Kudoa rousseauxii n. sp. (Cnidaria: Multivalvulida) Infects the Skeletal Muscles of the Freshwater Fish Brachyplatystoma rousseauxii in the Amazon River.

PURPOSE: Members of the genus Kudoa Meglitsch, 1947 are known to infect the muscles of commercially important fishes worldwide, including those in the order Siluriformes. This paper describes the occurrence of a new species of Kudoa in the catfish Brachyplatystoma rousseauxii based on morphological study and molecular analysis of the ribosomal RNA gene (SSU rDNA). METHODS: Fifteen specimens of Brachyplatystoma rousseauxii were purchased from fishing zones near Mosqueiro Island, Belém, Pará, Brazil. After necropsy, tissue samples and cysts were analyzed using a stereomicroscope, and fresh slides were viewed under a light microscope to confirm parasitic infection. The tissue fragments were removed and processed for molecular and histological analyses. RESULTS: Microscopic pseudocysts were found in the epaxial region of skeletal muscle fibers in 80% of the analyzed specimens. The myxospores were quadrangular with four shell valves (SV), pyriform polar capsules (PC), and internal symmetry. Phylogenetic analyses indicated that the new species formed a cluster with the species previously described in the Amazon, being close to two freshwater species. CONCLUSIONS: Morphological differences and molecular data of SSU rDNA support that Kudoa rousseauxii n. sp. is a new species that infects B. rousseauxii, a freshwater fish with intense migratory cycles that is widely captured and consumed in the Amazon.

PROTEOMIC ANALYSIS OF TAENIA SOLIUM CYST FLUID BY SHOTGUN LC-MS/MS.

Taenia solium cysts were collected from pig skeletal muscle and analyzed via a shotgun proteomic approach to identify known proteins in the cyst fluid and to explore host-parasite interactions. Cyst fluid was aseptically collected and analyzed with shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene alignment and annotation were performed using Blast2GO software followed by gene ontology analysis of the annotated proteins. The pathways were further analyzed with the Kyoto Encyclopedia of Genes and Genomes (KEGG), and a protein-protein interaction (PPI) network map was generated using STRING software. A total of 158 known proteins were identified, most of which were low-molecular-mass proteins. These proteins were mainly involved in cellular and metabolic processes, and their molecular functions were predominantly related to catalytic activity and binding functions. The pathway enrichment analysis revealed that the known proteins were mainly enriched in the PI3K-Akt and glycolysis/gluconeogenesis signaling pathways. The nodes in the PPI network mainly consisted of enzymes involved in sugar metabolism. The cyst fluid proteins screened in this study may play important roles in the interaction between the cysticerci and the host. The shotgun LC-MS/MS, gene ontology, KEGG, and PPI network map data will be used to identify and analyze the cyst fluid proteome of cysticerci, which will provide a basis for further exploration of the invasion and activities of T. solium.

Proteomic analysis of hydrolytic proteases in excretory/secretory proteins from Trichinella spiralis intestinal infective larvae using zymography combined with shotgun LC-MS/MS approach.

The critical step of Trichinella spiralis infection is that the muscle larvae (ML) are activated to intestinal infective larvae (IIL) which invade the intestinal columnar epithelium to further develop. The IIL excretory/secretory (ES) proteins play an important role in host-parasite interaction. Proteolytic enzymes are able to mediate the tissue invasion, thereby increasing the susceptibility of parasites to their hosts. The aim of the current study was to screen and identify the natural active proteases in T. spiralis IIL ES proteins using Western blot and gel zymography combined with liquid chromatography tandem mass spectrometry (LC-MS/MS). The T. spiralis ML and IIL ES proteins were collected from the in vitro cultures and their enzymatic acitvities were examined by gelatin zymography and azocasein degradation. The protease activities were partially inhibited by PMSF, E-64 and EDTA. Three protein bands (45, 118 and 165 kDa) of T. spiralis IIL ES proteins were identified by shotgun LC-MS/MS because they have hydrolytic activity to gelatin compared to the ML ES proteins. Total of 30 T. spiralis proteins were identified and they are mainly serine proteinases (19), but also metalloproteinases (7) and cysteine proteinases (3). The qPCR results indicated that transcription levels of four T. spiralis protease genes (two serine proteases, a cathepsin B-like cysteine proteinase and a zinc metalloproteinase) at IIL stage were obviously higher than at the ML stage. These proteolytic enzymes are directly exposed to the host intestinal milieu and they may mediate the worm invasion of enteral epithelium and escaping from the host's immune responses. The results provide the new insights into understanding of the interaction of T. spiralis with host and the invasion mechanism.

