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Pulmonary actinomycosis is described in 17 South Australian koalas necropsied between 2016 and 2019. From these cases, four koalas had secondary hypertrophic osteopathy. Plain radiographical and computed tomography images demonstrated periosteal reaction on multiple appendicular skeletal bones in all cases, including scapula, humerus, ulna, radius, ilium, femur, tibia, fibula, metacarpus, metatarsus and phalanx. Grossly, periosteal surfaces of the metaphyses and diaphyses of long bones were thickened and roughened; microscopically, this was characterised by bi-layered proliferation of well-differentiated trabecular bony spicules oriented perpendicular to the cortex (pseudocortices) and separated by vascular connective tissue, typical for hypertrophic osteopathy. Well characterised in domestic species and rarely reported in marsupials, this is the first radiographical and pathological characterisation of hypertrophic osteopathy in koalas, associated with pulmonary actinomycosis in all cases., (© 2021 Australian Veterinary Association.)

Although Chlamydia causes disease of the urethra and prostate of male koalas, its impact on the testis and epididymis has not been examined. This study describes chronic-active and granulomatous orchitis and epididymitis with interstitial fibrosis associated with infection by Chlamydia pecorum in 2 of 18 adult male koalas being euthanized at a koala hospital, 8 of which also had chlamydial prostatitis. By immunohistochemistry and transmission electron microscopy, chlamydial inclusions were demonstrated within Sertoli cells directly associated with mild inflammation surrounding intact seminiferous and epididymal tubules, marked pyogranulomatous inflammation around disrupted tubules, replacement of tubules by interstitial fibrosis, and aspermia. The presence of C. pecorum but not Chlamydia pneumoniae was detected by quantitative polymerase chain reaction of formalin-fixed tissues of the left and right testes and right epididymis in 1 animal. This is the first report of orchitis and epididymitis in a koala infected with C. pecorum., (© The Author(s) 2015.)

Major histocompatibility complex class II (MHCII) genes code for proteins that bind and present antigenic peptides and trigger the adaptive immune response. We present a broad geographical study of MHCII DA β1 (DAB) and DB β1 (DBB) variants of the koala (Phascolarctos cinereus; n=191) from 12 populations across eastern Australia, with a total of 13 DAB and 7 DBB variants found. We identified greater MHCII variation and, possibly, additional gene copies in koala populations in the north (Queensland and New South Wales) relative to the south (Victoria), confirmed by STRUCTURE analyses and genetic differentiation using analysis of molecular variance. The higher MHCII diversity in the north relative to south could potentially be attributed to (i) significant founder effect in Victorian populations linked to historical translocation of bottlenecked koala populations and (ii) increased pathogen-driven balancing selection and/or local genetic drift in the north. Low MHCII genetic diversity in koalas from the south could reduce their potential response to disease, although the three DAB variants found in the south had substantial sequence divergence between variants. This study assessing MHCII diversity in the koala with historical translocations in some populations contributes to understanding the effects of population translocations on functional genetic diversity.

Clinically normal koalas (n = 6) received a single dose of intravenous enrofloxacin (10 mg/kg). Serial plasma samples were collected over 24 h, and enrofloxacin concentrations were determined via high-performance liquid chromatography. Population pharmacokinetic modeling was performed in S-ADAPT. The probability of target attainment (PTA) was predicted via Monte Carlo simulations (MCS) using relevant target values (30-300) based on the unbound area under the curve over 24 h divided by the minimum inhibitory concentration (MIC) (fAUC0-24 /MIC), and published subcutaneous data were incorporated (Griffith et al., 2010). A two-compartment disposition model with allometrically scaled clearances (exponent: 0.75) and volumes of distribution (exponent: 1.0) adequately described the disposition of enrofloxacin. For 5.4 kg koalas (average weight), point estimates for total clearance (SE%) were 2.58 L/h (15%), central volume of distribution 0.249 L (14%), and peripheral volume 2.77 L (20%). MCS using a target fAUC0-24 /MIC of 40 predicted highest treatable MICs of 0.0625 mg/L for intravenous dosing and 0.0313 mg/L for subcutaneous dosing of 10 mg/kg enrofloxacin every 24 h. Thus, the frequently used dosage of 10 mg/kg enrofloxacin every 24 h subcutaneously may be appropriate against gram-positive bacteria with MICs ≤ 0.03 mg/L (PTA > 90%), but appears inadequate against gram-negative bacteria and Chlamydiae in koalas., (© 2013 John Wiley & Sons Ltd.)

