Experimental microcyst sarcocystis infection in lambs: pathology

Six 34- to 42-day-old lambs raised in coccidia-free conditions were inoculated with 70,000 sporocysts derived from sheep heart with microscopic sarcocysts. Fever and mild anorexia occurred between 25 and 33 days after inoculation. A transient anaemia was most marked 32 days after inoculation. Lambs were killed and examined 14, 25, 33, 42, 60 and 81 days after inoculation. Gross lesions were absent. First and second generation meronts were present in endothelial cells at 25 and 33 days after inoculation. Meronts were most numerous in kidney glomeruli. Developing sarcocysts were rare at 42 days after inoculation. Sarcocysts with a primary cyst wall 2 to 3 micron thick composed of palisade projections were common at 60 and 81 days after inoculation in striated muscle and brain. Mild to severe striated muscle myositis and non-suppurative encephalitis or encephalomyelitis with glial nodules were observed 25 to 81 days after inoculation. Sarcocyst frequency varied considerably; it was highest in myocardium, M vastus intermedius, M vastus medialis, M extensor carpi radialis and tongue muscle and was lowest in M masseter.     

Life cycle of Isospora suis in gnotobiotic and conventionalized piglets

Isospora suis had 3 asexual and 1 sexual intra-intestinal conventional life cycle. The first asexual generation was most prominent at 2 days p.i. (post inoculation) and produced 2–7 merozoites. The second-generation meronts were prevalent at 3–4 days p.i. and produced 2–12 large merozoites. At 4–5 days p.i. the third generation meronts were prominent and produced 4–24 small crescent shaped merozoites. Mature sexual stages were most prominent at 5–6 days p.i. The stages were most numeroous in the distal half of the small intestine. At 8–9 days p.i. stages morphologically similar to the second generation of meronts reappeared, followed by the further development into third generation merozoites and sexual stages. This was reflected in a prepatent period of 5 days and a biphasic patent period of 5–8 or 9, and 11–14 days p.i.Intraperitoneal injection of liver/spleen and intestinal lymph node homogenates, respectively, from piglets infected 24 and 48 h, previously with high doses of oocysts, resulted in a patent infection 10–12 days post inoculation of the donor piglets. No differences in the life cycle of I. suis were observed between conventionalized and germ-free piglets. An extra-intestinal life cycle of I. suis related to the second patent period was postulated.     

Ontogenetic Development of Creatine Phosphokinase in Skeletal Muscles and Heart from Pigs

Creatine Phosphokinase (CPK) in striated muscles shows only small changes in activity before birth. After birth and during the first month of extrauterine life the activity increases rapidly. The largest increase is seen in muscles with a glycolytic energy metabolism (m. long, dorsi) and the smallest in muscles with an oxydative energy metabolism (m. flexor dig. ped. sup.). The differences between these groups of muscles are statistically significant. In heart tissue the increase in CPK activity is lower, the levels amounting to 40 to 47 % of those in striated muscles.Early in fetal life only the BB isoenzyme is found in striated muscles. Synthesis of M subunits of GPK starts between day 76 and 65 before birth and increases rapidly after this time leading to disappearance of the BB isoenzyme 24 days prior to birth and of the MB isoenzyme at birth. In muscles with an oxydative as well as in muscles with a glycolytic metabolism all GPK activity after birth is caused by the MM isoenzyme.All three isoenzymes are present in heart tissue at the earliest prenatal stage investigated, the pattern being dominated by the BB isoenzyme. During further differentiation the MM isoenzyme increases and the BB isoenzyme decreases. The development is completed during the first month after birth with a final isoenzyme composition of 81 % MM and 19 % MB isoenzyme. kw|Keywords|k]pigs; k]ontogenesis; k]creatine phosphokinase; k]activity; k]isoenzymes     

Lesions in sheep inoculated with Sarcocystis tenella sporocysts from canine feces

