Comprehensive molecular, genomic and phenotypic analysis of a major clone of Enterococcus faecalis MLST ST40.

BACKGROUND: Enterococcus faecalis is a multifaceted microorganism known to act as a beneficial intestinal commensal bacterium. It is also a dreaded nosocomial pathogen causing life-threatening infections in hospitalised patients. Isolates of a distinct MLST type ST40 represent the most frequent strain type of this species, distributed worldwide and originating from various sources (animal, human, environmental) and different conditions (colonisation/infection). Since enterococci are known to be highly recombinogenic we determined to analyse the microevolution and niche adaptation of this highly distributed clonal type. RESULTS: We compared a set of 42 ST40 isolates by assessing key molecular determinants, performing whole genome sequencing (WGS) and a number of phenotypic assays including resistance profiling, formation of biofilm and utilisation of carbon sources. We generated the first circular closed reference genome of an E. faecalis isolate D32 of animal origin and compared it with the genomes of other reference strains. D32 was used as a template for detailed WGS comparisons of high-quality draft genomes of 14 ST40 isolates. Genomic and phylogenetic analyses suggest a high level of similarity regarding the core genome, also demonstrated by similar carbon utilisation patterns. Distribution of known and putative virulence-associated genes did not differentiate between ST40 strains from a commensal and clinical background or an animal or human source. Further analyses of mobile genetic elements (MGE) revealed genomic diversity owed to: (1) a modularly structured pathogenicity island; (2) a site-specifically integrated and previously unknown genomic island of 138 kb in two strains putatively involved in exopolysaccharide synthesis; and (3) isolate-specific plasmid and phage patterns. Moreover, we used different cell-biological and animal experiments to compare the isolate D32 with a closely related ST40 endocarditis isolate whose draft genome sequence was also generated. D32 generally showed a greater capacity of adherence to human cell lines and an increased pathogenic potential in various animal models in combination with an even faster growth in vivo (not in vitro). CONCLUSION: Molecular, genomic and phenotypic analysis of representative isolates of a major clone of E. faecalis MLST ST40 revealed new insights into the microbiology of a commensal bacterium which can turn into a conditional pathogen.

Pathogenesis of histomonosis in experimentally infected specific-pathogen-free (SPF) layer-type chickens and SPF meat-type chickens.

Several studies have shown differences in the course of histomonosis, the infection with the trichomonad parasite Histomonas meleagridis, in different chicken breeds. In the present study, 10 specific-pathogen-free (SPF) layer-type (LT) chickens and twelve SPF meat-type (MT) chickens were infected intracloacally with 200,000 H. meleagridis trophozoites. One and two weeks postinfection (p.i.), three birds of each group were euthanatized. The remaining birds were euthanatized 3 wk p.i. Infected birds showed severe gross lesions typical for histomonosis in ceca at the first and second week p.i., while livers showed necrotic foci at 2 and 3 wk p.i., but only very rarely at 1 wk p.i. Differences between groups in the severity of lesions were statistically insignificant. In histopathology, LT chickens showed a significantly more-severe necrosis and ablation of the cecal epithelium 1 wk p.i. Parasites without inflammation were also found in most investigated spleens and lungs but only in a few kidneys. Investigation of these organs for histomonal DNA by real-time PCR confirmed these results. In addition, the humoral immune response against histomonal actinin 1 and 3 was measured by an ELISA. The humoral immune response against actinin 1 started sooner and was significantly higher in LT chickens than in MT chickens. In conclusion, the results of the present study suggest that the genetic background of the birds influences the reaction to infection with H. meleagridis.

Liver pathology associated with increased mortality in turkey breeder and meat turkey flocks.

Between 2006 and 2011 a series of disease conditions characterized by raised mortality and liver disorders occurred in turkey breeder flocks and in meat turkey flocks in Germany. The flocks were between 12 and 23 wk of age, and mostly hens were affected. Clinical signs were nonspecific and accompanied by mortality varying between 1% and 7%. Affected birds displayed swollen livers that were marbled with black and red spots and yellowish areas. The pericardium was filled with an amber fluid, and the coronary groove was extensively filled with fat. Spleens were swollen, and a serous fluid that seemed to leak from the liver was present in the body cavity. Histopathological findings in all but one case included fatty degeneration of hepatocytes with parenchymal collapse and associated hemorrhages. Some animals showed cholangitis and hepatitis with intranuclear inclusion bodies. In three cases with breeders, electron microscopy detected virus particles that were between 23 and 30 nm and similar to parvo- or picornavirus. In addition, picornavirus RNA was detected in the livers of one meat turkey flock. Investigations by PCR for circovirus, polyomavirus parvovirus, and aviadenovirus yielded negative results in all cases, but an aviadenovirus was isolated from livers twice and a reovirus from the intestines once. Supplementation with vitamin E and selenium seemed to improve the situation. The most likely diagnosis is lipidosis, a metabolic disorder with complex etiology, which has rarely been described in turkeys.

