Hydrogen sulfide modulates IL-6/STAT3 pathway and inhibits oxidative stress, inflammation, and apoptosis in rat model of methotrexate hepatotoxicity.

Methotrexate (MTX) is a commonly used anticancer and immunosuppressive agent. However, MTX can induce hepatotoxicity due to oxidative stress, inflammation, and apoptosis. Hydrogen sulfide (H2S), the endogenous gaseous molecule, has antioxidant, anti-inflammatory, and anti-apoptotic effects. The present work explored the probable protective effect of H2S against MTX hepatotoxicity in rats and also the possible mechanisms underlying this effect. MTX was given at a single intraperitoneal (i.p.) dose of 20 mg/kg. Sodium H2S (56 µmol /kg/day, i.p.), as H2S donor, was given for 10 days, starting 6 days before MTX administration. H2S significantly reduced serum alanine aminotransferase, hepatic malondialdehyde, interleukin 6, nuclear factor κB p65, cytosolic cytochrome c, phosphorylated signal transducer and activator of transcription 3, and Bax/Bcl-2 ratio and significantly increased hepatic total antioxidant capacity and endothelial nitric oxide synthase (eNOS) in rats received MTX. In addition, H2S minimized the histopathological injury and significantly decreased the expression of STAT3 in liver tissue of MTX-challenged rats. The effects of H2S were significantly antagonized by administration of glibenclamide as KATP channel blocker, Nω-nitro-l-arginine, as eNOS inhibitor, or ruthenium red, as transient receptor potential vanilloid 1 (TRPV1) antagonist. It was concluded that H2S provided significant hepatoprotection in MTX-challenged rats through its antioxidant, anti-inflammatory, anti-apoptotic effects. These effects are most probably mediated by the ability of H2S to act as IL-6/STAT3 pathway modulator, KATP channel opener, eNOS activator, and TRPV1 agonist.

Investigation of the prevalence and severity of foot pad dermatitis at the slaughterhouse in fattening turkeys reared in organic production systems in Germany.

The present study shows the prevalence and severity of foot pad dermatitis (FPD) in turkeys reared in organic production systems assessed at slaughterhouses in Germany. The investigations of altogether 1,860 turkeys of the strains Kelly Broad Breasted Bronze (Kelly BBB; 540 toms, 540 hens) and British United Turkeys (B.U.T.) 6 and the Test Product 7 (TP 7; 780 hens) showed that 97.7% of the examined turkeys were diagnosed with different degrees of FPD. Only 4.6% of the toms and 1.3% of the hens had feet without lesions. Most frequent were necrotic lesions measuring up to 2 cm in diameter (64.3% of all turkeys). Extensive necrotic lesions of the foot pads (toms: 29.8%; hens: 12.4%) and necrosis of superficial scales (toms: 11.3%; hens: 7.6%) were less frequent. Plantar abscesses were rare findings (1.9%). In general, the feet of the Kelly BBB hens were more affected by foot pad lesions than those of the Kelly BBB toms. There were significant differences between the investigated flocks concerning the occurrence of foot pad lesions. The aim in rearing turkeys must be the reduction of FPD.

PI3K/Akt and Nrf2/HO-1 pathways involved in the hepatoprotective effect of verapamil against thioacetamide toxicity in rats.

Liver is a precious organ to maintain body life. Hepatotoxicity is a worldwide health problem that is still a challenge for research. Although countless pharmaceutical drugs and herbal compounds were screened for their hepatoprotective effects, the death from hepatotoxicity is increasing. Thus, there is continuous necessity of searching for the hepatoprotective effect of commonly used drugs. Accordingly, our aim was to examine a hepatoprotective potential for the antihypertensive drug, verapamil, and searching for new insights underlie its protective mechanism. Four groups of adult male rats were randomly arranged as controls, thioacetamide (TAA) hepatotoxic, and TAA + verapamil treated. Serum liver enzyme, hepatic antioxidant, lipid peroxidation, and inflammatory parameters were assessed. Gene relative expression for heme oxygenase-1 (HO-1), nuclear factor-erythroid 2-related factor 2 (Nrf2), phosphoinositide 3-kinase (PI3K), and serine/threonine-specific protein kinase (Akt) were quantified in hepatic tissue. TAA caused hepatic injury evident both histopathologically and biochemically by a decrease in all gene expressions. Verapamil alleviated the injury via its antioxidant and anti-inflammatory effects that were suggested to be via upregulation of the previous gene expressions. In conclusion, the calcium channel blocker, verapamil, that is used widely as antihypertensive exhibits a valuable hepatoprotective effect. The protection partially rests on activation of Nrf2/HO-1 and PI3K/Akt pathways.

