Prevalence of Sarcocystis spp. in Argentinean cattle

Sarcocystis cruzi, S. hirsuta and S. hominis are apicomplexan parasites that affect cattle worldwide with variable prevalence. The aim of the present study was to evaluate the prevalence of Sarcocystis spp. in Argentinean cattle comparing microscopic fresh examination and molecular methods. Blood, myocardium and loin samples were collected in five slaughterhouses from a total of 380 bovines. Origin of animals was representative of the major beef cattle production area of Argentina. Samples were analyzed by fresh microscopical examination, transmission electron microscopy (TEM), IFAT and PCR-RFLP. Thin walled sarcocysts corresponding with S. cruzi were found in 99.5% of heart samples. Sarcocysts were detected in 73.1% of loin samples; 71.5% had S. cruzi cysts and 23.1% had thick walled sarcocysts (S. hirsuta or S. hominis). TEM observation revealed the presence of characteristic S. hominis and S. hirsuta cyst walls in 7 and 1 loin samples respectively. Using IFAT, 379/380 animals had titers 25 or higher, showing a full agreement with fresh examination. Amplification products were detected in 35.5% (135/380) of loin samples; however Sarcocystis species could only be determined by RFLP in 29 samples. Agreement between fresh examination and PCR was low (Kappa value=0.262). This is the first report of S. hominis and S. hirsuta in Argentina. Further studies are needed to improve the sensitivity of molecular methods for species identification, especially for differentiation of S. cruzi and S. hirsuta from the zoonotic species S. hominis. The results of the present study and others focusing on sensitivity and specificity of Sarcocystis spp. diagnostic methods should contribute to improve food safety.     

Sarcocystis infection in the white-tailed deer (Odocoileus virginianus) in Montana: intensity and description of Sarcocystis odoi n sp

Sarcocystis infection was diagnosed in 27 of 36 (75%) samples of meat from white-tailed deer (Odocoileus virginianus) in Montana. Two structurally distinct thin- and thick-walled sarcocysts were found in the white-tailed deer; the thin-walled sarcocysts were those of S odocoileocanis. A new name, S odoi, was proposed for the thick-walled sarcocysts. Sarcocysts of S odoi were up to 1,050 microns long and 260 microns wide and contained a 6.5- to 12-microns thick wall. The ultrastructure of sarcocysts of S odocoileocanis was compared with that of S odoi. A cat fed meat containing thin- and thick-walled sarcocysts shed oocysts and sporocysts 24 days later. The sporocysts in the cat's feces were 13.3 X 9.6 microns and probably belonged to S odoi.     

Prevalence of Sarcocystis sarcocysts in nine-banded armadillos (Dasypus novemcinctus) from Florida.

The prevalence and identity of Sarcocystis spp. sarcocysts in the skeletal muscles of nine-banded armadillos (Dasypus novemcinctus) collected from Alachua County, FL, were determined. H & E stained sections of skeletal muscle from tongue and thigh were examined. Thirty nine of 63 (61.9%) armadillos examined contained Sarcocystis sarcocysts. Two species were identified, Sarcocystis dasypi and Sarcocystis diminuta. Sarcocystis dasypi sarcocysts were found in 38 of 63 (60.3%) and S. diminuta sarcocysts were found in 6 of 63 (9.5%). Sarcocysts of S. dasypi were larger, more densely packed with bradyzoites, and bradyzoites contained within the sarcocyst were smaller than those of S. diminuta. Mixed infections occurred in 5 of 63 (7.9%) armadillos examined.     

