Molecular characterization of tetracycline and vancomycin-resistant Enterococcus faecium isolates from healthy dogs in Egypt: a public health threat.

BACKGROUND: Vancomycin-resistant enterococci (VRE) are among the most common causative pathogens for nosocomial infections worldwide. Moreover, strains of VRE have been isolated from several domestic livestock in Egypt. METHODS: This study examined if healthy dogs are a potential source of VRE infection by isolating and characterizing Enterococcus faecium strains from stool samples on a morphological basis and biochemical activities. Subsequently, it was confirmed by genotypic characterization using polymerase chain reaction (PCR), followed by the detection of antibiotic resistance genes, virulence determinants, and genes contributing to enterocin production by PCR. Furthermore, the phylogenetic relationships among vanB and tetL genes were analyzed. RESULTS: All ten fecal samples were identified as E. faecium and confirmed by PCR. In addition, 90% of the isolates tested were positive for the virulence genes gelE and esp, and all the isolates tested were positive for the antibiotic resistance genes tetL and vanB. Only three of the five enterocin genes examined were detected. Ent As-48, bacteriocin 31, and Ent L50 were identified in 100%, 80%, and 60% of the samples, respectively. CONCLUSION: Dogs should be regarded as a reservoir of E. faecium that carries vancomycin resistance and virulence determinants that may affect public health in Egypt, considering a "One Health" task force approach to restrict their spread.

Host Size Selection in the Ectoparasite Tachaea chinensis (Isopoda: Corallanidae) under Laboratory Conditions.

Tachaea chinensis is a temporary ectoparasite infesting freshwater shrimps and prawns in eastern Asia. This study investigated the host size selection by T. chinensis across common freshwater shrimps under laboratory conditions. A total of 70 isopods were allowed to select between host shrimps of different size and different species in pairwise selection experiments. In treatments involving different sizes of the same host species, T. chinensis tended to select the medium host option in all of the four treatments. Similarly, T. chinensis demonstrated greater preference towards medium host (90%) when provided with mixed host options (medium Palaemon paucidens vs small Neocaridina spp.). However, despite the increase in the infestation proportion on medium Neocaridina spp., the isopod significantly selected the small P. paucidens when provided with a choice between medium Neocaridina spp. and small P. paucidens. In manca stage (1 day after hatch) T. chinensis treatment, the isopods showed no specific preference between large and medium Neocaridina spp. These results suggest that T. chinensis is likely to show size specificity according to the developmental stage, a size specificity that ultimately ensures adequate space for isopods' growth while maintaining a minimum risk of predation.

Molecular characterization and immunohistochemical localization of tilapia piscidin 3 in response to Aeromonas hydrophila infection in Nile tilapia.

The antimicrobial activity of tilapia piscidin 3 (TP3) was determined in vitro against a locally isolated Aeromonas hydrophila. A 388 bp fragment was amplified from the TP3 cDNA and sequenced. The coding sequence (CDS) of TP3 was estimated to be 231 bp codes for 76 amino acids long and stop codon. In silico analysis was performed to detect both the signal peptide and the prodomain cleavage sites to follow the amino acids number 22 and 70, respectively. Based on this, a peptide 23 amino acids long with a remarkably high computed antimicrobial probability was synthesized and used in the subsequent experiments. The antimicrobial activity of TP3 was determined with minimum inhibitory concentration (MIC) and minim um bactericidal concentration (MBC) methods. TP3 exhibited relatively weak antimicrobial activities against the tested bacteria. A challenge experiment was then performed in Nile tilapia with low and high doses of A. hydrophila, followed by timely recognition; after 3, 6, 24 h, and 7 days of the specific TP3 gene expression, immunohistochemical localization was also performed. Histopathological examination revealed provoked inflammatory responses and congestion in the same organs of TP3 expression. Immunohistochemical localization showed that A. hydrophila induced tilapia fish to express TP3 after 24 h within the gills, intestine, hepatopancreas, spleen, and posterior kidney. In quantitative real time (RT)-polymerase chain reaction analysis, the high dose showed higher mRNA expression levels than the low dose, and its expression levels increased in the A. hydrophila-infected fish. It was therefore concluded that TP3 plays an essential role in fish immunity.