Experimental infections with the protozoan parasite Histomonas meleagridis: a review.

In recent years, a number of studies about Histomonas meleagridis, and more specifically about experiments in vivo involving H. meleagridis to investigate the pathogenicity and efficacy of drugs or vaccines, have been published. Together with older publications, a considerable amount of information about experimental infections with H. meleagridis exist, which is helpful for planning future animal studies and can reduce the number of birds used in such studies toward better animal welfare. One hundred sixty-seven publications describing experimental infections with H. meleagridis were published in scientific journals between 1920 and 2012. One hundred forty-two of these publications describe infections of turkeys (Meleagris gallopavo) and 52 infections of chickens (Gallus gallus). In 18 studies, experiments involving other species were done. The most popular routes of infection were the intracloacal application of histomonal trophozoites from culture material, from lesions or from feces of infected birds, or using larvae of the cecal worm Heterakis gallinarum (83 studies) and the oral application of eggs or other stages of the cecal worm containing histomonal stages (83 studies). During the last 10 years, intracloacal application of trophozoites has become the most popular way to experimentally infect birds with H. meleagridis due to its high reproducibility and reliability. In most studies, infection doses of several 10,000 or 100,000 histomonal trophozoites were used for infection, and the resulting mortality in turkeys was more than 70 %. First mortality can occur as early as 6 days p.i.; peak mortality usually is 13-15 days p.i. Lower infection doses may delay mortality about 2 days. In chickens infected by the intracloacal route, mortality and clinical signs are rare, but infection rates are similar. Cecal lesions can be observed from 3 to 4 days p.i., lesions up to 3 weeks p.i.; liver lesions may be lacking completely or be present only in a small number of birds. In most studies infecting birds with Heterakis eggs containing histomonal stages, several 100 to 1,000 Heterakis eggs were used. However, lower doses might be sufficient, as infection with as few as 58 eggs per bird caused a mortality up to 90 % in turkeys. Clinical symptoms start 9 days p.i., and first mortality occurs after 12 days, while most of the infected birds die between 19 and 21 days p.i. The infectivity of Heterakis eggs containing histomonal stages for chickens is similar as for turkeys, but mortality and clinical signs are rare. Further infection was done by oral application of histomonal trophozoites either grown in culture or using lesions or feces of infected birds (26 studies). These yielded very mixed results, with infection rates between 0 and more than 80 % in turkeys and chickens. After successful oral infection of turkeys, mortality occurs at roughly the same time as after intracloacal infection. Further 18 studies employed seeder birds to infect in-contact birds. Other means of infection were exposure to contaminated soil or litter (22 studies), feeding contaminated earthworms (7 studies), intracecal inoculation (4 studies), or parenteral injection (4 studies). Main methods to assess the course of the infection were mortality, observation of clinical signs and pathological lesions, monitoring of the weight of the infected birds, and detection of the parasite by various methods.

A meta-analysis of the effect of Eimeria spp. and/or Clostridium perfringens infection on the microbiota of broiler chickens.

Coccidiosis in chickens is caused by Eimeria spp. The infection provides a growth advantage to Clostridium perfringens (CP), frequently leading to necrotic enteritis. One approach to alleviate the negative impacts of the diseases is to improve the bacterial composition in chickens, and many experiments investigating chicken enteric health in recent years include the characterization of the bacterial microbiota. This meta-analysis synthesized the data of studies investigating the intestinal microbiota after infection with coccidia and/or CP to provide a basis for future research. Inclusion criteria were that experiments contained a group infected with one or both pathogens and an uninfected control group, the use of 16SrRNA Illumina sequencing and the availability of raw data. A total of 17 studies could be included. Meta-analyses of 3 different data sets were performed: 1 on data of 9 experiments on chickens infected with coccidia only; the second on data of 4 studies on chickens infected with CP only; the third on raw data of 8 experiments with chickens infected with coccidia and CP. The meta-analysis of relative abundance and alpha diversity of the data sets was performed in R using the SIAMCAT and metafor packages. The number of families of interest identified by the analyses of experiments with infection with coccidia only, CP only and the combined infection were 23, 2, and 29, respectively. There was an overlap of 13 families identified by analyses of experiments with infection with coccidia only and of experiments with the combined infections. Machine learning was not able to find a model to predict changes of the microbiota in either 1 of the 3 analyses. Meta-analyses of functional profiles showed a more uniform reaction to the infections with the relative abundance of many pathways significantly altered. Alpha diversity was not affected by infection with either pathogen or the combination. In conclusion, the heterogeneity of these microbiota studies makes recognizing common trends difficult, although it seems that coccidia infection affects the microbiota more than an infection with CP. Future studies should focus on the bacterial functions that are changed due to these infections using metagenome techniques.

