Multilocus Sequence Typing of Eimeria maxima in Commercial Broiler Flocks.

About 35% of all broiler flocks in the United States receive an anticoccidial vaccine, but it is not possible to easily differentiate Eimeria vaccine strains from Eimeria field isolates. Being able to do that would allow using vaccines in a more targeted way. The objective of this study was to collect Eimeria maxima isolates from broiler flocks that received anticoccidial feed additives and flocks that had been vaccinated against coccidia and then test them with a multilocus sequencing typing (MLST) scheme developed for this study. Fecal samples were obtained from commercial broiler flocks in Alabama and Tennessee. Oocyst counts in samples tended to be lower in flocks receiving anticoccidial feed additives and higher in vaccinated flocks. Selected samples were screened for presence of E. maxima by quantitative PCR, and Eimeria spp. composition was investigated by next-generation amplicon sequencing (NGAS) in 37 E. maxima positive samples. Other detected Eimeria spp. besides E. maxima were Eimeria acervulina in 35 samples, Eimeria praecox in 23 samples, Eimeria mitis or Eimeria mivati in 17 samples, and Eimeria necatrix or Eimeria tenella in 10 samples. Six partial E. maxima genes (dnaJ domain containing protein, 70-kDa heat shock protein, prolyl endopeptidase, regulator of chromosome condensation domain containing protein, serine carboxypeptidase, and vacuolar proton-translocating ATPase subunit) of 46 samples were sequenced. The MLST scheme was able to differentiate two vaccines from each other. Three of 17 samples from vaccinated flocks differed from the vaccine used in the flock, while 16 of 29 samples from unvaccinated flocks differed from the vaccine. However, there was also a large number of low-quality, ambiguous chromatograms and negative PCRs for the selected genes. If and when more advanced, possibly next-generation sequencing-based methods will be developed, the genes should be considered as targets.

Tipificación por secuenciación multilocus de Eimeria maxima en parvadas comerciales de pollos de engorde. Alrededor del 35% de todas las parvadas de pollos de engorde en los Estados Unidos recibe una vacuna anticoccidial, pero no es posible diferenciar fácilmente las cepas vacunales de Eimeria de los aislados de campo de Eimeria. La posibilidad de diferenciar entre cepas vacunales y de campo permitiría usar vacunas de una manera más específica. El objetivo de este estudio fue recolectar aislamientos de Eimeria maxima de parvadas de pollos de engorde que recibieron aditivos alimenticios anticoccidiales y parvadas que habían sido vacunadas contra coccidia y luego analizarlos con un esquema de tipificación por secuenciación multilocus (MLST) desarrollado para este estudio. Las muestras fecales se obtuvieron de parvadas comerciales de pollos de engorde en Alabama y Tennessee. Los conteos de ooquistes en las muestras tendieron a ser más bajos en las parvadas que recibieron aditivos alimenticios anticoccidiales y más altos en las parvadas vacunadas. Las muestras seleccionadas se examinaron para determinar la presencia de E. maxima mediante PCR cualitativa, y Eimeria spp. la composición se investigó mediante secuenciación de amplicones de próxima generación (NGAS) en 37 muestras positivas de E. maxima. Además de E. máxima, otras Eimeria spp detectadas, fueron Eimeria acervulina en 35 muestras, Eimeria praecox en 23 muestras, Eimeria mitis o Eimeria mivati en 17 muestras, y Eimeria necatrix o Eimeria tenella en 10 muestras. Se secuenciaron seis genes parciales de E. maxima (proteína que contiene al dominio dnaJ, proteína de choque térmico de 70 kDa, prolil endopeptidasa, proteína que contiene al regulador del dominio de condensación cromosómica, serina carboxipeptidasa y la subunidad de ATPasa vacuolar translocadora de protones) de 46 muestras. El esquema MLST pudo diferenciar dos vacunas entre sí. Tres de 17 muestras de parvadas vacunadas diferían de la vacuna utilizada en la parvada, mientras que 16 de 29 muestras de parvadas no vacunadas diferían de la vacuna. Sin embargo, también hubo una gran cantidad de cromatogramas ambiguos y de baja calidad y PCR negativos para los genes seleccionados. En Cuando se desarrollen métodos más avanzados, posiblemente de próxima generación, basados en la secuenciación, estos genes deben considerarse como objetivos.