Cysticercosis and suicide - an example from a forensic collection.

In this case from 1937, the deceased was a 52-year-old female who was suffering from systemic cysticercosis, with prominent neurological and psychiatric symptoms. Given the protracted clinical course and autopsy findings it appears likely that the disease led the woman to commit suicide by ingesting lye, a corrosive substance, and the most common way to commit suicide in Belgrade at the time. The autopsy revealed many rounded transparent cysts, attached to the dura and pia-arachnoid, as well as encapsulated in the intercostal muscles, diaphragm and muscles of the arms, legs and the trunk. Solitary cysticercosis of muscles without involvement of the central nervous system is rare: most soft tissue and muscular cysticercal infections are associated with the central nervous system. Parasites usually lodge in the cerebral cortex or the subcortical white matter, due to the high vascular supply of these areas. Psychiatric symptoms in neurocysticercosis have been frequently reported, along with cognitive decline and intellectual deterioration, depressive disorders, behavioral disturbance and psychosis. Although sporadically, the disease is present even today, and neurocysticercosis is the leading cause of epilepsy in the developing world. To maintain its lifecycle, Taenia solium requires non-industrialized pig rearing conditions, consumption of undercooked pork, and low sanitation standards. Socioeconomic and sanitary improvement and educating people about food processing, the disease and antihelminthic therapy, are important factors contributing to a significant reduction in the prevalence of this potentially eradicable disease worldwide.

T. gondii infection induces IL-1R dependent chronic cachexia and perivascular fibrosis in the liver and skeletal muscle.

Cachexia is a progressive muscle wasting disease that contributes to death in a wide range of chronic diseases. Currently, the cachexia field lacks animal models that recapitulate the long-term kinetics of clinical disease, which would provide insight into the pathophysiology of chronic cachexia and a tool to test therapeutics for disease reversal. Toxoplasma gondii (T. gondii) is a protozoan parasite that uses conserved mechanisms to infect rodents and human hosts. Infection is lifelong and has been associated with chronic weight loss and muscle atrophy in mice. We have recently shown that T. gondii-induced muscle atrophy meets the clinical definition of cachexia. Here, the longevity of the T. gondii-induced chronic cachexia model revealed that cachectic mice develop perivascular fibrosis in major metabolic organs, including the adipose tissue, skeletal muscle, and liver by 9 weeks post-infection. Development of cachexia, as well as liver and skeletal muscle fibrosis, is dependent on intact signaling through the type I IL-1R receptor. IL-1α is sufficient to activate cultured fibroblasts and primary hepatic stellate cells (myofibroblast precursors in the liver) in vitro, and IL-1α is elevated in the sera and liver of cachectic, suggesting a mechanism by which chronic IL-1R signaling could be leading to cachexia-associated fibrosis.

Diagnosis of Porcine Cysticercosis at Necropsy: When Is Enough, Enough?

Changes in the incidence of porcine cysticercosis is used commonly in the assessment of efforts to control the transmission of Taenia solium, the cause of human neurocysticercosis. Although cysticerci may occasionally occur in tissues other than the striated muscles, particularly the brain, infection in pigs can be diagnosed reliably by assessment of muscle tissue alone.

Distribution of Toxoplasma gondii Tissue Cysts in Shoulder Muscles of Naturally Infected Goats and Lambs.

ABSTRACT: Toxoplasmosis has been recognized as a major public health problem worldwide. The consumption of uncooked or undercooked meat infected with Toxoplasma gondii tissue cysts is one of the main means of transmission of this parasite. Although sheep, goats, and pigs are commonly infected with T. gondii, little information is available on the distribution of T. gondii tissue cysts in naturally infected meat. In this study, we investigated the distribution of viable T. gondii tissue cysts in shoulder muscles of naturally infected lambs and goats. Hearts and shoulders of 46 lambs and 39 goats from a local grocery store were tested for T. gondii infection. Animals were evaluated for the presence of anti-T. gondii antibodies in heart blood and clots by the modified agglutination test. Fourteen of the 85 animals (seven lambs and seven goats) were seropositive. Six to 12 samples weighing 5, 10, and 50 g were obtained from shoulder muscles of each seropositive animal and used for bioassay in mice. The distribution of viable T. gondii differed according to the size of the sample analyzed, but in general larger sample sizes resulted in higher isolation rates (P < 0.05). Results of the study revealed an uneven distribution of T. gondii in muscle samples of lambs and goats and that T. gondii can be transmitted by consumption of very small servings (5 and 10 g) of meat when it is consumed raw or is undercooked.

