Infected Hens Research Articles

Colonization of internal organs by Salmonella Enteritidis in experimentally infected laying hens of four commercial genetic lines in conventional cages and enriched colony housing

The prevalence of Salmonella Enteritidis in commercial egg-laying flocks is a prominent public health concern because contaminated eggs cause human illness. Deposition of this pathogen inside eggs results from bacterial colonization of reproductive tissues in infected hens. Environmental conditions can influence avian Salmonella infections, but the food safety consequences of different poultry housing systems remain uncertain. The present study assessed the invasion of internal organs by Salmonella Enteritidis in groups of experimentally infected laying hens of four commercial genetic lines (designated as white egg lines W1 and W2 and brown egg lines B1 and B2). Groups of hens from each line were housed at 555 cm2 of floor space per bird in both conventional cages and colony units enriched with access to perches and nesting areas. All hens were orally inoculated with 5.75 × 107 colony-forming units of a two-strain Salmonella Enteritidis mixture. At 6 to 7 d post-inoculation, hens were euthanized, and samples of liver, spleen, ovary, oviduct, and intestinal tract were removed for bacteriologic culturing. The frequency of Salmonella Enteritidis recovery from intestinal samples was significantly (P < 0.05) greater for the two white egg lines combined than for the two brown egg lines combined in both conventional cage (72.2% vs. 50.0%) and enriched colony housing systems (66.7% vs. 37.5%). The frequency of intestinal Salmonella Enteritidis isolation from line B1 was significantly higher from hens in conventional cages (47.2%) than in enriched colonies (22.2%), but no differences were observed for other hen lines. Line W1 yielded more positive intestinal samples than either brown egg line in conventional cages, and line B2 had fewer positive intestinal samples than all other lines in enriched colonies. There were no significant differences between hen lines or housing systems in Salmonella Enteritidis isolation from other internal organs. These results demonstrate that Salmonella Enteritidis colonization of the intestinal tract can vary between genetic lines of egg-laying hens and that some lines are subject to housing system influences on Salmonella susceptibility.

Environmental Sampling Survey of H5N2 Highly Pathogenic Avian Influenza-Infected Layer Chicken Farms in Minnesota and Iowa.

Respiratory secretions, feces, feathers, and eggs of avian influenza-infected hens provide ample sources of virus which heavily contaminate barn and farm environments during a disease outbreak. Environmental sampling surveys were conducted in the Midwestern United States on affected farms during the 2015 H5N2 highly pathogenic avian influenza (HPAI) outbreak to assess the degree of viral contamination. A total of 930 samples were obtained from various sites inside and outside layer barns housing infected birds and tested with real-time reverse transcriptase PCR. The distribution and load of viral RNA in barns in which most birds were dead at the onset of depopulation efforts (high-mortality barns) were compared with those of barns in which birds were euthanatized before excess mortality occurred (normal-mortality barns). A statistically significant difference was seen between cycle threshold (Ct) values for samples taken of fans, feed troughs, barn floors, barn walls, cages, manure-associated locations, barn doors, egg belts, and the exterior of high-mortality vs. normal-mortality barns. In high-mortality barns, sample sites were found to be the most to least contaminated in the following order: cages, manure-associated locations, barn floors, egg belts, feed troughs, barn doors, barn walls, fans, exterior, and egg processing. Significant changes in Ct values over time following HPAI detection in a barn and depopulation of birds on an infected farm were observed for the manure-associated, barn floor, barn wall, and fan sampling sites. These results show that high mortality in a flock as a result of HPAI will increase contamination of the farm environment. The results also suggest optimal sampling locations for detection of virus; however, the persistence of RNA on highmortality farms may delay the determination that adequate sanitization has been performed for restocking to take place.

Ascaridia galli isolates with ITS1-5.8rRNA-ITS2 fragment homologous to Ascaridia columbae.