[Guideline for Long-Term Oxygen Therapy - S2k-Guideline Published by the German Respiratory Society]. / Leitlinie zur Langzeit-Sauerstofftherapie.

Long-term oxygen therapy is of great importance both for reducing mortality and for improving performance in patients with chronic lung diseases. The prerequisites for Long-term oxygen therapy are adequate diagnostics and clearly defined indication. A causal distinction into chronic hypoxaemic and hypercapnic respiratory failure is reasonable, from which the differential indication for non-invasive ventilation results.The revised guideline covers the diagnostics and indication of chronic lung and heart diseases, the role of oxygen in terminal illness and gives a detailed description of available oxygen devices. The guideline is intended to help avoid undersupply, oversupply and false prescriptions. Furthermore, the chapter "Postacute Oxygen Therapy" discusses the procedure, relevant in everyday life, but not yet clearly defined, for prescribing oxygen therapy for the home at the end of an inpatient stay. Another important point, the correct prescription of mobile oxygen systems, is also presented in the guideline. This document is a revised version of the guideline for longterm oxygen therapy and replaces the version of 2008.

Genotypic Characterization of Emerging Avian Reovirus Genetic Variants in California.

This study focuses on virus isolation of avian reoviruses from a tenosynovitis outbreak between September 2015 and June 2018, the molecular characterization of selected isolates based on partial S1 gene sequences, and the full genome characterization of seven isolates. A total of 265 reoviruses were detected and isolated, 83.3% from tendons and joints, 12.3% from the heart and 3.7% from intestines. Eighty five out of the 150 (56.6%) selected viruses for sequencing and characterization were successfully detected, amplified and sequenced. The characterized reoviruses grouped in six distinct genotypic clusters (GC1 to GC6). The most represented clusters were GC1 (51.8%) and GC6 (24.7%), followed by GC2 (12.9%) and GC4 (7.2%), and less frequent GC5 (2.4%) and GC3 (1.2%). A shift on cluster representation throughout time occurred. A reduction of GC1 and an increase of GC6 classified strains was noticed. The highest homologies to S1133 reovirus strain were detected in GC1 (~77%) while GC2 to GC6 homologies ranged between 58.5 and 54.1%. Over time these homologies have been maintained. Seven selected isolates were full genome sequenced. Results indicated that the L3, S1 and M2 genes, coding for proteins located in the virus capsid accounted for most of the variability of these viruses. The information generated in the present study helps the understanding of the epidemiology of reoviruses in California. In addition, provides insights on how other genes that are not commonly studied add variability to the reovirus genome.

Retrospective Study of Histomoniasis (Blackhead) in California Turkey Flocks, 2000-2014.