A New Species of ThelohanellusKudo, 1933 (Myxozoa: Bivalvulida) Infecting Skeletal Muscle of Blacktail Shiner, Cyprinella venusta Girard, 1856 (Cypriniformes: Cyprinidae) in the Chattahoochee River Basin, Georgia.

Thelohanellus magnacysta n. sp. (Bivalvulida: Myxobolidae) infects the skeletal muscle of blacktail shiner, Cyprinella venusta Girard, 1856 (Cypriniformes: Cyprinidae) in Bull Creek, Chattahoochee River Basin, eastern Georgia. Although numerous members of ThelohanellusKudo, 1933 have overlapping myxospore dimensions with the new species, it differs from all nominal congeners by polar filament coil number and polar capsule width as well as by lacking a mucous envelope, iodinophilic vacuole, and sutural markings. With the use of novel primers for Myxozoa, a phylogenetic analysis of the small subunit ribosomal DNA (SSU rDNA) suggests that the new species shares a recent common ancestor with a clade of cyprinid-infecting species of Myxobolus Bütschli, 1882 (Bivalvulida: Myxobolidae) and Thelohanellus. Consistent with other published research concerning the systematics of Thelohanellus, this result suggested that Thelohanellus and Myxobolus are polyphyletic and need revision. Histological sections of infected blacktail shiners confirmed that myxospores were only found within a plasmodium and only infected skeletal muscle and that plasmodia were encapsulated by a granuloma comprising varying degrees of acute granulomatous inflammation. The new species is the fourth of Thelohanellus reported from North America and the first reported from Cyprinella, as well as the first myxozoan described from the blacktail shiner.

Muscular Sarcocystosis in Sheep Associated With Lymphoplasmacytic Myositis and Expression of Major Histocompatibility Complex Class I and II.

Sarcocystosis is a protozoal disease affecting a wide range of animals. The aims of this study were to characterize the following in sheep: (1) the muscle pathology in Sarcocystis infection, (2) the inflammatory infiltrate and its relationship to severity of infection, and (3) immune markers expressed by parasitized muscle fibers and parasitic cysts. Skeletal muscle samples from 78 sheep slaughtered in southern Italy were snap frozen and analyzed by histopathology, immunohistochemistry, and immunofluorescence. Polymerase chain reaction (PCR) and sequencing were used for Sarcocystis species identification. All 40 muscle samples tested were PCR-positive for Sarcocystis tenella. Histologically, cysts were identified in 76/78 cases (97%), associated with an endomysial infiltrate of lymphocytes and plasma cells. The T cells were predominantly CD8+, with fewer CD4+ or CD79α+ cells. Eosinophils were absent. Notably, sarcolemmal immunopositivity for major histocompatibility complex (MHC) I and II was found in 76/78 cases (97%) and 75/78 cases (96%), respectively, both in samples with and in those without evident inflammatory infiltrate. The number of cysts was positively correlated with inflammation. In addition, MHC I was detected in 55/78 cyst walls (72%), and occasionally co-localized with the membrane-associated protein dystrophin. The findings suggest that muscle fibers respond to the presence of cysts by expression of MHC I and II. The possible role of MHC I and II in the inflammatory response and on the cyst wall is also discussed.

Evaluation of radiologic diagnostic criteria and treatment options in skeletal muscle hydatid cysts.

