A common Salmonella Enteritidis sequence type from poultry and human gastroenteritis in Ibagué, Colombia / Un tipo de secuencia común de Salmonella Enteritidis de origen aviar y de humano con gastroenteritis en Ibagué, Colombia

Salmonella Enteritidis es una de las mayores causas de salmonelosis en el mundo, siendo los huevos contaminados y la carne de pollo cruda sus principales fuentes de infección. En Ibagué, Colombia, se identificaron los principales serovares circulando en granjas, superficies de huevos y canales de pollo, sin embargo, se desconoce si esos serovares son responsables de gastroenteritis. Objetivo. Evaluar la relación genética entre aislamientos de Salmonella Enteritidis de aves de corral y humanos con gastroenteritis mediante multilocus sequence typing (MLST). Materiales y métodos. Se aisló Salmonella spp., de casos clínicos de gastroenteritis (n=110). Se realizó test de sensibilidad antibiótica, seguido de serotipificación y tipificación por medio de MLST y se comparó S. Enteritidis de humanos frente a S. Enteritidis de granjas ponedoras y de huevo comercializado (n=6). Resultados. Se aislaron 10 cepas de Salmonella spp., a partir de heces de humanos con gastroenteritis. Se obtuvo una prevalencia de Salmonella spp. de 9.09%, siendo S. Enteritidis (n=4), S. Typhymurium (n=2), S. Newport (n=1), S. Grupensis (n=1), S. Uganda (n=1) y S. Braenderup (n=1) los serotipos presentes en pacientes con gastroenteritis. El MLST indico que un tipo de secuencia común (ST11) de S. Enteritidis estuvo presente en todas las tres fuentes y mostraron el mismo patrón de resistencia antibiótica. Conclusión. S. Enteritidis ST11 constituye un vínculo entre el consumo/manipulación de huevos contaminados y gastroenteritis humana en Ibagué. Son necesarios estudios complementarios para conocer si otros serovares de Salmonella aislados de carne de pollo cruda también se asocian con la gastroenteritis humana.

Introducción. Salmonella Enteritidis es una de las mayores causas de salmonelosis en el mundo, siendo los huevos contaminados y la carne de pollo cruda sus principales fuentes de infección. En Ibagué, Colombia, se identificaron los principales serovares circulando en granjas, superficies de huevos y canales de pollo, sin embargo, se desconoce si esos serovares son responsables de gastroenteritis. Objetivo. Evaluar la relación genética entre aislamientos de Salmonella Enteritidis de aves de corral y humanos con gastroenteritis mediante multilocus sequence typing (MLST). Materiales y métodos. Se aisló Salmonella spp., de casos clínicos de gastroenteritis (n=110). Se realizó test de sensibilidad antibiótica, seguido de serotipificación y tipificación por medio de MLST y se comparó S. Enteritidis de humanos frente a S. Enteritidis de granjas ponedoras y de huevo comercializado (n=6). Resultados. Se aislaron 10 cepas de Salmonella spp., a partir de heces de humanos con gastroenteritis. Se obtuvo una prevalencia de Salmonella spp. de 9.09%, siendo S. Enteritidis (n=4), S. Typhymurium (n=2), S. Newport (n=1), S. Grupensis (n=1), S. Uganda (n=1) y S. Braenderup (n=1) los serotipos presentes en pacientes con gastroenteritis. El MLST indico que un tipo de secuencia común (ST11) de S. Enteritidis estuvo presente en todas las tres fuentes y mostraron el mismo patrón de resistencia antibiótica. Conclusión. S. Enteritidis ST11 constituye un vínculo entre el consumo/manipulación de huevos contaminados y gastroenteritis humana en Ibagué. Son necesarios estudios complementarios para conocer si otros serovares de Salmonella aislados de carne de pollo cruda también se asocian con la gastroenteritis humana.

Characterization of Salmonella from Commercial Egg-Laying Hen Farms in a Central Region of Colombia.

Salmonellosis affects humans more frequently than any other foodborne disease, and it causes severe economic losses in the poultry industry. A cross-sectional study was carried out to estimate the prevalence of Salmonella spp. in laying hen farms in the Tolima region of Colombia. Fifteen egg-laying hen farms were sampled, and a total of 589 samples were cultured to isolate Salmonella spp. A total of 14 isolates of Salmonella spp. were recovered from five farms, resulting in a prevalence of 33.33% (95%, confidence interval = 14%-53%) at the farm level. Salmonella spp. were recovered from eggshells (57.15%, n = 8), feed (28.57%, n = 4), and environmental samples (14.29%, n = 2). Farm practices, such as the milling of feed (odds ratio [OR] = 24) and the storage of eggs in the henhouses (OR = 11.25), in addition to the feed type (OR = 7.64) and the use of bamboo for construction of the facility (OR = 5.24), were identified as risk factors for Salmonella spp. The 14 isolates were identified as Salmonella Enteritidis (n = 6) and Salmonella Shannon (n = 8), and both serovars were resistant to a number of antibiotics. Pulsed-field gel electrophoresis presented three different XbaI macrorestriction patterns. The Salmonella Enteritidis isolates all presented a single pattern, whereas the Salmonella Shannon isolates were grouped into two distinct patterns. The results indicate that Salmonella spp. could be recovered from various sources at laying hen farms, and eggshell contamination is a particular concern.