The flagellate parasite Histomonas meleagridis causes a severe disease in turkeys. Since nitarsone, the last antiflagellate feed additive, was withdrawn from the market, H. meleagridis has gained increasing clinical and economic importance, and frequently entire turkey flocks are lost to the disease. Even before the antiflagellate market withdrawal, H. meleagridis has been recognized as a noteworthy disease in commercial turkeys. The aim of this study was to compile and analyze data on histomoniasis outbreaks diagnosed by the California Animal Health and Food Safety System in the years 2000 through 2014 while nitarsone was still available. Sixty-two cases were included in the study. In all cases, the parasite was detected by histopathology. Five cases were from breeder flocks, 44 from commercial meat turkey flocks, and 13 from other flocks such as backyard flocks or pet turkeys. There were between one and eight cases each year with no clear trend over time. Most cases occurred in the warmer months between April and October with a clear peak in August. Turkeys in the flocks with the disease were aged between 2 wk and 15 mo, with a median age of 9 wk. In cases for which mortality was reported, daily mortality ranged between 0.04% and 2% with an average of 0.34% and a median of 0.15%. Five-week mortality in infected houses on three infected ranches was between 1.3% and 68%. In 12 cases, the parasite was observed in organs other than ceca and liver. These included spleen, kidney, bursa of Fabricius, proventriculus, lung, pancreas, and crop. In 58 cases, histomoniasis was considered the most significant and primary diagnosis; in four cases histomoniasis was regarded as an incidental finding. In 14 other outbreaks, detailed information was available. In 11 cases, more than one house on the ranch was affected; in five cases the disease spread to all houses. Total mortality in turkeys from affected flocks ranged between 12% and 65%. In eight flocks, the birds were marketed with a weight that was considered too light. In conclusion, the present study shows that even with nitarsone available as antiflagellate feed, additive histomoniasis was a serious disease of turkeys in the United States with an epidemiology similar to that observed in Europe.

Biosecurity Assessment and Seroprevalence of Respiratory Diseases in Backyard Poultry Flocks Located Close to and Far from Commercial Premises.

Raising backyard chickens is an ever-growing hobby in the United States. These flocks can be a substrate for respiratory disease amplification and transmission to commercial facilities. Five hundred fifty-four chickens from 41 backyard flocks were sampled in this study. ELISA kits were used to detect antibodies against avian influenza (AI), infectious laryngotracheitis (ILT), Newcastle disease (ND), infectious bronchitis (IB), Ornithobacterium rhinotracheale (ORT), Mycoplasma gallisepticum (MG), and Mycoplasma synoviae (MS). All visited flock owners answered a biosecurity questionnaire that assessed biosecurity measures. The questionnaire revealed that backyard poultry owners lack simple biosecurity measures such as use of dedicated shoes, their chicken sources are unreliable, and few of them benefit from veterinary oversight. Only one flock had a clear vaccination history against ND and IB. ORT, ND, IB, MS, MG, and ILT were the most seroprevalent in backyard poultry flocks with 97% (41/42), 77.5% (31/40), 75% (30/40), 73% (31/42), 69% (29/42), and 45% (19/42), respectively. The vaccinated flock was not considered in these calculations. When examining the distance between backyard flocks and the nearest commercial poultry facility, ND and MG were significantly more likely to be found in backyard flocks close to (<4 miles) whereas ORT was significantly more likely in backyard chickens located far from (>4 miles) commercial poultry. Birds purchased directly from National Poultry Improvement Plan hatcheries showed a reduced ND, MG, and MS antibody prevalence. Wearing dedicated shoes decreased MS antibody-positive birds. Finally, history of wild bird contact had a clear effect on an increased seroprevalence of NDV and MG. Serological results suggest that backyard poultry flocks have the potential to serve as a reservoir or amplifier for poultry respiratory diseases. The information generated in this project should direct extension efforts toward emphasizing the importance of small flock biosecurity and chick acquisition sources.

Understanding Immune Resistance to Infectious Bronchitis Using Major Histocompatibility Complex Chicken Lines.

Genetic resistance or susceptibility to infectious diseases has been largely associated with the avian major histocompatibility complex (MHC) genes. Our goal was to determine resistance and susceptibility of MHC B haplotype in congenic and inbred chicken lines in order to establish a resistant-susceptible model. Eight congenic lines (253/B18, 254/B15, 330/B21, 312/B24, 331/B2, 335/B19, 336/B21, and 342/BO), two inbred lines (003/B17 and 077/B19), and three commercial lines (white leghorn, brown layers, and broilers) were used in two experiments. We analyzed and compared immunologic responses and the effect of challenge by measuring viral load, IgG and IgA humoral responses, histopathology and histomorphometry, clinical signs, and immune cell populations in the different MHC B haplotype lines. We found that respiratory signs, tracheal deciliation and inflammation, airsacculitis, viral shedding in tears, and local humoral responses were good parameters to determine resistance or susceptibility. Based on these results, we identified 331/B2 as the most resistant and 335/B19 as the most susceptible congenic chicken lines. These two lines will be used as an animal model in subsequent experiments to understand the mechanisms by which the immune system in chickens generates resistance to infectious bronchitis virus.

