Complete validation of a unique digestion assay to detect Trichinella larvae in horse meat demonstrates the reliability of this assay for meeting food safety and trade requirements.

A tissue digestion assay using a double separatory funnel procedure for the detection of Trichinella larvae in horse meat was validated for application in food safety programs and trade. The assay consisted of a pepsin-HCl digestion step to release larvae from muscle tissue and two sequential sedimentation steps in separatory funnels to recover and concentrate larvae for detection with a stereomicroscope. With defined critical control points, the assay was conducted within a quality assurance system compliant with International Organization for Standardization-International Electrotechnical Commission (ISO/IEC) 17025 guidelines. Samples used in the validation were obtained from horses experimentally infected with Trichinella spiralis to obtain a range of muscle larvae densities. One-, 5-, and 10-g samples of infected tissue were combined with 99, 95, and 90 g, respectively, of known negative horse tissue to create a 100-g sample for testing. Samples of 5 and 10 g were more likely to be positive than were 1-g samples when larval densities were less than three larvae per gram (lpg). This difference is important because ingested meat with 1 lpg is considered the threshold for clinical disease in humans. Using a 5-g sample size, all samples containing 1.3 to 2 lpg were detected, and 60 to 100% of samples with infected horse meat containing 0.1 to 0.7 lpg were detected. In this study, the double separatory funnel digestion assay was efficient and reliable for its intended use in food safety and trade. This procedure is the only digestion assay for Trichinella in horse meat that has been validated as consistent and effective at critical levels of sensitivity.

Herpesvirus infection in woodland caribou in Alberta, Canada.

Sera and genital swabs collected from 121 adult woodland caribou (Rangifer tarandus caribou) in five subpopulations in northern Alberta, Canada, between December 1997 and October 1999, were examined for evidence of infection with herpesviruses or pestiviruses. No virus was isolated from sera or swabs, and no antibodies against bovine viral diarrhea virus were detected. However, 63 (52%) of the 121 animals had neutralizing antibody titers against bovine herpesvirus 1. There was sufficient serum from 37 of the 121 caribou to allow parallel testing for antibodies against a new alphaherpesvirus isolated from an elk (Cervus elaphus nelsoni), and 20 animals had antibodies against this virus. Paired sera collected 11 mo apart from 14 caribou showed seroconversion in seven animals, indicating that an active herpesvirus infection was present. Virus neutralization data suggest that these caribou are infected with a distinct alphaherpesvirus.

Experimental Brucella abortus infection in wolves.

Four juvenile male wolves (Canis lupus) each received an oral dose of 1.6-1.7 x 10(12) colony-forming units of Brucella abortus biovar 1 isolated from a bison (Bison bison) in Wood Buffalo National Park (Canada), and two others served as negative controls. Infected wolves did not show clinical signs of disease but did develop high Brucella antibody titers. Small numbers of B. abortus were excreted sporadically in feces until day 50 postinoculation (PI). Very small numbers of the bacterium were isolated from urine of only one wolf late on the same day that it was infected, and very small numbers of colonies of B. abortus were obtained from buccal swabs of three wolves for up to 48 hr PI. Two infected wolves euthanized 6 mo after the start of the experiment had no lesions, and colonies of B. abortus were isolated from thymus and most major lymph nodes. The other two infected wolves euthanized 12 mo after the start of the experiment had no lesions, and smaller numbers of brucellae were recovered from fewer lymph nodes compared with the wolves killed 6 mo earlier. The sporadic excretion of very small numbers of brucellae by the wolves was insignificant when compared with the infective dose for cattle. Brucella abortus, brucellosis, Canis lupus, pathogenesis, serology, wolf.

Attenuation of a virulent type 2 bovine viral diarrhea virus.

