Melissa C. Libal

Rapid and Sensitive Detection of Mycobacterium avium subsp. paratuberculosis in Bovine Milk and Feces by a Combination of Immunomagnetic Bead Separation-Conventional PCR and Real-Time PCR

ABSTRACT Immunomagnetic bead separation coupled with bead beating and real-time PCR was found to be a very effective procedure for the isolation, separation, and detection of Mycobacterium avium subsp. paratuberculosis from milk and/or fecal samples from cattle and American bison. Samples were spiked with M. avium subsp. paratuberculosis organisms, which bound to immunomagnetic beads and were subsequently lysed by bead beating; then protein and cellular contaminants were removed by phenol-chloroform-isopropanol extraction prior to DNA precipitation. DNA purified by this sequence of procedures was then analyzed by conventional and real-time IS900-based PCR in order to detect M. avium subsp. paratuberculosis in feces and milk. By use of this simple and rapid technique, 10 or fewer M. avium subsp. paratuberculosis organisms were consistently detected in milk (2-ml) and fecal (200-mg) samples, making this sensitive procedure very useful and cost-effective for the diagnosis of clinical and subclinical Johnes disease (paratuberculosis) compared to bacteriological culture, which is constrained by time, labor, and expense under diagnostic laboratory conditions.

Fatal Pneumonia among Metalworkers Due to Inhalation Exposure to Bacillus cereus Containing Bacillus anthracis Toxin Genes

Bacillus cereus pneumonia is unusual in nonimmunocompromised hosts. We describe fatal cases in 2 metalworkers and the associated investigation. Anthrax toxin genes were identified in B. cereus isolates from both patients using polymerase chain reaction. Finding anthrax toxin genes in non-Bacillus anthracis isolates has, to our knowledge, only been reported once previously.

Neospora -like protozoan infection as a cause of abortion in dairy cattle

In 1989,29 of 240 drylot Holstein cows from a New Mexico dairy aborted over a 5-month period. Seven of the 9 fetuses examined had multifocal necrotizing encephalitis and multifocal myocarditis. Neospora-like protozoa were found in tissues from 3 of the fetuses. More recently, a protozoan that reacted positively with anti-N. caninum serum was identified as the most common cause of abortion in California dairy cattle. 1,2 Many of these abortions were epizootic, with multiple abortions over a 1 or 2-month period. 1 Abortions and congenital infections in calves infected by a Neosporalike protozoan have been reported in the United States, but these were isolated cases involving only a single calf in each herd. This protozoan has never been isolated, so its exact identity remains unknown. Recent studies indicate that the organism is closely related to N. caninum, but some ultrastructural and antigenic differences exist between the bovine parasite and N. caninum isolated from dogs. Except for a single case of abortion in a beef cow from Maryland, the cases of fetal and congenital neosporosis previously reported from the United States are from the western states of New Mexico, 11 Califomia and Washington. Nineteen additional cases of bovine abortion associated with Neosporalike infection that were submitted to the South Dakota Animal Disease Research and Diagnostic Laboratory (SDADRDL) are herein reported. Specimens from cases of bovine abortions that are submitted to the SDADRDL undergo a routine protocol of testing to determine the cause of the abortion. Entire animals are necropsied and samples of brain, heart, lung, liver, kidney, placenta, tissues with gross lesions, and, since February 1991, skeletal muscle are fixed for histopathologic examination. Abomasal fluid is cultured for aerobic and microaerophilic bacteria and fungi. Scrapings of placenta are digested with potassium hydroxide and examined for fungal hyphae by a direct, nonspecific fluorescent antibody (FA) technique. If hyphae are found, the placenta is cultured for fungi. Impression smears of kidney are examined by a direct FA technique for 6 serovars of Leptospira. Direct FA tests are performed on frozen sections of kidney and spleen for infectious bovine rhinotracheitis (IBR) virus and bovine viral diarrhea (BVD) virus. Pools of brain, lung, liver, kidney, and spleen are homogenized and inoculated onto primary cultures of fetal bovine lung and turbinate cells for virus isolation. When received, a homogenate of placenta is inoculated by itself onto the same cell lines. Formalin-fixed paraffin-embedded

Comparison of three treatment regimens for sheep and goats with caseous lymphadenitis.

