Ingestion of Neospora caninum tissue cysts by Mustela species.

Dogs are a definitive host of Neospora caninum, a protozoal parasite that causes abortion in cattle. Mustelids were tested to determine if they could also be definitive hosts. The procedures used were the same as those previously used to test dogs. Ermine (Mustela erminea), weasels (Mustela frenata) and ferrets (Mustela putorius) were fed N. caninum-infected mice. Neospora caninum oocysts were not observed. Mustelid faeces were fed to mice. The mice did not seroconvert and N. caninum was not detected in murine brains using tissue culture and PCR. The hypothesis that Mustela spp. are definitive hosts of N. caninum is not supported.

Repetitive abortion in Neospora-infected ewes.

Pregnant ewes inoculated with cultured Neospora caninum tachyzoites in 1995, 1996, or 1995 and 1996 aborted or delivered weak or clinically normal lambs in 1996. Nine of 11 ewes in the study had previously produced infected lambs or fetuses after being experimentally infected in 1995. Fetuses and lambs produced in 1996 showed histologic lesions and zoites indicative of Neospora. Serologic responses and production of infected fetuses/lambs indicated systemic neosporosis in the ewes during gestation, although tachyzoites could not be cultured from maternal tissues. The repetitive infection of fetuses, and resulting clinical and histopathologic findings in the present study are similar to those reported in naturally infected cattle, adding to the already established similarities of neosporosis between sheep and cattle.

Dogs are definitive hosts of Neospora caninum.

Dogs were investigated to determine if they are definitive hosts of Neospora caninum. Four dogs were fed N. caninum tissue cysts in infected mouse tissue, and two negative control dogs were fed uninfected mouse tissue. Dog faeces were examined daily for 30 days using a sucrose flotation technique. Three challenged dogs shed spherical to subspherical unsporulated oocysts, measuring 10 to 11 microns in diameter. Oocysts sporulated within 3 days and contained two sporocysts, each with four sporozoites. Outbred, inbred, and gamma-interferon knockout mice were inoculated with canine faecal extracts and monitored for evidence of neosporosis using a variety of morphologic, immunohistologic, serologic, and genetic analyses. Mice that received faeces from each dog observed to shed oocysts were demonstrated to have neosporosis by two or more techniques. One mouse was demonstrated to be infected with N. caninum by immunohistochemistry, ultrastructural analysis, and a species-specific PCR test. No evidence of neosporosis was observed in control animals. Based on this study, dogs are a definitive host of Neospora caninum.

Oral inoculation of cats with tissue cysts of Neospora caninum.

OBJECTIVE: To test the hypothesis that cats are definitive hosts of Neospora caninum. ANIMALS: 6 weaned male kittens obtained from 2 sources, and several dozen outbred mice. PROCEDURE: Cats were fed large numbers of 3 strains of N caninum: tissue cysts in buffered saline solution, mouse brain homogenates, and whole carcass homogenates from seropositive mice. Fecal specimens were examined for 4 weeks by use of flotation tests, and bioassays were performed in mice. One cat was inoculated parenterally with tachyzoites, to determine whether cats could respond serologically to N caninum. Tissue cysts from portions of oral inocula were cultured to verify viability. Indirect fluorescent antibody serologic testing, histologic and immunohistologic examinations, cell culture, and polymerase chain reaction procedures were performed 4 to 8 weeks after oral exposure, to seek evidence of infection of cats and mice. RESULTS: None of the cats or mice seroconverted to N caninum, with the exception of the single cat inoculated parenterally. Fecal shedding of oocysts was not observed, except for Isospora felis oocysts that were shed by 2 cats beginning prior to oral challenge exposure. Evidence of infection was not detected in tissues of cats or mice, with the exception of the parenterally inoculated cat. CONCLUSIONS: The hypothesis that cats are definitive hosts of N caninum is not supported. CLINICAL RELEVANCE: Extermination of cats in efforts to control bovine neosporosis is not warranted.

A protocol for the production of Neospora caninum tissue cysts in mice.

Identification of a definitive host for Neospora caninum has been inhibited by lack of an efficient method for producing bradyzoites, needed for oral infectivity trials. An improved protocol for producing bradyzoite-containing tissue cysts in mouse brains is described. Six variables, including mouse strain (Balb/C, CBA/Ca, and ICR), sex, N. caninum isolate (NC-2 and NC-Liverpool), tachyzoite inoculum dose, immunosuppression with methylprednisolone acetate (MPA), and sulfadiazine treatment were tested. Tissue cyst numbers were estimated using an immunohistologic staining procedure specific for bradyzoites. Male ICR mice (> or = 30 g) that were immunosuppressed with 2 mg MPA 7 days prior to and 2.5 mg MPA at the time of subcutaneous inoculation with 400,000 N. caninum tachyzoites produced the highest numbers of tissue cysts. Significant numbers were produced by methods using the NC-2 strain of N. caninum; however, protocols using NC-Liverpool produced greater numbers of tissue cysts. Sulfadiazine treatment did not appear to contribute to tissue cyst production. The procedure described is superior to previously described methods with regard to numbers of tissue cysts produced, protocol reproducibility, and survival of mice until tissue cyst formation.