Mechanisms underlying gastroprotective effect of paeonol against indomethacin-induced ulcer in rats.

Paeonol, a natural phenolic compound, possesses diverse beneficial effects including antioxidant and anti-inflammatory effects. Gastric ulcer is still the most prevalent irritant illness among the gastrointestinal diseases. The present study explored the protective effect of paeonol at two dose levels in indomethacin (IND)-induced gastric ulcer in rats. Forty-eight male Wistar rats were arranged into six groups: control, paeonol-treated, IND-treated, IND/paeonol (low and high doses)-treated, and ranitidine-treated groups. The oxidative status was evaluated by determining malondialdehyde level, superoxide dismutase activity, reduced glutathione content as well as hemoxygenase-1 (HO-1) gene expressions, and the antioxidant protein; NAD(P)H quinone oxidoreductase 1 (NQO1) immunostaining. The pro-inflammatory genes nuclear factor κB (NF-κB) and interleukin 1ß (IL-1ß) were estimated together with the proapoptotic gene of caspase 3. IND caused multiple gastric ulcers with evident oxidative damage and elevated pro-inflammatory and proapoptotic markers. Paeonol protected significantly, in a dose-dependent manner, the gastric mucosa from ulcerative lesion of IND similar to the reference drug ranitidine. Paeonol pretreatment diminished gastric oxidative stress and restored the gastric antioxidant capacity by elevating gastric gene expression of HO-1 and protein expression of NQO1. Paeonol also reduced NF-κB, IL-1ß, and caspase 3 gene expressions. In conclusion, paeonol offered a gastroprotection dependent on its antioxidant, anti-inflammatory, and antiapoptotic effects.

Investigations on aviadenoviruses isolated from turkey flocks in Germany.

During routine diagnosis in 2012, 69 samples of diseased turkey breeding and fattening flocks in Germany were examined for infection with aviadenoviruses by virus isolation using primary chicken embryo liver cells. In total, 21 aviadenovirus isolates, identified by a group-specific indirect immunofluorescence test, were obtained from 19 flocks. In almost all cases, molecular typing of these isolates based on partial hexon gene sequences revealed the presence of different types of turkey aviadenoviruses (TAdVs), including species Turkey aviadenovirus B (TAdV-B) with at least two different genotypes, as well as the species Turkey aviadenovirus C (TAdV-C) and Turkey aviadenovirus D (TAdV-D). Further analysis of DNA-dependent DNA polymerase gene sequences confirmed the classification of selected TAdV-C and TAdV-D isolates. Based on the results obtained for both genes, we suggest that TAdV-2, in addition to TAdV-4, belongs to the species TAdV-C. In contrast, amplification of the DNA polymerase gene fragment of nearly all investigated TAdV-B isolates failed due to unknown reasons. The results of sequence and phylogenetic analysis support the previously proposed classification of TAdVs into three different species and demonstrated how widely spread these viruses are in German turkey flocks. Analysis of case histories revealed a wide range of clinical and pathological changes; however an apparent link between types and disease conditions was not identified.

Introduction and enzootic of A/H5N1 in Egypt: Virus evolution, pathogenicity and vaccine efficacy ten years on.

It is almost a decade since the highly pathogenic H5N1 avian influenza virus (A/H5N1) of clade 2.2.1 was introduced to Egypt in 2005, most likely, via wild birds; marking the longest endemic status of influenza viruses in poultry outside Asia. The endemic A/H5N1 in Egypt still compromises the poultry industry, poses serious hazards to public health and threatens to become potentially pandemic. The control strategies adopted for A/H5N1 in Egyptian poultry using diverse vaccines in commercialized poultry neither eliminated the virus nor did they decrease its evolutionary rate. Several virus clades have evolved, a few of them disappeared and others prevailed. Disparate evolutionary traits in both birds and humans were manifested by accumulation of clade-specific mutations across viral genomes driven by a variety of selection pressures. Viruses in vaccinated poultry populations displayed higher mutation rates at the immunogenic epitopes, promoting viral escape and reducing vaccine efficiency. On the other hand, viruses isolated from humans displayed changes in the receptor binding domain, which increased the viral affinity to bind to human-type glycan receptors. Moreover, viral pathogenicity exhibited several patterns in different hosts. This review aims to provide an overview of the viral evolution, pathogenicity and vaccine efficacy of A/H5N1 in Egypt during the last ten years.