Persistence of Highly Pathogenic and Low Pathogenic Avian Influenza Viruses in Footbaths and Poultry Manure.

A questionnaire was designed in order to gather information about bedding material and footbath preparation and maintenance in different productive units across the state of California.This information was used to plan two experiments. In the first experiment, we tested the effectiveness of footbaths in inactivating highly pathogenic (HP) and low pathogenic (LP) avian influenza viruses (AIVs) on rubber boots. Surprisingly, quaternary ammonia- and quaternary ammonia + glutaraldehyde-based footbaths were not able to eliminate live HPAIV (H5N8) and LPAIV (H6N2) particles on boots, while a chlorine-based granulated disinfectant was able to destroy the virus at contact. These results demonstrated the potential of AIV, particularly the HPAIV isolate, to persist even if exposed to disinfecting footbaths, and suggest that footbaths, as a single tool, are not capable of preventing pathogen introduction into commercial flocks. In the second experiment, we investigated the persistence of HPAIV (H5N8) and LPAIV (H6N2) in bedding material and feces obtained from turkey, broiler, and egg-layer commercial productive units. Samples were collected at different times after spiking the bedding materials and feces. Results showed that HPAIV (H5N8) was more persistent than LPAIV (H6N2) in layer feces and bedding material obtained from commercial broilers and turkeys. Live HPAIV particles persisted 96 hr, the last time point measured, in layer feces and less than 60 hr in broiler and turkey bedding. In contrast, LPAIV persisted less than 24 hr after being spiked in all the different substrates. Further research in biosecurity practices such as footbath preparation and maintenance and better understanding of the mechanism of the increased persistence of AIV is warranted in order to identify effective litter treatments that destroy live virus in bedding material.

Diatoms and diatomaceous earth as novel poultry vaccine adjuvants.

Diatoms are single cell eukaryotic microalgae; their surface possesses a porous nanostructured silica cell wall or frustule. Diatomaceous earth (DE) or diatomite is a natural siliceous sediment of diatoms. Since silica has been proved to have adjuvant capabilities, we propose that diatoms and DE may provide an inexpensive and abundant source of adjuvant readily available to use in livestock vaccines.In a first experiment, the safety of diatoms used as an adjuvant for in-ovo vaccination was investigated. In a second experiment, we assessed the humoral immune response after one in-ovo vaccination with inactivated Newcastle Disease Virus (NDV) and DE as adjuvant followed by 2 subcutaneous boosters on d 21 and 29 of age. In both experiments, results were compared to Freund's incomplete adjuvant and aluminum hydroxide.No detrimental effects on hatchability and chick quality were detected after in-ovo inoculation of diatoms and DE in experiments 1 and 2 respectively. In experiment 2 no humoral responses were detected after the in-ovo vaccination until 29 d of age. Seven d after the second subcutaneous booster an antibody response against NDV was detected in chickens that had received vaccines adjuvanted with Freund's incomplete adjuvant, aluminum hydroxide, and DE. These responses became significantly higher 10 d after the second booster. Finally, 15 d after the second booster, the humoral responses induced by the vaccine with Freund's incomplete adjuvant were statistically higher, followed by comparable responses induced by vaccines containing DE or aluminum hydroxide that were significantly higher than DE+PBS, PBS+INDV and PBS alone. From an applied perspective, we can propose that DE can serve as a potential adjuvant for vaccines against poultry diseases.

