Maurice Pitesky

Bioaerosol Mass Spectrometry for Rapid Detection of Individual Airborne Mycobacterium tuberculosis H37Ra Particles

ABSTRACT Single-particle laser desorption/ionization time-of-flight mass spectrometry, in the form of bioaerosol mass spectrometry (BAMS), was evaluated as a rapid detector for individual airborne, micron-sized, Mycobacterium tuberculosis H37Ra particles, comprised of a single cell or a small number of clumped cells. The BAMS mass spectral signatures for aerosolized M. tuberculosis H37Ra particles were found to be distinct from M. smegmatis, Bacillus atrophaeus, and B. cereus particles, using a distinct biomarker. This is the first time a potentially unique biomarker was measured in M. tuberculosis H37Ra on a single-cell level. In addition, M. tuberculosis H37Ra and M. smegmatis were aerosolized into a bioaerosol chamber and were sampled and analyzed using BAMS, an aerodynamic particle sizer, a viable Anderson six-stage sampler, and filter cassette samplers that permitted direct counts of cells. In a background-free environment, BAMS was able to sample and detect M. tuberculosis H37Ra at airborne concentrations of >1 M. tuberculosis H37Ra-containing particles/liter of air in 20 min as determined by direct counts of filter cassette-sampled particles, and concentrations of >40 M. tuberculosis H37Ra CFU/liter of air in 1 min as determined by using viable Andersen six-stage samplers. This is a first step toward the development of a rapid, stand-alone airborne M. tuberculosis particle detector for the direct detection of M. tuberculosis bioaerosols generated by an infectious patient. Additional instrumental development is currently under way to make BAMS useful in realistic environmental and respiratory particle backgrounds expected in tuberculosis diagnostic scenarios.

Surveillance of Salmonella Enteritidis in Layer Houses: A Retrospective Comparison of the Food and Drug Administration's Egg Safety Rule (2010–2011) and the California Egg Quality Assurance Program (2007–2011)

SUMMARY. Between July 2007 and December 2011, 2660 environmental drag swab samples were collected in total from California layer flocks on behalf of the California Egg Quality Assurance Program (CEQAP), the egg safety rule (21 CFR Parts 16 and 118) of the Food and Drug Administration (FDA), or both. The samples were processed by the California Animal Health and Food Safety Lab, and positive or negative results for Salmonella enterica serovar Enteritidis (SE) were recorded. This study retrospectively compares the differences between the FDA and CEQAP programs with respect to their SE environmental sampling surveillance results. To accomplish this comparison, two different CEQAP (new and old) data sets representing different SE environmental surveillance approaches in the life of the flock were compared against each other and against the FDAs SE environmental testing plan. Significant differences were noted between the CEQAP and FDA programs with respect to the prevalence of SE in the farm environment. Analyses of the prevalence of SE at different stages in the flocks life cycle (chick papers, preproduction, midproduction, postmolt, and premarket) found the highest prevalence of SE in premarket (11.9%), followed by postmolt (3.5%) and midproduction (3.4%), and there was a tie between chick papers and preproduction (2.1%). To assess the main effects of the presence of SE in the farm environment, backwards binary logistic regression was used. Of six independent variables examined (age