Isolation, Structure Elucidation and in Vitro Anticancer Activity of Phytochemical Constituents of Goniothalamus wynaadensis Bedd. and Identification of α-Tubulin as a Putative Molecular Target by in Silico Study.

The phytochemical analysis of ethyl acetate and methanol extract of Goniothalamus wynaadensis Bedd. leaves led to an isolation of eight (1-8) known molecules, among them seven (2-8) isolated for the first time from this species, which includes (+)-goniothalamin oxide (2), goniodiol-7-monoacetate (3), goniodiol-8-monoacetate (4), goniodiol (5), (+)-8-epi-9-deoxygoniopypyrone (6) etc. The phytochemical modification by acetylation of 3 and 4 gave goniodiol diacetate (9) with absolute configuration (6R, 7R, 8R) confirmed by single crystal X-ray diffraction. Compounds 3-9 were cytotoxic against breast, ovarian, prostate and colon cancer cell lines with IC50 <10 µM. Cell cycle analysis and Annexin-V assay on MDA-MB-231 cell using goniodiol-7-monoacetate (3) exhibited apoptotic response as well as necrotic response and showed cell proliferation arrest at G2/M phase. An in silico target identification for these molecules was carried out with an α-tubulin protein target by covalent docking. To gain an in-depth understanding and identify the stability of these protein-ligand complexes on thermodynamic energy levels, further assessment of the isolated molecules binding to the Cys-316 of α-tubulin was performed based on reaction energetic analysis via DFT studies which hinted the isolated molecules may be α-tubulin inhibitors similar to Pironetin. Molecular dynamics reiterated the observations.

Synthesis, DFT analysis and in-vitro anti-cancer study of novel fused bicyclic pyranone isoxazoline derivatives of Goniodiol-diacetate-a natural product derivative.

Natural products, natural product-inspired molecules and natural product derivatives have contributed around 79% to the new chemotherapies against the most complex, deadly disease, cancer. In this study, a series of novel isoxazoline derivatives of Goniodiol diacetate (fused bicyclic pyranone isoxazoline derivatives)- a natural product derivative, were synthesized with quantitative yield as a single regioisomer by 1,3 - dipolar cycloaddition reaction with different aldoximes. The regiospecific product formed was confirmed by NOESY study and single-crystal X-ray diffraction. The regiospecificity of the product formation was further explained by coefficients of selected atomic orbitals in frontier molecular orbitals and natural population analysis (NPA in eV) of dipolarophile and dipole by density functional theory studies. All the derivatives have demonstrated anti-cancer activity selectively in human breast cancer (MDA-MB-231), ovarian cancer (SKOV3), prostate cancer (PC-3) and colon cancer (HCT-15) cell lines with EC50 < 10 µM. Additionally, Annexin V/PI assay and cell cycle analysis on selected potent compound 3 f exhibited tuned apoptotic response & necrosis compared to standard Vincristine and showed cell growth arrest at the S phase.

1,6-Addition of 1,2,3-NH triazoles to para-quinone methides: Facile access to highly selective N1 and N2 substituted triazoles.

The regioselective syntheses of N1 and N2 substituted triazoles through a 1,6-addition reaction of 1,2,3-NH triazoles to p-quinone methide were achieved under mild reaction conditions. The present reactions showed superior results in terms of selectivity, mild reaction conditions, short reaction time and broad substrate scope with good functional-group compatibility. Considering the high synthetic value of N1- and N2-substituted compounds and p-QM related research, the present strategy will greatly benefit researchers in various fields.

Expert Group Meeting on Aphasia: A Report.

A multidisciplinary team of experts took stock of the current state of affairs about many aspects of aphasia in India, including community burden, diagnostic assessment, therapy, rehabilitation, research, education, and advocacy. The broad spectrum of aphasiology was matched by the types of participants ranging from neurologists, speech-language pathologists, clinical psychologists, linguists, to experts in neuroimaging and computer sciences. Threadbare discussion in 16 sessions over 3 days leads to the identification of pressing problems and possible solutions. Many action plans have been envisaged and recommendations made. A few examples with high priority are community-based and hospital-based study incidence and prevalence of aphasia, development of test batteries for the assessment of many components of speech and communication in Indian languages which are validated on rigorous psychometric, and linguistic criteria, national registry for aphasia, educational modules about aphasia for different target groups, resources for advocacy and its training, a bank of research questions and outlines of research protocols for young professionals to pursue. The expert group will continue to oversee execution of some of the actionable plans in short and long term.