Brown-headed cowbirds (Molothrus ater) harbor Sarcocystis neurona and act as intermediate hosts

We tested the hypothesis that brown-headed cowbirds (Molothrus ater) harbor Sarcocystis neurona, the agent of equine protozoal myeloencephalitis (EPM), and act as intermediate hosts for this parasite. In summer 1999, wild caught brown-headed cowbirds were collected and necropsied to determine infection rate with Sarcocystis spp. by macroscopic inspection. Seven of 381 (1.8%) birds had grossly visible sarcocysts in leg muscles with none in breast muscles. Histopathology revealed two classes of sarcocysts in leg muscles, thin-walled and thick-walled suggesting two species. Electron microscopy showed that thick-walled cysts had characteristics of S. falcatula and thin-walled cysts had characteristics of S. neurona. Thereafter, several experiments were conducted to confirm that cowbirds had viable S. neurona that could be transmitted to an intermediate host and cause disease. Specific-pathogen-free opossums fed cowbird leg muscle that was enriched for muscle either with or without visible sarcocysts all shed high numbers of sporocysts by 4 weeks after infection, while the control opossum fed cowbird breast muscle was negative. These sporocysts were apparently of two size classes, 11.4+/-0.7 microm by 7.6+/-0.4 microm (n=25) and 12.6+/-0.6 microm by 8.0+/-0 microm (n=25). When these sporocysts were excysted and introduced into equine dermal cell tissue culture, schizogony occurred, most merozoites survived and replicated long term and merozoites sampled from the cultures with long-term growth were indistinguishable from known S. neurona isolates. A cowbird Sarcocystis isolate, Michigan Cowbird 1 (MICB1), derived from thin-walled sarcocysts from cowbirds that was passaged in SPF opossums and tissue culture went on to produce neurological disease in IFNgamma knockout mice indistinguishable from that of the positive control inoculated with S. neurona. This, together with the knowledge that S. falcatula does not cause lesions in IFNgamma knockout mice, showed that cowbird leg muscles had a Sarcocystis that fulfills the first aim of Koch's postulates to produce disease similar to S. neurona. Two molecular assays provided further support that both S. neurona and S. falcatula were present in cowbird leg muscles. In a blinded study, PCR-RFLP of RAPD-derived DNA designed to discriminate between S. neurona and S. falcatula showed that fresh sporocysts from the opossum feeding trial had both Sarcocystis species. Visible, thick-walled sarcocysts from cowbird leg muscle were positive for S. falcatula but not S. neurona; thin-walled sarcocysts typed as S. neurona. In 1999, DNA was extracted from leg muscles of 100 wild caught cowbirds and subjected to a PCR targeting an S. neurona specific sequence of the small subunit ribosomal RNA (SSU rRNA) gene. In control spiking experiments, this assay detected DNA from 10 S. neurona merozoites in 0.5g of muscle. In the 1999 experiment, 23 of 79 (29.1%) individual cowbird leg muscle samples were positive by this S. neurona-specific PCR. Finally, in June of 2000, 265 cowbird leg muscle samples were tested by histopathology for the presence of thick- and thin-walled sarcocysts. Seven percent (18/265) had only thick-walled sarcocysts, 0.8% (2/265) had only thin-walled sarcocysts and 1.9% (5/265) had both. The other half of these leg muscles when tested by PCR-RFLP of RAPD-derived DNA and SSU rRNA PCR showed a good correlation with histopathological results and the two molecular typing methods concurred; 9.8% (26/265) of cowbirds had sarcocysts in muscle, 7.9% (21/265) had S. falcatula sarcocysts, 1.1% (3/265) had S. neurona sarcocysts, and 0.8% (2/265) had both. These results show that some cowbirds have S. neurona as well as S. falcatula in their leg muscles and can act as intermediate hosts for both parasites.     

Experimental inoculation of domestic cats (Felis domesticus) with Sarcocystis neurona or S. neurona-like merozoites