of flock, year, season, owner, CEQAP membership, and analysis of pooled samples vs.. individual swabs), only age of flock, owner, and year were determined to be significant factors in the final model. Although CEQAP membership and pooling vs. individuals swabs were not included in the final model, Pearson chi-square tests did show significantly higher odds of SE for non-CEQAP member farms and higher odds of SE in pooled samples vs.. individual swabs. RESUMEN. Vigilancia de Salmonella Enteritidis en casetas de aves de postura: Comparación retrospectiva de la reglamentación de la Administración de Alimentos y Medicamentos en la seguridad del huevo (2010–2011) y del Programa de Aseguramiento de la Calidad del Huevo en California (2007–2011). Entre julio del 2007 y diciembre del 2011, se recolectaron 2660 muestras de hisopos de arrastre ambientales de parvadas completas de gallinas ponedoras de California bajo el Programa de Aseguramiento de la Calidad del Huevo en California (con las siglas en inglés CEQAP), bajo las reglamentaciones en seguridad del huevo (Código de Regulaciones Federales número 21, partes 16 y 118) de la Administración de Alimentos y Medicamentos (con las siglas en inglés FDA), o bajo ambos. Las muestras fueron procesadas en el Laboratorio de Salud Animal y Seguridad Alimentaria de California y se registraron los resultados positivos o negativos para Salmonella enterica serovar Enteritidis (SE). Este estudio retrospectivo comparó las diferencias entre los programas de la FDA y del CEQAP con respecto a sus resultados de muestreos ambientales de vigilancia. Para llevar a cabo esta comparación, dos conjuntos diferentes de datos del CEQAP (nuevos y antiguos) que representaban diferentes enfoques de muestreos de vigilancia ambiental para Salmonella enterica serovar Enteritidis durante toda la vida de la parvada se compararon entre sí y con el plan de muestreo ambiental para S. enterica serovar Enteritidis de la FDA. Se observaron diferencias significativas entre los programas de la CEQAP y de la FDA con respecto a la prevalencia de esta bacteria en el ambiente de la granja. Mediante los análisis de la prevalencia para S. enterica serovar Enteritidis en las diferentes etapas del ciclo de vida de la parvada (del papel que cubre las charolas de pollo, preproducción, a la mitad de la producción, postmuda, y previa a la comercialización) se encontró la mayor prevalencia de esta bacteria en pre-mercado (11.9%), seguido por el periodo postmuda (3.5%) y mitad de la producción (3.4%), y se observaron resultados similares entre las muestras de papeles de las charolas de pollitos y preproducción (2.1%). Para evaluar los principales efectos de la presencia de esta bacteria en el entorno de granja, se utilizó el método de regresión logística binaria inversa. De las seis variables independientes analizadas (edad de la parvada, año, estación, propietario, membresía al programa CEQAP y análisis de muestras combinadas vs hisopos individuales), sólo la edad de la parvada, el propietario, y el año se determinaron como factores significativos en el modelo final. Aunque la participación en el CEQAP y el análisis de las muestras combinadas en comparación con las muestras individuales no se incluyeron en el modelo final, la prueba de ji cuadrado de Pearson mostró una probabilidad significativamente más alta para para S. enterica serovar Enteritidis para las explotaciones que no son miembros CEQAP y mayores probabilidades en muestras combinadas en comparación con los hisopos individuales.