The purpose of this study was to produce an attenuated bovine viral diarrhea virus (BVDV) type 2 strain as a tool for identifying potential virulence markers in the BVDV2 genome. The attenuation of the virulent strain, BVDV2-24515, was accomplished by in vivo and in vitro passage. The strain was initially used to infect an elk (Cervus elaphus) [J. Wildl. Dis. 35 (1999) 671], re-isolated at 7 days post-inoculation from serum, and then subsequently passaged 56 times in cell culture. Two groups of calves were inoculated intranasally with either BVDV2-24515 or the putative attenuated virus, designated BVDV2-LATT. Calves inoculated with BVDV2-24515 had cumulative clinical scores which ranged from 6 to 53. Clinical signs in these calves consisted of anorexia, depression, dehydration, diarrhea (+/-bloody), and pneumonia. Several calves developed leukocytopenia, primarily a neutrocytopenia, and presented lesions of enteritis or pneumonia at necropsy. In contrast, cattle inoculated with BVDV2-LATT had cumulative clinical scores which ranged from 0 to 2. This was not significantly different from that of controls which received no virus (range: 0-1). Calves inoculated with BVDV2-LATT produced high neutralizing antibody titers against BVDV2. Thus, in addition to its potential use as a tool for identifying virulence markers, the attenuated virus is also worthy of further study as a candidate virus for inclusion in a modified-live vaccine.

Barkas effect for antiproton stopping in H2.

We report the stopping power of molecular hydrogen for antiprotons of kinetic energy above the maximum (approximately 100 keV) with the purpose of comparing with the proton one. Our result is consistent with a positive difference in antiproton-proton stopping powers above approximately 250 keV and with a maximum difference between the stopping powers of 21%+/-3% at around 600 keV.

[Oral contraceptive and breast cancer: a case-control study]. / Contraceptivos orais e câncer de mama: estudo de casos e controles.

OBJECTIVE: To investigate the association between breast cancer and the duration of use of oral contraceptives (OC), and age it started to be used in a population of Pelotas, Southern Brazil. METHODS: There were identified 250 incident cases of breast cancer in patients aged 20 to 60 years from records of pathology laboratories and there were enrolled 1,020 controls drawn from hospital and neighbourhood population. For 90 cases identified in Pelotas, 270 hospital controls and 270 neighbourhood controls were selected, for another 78 cases in Pelotas, 234 controls were selected, and for 82 cases from other municipalities, 246 hospital controls were selected. Controls were matched by age. Adjusted analysis was performed using conditional logistic regression. RESULTS: No association between oral contraceptive use and breast cancer was found (OR=1.1;CI95% 0.7 - 1.6 for hospital controls, and OR=0.9;CI95% 0.6 - 1.6 for neighbourhood controls) neither for different duration of use or starting age. To increase the test power, 250 cases and all 1020 controls were analyzed together, and an odds ratio of 1.6 (CI95% 1.0 - 2.4) was found for women older than 45 years of age who had been using oral contraceptives for five years or more. CONCLUSIONS: No evidence was found of a general association between oral contraceptive use and breast cancer. When analyzing the whole date set, with all neighbourhood and hospital controls together, for women older than 45 years of age who had been using oral contraceptives for more than 5 years, it was found an increased risk almost statistically significant (p=0.05).

Duration of bluetongue viremia in experimentally infected American bison.

Six yearling American bison (Bison bison bison) bulls and one yearling ewe (Ovis aries) were inoculated intradermally and subcutaneously with 2 x 10(5) plaque forming units (pfu) of bluetongue (BT) virus serotype 11. Two uninoculated yearling bison bulls served as negative controls. Blood samples were collected for serology and virus isolation on 0, 4, 7, 11 and 14 days post-inoculation (dpi) and every 2 wk thereafter to 127 dpi. Every 4 wk a new ewe was inoculated with a pooled sample of whole blood from the six infected bison, and each sheep was monitored for 28 days for clinical signs of BT and seroconversion. Bluetongue viremia was detected in all six inoculated bison starting at 4 to 28 dpi and was no longer detectable from 42 dpi onward. Pooled blood samples collected at 28, 56, 84 and 112 dpi from the six infected bison were not infectious for sheep. The six infected bison seroconverted by 11 to 28 dpi on a competitive enzyme-linked immunosorbent assay and by 28 dpi on the serum neutralization test, and all remained seropositive thereafter. No clinical signs or lesions attributable to BT were observed in the infected bison or controls. There was evidence that a small amount of epizootic hemorrhagic disease virus type 2 had been present in the BT virus inoculum; reasons are given for concluding that this did not affect the results of the BT study.