Variability Assessment of California Infectious Bronchitis Virus Variants.

On the basis of the data from the California Animal Health and Food Safety Laboratory System, 1444 infectious bronchitis (IB) cases were diagnosed between 1997 and 2012. Epidemiologic analyses demonstrated two major IB virus (IBV) outbreak peaks, affecting mainly 35-to-49-day-old broiler chickens. California variant 1737 (CA1737) and California variant 1999 (Cal 99) IBV types were the most prevalent genotypes during the analyzed period. To further understand the increased prevalence of these genotypes, we assessed and compared the variability of the S1 gene hypervariable region of CA1737 and Cal 99 with the variability of IBV strains belonging to the Massachusetts 41 (M41) and Arkansas (Ark) types during serial passages in embryonated chicken eggs. On the basis of the S1 nonsynonymous changes, seven different subpopulations were detected in M41. However, the predominant population of the field strain M41 before passages continued to be predominant throughout the experiment. In contrast, Ark passaging resulted in the detection of 13 different subpopulations, and the field sequence became extinct after the first passage. In IBV Cal 99, eight different subpopulations were detected; one of these became predominant after the second passage. In CA1737, 10 different subpopulations were detected. The field strain major sequence was not detected after the first passage but reappeared after the second passage and remained at low levels throughout the experiment. Compared with M41 and Ark, Cal 99 and CA1737 showed intermediate variability.

Runting Stunting Syndrome Associated with Transmissible Viral Proventriculitis in Broiler Chickens.

This report describes an outbreak of transmissible viral proventriculitis (TVP) associated with runting stunting syndrome (RSS) in 25- and 28-day-old broiler chickens, in which chicken proventricular necrosis virus (CNPV) was detected. Clinical signs included poor uniformity, very small birds for their age, increased mortality, and culling of smaller birds. Almost all birds necropsied exhibited moderate to severely enlarged proventriculi with diffusely pale serosa and thickened walls. Microscopically the proventriculi had lesions of degeneration and necrosis of the epithelium of the proventricular glands, accompanied by lymphocytic inflammation and glandular hyperplasia, with occasional formation of lymphoid nodules within the glandular parenchyma. Immunohistochemistry staining for CPNV was positive. Positive staining was generally found in the cytoplasm of glandular epithelial cells in the form of finely granular brown pigment. CPNV RNA was detected in the proventriculi by reverse transcriptase-PCR (RT-PCR). Other findings included mild enteritis in a few birds and small bursa of Fabricius. Direct electron microscopy performed on the intestinal samples was negative for viral particles. RT-PCR analysis of bursae was positive for infectious bursal disease virus (IBDV). In conclusion, this report associates TVP with RSS by describing an outbreak in which TVP attributable to CPNV was the most commonly found lesionin chickens with a clinical history compatible with RSS. Therefore, TVP should be considered as a possible differential diagnosis in cases compatible with RSS.

Retrospective Study on the Isolation of Ornithobacterium rhinotracheale from Chickens and Turkeys in Central California: 294 cases (2000-12).