Outbreak of immature paramphistomosis in Nellore Jodipi sheep.

An outbreak of immature paramphistomosis in Nellore Jodipi sheep was recorded in Nellore district of Andhra Pradesh in 158 sheep during the months of April-June-2012. The condition is diagnosed as immature paramphistomosis on the basis of symptoms, dung sample examination and necropsy findings. The infected sheep exhibited symptoms like persistent foetid, blackish diarrhoea, sub maxillary edema, dehydration and death. Necropsy findings include hemorrhagic thickening and necrosis of proximal duodenal mucosa, ascites, gelatinization of subcutaneous and renal fat. Duodenal scrapings revealed large number of immature paramphistomes under low power microscopy. Recovery of ailing sheep was observed after oral treatment of niclosamide @ of 100 mg/kg bwt along with supportive therapy.

Comparison of the salivary and the serum nitric oxide levels in chronic and aggressive periodontitis: a biochemical study.

BACKGROUND AND OBJECTIVES: Nitric oxide (NO) is a ubiquitous intercellular messenger molecule with important cardiovascular, neurological, and immune functions. In addition, it has been postulated that the pharmacological inhibition of NO or its actions may be therapeutically valuable in the disease management. The levels of nitric oxide may provide clues about the severity and the state of the underlying disease process. It could be an inflammatory biomarker that may enable clinicians to direct the environmentally based prevention or treatment programmes and to establish whether NO plays a role in the pathogenesis of periodontitis or not. Hence, the aim of the present study was to evaluate the salivary and the serum levels of NO in generalized chronic and aggressive periodontitis. The Study Design: Unstimulated whole saliva and serum samples were collected from a total of 60 subjects who were in the age group of 18-45 years, who participated in this study. They were divided into three equal groups with 20 subjects in each group; group A (healthy controls), group B (chronic periodontitis) and group C (aggressive periodontitis). The clinical parameters were assessed, based on the oral hygiene index simplified (OHI-S), the gingival index (GI), the probing pocket depth and the clinical attachment loss (CAL). A biochemical analysis was performed to evaluate and compare the salivary and the serum nitric oxide levels of the above groups. Statistical Analysis and Results: The statistical comparisons were done under the Griess Reaction. There were statistically significant salivary and serum levels of NO in the groups of periodontitis (group B and C) as compared to those in the healthy controls (group A). A significant positive correlation was found between the values of the salivary and the serum NO levels in chronic and aggressive periodontitis. CONCLUSION: Nitric oxide is a potent modulator of the inflammatory disease processes and under pathological conditions, NO has damaging effects. As there is a paucity in the studies which have compared chronic and aggressive periodontitis, this study paved an interest for combining the serum and the salivary analysis in comparing the levels of nitric oxide in chronic and aggressive periodontitis.

Sarcocystis neurona infection in gamma interferon gene knockout (KO) mice: comparative infectivity of sporocysts in two strains of KO mice, effect of trypsin digestion on merozoite viability, and infectivity of bradyzoites to KO mice and cell culture.

The protozoan Sarcocystis neurona is the primary cause of Equine Protozoal Myeloencephalitis (EPM). EPM or EPM-like illness has been reported in horses, sea otters, and several other mammals. The gamma interferon gene knockout (KO) mouse is often used as a model to study biology and discovery of new therapies against S. neurona because it is difficult to induce clinical EPM in other hosts, including horses. In the present study, infectivity of three life cycle stages (merozoites, bradyzoites, sporozoites) to KO mice and cell culture was studied. Two strains of KO mice (C57-black, and BALB/c-derived, referred here as black or white) were inoculated orally graded doses of S. neurona sporocysts; 12 sporocysts were infective to both strains of mice and all infected mice died or became ill within 70 days post-inoculation. Although there was no difference in infectivity of sporocysts to the two strains of KO mice, the disease was more severe in black mice. S. neurona bradyzoites were not infectious to KO mice and cell culture. S. neurona merozoites survived 120 min incubation in 0.25% trypsin, indicating that trypsin digestion can be used to recover S. neurona from tissues of acutely infected animals.