Antimicrobial Susceptibility Patterns of Enterococcus faecalis and Enterococcus faecium Isolated from Poultry Flocks in Germany.

Between 2010 and 2011, 145 Enterococcus isolates (Enterococcus faecalis, n = 127; Enterococcus faecium, n = 18) were collected during routine bacteriologic diagnostics from broilers, layers, and fattening turkeys in Germany showing various clinical signs. The susceptibility to 24 antimicrobial agents was investigated by broth microdilution test to determine minimum inhibitory concentrations (MICs). All E. faecalis isolates (n = 127) were susceptible to the beta-lactam antibiotics ampicillin, amoxicillin-clavulanic acid, and penicillin. Corresponding MIC with 50% inhibition (MIC50) and MIC with 90% inhibition (MIC90) values of these antimicrobial agents were at the lower end of the test range (≤ 4 µg/ml). In addition, no vancomycin-resistant enterococci (VRE) were found. High resistance rates were identified in both Enterococcus species for lincomycin (72%-99%) and tetracycline (67%-82%). Half or more than half of Enterococcus isolates were resistant to gentamicin (54%-72%) and the macrolide antibiotics erythromycin (44%-61%) and tylosin-tartate (44%-56%). Enterococcus faecalis isolated from fattening turkeys showed the highest prevalence of antimicrobial resistance compared to other poultry production systems. Eighty-nine out of 145 Enterococcus isolates were resistant to three or more antimicrobial classes. Again, turkeys stood out with 42 (8 1%) multiresistant isolates. The most-frequent resistance patterns of E. faecalis were gentamicin, lincomycin, and tetracycline in all poultry production systems.

Occurrence of vancomycin-resistant enterococci in turkey flocks.

In the present study, the prevalence of vancomycin-resistant enterococci (VRE) in turkeys in the southwest of Germany was investigated. For this purpose, 200 cloacal swab samples and 5 environmental dust samples (tested as a pooled sample) of each of the 20 flocks (10 female and 10 male flocks) included in this study were examined. The VRE could be isolated by means of a procedure combining bacterial cultivation in an enrichment broth and on a selective solid media. Enterococci were identified biochemically and subsequently tested on the presence of the vancomycin resistance genes vanA, vanB (B1/B2/B3), and vanC (C1/C2/C3) using real-time PCR assays. In 54 (27%) turkeys originating from 11 (55%) flocks and in 14 (70%) of the dust samples, exclusively vanA and vanC1 genes could be detected. Of the turkeys examined, 46 were colonized with VRE bearing the resistance gene vanC1 and 8 vanA, originating from 9 and 2 flocks, respectively. None of the birds carried vanB, vanC2, or vanC3 positive VRE. The results obtained from the birds are largely confirmed by the dust samples originating from 4 vanA and 10 vanC1 positive flocks. However, one flock housing animals colonized with vanC1 positive VRE could not be confirmed by the dust samples that revealed vanA bearing VRE. However, in one case vanA and in 3 cases vanC1 carrying VRE could be detected in dust samples of the turkey houses, but not in the turkeys of the associated flock. In 5 flocks the turkeys as well as the dust samples were free of VRE.

Persistence of Histomonas meleagridis in or on materials used in poultry houses.

Histomonas meleagridis is the causative agent of blackhead disease or histomonosis in turkeys, and previous research suggests that this parasite survives poorly outside of hosts except within heterakid nematodes. However, we investigated the viability of H. meleagridis in or on several artificially contaminated materials kept at ambient room temperature (22 +/- 2 C) to mimic the situation in the field. The protozoan survived for up to 1 hr on wood, rubber, and metal; up to 3 hr on egg-tray cartons, egg shells, and bricks; up to 6 hr on straw, turkey feathers, and feed; and up to 9 hr in nonchlorinated tap water and fecal matter. Therefore, contaminated water, fresh fecal matter, or both could play a role in transmission of the parasite within and among poultry houses rather than other materials tested in this study.