Buffering Capacity Comparison of Tris Phosphate Carbonate and Buffered Peptone Water Salmonella Pre-Enrichments for Manufactured Feed and Feed Ingredients.

Various culture-based methods to detect Salmonella in animal feed have been developed due to the impact of this bacterium on public and animal health. For this project, tris phosphate carbonate (TPC) and buffered peptone water (BPW) buffering capacities were compared as pre-enrichment mediums for the detection of Salmonella in feed ingredients. A total of 269 samples were collected from 6 feed mills and mixed with the pre-enrichments; pH was measured before and after a 24 h incubation. Differences were observed when comparing pH values by sample type; DDGS and poultry by-product meal presented lower initial pH values for TPC and BPW compared to the other samples. For both TPC and BPW, meat and bone meal presented higher final pH values, while soybean meal and peanut meal had lower final pH values. Furthermore, for BPW, post cooling, pellet loadout, and wheat middlings reported lower final pH values. Additionally, most feed ingredients presented significant differences in pH change after 24 h of incubation, except DDGS. From meat and bone meal samples, four Salmonella isolates were recovered and identified: three using BPW and one using TPC. TPC provided greater buffer capacity towards neutral pH compared to BPW, but BPW was more effective at recovering Salmonella.

Meat quality of broiler chickens processed using electrical and controlled atmosphere stunning systems.

Increased consumer concern for animal welfare has led some poultry producers to alter their stunning methods from electrical to controlled atmosphere stunning. The potential for different impacts on meat quality between commercially applied controlled atmosphere stunning (CAS) and electrical stunning (ES) using current US parameters needs further evaluation. Three trials were conducted in a commercial broiler processing facility that uses separate processing lines for ES and CAS. Blood glucose concentrations were measured from broilers stunned by either CAS or ES at: 1) lairage, 2) pre-stunning, and 3) post-stunning, using a glucose monitor. Occurrence of visible wing damage was evaluated post-defeathering and breast fillet meat quality was evaluated through measurement of pH, color, and drip loss at deboning and after 24 h. Data were analyzed using GLM or chi-square with a significance at P ≤ 0.05 and means were separated by Tukey's HSD. Blood glucose concentrations (mg/dL) from CAS and ES birds were not different at lairage (284, 272, P = 0.2646) or immediately prior to stunning (274, 283, P = 0.6425). Following stunning and neck cut, circulating blood glucose from birds stunned by CAS was higher than ES (418, 259, P < 0.0001). CAS carcasses had more visible wing damage than ES carcasses (3.6%, 2.2%, P < 0.0001). Breast fillet pH was lower, L* was higher, and a* was lower at debone for CAS fillets (5.81, 54.65, 1.96) compared to ES fillets (5.92, 53.15, 2.31, P < 0.0001, P = 0.0005, P = 0.0303). Drip loss did not differ between breast fillets from CAS or ES broilers (4.83, 4.84; P = 0.0859). The implications of increased blood glucose concentration post-CAS are unknown and require further evaluation. However, the increase in visible wing damage observed post-defeathering from CAS carcasses indicated a need for equipment parameter adjustments during the process from stunning through defeathering when using CAS for broiler stunning. Although differences were observed in breast fillet attributes at deboning, these differences would have minimal practical application and were no longer present at 24 h. Overall, use of CAS in a commercial facility resulted in differences in subsequent product quality when compared to ES.

Description of Eimeria pavonina (coccidia) of peafowl in Germany.

There are only a few reports about the occurrence of coccidia in peafowl and no reports about the occurrence of Eimeria spp. in peafowl kept in Europe. Here, we describe the occurrence of Eimeria pavonina in diseased peafowl from Germany. In January 2011, one young peacock kept in an aviary showed a marked depression. No parasites were detected in samples from the diseased bird, but in samples of birds from the same and other aviaries, coccidian counts were between 400/g and 66,000/g. All peacocks were treated with toltrazuril. After treatment, the clinical condition of the diseased bird improved but, two weeks afterwards, other birds in the aviary were still shedding coccidia in their feces. Based on morphology, the coccidia were identified as E. pavonina. Parts of the 18s rRNA gene and the cytochrome oxidase subunit 1 (cox-1) gene were sequenced. A phylogenetic tree based on the 18s rRNA sequence placed the Eimeria sp. from peafowl closest to Eimeria spp. found in pheasants and partridges as well as to Eimeria meleagrimitis. A phylogenetic tree based on the sequence of cox-1 in contrast suggested a closer relationship to Eimeria necatrix and Eimeria tenella.