OBJECTIVE To compare the effectiveness of 3 treatment regimens for small ruminants with caseous lymphadenitis. DESIGN Randomized clinical trial. ANIMALS 44 client-owned sheep and goats. PROCEDURES Aspirates were obtained from 48 lesions of 44 enrolled animals and submitted for bacterial culture. Animals were randomly assigned to 1 of 3 treatment groups. Treatment for group A (n = 15 lesions) consisted of opening, draining, and flushing the lesions and SC administration of procaine penicillin G. Treatment for group B (n = 15 lesions) consisted of closed-system lavage and intralesional administration of tulathromycin. Treatment for group C (n = 18 lesions) consisted of closed-system lavage and SC administration of tulathromycin. All animals were reexamined approximately 1 month after treatment, unless treatment failure was detected prior to that time. RESULTS 43 animals with lesions had positive results (Corynebacterium pseudotuberculosis) for bacterial culture. Proportions of lesions that had resolution of infection by 1 month after treatment did not differ significantly among the treatment groups (group A, 13/14 [92.9%]; 95% confidence interval [CI], 69.5% to 99.6%; group B, 10/12 [83.3%]; 95% CI, 54.9% to 97.1%; and group C, 14/17 [82.4%]; 95% CI, 59.1% to 95.3%). CONCLUSIONS AND CLINICAL RELEVANCE Acceptable alternatives to opening, draining, and flushing of lesions may exist for treatment of sheep and goats with caseous lymphadenitis. Use of tulathromycin and penicillin in this study constituted extralabel drug use, which would require extended withholding times before milk or meat of treated sheep and goats can be sold for human consumption.

Growth and Metabolic Characterization of Macrorhabdus Ornithogaster

Macrorhabdus ornithogaster (M. ornithogaster) is an anamorphic ascomycetous yeast found only in the stomach of birds. Infection is often benign but has also been associated with disease in some species of birds under some circumstances. In vitro efforts to grow M. ornithogaster have been largely unsuccessful. In this report, multiple liquid and solid media of varying pH, sugar concentration, and fetal bovine serum (FBS) concentrations, incubated at various temperatures in room air or microaerophilic conditions, were examined for their ability to support the growth of M. ornithogaster, obtained from a budgerigar (Melopsittacus undulatus). Optimum growth conditions were found to be Basal Medium Eagles, pH 3 to 4, containing 20% FBS, and 5% glucose or sucrose under microaerophilic conditions at 42°C. Using these conditions, M. ornithogaster was repeatedly passaged without loss of viability. Polyclonal isolates of M. ornithogaster consistently assimilated glucose, sucrose, and trehalose. M. ornithogaster did not grow with prolonged exposure to atmospheric oxygen, but growth in microaerophilic conditions was moderately enhanced by preincubation with atmospheric oxygen for 24 hours. An isolate of M. ornithogaster was found to be infective to day-old chickens, reduce their rate of weight gain, and induce a mild to moderate heterophilic inflammation of the isthmus. M. ornithogaster was reisolated from the chicks 7 days after infection, fulfilling Kochs postulates. A 761-bp sequence of 18S rDNA from this isolate was compared to the originally reported M. ornithogaster sequence and was found to be 97% identical.

In vitro antimicrobial activity of gallium maltolate against virulent Rhodococcus equi.