Prevalence of types of methicillin-resistant Staphylococcus aureus in turkey flocks and personnel attending the animals.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) have been isolated from a number of livestock species and persons involved in animal production. We investigated the prevalence of LA-MRSA in fattening turkeys and people living on farms that house fattening turkeys. Eighteen (90%) of 20 investigated flocks were positive for MRSA, and on 12 of the farms 22 (37·3%) of 59 persons sampled were positive for MRSA. People with frequent access to the stables were more likely to be positive for MRSA. In most flocks MRSA that could be assigned to clonal complex (CC) 398 were detected. In five flocks MRSA of spa-type t002 that is not related to CC398 were identified. Moreover, other methicillin-resistant Staphylococcus spp. were detected on 11 farms and in eight people working on the farms.

Polymelous layer chick displaying additional malformations of the hind gut: case report and in-depth review of related literature.

A case report of a male 6-day-old male layer chick featuring incomplete polymelia of the hind limbs and hindgut malformations is presented. The chick was submitted to computed tomography (CT) examination and subsequent anatomical dissection. Apart from the two supernumerary hind limbs, the anatomical dissection revealed additional hindgut alterations: three uniform-sized caeca flanked the ileum, and the rectum branched into paired cloacae. The supernumerary hind limbs were localized caudal to the normal hind limbs in an inverted position and were attached to pelvic girdle elements and to a curtate pygostyle. They featured a prominent unpaired femur besides paired tibiotarsi, tarsometatarsi and species-specific phalanges of the toes. Additionally, two separate bones attached to the caudoventral aspect of the regular hip bones were developed. The supernumerary limbs were in part mobile and received nerve and vascular supply. Digital 3D-reconstruction based on the CT datasets revealed the osseous components of the malformed body parts. The possible morphogenesis including an in-depth literature review and the clinical implications of the reported malformations are discussed.

Elucidation of colonization time and prevalence of thermophilic Campylobacter species during turkey rearing using multiplex polymerase chain reaction.

Two turkey flocks (male and female) and the environment of their house were investigated for the presence of thermophilic Campylobacter. Sample DNA was extracted directly from fecal material and environmental samples. Bacterial identification was done using a modified Campylobacter species specific multiplex PCR. The times needed for colonization and prevalence in male and female turkeys were determined independently. All environmental samples collected before restocking were negative in the PCR analysis, showing a good hygiene and biosecurity system. The first positive PCR results were obtained in drinking water samples at 6 d of age. Colonization occurred between the second and third week of age, starting in female birds and then followed by the males. Campylobacter jejuni was detected by multiplex PCR at first; later on, Campylobacter coli and mixtures of both were seen. After the 9 wk of age, the colonization of the flocks was completed. Great attention should be given to drinking water as a supposed source of Campylobacter contamination. Multiplex PCR proved to be a rapid, sensitive, and cheap tool for the diagnosis of Campylobacter contamination.

One-step RT-qPCR with an internal control system for the detection of turkey rotaviruses in faecal samples.

Turkey rotaviruses are one of the major pathogens responsible for the poult enteritis syndrome (PES). In this study a one step real-time reverse transcription polymerase chain reaction (RT-qPCR) assay targeting the rotaviral non-structural protein 4 (NSP4) was developed. The NSP4 is a highly conserved gene inside the turkey rotavirus genome and contains an internal control system to monitor any potential RT-qPCR inhibitors. The detection limit of the optimized NSP4-RT-qPCR assay ranged from 8.15 to 8.15 × 10(5) copy numbers. In total 149 faecal samples were collected from eight different flocks of commercial turkey farms. Faecal samples from hens and toms were collected separately at 2-week intervals from the 2nd week of age through the 16th and 20th week of age (age of slaughter for female and male, respectively) and tested. One farm reared only hens. The samples were tested previously using conventional RT-PCR targeting the same gene. When the conventional RT-PCR was compared with the developed NSP4-RT-qPCR, the results revealed that 11% of the samples of the conventional RT-PCR were false negative. The results indicate that this NSP4-RT-qPCR is highly sensitive for the detection of turkey rotaviruses in faeces. In addition, it could be suitable for the development of high-throughput screening.

Recurring histomonosis on an organic farm.

