Pathologic Findings and Viral Antigen Distribution During Natural Infection of Ring-Necked Pheasants With H5N2 Highly Pathogenic Avian Influenza Virus A.

Highly pathogenic avian influenza (HPAI) is a major viral disease of poultry characterized by acute onset, systemic infection, and rapid death. In January 2015, H5N2 HPAI was identified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and gene sequencing as the cause of rapid death in 40 of 390 ring-necked pheasants (approximately 10% mortality), raised in a game bird farm in Washington State. We report clinicopathologic findings and viral antigen distribution in pheasants that died during the outbreak. Affected birds were depressed with reluctance to move, ruffled feathers, and drooping heads. Congestion of the cerebellar meningeal blood vessels was the only consistent gross pathologic finding. Meningoencephalitis with vasculitis and necrosis in the spleen and heart were the major microscopic lesions in the birds. Viral antigen was consistently detected in the brain, heart, and ovary with variable presence in other organs.

Systemic Scuticociliatosis (Philasterides dicentrarchi) in sharks.

Scuticociliatosis is an economically important, frequently fatal disease of marine fish in aquaculture, caused by histophagous ciliated protozoa in the subclass Scuticociliatida of the phylum Ciliophora. A rapidly lethal systemic scuticociliate infection is described that affected aquarium-captive zebra sharks (Stegostoma fasciatum), Port Jackson sharks (Heterodontus portusjacksoni), and a Japanese horn shark (Heterodontus japonicus). Animals died unexpectedly or after a brief period of lethargy or behavioral abnormality. Gross findings included necrohemorrhagic hepatitis and increased volumes of celomic fluid. Histologically, 1 or more of a triad of necrotizing hepatitis, necrotizing meningoencephalitis, and thrombosing branchitis were seen in all cases, with necrotizing vasculitis or intravascular fibrinocellular thrombi. Lesions contained variably abundant invading ciliated protozoa. Molecular identification by polymerase chain reaction from formalin-fixed tissues identified these as the scuticociliate Philasterides dicentrarchi (syn. Miamiensis avidus), a novel and potentially emergent pathogen in sharks.

Coxiella-like infection in psittacines and a toucan.

Seven psittacine birds and a toucan (Ramphastos toco) were diagnosed as infected with Coxiella-like bacteria, based on polymerase chain reaction and bacterial 16S rRNA gene sequence obtained from each bird's liver tissue. Most of the birds exhibited lethargy and weakness for several days prior to death. Gross lesions included mild to moderate emaciation and severely enlarged and mottled pale livers and spleens. Microscopically, there was multifocal necrosis of hepatocytes with infiltration of a mixed population of inflammatory cells, including lymphocytes, heterophils, plasma cells, and macrophages randomly scattered throughout in most birds. In several birds within the macrophages there were vacuoles containing basophilic small cocco-bacilli organisms measuring about 0.5-1 microm. The spleens had increased numbers of mononuclear phagocytic system cells, some of which had vacuoles that contained similar organisms, as observed in the liver. There was inflammation in the epicardium and endocardium, interstitium of the lungs, kidney, adrenal and thyroid glands, lamina propria of the intestine, and in occasional birds in the brain, bursa of Fabricius, and bone marrow associated with similar organisms in the macrophages. Transmission electron microscopy of the liver and lungs in most birds and in the thyroid glands of one bird revealed pleomorphic round to elongated bacteria measuring about 0.45 microm in diameter and more than 1.0 microm in length. Most of these organisms contained a peripheral zone of loosely arranged electron dense material that was located immediately beneath a trilaminar membrane. Occasional organisms contained nucleoids. This is the first documentation of disease presumptively associated with Coxiella-like bacteria in birds.

Fatal coxiellosis in Swainson's Blue Mountain Rainbow Lorikeets (Trichoglossus haematodus moluccanus).

Three Swainson's Blue Mountain Rainbow Lorikeets (Trichoglossus haematodus moluccanus), ranging from 6 to 8 months of age, presented with lethargy, emaciation, and progressive neurologic signs. The first one died 24 hours after the onset of clinical signs, and the other two were euthanized 10 to 14 days after the onset of progressive neurologic disease. Clinical signs in these lorikeets included head pressing, hemiparesis, seizures, obtunded mentation, weakness, and lethargy. Two of the lorikeets had hepatomegaly, and one had splenomegaly on gross examination. Histopathology revealed disseminated microgranulomas in the liver, spleen, and brain, and lymphohistocytic perivascular encephalitis and cephalic vasculitis. Electron microscopic examination of macrophages in brain lesions revealed spherical to rod-shaped prokaryotic organisms with a trilaminar cell wall. Molecular analysis revealed a novel species of Coxiella. This is believed to be the first report of a Coxiella sp. causing disease in a lorikeet.