Detection and typing of Ornithobacterium rhinotracheale from German poultry flocks.

Ornithobacterium rhinotracheale (ORT) is a gram-negative staining rod. In chickens and turkeys ORT causes a respiratory disease. Between 2009 and 2011 some 714 dry swabs taken from diseased turkeys, broilers, broiler breeders, layers, or from unknown origin were investigated by PCR for the presence of ORT. Swabs that tested positive numbered 197 out of 481 from turkeys (41.0%), 10 out of 144 from broilers or broiler breeders (6.9%), 17 out of 28 from layers (60.7%), and 26 out of 61 from unknown origin (42.6%). The results of three swabs from turkeys were suspect. Furthermore, 310 isolates from turkeys and 62 isolates from unknown origin were typed using an agar gel precipitation (AGP) test. Of the isolates from turkeys, 56.1% belonged to serotype A and 20.6% to serotype E. The prevalence of other isolates was below 10%. Serotypes D, F, and K were not detected. Eleven isolates were not typable with reference sera against serotypes A-L. The three serotypes most often found in the isolates from unknown origin were A (35.5%), B (19.4%), and C (12.9%). The prevalence of other isolates was below 10%. Serotypes F and K were not detected. Seven isolates were not typable with reference sera A-L. Cross-reactions, especially of serotype A isolates with serotypes I, H, and J, were common. Additionally, the partial 16S ribosomal RNA (rRNA) and the complete Or01 genes of reference strains A-H and of nine field isolates were cloned and sequenced. Identity scores of 16S rRNA fragments were between 98% and 100%. Identities of the Or01 sequences were between 94% and 100%. Phylogenetic trees of both genes showed similarities. However, there was no apparent correlation between reference strains and isolates belonging to one serotype, so sequencing of 16S rRNA or of the Or01 gene does not seem to be a suitable method to replace the AGP for serotyping. Further investigations are necessary to clarify the cross-reactions between different serotypes and their real role in the pathogenicity and in consideration of vaccine production.

Type C botulism in a commercial turkey farm: a case report.

Botulism is an intoxication caused by exotoxins of Clostridium botulinum. The case of botulism described here occurred on a commercial meat turkey farm with two houses. Toms and hens were maintained in two separate houses, toms in house A and hens in house B. At 10 wk of age, an increase in mortality was observed in the toms located in house A. Clinically the animals presented with paralysis of the legs, wings, and neck. Affected birds were sitting and reluctant to move. Necropsy failed to find any specific lesions. In liver, heart, muscles, crop, and gizzard as well as in intestinal contents, DNA of C. botulinum type C was detected by PCR. The result was confirmed by a mouse lethality neutralization test. During the 2 wk after the onset of the clinical signs the mortality was about 12%. The hens kept in house B did not show any symptoms and remained healthy. Investigations of environmental samples to detect the source of the toxin were not successful. After 2 wk clinical signs and mortality abated. At 16 wk of age, toms again showed the same clinical signs accompanied by raised mortality. Again C. botulinum toxin type C was detected. Within 2 wk the total mortality reached roughly 50%. Based on the "precautionary principle" and in agreement with the local authorities, the birds were euthanatized using CO2 in order to not compromise food safety.

DETECTION OF COCCIDIA OOCYSTS IN LITTER AND FECES OF BROILERS IN A FLOOR PEN TRIAL.

Counting oocysts in feces or litter is 1 method to monitor infection levels with Eimeria spp. in chickens after experimental infection or in commercial flocks. Counts of oocysts shed in the feces are thought to follow a typical pattern, with clear peaks representing infection cycles while oocyst counts in litter are representative for at least 2 wk before they begin to deteriorate. The objective of the study was to compare oocyst counts in fresh feces and litter of broilers kept in floor pens with fresh pine shavings as litter material for 42 days. The birds were spray-vaccinated against coccidia in the hatchery. Every 2 to 3 days, 7 pens were sampled by collecting fresh feces and litter from at least 3 locations per pen. Oocysts were counted using a McMaster chamber (Vetslides, Park City, Utah). There were significant differences between pens in oocyst counts in feces as well as in litter, and there were no obvious shedding patterns. Overall, the geometric mean of oocyst counts on day 8 and later was 1,300 oocysts per gram (opg) in feces and 2,700 opg in litter. The variability was lower in litter samples, and the number of pens required to find significant differences between groups in a hypothetical experiment was unrealistically high on most days. Investigating individual fecal samples showed large differences within the pens in addition to the differences between the pens.

Network meta-analysis comparing the effectiveness of anticoccidial drugs and anticoccidial vaccination in broiler chickens.