Sarcocystis neurona is the parasite most commonly associated with equine protozoal myeloencephalitis (EPM). Recently, cats (Felis domesticus) have been demonstrated to be an experimental intermediate host in the life cycle of S. neurona. This study was performed to determine if cats experimentally inoculated with culture-derived S. neurona merozoites develop tissue sarcocysts infectious to opossums (Didelphis virginiana), the definitive host of S. neurona. Four cats were inoculated with S. neurona or S. neurona-like merozoites and all developed antibodies reacting to S. neurona merozoite antigens, but tissue sarcocysts were detected in only two cats. Muscle tissues from the experimentally inoculated cats with and without detectable sarcocysts were fed to laboratory-reared opossums. Sporocysts were detected in gastrointestinal (GI) scrapings of one opossum fed experimentally infected feline tissues. The study results suggest that cats can develop tissue cysts following inoculation with culture-derived Sarcocystis sp. merozoites in which the particular isolate was originally derived from a naturally infected cat with tissue sarcocysts. This is in contrast to cats which did not develop tissue cysts when inoculated with S. neurona merozoites originally derived from a horse with EPM. These results indicate present biological differences between the culture-derived merozoites of two Sarcocystis isolates, Sn-UCD 1 and Sn-Mucat 2.     

Incidence of sarcocysts in skeletal muscles of horses.

The incidence of sarcocysts was examined in postural, propulsive and respiratory muscles from 74 horses ranging in age from mid-gestation to 14 years post-natal. Cryostat sections were stained for myosin adenosine triphosphatase (ATPase) at pH 9.5 and the type of muscle fibre containing sarcocysts was identified. Sarcocysts were found in muscles from three animals, all aged 1 year or more. Counts showed that they displayed no preference for any particular muscle. However, fibres with a high activity for myosin ATPase were preferentially colonized. Transverse sectional profiles of sarcocysts showed a wide variation in size, shape and wall thickness. Both the proportion of horses infected and the intensity of infection per animal were considerably lower than those reported in other studies.     

Physeal Fractures in the Horse:I. Classification and Incidence

Seventy appendicular skeletal physeal fractures in 67 horses were reviewed and classified using the Salter-Harris classification. All the horses were less than 2 years old (mean age at injury 6.2 months). The mean age at injury for pressure physeal fractures (5.2 months) was significantly less (p < .05) than for traction physeal fractures (8.3 months). The majority (67.2%) of the horses were female. Forty-eight (69%) pressure physes and 22 (31%) traction physes were affected. The most common pressure and traction physeal fracture sites were the proximal femoral physis and the proximal ulnar physis, respectively. Sixty-seven physeal fractures were classified: 14 as Type I (20.9%), 42 as Type II (62.7%), six as Type III (8.9%), and five as Type IV (7.5%). Forty-six pressure physeal fractures were classified: six as Type I (13.0%), 30 as Type II (65.2%), five as Type Hi (10.9%), and 5 as Type IV (10.9%).     

Heterogeneity of Hypertrophy in Feline Hypertrophic Heart Disease

Eighty-six cats with non-dilated left ventricular myocardial hypertrophy were studied retrospectively. Cats were categorized by two-dimensional echocardiography as having symmetric ventricular hypertrophy (Type I), asymmetric with predominant septal thickening hypertrophy (Type II), and asymmetric hypertrophy with predominant free-wall thickening (Type III). The distribution of hypertrophy was judged subjectively and objectively. Subjective and objective results were similar (P= 0.03) although overlap existed between groups. Morphologic patterns (Types I, II, and III) were compared with breed, age, sex, heart rate, percent fractional shortening, left atrial size, serum creatinine concentration, and the presence (yes/no) of pleural effusion, pulmonary edema, pericardial effusion, heart murmur, dyspnea, thromboembolism, hyperthyroidism, and being alive at the time of study. Interventricular septal thickness, left ventricular free wall thickness, percent fractional shortening, and left atrial size additionally were compared to 3-month survival. Cats with Type HI hypertrophy were more likely to experience thromboembolism than cats with Type II hypertrophy (P= 0.05) and cats with Type I hypertrophy were more likely to have heart murmurs than cats with Type III (P= 0.02). No other significant associations were found in comparison to pattern of hypertrophy. Both left atrial size and percent fractional shortening significantly correlated with 3-month survival (P < 0.001 for each). The degree of interventricular septal wall thickness was associated with 3-month survival (P= 0.02) when known hyperthyroid cats were excluded from the study group, while left ventricular free wall thickness consistently was not associated with survival. This study demonstrates the heterogeneity of hypertrophy in cats with hyper-trophic heart disease and provides predictors of survival (left atrial size, percent fractional shortening, and interventricular septal wall thickness when compared with euthanasia/spontaneous death data).     