Sarcocystosis was studied in 37 sheep after oral inoculation with 10(4)-5 x 10(7) sporocysts of Sarcocystis tenella from canine feces. Two sheep inoculated with 2.5 x 10(7) and 5 x 10(7) sporocysts became moribund 16 and 19 days post-inoculation (DPI), respectively, due to occlusion of arteries of gut and mesentery by first generation meronts. Sheep inoculated with 10(7) sporocysts remained clinically normal until 21 DPI and those inoculated with 10(5)-10(6) became ill 24-28 DPI due to anemia coincident with maturation of second generation meronts. Inflammation, hepatitis and myocarditis were the main lesions of acute and subacute ovine sarcocystosis. Inflammation began to subside by the time (75 DPI) sarcocysts matured. Sarcocystis-induced encephalitis was distinguished from naturally occurring myelomalacia in sheep caused by an unidentified sporozoan.     

Myopathy and encephalopathy in chick embryos experimentally infected with Akabane virus

Chick embryos infected with Akabane virus by the yolk sac route at 6 days of incubation developed polymyositis and encephalitis. At 3 to 7 days after inoculation, skeletal muscles had myotubule degeneration, clumping of muscle cell nuclei, and infiltration of heterophils; dysplasia and aplasia were evident at 9 to 15 days after inoculation. Changes in the cerebral neostriatum and optic lobes at 2 to 11 days after inoculation included necrosis of primordial nervous tissue, hemorrhages, and hyperplasia of the vascular endothelial cells. Cavities were in nervous tissue subsequent to encephalitis. Hydranencephaly and vascular wall thickening were found 13 and 15 days after inoculation. Embryos infected intravenously at 15 days incubation had foci of encephalitis 3 to 6 days after inoculation, including neuronal degeneration, neuroglial hyperplasia, vascular endothelial proliferation, and heterophil infiltration.     

Equine protozoal myeloencephalitis in a pony

A 10-year-old pony died 5 days after the onset of a nervous disorder. Necropsy revealed a yellowish area of discoloration (1.5 by 1 cm) in the medulla oblongata. Microscopically, necrosis and nonsuppurative myeloencephalitis were found in the medulla oblongata. Immature and mature meronts (25 by 10 microns) were seen in neural tissue and in capillaries of the brain stem. Organisms were similar structurally to those seen in equine protozoal myeloencephalitis of horses.     

Myodegeneration in Kentucky white-tailed deer

Skeletal or cardiac myodegeneration, or both, was observed in 4 free-ranging female white-tailed deer that were found dead or recumbent and unable to rise. Gross pathologic findings included white or chalky streaks in heart or skeletal musculature. Degeneration, necrosis, and mineralization were observed microscopically in affected striated muscles. Although the precise cause of myodegeneration was not determined, changes were considered compatible with nutritional or exertional myopathy.     

Pathology of specific-pathogen-free chickens inoculated with H5N1 avian influenza viruses isolated in Japan in 2004

Specific-pathogen-free chickens inoculated with H5N1 highly pathogenic avian influenza (HPAI) viruses isolated in Japan in 2004 were investigated pathologically. The chickens inoculated intravenously with the viruses died within 26 hr after inoculation. Macroscopically, minimal necrosis of the tip of the comb, and hemorrhages of the palpebral conjunctiva, liver, cerebellum, and muscles were rarely observed. Histologically, dead chickens had minimal focal necrosis of hepatocytes with fibrinous thrombi in sinusoids, mild necrosis of splenic ellipsoids with fibrinous exudation, minimal necrosis of the brain, mild necrosis of epidermal cells of the comb with congestion of the lamina propria, and hemorrhages and edema of the lamina propria of the conjunctiva. Virus antigens were seen in the sinusoidal endothelial cells and hepatocytes in the liver, the capillary endothelial cells of the spleen, the capillary endothelial cells and cardiac myocytes in the heart, the capillary endothelial cells and necrotic nerve cells in the brain, the capillary endothelial cells in the lamina propria of the comb, the renal tubular epithelial cells, and the pancreatic acinar cells. The chickens inoculated by natural infectious routes died within 1-4 days after inoculation. Macroscopically, some chickens had hemorrhages in the conjunctiva, edematous swelling of the face and wattles, hydropericardium, hemorrhages of the proventriculus and bursa of Fabricius, increased secretion of tracheal mucus, and congestion and edema of lungs. Histologic lesions by natural infectious routes were similar to those by intravenous inoculation, except for the pancreatic necrosis. This study suggests H5N1 HPAI viruses isolated in Japan in 2004 cause pathologic conditions similar to natural cases.     