With the trend to organic production and concerns about using antibiotic feed additives, the control of infections with Eimeria spp. in broiler flocks has become more difficult. Vaccination against coccidia is an alternative, but there are concerns that the live vaccines used might have negative effects on production parameters and intestinal health. Reports of experiments directly comparing anticoccidial drugs and anticoccidial vaccines are rare. This network meta-analysis (NMA) identified and analyzed 61 articles reporting 63 experiments testing anticoccidial drugs and anticoccidial vaccines under conditions resembling commercial broiler production. The effect sizes were mean differences in body weight/body weight gain (BW/BWG) and feed conversion rate (FCR) between the 175 included groups. The results show that groups vaccinated against coccidia have a similar BW/BWG and FCR at processing age compared to groups given anticoccidial drugs. However, the results tended to be more favorable for anticoccidial drugs than for vaccines. The analysis of eight subsets, containing only groups (1) groups that had not received an AGP in addition to an anticoccidial drug, (2) groups that had not received ionophores, (3) groups that had not received chemicals, (4) groups that had not received an attenuated vaccine, (5) groups that had not received a fully virulent vaccine, (6) groups that were not additionally challenged with bacteria or not challenged, (7) groups that had received a severe challenge as defined by a total infection dose of more than 100,000 oocysts or were not challenged, (8) groups that were challenged on day 15 or earlier or not challenged brought similar results and confirmed the robustness of the NMA. In addition, the analysis exposes unnecessary, as well as inherent, problems with data quality, which every researcher working with coccidia should carefully consider, and identifies under-researched areas that should be addressed in future research.

Analysis of Information for Backyard Flock Owners Provided by Extension Websites.

Early recognition and prevention of infectious diseases in poultry flocks are essential to reduce spread from bird to bird, to prevent zoonoses, and to keep losses low. Backyard flock owners often have little knowledge about poultry health, and specialized veterinarians are difficult to find. Alternative sources for support, education, and training for noncommercial poultry are the websites of cooperative extension services offering online webinars, presentations, and programs about poultry health and diseases. The aim of this investigation was to survey 23 websites of the extension services of the top 13 states in poultry production for information on backyard poultry health. The eXtension website by the United States Cooperative Extension System was added as a nation-wide source of information. Structure, content, and presentation form were compared and analyzed. The results displayed large differences between the investigated webpages and identified opportunities for improving the sites, especially in completeness, accessibility, and presentation of the information. From 23 extension websites, 13 provided none to very limited online information and 5 websites covered almost all of the investigated content. The primary media used were articles with pictures, and only three universities added videos and webinars. Based on these results and according to the increased need for online sources about poultry health, the extension websites should provide complete and correct information or at least links to approved sources. Videos, podcasts, and webinars can increase outreach and learning achievement. This survey may help to improve the presentation and content of poultry health-related extension websites.

Análisis de la información disponible para propietarios de parvadas de traspatio proporcionada por páginas de extensión en la red internet. El reconocimiento temprano y la prevención de enfermedades infecciosas en las parvadas avícolas son esenciales para reducir su propagación entre aves, para prevenir las zoonosis y para mantener posibles pérdidas bajo control. Los propietarios de parvadas de traspatio a menudo tienen poco conocimiento sobre salud avícola y es difícil encontrar veterinarios especializados. Las fuentes alternativas de apoyo, educación y capacitación para aves de corral no comerciales son las páginas en la red internet preparadas por los servicios de extensión cooperativa que ofrecen seminarios en línea, presentaciones y programas sobre salud y enfermedades de las aves de corral. El objetivo de esta investigación fue analizar 23 páginas en la red internet de los servicios de extensión de los 13 estados principales en producción avícola para obtener información sobre la salud de las aves de corral de traspatio. La página de internet eXtension del Sistema de Extensión Cooperativa de los Estados Unidos se agregó como fuente de información a nivel nacional. Se compararon y analizaron la estructura, el contenido y la forma de presentación. Los resultados mostraron grandes diferencias entre las páginas de internet investigadas y se identificaron oportunidades para mejorar los sitios, especialmente en cuanto a la profundidad, accesibilidad y presentación de la información. De 23 páginas de extensión en la internet, 13 proporcionaron ninguna o muy poca información en línea y cinco páginas cubrieron casi la totalidad del contenido investigado. Los medios principales utilizados fueron artículos con imágenes y solo tres universidades agregaron videos y seminarios en línea. Con base en estos resultados y de acuerdo con la creciente necesidad de fuentes en línea sobre salud aviar, las páginas de extensión en la internet deben proporcionar información completa y verídica o al menos enlaces con fuentes aprobadas. Los videos, cápsulas informativas en línea también denominadas "podcasts" y seminarios en línea pueden aumentar el alcance y los logros de aprendizaje. Esta encuesta puede ayudar a mejorar la presentación y el contenido de las páginas de extensión en la internet que están relacionadas con la salud avícola.