Ascaridia (A.) galli is one of the most commonly occurring nematodes in poultry worldwide, often in hens and broiler chickens. The infection with Ascaridia galli in free-range chickens was even 70%. There is not much information about A. galli genetic features. The present study was conducted to assess the genetic diversity of A. galli isolated from hens in Poland by analyzing the nucleotide sequence of the region ITS1-5.8rRNA-ITS2 and to define its homology within the family Ascaridiidae. Adult A. galli were collected from the intestines of naturally infected hens from two flocks of free-run laying hens from the Wielkopolska region in Poland. From all parasites an identical ITS1-5.8rRNA-ITS2 sequence was obtained, which was homologous in 99% with A. columbae (JQ995321.1) sequence. The high homology sequences of A. galli (KX683286) from Poland and A. columbae (JQ995321.1) isolate from the USA, support the observations of other authors suggesting that A. galli and A. columbae might be closely related. It is the first whole ITS1-5.8rRNA-ITS2 of A. galli in the GenBank database, so there is not enough data for detailed phylogenetic analysis of A. galli. Detailed genetic analysis is necessary to get better insight into the birds' Ascaridia species.

Performance, egg quality, and liver lipid reserves of free-range laying hens naturally infected with Ascaridia galli.

A study was conducted to determine the performance, egg quality, and liver lipid reserves of laying hens exposed to ranges contaminated with Ascaridia galli. Sixteen-week-old Lohmann Brown laying hens (n = 200) were divided into 4 treatments with 5 replicates containing 10 hens per pen. Hens of treatment 1 [negative control (NC)] ranged on a decontaminated area, and hens of treatments 2 (low infection) and 3 (medium infection) ranged on areas previously contaminated by hens artificially infected with 250 and 1,000 embryonated A. galli eggs, respectively. The hens of treatment 4 [positive control (PC)] ranged on areas previously contaminated by hens artificially infected with 2,500 embryonated A. galli eggs, and in addition these hens were orally inoculated with 1,000 embryonated eggs. Results indicated that hens of the medium infection group had a higher number of intestinal A. galli worms and A. galli eggs in the coprodeum excreta (43.9 ± 4.0 and 3,437± 459 eggs/g) compared to hens of the low infection group (23.8 ± 4.0 and 1,820± 450 eggs/g) (P < 0.01) and similar worm counts to PC hens (34.4 ± 4.0 and 2,918± 474) (P>0.05). Egg production, egg mass, feed intake, and feed conversion ratio (FCR) were not affected by A. galli infection (P > 0.05). Egg quality parameters (egg weight, shell reflectivity, shell weight, shell thickness, shell percentage, shell breaking strength, deformation, albumen height, Haugh unit, and yolk score) were not affected by A. galli infection (P > 0.05). Highly infected hens had lower liver lipid content (2.72± 0.51g) compared to uninfected hens (4.46± 0.58g, P < 0.01). The results indicate that exposure to ranges contaminated with A. galli resulted in infection of the ranging hens, but this did not affect egg production or egg quality. Infection with A. galli lowered the liver lipid reserves of the host significantly, suggesting infected hens use more energy reserves for maintenance and production.

Morphological Differences of Pancreatic Lesions in Mute Swans and Hens Naturally Infected with Highly Pathogenic Avian Influenza Virus H5N8

Abstract During the epizootic of highly pathogenic avian influenza subtype H5N8 in Serbia in the winter of 2016-2017, the highest percent of mortality due to this infection was recorded in mute swans (Cygnus olor). Besides mute swans, avian influenza virus subtype H5N8 was also diagnosed in a small number of hens in rural households. Pancreatic tissues from avian influenza H5N8 positive mute swans and hens that died during this outbreak were collected to determine the character of morphological lesions and the distribution of the viral antigen in this organ. Macroscopic examination of the pancreas of mute swans revealed hemorrhages as well as necrosis, while there were no macroscopic visible lesions in the pancreas of infected hens. Despite the different macroscopic finding, microscopic examination of the pancreas of both infected bird species revealed lesions in the form of acute pancreatitis and multifocal acinar necrosis. The viral antigen was abundantly expressed in the cytoplasm and nucleus of necrotic cells, as well as in macrophages in both examined bird species. Immunohistochemical expression of the viral antigen in the pancreas was strongly consistent with histological lesions. According to the above described findings, it could be concluded that highly pathogenic avian influenza virus H5N8 has a high affinity to pancreatic tissue in both mute swans and hens and the distribution and the character of the lesions in the pancreas are similar in both bird species.