Ornithobacterium rhinotracheale is a rod-shaped, gram-negative, and mostly oxidase-positive bacterium that causes respiratory infections in chickens and turkeys worldwide and can also spread to nonrespiratory organs. The present report analyzes 294 cases in which O. rhinotracheale was isolated from turkeys or chickens in central California in the years 2000 through 2012. Two hundred sixteen cases were from turkey flocks and 78 from chicken flocks. The median age of turkey flocks was 8.7 wk; the median age of chicken flocks was 6.4 wk. From turkeys, O. rhinotracheale was more often isolated from August to January than during the rest of the year. Chickens cases were more evenly distributed throughout the year. The organs with the highest isolation rate were the infraorbital sinus and trachea, followed by lungs and air sacs. Isolation from other organs was rare. Pure cultures were obtained from relatively more turkey organs than chicken organs. The organ from which there was the highest chance to obtain a pure culture was the air sac. In 108 turkey flocks (50.0%) and 64 chicken flocks (82.1%) at least one other respiratory pathogen was detected. The most common gross lesions were increased mucus in trachea, caseous or fibrinous exudate in the air sacs, consolidated lungs indicating pneumonia, congested and edematous lungs, and a flattened trachea. For most types of lesions, the percentage of affected turkeys was higher than the percentage of affected chickens. The percentage of birds with lesions was higher if other respiratory pathogens were present. Overall, the host species (turkey or chicken) was a more important factor for the prevalence of most lesions than the detection of other respiratory pathogens. The most common histopathologic lesions in the sinus and trachea were heterophilic or mononuclear inflammatory cell infiltration. In the lungs and air sacs, the inflammation was characterized by heterophilic infiltration and/or fibrin accumulation. These results are helpful in selecting the most appropriate samples for isolation of O. rhinotracheale. In addition, they show the incidence of the bacterium in turkeys and chickens and which lesions can be expected after infection with O. rhinotracheale, and they indicate that in some cases O. rhinotracheale can be the primary, or at least the major, pathogen.

Retrospective study of turkey viral hepatitis in California turkey flocks, 2000-2012.

Turkey viral hepatitis (TVH) is a disease characterized by an inflammation of the liver, and occasionally of the pancreas, of turkeys. Little is known about the occurrence of TVH in turkey flocks; thus, the aim of the present article is to summarize retrospectively 76 cases of TVH diagnosed at the California Animal Health and Food Safety System (CAHFS), University of California, Davis, in the years 2000 through 2012. Flocks diagnosed with TVH were between 7 and 61 days old, with an average age of 29.4 days and a median age of 28 days. In the majority of cases, increased mortality was reported. In 55 cases, gross lesions were present in the liver; the most common lesions were a few to numerous pale white foci in 35 cases. In livers of 74 cases, histologic lesions were observed. Multifocal necrosis with inflammation was observed in livers of 42 cases, inflammation but no necrosis in 22 cases, and necrosis without inflammation in 8 cases. In 17 cases, pale white foci were found in the pancreas; in 4 cases, larger areas of the organ were pale. In 33 cases, histologic changes were observed in the pancreas. Necrosis with inflammation was observed in the pancreas of 17 cases, necrosis without inflammation in 7 cases, and inflammation without necrosis in 9 cases. No Salmonella was isolated from any of the livers cultured. Transmission electron microscopy of liver and pancreas demonstrated viral particles between 23 and 25 nm in two cases. Concurrent diseases were mostly poult enteritis (65 cases), but also respiratory diseases and colibacillosis. TVH occurred more often in turkeys during winter months. In conclusion, TVH was observedon a regular basis in California turkey flocks during the last 12 yr.

Recurring histomonosis on an organic farm.

This report describes outbreaks of histomonosis, a severe disease caused by the protozoan parasite Histomonas meleagridis, which occurred over a period of 3 yr on an organic farm in southern Germany. Among other species, the farm houses layers, broilers, and turkeys. In August 2005 one group of turkeys was naturally infected with H. meleagridis. The strain causing infection was typed by C-profiling as genotype B. A second outbreak occurred 3 yr later. Again, a group of turkeys was naturally infected. The strain causing the infection belonged to genotype A. Two months later one group of broilers became infected with H. meleagridis type B and a group of turkeys with H. meleagridis type A. Four weeks later two further groups of broilers showed symptoms. DNA of H. meleagridis was detected but genotyping was not possible. In conclusion, genotyping of the histomonal strains causing the disease showed that at least two different histomonal strains caused the outbreaks and that the strains circulated on the farm at the same time.

Factors influencing the activity of tiamulin against Histomonas meleagridis in vitro.