Plastinated fetus: 3D CT scan (VRT) evaluation.

OBJECTIVE: The intent of this study was to evaluate the effect of plastination on the morphology and structure of stored organs, to find out how much accuracy a plastinated specimen has, and to look into the changes that occurred because of plastination. MATERIALS AND METHODS: A human fetus of gestational age 24 weeks was plastinated, and 3D CT scan evaluation of the fetus was done. RESULTS: The results showed normal, well-defined, clearly identifiable organs, with no alteration in morphology and structure of organs. CONCLUSION: In our opinion, plastinated specimens are better way of visualization of morphology and structure of stored organs, which is a useful tool for teaching as well as for research purposes.

Multi-locus DNA sequencing of Toxoplasma gondii isolated from Brazilian pigs identifies genetically divergent strains.

Five Toxoplasma gondii isolates (TgPgBr1-5) were isolated from hearts and brains of pigs freshly purchased at the market of Campos dos Goytacazes, Northern Rio de Janeiro State, Brazil. Four of the five isolates were highly pathogenic in mice. Four genotypes were identified. Multi-locus PCR-DNA sequencing showed that each strain possessed a unique combination of archetypal and novel alleles not previously described in South America. The data suggest that different strains circulate in pigs destined for human consumption from those previously isolated from cats and chickens in Brazil. Further, multi-locus PCR-RFLP analyses failed to accurately genotype the Brazilian isolates due to the high presence of atypical alleles. This is the first report of multi-locus DNA sequencing of T. gondii isolates in pigs from Brazil.

Two new species of Sarcocystis (Apicomplexa: Sarcocystidae) infecting the wolverine (Gulo gulo) from Nunavut, Canada.

Infection with Sarcocystis species is common in many species of animals, but it has not yet been reported in wolverines (Gulo gulo). Histological sections of tongues from 41 wolverines in the Kitikmeot Region, Nunavut, Canada, were examined for sarcocysts. Sarcocysts were found in 33 (80.4%) wolverines. Two structurally distinct types of sarcocysts were found. Type A sarcocysts were thin (<1 µm thick) walled. Ultrastructurally, the parasitophorous vacuolar membrane (Pvm) had minute undulations, but it lacked villar protrusions and was not invaginated into the granular layer. The bradyzoites were slender, about 5 × 1 µm in size. Structurally, these sarcocysts were distinct from known species of Sarcocystis and possessed a novel 18S and ITS-1 sequence, sharing 98% and 78% sequence similarity with Sarcocystis canis . A new species name, Sarcocystis kalvikus, is proposed for type A sarcocysts. In contrast, type B sarcocysts had relatively thicker (about 2 µm) cyst walls and larger bradyzoites, each about 10 × 2-3 µm. Ultrastructurally, the Pvm on the sarcocyst wall had villar protrusions that were either mushroom-like or sloping. Molecular analysis identified a unique 18S and ITS-1 sequence that placed them in a clade within the Sarcocystidae. Based on histology, TEM, and genetic data, the new name, Sarcocystis kitikmeotensis, is proposed. Sarcocystis kalvikus was found in 14 (34.1%), S. kitikmeotensis was found in 7 (17%), and both species were found in 12 (29.2%) of 41 wolverines.

Non-archetypal Type II-like and atypical strains of Toxoplasma gondii infecting marsupials of Australia.

Australia is geographically isolated and possesses a remarkable diversity of wildlife species. Marsupials are highly susceptible to infection with the cosmopolitan parasite Toxoplasma gondii. Of 46 marsupials screened for T. gondii by multilocus PCR-DNA sequencing at polymorphic genes (B1, SAG3, GRA6, GRA7), 12 were PCR-positive; the majority (67%; 9/12) were infected by non-archetypal Type II-like or atypical strains. Six novel alleles were detected at B1, indicating greater diversity of genotypes than previously envisaged. Two isolates lethal to marsupials, were avirulent to mice. The data support the conclusion that Australia's isolation may have favoured the persistence of non-archetypal ancestral genotypes.

Molecular studies on Abeta(42) induced genomic instability in aged rabbit brain.