Detection of Ascaridia galli infection in free-range laying hens

Reliable methods for detection of A. galli infection using excreta egg count (EEC) and ELISA assays to determine A. galli specific IgY levels in serum and yolk samples were compared from hens infected naturally and artificially. Artificially infected hens were used to generate samples for analysis of preferred detection methods and to generate contaminated ranges for use in the naturally acquired infection study in which Lohmann Brown hens (n = 200) at 16 weeks of age were randomly assigned to four treatments with five replicate pens. Hens of negative control (NC) ranged on a decontaminated area, hens of low infection, medium infection and positive control (PC) ranged on the areas previously contaminated by hens artificially infected with 250, 1000 and 2500 A. galli eggs/hen, respectively. Additionally, hens of PC were orally infected with 1000 A. galli eggs/hen. Anti A. galli antibody levels in hen serum (SIgY) and yolk (YIgY) were measured before range access, and 2, 7 and 12 weeks after access to the contaminated ranges. In a natural infection study, eggs were detected in the excreta of all hens 4 weeks after range access, with the exception of NC in which no eggs were detected. EEC increased to reach maximum value (2204 ± 307 eggs/g) after 11 weeks of range access and then declined at 12 weeks (905 ± 307eggs/g) (p < 0.01). While SIgY OD values were not different in hens between any groups before range access, after 2 weeks, both SIgY and YIgY gradually increased in hens of PC (1.17 ± 0.03 and 0.88 ± 0.04) and medium infection (1.07 ± 0.03 and 0.96 ± 0.04) compared to low infection (0.38 ± 0.03 and 0.29 ± 0.04) (p < 0.01) and NC. After 12 weeks, SIgY were similar in hens of PC, medium and low groups whereas YIgY was higher in hens of low infection group (p < 0.01). Sensitivity of the serum and egg yolk antibody levels assay to detect A. galli infection was 100% and 96%, respectively, whereas the pooled EEC method yielded a sensitivity of 93%. The results of this study suggest that hens naturally infected with A. galli produce both SIgY and YIgY at different levels depending on the infection intensity and duration of exposure which allows the diagnosis of prior infection or early diagnosis of current infection. Use of the practical and non-invasive method of yolk sample analysis for detecting IgY can be just as informative as using serum samples to detect A. galli infection.

Effect of an artificial Ascaridia galli infection on egg production, immune response, and liver lipid reserve of free-range laying hens

This study was conducted to determine the effect of Ascaridia galli infection on free-range laying hens. Lohmann Brown laying hens (n = 200) at 17 wk of age were allocated to 4 treatment groups (n = 50 per group), each with 5 replicate pens of 10 hens. Hens in 3 treatment groups were orally inoculated with different doses of embryonated A. galli eggs: low (250 eggs), medium (1,000 eggs), and high (2,500 eggs) levels, whereas hens of the control group were not infected. Infection levels were monitored using excreta egg counts and mature A. galli worm counts in the intestine. Anti A. galli antibody titers (IgY) in the serum were measured prior to infection, and at 6, 11, 15, and 20 wk post infection (PI) and in egg yolk at 11 and 20 wk PI. Parameters evaluated included feed intake, egg production, egg weight, egg mass, FCR, liver weight, liver fat, and intra epithelial immune cell infiltration. The results showed no difference in feed intake, body weight, or FCR among any treatment groups (P > 0.05). Egg production was lower in the low infection group compared to other groups at 20 wk of age (P < 0.01). Serum IgY was higher in the infected groups’ hens at 20 wk PI compared to control group hens (P < 0.01). Yolk IgY increased significantly over time and was higher in infected hens compared to hens of the control group at 11 and 20 wk PI (P < 0.001). No differences were observed in liver lipid content or intraepithelial lymphocytes infiltration among treatment groups. Ascaridia galli eggs in the coprodeum content and adult A. galli worm count were higher in infected hens compared to hens of the control group (P < 0.01). In conclusion, the effects of artificial infection with A. galli on the parameters investigated were minor, and egg yolk antibody may be a more reliable indicator of A. galli infection than serum antibody or excreta egg count.