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

An overview of the epidemic of highly pathogenic H5N1 avian influenza virus in Egypt: epidemiology and control challenges.

Emergence of the highly pathogenic avian influenza (HPAI) H5N1 virus in Egypt in mid-February 2006 caused significant losses for the poultry industry and constituted a potential threat to public health. Since late 2007, there has been increasing evidence that stable lineages of H5N1 viruses are being established in chickens and humans in Egypt. The virus has been detected in wild, feral and zoo birds and recently was found in donkeys and pigs. Most of the outbreaks in poultry and humans occurred in the highly populated Nile delta. The temporal pattern of the virus has changed since 2009 with outbreaks now occurring in the warmer months of the year. Challenges to control of endemic disease in Egypt are discussed. For the foreseeable future, unless a global collaboration exists, HPAI H5N1 virus in Egypt will continue to compromise the poultry industry, endanger public health and pose a serious pandemic threat.

Unusual biphasic disease in domestic pigeons (Columba livia f. domestica) following experimental infection with Sarcocystis calchasi.

A novel Sarcocystis species has recently been reported in the domestic pigeon (Columba livia f. domestica) as intermediate host, causing severe central nervous signs similar to Paramyxovirus-1 or Salmonella Typhimurium var. cop. infection. Transmission of the parasite via the northern goshawk (Accipiter gentilis) as definitive host has been established. Experimental infection of domestic pigeons with sporocysts excreted by experimentally infected northern goshawks reproduced the natural infection in the pigeon, proving the causative role of the parasite in the disease. Here, we describe in greater detail the course of the fulminant biphasic disease depending on the infectious dose. Pigeons infected with 10(3) or 10(4) sporocysts showed clinical signs of polyuria and apathy around 10-11 days postinfection (dpi) and sudden neurological signs 51-57 dpi as a second phase of disease. Pigeons infected with higher doses died within 7-12 dpi, also showing polyuria and apathy but without nervous signs. At necropsy, livers and spleens had multifocal necroses and infestations with parasitic stages, namely, schizonts. Moreover, lesions and schizonts were also found in the lung, bone marrow, and next to blood vessels in the connective tissue of various organs. Pigeons infected with 102 sporocysts remained symptomless until 58-65 dpi, when sudden central nervous signs occurred. Major histopathologic findings of pigeons with neurological signs were encephalitis and myositis of virtually every skeletal muscle with high infestations of sarcocysts. Only mild myocarditis and very few cysts were found in the heart muscles. Importantly, a sentinel pigeon developed identical lesions when compared to those of low-dose infected pigeons, suggesting a risk of mechanical transmission of sporocysts from freshly infected to uninfected pigeons in a flock. By contrast, chickens failed to develop any clinical signs or pathologic lesions in the same experiment. The findings further characterize the new highly pathogenic disease in domestic pigeons, which clinically mimics paramyxovirosis and salmonellosis in both phases of the disease and exclude chickens as further intermediate host species.

Isolation of highly pathogenic avian influenza H5N1 from table eggs after vaccinal break in commercial layer flock.

In May 2009, during routine monitoring of a commercial layer flock of about 87,000 birds kept in cages in 4 different houses that had been vaccinated 3 times with an inactivated H5N1 vaccine at weeks 1, 7, and 16, highly pathogenic avian influenza (HPAI) virus of subtype H5N1 was isolated and detected by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in tracheal and cloacal swabs collected from houses 3 and 4; 7 days after onset of clinical signs, there was an increase in mortality accompanied by a decrease in egg production and egg quality. In addition, using RT-PCR, the viral RNA could be detected from albumin and eggshell as well. Seven days after the onset of the clinical signs, the hemagglutination inhibition (HI) titers in the affected houses were 3.2 and 1.9 log2. In the other two houses, there were no clinical signs, and all tested samples were negative using virus isolation and real-time RT-PCR. The HI titers were 6.6 and 7.0 log2 in nonaffected houses. The isolated virus from egg albumin showed high nucleotides and amino-acid identities and clustered with viruses from recently H5N1-confirmed human infections and poultry from different places in Egypt. Moreover, several amino-acid substitutions of viral H5 protein were observed. The vaccinal break seems to be associated with immune escape mutants and/or improper vaccination. The role of contaminated eggs as a source of infection and as a vehicle for spread of the virus should be considered in area with avian influenza outbreaks.

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

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

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

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