Separation and cryopreservation of Neospora caninum tissue cysts from murine brain.

A protocol was developed for the separation, concentration enumeration, and cryopreservation of Neospora caninum tissue cysts from mouse brains. Brains from chronically infected mice were homogenized and tissue cysts counted in 10-microliters aliquots. Tissue cysts were separated from brain homogenates by centrifugation at 4,400 g on 35% (v/v) Percoll/phosphate-buffered saline (PBS) continuous-density gradients. After removal of the brain layer, the separated tissue cysts were concentrated by diluting the remaining solution with PBS and centrifuging at 500 g. The pellet was resuspended in PBS and tissue cysts were enumerated. Fifty percent of tissue cysts were recovered from brains centrifuged once and 64% from brains centrifuged twice. Tissue cysts were preserved with 7.5% dimethyl sulfoxide in horse serum at -60 C. After thawing, bradyzoites were digested in an acid/pepsin solution and placed onto Vero cell cultures. Neospora caninum tachyzoites were recovered from cell cultures, indicating that bradyzoites retained viability after concentration and cryopreservation. Separated tissue cysts ranged in diameter from 107 microns to 15 microns (average = 31 microns), and the average bradyzoite dimensions were 2 x 7.5 microns. These methods make it possible to store viable N. caninum tissue cysts for oral-infectivity trials and other studies.

Experimental neosporosis in pregnant ewes and their offspring.

Six groups of six pregnant ewes each were inoculated with 170,000 or 1,700,000 tachyzoites of Neospora caninum on gestation day 65, 90, or 120. All ewes seroconverted, and none showed signs of illness other than abortion. Regardless of the inoculum dose, all ewes inoculated on gestation day 65 aborted; ewes inoculated on gestation day 90 aborted, gave birth to weak lambs, or gave birth to clinically normal lambs; and all ewes inoculated on gestation day 120 gave birth to clinically normal lambs. Using an immunohistological procedure that stains bradyzoites, we observed protozoal cysts in brains of 11 of 29 (38%) aborted fetuses, in one of four (25%) weak lambs, and in seven of 18 (39%) clinically normal lambs. Cysts were not observed in extraneural tissues from two clinically normal lambs that had cysts in the brain. No evidence of infection was observed in tissues of five ewes examined using an immunohistological procedure that stains N. caninum tachyzoites and bradyzoites. Multifocal nonsuppurative encephalitis was observed in 46 of 51 (90%) aborted, weak, or clinically normal lambs. Cerebral necrosis, dystrophic mineralization, and meningitis were also commonly identified in live and aborted lambs (even when severely autolyzed). Nonsuppurative, necrotizing placentitis was observed in 15 of 17 (88%) placentas. Nonsuppurative myositis was common in fetuses but not in live lambs. Inflammation occurred less frequently in liver and lung. Clinical and pathological features of neosporosis in sheep closely resemble those of bovine neosporosis and ovine toxoplasmosis. Although abortion caused by naturally occurring neosporosis in sheep has not been reported, diagnosticians should carefully distinguish between neosporosis and toxoplasmosis in cases of ovine protozoal abortion unless future investigations exclude the likelihood of naturally acquired neosporosis in sheep.

Prevalence and epidemiology of trichostrongylids in Wyoming cattle with consideration of the inhibited development of Ostertagia ostertagi.

During 1988, monthly collections of abomasa and the cranial portion of the small intestine of 208 native Wyoming cattle were examined for adult and larval helminth parasites. Egg counts were performed on colonic faecal specimens. The animals ranged from 7 months to 8 years of age and were sampled at slaughter houses or diagnostic laboratories from five different counties in the State. Most of the cattle had ranged for a long time on non-irrigated, high altitude grass plains from about 1250 to 2500 m above sea level. All animals examined were found infected. Nematode genera (and number of species) found included Ostertagia (4), Trichostrongylus (2), Haemonchus (1), Cooperia (4) and Nematodirus (2). One trematode, Fasciola hepatica, and a cestode, Moniezia benedeni, also were found. Trichostrongylus axei, Trichostrongylus longispicularis and Cooperia lyrata were found for the first time in Wyoming, Ostertagia circumcincta was found for the first time in cattle in Wyoming, and Haemonchus placei, tentatively identified in a previous survey, was definitively confirmed. Predominant species (and prevalence) were Ostertagia ostertagi (98%), Cooperia oncophora (60.6%), Ostertagia bisonis (41.8%), Cooperia bisonis (33.6%) and T. Axei (27.9%). One or more of the four species of Ostertagia were found in all animals. Inhibited O. ostertagi and O. bisonis larvae predominated from November to April and reached a peak in January to comprise 88% of the total nematode burden for that month. The adult population of abomasal nematodes peaked in April and predominated thereafter until October. Haemonchus placei also spent the autumn-winter period in an inhibited state of development. Faecal egg counts were lowest during winter months, when the highest number of worms were present as L4 larvae, as determined by luminal and mucosal counts. Nematode populations in the small intestine peaked in June and persisted until November. The results of this study showed the magnitude and species composition of nematode parasitism in cattle raised on high altitude grass plains, underscored the inaccuracy of faecal egg counts as an indicator of worm burden during fall and winter seasons, and provided information on which anthelmintic treatment timing would be most efficacious.