Clinically normal koalas (n = 19) received a single dose of intravenous (i.v.) chloramphenicol sodium succinate (SS) (25 mg/kg; n = 6), subcutaneous (s.c.) chloramphenicol SS (60 mg/kg; n = 7) or s.c. chloramphenicol base (60 mg/kg; n = 6). Serial plasma samples were collected over 24-48 h, and chloramphenicol concentrations were determined using a validated high-performance liquid chromatography assay. The median (range) apparent clearance (CL/F) and elimination half-life (t(1/2)) of chloramphenicol after i.v. chloramphenicol SS administration were 0.52 (0.35-0.99) L/h/kg and 1.13 (0.76-1.40) h, respectively. Although the area under the concentration-time curve was comparable for the two s.c. formulations, the absorption rate-limited disposition of chloramphenicol base resulted in a lower median C(max) (2.52; range 0.75-6.80 μg/mL) and longer median tmax (8.00; range 4.00-12.00 h) than chloramphenicol SS (C(max) 20.37, range 13.88-25.15 μg/mL; t(max) 1.25, range 1.00-2.00 h). When these results were compared with susceptibility data for human Chlamydia isolates, the expected efficacy of the current chloramphenicol dosing regimen used in koalas to treat chlamydiosis remains uncertain and at odds with clinical observations., (© 2012 John Wiley & Sons Ltd.)

Objective: To document the application of diagnostics and treatments at one rehabilitation facility over 10 years and their effects on recovery and post-release survival of 88 koalas treated for chlamydiosis, and to highlight associated wildlife care issues with potential significance to animal welfare and disease ecology., Design: Using a retrospective analysis of medical records, we identified risk factors for successful release using a logistic regression model and descriptive statistics., Procedure: We examined the clinical presentation, signalment, diagnostics, treatments, outcomes and whether released koalas were re-presented by the end of 2008 indicating post-release survival., Results: Records of 88 koalas were included. Treatments and diagnostics were directed at the anatomical site displaying clinical signs. Younger age and use of ancillary treatments were associated with successful release. The type, route and duration of the treatments used were not those theorised to result in microbial cure. Despite this, approximately 50% of koalas were released and many survived in the wild for extended periods., Conclusions: Wildlife rehabilitators' records can guide research priorities and the development of care facilities and policies. This study identified the need for more accessible chlamydial diagnostic tests and veterinary support of carers, and the need for a more rigorous assessment of novel therapies. Current treatment regimens appear to be moderately successful in terms of clinical improvement, but it is unclear which aspects are responsible for the success or whether microbial cure is achieved. The long-term effect of released koalas on wild populations requires further study to assess its contribution to the conservation of koala populations., (© 2012 The Authors. Australian Veterinary Journal © 2012 Australian Veterinary Association.)