Frequency and Duration of Fecal Shedding of Salmonella Serovars Heidelberg and Typhimurium by Experimentally Infected Laying Hens Housed in Enriched Colony Cages at Different Stocking Densities.

Eggs contaminated with Salmonella Enteritidis are leading sources of human salmonellosis, but Salmonella Heidelberg and Salmonella Typhimurium are also egg-associated pathogens. The management practices and housing facilities characterizing different systems for housing commercial egg flocks can influence Salmonella persistence and transmission. Animal welfare aspects of poultry housing have been widely debated, but their food safety ramifications are not thoroughly understood. The present study assessed the effects of two different bird stocking densities on the frequency and duration of fecal shedding of strains of Salmonella Heidelberg and Salmonella Typhimurium in groups of experimentally infected laying hens housed in colony cages enriched with perching and nesting areas. In separate trials, laying hens were distributed into two groups housed in enriched colony cages at stocking densities of 648 and 973 cm2/bird, and a third group was housed in conventional cages at 648 cm2/bird. All hens were orally inoculated with doses of 108 colony-forming units (CFU) of either Salmonella Heidelberg or Salmonella Typhimurium. At eight weekly postinoculation intervals, samples of voided feces were collected from beneath each cage and cultured to detect Salmonella. Fecal shedding of Salmonella Heidelberg continued for 8 wk in all housing groups, but Salmonella Typhimurium shedding ceased after as little as 5 wk in enriched colony cages at low stocking density. After Salmonella Heidelberg infection, the overall frequency of positive fecal cultures for all sampling dates combined was significantly (P < 0.05) greater from either conventional cages (51.0%) or enriched colony cages (46.5%) at high stocking density than from enriched colony cages at low stocking density (33.3%). No significant differences in Salmonella Typhimurium fecal isolation were identified between housing groups. These results demonstrate that stocking density can affect intestinal colonization and fecal shedding in laying hens for some (but not necessarily all) Salmonella serovars or strains.

Colonization of internal organs by Salmonella serovars Heidelberg and Typhimurium in experimentally infected laying hens housed in enriched colony cages at different stocking densities

Contaminated eggs produced by infected commercial laying flocks are often implicated as sources of human infections with Salmonella Enteritidis, but Salmonella serovars Heidelberg and Typhimurium have also been associated with egg-transmitted illness. Contamination of the edible contents of eggs is a consequence of the colonization of reproductive tissues in systemically infected hens. In recent years, the animal welfare implications of diverse poultry housing and management systems have been vigorously debated, but the food safety significance of laying hen housing remains uncertain. The present study evaluated the effects of 2 different bird stocking densities on the invasion of internal organs by Salmonella serovars Heidelberg and Typhimurium in groups of experimentally infected laying hens housed in colony cages enriched with perching and nesting areas. Laying hens were distributed at 2 different stocking densities (648 and 973 cm2/bird) into colony cages and (along with a group housed in conventional cages at 648 cm2/bird) orally inoculated with doses of 107 cfu of 2-strain cocktails of either Salmonella Heidelberg or Salmonella Typhimurium. At 5 to 6 d post-inoculation, hens were euthanized and samples of internal organs (cecum, liver, spleen, ovary, and oviduct) were removed for bacteriologic culturing. The overall frequency of Salmonella isolation from ceca after inoculation with strains of serovar Heidelberg (83.3%) was significantly (P < 0.001) greater than the corresponding value for strains of serovar Typhimurium (53.8%), whereas Salmonella was recovered significantly more often from both livers (85.2% vs. 53.7%; P < 0.0001) and spleens (78.7% vs. 56.5%; P = 0.0008) after inoculation with strains of serovar Typhimurium than strains of serovar Heidelberg. However, there were no significant differences (P > 0.05) between stocking densities or cage systems in the frequencies of isolation of either Salmonella serovar from any of the five sampled tissues. These results contrast with prior studies, which reported increased susceptibility to internal organ invasion by Salmonella Enteritidis among hens in conventional cages at higher stocking densities.