Characterization of the recent outbreak of foot-and-mouth disease virus serotype SAT2 in Egypt.

An outbreak of foot-and-mouth disease (FMD) in Egypt affected approximately 40,000 cattle and water buffaloes and killed more than 4,600 animals during February-March 2012. To investigate the etiology of the 2012 outbreak, specimens were collected from six governorates and analyzed using universal primers to amplify the 5' untranslated region (UTR) by reverse-transcription polymerase chain reaction. Only FMDV-SAT2 was detected, with an overall detection rate of 80.3 %. Complete VP1- and leader-proteinase-coding sequences, obtained from three isolates from three different governorates, were compared with previously reported sequences. Phylogenetic analysis of these sequences indicated that the circulating viruses were homogeneous and were closely related to topotype VII. Importantly, the newly emerged viruses were genetically closely related to strains isolated from Saudi Arabia, Sudan, Eritrea and Cameroon between 2000 and 2010, suggesting the dominant nature of this virus and underscoring the need for worldwide intensive surveillance to minimize its devastating consequences.

Host selection and potential predation in the host-parasite interaction between the isopod Tachaea chinensis and freshwater host species.

Tachaea chinensis is an ectoparasite commonly found on diverse ecologically and commercially valuable freshwater shrimps and prawns. Previous studies on this parasite have focused on its distribution and taxonomical identification, while its host preference and/or the potential predation in this host-parasite interaction remained poorly understood. In this study, we investigate the host preference and potential predation of the isopod T. chinensis using manipulative choice and predation experiments under laboratory settings. The preference toward a broad range of host decapods in single-host treatments, indicates a low host specificity, which ultimately aids in the survival of this parasite in the natural environment. Tachaea chinensis responded well to the shrimp Palaemon paucidens when presented with uncommon host species in all three treatments. In host-parasite predation treatments, all the tested P. paucidens shrimp, the prawn Macrobrachium nipponense, and the crayfish Procambarus clarkii were able to consume the isopod-especially the invasive crayfish P. clarkii, which consumed a greater percentage in a considerably shorter time frame (Fisher's exact test, P < 0.01). This study demonstrated for the first time the ability of larger freshwater decapods to prey upon T. chinensis. Despite the large difference in the maximum attainable size of those freshwater species, a high predation pressure by the invasive crayfish on the isopod is anticipated, if they are present in the same environment.

Morphomolecular identification and considerations of the infestation site adaptations of Pricea multae (Thoracocotylidae: Priceinae) from Scomberomorus commerson, off Arabian Gulf, Saudi Arabia.

Monogeneans Pricea multae naturally infested 42 of the 120 (35%) mackerel fish (Scomberomorus commerson) examined. For the first time, an infestation was discovered off the coast of Jubil in the Arabian Gulf of Saudi Arabia. Based on the structure clarified through light and electron microscopy of mounted specimens and molecular analysis of rDNA and measurements of this monogenean parasite was identified as P. multae. The tegumental surface of the parasite was characterized by tegumental ridges running transversally, generating folds in both the dorsal and ventral surfaces of the body at regular intervals. The study clarified the importance and function of the micro-structures, such as tegumental folds, perforations, sensory ganglia present on the parasite's surface, and the larger hamulus supported by a relatively unmodified internal spine. This monogenean parasite has adapted to its host infestation site uniquely.

Pathological and immunohistochemical studies following the experimental infection of ayu (Plecoglossus altivelis) by Edwardsiella ictaluri.