Prevalence of Select Intestinal Parasites in Alabama Backyard Poultry Flocks.

Keeping chickens as backyard pets has become increasingly popular in the United States in recent years. However, biosecurity is generally low in backyard flocks. As a consequence, they can serve as reservoirs for various pathogens that pose a risk for commercial poultry or human health. Eighty-four fecal samples, 82 from chickens and two from turkeys, from 64 backyard flocks throughout the state of Alabama were collected in the summers of 2017 and 2018. Coccidia oocysts were seen in 64.1% of flocks with oocyst counts in most samples below 10,000 oocysts per gram. Eggs of Ascaridia spp. or Heterakis gallinarum were observed in 20.3% of the flocks, and eggs of Capillaria spp. in 26.6% of the flocks. Egg counts were low, rarely exceeding 1000 eggs per gram. DNA extracted directly from fecal samples was investigated by PCR for other relevant parasites. The results showed that 4.7% of flocks were positive for Histomonas meleagridis, 18.8% of flocks for Tetratrichomonas gallinarum, 18.8% of flocks for Cryptosporidium spp. and 87.5% of flocks for Blastocystis spp. The results will help to provide information that can be used to design outreach programs to improve health and wellbeing of birds in backyard flocks.

A Survey of Coccidia and Nematodes in Pastured Poultry in the State of Georgia.

The aim of this study was to evaluate and quantify the parasitological challenge in pastured poultry production in the state of Georgia. Over the course of 1 yr, fecal samples from six turkey flocks, 10 broiler flocks, and 13 layer flocks were collected on a pastured farm in 2-wk intervals to determine counts of Eimeria oocysts and nematode eggs. Average coccidia counts were 10,198 oocysts per gram of feces (OPG) in broiler flocks, 1470 OPG in layer flocks, and 695 OPG in turkey flocks. The means in broiler and turkey flocks were higher at their first week on pasture. Counts in broilers and layers were significantly higher in spring than in winter and summer. Coccidia counts in broilers were lower than published numbers in conventionally reared poultry, indicating the rotation system of the pastures might effectively reduce the infection pressure. Next-generation sequencing of PCR products showed the presence of most described Eimeria spp. in broilers, layers, and turkeys. In addition, operational taxonomic units (OTUs) x, y, and z were found. The frequency of species was similar for broilers and layers, with the exception that Eimeria praecox and OTU z were more common in layers. In layer flocks, the average count of roundworm eggs per gram of feces (EPG) was 509 EPG with 80% of the samples being positive. The mean counts had no clear pattern related to age. There was an increase of EPG with the increase of temperatures during spring and summer with the peak at midfall. Worm eggs from laying hens were identified as Ascaridia galli. The seasonal differences suggest that higher temperatures might result in an increase of egg survival and sporulation in the environment.

Artículo regular­Muestreo de coccidias y nematodos en aves en pastoreo en el estado de Georgia El objetivo de este estudio fue evaluar y cuantificar el desafío parasitológico en la producción avícola en pastoreo en el estado de Georgia. En el transcurso de un año, se recolectaron muestras fecales de seis parvadas de pavos, 10 parvadas de pollos de engorde y 13 parvadas de gallinas de postura en una granja de pastoreo en intervalos de dos semanas para determinar los conteos de ooquistes de Eimeria y huevos de nematodos. Los recuentos promedio de coccidias fueron 10,198 ooquistes por gramo de heces (OPG) en parvadas de pollos de engorde, 1470 ooquistes por gramo de heces en parvadas ponedoras y 695 ooquistes por gramo de heces en parvadas de pavos. Los promedios en las parvadas de pollos de engorde y pavos fueron más altos en su primera semana en pastoreo. Los conteos en pollos de engorde y ponedoras fueron significativamente más altos en primavera que en invierno y verano. Los recuentos de coccidios en pollos de engorde fueron más bajos que los números publicados en aves criadas de manera convencional, lo que indica que el sistema de rotación de pastizales podría reducir efectivamente la presión de infección. La secuenciación de próxima generación de los productos de PCR mostró la presencia de la mayoría de las especies de Eimeria spp descritas en pollos de engorde, gallinas de postura y pavos. Además, se encontraron unidades taxonómicas operativas (OTU) x, y, z. La frecuencia de especies fue similar para pollos de engorde y gallinas de postura, con la excepción de que Eimeria praecox y las unidades taxonómicas operativas z fueron más comunes en gallinas de postura. En las parvadas de gallinas de postura, el recuento promedio de huevos de helmintos intestinales por gramo de heces (EPG) fue de 509 EPG, con el 80% de las muestras positivas. Los recuentos medios no tenían un patrón claro relacionado con la edad. Hubo un aumento de huevos de helmintos intestinales por gramo de heces con el aumento de las temperaturas durante la primavera y el verano con el pico a la mitad del otoño. Los huevos de helmintos de las gallinas de postura se identificaron como Ascaridia galli. Las diferencias estacionales sugieren que las temperaturas más altas podrían resultar en un aumento de la supervivencia de los huevos y su esporulación en el medio ambiente.