Variations of the Extensor Digitorum Brevis Muscle in the Dog

The variation of the extensor digitorum brevis (EDB) muscle was examined in 113 hindlimbs of dogs. Four types were discriminated in this muscle. Of the three heads of the muscle, the medial head went to the 2nd digit in types I and II, and to the 2nd and 3rd digits in types III and IV. The middle head went to the 3rd digit in types I and IV, and to the 3rd and 4th digits in types II and III. The lateral head went to the 4th digit in all the types. Type II was the most common of the four (78.76 %). Type III was found in 8.85%, type 1 in 973% and type IV in 2.66%.     

Prevalence of dog-derived Sarcocystis spp. in some New Zealand lambs

Pepsin: hydrochloric acid digestion and histological examination of the myocardium of 51 hearts from five to nine month-old lambs from the southern half of the North Island, showed that 47 (92%) were infected with microscopic sarcocysts that are presumed to be dog-derived. Of 157 sarcocysts detected histologically, 32 corresponded morphologically with Sarcocystis ovicanis and 120 with Sarcocystis tenella (Syn. S. arieticanis). The remainder could not be identified as to type.     

Morphological studies on the vascular architecture in the boar spermatic cord

The morphological features of the testicular artery and the pampiniform plexus in the boar spermatic cord were evaluated by light microscopy, corrosion cast technique combined with scanning electron microscopy and by transmission electron microscopy. The testicular veins could be subdivided into 4 types according to their perivascular elements and their location to the testicular artery. Type I vein consisted of large veins and gave rise to type II and III veins. Type II vein was composed of a single layer of veins and ran along the testicular artery, while type III vein consisted of several layers of veins and was located between type II veins. Type IV vein, which was regarded as a venous portal system, was composed of small muscular and pericytic venules and was located in the tunica adventitia of type II vein and the testicular artery. Occasionally, type IV vein penetrated deep into the tunica media of the testicular artery, accompanied by a fenestrated endothelium in its thin portion. The direct arterio-venous anastomosis between the pampiniform plexus and the testicular artery was not found. Type IV vein was considered to be the most important vessel in reducing the capacity of the barrier between the testicular artery and the veins (type II and III). It is, therefore, suggested that type IV venous network may play a role in transferring the substances between the artery and the veins.     

Experimental infection with Sarcocystis medusiformis in sheep

The development of the parasite was studied in 48 sheep killed between 188 and 1132 days after experimental inoculation with Sarcocystis medusiformis sporocysts from cats. Immature sarcocysts were present at 188 days post inoculation (d.p.i.). At 331 d.p.i. macroscopic sarcocysts with an elongate fusiform appearance were seen in the laryngeal, abdominal and diaphragm musculature. The largest cyst measured 2 mm in length by 0.5 mm in width at 331 d.p.i.; histologically they contained metrocytes at the periphery of the cyst with more densely staining merozoites in the central region. By 443 d.p.i. typical 'thin' cysts 2-3.5 mm in length were seen in the flank and external thoracic muscles. By 765 d.p.i. sarcocysts were 5 mm in length. The ultrastructure of the cyst wall of these cysts resembled that of S. medusiformis. At 1132 d.p.i. sarcocysts measured 4 mm X 0.5 mm.     

Morphological and molecular identification of three species of Sarcocystis in reindeer (Rangifer tarandus tarandus) in Iceland