In vivo visualization of abdominal malignancies with acoustic radiation force elastography.

The utility of acoustic radiation force impulse (ARFI) imaging for real-time visualization of abdominal malignancies was investigated. Nine patients presenting with suspicious masses in the liver (n = 7) or kidney (n = 2) underwent combined sonography/ARFI imaging. Images were acquired of a total of 12 tumors in the nine patients. In all cases, boundary definition in ARFI images was improved or equivalent to boundary definition in B-mode images. Displacement contrast in ARFI images was superior to echo contrast in B-mode images for each tumor. The mean contrast for suspected hepatocellular carcinomas (HCCs) in B-mode images was 2.9 dB (range: 1.5-4.2) versus 7.5 dB (range: 3.1-11.9) in ARFI images, with all HCCs appearing more compliant than regional cirrhotic liver parenchyma. The mean contrast for metastases in B-mode images was 3.1 dB (range: 1.2-5.2) versus 9.3 dB (range: 5.7-13.9) in ARFI images, with all masses appearing less compliant than regional non-cirrhotic liver parenchyma. ARFI image contrast (10.4 dB) was superior to B-mode contrast (0.9 dB) for a renal mass. To our knowledge, we present the first in vivo images of abdominal malignancies in humans acquired with the ARFI method or any other technique of imaging tissue elasticity.

Conventional crops and organic amendments for Pb, Cd and Zn treatment at a severely contaminated site.

The ability of selected plants and amendments to treat Pb, Cd and Zn accumulations from a metalliferous waste disposal site was studied both in the greenhouse and field. Spinach (Spinacea oleracea), cabbage (Brassica oleracea), and a grass-legume mix (red fescue, Festuca rubra; ryegrass, Lolium perenne); and bean (Vicia faba) were grown in the greenhouse on blast furnace slag or baghouse dust amended with composted peat (CP). All plant species accumulated Pb, Cd and Zn to varying degrees. Total soil metal concentrations had a marked influence on plant uptake. Topdressing versus incorporating CP had a significant (p<0.05) effect on spinach and cabbage tissue metal concentrations. Soil Pb and Zn tended to shift towards less bioavailable forms after treatment with CP. Field plots were treated with CP, farmyard manure (FYM), or inorganic fertilizer. Dry matter production of spinach, cabbage and a grass-legume mix was greatest on either the CP or FYM treatments. Phytostabilization in combination with organic amendments may be the most appropriate technology to ensure stabilization of soil metals at this site.

Spinal intradural Mycobacterium haemophilum granuloma in an American Bison (Bison bison).

Mycobacterium haemophilum, a nontuberculous mycobacterium, is a pathogen in immunocompromised human patients. We report a case of M haemophilum-induced granuloma in the spinal cord of an American Bison (Bison bison). M haemophilum infection was diagnosed by sequencing a portion of the 16 S ribosomal DNA and comparing the amplicon sequence with sequences in GenBank.

Sensitivity and specificity of the complement fixation test for detection of cattle persistently infected with Anaplasma marginale.

The complement fixation (CF) test commonly is used to identify cattle infected with Anaplasma marginale prior to interstate or international movement. Estimates of the accuracy of the CF test in detecting animals persistently infected with A. marginale vary widely. In this study, the sensitivity and specificity of the CF test for detection of carrier animals was determined using serum from 232 cattle previously defined as A. marginale positive or negative by nested polymerase chain reaction methods and hybridization. Considering results from 2 independent laboratories and interpreting a 1:5 suspect reaction as positive, the best estimate of CF test sensitivity was 20%, with a specificity of 98%. Using a 1:10 cutoff, sensitivity decreased to 14% and specificity increased to 99%. Results of this study indicate that the CF test is ineffective for identifying cattle persistently infected with A. marginale and thus is inadequate for anaplasmosis regulatory and surveillance programs.

Learning in year-old female autoimmune BXSB mice.

BXSB/ MpJ-Yaa and NZB/BINJ mice have been used as animal models for both developmental learning disability and systemic autoimmune disease. Approximately 40-60% of these animals show ectopic clusters of neurons in Layer I of cortex similar to those found in postmortem analyses of human dyslexics, and all exhibit an autoimmune condition similar to systemic lupus erythematosus (SLE) in humans. The expression of immune disease in the BXSB strain, unlike in humans, is more severe in males than females. Most previous studies have examined the behavioral sequelae of neocortical ectopias at a relatively young age, when the BXSB females (unlike the male BXSB and female and male NZBs) are not yet showing high titers of autoantibodies associated with their lupus-like form of autoimmune disease. This study examined the behavior of BXSB females at an age subsequent to autoimmune disease onset. When contrasted with younger animals, year-old BXSB females showed good learning behavior, with no differences in Lashley maze learning and remarkably good performance in a visual discrimination learning task. These results are consistent with other data which indicate that many types of learning behavior are apparently unperturbed by systemic autoimmune disease. Results also showed significant interactions between a measure of lateral paw preference and the presence or absence of ectopias in Lashley maze learning. Animals without ectopias that exhibited a right lateral paw preference showed the greatest number of errors on a number of test measures. These findings support previous results indicating that behavioral effects associated with ectopias may vary based upon the behavioral laterality of affected animals.