Antigenic and molecular characterization of a herpesvirus isolated from a North American elk.

OBJECTIVE: To determine whether a herpesvirus isolated from the semen of a North American elk was related to bovine herpesvirus 1 (BHV-1). SAMPLE POPULATION: Semen from 1 healthy bull elk and 2 subtypes of BHV-1 (BHV-1.1 and BHV-1.2). PROCEDURES: A virus with cytopathic and electron microscopic characteristics consistent with an alpha-herpesvirus was isolated from elk semen, using fetal bovine kidney cells. Cross-neutralization assays were performed with antisera against BHV-1 and the elk herpesvirus (EIkHV). Restriction endonuclease digests of EIkHV DNA were compared with digests of BHV-1.1 and BHV-1.2 DNA. A portion of the ElkHV DNA polymerase gene was amplified with consensus primers by use of the polymerase chain reaction and sequenced. Sequence was compared with known sequences of other herpesviruses. An immunoperoxidase monolayer assay was used to determine reactivities of 22 BHV-1-specific monoclonal antibodies (mAb) against ElkHV. In vitro neutralizing activities of the reactive mAb were determined by use of a microneutralization assay. RESULTS: Results of cross-neutralization assays indicated that ElkHV was serologically related to BHV-1. Endonuclease digestion of ElkHV DNA generated fragments that were distinct from those of BHV-1. Nucleotide sequencing confirmed that ElkHV is an alphaherpesvirus closely related to but distinct from BHV-1. Six of 22 BHV-1-specific mAb reacted against ElkHV; 2 of these 6 also neutralized in vitro infectivity of ElkHV. CONCLUSIONS AND CLINICAL RELEVANCE: ElkHV is antigenically and genetically distinguishable from BHV-1. However, the viruses are serologically related and share at least 6 antigenic determinants, one of which is a major neutralizing determinant.

Viremia and virus shedding in elk infected with type 1 and virulent type 2 bovine viral diarrhea virus.

In order to determine whether elk (Cervus elaphus) could be infected with and shed bovine viral diarrhea virus (BVDV) and to determine whether BVDV could cause disease in elk, two groups of five yearling elk each and two control cattle were experimentally inoculated intranasally with type 1 Singer strain or a virulent type 2 isolate of BVDV, strain 24515. Virulence of the type 2 isolate was confirmed by inoculation of a control bovine cow which developed diarrhea, dehydration, severe thrombocytopenia, hemorrhages, and enteritis with intestinal necrosis. None of the elk inoculated with type 1 or type 2 BVDV developed clinical signs of illness. However, all elk became infected as demonstrated by viremia, nasal shedding, and/or seroconversion. One uninoculated, in-contact elk contracted type 1 BVDV and seroconverted. Thus, although BVDV does not appear capable of producing disease in nonpregnant elk, the species is susceptible to infection and can shed and transmit BVDV.

The immunological response of llamas (Lama glama) following experimental infection with Mycobacterium bovis.