AIM: To investigate the diagnosis and treatment methods of soft tissue involvement of hydatid cysts (HCs). MATERIALS AND METHODS: Eleven patients who were diagnosed as having HC with muscular tissue (soft tissue) involvement between 2010 and 2016 were evaluated retrospectively. Seven patients had typical HC magnetic resonance imaging (MRI) and four patients had cysts with an unusual appearance. We evaluated how to diagnose the cysts using imaging methods, their characteristic radiologic images, and treatment alternatives against the disease. The patients were treated with antihelminthic chemotherapy preoperatively and postoperatively. RESULTS: Of the 11 patients who underwent treatment, 7 were diagnosed using MRI and 4 were diagnosed with histopathologic examinations. The mean follow-up period was 16 (range, 6-24) months, and the mean age was 39.4 (range, 24-56) years. In seven patients, multivesicular appearance with specific MRI findings, T2-hypointense rim appearance, double-rim sign, membrane dissociation, and appearance of daughter cysts were identified as diagnostic criteria. Two patients underwent ultrasound assisted percutaneous aspiration-injection-reaspiration (PAIR) treatment. Seven patients underwent total pericystectomy, and two patients underwent subtotal pericystectomy with serum saline injection into the cyst. Two patients showed signs of mild anaphylaxis, one during the diagnosis and one during treatment. CONCLUSION: There may be difficulties in the diagnosis and treatment of HCs of the musculoskeletal system. It should be known that there are alternative methods in the treatment (cyst excision and PAIR treatment). Clinical, serologic, and radiologic findings should be used in the diagnosis. To avoid complications during the histopathologic diagnosis, MRI should be examined in detail. It is thought that atypical cysts can be diagnosed (double-layer appearance and peripheral rim sign) in addition to typical cysts (detached membrane and multivesicular appearance), and diagnosis and treatment can be planned without anaphylactic complications.

Visceral larva migrans detection using PCR-RFLP in BALB/c mice infected with Toxocara canis.

Toxocara canis is an important zoonotic roundworm distributed worldwide. The infective larvae of T. canis are one of the causes of visceral larva migrans (VLM), a clinical syndrome in humans. Diagnosing VLM is difficult, and the differential diagnosis of the larval development stage is limited. Therefore, this experimental research aimed to diagnose T. canis larvae using a molecular method, not only in liver tissue, which is the most commonly affected tissue, but also in the limb muscles, lungs and brain tissues. For this purpose, 24 BALB/c mice were infected with 1000 embryonated T. canis eggs. Necropsies were performed on the second, fourth, seventh and 14th days post-infection. While a part of the samples were digested with pepsin-HCl, the molecular method was used for the remainder of the samples to replicate the mitochondrial DNA adenosine triphosphate (ATP) synthase subunit-6 gene region of T. canis. BbsI, a restriction endonuclease, was used to determine the specificity of the amplicons obtained from Polymerase chain reaction (PCR). The detection limit for embryonated eggs was recorded. The PCR results showed that the sensitivity of the PCR analysis was 83.3% in the liver (with 88.8% accuracy), 87.5% in the lungs (with 91.6% accuracy) and 75.0% in the brain, forelimb and hindlimb muscles (with 83.3% accuracy). In all tissues, the test specificity was determined to be 100%. In this study, the molecular method was applied to only experimentally infected BALB/c mice tissues; thus, it is suggested that it can be also employed in different paratenic hosts and materials possibly infected with T. canis.

Organometallic Gold(III) Complex [Au(Hdamp)(L14)]+ (L1 = SNS-Donating Thiosemicarbazone) as a Candidate to New Formulations against Chagas Disease.

Chagas disease remains a serious public health concern with unsatisfactory treatment outcomes due to strain-specific drug resistance and various side effects. To identify new therapeutic drugs against Trypanosoma cruzi, we evaluated both the in vitro and in vivo activity of the organometallic gold(III) complex [Au(III)(Hdamp)(L14)]Cl (L1 = SNS-donating thiosemicarbazone), henceforth denoted 4-Cl. Our results demonstrated that 4-Cl was more effective than benznidazole (Bz) in eliminating both the extracellular trypomastigote and intracellular amastigote forms of the parasite without cytotoxic effects on mammalian cells. In in vivo assays, 4-Cl in PBS solution loses the protonation and becomes the 4-neutral. 4-Neutral reduced parasitaemia and tissue parasitism in addition to protecting the liver and heart from tissue damage at 2.8 mg/kg/day. All these changes resulted in the survival of 100% of the mice treated with the gold complex during the acute phase. Analyzing the surviving animals of the acute infection, the parasite load after 150 days of infection was equivalent to those treated with the standard dose of Bz without demonstrating the hepatotoxicity of the latter. In addition, we identified a modulation of interferon gamma (IFN-γ) levels that may be targeting the disease's positive outcome. To the best of our knowledge, this is the first gold organometallic study that shows promise in an in vivo experimental model against Chagas disease.

[Pig-slaughtering at home. Customs do not change. An infectological case report from a historical point of view]. / Családi disznótor, avagy a szokások nem változnak. Hagyományorzo infektológiai esetbemutatás.