In this study, we investigated the activity of tiamulin fumerate against three strains of Histomonas meleagridis in vitro under different conditions. Tiamulin reduced histomonal growth of all three strains at concentrations of 20 ppm and higher. Cultures in phosphate-buffered saline-based medium were more susceptible than cultures in traditional Dwyers medium. When the cultures were inoculated with higher numbers of histomonads, the activity of tiamulin was reduced. Bacteria present in the cultures were resistant against tiamulin.

Serologic response against fowl poxvirus and reticuloendotheliosis virus after experimental and natural infections of chickens with fowl poxvirus.

Two infection studies in chickens were done to investigate the humoral immune response against fowl poxvirus (FPV) and reticuloendotheliosis virus (REV) after intradermal infection with different passages of a field isolate and with the vaccine strain HP B. The field isolate in a low passage carried the near-full-length REV provirus and induced antibodies to REV, but not to FPV. The vaccine strain carried only remnants of the long terminal repeat and induced antibodies against FPV, but not against REV. The field isolate lost the provirus after 36 passages in vitro, and it induced few antibodies against FPV and no antibodies against REV. Intravenous challenge with the low passage field isolate caused some antibody development against FPV in the birds that had previously been infected with the field isolate, but it caused no antibodies against REV in the previously vaccinated birds. REV proviral DNA was found in peripheral blood mononuclear cells of most birds that had been infected with the low passage field isolate. However, FPV DNA was found only once. The findings showed that the integrated REV provirus had an effect on the pathogenesis of fowlpox and that the tested vaccine strain is effective against FPV strains carrying REV provirus. Investigation of sera from FPV diseased flocks and flocks vaccinated against FPV showed a similar proportion of sera with antibodies against FPV. Sera from all diseased flocks but only from two of 10 vaccinated flocks had antibodies against REV. This indicated that the integrated REV provirus is common in FPV field strains.

Quantitative PCR as a tool to determine the reticuloendotheliosis virus-proviral load of fowl poxvirus.

Sequences of the reticuloendotheliosis virus (REV), an avian retrovirus, are integrated into the genome of fowl poxvirus (FPV). We developed and evaluated a quantitative multiplex real-time PCR (multiplex qPCR) assay to determine the REV-proviral load of FPV strains. Amplification efficiencies were 98.7% for the amplification of the FPV DNA and 88.7% for the amplification of REV-proviral DNA. The ratio between FPV DNA and REV-proviral DNA was calculated from the PCR efficiencies and the threshold cycle deviation of the unknown samples vs. a standard. The intraassay variation was determined by investigating triplets of different dilutions of the standard. The coefficient of variation between the threshold cycles was below 0.05 in all tested dilutions. The ratios of the triplet had a coefficient of variation of 0.201. Generally, the method overestimated the relative amount of REV-proviral DNA. Skin lesions from fowlpox outbreaks were investigated with the multiplex qPCR. The FPV:REV ratio was between 1:0.803 and 1:1.411 in samples with sufficient DNA to allow a conclusion. In addition, the investigation of cell culture material of several passages of a FPV field isolate showed a complete loss of the REV provirus after 36 passages. The loss rate of the REV provirus was approximately 50% per passage. In conclusion, we established the multiplex qPCR assay as a convenient and reliable method to determine the REV-proviral load of FPV. The first results we obtained with it show that it is of value for further investigations about the significance of the integration of the REV provirus into the genome of FPV.

Detection of Histomonas meleagridis DNA in different organs after natural and experimental infections of meat turkeys.