Historical, Spatial, Temporal, and Time-Space Epidemiology of Very Virulent Infectious Bursal Disease in California: A Retrospective Study 2008–2011

SUMMARY. In December of 2008 very virulent infectious bursal disease virus (vvIBDV) was identified in a commercial flock in northern California. Since then several other backyard and commercial facilities in California have had flocks affected by the same strain and other unique (previously unseen) strains of IBDV. Previous to this incident, very virulent infectious bursal disease (vvIBD) had never been identified in North America. Following the initial outbreak in 2008, California became the first state to undertake a voluntary surveillance effort to try to determine the geographical prevalence of vvIBD based on sequencing of a portion of the segment A region of the vvIBDV genome. To date we have complete geographical information on approximately 500 separate accessions representing approximately 1500 birds from over 200 commercial (∼85% of the facilities) and backyard facilities (∼15% of the facilities) throughout the state. Sequencing of targeted regions of both the segment A and segment B regions of the genome has revealed three distinct types of IBDV in California chickens. One type is genetically and in pathogenically consistent with vvIBDV. The second and third types only have a segment A region consistent with vvIBDV. Geographic information system mapping coupled with spatial-temporal cluster analysis identified significant spatial and time-space clustering; however, no temporal clustering was noted. The lack of temporal clustering coupled with negative vvIBDV results in tested avian wildlife implies that avian wildlife in California do not currently appear to play a significant role in vvIBDV transmission. In the voluntary surveillance that was done in the Central Valley of California, which has a high density of commercial poultry, no positive farms were found when 142 of 504 farms were sampled. Given this level of sampling, the confidence (probability) of detecting an affected commercial flock was calculated to be between 28% and 81% depending on whether one or five hypothetically affected farms were affected. RESUMEN. Epidemiología histórica, espacial y espacio-temporal del virus muy virulento de la enfermedad infecciosa de la bolsa en California: Un estudio retrospectivo 2008–2011. En diciembre del 2008, se identificó un virus muy virulento de la enfermedad infecciosa de la bolsa (con las siglas en inglés vvIBDV) en un lote comercial en el norte de California. Desde entonces, varias instalaciones avícolas comerciales y de traspatio en California han tenido parvadas afectadas por la misma cepa y otra cepa única (no detectada anteriormente) del virus de Gumboro. Antes de este incidente, el virus muy virulento de la enfermedad infecciosa de la bolsa nunca había sido identificado en América del Norte. Después del brote inicial en el año 2008, California se convirtió en el primer estado en realizar un esfuerzo voluntario de vigilancia para determinar la prevalencia geográfica del virus muy virulento de Gumboro basado en el análisis de secuencias de una porción del segmento A del genoma de este virus muy virulento. Hasta la fecha se cuenta con información geográfica completa de aproximadamente 500 registros de diagnóstico diferentes que representan aproximadamente 1,500 aves de más de 200 instalaciones comerciales (aproximadamente el 85% de las instalaciones) y de las instalaciones de traspatio (aproximadamente el 15% de las instalaciones) en todo el estado. El análisis de las secuencias de las regiones blanco de los segmentos A y B del genoma ha revelado tres tipos distintos de cepas del virus de Gumboro en pollos de California. Un tipo es genética y patogénicamente similar con el virus muy virulento de Gumboro. El segundo y tercer tipos solo tienen un segmento A similar a las cepas virulentas. El mapeo por sistemas de información geográfica (con las siglas en inglés GIS), junto con el análisis de conglomerados espacio-temporales identificaron agrupamientos espaciales y espacio-temporales significativos, sin embargo, no se observó agrupación temporal. La falta de agrupación temporal junto con los resultados negativos para la presencia de cepas muy virulentas en la fauna silvestre aviar implica que esta fauna silvestre de California no parece jugar un papel importante en la transmisión de las cepas muy virulentas del virus de Gumboro. En la vigilancia voluntaria que se llevó a cabo en el Valle Central de California, que tiene una alta densidad de avicultura comercial, no se detectaron granjas positivas cuando se muestrearon 142 granjas de un total de 504. De acuerdo a este nivel de muestreo, la confianza (probabilidad) de detección de una parvada afectada se calculó entre 28% y 81% dependiendo de si una o cinco granjas afectadas hipotéticamente resultaron afectadas.

Non-DNA Methods for Biological Signatures

Publisher Summary This chapter focuses on the methods that can determine chemical or structural features of biological agent particles that are signatures of particular methods of growth and post-growth processing (often referred to as “weaponization”). The detection of these signatures in a sample of a bio-weapon (BW) agent can aid the attribution by indicating: (1) the level of sophistication of the producer, (2) the access to particular types of agent weaponization information, (3) the likelihood that the material could be or has been produced at a significant scale, (4) and by providing essential sample matching data for ascertaining a putative relationship with other samples obtained in other venues. An example of the use of biologicals in forensic science is DNA, amplied by the Polymerase Chain Reaction (PCR) technique, legally admissible in courtas evidence. DNA evidence is successfully used in the court to convict or clear people of crimes because each persons DNA is unique. High-resolution techniques are being applied to investigations; such as Environmental scanning electron microscopy (ESEM) is used for taking high-resolution images under hydrated conditions; this avoids any artifacts associated with the critical point drying process that is required under normal Scanning Electron Microscopy (SEM) operations. ESEM is also equipped with Energy Dispersive X-ray (EDX) microanalysis and Backscatter capabilities. SEM is a standard “workhorse” technique for characterizing particulate samples, found in many laboratories worldwide. It provides excellent imaging of the surfaces of agent particles and other material in a sample, and is used for identifying likely agent particles for analysis by other instruments. When combined with EDX, the elemental composition of the material in the imaged region can be determined. These techniques continue to signature libraries of correlations between analyses and growth and processing conditions of growth, it will be necessary to develop an information system which combines types of data to determine unique signatures.