Hepatic Perisinusoidal Myofibroblast Proliferation and Systemic Inflammatory Response Precedes Sep/Tox Hepatitis in Broilers.

Septicemia-toxemia (sep/tox) falls under U.S. Department of Agriculture (USDA) food safety Category 1 and is the most common and economically significant cause of broiler carcass condemnations. Hepatic lesions are considered a possible consequence of septicemia and associated bacterial contamination of the carcass. Thus, these lesions are considered an indicator of sep/tox (sep/tox hepatitis). This study was undertaken to analyze the histologic lesions preceding grossly visible liver lesions leading to condemnation because of sep/tox at the processing plant. Livers from carcasses of broilers condemned by USDA inspectors for sep/tox were used to establish microscopic and gross criteria of end-stage sep/tox hepatitis. Following the characterization of sep/tox hepatitis, broilers from a farm with a history of sep/tox condemnations were submitted for postmortem examination and bacteriologic investigation at four intervals during the final 20 days of production. Five healthy and five clinically ill chickens were submitted from four houses at 18, 25, 32, and 38 days of production (160 total). Microscopic lesions representing hepatic perisinusoidal myofibroblast proliferation (HPMP), periportal extramedullary granulopoiesis (PEMG), splenic follicular histiocytosis, and bone marrow cellularity (BMC) were graded subjectively for each bird, and subjective grading was evaluated with digital quantitative techniques. Perisinusoidal hepatic stellate cell morphology and progressive transformation of these cells into myofibroblasts was confirmed by immunohistochemistry for smooth muscle actin and desmin. Aerobic cultures of livers and gall bladders from sep/tox birds yielded no growth of bacteria associated with septicemia. Mild to severe HPMP was observed in all age groups, representing 28% of examined birds. Increases in inflammatory cells observed by PEMG and BMC were positively correlated with progressive HPMP and end-stage sep/tox hepatitis in broiler chickens.

Artículo regular­Proliferación de miofibroblastos perisinusoidales hepáticos y respuesta inflamatoria sistémica que precede a la hepatitis por septicemia y toxemia (sep/tox) en pollos de engorde. La septicemia-toxemia (sep/tox) se incluye en la Categoría 1 de seguridad alimentaria del Departamento de Agricultura de los Estados Unidos. (USDA) y es la causa más común y económicamente significativa de decomisos de canales de pollos de engorde. Las lesiones hepáticas se consideran una posible consecuencia de la septicemia y de la contaminación bacteriana asociada con la canal. Por lo tanto, estas lesiones se consideran un indicador de septicemia/toxemia (hepatitis sep/tox). Este estudio se llevó a cabo para analizar las lesiones histológicas que preceden a las lesiones hepáticas muy visibles que conducen a los decomisos debido a septicemia/toxemia en la planta de procesamiento. Se utilizaron hígados de canales de pollos de engorde decomisados por los inspectores del USDA por septicemia/toxemia para establecer criterios microscópicos y generales de hepatitis en etapa terminal de la septicemia/toxemia. Después de la caracterización de la hepatitis por septicemia/toxemia, los pollos de engorde de una granja con un historial de decomisos por septicemia/toxemia se sometieron a examen post mortem e investigación bacteriológica en cuatro intervalos durante los últimos 20 días de producción. Se enviaron cinco pollos sanos y cinco clínicamente enfermos de cuatro casetas a los 18, 25, 32 y 38 días de producción (160 en total). Las lesiones microscópicas que representan la proliferación de miofibroblastos perisinusoidales hepáticos (HPMP), la granulopoyesis extramedular periportal (PEMG), la histocitosis folicular esplénica y la celularidad de la médula ósea (BMC) se clasificaron subjetivamente para cada ave, y la clasificación subjetiva se evaluó con técnicas cuantitativas digitales. La morfología de las células estrelladas hepáticas perisinusoidales y la transformación progresiva de estas células en miofibroblastos se confirmó mediante inmunohistoquímica para actina y desmina del músculo liso. Los cultivos aeróbicos de hígados y vesícula biliar de aves con septicemia/toxemia no produjeron crecimiento de bacterias asociadas con la septicemia. Se observó proliferación de miofibroblastos perisinusoidales hepáticos de leve a severa en todos los grupos de edad, lo que representa el 28% de las aves examinadas. Los aumentos en las células inflamatorias observados por granulopoyesis extramedular periportal y celularidad de la médula ósea se correlacionaron positivamente con proliferación progresiva de miofibroblastos perisinusoidales hepáticos y con hepatitis por septicemia/toxemia en etapa terminal en pollos de engorde.