In recent decades, several mass mortalities were recorded in riverine ayu (Plecoglossus altivelis) in Tokyo Metropolis, Hiroshima Prefecture, and Yamaguchi Prefecture, Japan; in these outbreaks, microbiological and pathological examinations revealed Edwardsiella ictaluri as the causative agent. In this study, histopathological findings and immunohistochemical localization of the bacteria following experimental infection of ayu were discussed. Infection experiments were performed using 44 healthy cultured ayu fingerlings using E. ictaluri isolate (H90). The fish were injected with the isolate intraperitoneally with a dose of 5.1 × 105 cfu/fish, while the control fish were injected with sterile phosphate buffered saline. The fish were observed for clinical signs, with daily collection of dead fish, and isolation of bacteria from the posterior kidney was performed and confirmed to be E. ictaluri by slide agglutination using anti-PH0744 serum. Daily collection of five moribund fish for necropsy and tissue specimens collection from hepatopancreas, spleen, posterior kidney, gills, brain, heart, and intestine for histopathological and immunohistochemical examination. Post-mortem lesions were recorded as exophthalmia, bloody ascitis, hemorrhagic kidney and distended gallbladder, meningio-encephalitis, hemorrhagic vent, and petechial hemorrhages on viscera. Histopathological examination revealed diffuse severe congestion in blood vessels and several degenerative and necrotic changes inconcurrent with positive antigenic staining by immunohistochemistry.

Phylogeny and ultrastructure of Henneguya fusiformis Kostoïngué, Fall, Faye and Toguebaye 1999, a newly recorded species infecting the ovaries of Clarias gariepinus in Egypt.

The genus Henneguya is the second largest within the class Myxosporea, which infects marine and freshwater fish. One hundred Clarias gariepinus specimens were collected alive from a branch of the Nile River in Kafrelsheikh, Egypt. Microscopic and molecular procedures were used to describe how Henneguya fusiformis infects the ovaries of C. gariepinus. The infected fish showed no pathogenic changes except for macroscopic creamy whitish nodules in their ovaries with the highest prevalence during the spring season. The mature spores are spindle-shaped. The total spore length, spore body length and width are 53.4 ± 0.8 (52.5-54.3) µm, 29.8 ± 0.5 (29.2-30.4) µm and 6.5 ± 0.3 (6.1-6.9) µm, respectively. The spore anterior end consisted of two equal polar capsules, located in a tandem position, each one measuring 4.2 × 2.1 µm. The polar filament formed a coil with 6-8 turns. The measurement of the spore end with two extended processes was 24.3 ± 0.4 (23.9-24.8) µm. Phylogenetic analyses of the 18S ribosomal RNA gene sequence revealed that H. fusiformis are clustered together with other myxobolids that are histozoic in channel catfish, Ictalurus punctatus, and Asian Redtail catfish, Hemibagrus nemurus" (Clariidae) in the United States and Malaysia, respectively. To the best of our knowledge, this is the first record of H. fusiformis in Egypt. Additionally, our study is the first record of H. fusiformis in the ovaries of C. gariepinus.

Immunosuppressive effect of cyclophosphamide in Nile tilapia (Oreochromis niloticus).

Cyclophosphamide, a common chemotherapeutic and immune suppressor agent, is regularly used in research to weaken the immune system in laboratory animal models. Nile tilapia (Oreochromis niloticus) is a widely used experimental fish model for immune-modulatory research; with the lack of knowledge about the immune-compromised tilapia model, an urgent need is to develop and optimise such a model. Sixty healthy Oreochromis niloticus fish, average weight 50 ± 10 g, were divided into four experimental groups. Fish in group I, negative control group, were injected with phosphate-buffered saline only, and fish in groups II, III and IV were injected with cyclophosphamide (CP) at 100, 200 and 400 mg/kg body weight (BW), respectively, via the intraperitoneal route. Different immune-related parameters were investigated 3 weeks after CP injection. The results have revealed a significant decrease in total red blood cells (RBCs), white blood cells (WBCs) and thrombocyte counts and reduced haemoglobin and haematocrit values in CP-treated fish, especially those injected with 200 mg/kg BW compared with the control group (p < 0.05). Also, significantly lower levels of serum proteins (total protein, albumin, α1- and γ-globulins) were observed in CP-treated fish, especially those injected with 200 mg/kg BW in comparison with the control group (p < 0.05). Furthermore, CP-treated fish showed a reduction in the expression of immune-related genes (interleukin-1, and tumour necrosis factor-α in spleen and tumour necrosis factor-α and transforming growth factor ß-1 in head-kidney), especially those injected with 200 mg/kg BW compared with the control group (p < 0.05). In conclusion, the Oreochromis niloticus immune-suppressed model can be induced by intraperitoneal CP injection at 200 mg/kg BW.

