Biomedical subjects

D E Hill

Publications and source records attributed to D E Hill.

At least 19 recordsLinked to original sources

PubMed2006

A Trichinella murrelli infection in a domestic dog in the United States.

Trichinella murrelli infection was diagnosed in a naturally infected Beagle bitch from VA, USA, where encapsulated larvae were found in histological sections of several skeletal muscles. A laboratory reared dog fed infected muscles resulted in viable muscle larvae that were subsequently infective to Swiss-Webster mice. Multiplex PCR using larvae from the experimentally infected dog demonstrated two distinct bands migrating at 127 bp and 316 bp which together are diagnostic for T. murrelli; the isolate was assigned the ISS code: ISS1608 by the International Trichinella Reference Centre. This is the first report of T. murrelli infection in a companion animal.

Animals↗

PubMed2006

Effects of time and temperature on the viability of Toxoplasma gondii tissue cysts in enhanced pork loin.

Enhanced or pumped pork products represent a significant proportion (40 to 50%) of the commercially available pork cuts available to consumers at the retail level. In a previous study, pork loins containing viable Toxoplasma gondii tissue cysts were pumped with solutions containing 2% sodium chloride or 1.4% or higher potassium or sodium lactate and stored at 4 degrees C for 7 days. This treatment prevented transmission of T. gondii to cats. In the present study, enhanced pork loins were stored for 0, 8, 16, 24, 32, or 40 h at 4 degrees C and then fed to T. gondii-seronegative cats to determine how quickly the loss of tissue cyst viability occurred. In a second experiment, pork loins collected from pigs experimentally infected with T. gondii were stored at temperatures found in retail meat cases and then fed to T. gondii-seronegative cats to determine the effect of typical meat case storage temperatures on tissue cyst viability. In both experiments, cat feces were examined for 14 days after the infected meat meal to assess oocyst shedding. The results indicate that solutions containing 2% sodium chloride or 1.4% potassium or sodium lactate are effective within 8 h of injection for killing T. gondii tissue cysts in pork loins and that storage at meat case temperatures at or below 0 degrees C (32 degrees F) for 7 days also killed T. gondii tissue cysts in pork loins.

Animals↗

PubMed2005

Trichinella nativa in a black bear from Plymouth, New Hampshire.

A suspected case of trichinellosis was identified in a single patient by the New Hampshire Public Health Laboratories in Concord, NH. The patient was thought to have become infected by consumption of muscle larvae (ML) in undercooked meat from a black bear killed in Plymouth, NH in October 2003 and stored frozen at -20 degrees C fro 4 months. In January 2004, a 600 g sample of the meat was thawed at 4 degrees C, digested in hydrochloric acid and pepsin, and larvae were collected by sedimentation. Intact, coiled, and motile ML were recovered (366 larvae per gram (l pg) of tissue), which were passed into mice and pigs. Multiplex PCR revealed a single 127 bp amplicon, indicative of Trichinella nativa. The Reproductive Capacity Index (RCI) for the T. nativa-Plymouth isolate in mice was 24.3. Worm burdens in the diaphragms of two 3-month-old pigs given 2,500 ML were 0.05 and 0.2l pg by 35 days post-inoculation, while 2.2 and 0.75 l pg were recovered from two 3-month-old pigs given 10,000 ML; no larvae were recovered from four 1-year-old pigs given 2,500 ML (n=2) or 10,000 ML (n=2). Viable larvae were also recovered from frozen black bear meat harvested at two additional locations, one in southern Ontario, Canada, and one in upstate New York, USA. Multiplex PCR using genomic DNA from these parasite samples demonstrated that both isolates were T. nativa. This is the first report of the freeze-resistant species, T. nativa, within the continental United States.

Animals↗

PubMed2005

Trichinella pseudospiralis from a wild pig in Texas.

In December 2001, the routine inspection of a wild boar intended for human consumption revealed the presence of Trichinella ssp. larvae. Biological, morphological and genetic analyses demonstrated the parasite to be Trichinella pseudospiralis. This is the second report of T. pseudospiralis in the United States and the first report of the parasite in a food animal species in the U.S.

Animals↗

PubMed2005

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.

Agglutination Tests↗

PubMed2005

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.

Animals↗

PubMed2005

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.

Animals↗

PubMed2005

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.

Agglutination Tests↗

PubMed2005

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.

Animals↗

PubMed2004

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.

Animals↗

PubMed2004

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.

Animals↗

PubMed2004

First isolation of Neospora caninum from an aborted bovine fetus in Spain.

Neospora caninum was isolated from the brain of a 6-mo-old aborted bovine fetus from Galicia, Spain. The fetal brain homogenate was inoculated intraperitoneally into cortisonized mice. The peritoneal exudate from the infected mice, along with mouse sarcoma cells (Tg180), was inoculated into a second group of mice, and parasites were harvested from the peritoneal exudate. The parasites were adapted to in vitro growth in Vero monolayers. The tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies on indirect fluorescent antibody test. The tachyzoites were lethal to interferon gamma gene knock out (KO) mice and could be identified immunohistochemically in the tissues. The identity of the parasite was also confirmed by polymerase chain reaction amplification of N. caninum-specific fragments. The sequences of the amplified gene 5 fragments (GenBank AY494944) were found to be identical to that of an Austrian isolate of N. caninum but not to that of NC-1. This is the first isolation of viable N. caninum from Spain.

Abortion, Veterinary↗

PubMed2004

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.