Infection of koalas by Chlamydophila pecorum is very common and causes significant morbidity, infertility and mortality. Fundamental to management of the disease is an understanding of the importance of multi-serotype infection or pathogen virulence in pathogenesis; these may need consideration in plans involving koala movement, vaccination, or disease risk assessment. Here we describe diversity of ompA VD1-3, and ORF663 hypothetical gene tandem repeat regions, in a single population of koalas with diverse disease outcomes. We PCR amplified and sequenced 72 partial ompA segments and amplified 25 tandem repeat segments (ORF663 hypothetical gene) from C. pecorum obtained from 62 koalas. Although several ompA genotypes were identified nationally, only one ompA genotype existed within the population studied, indicating that severe chlamydial disease occurs commonly in free-ranging koalas in the absence of infection by multiple MOMP serotypes of C. pecorum. In contrast, variation in tandem repeats within the ORF663 hypothetical gene was very high, approaching the entire range reported for pathogenic and non-pathogenic C. pecorum of European ruminants; providing an impetus for further investigation of this as a potential virulence trait., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Nine mature koalas with chlamydiosis, typically keratoconjunctivitis and/or urogenital tract infection, were treated with daily subcutaneous injections of chloramphenicol at 60 mg/kg for 45 days (five koalas), or for a shorter duration (four koalas). All koalas were initially positive for Chlamydia pecorum as determined by real-time polymerase chain reaction (qPCR). Plasma chloramphenicol concentrations were determined at t = 0, 1, 2, 4, 8, and 24 h after the day 1 injection (nine koalas) and after the day 15 injection (seven koalas). Chloramphenicol reached a median (and range) maximum plasma concentration of 3.03 (1.32-5.03 μg/mL) at 4 (1-8 h) after the day 1 injection and 4.82 (1.97-27.55 μg/mL) at 1 (1-2 h) after day 15. The median (and range) of AUC(0-24) on day 1 and day 15 were 48.14 (22.37-81.14 μg·h/mL) and 50.83 (28.43-123.99 μg·h/mL), respectively. The area under the moment curve (AUMC)(0-24) median (and range) for day 1 and day 15 were 530.03 (233.05-798.97 h) and 458.15 (291.72-1093.58 h), respectively. Swabs were positive for chlamydial DNA pretreatment, and all koalas except one, produced swabs negative for chlamydial DNA during treatment and which remained so, for 2-63 days after treatment, however whether chloramphenicol treatment prevented long-term recrudescence of infection was not established. At this dose and dosing frequency, chloramphenicol appeared to control mild chlamydial infection and prevent shedding, but severe urogenital disease did not appear to respond to chloramphenicol at this dosage regime. For koalas affected by severe chlamydiosis, antibiotics alone are not sufficient to effect a cure, possibly because of structural or metabolic changes associated with chronic disease and inflammation., (© 2011 Blackwell Publishing Ltd.)

Koalas (n = 43) were treated daily for up to 8 weeks with enrofloxacin: 10 mg/kg subcutaneously (s.c.), 5 mg/kg s.c., or 20 mg/kg per os (p.o.); or marbofloxacin: 1.0-3.3 mg/kg p.o., 10 mg/kg p.o. or 5 mg/kg s.c. Serial plasma drug concentrations were determined on day 1 and again at approximately 2 weeks, by liquid chromatography. The median (range) plasma maximum concentrations (C(max) ) for enrofloxacin 5 mg/kg s.c. and 10 mg/kg s.c. were 0.83 (0.68-1.52) and 2.08 (1.34-2.96) μg/mL and the median (range) T(max) were 1.5 h (1-2) and 1 h (1-2) respectively. Plasma concentrations of orally dosed marbofloxacin were too low to be quantified. Oral administration of enrofloxacin suggested absorption rate limited disposition pharmacokinetics; the median (range) C(max) for enrofloxacin 20 mg/kg p.o. was 0.94 (0.76-1.0) μg/mL and the median (range) T(max) was 4 h (2-8). Oral absorption of both drugs was poor. Plasma protein binding for enrofloxacin was 55.4 ± 1.9% and marbofloxacin 49.5 ± 5.3%. Elevations in creatinine kinase activity were associated with drug injections. Enrofloxacin and marbofloxacin administered at these dosage and routes are unlikely to inhibit the growth of chlamydial pathogens in vivo., (© 2010 Blackwell Publishing Ltd.)

Gastrointestinal helminths were collected from pademelons of the genus Thylogale (Marsupialia: Macropodidae) in eastern Australia and Papua New Guinea. Examined were 12 Thylogale stigmatica stigmatica and 13 T. s. wilcoxi, the latter subdivided into eight specimens from the northern limit of their distribution and five from southern areas, all from eastern Queensland, Australia, one T. s. oriomo from Papua New Guinea and ten T. thetis from southeastern Queensland and northern New South Wales, Australia. Six species of cestodes and 40 species of nematodes were found. The helminth community of T. s. stigmatica was similar to that found in northern specimens of T. s. wilcoxi, while differences from the helminth community present in southern T. s. wilcoxi could be accounted for by parasites acquired from sympatric T. thetis. Thylogale thetis harboured a community of helminths distinct from but related to that in T. stigmatica. The evidence suggests that all subspecies of T. stigmatica examined share a common helminth community, but that in areas of sympatry, T. stigmatica and T. thetis share some of their parasites.

