Laboratory evaluation of an outbreak of nocardiosis in immunocompromised hosts.

Seven patients in a renal unit were proved to have nocardiosis in an interval of nine months. Six of these patients had received renal transplants. Serologic investigation suggested that two additional cases of undiagnosed pulmonary disease were also nocardial, and that there were no subclinical cases in patients or staff. Clinical-serologic correlations indicate that serologic evaluation may be a useful adjunct in diagnosis of nocardiosis, if used early and repeatedly, and to follow response to therapy. Epidemiologic investigations yielded cultures of Nocardia asteroides from air and dust inside the unit and elsewhere in the hospital. Biochemical, metabolic, physical and immunologic characterization of the isolates indicated that those from patients and those from the unit environment were identical, whereas some from outside the unit could be differentiated from these. The "epidemic strain" had type III antigen, which surveys indicated is not the most common type in human nocardiosis (it occurs in association with a minority of human cases). The isolates were of subgroup B, which has been associated with virulence. The characterization methods employed could be useful in studies of nocardial epidemiology. The laboratory studies indicate epidemic spread within the unit of a single organism, and current epidemiologic guidelines, which do not recommend respiratory isolation of cases of pulmonary nocardiosis, may need reconsideration particularly when there are immunocompromised hosts in the environment.

Major biological consequences of aflatoxicosis in animal production.

Aflatoxins, a family of closely related, biologically active mycotoxins, have been known as a prominent cause of animal disease for 30 yr. The toxins occur naturally on several key animal feeds, including corn, cottonseed, and peanuts. Occurrence of aflatoxin on some field crops tends to spike in years when drought and insect damage facilitate invasion by the causative organisms, Aspergillus flavus and A. parasiticus, which abound in the crop's environment. Acute aflatoxicosis causes a distinct overt clinical disease marked by hepatitis, icterus, hemorrhage, and death. More chronic aflatoxin poisoning produces very protean signs that may not be clinically obvious; reduced rate of gain in young animals is a sensitive clinical register of chronic aflatoxicosis. The immune system is also sensitive to aflatoxin, and suppression of cell-mediated immune responsiveness, reduced phagocytosis, and depressed complement and interferon production are produced. Acquired immunity from vaccination programs may be substantially suppressed in some disease models. In such cases the signs of disease observed are those of the infectious process rather than those of the aflatoxin that predisposed the animal to infection. Mixtures of aflatoxin with other mycotoxins can result in greatly augmented biological responses in terms of rate of gain, lethality, and immune reactivity. Because of its great biological activity, its wide-spread potential presence in areas where critical feed crops are grown, and its propensity to spike in problem years, aflatoxin promises to be a continuing problem in animal production.

Determination of sensitivity and specificity of the Nocardia asteroides skin test for detection of bovine mammary infections caused by Nocardia asteroides and Nocardia caviae.

A total of 116 cows from 4 dairy herds in California were used to determine the sensitivity and specificity of extracellular antigen of Nocardia astero des for possible detection of bovine mastitis caused by N asteroides and N caviae. Three different positive criteria were used. These criteria gave different sensitivity and specificity to the skin test. The highest sensitivity of 80% and highest specificity of 96.04% were obtained by combining 2 criteria. The study found no cross-reaction between N asteroides antigen and Mycobacterium fortuitum or N asteroides and tuberculin skin tests. There was cross-reaction between N asteroides antigen and N caviae infection.

Penitrem A intoxication of calves: blood chemical and pathologic changes.

An isolate of Penicillium puberulum, obtained from moldy silage, was found to produce a tremorgenic mycotoxin, penitrem A. Dried ground mycelium of this isolate was administered orally to calves either in increasing daily doses or in a single dose that equalled the amount given over a 3-day period in the increasing daily-dose schedule. Signs of intoxication included tremor, ataxia, muscular rigidity, and convulsive episodes. Marked increases in plasma concentrations of lactic acid, pyruvic acid, glucose, and creatine phosphokinase activity were found coincident with the development of severe tremor. The only gross or microscopic change observed in tissues of intoxicated animals was an accumulation of fat in the liver. Changes in the various plasma constituents were interpreted as a secondary effect of the intoxication.