Experimental microcyst sarcocystis infection in lambs: serology and immunohistochemistry

Density gradient centrifugation using a performed self generated gradient of colloidal silica enabled the isolation of microscopic sheep sarcocystis cystozoites, free from heart muscle contamination. The efficiency of separation of cystozoites from residual heart muscle after digestion in pepsin and hydrochloric acid was 63 to 92 per cent. Antigens from cystozoites were used on enzyme-linked immunosorbent assays (ELISA) of plasma from six coccidia-free lambs infected once orally with 70,000 microcystic sheep sarcocystis sporocysts and for raising antisera in rabbits. Use of an anti-sheep IgM conjugate in the ELISA showed that anti-sarcocystis IgM production was transitory, appearing five to 10 days after infection, peaking in concentration at 42 days and following the peak of the acute phase of infection (32 and 33 days) in the lambs. In contrast, total anti-sarcocystis immunoglobulins, detected by ELISA, increased from five to 21 days after infection and continued to increase until the lambs were killed (the last at 81 days) and was more useful in diagnosing chronic infection. No cross reactions between microcystic sheep sarcocystis and Toxoplasma gondii or Eimeria species of sheep were observed. A peroxidase anti-peroxidase test, using rabbit anti-sarcocystis sera, detected second generation meronts and sarcocysts in fixed tissues from infected lambs making it useful for the diagnosis of acute or chronic disease post mortem.     

Bluetongue virus-induced hydranencephaly in cattle

Direct inoculation of bluetongue virus into 125-day bovine fetuses resulted in development of hydranencephaly. The earliest lesions after virus inoculation were a severe necrotizing encephalitis, which was most prominent in the cerebrum, and an associated nonsuppurative meningitis. At birth, the brains of infected fetuses had thin-walled cerebral hemispheres, dilated lateral ventricles, and cerebral cysts. No gross lesions were observed in the brain stem or cerebellum. Two morphologically different lesions were present in the brain of a fetus sacrificed 20 days after virus inoculation. There were discrete foci of hemorrhagic cerebral necrosis that resembled infarcts and widespread microcavitations of the intermediate and subventricular zones. Changes consistent with vascular damage were present in the brains of fetuses sacrificed 12 and 20 days after virus inoculation. Calves with bluetongue virus-induced hydranencephaly would have poor viability, but they would not be expected to have any significance as virus reservoirs.     

Lymphocytic depletion of bursa of Fabricius and thymus in chickens inoculated with Escherichia coli