Eosinophilic myositis and muscular sarcocystosis in the carcasses of slaughtered cattle and lambs.

Fifty-three bovine and 7 ovine carcasses condemned for having eosinophilic myositis were evaluated. Four (7.3%) of the bovine carcasses had a few, large local lesions in skeletal muscles (category A), and 49 (92.7%) of the bovine carcasses and 7 (100%) of the ovine carcasses had multiple, small, disseminated lesions in tongue, esophagus, heart, diaphragm, or skeletal muscles (category B). Tissue from carcasses of category B were evaluated for bacteria, viruses, selenium, and pathologic changes. Pathogenic bacteria and viruses were not isolated and selenium concentrations were normal. In category B, all carcasses had granulomas; of the 49 bovine carcasses and 7 ovine carcasses, 38 (77.6%) and 7 (100%), respectively, had one or more granulomas with opened dead sarcocysts. The data indicated that opened sarcocysts killed the host myocyte and adjacent myocytes and stroma, thereby initiating granuloma formation.

Encephalitic sarcocystosis in a newborn calf.

Sporozoan schizonts were seen in histologic sections of cerebrum from a Hereford calf that died immediately after birth. Schizonts appeared in endothelial cells of small vessels in the gray and white matter. Rosette and palisade configurations of merozoites in schizonts, as well as the size of schizonts (15 to 40 X 21 microns) and merozoites (8 microns), resembled Sarcocystis stages described in cattle, and differentiated the organisms from Toxoplasma that infects nerve cells and undergoes endodyogeny. Specific identification of the infecting agent was not successful because tests of sera from the calf were not possible. Developmental, morphologic, and ultrastructural differences in schizogonic stages of the heteroxenous sporozoan species infecting cattle are poorly known and are presently unreliable criteria for species identification. Encephalitis, meningitis, and necrosis occurred in cerebral, cerebellar, and brain stem gray and white matter infiltrated with plasmacytes, lymphocytes, and macrophages. Microthrombi were often seen in small blood vessels within the reaction foci. Herd-mate sera tested against S cruzi were negative. The herd from which the calf was born was exposed to a variety of free-ranging domestic, feral, and wild scavengers with free access to dead range animals. Consequently, the cattle were undoubtedly exposed to infective cysts in feed or water contaminated by feces from carnivores.

Experimental infection of dogs with Sarcocystis from wapiti.

Ten domestic dogs became infected with Sarcocystis when fed simple portions of heart, esophagus and diaphragm from a two-year-old female wapiti (Cervus canadensis). The prepatent period was 14 days in all exposed dogs; the patent period ranged from 8 to 20 days. Neither the 10 control dogs, nor two dogs fed sporocysts collected from the infected dogs passed sporocysts within the study period. Sporocysts averaged 16.5 by 11.1 micron in size.

Formation of sulfhydryl groups in the walls of Eimeria stiedai and E. tenella oocysts subjected to in vitro excystation.

Eimeria stiedai or Eimeria tenella oocysts were incubated in aqueous cysteine hydrochloride (cysHCl) under carbon dioxide (CO2), aqueous cysHCl under air, water under CO2 or water under air, and analyzed for sulfhydryl (-SH) groups. The cysHCl-CO2 treatment produced more -SH groups than the other treatments and was effective in allowing activation of intact and sodium hypochlorite (NaOCl)-treated E. stiedai oocysts as well as NaOCl-treated E. tenella oocysts. The CO2-cysHCl complex may act directly on the oocyst wall, especially in the micropylar region, to unmask lipid-shielded disulfide bridges, which are reduced to -SH groups. The reduction apparently disturbs the protein superstructure of the oocyst wall, promotes opening of the micropyle, and changes the impermeable state of the sporulated oocyst.

The effects of selected gases on excystation of coccidian oocysts.

The mechanism of CO2 action in changing coccidian oocyst wall permeability was indirectly studied by substituting NO, NO2, N2O, H2S, SO2, CH4, NH3, and 8M urea in place of CO2 in an established excystation procedure. Changes in oocyst wall permeability of Eimeria stiedai, E. bovis, and E. tenella were determined by incubation in test gases and cysteine HCl followed by attempted activation of sporozoites by trypsin and bile and staining of intraoocyst components with methylene blue. The gases CH4, NO2, and N2O were negative for all 3 tests, as were SO2, NH3, and 8M urea which, in addition, were toxic to the oocysts. Both H2S and NO were capable of mimicking the action of CO2 and are related chemically to the reducing agent, and thus tend to underscore its importance in excystation. It now appears that the role of CO2 is that of an allosteric effector enhancing the action of the reducing agent.