DNA stability and conformation are important in the life cycle of an organism. The DNA instability is postulated to be one of the risk factors for neuronal death in neurodegenerative disorders. Among all other risk factors, amyloid is one of the most important risk factor for neurodegeneration. Abeta(42) is implicated in Alzheimer's disease (AD). Studies from our lab and elsewhere have shown that Abeta(42) could cause DNA damage and alter DNA stability in vitro and there are no mechanistic studies to understand Abeta induced genomic instability under in vivo condition. The present study aims to characterize Abeta(42) induced DNA instability and also to map the changes in DNA conformation in vivo and its correlation to brain structural changes. The aged (4yr) New Zealand rabbits are intracisternally injected with Abeta(42) and are sacrificed after 25 days, when the rabbits developed AD like behavior. Genomic DNA is isolated from frontal cortex (FC), hippocampus (H) and midbrain (M) regions of Abeta(42) injected and control rabbit brain. The DNA stability parameters are analyzed. And the results showed that DNA is damaged in FC and H; where as in M, DNA is in condensed state. The DNA conformation study evidenced the presence of C-, pi- and psi-type DNA in conformations in FC, H and M of Abeta injected rabbit brain regions respectively. But in control rabbit brain, DNA is in B-conformation in all the brain regions studied. Magnetic resonance imaging (MRI) studies showed no significant changes in brain structure between control and Abeta(42) injected aged rabbit brain regions. The mechanism of Abeta(42) induced neurodegeneration through genomic instability is discussed in detail.

Implementation of an electromagnetic imaging system to facilitate nasogastric and post-pyloric feeding tube placement in patients with and without critical illness.

BACKGROUND: Artificial nutrition support is required to optimise nutritional status in many patients. Traditional methods of placing feeding tubes may incur clinical risk and financial costs. A technique facilitating placement of nasogastric and post-pyloric tubes via electromagnetic visual guidance may reduce the need for X-ray exposure, endoscopy time and the use of parenteral nutrition. The present study aimed to audit use of such a system at initial implementation in patients within an acute NHS Trust. METHODS: A retrospective review was undertaken of dietetic and medical records for the first 14 months of using the Cortrak system. Data were collected on referral origin, preparation of the patient prior to insertion, placement success rates and need for X-ray. Cost analysis was also performed. RESULTS: Referrals were received from primary consultants or consultant intensivists, often on the advice of the dietitian. Fifty-nine percent of patients received prokinetic therapy at the time of placement. Thirty-nine tube placements were attempted. Sixty-nine percent of referrals for post-pyloric tube placement resulted in successful placement. X-ray films were requested for 22% of all attempted post-pyloric placements. Less than half of nasogastric tubes were successfully passed, although none of these required X-ray confirmation. The mean cost per tube insertion attempt was 111 pounds. CONCLUSIONS: This system confers advantages, particularly in terms of post-pyloric tube placement, even at this early stage of implementation. A reduction in clinical risk and cost avoidance related to X-ray exposure, the need for endoscopic tube placement and parenteral nutrition have been achieved. The implementation of this system should be considered in other centres.

Isolation and characterization of viable Toxoplasma gondii isolates revealed possible high frequency of mixed infection in feral cats ( Felis domesticus) from St Kitts, West Indies.

Cats are essential in the epidemiology of Toxoplasma gondii because they are the only hosts that can excrete the environmentally resistant oocysts in nature. Samples of serum, feces, and tissues from feral cats from St Kitts, West Indies were examined for T. gondii infection. Antibodies to T. gondii were assayed by the modified agglutination test, and found in 71 of 96 (73.9%) of cats with titres of 1:10 in six, 1: 20 in six,1:40 in seven,1: 80 in three, 1: 160 in 10, 1:320 in 13, 1:640 in nine, and 1:1,280 or higher in 17. Tissues of 10 cats were bio-assayed in mice. Toxoplasma gondii was isolated from tissues of 7 cats; from hearts of 6, from tongue of 5, and brains of 3 cats. All 7 isolates were avirulent for mice. Toxoplasma gondii oocysts were not found in the feces of 51 cats. Genotyping of these 7 T. gondii isolates by 10 multi-locus PCR-RFLP markers, including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and an apicoplast marker, Apico, revealed 4 genotypes, including clonal Type II, Type III and 2 unique genotypes. Five of the 7 cats had infection with 2 genotypes, indicating high frequency of mixed infection in the cat population on the St Kitts island.