Six Sarcocystis species have previously been described from reindeer in Norway based on sarcocyst morphology and DNA sequencing. The aim of this study was to determine whether reindeer in Iceland, which descend from reindeer imported from Norway in 1787, also were infected with Sarcocystis, and to identify and genetically characterise any species present. Muscle tissue from the heart, diaphragm and/or oesophagus was collected from 36 reindeer in Iceland. Pieces of all tissue samples were examined histologically. Frozen/thawed samples of cardiac muscle, oesophagus and/or diaphragm from 11 of the 36 reindeer were also examined under a stereoscopic microscope and sarcocysts present were identified to species either in situ or under a light microscope. Two cysts of each species, originating from two different reindeer were randomly selected for DNA analyses. The complete ssu rRNA gene was amplified by the polymerase chain reaction (PCR) and sequenced. In addition, two sarcocysts that could not be classified by microscopic examination were selected for partial ssu rRNA gene sequence analysis. By histology, sarcocysts were found in the diaphragm and/or oesophagus of 8 of 36 (22.2%) animals. By examination of fresh tissue, sarcocysts of Sarcocystis rangi, S. tarandivulpes and S. hardangeri were found in the oesophagus of seven of nine (77.8%) animals, suggesting a high prevalence of Sarcocystis in the Icelandic reindeer population. Cyst morphology and the ssu rRNA gene sequence of each of the three species were identical to isolates of the same species from Norwegian reindeer. DNA sequencing was useful in order to identify cysts with an ambiguous morphology. This is the first record of these Sarcocystis species in reindeer outside Norway.     

Naturally occurring Sarcocystis neurona-like infection in a dog with myositis

Tissue stages similar to those of Sarcocystis neurona, the causative agent of equine protozoal myeloencephalitis, were identified in skeletal muscles of a dog. The dog, a 6-year-old Labrador retriever, was seropositive for Toxoplasma gondii infection and euthanized due to a history of polymyositis and progressive muscular atrophy. Histologically, 30, variably sized, microscopic, intracellular sarcocysts were observed in 60 sections of skeletal muscles taken from the neck, fore limbs and hind limbs. The cysts were only observed in inflamed skeletal muscles, but were mostly in myocytes at the periphery of areas infiltrated with leukocytes. Ultrastructurally, the cyst wall had villar protrusions consistent with sarcocysts. Immunohistochemistry with monoclonal S. neurona antibodies demonstrated positive labeling of zoites in merozoites or schizonts in the skeletal muscle interstitium, but no labeling of the sarcocysts. Initial PCR analysis with primers amplifying a genetic sequence encoding Apicomplexan 18s rRNA, and subsequent PCR analysis with differentiating primers indicated that the genetic sequences had 100% identity with sequences reported for S. neurona.     

Ultrastructure of sarcocysts from water buffalo in India

The ultrastructure of sarcocysts of macro- and microscopic species of Sarcocystis was compared from naturally infected water buffalo from India. Grossly visible sarcocysts had walls consisting of cauliflower-like villar protrusions, typical of S. fusiformis. The sarcocyst wall of the microscopic species of Sarcocystis was 6.4 microns thick and consisted of tightly packed conical villar protrusions that were 9.6 microns long and 3.7 microns wide at the base. At approximately 3 microns above the base, the distal two-thirds of the villar protrusion became conical shaped and was bent laterally at an angle of 45 degrees to the sarcocyst surface. The granular layer beneath the villar protrusions was 0.9 microns thick. In S. levinei the granular layer was 1.9 microns thick, the villar protrusions were narrow and it had a highly undulating primary cyst wall. Whether the microscopic S. levinei-like sarcocysts of Indian and Malaysian water buffalo are distinct species of Sarcocystis will require further investigation.     

Ultrastructural differentiation between sarcocysts of Sarcocystis hirsuta and Sarcocystis hominis

The ultrastructure of macroscopic (2-7 mm) Sarcocystis hirsuta sarcocysts from naturally infected cattle from New Zealand was compared with the ultrastructure of 222-day-old S. hominis in experimentally infected cattle in the United States. The villar protrusions of S. hirsuta were approximately 8 microns long, constricted at the base, expanded laterally in the mid-region and tapered distally. Some of the villar tips were folded to form two to four conical projections. The distal portion of the villar protrusions was bent at an angle of 45-90 degrees to the sarcocyst surface. The villar core contained numerous microfilaments and rows of electron-dense granules. The villar protrusions of S. hominis were cylindrical, oriented nearly perpendicularly to the sarcocyst surface, not constricted at their base and contained relatively few electron-dense granules. Although the sarcocysts of S. hirsuta were indistinguishable from those of S. hominis by light microscopy, they were distinguishable ultrastructurally.     

