) mice. N. caninum was isolated from the tissues of three deer and all three isolates were mildly virulent to KO mice. Only one of the isolates could be adapted to in vitro growth. Protozoa in the tissues of KO mice reacted with N. caninum-specific polyclonal antibodies and N. caninum DNA was demonstrated in infected tissues by PCR assays; sequences of portions of the ITS-1 and gene 5 loci were identical to those in the public database. This is the first record of in vitro isolation of N. caninum from white-tailed deer and lends credence to the white-tailed deer as an intermediate host for this parasite.

First biologic and genetic characterization of Toxoplasma gondii isolates from chickens from Africa (Democratic Republic of Congo, Mali, Burkina Faso, and Kenya).

The prevalence of Toxoplasma gondii in free-ranging chickens (Gallus domesticus) is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. In the present study, prevalence of T. gondii in chickens from Democratic Republic of Congo, Mali, Burkina Faso, and Kenya is reported. The prevalence of T. gondii antibodies in sera of 50 free-range chickens from Congo was 50% based on the modified agglutination test (MAT); antibody titers were 1:5 in 7, 1:10 in 7, 1:20 in 6, 1:40 in 1, and 1:160 or more in 4 chickens. Hearts, pectoral muscles, and brains of 11 chickens with titers of 1:20 or more were bioassayed individually in mice; T. gondii was isolated from 9, from the hearts of 9, brains of 3, and muscles of 3 chickens. Tissues of each of the 14 chickens with titers of 1:5 or 1:10 were pooled and bioassayed in mice; T. gondii was isolated from 1 chicken with a titer of 1:10. Tissues from the remaining 25 seronegative chickens were pooled and fed to 1 T. gondii-free cat. Feces of the cat were examined for oocysts, but none was seen. The results indicate that T. gondii localizes in the hearts more often than in other tissues of naturally infected chickens. Genotyping of these 10 isolates using the SAG2 locus indicated that 8 were isolates were type III, 1 was type II, and 1 was type I. Two isolates (1 type I and 1 type III) were virulent for mice. Toxoplasma gondii was isolated by mouse bioassay from a pool of brains and hearts of 5 of 48 chickens from Mali and 1 of 40 chickens from Burkina Faso; all 6 isolates were avirulent for mice. Genetically, 4 isolates were type III and 2 were type II. Sera were not available from chickens from Mali and Burkina Faso. Toxoplasma gondii antibodies (MAT 100 or more) were found in 4 of 30 chickens from Kenya, and T. gondii was isolated from the brain of 1 of 4 seropositive chickens; this strain was avirulent for mice and was type II. This is the first report on isolation and genotyping of T. gondii from any source from these 4 countries in Africa.

Prevalence of viable Toxoplasma gondii in beef, chicken, and pork from retail meat stores in the United States: risk assessment to consumers.

The prevalence of viable Toxoplasma gondii was determined in 6,282 samples (2,094 each of beef, chicken, and pork) obtained from 698 retail meat stores from 28 major geographic areas of the United States. Each sample consisted of a minimum of 1 kg of meat purchased from the retail meat case. To detect viable T. gondii, meat samples were fed to T. gondii-free cats and feces of cats were examined for oocyst shedding. Initially, 100 g of meat from 6 individual samples of a given species were pooled (total, 600 g), fed to a cat over a period of 3 days, and feces were examined for oocysts for 14 days; the remaining meat samples were stored at 4 C for 14 days (until results of the initial cat fecal examination were known). When a cat fed pooled samples had shed oocysts, 6 individual meat samples from each pool were bioassayed for T. gondii in cats and mice. Toxoplasma gondii isolates were then genetically characterized using the SAG2 locus and 5 hypervariable microsatellite loci. In all, 7 cats fed pooled pork samples shed oocysts. Toxoplasma gondii oocysts were detected microscopically in the feces of 2 of the cats; 1 isolate was Type II and the second was Type III. Analyzed individually, T. gondii was detected by bioassay in 3 of the 12 associated samples with genetic data indicating T. gondii isolates present in 2. The remaining 5 pooled pork samples had so few oocysts that they were not initially detected by microscopic examination, but rather by mouse bioassay of cat feces. Two were Type I, 1 was Type II, and 2 were Type III. None of the cats fed chicken or beef samples shed oocysts. Overall, the prevalence of viable T. gondii in retail meat was very low. Nevertheless, consumers, especially pregnant women, should be aware that they can acquire T. gondii infection from ingestion of undercooked meat, and in particular, pork. Cooking meat to an internal temperature of 66 C kills T. gondii.