A comprehensive evaluation of an ELISA for the diagnosis of the two most common ascarids in chickens using plasma or egg yolks

BackgroundClassical faecal egg counts (FEC) provide less reliable diagnostic information for nematode infections in chickens. We developed an ELISA based on Ascaridia galli antigens and tested two hypotheses, as follows: (i) IgY antibodies developed against A. galli will also be useful to identify Heterakis gallinarum infections, and (ii) circulating antibodies stored in egg yolks are as good as plasma samples, so a non-invasive diagnosis is possible. The aim of this study, therefore, was to compare the diagnostic accuracy of the ELISA system with FEC, using both plasma and egg yolks from experimentally infected hens. In addition, naturally infected animals were evaluated to validate the assay.ResultsThe assay quantified large differences (P < 0.001) in plasma or in egg-yolk IgY concentrations between infected and uninfected animals in two experiments, each performed with either of the nematode species. The assay performed with high accuracy as quantified with the area under the ROC curve (AUC) values of > 0.90 for both nematodes using either plasma or egg yolks. Sensitivity of the assay was 94 and 93% with plasma and egg yolk samples, respectively, whereas FEC yielded in a sensitivity of 84% in A. galli experiment. Total test accuracy of the assay with plasma samples (AUC = 0.99) tended to be higher (P = 0.0630) than FEC (AUC = 0.92) for A. galli, while the assay with either sample matrix performed similar to FEC (AUC ≥ 0.91) for H. gallinarum. Among the three tests, the FECs correlated better with A. galli burden than the ELISA. Although 90% of naturally infected hens were correctly identified by the ELISA, 45% of the infected hens tested negative with FEC, indicating the validity of the higher test accuracy of the ELISA.ConclusionsAntigens of A. galli can be used successfully to identify H. gallinarum-infected animals, indicating that chickens develop cross-reactive antibodies against the two closely related species. Egg yolks are as informative as plasma samples, so that animal welfare-friendly sampling is possible. Although the assay with plasma samples reveals qualitative information of higher quality than FECs on the infection status of naturally infected birds, the latter is still a better tool to assess the intensity of A. galli but not of H. gallinarum infections.

Frequency and Duration of Fecal Shedding of Salmonella Enteritidis by Experimentally Infected Laying Hens Housed in Enriched Colony Cages at Different Stocking Densities.

Human infections with Salmonella Enteritidis are often attributed to the consumption of contaminated eggs, so the prevalence of this pathogen in egg-laying poultry is an important public health risk factor. Numerous and complex environmental influences on Salmonella persistence and transmission are exerted by management practices and housing facilities used in commercial egg production. In recent years, the animal welfare implications of poultry housing systems have guided the development of alternatives to traditional cage-based housing, but their food safety consequences are not yet fully understood. The present study assessed the effects of different bird stocking densities on the frequency and duration of fecal shedding of S. Enteritidis in groups of experimentally infected laying hens housed in colony cages enriched with perching and nesting areas. In two trials, groups of laying hens were distributed at two stocking densities (648 and 973 cm2/bird) into enriched colony cages and (along with a group housed in conventional cages at 648 cm2/bird) orally inoculated with doses of 1.0 × 108 cfu of S. Enteritidis. At 10 weekly postinoculation intervals, samples of voided feces were collected from beneath each cage and cultured to detect S. Enteritidis. Fecal shedding of S. Enteritidis was detected for up to 10 weeks postinoculation by hens in all three housing treatment groups. The overall frequency of positive fecal cultures was significantly (P < 0.05) greater from conventional cages than from enriched colony cages (at the lower stocking density) for the total of all sampling dates (45.0 vs. 33.3%) and also for samples collected at 4–9 weeks postinfection. Likewise, the frequency of S. Enteritidis isolation from feces from conventional cages was significantly greater than from enriched colony cages (at the higher hen stocking density) for the sum of all samples (45.0 vs. 36.7%) and at 6 weeks postinoculation. Moreover, the frequency of S. Enteritidis fecal recovery from enriched colony cages at the higher hen stocking was significantly greater than from similar cages at the lower stocking density for all 10 sampling dates combined (39.4 vs. 33.3%). These results suggest that stocking density can affect S. Enteritidis intestinal colonization and fecal shedding in laying hens, but some other difference between conventional and enriched colony cage systems appears to exert an additional influence.