Piscirickettsia-like organisms as a cause of acute necrotic lesions in Colombian tilapia larvae.

Rickettsial organisms are well-known fish pathogens in both natural and culture environments. This study reports an outbreak of disease in red tilapia larvae caused by piscirickettsia-like organisms (PLOs), which lasted from June until October 2009. Severe mortality was recorded almost exclusively in larvae and postlarvae aged 1-22 days old. Although clinical or gross findings were not evident in diseased fish, histopathology revealed severe necrosis of the epidermis and gill epithelium, with concomitant changes in the underlying skeletal muscle as being the most relevant microscopic lesions. Although PLOs were visible with the routine hematoxylin eosin technique, they were better observed with Giemsa and toluidine blue stains. Transmission electron microscopy revealed that the bacterium was located within the cytoplasm and phagolysosoma-like structures of epithelial cells from the gills and the skin. The bacteria measured 0.9 ± 0.2 µm × 2.1 ± 0.6 µm and had a double cell membrane (the outer one having undulating projections), with variable electron-dense and electron-lucent areas. Ultrastructurally, abundant myelin figures surrounded the microorganisms within host cell cytoplasm. Results indicated that Piscirickettsia-like organisms can cause massive epithelial cell damage associated with concomitant alteration of the electrolyte balance.

Production and efficacy of an Aeromonas hydrophila recombinant S-layer protein vaccine for fish.

A recombinant protein for the S-layer protein of Aeromonas hydrophila was produced and its ability to protect common carp Cyprinus carpio L. against six virulent isolates of A. hydrophila was assessed. A group of 120 carp (30-40 g) were vaccinated intra-peritoneally with 0.1 ml of adjuvanted vaccine (30 microg protein per fish). Another group of 120 carp were injected with 0.1 ml of PBS-adjuvant mixture to serve as controls. Twenty fish from each group were challenged with each one of six virulent isolates of A. hydrophila 35 days post-vaccination. The fish were maintained in 12 separate tanks before terminating the experiment at 16 days post-challenge. The relative percentage survival (RPS) for the six isolates of A. hydrophila ranged from 56 to 87%. The difference in survival rate of fish challenged with four of the isolates was statistically significant in vaccinated fish compared to control fish, when analysed using a Chi-square test. The results of the study suggest that the recombinant S-layer protein of A. hydrophila could be useful as a vaccine antigen to protect fish against different isolates of this pathogenic bacterium.

Enhanced survival of shrimp, Penaeus (Marsupenaeus) japonicus from white spot syndrome disease after oral administration of recombinant VP28 expressed in Brevibacillus brevis.

White spot syndrome virus (WSSV) disease is a major threat to shrimp culture worldwide. Here, we assessed the efficacy of the oral administration of purified recombinant VP28, an envelope protein of WSSV, expressed in a Gram-positive bacterium, Brevibacillus brevis, in providing protection in shrimp, Penaeus japonicus, upon challenge with WSSV. Juvenile shrimp (2-3g in body weight) fed with pellets containing purified recombinant VP28 (50microg/shrimp) for 2 weeks showed significantly higher survival rates than control groups when challenged with the virus at 3 days after the last day of feeding. However, when shrimp were challenged 2 weeks after the last day of feeding, survival rates decreased (33.4% and 24.93%, respectively). Survival rate was dose-dependent, increasing from 60.7 to 80.3% as the dose increased from 1 to 50microg/shrimp. At a dose of 50microg/shrimp, the recombinant protein provided protection as soon as 1 day after feeding (72.5% survival). Similar results were obtained with larger-sized shrimp. These results show that recombinant VP28 expressed in a Gram-positive bacterium is a potential oral vaccine against WSSV.

A soluble nonglycosylated recombinant infectious hematopoietic necrosis virus (IHNV) G-protein induces IFNs in rainbow trout (Oncorhynchus mykiss).