We report a case of a 41-year-old female patient presenting with watery diarrhoea and myalgia in the winter-season. Before her symptoms started she had participated in a pig slaughtering with her family. Some of the family members also became ill. On her physical examination periorbital odema and myalgia were found. Eosinophilia, hypalbuminaemia, elevated lactate dehydrogenase and creatin kinase levels were detected on laboratory investigations. The clinical picture, the laboratory findings and background epidemiological data implied the diagnosis of trichinellosis and albendazol was started. Serum gained on the 22nd post-infectious day turned out to be equivocal for trichinellosis. For this reason and because of the refractory fever a muscle-biopsy was done. Granulomatous myositis described by histology and Trichinella seropositivity from the repeated serum sample on the 62nd post-infectious day finally confirmed the diagnosis. During the course of the disease, we experienced elevation of troponin I suggesting myocarditis, but it was accompanied neither with abnormal ECG signs nor characteristic symptoms. Almost a century ago, a case report was published in Hungarian with a similar introduction. Trichinellosis in that epidemic setting led to the death of five people. Orv Hetil. 2019; 160(24): 952-957.

Kudoa sp. (Myxozoa, Multivalvulida): first report in five commercial fish species from the Canary Islands-FAO 34 (Macaronesia-Spain).

Kudoid myxozoans have been reported causing serious chronic problems in marine fisheries, by reducing the market value of infected fish through pathological damage to the host musculature. We report here the overall prevalence of a Kudoa species in 84/277 (30.3%) fishes from 20 different species of high commercial value captured between October 2011 and December 2013 from the United Nations Food and Agriculture Organization (FAO) 34 commercial fishing area, near the coast of the Canary Islands (Spain). Macroscopic examination showed myxozoan-like cysts in skeletal muscle from 5 of the 20 fish species examined, with the following prevalences: Pagellus acarne (86.7%), Pagellus erythrinus (46.5%), Serranus cabrilla (27.8%), Spondyliosoma cantharus (19.4%), and Sarpa salpa (28.6%). Infection intensity was determined based on spore counts following muscle tissue digestion. Morphometric studies to characterize the species and DNA sequence analysis results suggest that these infections are attributable to a Kudoa species closely related to Kudoa trachuri. This paper reports the first study on a multivalvulidan species to be identified from the Canary Islands. Furthermore, this is the first report of Kudoa parasites in all of the hosts mentioned above, with the exception of P. acarne.

Morphological and molecular description of Sarcocystis ratti n. sp. from the black rat (Rattus rattus) in Latvia.

Rodents have been widely studied as intermediate hosts of Sarcocystis; however, only a few reports on these parasites in the black rat (Rattus rattus) are known. Having examined 13 black rats captured in Latvia, sarcocysts were found in skeletal muscles of two mammals and were described as Sarcocystis ratti n. sp. Under a light microscope, sarcocysts were ribbon-shaped, 0.9-1.3 × 0.09-0.14 mm in size and had a thin (0.8-1.3 µm) and smooth cyst wall. The lancet-shaped bradyzoites were 8.3 × 4.3 (7.5-9.3 × 3.9-4.8) µm. Under a transmission electron microscope, the cyst wall was up to 1.3 µm thick, wavy, the ground substance appeared smooth, type 1a-like. Morphologically, sarcocysts of S. ratti were somewhat similar to those of S. cymruensis, S. rodentifelis, and S. dispersa-like previously identified in the brown rat (Rattus norvegicus). On the basis of 18S rDNA, 28S rDNA, and cox1, significant genetic differences (at least 2.3, 4.5, and 5.8%, respectively) were observed when comparing S. ratti with other Sarcocystis species using rodents as intermediate hosts. While ITS1 sequences of S. ratti were highly distinct from other Sarcocystis species available in GenBank. Phylogenetic and ecological data suggest that predatory mammals living near households are definitive hosts of S. ratti.

A pathological study of Leishmania infantum natural infection in European rabbits (Oryctolagus cuniculus) and Iberian hares (Lepus granatensis).