Animals↗

PubMed2004

Effect of commonly used enhancement solutions on the viability of Toxoplasma gondii tissue cysts in pork loin.

Retail meat cuts of pork are frequently enhanced with salt solutions to improve flavor and texture and to extend shelf life through reductions in microbial contamination. A study of the effect of commonly used meat enhancement solutions on the viability of Toxoplasma gondii tissue cysts was performed using tissues from experimentally infected mice and pigs. Brains of T. gondii-infected mice were injected to 110% of the original weight of the brain with solutions containing sodium chloride (1 and 2%), sodium diacetate (0.1 and 0.2%), sodium tripolyphosphate (0.25 and 0.5%), potassium lactate (1.4 and 1.96%), or sodium lactate (1.4, 1.5, and 2.0%) alone or in combination and stored at 4 degrees C for 7 days before feeding to T. gondii-seronegative cats. Loins were collected from pigs experimentally infected with T. gondii and injected as above and stored for 7, 28, or 45 days at 4 degrees C before feeding to T. gondii-seronegative cats. Cat feces were examined for 14 days to assess oocyst shedding. The present study demonstrated that injection of mouse brains or pork loins with solutions containing 2% sodium chloride or > or =1.4% potassium or sodium lactate, alone or in combination with other components, prevented transmission of T. gondii to cats.

Animals↗

PubMed2003

Detection of Hammondia heydorni-like organisms and their differentiation from Neospora caninum using random-amplified polymorphic DNA-polymerase chain reaction.

Neospora caninum and Hammondia heydorni are morphologically and phylogenetically related coccidians that are found in dogs. New diagnostic genetic loci, based on random-amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), were developed to aid in the detection of H. heydorni-like parasites and to discriminate them from N. caninum and other related coccidians of dogs. On the basis of the data obtained from 5 random decamers, H. heydorni (Manhattan-1) and N. caninum (NC1) were characterized by distinct banding patterns (similarity index = 0.068). High-stringency PCR assays were developed from the sequences of 2 cloned bands (GenBank BZ592549 and BZ592593), uniquely amplified from H. heydorni. Interestingly, using these primers, PCR amplification was achieved only from 2 of the 5 isolates presumed to represent H. heydorni. The same result was obtained from these 5 isolates using a recently described PCR assay directed to the H. heydorni internal transcribed spacer-1. It is concluded that H. heydorni and N. caninum are genetically distinct and that such tools may be useful for more detailed characterization of the diversity of related parasites occurring in dogs.

Animals↗

PubMed2002

Biological and genetic characterisation of Toxoplasma gondii isolates from chickens (Gallus domesticus) from São Paulo, Brazil: unexpected findings.

In spite of a wide host range and a world wide distribution, Toxoplasma gondii has a low genetic diversity. Most isolates of T. gondii can be grouped into two to three lineages. Type I strains are considered highly virulent in outbred laboratory mice, and have been isolated predominantly from clinical cases of human toxoplasmosis whereas types II and III strains are considered avirulent for mice. In the present study, 17 of 25 of the T. gondii isolates obtained from asymptomatic chickens from rural areas surrounding São Paulo, Brazil were type I. Antibodies to T. gondii were measured in 82 chicken sera by the modified agglutination test using whole formalin-preserved tachyzoites and mercaptoethanol and titres of 1:10 or more were found in 32 chickens. Twenty-two isolates of T. gondii were obtained by bioassay in mice inoculated with brains and hearts of 29 seropositive (> or =1:40) chickens and three isolates were obtained from the faeces of cats fed tissues from 52 chickens with no or low levels (<1:40) of antibodies. In total, 25 isolates of T. gondii were obtained by bioassay of 82 chicken tissues into mice and cats. All type I isolates killed all infected mice within 4 weeks whereas type III isolates were less virulent to mice. There were no type II strains. Tissue cysts were found in mice infected with all 25 isolates and all nine type I isolates produced oocysts. Infected chickens were from localities that were 18-200 km apart, indicating no common source for T. gondii isolates. This is the first report of isolation of predominantly type I strains of T. gondii from a food animal. Epidemiological implications of these findings are discussed.

Agglutination Tests↗

PubMed2002

Establishment of Besnoitia darlingi from opossums (Didelphis virginiana) in experimental intermediate and definitive hosts, propagation in cell culture, and description of ultrastructural and genetic characteristics.

Besnoitia darlingi from naturally infected opossums (Didelphis virginiana) from Mississippi, USA, was propagated experimentally in mice, cats, and cell culture and was characterised according to ultrastructural, genetic, and life-history characteristics. Cats fed tissue cysts from opossums shed oocysts with a prepatent period of nine or 11 days. Oocysts, bradyzoites, or tachyzoites were infective to outbred and interferon-gamma gene knockout mice. Tachyzoites were successfully cultivated and maintained in vitro in bovine monocytes and African green monkey cells and revived after an 18-month storage in liquid nitrogen. Schizonts were seen in the small intestinal lamina propria of cats fed experimentally-infected mouse tissues. These schizonts measured up to 45 x 25 microm and contained many merozoites. A few schizonts were present in mesenteric lymph nodes and livers of cats fed tissue cysts. Ultrastructurally, tachyzoites and bradyzoites of B. darlingi were similar to other species of Besnoitia. A close relationship to B. besnoiti and an even closer relationship to B. jellisoni was indicated for B. darlingi on the basis of the small subunit and ITS-1 portions of nuclear ribosomal DNA.

Animals↗

PubMed2002

Redescription of Neospora caninum and its differentiation from related coccidia.

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums.

Animals↗