Specific-pathogen-free 10-week-old chickens were inoculated via the air sac with Escherichia coli and showed lymphocytic depletion of bursa of Fabricius and thymus. In experiment I, chickens were necropsied at 12 and 24 hours, 2, 3, and 5 days after inoculation. At 12 hours after inoculation there was lymphocytic depletion in the medulla of lymphoid follicles of the bursa. At 24 hours after inoculation there was lymphocytic depletion also in the cortex of follicles and edema in interfollicular interstitium and follicular medulla. At 2 and 3 days after inoculation there were more marked lymphocytic depletion in medulla and cortex, and fibrosis in interfollicular interstitium. Partial repopulation of follicles with lymphocytes was seen at 5 days after inoculation. In the thymus, lymphocytic depletion occurred in the cortex. At 12 hours after inoculation, lymphocytic necrosis increased in number more than that of control chickens. The width of the cortex and medulla decreased. At 24 hours after inoculation, lymphocytic necrosis increased further. At 2 to 5 days after inoculation, the boundary between the cortex and medulla of lobules was obscure and cellular elements of the cortex and medulla were mingled. In experiment II, chickens were necropsied as in experiment I and also at 8 and 14 days after inoculation. The relative weights of the bursa and thymus reduced rapidly to minimal relative weights at 8 days after inoculation. At 14 days after inoculation, both bursa and thymus had normal relative weights and histological structures. These findings indicate that E. coli infection may induce transient lymphocytic depletion of lymphoid tissues in the chicken.     

Pathology of cloacal bursae of gnotobiotic turkeys orally inoculated with Escherichia coli

Cloacal bursae from three-week-old gnotobiotic turkeys were examined by light and electron microscopy and bacteriologic techniques at 1, 2, 5, 8, 12, and 16 days after oral inoculation of highly virulent (group 1) and weakly virulent (group 2) Escherichia coli. In both groups a significant decrease in follicular volume and increase in interstitial volume were associated with infection. Follicular cortical, follicular medullary and total follicular transectional areas increased with time in inoculated and control turkeys. In group 1, granulocytic inflammation developed in bursae on day 1 and diminished by day 8 after inoculation. Microabscesses were present on days 5 and 8 after inoculation in less than 1% of follicles. Bacteria were seen in few follicular medullae on days 5 and 8 after inoculation; bacteremia was detected on days 1, 2, and 5 after inoculation. In group 2, pyogranulomatous bursitis was first seen at five days after inoculation and became progressively more severe with time. Follicular alterations in group 2 included abscessation, lymphocyte necrosis, reticuloepithelial hyperplasia and perifollicular fibroplasia. Ultrastructurally, follicular pads had degenerate and necrotic epithelial cells, intercellular edema, and cystic spaces that contained acidic mucosubstances and laminar deposits of calcium salts. Bacteria were seen within necrotic centers of follicular abscesses, in phagosomes of macrophages and multinucleate giant cells and within extracellular spaces of follicular pads and follicular medullae from day 5 to day 16 after inoculation; no bacteremia was detected. We conclude that E. coli passes through the bursal follicle-associated epithelium and replicates within follicular medullae, that extensive follicular necrosis is associated with persistence of E. coli in follicular medullae, and that E. coli of low virulence may cause severe pyogranulomatous bursitis in young turkeys without causing the respiratory or systemic diseases which are commonly associated with organisms of high virulence.     

Plasma cell quantitation in the gland of Harder during infectious bursal disease virus infection of 3-week-old broiler chickens

Histopathologic changes in the gland of Harder (GH) and bursa of Fabricius (BF) were studied during and after infection of 3-week-old broiler chickens with a pathogenic strain of infectious bursal disease virus (IBDV). Plasma cell (PC) necrosis in the GH was seen from 5 to 14 days postinoculation (PI), BF follicular necrosis was observed from 1 to 7 days PI. PC numbers within the GH, counted for 28 days after inoculation, declined and were reduced (P less than 0.01) by 51% at 7 days after inoculation, which coincided with PC necrosis and heterophil infiltration. After 14 days PI, however, PC numbers were equal to those in uninfected controls. Since the GH is a major antibody-producing site in the paraocular area, the reduction in PC number at 7 days PI might indicate compromise of local immunity in the paraocular region and upper respiratory tract associated with IBD.     