In this study, we describe the pathology of Leishmania infantum infection in naturally infected wild Leporidae and compare diagnosis of infection using histopathology, direct fluorescent antibody (DFA) assay, immunofluorescence antibody test (IFAT) and quantitative real-time PCR (qPCR). Tissues were analysed from 52 European rabbits (Oryctolagus cuniculus) and 7 Iberian hares (Lepus granatensis) from the Community of Madrid (Spain). Our results show that L. infantum infection is associated with only minimal histopathological lesions and that L. infantum amastigotes can be detected by DFA assay in all tissues types tested, including skin. These results were confirmed by qPCR on fresh frozen tissues in 13% of rabbits and 100% of hares. However, L. infantum DNA could not be detected by qPCR on paraffin-embedded tissue obtained by laser capture microdissection. Using the DFA assay to diagnose L. infantum, infection may provide further insights into this disease in wild animals and may allow the precise tissue localization of L. infantum, thereby guiding follow-up tests with more accurate qPCR.

Isolation and Genetic Characterization of Toxoplasma gondii from Tissues of Wild Turkeys (Meleagris gallopavo) in Pennsylvania.

Toxoplasmosis in wild turkeys (Meleagris gallopavo) is of epidemiological interest because turkeys feed from the ground, and detection of infection in turkeys indicates contamination by oocysts in the environment. During the 2018 spring hunting season in Pennsylvania, fresh (unfixed, not frozen) samples were obtained from 20 harvested wild turkeys and tested for Toxoplasma gondii infection. Hearts from all wild turkeys and skeletal muscle from 1 were bioassayed for T. gondii by inoculation in outbred Swiss Webster (SW) and interferon-gamma gene knockout (KO) mice. Antibodies to T. gondii were detected in 1:5 dilution of neat serum from 5 of 15 wild turkeys and in fluid from the heart of 1 of 4 wild turkeys with the modified agglutination test (MAT); neat serum was not available from 4 wild turkeys. Viable T. gondii was isolated from hearts of 5 wild turkeys, 1 with MAT of 1:10, 1 with MAT of 1:5, and 3 seronegative (MAT < 1:5). Toxoplasma gondii was isolated from both heart and skeletal muscle in the 1 wild turkey that had skeletal muscle submitted. The KO mice inoculated with tissue from all 5 infected wild turkeys died or were euthanatized when ill, 7-21 days post-inoculation (PI). Tachyzoites were detected in lungs of all KO mice, and the T. gondii strains were successfully propagated in cell culture. The SW mice inoculated with tissues of wild turkeys remained asymptomatic, and tissue cysts were seen in the brains of infected mice when euthanatized in good health at 46 days PI; 1 of the 2 SW mice inoculated with the heart of 1 turkey died on day 26, and tachyzoites were detected in its lung. Genetic typing on DNA extracted from culture-derived tachyzoites using the PCR restriction fragment length polymorphism with 10 genetic markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) revealed that 4 isolates belonged to ToxoDB PCR-RFLP genotype #5 and 1 was genotype #216.

Toxoplasma gondii tissue cyst formation and density of tissue cysts in shoulders of pigs 7 and 14 days after feeding infected mice tissues.

Among the meat sources of Toxoplasma gondii, pork is considered important in the epidemiology of toxoplasmosis in the USA. How soon after infection T. gondii forms tissue cysts in pork is unknown. In the present study, eight serologically negative ˜3 months old pigs were fed mouse tissues infected with VEG (Type III) strain of T. gondii and euthanized 7 (4 pigs) and 14 days (4 pigs) post-inoculation (p.i.). Meat from the right shoulder of each pig was bioassayed in mice for T. gondii tissue cysts by peptic digestion. From each pig, the shoulder muscle was cut at random spots into 5 g, 10 g and 50 g portions. Extreme care was taken to use different scalpels and forceps to minimize cross contamination among 17 samples (6 replicates of each 5 g and 10 g portions and 5 replicates of 50 g). From the four pigs euthanized at 7 days p.i., a composite of ˜200 g of leftover meat from each shoulder was bioassayed in cats and their feces were tested for oocyst excretion. All eight pigs developed T. gondii antibodies (modified agglutination test, MAT, 1: 80 or higher) and viable T. gondii was isolated from shoulder meat of each pig. All four cats fed pork from excreted T. gondii oocysts. The density of T. gondii, based on mouse infectivity, varied within 5-50 g samples each pig, and between pigs within the same group, day 7 versus day 14 p.i. There were no significant differences in mouse bioassay results obtained with day 7 versus day 14 infected pigs. Overall, the rate of isolation of T. gondii increased with sample size of meat bioassayed. Results demonstrate that tissue cysts are formed early in infection and they are unevenly distributed.