Deterioration of eggshell quality is related to calbindin in laying hens infected with velogenic genotype VIId Newcastle disease virus

The aim of this study was to determine the mechanism by which Newcastle disease virus (NDV) affects eggshell quality. Thirty-week-old specific pathogen free (SPF) egg-laying hens were inoculated with the velogenic genotype VIId NDV strain (infected group) or with inoculating media without virus (control group) by combined intraocular and intranasal routes. The levels of CaBP-D28k mRNA expression in the uterus, a gene related to eggshell quality, were examined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The quality of eggshells was analyzed by scanning electron microscopy (SEM). The infected group showed a marked decline in egg production when compared to the control group. The NDV antigen was found more abundantly in the glandular epithelium of the infected hens' uteri from 1 to 15 d post-inoculation (dpi). The levels of CaBP-D28k mRNA expression in the uteri of infected hens were significantly lower than in the control hens from 3 to 15 dpi (P < 0.05). The changes in the Ca concentrations in the eggshells were consistent with the expression of CaBP-D28k mRNA in the infected hens. Ultrastructural examination of eggshells showed significantly reduced shell thickness in the infected hens from 1 to 15 dpi (P < 0.05). Furthermore, obvious changes in the structure of the external shell surface and shell membrane were detected in the infected hens compared with the control hens. In conclusion, the current study confirmed that velogenic genotype VIId NDV infection is associated with the deterioration of the eggshell quality of the laying hens.

Fowl typhoid in laying hens cause hepatic oxidative stress.

The aim of this study was to analyses nitric oxide, antioxidant status, and oxidative profile in the liver of laying hens naturally infected by Salmonella enterica subsp enterica serovar Gallinarum (S. Gallinarum). The nitrite/nitrate (NOx), reactive oxygen species (ROS), thiobarbituric acid-reactive substances (TBARS), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities were measured in liver samples, as well the biomarkers of hepatic function (alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), total protein and albumin levels measured in serum. NOx levels and CAT activity were reduced in hepatic tissue of infected hens. On the other hand, TBARS and ROS levels, GR, GPx and GST activities were higher in infected animals. On biomarkers of tissue damage, ALT, AST, GGT and total protein levels were higher in serum of infected hens, and showed decreased albumin levels. In summary, ROS and TBARS production lead to damage on the membrane lipids that alter activities of antioxidant enzymes CAT, GR, GPx and GSH, an adaptive response against S. Gallinarum infection, contributing to the pathophysiology and clinical signs of the disease.

Comparative pathomorphological, bacteriological and serological examination of broiler breeders and pheasants experimentally infected with Ornithobacterium rhinotracheale

ABSTRACTThe aim of the investigations was to determine the influence of Ornithobacterium rhinotracheale (ORT) on the development of pathomorphological lesions in the respiratory organs and on the health status of experimentally infected broiler breeders and pheasants from the rearing stage. There was no evidence of clinical signs in infected broiler breeder hens nor in the group of infected pheasants except for one bird in the latter group which exhibited slower movement and gasping. The frequency and intensity of pathomorphological lesions were higher in pheasants. The gross pathology findings were characterized mainly by redness of the mucosa of the upper respiratory tract and accumulation of mucous content in the nasal cavities, infraorbital sinuses, larynx and trachea. Histopathology confirmed the presence of inflammation of the upper respiratory tract. Lesions in the lungs included hyperaemia, granulomatous and fibrinous pneumonia. ORT was reisolated only from the group of infected pheasants. Reisolation was successful from the respiratory organs (trachea, larynx, infraorbital sinuses, and lungs) of eight out of 10 infected birds. The serological response in both species was characterized by rapid production of specific antibodies that reached a maximum level in the blood in the first week after experimental infection. The antibody titres decreased gradually and were maintained at a stable level until the 12th week after inoculation. Fourteen weeks post-inoculation specific antibodies could not be detected by enzyme-linked immunosorbent assay.