Ultrastructural and transmission evidence of Sarcocystis cruzi associated with eosinophilic myositis in cattle.

Skeletal muscle, diaphragm, tongue, esophagus and heart of beef carcasses that were condemned for eosinophilic myositis and those that were unaffected were collected at an abattoir in Colorado and studied to determine the involvement of Sarcocystis spp. All affected carcasses contained similar granulomatous lesions with adjacent infiltrations of leukocytes. Intact or fragments of sarcocysts were found within 32 of 363 granulomas, and whole sarcocysts were present in nearby unaffected muscle cells. Light and electron microscopic examinations revealed that sarcocysts, affected or unaffected by cellular response in condemned carcasses, as well as those found in unaffected carcasses, were consistent with those of S. cruzi. Transmission experiments confirmed that S. cruzi were present in all carcasses, and that dogs, but not cats, were the definitive hosts. The results of pepsin-HCl digestion assays showed that unaffected carcasses that were approved for human consumption generally contained more infective parasites than carcasses that were condemned for eosinophilic myositis. This study provides evidence to support the suggestion that dogs, rather than cats, and unaffected rather than eosinophilic myositis-affected carcasses, have greater potential for contributing to the perpetuation of eosinophilic myositis in the cattle industry.     

Genetic diversity among sea otter isolates of Toxoplasma gondii

Sea otters (Enhydra lutris) have been reported to become infected with Toxoplasma gondii and at times succumb to clinical disease. Here, we determined genotypes of 39 T. gondii isolates from 37 sea otters in two geographically distant locations (25 from California and 12 from Washington). Six genotypes were identified using 10 PCR-RFLP genetic markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico, and by DNA sequencing of loci SAG1 and GRA6 in 13 isolates. Of these 39 isolates, 13 (33%) were clonal Type II which can be further divided into two groups at the locus Apico. Two of the 39 isolates had Type II alleles at all loci except a Type I allele at locus L358. One isolate had Type II alleles at all loci except the Type I alleles at loci L358 and Apico. One isolate had Type III alleles at all loci except Type II alleles at SAG2 and Apico. Two sea otter isolates had a mixed infection. Twenty-one (54%) isolates had an unique allele at SAG1 locus. Further genotyping or DNA sequence analysis for 18 of these 21 isolates at loci SAG1 and GRA6 revealed that there were two different genotypes, including the previously identified Type X (four isolates) and a new genotype named Type A (14 isolates). The results from this study suggest that the sea otter isolates are genetically diverse.     

Prevalence of Sarcocystis species in sheep and goats in northern Nigeria.

Tissue samples comprising the oesophagus and diaphragm were collected from 400 sheep and 400 goats slaughtered at the abattoirs in the study area. Out of this number, 36 were positive for Sarcocystis cysts (sarcocysts) in sheep and 56 in goats. The sarcocysts in sheep measured 35.7 to 500 microns lengthwise and the cyst-wall 2.4 microns. They were identified to be Sarcocystis tenella. The cysts in goats measured 98 to 700 microns and the cyst-wall 2.7 microns. They were identified to be Sarcocystis capracanis. In both animals species, the sarcocysts were more frequent in the oesophagus than in the diaphragm. All sarcocysts seen were microscopic.     

Atresia ani in the dog: a retrospective study

Congenital anomalies of the rectum and anus are rare in dogs. The most frequently reported anomaly is atresia ani. Four types of atresia ani have been reported, including congenital anal stenosis (Type I); imperforate anus alone (Type II) or combined with more cranial termination of the rectum as a blind pouch (Type III); and discontinuity of the proximal rectum with normal anal and terminal rectal development (Type IV). An increased incidence was found in females and in several breeds, including miniature or toy poodles and Boston terriers. Surgical repair is the treatment of choice, but postoperative complications can occur, including fecal incontinence and colonic atony secondary to prolonged preoperative distension.