Counterimmunoelectrophoresis (immunoelectroosmosis) and serum electrophoretic pattern in serologic diagnosis of canine blastomycosis.

Counterimmunoelectrophoresis (CIEP) with blastomyces and histoplasma antigens was used in a serologic study of 181 dogs clinically suspected of having blastomycosis and of 8 dogs with confirmed blastomycosis or histoplasmosis. Thirteen of the 181 dogs, positive by CIEP, were euthanatized, and the diagnosis was confirmed by cultivation and/or microscopic detection of Blastomyces dermatitidis. Additional CIEP-positive dogs were confirmed by staining of aspirates collected in vivo. Radiographic support for the diagnosis was reported in 4 other dogs in which histoplasmosis was excluded by a negative CIEP with histoplasma antigen. The precipitating antibody may disappear during the course of the disease, as it did in 1 dog treated with amphotericin B, but not cured. This dog reverted from CIEP-positive to CIEP-negative within 17 months of treatment (with a weak reaction after 10 months of treatment). The CIEP-detectable antibody was present only in 1 dog without a confirmation by histopathologic findings or cultivation among 24 well-documented cases and 181 total tested sera. The CIEP was more sensitive and specific than was the gel-diffusion precipitin test, eliminated the problems of anticomplementarity that often affected the results of complement-fixation tests with canine sera, and served well in detecting dogs with blastomycosis. Electrophoretic pattern of sera from CIEP-positive dogs with blastomycosis showed a decrease in albumin and an increase in alpha 2- and often in beta- and gamma-globulins, with a substantial decrease of the albumin/globulin ratio.

Effects of aflatoxin consumption on the clinical course of swine dysentery.

Specific-pathogen-free pigs were examined for susceptibility to swine dysentery after daily treatment with aflatoxin B1 (0.07 to 0.14 mg/kg). In the pigs (group II) given both aflatoxin and Treponema hyodysenteriae, the incubation period before the onset of swine dysentery was shorter than that in pigs (group III) given T hyodysenteriae alone. Also, the number of days in which infected pigs showed signs of diarrhea and dysentery was greater in the group given aflatoxin and T hyodysenteriae than in the group given T hyodysenteriae alone. Four of 8 pigs given aflatoxin and T hyodysenteriae died during the experiment, whereas only 1 of 8 infected pigs in the other group died. Pigs in both groups, convalescent from swine dysentery, were immune to rechallenge exposure. Subclinical effects of aflatoxin consumption were evident at necropsy, but clinical signs and lesions of swine dysentery were not observed in group III pigs (given aflatoxin only).

Use of bacteria and yeasts to identify T lymphocytes in peripheral blood and lymphoid tissues of healthy guinea pigs and guinea pigs fed aflatoxin.

Fifty-four bacterial and 10 yeast isolates were screened to identify strains that were consistently bound by peripheral blood lymphocytes (PBL) of guinea pigs. None of the yeast isolates examined was bound by guinea pig PBL. Of 54 bacterial isolates, 10 were bound by greater than 5% of PBL. Potential lymphocyte markers from these bacteria were chosen for further study. Sodium azide inhibited the binding of bacteria by PBL. Preparations of T lymphocytes indicated that Salmonella schottmuelleri was bound by most T lymphocytes; Yersinia enterocolitica was bound by a subpopulation of T lymphocytes. Patterns of binding of bacteria by lymphocytes from thymus, lymph node, or spleen differed from binding patterns obtained using PBL. Aflatoxin did not affect the total WBC, differential leukocyte count, absolute lymphocyte count, or relative percentage of PCV of guinea pigs given as much as 0.060 mg of aflatoxin B1 equivalents/kg of body weight/day for 3 weeks. Changes in absolute numbers of peripheral blood T lymphocytes were not observed in guinea pigs given aflatoxin when immunofluorescence or bacterial binding was used to identify lymphocyte populations, except that the number of S schottmuelleri-binding lymphocytes (tentatively identified as T lymphocytes) in guinea pigs given the highest dose of aflatoxin (0.060 mg of aflatoxin B1 equivalents/kg/day) was less than the number of T lymphocytes identified by immunofluorescence.