The objective of this study was to determine the in vitro antimicrobial activity of gallium maltolate (GaM) against Rhodococcus equi. A total of 98 virulent bacterial isolates from equine clinical cases were examined, of which 19 isolates were known to be resistant to macrolides and rifampin. Isolates were cultured with various concentrations of GaM and minimal inhibitory concentration (MIC) values were determined after 24 and 48 h. Both the MIC(50) and the MIC(90) after 24h of growth were 558 ng/mL (8 μM) and after 48 h of growth were 2230 ng/mL (32 μM). There were no apparent differences between MICs of macrolide-resistant and macrolide-susceptible isolates.

Antimicrobial activity of gallium maltolate against Staphylococcus aureus and methicillin-resistant S. aureus and Staphylococcus pseudintermedius: An in vitro study

Gallium is a trivalent semi-metallic element that has shown antimicrobial activity against several important human pathogens. This antimicrobial activity is likely related to its substitution in important iron-dependent pathways of bacteria. The genus Staphylococcus, which includes human and animal pathogens that cause significant morbidity and mortality, requires iron for growth and colonization. In this study, gallium maltolate, at various concentrations between 50 and 200μM, inhibited the in vitro growth of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) at time-points between 8 and 36h after inoculation. The inhibitory activity of gallium maltolate against clinical isolates of MRSA and methicillin-resistant Staphylococcus pseudintermedius (MRSP) from a veterinary teaching hospital was determined.

Systemic Pasteurella haemolytica infection as a rare sequel to avirulent live Pasteurella haemolytica vaccination in cattle

Eleven cases of systemic Pasteurella haemolytica infection in cattle were identified from routine diagnostic laboratory submissions during the falls of 1988, 1989, and 1991. All cases came with a history of recent vaccination with an avirulent live culture P. haemolytica product. Nine of 11 cases involved cattle vaccinated between 2 and 18 days previously with this product. Ten of 11 cases involved 182-227-kg beef calves that were vaccinated between September and November during routine processing for entry into feedlots. The morbidity and mortality was generally low. The major pathologic findings included meningitis, injection site abscessation and/or cellulitis, and polyarthritis. Systemic infection was indicated in all cases by the isolation of P. haemolytica from 2 or more organs or distinct anatomical sites. In 6 cases, the vaccine injection site was cultured, and in all 6 cases, P. haemolytica was isolated. Three separate P. haemolytica isolates from 2 cases were further studied by restriction enzyme analysis (REA). These isolates were from tissues with suppurative inflammation, including the brain, joint, and injection site. The REA patterns of each of these 3 isolates were identical to the REA pattern of the vaccine masterseed, which strongly suggested that the organisms causing systemic infection were the same as the organism used to produce the vaccine. Because the overall incidence was quite low, other factors, such as stress, probably played a major role in the expression of this syndrome.

Pulmonary Phialemonium curvatum phaeohyphomycosis in a Standard Poodle dog.

Phialemonium curvatum, frequently misidentified as an Acremonium species, is reported here as a new agent of pulmonary phaeohyphomycosis in a Standard Poodle dog, and added as a new species in the genus to cause mycoses in canines. In vitro susceptibility data, for both human and animal isolates, suggests resistance to amphotericin B and susceptibility to the triazole agents itraconazole, voriconazole, and posaconazole.

Septicemia in a Neonatal Calf Associated with Chromobacterium violaceum

Chromobacterium violaceum infections are highly fatal and rarely reported in domestic animals. This report describes a fatal case of C. violaceum septicemia in a 7-day-old female beef calf. The calf had necrosuppurative omphalophlebitis, necrotizing interstitial pneumonia, necrosuppurative hepatitis and splenitis, anterior uveitis with hypopyon, suppurative polyarthritis, and disseminated hemorrhagic meningitis with multifocal necrotizing encephalitis. Histologically, clusters of gram-negative bacilli were found in many of the lesions. C. violaceum was isolated in high numbers from the lungs, liver, spleen, carpus, and in pure culture from the cerebrospinal fluid. To our knowledge, this is the first case report of chromobacteriosis in a calf.