Llamas were experimentally infected with Mycobacterium bovis in order to evaluate the axillary skin test and the ELISA as diagnostic procedures for tuberculosis in llamas (Lama glama). Six llamas were given a single intratracheal challenge with 1 of 2 doses of a recent field isolate of M. bovis and 2 llamas were left as noninfected controls. This resulted in a progressive disease in some animals with 1 mortality as early as 68 d post-infection (PI). The tuberculin skin test, at the axillary site, was positive in 4 of 5 infected llamas at 80 d PI. At 143 d PI, all 3 surviving lamas were positive, including the one which had not responded at 80 d PI. The application of skin and serological tests throughout the course of this experiment adds support for the need to further evaluate the skin test and its anamnestic effect on serodiagnosis since serological responses were generally not observed in the absence of skin testing or antibiotic treatment. The wide variation in M. bovis antigens recognized by the serological response would indicate that a diagnostic panel should include multiple antigens such as MPB70 and lipoarabinomannan (LAM). While skin testing or serology alone may be of limited value to diagnose tuberculosis in llamas, together they may offer an enhanced potential for immunodiagnosis of tuberculosis.

Infection of cattle with Brucella abortus biovar 1 isolated from a bison in Wood Buffalo National Park.

An experiment was conducted to determine if cattle could be infected with a strain of Brucella abortus biovar 1 isolated from a bison in Wood Buffalo National Park. Three pregnant cows inoculated conjunctivally with 5.7 x 10(8) cfu of the bacterium, and their subsequent calves, showed seroconversion on standard serological tests for bovine brucellosis, and large numbers of the bacterium were isolated from numerous tissues at necropsy. A 4th cow that was moved into the pen that previously contained the inoculated cows subsequently showed seroconversion, and the same strain of B. abortus biovar 1 was isolated from numerous tissues. Although this strain from bison in Wood Buffalo National Park has existed in isolation from cattle for over 60 years, it remains infectious and contagious for cattle.

Experimental studies on Brucella abortus in moose (Alces alces).

Four moose (Alces alces) were inoculated conjunctivally with B. abortus biovar 1 to determine their susceptibility to brucellosis, and to describe the serology, bacteriology, hematology, clinical chemistry, and pathology associated with infection. All moose became infected. Two moose were killed at day 70 post-exposure, one (83F) died acutely at day 85, and one was killed at day 166. None of the moose had clinical signs, except for 83F immediately before death. Infected moose were readily detected serologically by the buffered antigen plate test, Brewer card test, standard tube agglutination test, and complement fixation test as used for brucellosis in cattle. With the exception of samples from 83F taken 24 hours before death, clinical chemistry, and hematology results were stable for all moose, and similar to normal values seen in cattle. Lesions seen in all moose were indicative of endotoxemia, and moose 83F died of acute endotoxic shock. Brucella abortus biovar 1 was isolated from several tissues in all moose, most notably from lymph nodes where counts often exceeded 4 x 10(4) colony forming units per g of tissue. Thus infection with B. abortus will kill moose, and progression of the disease is likely rapid under field conditions. Moose appear to be a dead-end host for brucellosis.

Susceptibility of mule deer (Odocoileus hemionus) and two species of North American molluscs to Elaphostrongylus cervi (Nematoda: Metastrongyloidea).

An experimental study was done to determine whether Elaphostrongylus cervi can be transmitted to common intermediate and definitive hosts indigenous to North America. First-stage larvae of E. cervi obtained from red deer (Cervus elaphus elaphus) in New Zealand developed to the infective third stage in snails (Triodopis multilineata) and slugs (Deroceros reticulatum). Two mule deer (Odocoileus hemionus) were inoculated orally with 102 or 406 third-stage larvae. One animal developed a patent infection on day 121 postinoculation (PI), and the other had numerous first-stage larvae in its lungs and colonic feces on 128 days PI when it was killed. A control red deer inoculated with 100 larvae began passing larvae on 119 days PI. Larval excretion was low and intermittent in the mule deer and during the first 4 wk of patency in the red deer. Both mule deer developed progressive neurological disease beginning on day 104 PI and had to be killed 4 or 7 wk later, respectively. The red deer remained clinically normal.

The morphology and pathology of Besnoitia sp. in reindeer (Rangifer tarandus tarandus).