Toxoplasmosis in captive dolphins (Tursiops truncatus) and walrus (Odobenus rosmarus).

Toxoplasma gondii infection in marine mammals is intriguing and indicative of contamination of the ocean environment and coastal waters with oocysts. Toxoplasma gondii infection was detected in captive marine mammals at a sea aquarium in Canada. Antibodies to T. gondii were found in all 7 bottlenose dolphins (Tursiops truncatus) tested. Two of these dolphins, as well as a walrus (Odobenus rosmarus) at the facility, died. Encephalitis and T. gondii tissue cysts were identified in histological sections of the brain of 1 dolphin (dolphin no. 1). Another dolphin (dolphin no. 2) had mild focal encephalitis without visible organisms, but viable T. gondii was isolated by bioassay in mice and cats from its brain and skeletal muscle; this strain was designated TgDoCA1. The PCR-RFLP typing using 11 markers (B1, SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) identified a Type II strain. The DNA sequencing of B1 and SAG1 alleles amplified from TgDoCA1 and directly from the brains of dolphin no. 1 and the walrus showed archetypal alleles consistent with infection by a Type II strain. No unique polymorphisms were detected. This is apparently the first report of isolation of T. gondii from a marine mammal in Canada.

Studies on genomic DNA stability in aluminium-maltolate treated aged new zealand rabbit: relevance to the alzheimers animal model.

BACKGROUND: Alzheimers disease (AD) is a devastative neurodegenerative disorder. Lack of substantial animal model that can unravel molecular underpinnings has been a major lacuna which limited the understanding of the etiology of the disease in turn limiting the employment of potential therapeutic strategies to combat the disease for a few decades. Our studies for the first time provided substantial animal model and tattered the etiology of the disease at a molecular level. METHODS: In this study DNA was isolated from Hippocampus (H), Midbrain (M) and Frontal Cortex (Fc) of control and aluminium maltolate (Al-M) treated aged New Zealand rabbit brain. DNA damage has been studied using Agarose gel electrophoresis, Ethidium Bromide (EtBr) binding and Melting temperature techniques. RESULTS: Al-M treated aged New Zealand rabbit's H and M showed higher DNA damage compared to corresponding controls, where as Fc showed mild DNA damage compared to corresponding controls. CONCLUSIONS: This study tangibly provides substantial molecular level understanding of the disease in turn providing an adequate platform to streamline potential therapeutic strategies. KEYWORDS: Alzheimer's disease; Aluminium maltolate; Animal model; DNA damage.

Trichinella murrelli in scavenging mammals from south-central Wisconsin, USA.

Tissues and serum from 59 raccoons (Procyon lotor), 42 coyotes (Canis latrans), and seven Striped Skunks (Mephitis mephitis) collected in Dane and Iowa Counties, Wisconsin, USA, between October 2005 and March 2006 were microscopically and serologically examined for the presence of Trichinella spp. Encapsulated larvae were found on compression slides prepared from tongue tissues from a few animals. Complete tissue digestion of tongues revealed that 19% of the raccoons, 26% of the coyotes, and none of the seven skunks tested were infected with Trichinella spp. Cats were subsequently experimentally infected by feeding them the raccoon tissues containing muscle larvae, and muscle larvae isolated from the collected tongues were experimentally transmitted to mice. Multiplex polymerase chain reaction analysis of the isolated muscle larvae demonstrated two distinct bands migrating at 127 base pairs (bp) and 316 bp in all samples, which together are diagnostic for Trichinella murrelli; the isolates were assigned Istituto Superiore di Sanita (ISS) codes ISS1656 through ISS1667, and ISS1708 through ISS1710 by the International Trichinella Reference Centre. These findings extend the geographic range of T. murrelli into Wisconsin, USA.

Isolation of Toxoplasma gondii from bottlenose dolphins (Tursiops truncatus).