The histopathogenesis of paralytic rabies in six-week-old C57BL/6J mice following inoculation of the CVS-11 strain into the right triceps surae muscle

A fatal encephalomyelitis was developed after intracerebral and hind limb inoculation of in 6-week-old C57BL/6J mice by the inoculation of fixed rabies virus (CVS-11 strain), intracerebrally and into hind. After the intracerebral inoculation, virus antigens were detected in the cerebral cortex and hippocampus at 2 days postinoculation (PI), and later spread centrifugally to thalamus, brain stem, cerebellum, spinal cord and spinal ganglia. At 4 days PI, severe apoptosis and DNA fragmentation were observed in the hippocampus and cerebral cortex. All mice infected intracerebrally were dead without limb paralysis at from 10 to 11 days PI. In contrast, mice infected with virus intramuscularly were persistently observed virus antigens in the myocytes at the site of inoculation from 2 days PI. At 4 days PI, the antigens were demonstrated in the spinal dorsal root ganglia, spinal cord and muscle spindles without their detection in the cerebrum and hippocampus. There were no apoptosis in the spinal cord and dorsal root ganglia, however hind limb paralysis was found in all infected mice. Hind limb paralysis was progressed to quadriparalysis, and mice were dead from 11 to 13 days PI. From 4 days PI, necrosis of neuron was observed in the the spinal and dorsal ganglia with infiltration of lymphocyte. This study suggested that the necrosis of spinal neurons was more important to cause the paralysis of hind limb rather than the severe cerebral infection and apoptosis in C57BL/6J mice infected with CVS-11 strain. The virus primarily replicated in the muscles was ascended the spinal cord via afferent fibers and retrogradely invaded the cerebrum, and with subsequent spread to muscle spindles.     

Studies on experimental enteric salmonellosis in ponies.

Clinical, bacteriological, serological and haematological observations were made on 13 adult ponies orally inoculated with Salmonella typhimurium. The results were compared to two control ponies and four others infected by accidental transmission. The clinical responses in inoculated ponies included pyrexia lasting four days and neutropaenia during the first five days after inoculation followed by a neutrophilia. Pyrexia and neutropaenia was associated with maximal shedding of organisms in the rectal faeces. Changes in the character of the faeces occurred between one and two days after inoculation and appeared to be associated with the serological response. Serological responses occurred in all the infected ponies except one. At necropsy, of the 14 ponies with positive cultures in the colon, seven had negative cultures in the rectal faeces. Serological studies performed on 43 clinically normal horses indicated a correlation between age and salmonella agglutination titre.     

Pulmonary lesions in lambs experimentally infected with ovine adenovirus 5 strain RTS-42

Twelve lambs were inoculated transtracheally and intranasally with Mastadenovirus ovi 5 strain RTS-42 and killed sequentially. Pulmonary lesions were studied by light and electron microscopy. Four lambs served as sham inoculated controls. Pulmonary lesions consisted of multifocal areas of bronchiolitis and alveolitis associated with necrosis and sloughing of isolated type I and type II alveolar epithelial cells and nonciliated bronchiolar epithelial cells. This was followed rapidly by hyperplasia of the remaining epithelium and repair of the damage. A cellular infiltrate of neutrophils and macrophages began at 2 days after inoculation, peaked at 4 days after inoculation, gradually diminished until minimal at 12 days after inoculation, and was resolved at 21 days after inoculation. Surfactant was abundant and, along with debris, was removed from the alveoli by macrophages. Clinical disease was not seen, but lesions were believed to be sufficient to allow bacteria to colonize the lungs and cause severe disease.     

Failure to detect prion protein (PrPres) by immunohistochemistry in striated muscle tissues of animals experimentally inoculated with agents of transmissible spongiform encephalopathy

Transmissible spongiform encephalopathies (TSEs) are fatal neurologic diseases. Infection by the causative agent, a prion, induces accumulations of an abnormal form of prion protein (PrP(res)) in tissues of nervous and lymphoid systems. Presence of characteristic histopathologic changes (spongiform encephalopathy) and detection of protease-resistant PrP(res) in neural and lymphoid tissues are the basis of currently available methods for diagnosis of TSEs. In this study, samples of striated muscle tissues (tongue, heart, diaphragm, and masseter muscle) from 20 animals (cattle, sheep, elk, and raccoons) were examined for PrP(res) by immunohistochemistry (IHC). All the animals had developed a TSE after experimental inoculation. PrP(res) was found by IHC in the brain but not in the muscle tissues of all the animals examined. These findings are contradictory to recently published reports of laboratory animals with TSEs, where these altered prion proteins were detected in tongue and other striated muscles. Further testing of muscle tissues is needed to confirm the findings of the present study.     