Comparison of effects of dietary T-2 toxin on growth, immunogenic organs, antibody formation, and pathologic changes in turkeys and chickens.

The effect of T-2 toxin (trichothecene mycotoxin) consumption on Broad-Breasted White turkey poults and White Leghorn chicks was studied. Groups of ten 8-day-old poults were fed rations containing T-2 at 10 ppm, 2ppm, or 0 ppm (controls) for a period of 4 weeks; a 4th group (inanition control) was fed control rations equal to the amount consumed by the group fed rations containing T-2 at 10 ppm during the previous 24 hours. A similar experimental design was used to study the effect of the toxin on 1-day-old chicks. The thymus glands of the poults given the feed containing 10 ppm were markedly decreased in size compared with thymus glands from poults in the control group, 0.182 vs 0.331 (percentage of body weight). There was no significant (P less than or equal to 0.05) decrease in thymus gland size in poults given 2 ppm or in the inanition controls. Dietary treatment did not appear to affect the size of the bursa or spleen of the poults. Histopathologic examination of thymus glands from poults given 10 ppm of T-2 revealed a depletion of cortical lymphocytes. Chicks appeared less sensitive to T-2 toxin than did the poults. There was no effect by any dietary treatment on the size of the thymus gland, bursa, or spleen of chicks. Reductions were noticed in feed efficiency and weight gain. There was no effect of T-2 toxin on agglutinating antibody formation to Pasteurella multocida bacterin.

Effects of aflatoxin on the development of acquired immunity to swine erysipelas.

The effect of aflatoxin consumption on the development of acquired immunity to swine erysipelas was studied. Twenty-four pigs were divided into 4 groups (6 pigs/group). Two groups were fed a normal diet and 2 groups were fed the same diet but containing aflatoxin. One group from each diet treatment was given a single injection of an erysipelas bacterin, and 21 days later all pigs were given a challenge inoculum of virulent Erysipelothrix rhusiopathiae organisms. On the basis of the response to the challenge inoculation, pigs were classified as immune, partially immune (PI), or susceptible. Three of the vaccinated pigs fed the normal diet were immune, 2 were PI, and 1 was susceptible, whereas none of the vaccinated pigs given the aflatoxin diet were immune, only 1 was PI, and the remainder were susceptible. Two of the nonvaccinated pigs fed the normal diet were PI and 4 were susceptible; all of the nonvaccinated pigs fed the aflatoxin diet were susceptible. It was concluded that aflatoxin consumption interfered with the development of acquired immunity and apparently increased the severity of the E rhusiopathiae infection in unvaccinated pigs.

Effects of dietary aflatoxin on existing bacterial intramammary infections of dairy cows.

Aflatoxin, a naturally occurring mycotoxin with immunosuppressive properties, was given to 4 lactating dairy cows that had experimentally induced or naturally occurring mammary infections in 2 of their 4 mammary quarters. Aflatoxin was given orally in daily doses approximating 0.3 mg of B1 activity/kg of body weight for periods of 12 to 14 days. Infecting organisms were Streptococcus agalactiae, Staphylococcus aureus, and Staphylococcus hyicus. The 4 cows were monitored for clinical signs of disease and signs of mastitis for periods before, during, and after aflatoxin consumption. The cows developed inappetence, lost body weight, and had variable reductions in daily milk yield. Serum ornithine-carbamoyl transferase and sorbitol dehydrogenase activities were elevated during and 1 to 3 weeks after aflatoxin consumption. Exacerbations of acute clinical mastitis did not occur in any of the infected quarters; however, bacterial populations in the milk increased in 3 of the 4 cows during aflatoxin consumption. Mean California mastitis test scores were generally highest in the infected quarters in the period after the last dose of aflatoxin was given. Aflatoxin M1 appeared in milk 3 to 6 hours after aflatoxin consumption began, and persisted for 72 hours or longer after the last dose of aflatoxin was given. Aflatoxin B1 and M1 were present in urine samples taken at 6 hours after aflatoxin consumption began and persisted for 72 through 120 hours after the last dose of aflatoxin was given.

Vaccination studies of aspergillosis in turkeys: subcutaneous inoculation with several vaccine preparations followed by aerosol challenge exposure.