Assessing Salmonella typhimurium persistence in poultry carcasses under multiple thermal conditions consistent with composting and wet rendering

Mitigation of Salmonella associated with poultry carcasses is primarily accomplished by rendering or carcass composting. While rendering temperatures and pressures are well established for pathogen inactivation in poultry carcasses, parameters controlling composting processes are less defined in part because multiple conditions and procedures are utilized. Consequently, limited knowledge exists describing the impacts of composting with varying temperature and mixing protocols with respect to the inactivation of Salmonella in poultry carcasses. To improve the existing knowledge of Salmonella survival in poultry carcasses, inactivation of Salmonella enterica serovar Typhimurium (ST) LT2 was investigated. The impacts of various composting temperatures (55, 62.5°C) and low-rendering (i.e., pasteurization) temperatures (70, 78°C) on Salmonella inactivation were tested in a bench-top setting using a ground carcass slurry and whole birds under mixed and non-mixed conditions. Results showed that the ground carcass slurry and the whole carcass exposed to temperatures consistent with composting had no detectable Salmonella after 110 h with a level of detection of one CFU/mL of ground carcass slurry and one CFU/g of whole carcasses, respectively. In addition, grinding of carcasses as opposed to whole carcasses was more predictable with respect to Salmonella heat inactivation. Furthermore, results showed that constant mixing decreased the overall time required to eliminate Salmonella under composting and low-rendering temperatures.

Descriptive survey and Salmonella surveillance of pastured poultry layer farms in California

&NA; While pasture‐raised poultry comprises a small portion of the commercial poultry industry in North America, these alternative rearing systems have become increasingly popular. As such, it is critical to improve our understanding of husbandry practices and prevalence of zoonotic and epizoonotic diseases in these systems. This research reviews the results of a survey sent to 82 commercial pastured poultry farms in California. While the survey response was low (13.4%), it was enhanced by detailed in‐person interviews and farm visits. In addition, we conducted drag swabs for Salmonella Enteritidis. On average, farms utilized 12.3% of their total farmland for pastured poultry operations, which often coexisted with other livestock (45%), touch crops (27%), and non‐touch crops (45%). While the mean (44.6 sq. ft./hen) and median (22.2 sq. ft./hen) pasture stocking densities were within auditing guidelines, the mean (1.2 sq. ft./hen) and median (0.5 sq. ft./hen) coop stocking densities were below the pending USDA (2016) guidelines recommended in 7 CFR Part 205. Drag swab results showed the presence of Salmonella Enteritidis (SE) in the environment of one of the 11 farms (9.1%). In addition, Salmonella Pullorum (SP) whole blood agglutination tests were used to understand the prevalence of Salmonella spp. in laying hens within the studied farms. Results showed the presence of antibodies in flocks at six of the seven non‐SE vaccinated farms, with a mean on‐farm prevalence of 25.6% in laying hens. Logistic regression was used to determine risk factors for Group D Salmonella exposure in non‐vaccinated flocks, using the SP blood agglutination data as the dependent variable and the survey questions as the independent variables. Statistically significant (P < 0.05) risk factors included exposed wire floors and flock size. These results improve our understanding of Salmonella prevalence and husbandry practices on commercial pastured poultry farms in California.

Marek's Disease in Backyard Chickens, A Study of Pathologic Findings and Viral Loads in Tumorous and Nontumorous Birds

SUMMARY Mareks disease (MD) is a major cause of mortality in backyard chickens. The diagnosis of MD is complex, however, and knowledge of Mareks disease virus (MDV) in spontaneous field cases such as in backyard chickens is largely unknown. In this study, 40 backyard chickens with a presumptive MD diagnosis based on histologic lymphoid infiltrations in peripheral nerves with and without lymphomas were investigated. Twenty-eight of the birds were submitted to the diagnostic laboratory for disease explorations, and 12 chickens were from a flock in which some members demonstrated anisocoria and pupil irregularities compatible with ocular MD. Histologic scores were established for brain, peripheral nerves, heart, lung, liver, kidney, and gonad sections, ranging from mild (+) to severe (+++) lymphoid infiltrations. Twelve chickens had gross lymphomas, and all but two chickens had mild to severe peripheral nerve lymphoid infiltrates. There were no age or breed predispositions in the study group. Quantification of serotypes MDV-1, −2, and −3 performed with real-time PCR demonstrated high correlation (R2 = 0.94) between fresh and fixed spleen specimens, as well as between histopathology scores and MDV-1 viral loads. MDV-2 DNA was detected in a portion of the chickens, likely consistent with naturally occurring virus, whereas the vaccine strain MDV-3 was rarely detected. Significant differences in MDV-1 viral loads between tumorous and nontumorous chickens were observed, in which a ratio of MDV-1 glycoprotein B/glyceraldehyde-3-phosphate dehydrogenase ≥ 0.5 was suggestive of gross tumors in this study. We propose that real-time PCR may be a good tool for MD diagnosis in backyard chickens.