Four of five reindeer (Rangifer tarandus tarandus) obtained from a Besnoitia sp.- infected herd at the Assiniboine Park Zoo in Winnipeg, Manitoba, Canada, in October 1989, had evidence of mild dermatitis over the articular surfaces of carpal and tarsal joints. Cysts of Besnoitia sp., either surrounded by inflammatory reactions or without evident host response, were present within the dermis, submucosa of the nasal turbinates, periosteum, tendons, testes and hooves. The light microscopic and histochemical features of Besnoitia sp. from reindeer were indistinguishable from those of other Besnoitia spp. described in cattle, rodents and horses. The Besnoitia sp. cysts and organisms from reindeer were unique in that bradyzoite membrane micropores and cytoplasmic enigmatic bodies were not observed. Two cats were fed cysts of Besnoitia sp. but no oocysts were detected in feces for 90 days post-infection.

An abattoir study of tuberculosis in a herd of farmed elk.

The purpose of this study was to examine the prevalence and distribution of grossly visible lesions of tuberculosis in a herd of 344 North American elk (Cervus elaphus) depopulated during a three-month period in 1991. Abattoir inspection detected mycobacterial lesions in 134 (39.8%) of the 337 animals received for slaughter. The prevalence of lesions increased with the age of the animals. Lesions were predominantly suppurative rather than caseous, and mineralization was less evident than in tuberculous lesions in cattle. Lesions occurred predominantly in lymph nodes, and lungs were the only organs in which mycobacterial lesions were found. The distribution of lesions suggested that aerosol transmission was the most significant means of spread of the disease within the herd. Giant liver flukes (Fascioloides magna) were observed in approximately 80% of the adult elk.

Diagnosis of Elaphostrongylus cervi infection in New Zealand red deer (Cervus elaphus) quarantined in Canada, and experimental determination of a new extended prepatent period.

A modified Baermann assay was used to recover dorsal-spined, first stage larvae of Elaphostrongylus cervi from feces and lungs of red deer (Cervus elaphus elaphus) from three of four herds imported from New Zealand into Canadian quarantine facilities. Tests done on a series of fecal collections showed that larval output from infected red deer was low and sporadic, casting doubt on the efficacy of the Baermann assay to detect all infected individuals in the herds. The animals had passed repeated preembarkation Baermann tests for E. cervi in New Zealand. Seven larvae recovered from these red deer were used to establish a patent infection in a naive red deer. The prepatent period was 206 days and larval shedding was intermittent. Elaphostrongylus cervi is a foreign animal parasite in continental North America, which could become irrevocably established if it were introduced. The data reported indicates that there is currently no reliable method for the detection of E. cervi infection.

The brucellosis and tuberculosis status of wood bison in the Mackenzie Bison Sanctuary, Northwest Territories, Canada.

Postmortem examinations were done on 51 wood bison (Bison bison athabascae) killed as part of a multidisciplinary research project in the Mackenzie Bison Sanctuary, Northwest Territories, Canada, between 1986 and 1988. There was no gross, histological or bacteriological evidence of brucellosis or tuberculosis in these bison. Traumatic lesions were seen in one calf that had been attacked by wolves and a second calf that had been gored. Antibody titers to Brucella abortus were not found in sera from these 51 animals or an additional 112 wood bison that were chemically-immobilized or killed in the Sanctuary between 1986 and 1990. The combined prevalence of the diseases in the population could not have exceeded 5.95% for the necropsy survey to have missed finding at least one infected animal, and the prevalence of brucellosis in the population would have had to be less than 1.95% for the broader serological survey to have failed to find at least one reactor animal on the battery of tests. These results, and the cumulative epidemiological information on brucellosis and tuberculosis in bison, indicate that bovine brucellosis and tuberculosis are not enzootic in the wood bison population in and around the Mackenzie Bison Sanctuary, and suggest that the population is free of these diseases. However, this expanding population is at risk of contracting both diseases from the infected bison population in and around nearby Wood Buffalo National Park.