Viral glycoproteins interact with cell-surface receptors to mediate virus entry and innate immune system activation. We found that a soluble recombinant infectious hematopoietic necrosis virus G-protein (rIHNV-G) stimulated an early innate immune response mediated by proinflammatory cytokines, IFN1 and IFN-gamma in rainbow trout (Oncorhynchus mykiss) fry. Expression of both IFN1 and IFN-gamma mRNA transcripts was an early event and was rIHNV-G dose-dependent. In addition, preliminary evidence revealed that the innate immune response induced by rIHNV-G protein could protect rainbow trout fry from a subsequent IHNV virus challenge. Finally, the binding and distribution of FITC-rIHNV-G protein on rainbow trout spleen and head kidney leukocytes resemble morphological changes which occur on the cell membrane during antigen-receptor interaction including membrane reorganization, patching, polarization and capping. Thus a soluble nonglycosylated rIHNV-G protein could mediate the activation of rainbow trout leukocytes, with concomitant production of proinflammatory cytokines and IFNs.

Innate immunomodulation with recombinant interferon-alpha enhances resistance of rainbow trout (Oncorhynchus mykiss) to infectious hematopoietic necrosis virus.

We examined the in vivo immunostimulatory effects of a recombinant Atlantic salmon (Salmo salar) interferon-alpha2 (rSasaIFN-alpha2). The mature rSasaIFN-alpha2, expressed and purified from Escherichia coli, was administered to rainbow trout (Oncorhynchus mykiss) via the oral, immersion, or intraperitoneal (IP) injection route. Injection of rSasaIFN-alpha2 at 0.1microg/g fish gave significantly greater protection than a phosphate buffered saline (PBS) injection against a lethal challenge of infectious hematopoietic necrosis virus (IHNV), with a relative percent survival of 39%. Relative percent survival (RPS) increased significantly to 92% when the fish were injected with rSasaIFN-alpha2 at 1microg/g fish. Antiviral protection was evident for up to 7 days post-injection of rSasaIFN-alpha2. Administration of rSasaIFN-alpha2 by the oral or immersion route was not protective, and the fish succumbed to virus infection. The level of systemic IFN-induced expression of the Mx1 gene was significantly greater (p<0.01) in the IFN-injected group than in the PBS-injected group, and this was correlated with the fish survival rates in the challenge study. We used relative quantitative real-time polymerase chain reactions to examine the systemic expression of several other IFN-induced genes (including genes for IFN1, IFN regulatory factors 1 and 2, MHC-I, STAT1, vig-1, and GBP) and found that their expression was significantly increased 1-day post-rSasaIFN-alpha2 injection. Expression of the IFN-gamma and interleukin-1beta genes was not significantly increased. Thus, a salmonid rIFN-alpha can modulate the innate immune response of rainbow trout and mediate early antiviral protection against IHNV.

Biological characterisation of a recombinant Atlantic salmon type I interferon synthesized in Escherichia coli.

Type I (alpha/beta) interferons (IFNs) are a family of cytokines that stimulate the expression of numerous proteins that mediate an antiviral state in uninfected cells. Two Atlantic salmon (Salmo salar) IFN-alpha (SasaIFN-alpha1 & 2) genes have previously been cloned and both were found to contain a putative N-linked glycosylation site. Recombinant SasaIFN-alpha1 (rSasaIFN-alpha1) produced in eukaryotic systems has repeatedly been shown to confer antiviral properties. However, different IFN-alpha subtypes may exhibit differential antiviral activities and be subject to glycosylation. To evaluate the potential therapeutic impact of a rSasaIFN-alpha, the mature form of the SasaIFN-alpha2 protein was produced in a high-level Escherichia coli expression system. Expression of the rSasaIFN-alpha2 was detected by SDS-PAGE and Western blot, and its identity was confirmed by mass spectrometry. In the homologous Chinook salmon embryonic (CHSE-214) cell line, the rSasaIFN-alpha2 incited early expression of the IFN-induced Mx protein and exhibited high antiviral activity of about 2.8 x 10(6) U mg(-1) against infectious pancreatic necrosis virus (IPNV). Conversely, antiviral protection by rSasaIFN-alpha2 was not observed in a heterologous Japanese flounder embryo (HINAE) cell line. Hence, a biologically active form of rSasaIFN-alpha2 was successfully produced using a glycosylation-deficient prokaryotic system and purified to homogeneity, suggesting that glycosylation is not required for its antiviral activity.

Genetic loci of major antigenic protein genes of Edwardsiella tarda.

Seven antigenic proteins of Edwardsiella tarda were identified by using a rabbit polyclonal antiserum. Four of these proteins also reacted with a Japanese flounder antiserum. The amino acid sequences had identity to lipoproteins, periplasmic proteins, and exported and secreted proteins with roles in transport of metabolites across the cell membrane, stress response, and motility. These genes and their products are useful for developing DNA or recombinant subunit vaccines to control edwardsiellosis.