Focal duodenal necrosis (FDN) is a poorly understood intestinal disease of egg layers, and has been associated with drops in egg production and decreased egg weights. The etiology of this disease is still unknown, but the condition has been associated with Clostridium colinum and Clostridium perfringens. In order to investigate the etiology, duodenal samples were taken from hens with FDN. The hens originated from table egg layer farms in three states. The samples were examined by histopathology, bacteriology, and immunohistochemistry. Macroscopically, all samples contained focal to multifocal, variably sized, reddened or brownish gray areas of mucosal erosion. Histopathology revealed mild to severe heterophilic and lymphoplasmacytic enteritis with loss of enterocytes at the villous tips, luminal fibrinonecrotic exudate, and variable numbers of Gram-positive and Gram-negative rod-shaped bacteria within the lesions in 16/30 samples. Clostridium perfringens was isolated by anaerobic bacteriology from 4/13 samples that had characteristic microscopic lesions of FDN. Polymerase chain reaction (PCR) revealed that all four isolates were Type A C. perfringens, positive for beta2 gene and negative for necrotic enteritis toxin B and enterotoxin genes. PCR for Clostridium colinum applied to DNA extracted from frozen intestinal samples yielded negative results in 14/14 duodenal samples. Immunohistochemistry (IHC) for 7C. perfringens, alpha and beta2 toxins stained a few to numerous long rod-shaped bacteria present in the lesions. IHC for alpha and beta2 toxins also stained enterocytes at the villous tips, inflammatory cells in the lamina propria, as well as degenerated and sloughed enterocytes present within the luminal exudate. These findings suggest that C. perfringens may play a role in the development of FDN. Experimental challenge studies with these isolates still need to be performed in order to reproduce the disease and fulfill Koch's postulates.

Mallards are widely recognized as reservoirs for Influenza A viruses (IAV); however, host factors that might prompt seasonality and trends in subtype diversity of IAV such as adaptive heterosubtypic immunity (HSI) are not well understood. To investigate this, we inoculated mallards with a prevailing H3N8 low pathogenic avian influenza virus (LPAIV) subtype in waterfowl to determine if prior infection with this virus would be protective against heterosubtypic infections with the H4N6, H10N7 and H14N5 LPAIV subtypes after one, two and three months, respectively. Also, we investigated the effect of cumulative immunity after sequential inoculation of mallards with these viruses in one-month intervals. Humoral immunity was assessed by microneutralization assays using a subset of representative LPAIV subtypes as antigens. Our results indicate that prior inoculation with the H3N8 virus confers partial protective immunity against subsequent heterosubtypic infections with the robustness of HSI related to the phylogenetic similarity of the HA protein of the strains used. Furthermore, induced HSI was boosted and followed by repeated exposure to more than one LPAIV subtype. Our findings provide further information on the contributions of HSI and its role in the dynamics of IAV subtype diversity in mallards.

Two groups of Mycoplasma gallisepticum (MG) isolates (n = 24) from Jordan were analyzed by molecular methods and compared with other Middle Eastern isolates, related international isolates, and reference strains. The first group (n = 19) was isolated from July 2004 to January 2005 (isolation period A), and the newer group (n = 5) from June 2007 to April 2008 (isolation period B). The groups of isolates are from chicken flocks from northern Jordan, but are not from the same farms. None of the flocks were vaccinated for MG. Random amplified polymorphic DNA analysis, targeted sequencing of the partial MG cytadhesin 2 (mgc2), and the MG 16S-23S rRNA intergenic spacer region (IGSR) divided the Jordanian isolates into two groups. All of the 19 isolates from time period A, in addition to two isolates from time period B, were indistinguishable from the F strain. Three of five isolates from time period B were characterized as wild types and were indistinguishable from each other. The wild-type field strain was readily distinguished from the F strain. It was 91% and 96.4% similar to the F strain based on Clustal-W alignments of sequences of mgc2 and IGSR, respectively. Sequence similarity of mgc2 gene of the Jordan wild-type strain to isolates from Israel and Egypt ranged from 96.5% to 100%, whereas for IGSR it was 99.4%-100%. We theorize that the F-strain live MG vaccine, commonly used in Jordan prior to 2007, was transmitted to nonvaccinated poultry in the region and was a predominant genotype during time period A.

Heavy metals have been implicated for their ability to increase antibiotic resistance in bacteria collected from polluted waters, independent of antibiotic exposure. Specific-pathogen-free Leghorn chickens were therefore given Pb acetate in the drinking water to expose the enteric bacteria to Pb and to determine if antibiotic resistance changed in these bacteria. Concentrations of Pb used were 0.0, 0.01, 0.1, 1.0, or 10.0 mM; birds given the highest 2 concentrations showed signs of moribundity and dehydration and were removed from the study. Vent culture samples were collected for bacterial cultures on d 0 before Pb exposure, d 7 and 14, and then birds were euthanized by CO2 gas for necropsy on d 14, at which time intestinal contents were also collected for bacterial cultures. Fecal swabs but not intestinal samples from Pb-exposed birds contained isolates that had significantly elevated antibiotic resistance. Some of the isolates contained bacteria that were resistant to up to 20 antibiotics. These results suggest the need for repeated studies in chickens infected with zoonotic pathogens.

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Focal duodenal necrosis (FDN) is an intestinal disease of egg-layer chickens characterized by multifocal necrosis of the duodenal loop and proximal jejunum. Affected flocks usually have decreased egg weights and drops in egg production. Previous studies have associated this condition with Clostridium perfringens infection. We tried to reproduce FDN by experimental infection of egg-laying chickens using different netB-positive and netB-negative C. perfringens strains, and duodenal homogenate obtained from FDN lesions. Chickens challenged with C. perfringens and/or duodenal homogenate developed duodenitis after challenge. Gross lesions included mucosal erosions, hyperemia, mucosal hemorrhages, and watery intestinal content. Microscopic lesions included mild enterocyte degeneration and necrosis, and mild-to-moderate hemorrhage and lymphoplasmacytic and heterophilic infiltration of the lamina propria. Two netB-positive C. perfringens strains closely related to necrotic enteritis pathogenic strains, by genomic composition, were re-isolated from lesions. Necrosis of intestinal crypts was observed in chickens challenged with duodenal homogenate with or without C. perfringens coinfection. Characteristic microscopic FDN lesions with significant necrosis and loss of villus enterocytes were not reproduced.

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