We have examined the correlations between direct surface-finish metrology techniques and normalincidence, soft x-ray reflectance measurements of highly polished x-ray multilayer mirrors. We find that, to maintain high reflectance, the rms surface roughness of these mirrors must be less than ~ 1 Å over the range of spatial frequencies extending approximately from 1 to 100 µm(-1)1 (i.e., spatial wavelengths from 1 µm to 10 nm). This range of spatial frequencies is accessible directly only through scanning-probe metrology. Because the surface-finish Fourier spectrum of such highly polished mirrors is described approximately by an inverse power law (unlike a conventional surface), bandwidth-limited rms roughness values measured with instruments that are sensitive to only lower spatial frequencies (i.e., optical or stylus profileres) are generally uncorrelated with the soft x-ray reflectance and can lead to erroneous conclusions regarding the expected performance of substrates for x-ray mirrors.

The recognition of a reversible cause for acute respiratory failure (ARF) is frequently difficult in patients with severe chronic obstructive pulmonary disease (COPD). We sought to identify clinical findings present at the time of tracheal intubation that were associated with successful weaning and short-term survival among a population of male veterans with severe COPD. Over a 5-year period (1987 to 1991), 39 episodes of ARF requiring mechanical ventilation (MV) were identified in 33 men with severe COPD. All the patients had a baseline FEV1 < 1 L. Univariate analysis suggested a higher serum albumin level and absence of pulmonary infiltrates on chest radiography distinguished survivors (weaned from MV for 72 h) from nonsurvivors (died while undergoing MV or within 72 h of weaning). Multivariate analysis revealed the absence of pulmonary infiltrates on initial chest radiography was the strongest correlate for survival. To examine the significance of these correlates in ARF complicating milder COPD, 19 patients with lesser degrees of chronic airways obstruction and ARF were also studied. Unlike patients with severe COPD, the presence or absence of pulmonary infiltrates on chest radiography was not correlated with survival in patients with milder chronic airways obstruction. Analyzing all COPD patients with ARF, the mortality risk associated with the presence of pulmonary infiltrates on chest radiography increased dramatically with declining baseline lung function. Mortality risk ratio analysis revealed the greatest likelihood for survival was predicted by a higher baseline FEV1 and the absence of pulmonary infiltrates on chest radiography. The extent of baseline airways obstruction alone was not correlated with short-term survival in either group. These observations suggest that in the subset of patients with severe COPD and ARF, the presence of pulmonary infiltrates on chest radiography at the time of tracheal intubation may be associated with less likelihood for survival. An exacerbation of COPD may infrequently be the terminal illness in these patients.

Much of the tissue damage associated with emphysema and other inflammatory diseases has been attributed to the proteolytic activity of neutrophil elastase, a major component of the azurophil granule. Recently, two additional azurophil granule proteins with NH2-terminal sequence homology to elastase were isolated (Gabay, J. E., Scott, R. W., Campanelli, D., Griffith, J., Wilde, C., Marra, M. N., Seeger, M., and Nathan, C. F. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5610-5614) and designated azurophil granule protein 7 (AGP7) and azurocidin. Azurocidin and AGP7 represent significant protein components of the azurophil granule, together comprising approximately 15% of the acid-extractable protein as judged by reverse-phase high performance liquid chromatography analysis. AGP7 migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as four distinct glycoforms of molecular mass 28-34 kDa, whereas azurocidin exhibits three predominant bands with molecular mass of 28-30 kDa. Treatment of intact azurophil granules with [3H]diisopropyl fluorophosphate resulted in labeling of elastase, cathepsin G, and AGP7, whereas azurocidin was not labeled. Tryptic mapping of 3H-labeled AGP7 allowed us to identify and sequence the active-site polypeptide that has 70% identity to elastase over 20 residues. The active site peptide of azurocidin was also identified by sequence analysis of tryptic fragments and showed 65% identity to the active site of elastase. Surprisingly, the catalytic serine of azurocidin is replaced by glycine, explaining its inability to label with [3H]diisopropyl fluorophosphate. Thus, we have identified two azurophil proteins closely related to neutrophil elastase, one of which has apparently lost its proteolytic activity due to mutation of the catalytic serine.