Five vaccines were prepared from an isolate of Aspergillus fumigatus: a culture filtrate vaccine (I), a spore vaccine (II), a mycelial vaccine (III), and 2 germling vaccines (IV and V). The 2 germling vaccines were produced on different media. Two experiments were conducted to test the vaccines for efficacy in groups of 21 turkey poults by giving each bird 2 subcutaneous inoculations of the respective vaccine at 1 and 2 weeks of age. The birds were challenge exposed to an aerosol of spores of A fumigatus at 1 month of age. The 2 experiments differed only in the challenge exposure. In experiment 1, 38% of the poults were protected when vaccinated with an A fumigatus germling vaccine (IV) and challenge exposed with a dose of aerosolized A fumigatus spores that killed 100% of the nonvaccinated controls. Onset of postchallenge-exposure deaths were delayed by 2 days in the turkeys given vaccine IV. In experiment 2, 48% of the poults were protected when given germling vaccine IV, whereas 62% to 86% of the poults in the nonvaccinated and 3 other vaccine groups died. In experiment 2, 50% of the poults given a mycelial vaccine (III) were protected. Torticollis was a frequent sign of the disease in experiment 2.

Fate of aflatoxins in tissues, fluids, and excrements from cows dosed orally with aflatoxin B1.

A study was conducted to determine aflatoxins in tissues and non-tissues of 2 Holstein cows given oral doses of 0.35 mg of purified aflatoxin B1/kg of body weight/day for 3 consecutive days. Cow 1 was slaughtered 24 hours after the 3rd dose, and cow 2, after day 3, was fed aflatoxin-free rations for 7 additional days before slaughter. Tissue samples of brain, gallbladder and bile, heart, intestine, kidney, liver, lung, mammary gland, skeletal muscle, spleen, supramammary lymph nodes, thymus, and tongue, and nontissue samples of blood, feces, milk, rumen content, and urine were examined. Aflatoxins B1 and M1 were found in all samples of cow 1, except the thymus. Kidney, liver, and mammary gland had the highest concentrations of total aflatoxins (57.9, 13.2, and 25.1 ng/g, respectively), with the aflatoxin M1 concentration 40 times more than the aflatoxin B1 level in kidney. Aflatoxin residues were present (0.02 to 0.11 ng/g) only in kidney, liver, and intestine of the tissues from cow 2 (fed aflatoxin-free feed for 7 additional days). Aflatoxin B1 was not present in nontissue samples, but aflatoxin M1 (0.10 and 1.5 ng/ml) was found in the last milk and urine samples from the same cow. Urine assays are a possible way to monitor the presence of aflatoxin residues in meat tissues.

Effect of feeding corn naturally contaminated with aflatoxin on feed efficiency, on physiologic, immunologic, and pathologic changes, and on tissue residues in steers.

Two of 3 groups of Holstein-Friesian steers (groups II and III; n = 5 each) were fed a ration containing corn naturally contaminated with 800 ng of aflatoxin/g. The other group of steers (group I; n = 5) was fed a ration containing noncontaminated corn. The respective rations were fed for 17.5 weeks, except the ration given to group III; the latter's first diet (contaminated with aflatoxin) was changed to a noncontaminated diet after 15 weeks, continuing for the remaining 2.5 weeks. All steers were killed and tissues and fluids were obtained for aflatoxin analysis. Although aflatoxin B1 and M1 could be detected in blood and urine at several sampling times during the experimental period in groups II and III steers (given the diets containing aflatoxin), there appeared to be no effects on body weight gains and immune phenomena, such as lymphoblastogenesis and antibody production, but there was a waning of the delayed cutaneous hypersensitivity in steers given aflatoxin-contaminated diets. In group III animals (diet was changed to noncontaminated ration at 15 weeks), aflatoxin B1 and M1 disappeared from urine before they were slaughtered. All tissues and fluids, except the rumen contents from these group III steers, were void of detectable aflatoxins B1 and M1 at necropsy. The concentrations of aflatoxin B1 in the rumen content of the latter steers were low. All tissues collected at necropsy from the group II steers fed the aflatoxin diet throughout the 17.5 weeks had detectable aflatoxins B1 or M1 present.