Hepatic cholinesterase of laying hens naturally infected by Salmonella Gallinarum (fowl typhoid)

Salmonella is a facultative intracellular pathogen that may cause foodborne gastroenteritis in humans and animals consisting of over 2000 serovars. The serovar Salmonella Gallinarum is an important worldwide pathogen of poultry. However, little is known on the mechanisms of pathogenesis of Salmonella in chickens. The aim of this study was to evaluate cholinesterase and myeloperoxidase activities in hepatic tissue of laying hens naturally infected by S. Gallinarum. Twenty positive liver samples for S. Gallinarum were collected, in addition to seven liver samples from healthy uninfected laying hens (control group). The right liver lobe was homogenized for analysis of acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and myeloperoxidase (MPO), and the left lobe was divided into two fragments, one for histopathology and the other for Salmonella isolation. The results showed changes in AChE and BchE activity in the liver of infected laying hens compared to the control group (P < 0.05), i.e. reduced AChE and increased BChE activities in liver samples. Infected animals showed increased MPO activity compared to healthy animals (P < 0.05). Furthermore, the histopathological findings showed fibrinoid necrosis associated to the infiltration of lymphocytes, plasma cells, macrophages,heterophils in the liver of infected hens. These findings suggest that the inflammatory process was attenuated providing a pro-inflammatory action of both enzyme analyzed in order to reduce the free ACh, a molecule which has an anti-inflammatory action. Therefore, our results lead to the hypothesis that cholinesterase plays an important role on the modulation of immune response against S. Gallinarum with an inflammatory effect, contributing to the response against this bacterium. This study should contribute to a better understanding on the pathogenic mechanisms involved in laying hens infected by S. Gallinarum.

Anaemia, Serum Iron Concentrations and δ-Aminolevulinate Dehydratase Activity in Laying Hens Infected Naturally by Salmonella Gallinarum

The aim of this study was to evaluate anaemia, serum iron concentrations and δ-aminolevulinate dehydratase (ALA-D) activity in laying hens infected naturally by Salmonella Gallinarum and having severe hepatic lesions. Liver and serum samples were collected from 27 laying hens (20 infected and seven uninfected). The δ-ALA-D activity, haematocrit and serum iron concentrations were evaluated. There were significant decreases in δ-ALA-D activity, haematocrit and serum iron concentrations (P<0.01) in birds infected by S. Gallinarum when compared with uninfected birds. There was a positive correlation (P<0.001) between serum iron concentration, haematocrit (r(2)=0.82) and δ-ALA-D activity (r(2)=0.75). A positive correlation was also observed between δ-ALA-D activity and haematocrit (r(2)=0.78; P<0.01). Liver samples showed moderate focal coagulative necrosis associated with infiltration of lymphoplasmacytic cells, macrophages and heterophils. The anaemia in the infected hens may be related to reduction in δ-ALA-D activity and serum iron concentrations, since both are important for haemopoiesis.

Colonization of internal organs by Salmonella Enteritidis in experimentally infected laying hens housed in enriched colony cages at different stocking densities

Epidemiologic analyses have linked the frequency of human infections with Salmonella enterica subspecies enterica serovar Enteritidis to the consumption of contaminated eggs and thus to the prevalence of this pathogen in commercial egg-laying flocks. Contamination of the edible contents of eggs by Salmonella Enteritidis is a consequence of the colonization of reproductive tissues in systemically infected hens. The animal welfare implications of laying hen housing systems have been widely debated, but no definitive consensus has yet emerged about the food safety significance of poultry housing options. The present study sought to determine the effects of two different bird stocking densities on the invasion of internal organs by Salmonella Enteritidis in groups of experimentally infected laying hens housed in colony cages enriched with perching and nesting areas. In two trials, groups of laying hens were distributed at two different stocking densities into colony cages and (along with a group housed in conventional cages) orally inoculated with doses of 1.0 × 107 cfu of Salmonella Enteritidis. At 5 to 6 d post-inoculation, hens were euthanized and samples of internal organs were removed for bacteriologic culturing. For both trials combined, Salmonella Enteritidis was recovered at a significantly (P < 0.05) greater frequency from hens in enriched colony cages at the higher stocking density than at the lower density from livers (75.0% vs. 51.4%) and ovaries (51.4% vs. 30.6%). However, spleens from hens in enriched colony cages at the higher stocking density were significantly less often positive for Salmonella Enteritidis than from hens in conventional cages at that same density (90.3% vs. 68.1%). These results suggest that stocking density can influence the susceptibility of hens to Salmonella Enteritidis, but other housing systems parameters may also contribute to the outcome of infections.