Neutrophil granules contain proteins important in host defense against bacterial pathogens. Granule proteins released from activated neutrophils facilitate opsonization, phagocytosis, tissue digestion, and antimicrobial activity. Three similar, if not identical, neutrophil proteins, bactericidal/permeability-increasing protein (BPI), 57,000 m.w. cationic antimicrobial protein, and bactericidal protein have been described that specifically kill gram negative bacteria. Since LPS is a structure common to all gram-negative bacteria, we investigated whether the microbicidal protein BPI affects biologic activity of LPS in vitro. Human neutrophils can be activated both in vitro and in vivo by LPS. Upon stimulation, surface expression of CR1 and CR3 increases markedly. Using flow microfluorimetry, we analyzed surface expression of CR1 and CR3 as a measure of neutrophil stimulation in response to LPS. CR up-regulation on neutrophils was TNF independent, suggesting direct LPS stimulation of neutrophils in this system. Purified BPI completely inhibited CR up-regulation on neutrophils stimulated with both rough and smooth LPS chemotypes at 1.8 to 3.6 nM (100 to 200 ng/ml). By comparison, the polypeptide antibiotic polymyxin B completely inhibited the same dose of LPS at 0.4 nM. The inhibitory activity of BPI appeared to be specific for LPS because neutrophil stimulation by formylated peptide or TNF was unaffected. The specificity of BPI for LPS was further demonstrated by inhibition of LPS activity in the limulus amebocyte lysate assay. Therefore, the role of BPI in infection may not be limited to its microbicidal activity, but it may also regulate the neutrophil response to LPS.

The American education system is being challenged to raise the academic achievement of students to prepare them for the jobs of the future. Yet many demographic, as well as economic and social factors, are making the task more difficult. Low birth rates, especially among non-Hispanic whites, along with high immigration rates, have increased the share of minority and non-English students in public schools. The rise in single-parent families has increased the number of poor students and migration from the cities to the suburbs has concentrated poor and minority students in inner city schools. These same children will make up a greater share of the future labor force. At the same time, the aging of the general population may lessen the commitment of homeowners- -whose taxes pay between 1/3 and 1/2 of education costs. The aging labor force may bring a shortage of qualified teachers, particularly in specialized subject areas. Poor and minority students generally have below average academic skills and are more likely to drop out of high school than non-minority students. However, the skills of American students rank below those of most other industrialized nations, calling into question the ability of Americans to succeed in an increasingly international economic system. How can schools be improved and minority student achievement be enhanced? Reforms of education finance systems, court-ordered integration, and stiffer requirements for teachers and for graduation from high school are among many attempts to meet the immense challenges faced by American schools.

Previous reports found the 67Ga scan highly accurate in staging lung cancer. In the present study the cost-effectiveness of the 67Ga scan was measured and compared with that of routine tests (radionuclide liver and bone scans, brain CT scan) used to stage lung cancer. In 160 patients, the 67Ga scan had a lower sensitivity, specificity, and negative predictive value than the combination of routine tests in detecting metastatic disease. The 67Ga scan was less accurate than the appropriate routine test in establishing the presence of liver, bone, and brain metastases. In the mediastinum the 67Ga scan was not more accurate than the chest radiograph. The average cost to accurately stage a patient by 67Ga scan was $812.12 and by routine tests was $737.60. The cost for metastatic disease was $1,417.70 by 67Ga scan and $1,287.70 by routine tests. It is concluded that at our institution the use of 67Ga scan to stage lung cancer is not cost-effective.

A patient with miliary blastomycosis had acute fulminating respiratory failure requiring prolonged external ventilatory support. Treatment consisted of antifungal chemotherapy with two drugs and administration of corticosteroids. Restrictive ventilatory impairment and exercise-induced hypoxemia persist at one year after completion of therapy.