Detection of DNA of Histomonas meleagridis and Tetratrichomonas gallinarum in German poultry flocks between 2004 and 2008.

Between 2004 and 2008, 338 samples from 156 German turkey, chicken, and peacock flocks with suspected histomonosis (histomoniasis) were sent to the Institute for Poultry Diseases of the Free University Berlin. Most samples were from ceca or livers; the other samples were organ pools or were taken from other organs or the environment. In 108 samples from 65 flocks, histomonal DNA was detected by polymerase chain reaction (PCR). Tetratrichomonas gallinarum DNA was found in 5.3% of investigated samples from flocks infected with Histomonas meleagridis and in 27.4% of investigated samples from flocks that were not infected with H. meleagridis. For subtyping of the strains, the C-profiling method, a method used to analyze the internal transcribed spacer-1 (ITS-1) of the rRNA gene, was modified to be more specific for H. meleagridis. Results showed the presence of more than the three subtypes described so far. There was no clear correlation between the subtype and the host. By C-profiling the clonal cultures, heterogeneous ITS-1 sequences were shown to probably result from intragenomic differences between rRNA genes.

Evaluation of nifurtimox for potential use in control of histomoniasis in turkeys.

Nifurtimox (NFX), a compound with known antiprotozoal activity, was evaluated for potential use in the prevention or treatment of histomoniasis in turkeys. A test of NFX in vitro showed that the compound was progressively active at concentrations of 12.5-200 ppm. Lower concentrations appeared only to delay growth of histomonads, while a concentration of 200 ppm was completely inhibitory. A series of tests in turkey poults showed that NFX had significant (P < 0.05) efficacy at 300-400 ppm when given in the feed throughout a 14-day experimental infection period. The beneficial effect was most prominent in the reduction of mortality and the suppression of liver lesions. Cecal lesions appeared less affected. Treatment with 400 ppm for a 3-day period after inoculation of turkeys was partially effective. In all tests, liver lesions were suppressed more effectively than cecal lesions, indicating that the concentration of the compound in the liver during metabolic excretion was important in the observed efficacy of this compound. Lack of any effect on growth or feed consumption in uninfected turkeys during a medication period of 16 days indicated that this compound was well tolerated by turkeys at 400 ppm in the feed and might be of benefit in the prevention or treatment of histomoniasis in turkeys.

Production of H5-specific monoclonal antibodies and the development of a competitive enzyme-linked immunosorbent assay for detection of H5 antibodies in multiple species.

The hemagglutinin gene of an avian influenza virus (AIV) A/duck/NC/674964/07 (H5N2) was cloned and expressed in a baculovirus system (H5-Bac). In parallel, a recombinant hemagglutinin of A/Vietnam/1203/04 (H5N1) was expressed in mammalian cells, purified, and used for generation of H5-specific monoclonal antibodies (MAb). The purified H5-Bac was used to develop a competitive enzyme-linked immunosorbent assay (cELISA) to detect H5 antibodies in a species-independent approach using one of the established H5-specific MAbs as the competitor antibody. The cELISA performed with influenza antibody-free sera or with sera of animals infected with other than H5-encoding AIV showed no significant inhibition of H5-MAb binding, indicating high test specificity. In contrast, sera of poultry (chickens, turkeys, ducks) experimentally infected with H5-encoding AIV were able to significantly inhibit the binding of the MAb in a species-independent approach. Comparison of the results of the cELISA with results obtained by a hemagglutination inhibition assay showed a gradient of the sensitivity (turkeys > ducks > chicken). The described results show that H5-specific antibodies in sera can be detected in a species-independent approach by using a recombinant protein.

Articular aspergillosis of hip joints in turkeys.

Aspergillosis is an important cause of morbidity and mortality in birds. Turkey poults are known to be particularly susceptible to fungal infection. Although the respiratory tract is the most commonly affected, dissemination can occur into virtually any organ. Here, we report an unusual outbreak of articular aspergillosis in a flock of meat turkeys with clinical signs of lameness. Between 7 and 11 weeks of age, turkeys had severe granulomatous osteoarthritis of the hip joints with necrosis of the femur head. Fungal morphology and PCR amplification and sequencing of the first ITS1-5.8S-ITS2 rDNA region identified Aspergillus fumigatus as the infectious agent. Concurrently, Staphylococcus spp. was isolated from the hip joints, which may have promoted the tropism of the fungus. Mild respiratory tract aspergillosis was observed in only one case. The findings suggest that fungal arthritis may present a specific disease entity in turkeys and should be considered as further cause of lameness in turkeys.