Learning and memory in the autoimmune BXSB mouse: effects of neocortical ectopias and environmental enrichment.

Approximately 40-60% of BXSB mice have ectopic cell clusters in layer 1 of neocortex. Prior studies have shown distinct behavioral differences between those with ectopias and their non-ectopic littermates. In this study, female BXSB mice were reared after weaning in either enriched environments or standard cages. Following an initial round of behavioral testing, all mice were housed in standard cages and retested. Enriched cage mice (both ectopic and non-ectopic) showed increased activity, greater speed, and enhanced learning scores across a variety of tests. Additionally, prior test experience itself had significant positive effects on Hebb-Williams maze learning. The presence of ectopias resulted in better Morris maze learning for standard cage reared mice. Further, ectopic mice, regardless of their housing condition, showed better long-term retention in the Morris maze than did their non-ectopic counterparts. These findings show that abnormalities in corticogenesis need not always result in functional deficit.

Perspectives on interactions with QA--the regulatory perspective.

It has been my experience that the quality assurance unit (QAU) normally serves as the facility point-of-contact with the agency on matters relating to a Good Laboratory Practice (GLP) compliance inspection. The QAU usually receives the notification letter, does most of the coordinating for the GLP inspection, and often bears the criticism for deficiencies found during the inspection. In general, as inspectors, we have found it difficult to get management to show any interest in the inspection and, in many facilities, have been forced to communicate almost entirely with the QAU. The evolution of the relationship between regulators and the QA community, which parallels the evolution of the image of quality assurance personnel from inexperienced technicians to recognized professionals, is discussed. Also discussed are both typical and atypical examples of interaction between government inspectors and quality assurance units.

Laboratory accreditation.

An investigation of alleged data fraud at a pesticide analytical laboratory led EPA to take a closer look at the Good Laboratory Practice (GLP) inspection program. There was special focus on changes which might be made in the program to enhance the chances of detecting fraud in regulated studies. To this end, the Assistant Administrator of the Office of Prevention, Pesticides and Toxic Substances (OPPTS) requested EPA's Office of Inspector General (OIG) to examine the GLP program. Several reports were issued by the OIG, including the recommendation that a laboratory accreditation program be adopted. EPA has been examining ways to implement the OIG's recommendations, including (1) laboratory accreditation consisting of three components: document submission and assessment, site visit and assessment, and proficiency assessment; and (2) mandatory registration of all facilities participating in GLP-regulated studies, based on document submission and assessment. These two alternatives are compared, and the advantages and disadvantages of each are discussed.

Evaluation of a kinetic enzyme-linked immunosorbent assay for detection of caprine arthritis-encephalitis virus-specific antibodies.

A kinetic indirect enzyme-linked immunosorbent assay (k-ELISA) was evaluated for detection of antibody to caprine arthritis-encephalitis virus (CAEV), using sodium dodecyl sulfate-treated CAEV-63 as antigen. Two hundred fifteen caprine sera submitted to the diagnostic laboratory were tested for CAEV antibody by the k-ELISA and by immunoprecipitation of [35S]-methionine-labeled CAEV. A k-ELISA positive cutoff point of 80 yielded a sensitivity of 94.4% and a specificity of 100%, as compared with immunoprecipitation. A k-ELISA cutoff point of 50 resulted in a sensitivity of 100%, with 95.6% specificity. When sera with k-ELISA scores between 50 and 80 were considered suspect, testing of 1,001 diagnostic sera resulted in < 1.5% suspect reactions. Using the 80 cutoff point, the CAEV k-ELISA had good sensitivity and specificity, with the added advantages of quick turn-around time, few suspect reactions, and adaptability to large numbers of samples

Evaluation of agar gel immunodiffusion serology using caprine and ovine lentiviral antigens for detection of antibody to caprine arthritis-encephalitis virus.