Transmission of an H5N8-Subtype Highly Pathogenic Avian Influenza Virus from Infected Hens to Laid Eggs.

We showed here that an H5N8-subtype highly pathogenic avian influenza virus (HPAIV) was transmitted to both the internal contents and shells of eggs laid by white leghorn hens experimentally infected with the virus. Seven of eight HPAIV-infected hens laid eggs until 4 days postinoculation (dpi). The mean number of eggs laid per head daily decreased significantly from 0.58 before inoculation to 0.18 after viral inoculation. The virus was detected in the eggs laid by three of the seven hens. Viral transmission was detectable beginning on 3 dpi, and virus titers in tracheal and cloacal swabs from the hens that laid the contaminated eggs exceeded 2.9 log10 EID50. The level of viral replication and its timing when virus replicates enough to be detected in oviduct after virus inoculation appear to be key factors in the transmission of H5N8 HPAIV from infected hens to laid eggs.

Morphology of immune organs after very virulent plus strain of Marek's disease virus infection in vaccinated hens.

Marek's disease (MD) outbreaks in poultry flocks may be associated with overriding of vaccine immune protection by very virulent (vvMDV) or very virulent plus (vv+MDV) strains. This paper presents the study on lymphoid organ morphology in the latent phase of MD caused by vv+MDV which break post-vaccinal protection in hens. We also immunohistochemically examined B and T populations as well as B/T and CD4+/CD8+ ratio of lymphocytes in lymphatic organs and, as a background, in MD lymphomas from non-lymphatic organs. The number of antigen expressed cells was evaluated as a percentage of positive cells in the one power field. Organ samples were collected from 24 dead reproductive hens (Ross 308 line) in age between 35-56 weeks, infected with vv+MDV. The hens originated from farms with MD outbreaks, despite earlier routine vaccination with CVI988/Rispens + HVT. The control organ samples originated from 15 clinically healthy hens at the same age and line, subjected to the same vaccination schedule. The number of CD3+, CD8+ and TCRγδ+ cells was significantly lower in MDV infected thymus, spleen and cecal tonsils in comparison to that found in the control organs. The proportion of CD4+ was also distinctly reduced in the thymus and limited in the spleen of MDV infected hens. This study revealed that infection with field vv+MDV isolates might break post-vaccinal protection and influence the central and peripheral immune system. The decrease in CD8+ and TCRγδ+ cell number in the thymus, spleen and cecal tonsils suggests that primarily these cells are involved in cell-mediated cytotoxicity against MDV transformed cells during latency.

Deterioration of eggshell quality in laying hens experimentally infected with H9N2 avian influenza virus.

This study aimed to determine the mechanism by which H9N2 avian influenza virus (AIV) affects eggshell quality. Thirty-week-old specific pathogen free egg-laying hens were inoculated with the chicken-origin H9N2 AIV strain (A/Chicken/shaanxi/01/2011) or with inoculating media without virus by combined intraocular and intranasal routes. The time course for the appearance of viral antigen and tissue lesions in the oviduct was coincident with the adverse changes in egg production in the infected hens. The viral loads of AIV have a close correlation with the changes in the uterus CaBP-D28k mRNA expression as well as the Ca concentrations in the eggshells in the infected hens from 1 to 7 days post inoculation (dpi). Ultrastructural examination of eggshells showed significantly decreased shell thickness in the infected hens from 1 to 5 dpi (P < 0.05). Furthermore, obvious changes in the structure of the external shell surface and shell membrane were detected in the infected hens from 1 to 5 dpi as compared with the control hens. In conclusion, this study confirmed that H9N2 AIV strain (A/Chicken/shaanxi/01/2011) infection is associated with severe lesions of the uterus and abnormal expression of CaBP-D28k mRNA in the uteri of the infected hens. The change of CaBP-D28k mRNA expression may contribute to the deterioration of the eggshell quality of the laying hens infected with AIV. It is noteworthy that the pathogenicity of H9N2 AIV strains may vary depending on the virus strain and host preference.