Mucormycosis is an opportunistic invasive infection caused by fungi of the order Mucorales. Rhizopus, Absidia, and Mucor are the most commonly encountered genera. Disease is characterized by vascular invasion, thrombosis, and tissue necrosis. Rhinocerebral disease is the most common manifestation but pulmonary, cutaneous, gastrointestinal, and widely disseminated forms have been reported. Pulmonary and disseminated disease are usually seen in neutropenic patients with leukemia or lymphoma. Both present as fever and unexplained pulmonary infiltrates unresponsive to antibacterials and corticosteroids. Disease is usually fulminant and has a high mortality rate. Diagnosis is most commonly made at autopsy. A single case of disseminated disease is reported that is unusual in its subacute course and its occurrence in an otherwise healthy non-neutropenic diabetic male.

Sorghum samples were either untreated or oxidized with performic acid (PA) before hydrolysis, and their amino acid contents were determined by cation exchange chromatography using an amino acid analyzer. HCl was used to destroy excess PA. Oxidative pretreatment of the samples resulted in increased yields of Cys (as cysteic acid), Met (as Met dioxide), and His, destroyed Tyr and Phe, and resulted in the appearance of an extraneous peak which most likely consisted of halogenation by-products (HBP) of Tyr and Phe. The destruction of Tyr and Phe occurred despite the presence of phenol, a halogen scavenger, in both the PA and hydrolysis reagents. The higher His values observed in all oxidized samples most likely resulted from the co-elution of His with Tyr and Phe HBP. It was concluded that the complete (except Trp) amino acid content of a feedstuff cannot be accurately determined from only one oxidized hydrolysate preparation by using this particular procedure.

Primary (azurophil) granules of neutrophils contain proteins which play a major role in the killing and digestion of bacteria in the phagolysosome. We have isolated and characterized a novel antimicrobial peptide from the azurophil granule fraction of discontinuous Percoll gradients. We have named this peptide human neutrophil peptide 4 (HNP-4) based on its structural similarity to a group of antimicrobial polypeptides known as defensins (HNP 1-3). Using size exclusion and reverse-phase high performance liquid chromatography, HNP-4 was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequence analysis. The amino acid sequence determined from isolated HNP-4 and from tryptic fragments of reduced and alkylated peptide is: NH2-Val-Cys-Ser-Cys-Arg-Leu-Val-Phe-Cys-Arg-Arg-Thr-Glu- Leu-Arg-Val-Gly-Asn-Cys-Leu-Ile-Gly-Gly-Val-Ser-Phe-Thr-Tyr-Cys-Cys-Thr- Arg-Val - COOH. Based on this sequence, HNP-4 has a calculated molecular weight of 3715 and a theoretical pI of 8.61. HNP-4 shows structural similarity to the family of three human defensins. HNP-4 and the defensins have identical cysteine backbones and, like the defensins, HNP-4 is rich in arginine (15.2 mol %). However, the amino acids at 22 of the 33 positions differ between HNP-4 and human defensins. Further, HNP-4 is significantly more hydrophobic than the defensins, as determined by its retention time on reverse-phase high performance liquid chromatography. In vitro, purified HNP-4 was shown to kill Escherichia coli, Streptococcus faecalis, and Candida albicans. Compared to a mixture of the other human defensins, HNP-4 was found to be approximately 100 times more potent against E. coli and four times more potent against both S. faecalis and C. albicans.

Corn, peanut meal, and soybean meal samples were either untreated or oxidized with performic acid before hydrolysis; the amino acids were determined by cation exchange high performance liquid chromatography (LC) and conventional cation exchange LC using an amino acid analyzer (AAA). Reproducibility of each procedure was assessed by repeated injections of the same calibration standard solution over a period of several days. LC data were more precise with regard to coefficients of variation for amino acid retention times, but were more variable with regard to peak areas. Although some significant differences between methods were noted, feedstuff amino acid values obtained by LC and AAA compared very well. The only consistent differences observed within each feedstuff were that Phe and Tyr values were significantly lower when analyzed by LC compared with AAA. Results of this study suggest that modular LC instrumentation can be used to accurately and reproducibly analyze amino acids in feedstuff hydrolysates. Advantages of using ninhydrin derivatization for feedstuff analysis, as opposed to using o-phthalaldehyde or dansyl chloride, are discussed.