Biologic and molecular characteristics of Toxoplasma gondii isolates from striped skunk (Mephitis mephitis), Canada goose (Branta canadensis), black-winged lory (Eos cyanogenia), and cats (Felis catus).

Toxoplasma gondii isolates can be grouped into 3 genetic lineages. Type I isolates are considered virulent to outbred mice, whereas Type II and III isolates are not. In the present report, viable T. gondii was isolated for the first time from striped skunk (Mephitis mephitis), Canada goose (Branta canadensis), and black-winged lory (Eos cyanogenia). For the isolation of T. gondii, tissues were bioassayed in mice, and genotyping was based on the SAG2 locus. Toxoplasma gondii was isolated from 3 of 6 skunks, 1 of 4 Canada geese, and 2 of 2 feral cats (Felis catus) from Mississippi. All donor animals were asymptomatic. Viable T. gondii was also isolated from 5 of 5 lories that had died of acute toxoplasmosis in an aviary in South Carolina. Genotypes of T. gondii isolates were Type III (all skunks, lories, and the goose) and Type II (both cats). All 5 Type III isolates from birds and 2 of the 3 isolates from skunks were mouse virulent.

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.

Redescription of Besnoitia tarandi (Protozoa: Apicomplexa) from the reindeer (Rangifer tarandus).

Besnoitia tarandi tissue cysts were found in naturally-infected reindeer (Rangifer tarandus) from Finland. Infectivity of its tissue cysts, bradyzoites, and tachyzoites to animals and cell culture was studied. The bradyzoites and tissue cysts were not infectious to out-bred mice, rabbits or gerbils. When fed tissue cysts, neither cats nor dogs excreted oocysts. However, the parasite was lethal to interferon-gamma gene knock out mice irrespective of the route of inoculation. The parasite was grown successfully in African Green Monkey cells from tissues of two reindeer for the first time. Non-dividing, uninucleate tachyzoites from smears from cell cultures were 5.6 x 1.4 microm (4.5-7.4 x 1.0-1.9, n=50) in size. Longitudinally-cut bradyzoites in tissue sections measured 7.4 x 1.3 microm (6.5-7.8 x 1.0-1.6, n=30). Ultrastructurally, tachyzoites and bradyzoites were similar to those in other Besnoitia species, and in particular to parasites described from cattle (Besnoitia besnoiti) and equids (Besnoitia bennetti) in that their bradyzoites lacked enigmatic bodies. Based on comparative analysis of three portions of nuclear ribosomal DNA (the small and large subunits and the first internal transcribed spacer) B. tarandi was found to be more closely related to the other congeners described from ungulates. The parasite was formally redescribed and specimens deposited in the US National Parasite Collection.

Biologic, morphologic, and molecular characterisation of Neospora caninum isolates from littermate dogs.

Isolation and biologic and molecular attributes of Neospora caninum from three littermate dogs are described. Tissue cysts were confined to the brain and striated muscles. N. caninum was isolated (isolates NC-6, NC-7, and NC-8) in rodents and cell culture that had been inoculated with brain tissue from the dogs. Schizont-like stages reactive with N. caninum antibodies were seen in cell cultures seeded with bradyzoites released from Percoll-isolated N. caninum tissue cysts from the brain of one dog. Tissue cysts were infective orally to mice and gerbils, but not to cats and dogs. The isolates were also identified as N. caninum by PCR and sequence analysis.

Shedding of Neospora caninum oocysts by dogs fed tissues from naturally infected water buffaloes (Bubalus bubalis) from Brazil.

Attempts were made to isolate Neospora caninum from naturally infected water buffaloes (Bubalus bubalis) from Brazil. Brains from six buffaloes with indirect fluorescent antibodies (>1:100) to N. caninum were used to isolate the parasite by bioassay in dogs and gerbils followed by in vitro culture. Shedding of Neospora-like oocysts was noticed in dogs fed brains from three buffaloes (isolate designation NcBrBuf-1, 2 and 4). Two more isolates (NcBrBuf-3 and 5) were obtained by in vitro culture of the brains of gerbils previously infected with brains of two other buffaloes. The identity of the isolates was confirmed by biological and molecular methods. The isolates were found to be non-pathogenic to gerbils. All five isolates amplified the gene 5 amplicons using Neospora-specific PCR assay. The sequences of gene 5 fragments and the common toxoplasmatiid ITS-1 fragments were analyzed. The dynamics of oocyst production in the dogs indicate that water buffaloes are natural intermediate hosts for N. caninum. This is the first report of isolation of N. caninum from water buffaloes.