Parasitism of Toxocara canis larvae in Japanese quails by inoculation of the ascarid eggs.

The distribution of T. canis larvae and pathological changes caused by them were studied in Japanese quails orally inoculated with 1,500, 4,000 or 15,000 embryonated eggs. Larvae were distributed mainly in the liver and, to lesser extent, in the muscles, brain, eyes and other organs. The number of larvae varied from 7 to 3,346, and from 1 to 288 in the liver and muscles (breast and legs), respectively. A small number of larvae were also recovered from the heart, gizzard, brain and eyes. In the groups of quails inoculated with 4,000 or 15,000 eggs, small white foci were observed on the surface of the liver 6 or 12 hr after inoculation. Histopathological examinations revealed necrotic lesions, leukocytic infiltration, granuloma and nodular lesions. The pathological changes became more serious with the large size of inoculum and days after inoculation.     

Experimental bovine respiratory syncytial virus infection in conventional calves: ultrastructural respiratory lesions

The morphogenesis and repair of airway and alveolar injury induced by bovine respiratory syncytial virus (BRSV) was studied ultrastructurally in conventional calves to characterize pulmonary cell types susceptible to viral infection and cytopathologic changes associated with infection. Viral nucleocapsids and budding virions were present in tracheal and bronchial ciliated and nonciliated epithelial cells and mucous cells 3, 5, and 7 days after inoculation and in bronchiolar ciliated and nonciliated epithelial cells 5 days after inoculation. Mild interstitial pneumonia was observed 5 days after inoculation and was characterized by swelling of type 1 and type 2 alveolar epithelial cells, interstitial edema, and infiltration by lymphocytes and macrophages. Viral assembly and release in tracheal and bronchial epithelial cells was associated with loss of cilia from ciliated cells, formation of syncytial epithelial cells, swelling of mitochondria and endoplasmic reticulum, and cell necrosis. Neutrophils, lymphocytes, and macrophages were present in close association with the viral-infected and damaged epithelial cells. There was intercurrent hyperplasia of basal epithelial cells that, in association with other epithelial lesions, resulted in the loss of normal ciliated epithelium in these airways 5 and 7 days after inoculation. Regeneration of airway epithelium was largely completed by 10 days after inoculation, except in 1 of 4 calves that had failure of epithelial repair and that developed secondary bacterial pneumonia. Pulmonary ultrastructure in BRSV-inoculated calves 30 days after inoculation was indistinguishable from that in controls. The results demonstrated that BRSV can induce reversible alterations in airway epithelium, which may cause depression of mucociliary clearance and thereby enhance susceptibility to bacterial infection.     

Ultrastructure of the intestinal mucosa in pigs experimentally inoculated with an edema disease-producing strain of Escherichia coli (0139:K12:H1)

The intestinal tissues from 11 pigs orally inoculated with Escherichia coli (E. coli, 0139:K12:H1) were examined by transmission electron microscopy. The colonization of E. coli along the small intestinal mucosa was found in seven principals without any major changes in the enterocytes from day 2 to day 7 after inoculation when the experiment was terminated. Lesions of vessels of the intestinal mucosa could be detected as early as two days after inoculation and persisted until the experiment was terminated. Lesions consisted of endothelial swelling and vacuolation, subendothelial fibrin deposition, perivascular edema, microthrombus formation, endothelial proliferation, and necrosis of the tunica media. The possible pathogenesis of the disease is discussed.