Toxoplasma gondii infection in marine mammals is intriguing and indicative of contamination of the ocean environment and coastal waters with oocysts. In previous serological surveys, >90% of bottlenose dolphins (Tursiops truncatus) from the coasts of Florida, South Carolina, and California had antibodies to T. gondii by the modified agglutination test (MAT). In the present study, attempts were made to isolate T. gondii from dead T. truncatus. During 2005, 2006, and 2007, serum or blood clot, and tissues (brain, heart, skeletal muscle) of 52 T. truncatus stranded on the coasts of South Carolina were tested for T. gondii. Antibodies to T. gondii (MAT 1:25 or higher) were found in 26 (53%) of 49 dolphins; serum was not available from 3 animals. Tissues (heart, muscle, and sometimes brain) of 32 dolphins (26 seropositive, 3 seronegative, and 3 without accompanying sera) were bioassayed for T. gondii in mice, or cats, or both. Tissues of the recipient mice were examined for T. gondii stages. Feces of recipient cats were examined for shedding of T. gondii oocysts, but none excreted oocysts. Toxoplasma gondii was isolated from hearts of the 3 dolphins (2 with MAT titers of 1:200, and 1 without accompanied serum) by bioassay in mice. Genotyping of these 3 T. gondii isolates (designated TgDoUs1-3) with the use of 10 PCR-RFLP markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) revealed 2 genotypes. Two of the 3 isolates have Type II alleles at all loci and belong to the clonal Type II lineage. One isolate has a unique genotype. This is the first report of isolation of viable T. gondii from T. truncatus.

Assessment of serum macro and trace element homeostasis and the complexity of inter-element relations in bipolar mood disorders.

BACKGROUND: Bipolar disorders are complex neuropsychiatric in nature and are clinically classified as Type I, Type II, and Type V. The etiological factors include environmental-genetic inter-relations. Trace metals play a significant role in neurological disorders. There is very limited information on the role of macro and trace elements in bipolar disorders. METHODS: Trace elements namely Na, K, S, Ca, Mg, P, Cu, Fe, Zn, Mn and Al were analyzed in serum samples of 3 bipolar types: bipolar I, bipolar II and bipolar V with a control group using inductively coupled plasma-atomic emission spectrometry (ICP-AES). The patients were assessed as per the standard diagnostic criteria and classified into the bipolar type I, II hypomanic, II depressives and V. RESULTS: In bipolar I (mania), Na, K, P, Cu, Al and Mn were increased significantly (p<0.001). In bipolar II hypomania, Na, S, Al and Mn were increased significantly (p<0.02), while in bipolar II depression, Na, K, Cu and Al were increased (p<0.001). In bipolar V, Na, Mg, P, Cu, and Al were increased significantly (p<0.002), though S (p<0.00001), Fe (p<0.002) and Zn (p<0.004) were decreased in all 3 bipolar groups. CONCLUSIONS: There is a disturbance in the charge distribution and element-element interdependency in bipolar serum when compared to controls. These results suggest that there is a definite imbalance in macro and trace element homeostasis as evidenced by element inter-relationships in serum samples of bipolar groups when compared to controls.

Endemic toxoplasmosis in pigs on a farm in Maryland: isolation and genetic characterization of Toxoplasma gondii.

The prevalence of Toxoplasma gondii was investigated on a poorly managed pig farm in Maryland. Serum and tissue samples from 48 of the 100 pigs on the farm were available for T. gondii evaluation. Serological testing was performed using both ELISA and the modified agglutination test (MAT). Antibodies to T. gondii were detected by ELISA in 12 of 48 animals, while antibodies were detected in 34 of 48 pigs by MAT with titers of 1:10 in 1, 1:20 in 4, 1:40 in 7, 1:80 in 3, 1:160 in 8, 1:320 in 3, 1:640 in 4, and 1:1,280 in 4. Hearts of 16 pigs with MAT titers of 1:10 or higher were bioassayed for T. gondii in cats; 11 cats shed T. gondii oocysts. Hearts of 22 pigs were autolyzed and bioassayed only in mice; T. gondii was isolated from 3 of these 22 pigs. Genetic typing of the 14 T. gondii isolates using the SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico loci revealed 4 genotypes; 10 isolates belonged to type II lineage (genotypes 1 and 2), 3 belonged to genotype 3, and 1 belonged to genotype 4. Genotype 1 and 2 have type II alleles at all genetic loci, except the former has type II allele and the latter has a type I allele at locus Apico. Both genotypes 1 and 2 are considered to belong to the clonal type II lineages. Genotype 3 and 4 are nonclonal isolates. Results document high prevalence of T. gondii in pigs on a farm in Maryland.