Histomonas meleagridis infection of turkeys is usually accompanied by a severe disease with unspecific clinical symptoms but with distinct pathological lesions in the ceca and liver. In the literature some macro- and microscopic evidence of the spread of histomonads to the other organs has been provided. The aim of the present investigations was to use real-time polymerase chain reaction (PCR) to demonstrate the dissemination of H. meleagridis DNA to different organs after natural and experimental infection of meat turkeys. Samples from several organs were collected from a meat-turkey flock, which proved to be naturally infected with histomoniasis, and examined for histomonad DNA by real-time PCR. Histomonad DNA was detected in all investigated ceca, livers, spleens, kidneys, and pooled brain swabs. Additionally it was found in 75% of investigated samples from bursae of Fabricius, in 50% of investigated duodenums, and in 40% of investigated jejunum samples. After experimental intracloacal infection of 3-wk-old turkey poults with 147,500 histomonads, similar samples were collected from all turkeys that died. After a 3-wk observation period the surviving birds, as well as the noninfected control group, were euthanatized and samples were taken. During the entire experimental period, 10 birds out the 20 infected birds died. Histomonad DNA was detected in all investigated ceca, livers, lungs, and hearts (100%) and almost all kidneys (90%) and bursae of Fabricius (80%). On the other hand, only 30% of examined spleens and 10% of brain samples revealed positive results. Surviving infected birds were euthanatized and necropsied; histomonad DNA was found in one out of 10 livers but not in any ceca. Also, histomonad DNA could not be detected in examined cecal and lung samples from the noninfected control group.

Metacognitive deficits in frontotemporal dementia.

OBJECTIVES: To investigate whether metacognitive impairments in self-awareness and self-monitoring occur in patients with frontotemporal dementia (FTD), particularly among those with prominent social and dysexecutive impairments. METHODS: Patients diagnosed with FTD were divided by clinical subtype (social-dysexecutive (n = 12) aphasic (n = 15), and constituent subgroups of progressive non-fluent aphasia and semantic dementia) and compared with subjects with probable Alzheimer's disease (AD, n = 11) and age-matched healthy controls (n = 11). All subjects completed comprehensive behavioural ratings scales, which were compared with caregiver ratings. Subjects also rated their test performances in verbal associative fluency, word list learning, and memory task with comparisons made between actual and judged performance levels. RESULTS: The FTD sample as a whole showed significantly less behavioural self-awareness and self-knowledge than the AD and healthy control samples. FTD patients with prominent social and dysexecutive impairments demonstrated the most extensive loss of self-awareness and self-knowledge, significantly overrating themselves in multiple social, emotional, and cognitive domains, and failing to acknowledge that any behavioural change had occurred in most areas. The remaining clinical samples showed select and minimal discrepancies. All clinical groups were significantly unaware of their apathy levels. Most FTD patients judged episodic cognitive test performance adequately, with partial difficulties observed in the socially impaired and progressive non-fluent aphasia subgroups. CONCLUSIONS: FTD patients, particularly those with prominent social and dysexecutive impairments, exhibit profound metacognitive anosognosia that may represent a loss of self-awareness, self-monitoring, and self-knowledge, likely related to significant prefrontal pathophysiology. Other FTD clinical groups and AD patients showed less pervasive and more select metacognitive deficiencies.

Comparison of the specificity and sensitivity of PCR, nested PCR, and real-time PCR for the diagnosis of histomoniasis.

Blackhead, also known as enterohepatitis, is caused by a protozoan parasite called Histomonas meleagridis. Clinical symptoms are nonspecific. Until now, diagnosis has been mainly based on postmortem lesions and microscopical and histopathological examination. In many cases, especially in layer flocks, these conventional methods are not sufficient, as the lesions are sometimes not clear. The technique for isolation of histomonads in vitro offers many advantages, but the confirmation of histomonads growing in culture may require a time-consuming procedure of rectal inoculation of culture material into chickens or turkeys. The aim of our investigation was to establish a conventional polymerase chain reaction (PCR), a nested PCR, and a real-time PCR, and to examine their specificity as well as sensitivity in the diagnosis of histomoniasis. The obtained results have shown that the conventional PCR is more sensitive than the real-time PCR. Furthermore, the sensitivity of the PCR can be increased by adding the nested PCR. However, the real-time PCR is more specific.