A cooperative approach to animal disease response activities: Analytical hierarchy process (AHP) and vvIBD in California poultry.

Very virulent infectious bursal disease virus (vvIBDv) was first detected in the United States at the end of 2008. Since its detection, Federal and State animal health officials, the poultry industry and the research/academic community have led response activities through a collaborative effort. By June 2011, much still remained unknown regarding the basic epidemiology and ecology of vvIBD in California, although there were a number of potential activities to fill this information gap. Available resources limited the ability to pursue all the activities, and responsible parties and stakeholders recognized the need to prioritize the activities. The analytic hierarchy process (AHP) is a useful multi-criteria decision making methodology that incorporates qualitative information (in the form of judgments) with available quantitative information. This is especially useful when there is very limited quantitative information, such as in the situation with vvIBD in California. A commercial package that allows ready use of the AHP model was utilized for prioritizing activities, incorporating input from members from the three stakeholder groups: State and Federal animal health officials, poultry industry, and research/academia. Based on their inputs on 17 potential activities, the participants identified three priority activities; specifically determination of risk factors for re-emergence or re-introduction at affected premises, development of a laboratory diagnostic test to screen for segment B of the vvIBDV genome and surveillance of other potential reservoirs (mealworms, rodents, beetles). In order to evaluate the ability of the AHP to respond to differences, a sensitivity analysis was done in order to evaluate changes in prioritization of activities. Changes in prioritization were noted demonstrating the plasticity of the model under different conditions. However, a 50% increase or decrease in weighting was necessary to affect the order of the three highest scoring activities. The use of a tool such as the AHP enables the development of a transparent, repeatable and flexible decision process, which can be useful in certain animal health response situations including the re-emergence of a previously eliminated disease or the introduction of a foreign animal disease.

Validation of Single and Pooled Manure Drag Swabs for the Detection of Salmonella Serovar Enteritidis in Commercial Poultry Houses.

SUMMARY Single swabs (cultured individually) are currently used in the Food and Drug Administration (FDA) official method for sampling the environment of commercial laying hens for the detection of Salmonella enterica ssp. serovar Enteritidis (Salmonella Enteritidis). The FDA has also granted provisional acceptance of the National Poultry Improvement Plan’s (NPIP) Salmonella isolation and identification methodology for samples taken from table-egg layer flock environments. The NPIP method, as with the FDA method, requires single-swab culturing for the environmental sampling of laying houses for Salmonella Enteritidis. The FDA culture protocol requires a multistep culture enrichment broth, and it is more labor intensive than the NPIP culture protocol, which requires a single enrichment broth. The main objective of this study was to compare the FDA single-swab culturing protocol with that of the NPIP culturing protocol but using a four-swab pool scheme. Single and multilaboratory testing of replicate manure drag swab sets (n  =  525 and 672, respectively) collected from a Salmonella Enteritidis–free commercial poultry flock was performed by artificially contaminating swabs with either Salmonella Enteritidis phage type 4, 8, or 13a at one of two inoculation levels: low, x¯  = 2.5 CFU (range 2.5–2.7), or medium, x¯  = 10.0 CFU (range 7.5–12). For each replicate, a single swab (inoculated), sets of two swabs (one inoculated and one uninoculated), and sets of four swabs (one inoculated and three uninoculated), testing was conducted using the FDA or NPIP culture method. For swabs inoculated with phage type 8, the NPIP method was more efficient (P < 0.05) for all swab sets at both inoculation levels than the reference method. The single swabs in the NPIP method were significantly (P < 0.05) better than four-pool swabs in detecting Salmonella Enteritidis at the lower inoculation level. In the collaborative study (n  =  13 labs) using Salmonella Enteritidis phage type 13a inoculated swabs, there was no significant difference (P > 0.05) between the FDA method (single swabs) and the pooled NPIP method (four-pool swabs). The study concludes that the pooled NPIP method is not significantly different from the FDA method for the detection of Salmonella Enteritidis in drag swabs in commercial poultry laying houses. Consequently based on the FDA’s Salmonella Enteritidis rule for equivalency of different methods, the pooled NPIP method should be considered equivalent. Furthermore, the pooled NPIP method was more efficient and cost effective.

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

SUMMARY 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.