Evaluation of histopathologic and physiologic changes in cows having premature births after consuming Ponderosa pine needles.

Premature calving, typified by early expulsion (17 to 43 days) of weak or dead calves and accompanied by retained placentas, was induced in 8 of 9 pregnant cows fed a mixture of Ponderosa pine needles and alfalfa hay. Five control cows of comparable gestation age fed only alfalfa hay maintained normal pregnancies until they were euthanatized at the time the pine needle-treated cows were producing premature calves. Serum specimens from all cows were assayed for progesterone concentration and ovaries and placentomes were examined for histopathologic changes. There were no bacterial, fungal, chlamydial, or viral agents determined to be associated with the premature births. Serum progesterone concentration in the treated cows decreased progressively and were 0.4 to 1.5 ng/ml at the time of premature calving. Histopathologic changes were evident in the placenta and corpora lutea of treated cows only. The number of binucleate trophoblastic giant cells in placentomes was less than normal and the number of necrotic luteal cells in corpora lutea was greater than normal.

Mucormycotic dermatitis in captive adult Wyoming toads.

During late May 1995, 50 adult captive endangered Wyoming toads (Bufo baxteri) were brought out of hibernation. Approximately 3 to 10 days after hibernation emergence, all toads were hormonally induced to breed, and paired. Each pair was placed in their own breeding tank. Four toads developed clinical signs of disease which included lethargy and multiple (4 to 12) small (2 mm) raised hyperemic nodules with white fuzzy caps on the ventral skin. The condition progressively worsened until death occurred, within 3 to 6 days. Mycotic dermatitis caused by Mucor sp. was diagnosed in the four toads through histology and isolation of the organism. This is the first case report of a Mucor sp. causing a fatal dermatitis in an amphibian without significant inflammatory response and without systemic involvement.

Causes of mortality of the Wyoming toad.

Wyoming toads (Bufo baxteri) that died from January 1989 to June 1996 were submitted to the Wyoming State Veterinary Laboratory (Laramie, Wyoming, USA) for postmortem evaluation. These consisted of 108 free-ranging toads and 170 animals from six captive populations. Ninety-seven (90%) of 108 free-ranging toad carcasses were submitted during September and October. From 1989 to 1992, 27 (77%) of 35 mortalities in the captive populations occurred in October, November, and December. From 1993 to 1996, mortality in captive toads occurred without a seasonal pattern and coincided with changes in hibernation protocols that no longer mimicked natural cycles. Cause of mortality was determined in 147 (53%) of the 278 cases. Mycotic dermatitis with secondary bacterial septicemia was the most frequent diagnosis in 104 (71%) of 147 toads. Basidiobolus ranarum was found by microscopic examination of skin sections in 100 (96%) of 104 of these mortalities. This fungus was isolated from 30 (56%) of 54 free-ranging and 24 (48%) of 50 captive toads. This research documents the causes of mortality for both free-ranging and captive endangered Wyoming toads over a 7 yr period.

Implications of mycotoxins in animal disease.

Mycotoxins are formed on animal feeds when conditions of moisture and temperature permit the growth of naturally occurring toxigenic fungi. In general, mycotoxins are low molecular weight, nonantigenic substances, many of which are relatively heat stable. Mycotoxins can cause acute disease episodes when animals consume critical quantities of them. Specific toxins affect specific organs or tissues such as the liver, kidney, oral and gastric mucosa, brain, or reproductive tract. In acute mycotoxicoses, the signs of disease often are marked and directly referable to the affected target organs. Most frequently, however, concentrations of mycotoxin in feeds are below those that cause acute disease. At lower concentrations, the effects of mycotoxins are more protean. They reduce the growth rate of young animals, and some interfere with native mechanisms of resistance and impair immunologic responsiveness, making the animals more susceptible to infection. These effects on immunity and resistance are difficult to recognize because the signs of disease are associated with the infection rather than with the mycotoxin that predisposed the animal to infection. Animals vary in their susceptibility to some mycotoxins, according to the species and age of animal; young growing animals are more susceptible to certain mycotoxins than are adults. The major effects, sources, and dose response relationships of mycotoxins important to the health of food-producing animals are presented in accompanying tables.