The sensitivity of the agar gel immunodiffusion (AGID) test for the detection of antibody to caprine arthritis-encephalitis virus (CAEV) was investigated with CAEV or ovine progressive pneumonia virus (OPPV) as the source of antigen. A total of 218 goat serum specimens were tested for anti-CAEV antibody by AGID and immunoprecipitation of [35S]methionine-labeled CAEV. In comparison with that of immunoprecipitation, the sensitivity of the CAEV AGID test was 0.91, and that of the OPPV AGID test was 0.56. The AGID test with either antigen was 100% specific. The lower sensitivity of the OPPV AGID test in detecting caprine antibody to CAEV indicates that OPPV antigen is of limited value for use in CAEV diagnosis and control programs.

Antagonism of estrogenic effects on feeding behavior by central implants of anisomycin.

The following experiment determined whether the estrogenic suppression of food intake is dependent upon changes in protein synthesis within neurons of the paraventricular nucleus of the hypothalamus (PVN). Ovariectomized rats were treated centrally with anisomycin-filled or empty (control) cannulae in the PVN. Females were injected with either 2.0 micrograms of estradiol benzoate (EB) or the oil vehicle and the inner cannulae were removed 2 h later. EB injections significantly lowered food and water intake in the central control group but not in animals given PVN implants of anisomycin. Body weight gain decreased for all females. EB induced comparable levels of female sexual behavior in both groups, demonstrating that anisomycin implants did not affect the ability of estradiol to stimulate lordosis. These findings indicate that the effects of estradiol on food intake require the activation of protein synthesis in estrogen-sensitive PVN neurons.

Modulation of the satiety effect of cholecystokinin by estradiol.

Data obtained from a wide variety of mammalian species indicate that feeding behavior can be influenced by changes in endogenous estrogens and by exogenous estrogenic treatments. The present experiment represents an initial investigation of the hypothesis that the suppression of food intake by estradiol is mediated by an enhancement of the satiety effect of cholecystokinin (CCK). Twenty-four female rats were ovariectomized and implanted either with a 5% estradiol silastic capsule or an empty capsule on the day of surgery. Three weeks later, animals received IP injections of CCK-octapeptide (5.0 or 10.0 micrograms/kg) or saline after 24-h food deprivation. Food and water intake were measured 60 min after treatment. Although CCK suppressed feeding in all subjects, the effects on food intake were greater in estradiol-treated females. CCK injections also reduced water intake, but there was no interaction between estradiol and CCK on drinking. These findings indicate that the inhibitory effect of CCK on food intake is enhanced in females treated with a physiological dose of estradiol, and suggest that the effects of estradiol on feeding behavior may be mediated by a potentiation of the satiety effect of CCK.

Worker exposure to ethion in Florida citrus.

Dermal and respiratory exposure to ethion was determined for 17 men in eight spray crews in Florida citrus spray operations. Respiratory exposure was less than 1% of the total exposure. Hands represented 42% of the total body exposure for applicators and 76% for suppliers. At one location, suppliers exhibited a larger decrease in ChE activity than applicators. This difference appeared related to the higher mean dermal ethion exposure to suppliers. Acute symptoms of organophosphorous poisoning were not observed. The total percent/hr of the probable human dermal LD50 was very low in all cases. These data indicate a relatively low potential acute hazard to workers applying ethion with air blast equipment under the conditions of this study.

The recognition of diazinon, an organophosphorus pesticide, when found in samples in the form of decomposition products.

The contents of a bottle, from which a human being was reported to have drunk and which were believed responsible to an organophosphorus poisoning, were submitted for chemical analysis. Initial screening by gas chromatography with phosphorus, sulfur, and nitrogen specific detectors failed to identify any intact organophosphorus pesticide. Mass spectrometric techniques were applied to the identification. Field ionization, field desorption, chemical ionization, exact mass measurements at high resolution, and GC/low resolution mass spectrometry were used to help define the qualitative and partial quantitative nature of the sample components. Results of this study were consistent with the virtually complete conversion of the pesticide, diazinon [Chemical Abstracts reference number 333-41-5], into a mixture of at least twenty-six chemically distinct products or impurities. The most abundant chemical compounds found in the sample included: 2-isopropyl-4-methyl-6-hydroxypyrimidine [2814-20-2]; 2-isopropyl-4-methyl-6-mercaptopyrimidine; 6,6'-dithiobis-(2-isopropyl-4-methylpyrimidine); 6,6'-dithiobis-(2-isopropyl-4-methylpyrimidine); 4-ethoxy-2-isopropyl-6-methylpyrimidine [72799-31-6]; 4-thioethoxy-2-isopropyl-6-methylpyrimidine; triethylphosphorothiolate and triethylphosphorothiolate. Also found were several potent acetylcholinesterase inhibitors: monothionotetraethylpyrophosphate; dithionotetraethylpyrophosphate [3689-24-5]; tetraethylpyrophosphate. Model decomposition studies verified the formation of these compounds. These results were then used to identify compounds in two other samples.