Isolation, identification and virulence determinants of Streptococcus agalactiae from bovine subclinical mastitis in Egypt.

INTRODUCTION: This study aimed to investigate the prevalence of contagious mastitis caused by Streptococcus agalctiae (S. agalactiae) in cattle from households and small-scale dairy farms in Egypt. Molecular characterization of S.agalactiae isolates was described including the genetic determinants of virulence to assess the genetic variation in isolated strains of S. agalactiae. METHODOLOGY: Three hundred and sixty milk samples were collected from 90 apparently healthy dairy cows randomly selected from household and small-scale dairy farms were examined by Somatic Cell Count (SCC) as an indicator for subclinical mastitis. S.agalactiae isolates were bacteriologically and molecularly identified followed by identification of virulence genes using PCR. RESULTS: A total of 172 milk samples (47.77%) were positive with SCC > 200×103/ml. Bacteriological examination of the positive SCC milk samples revealed that 28 (16.28%) of the isolates were S.agalactiae. Molecular examination using PCR confirmed only 22 isolates of S. agalactiae (12.8%). Moreover, we used the pattern of virulence genes to address the genetic variation of S. agalactiae strains isolated from cases of contagious mastitis in cattle in Egypt. Virulence genes hylB, cylE, iagA, and bac were determined in 100%, 68.2%, 13.6% and 100% of isolates respectively. CONCLUSIONS: The use of molecular methods for the identification of the causative agent in mastitis confirmed that, in Egypt, Streptococcus agalactiae is considered as one of the predominant infectious agents among contagious mastitis causing pathogens. The pattern of virulence genes presented the genetic diversity of highly virulent S. agalactiae strains isolated from cases of contagious mastitis in cattle in Egypt.

Comparative immunohistological study on using capsaicin, piperine, and okadaic acid for the transepithelial passage of the inactivated viral and bacterial vaccines in fish.

The practical difficulty of parenteral application of fish vaccines against devastating fish diseases diverted the interest toward oral vaccination. Search for effective methods to enhance the oral uptake of viral and bacterial vaccines is continuing. The current research focus on a new role of mucosal fish vaccine adjuvants inducing the antigen uptake by enhancing vascularity or increasing intestinal permeability. Some inflammatory substances cause reversible pathology to the intestinal epithelium, which could be employed for the transepithelial passage of vaccine particles. The natural inflammatory substances used were capsaicin, piperine, and okadaic acid as 1 mg, 2 mg, and 1 µg/fish, respectively. Two inactivated vaccines were used as antigens to test the effect of these inflammatory substances in two different fish hosts. Tested vaccines were inactivated redspotted grouper nervous necrosis virus vaccine in sevenband grouper (Epinephelus septemfasciatus) and inactivated Edwardsiella tarda vaccine in red sea bream (Pagrus major) fish models. The inflammatory substances and each vaccine were anally intubated to fish. Capsaicin proved to be effectively aiding the transepithelial passage of vaccine particles more than piperine, while okadaic acid had no detectable effect.

Piscidin 4: Genetic expression and comparative immunolocalization in Nile tilapia (Oreochromis niloticus) following challenge using different local bacterial strains.

The antimicrobial activity of tilapia piscidin 4 (TP4) was determined in vitro against four bacterial strains, Aeromonas hydrophilla, Pseudomonas fluorescens, Streptococcus iniae and Vibrio anguillarum. Nile tilapia were infected with low and high doses of the tested pathogens; after 3, 6, 24 h and 7 days of the specific TP4 gene expression, tissue immunolocalization was also performed. Histopathological examination revealed septicaemia and necrosis of hemopoietic tissue for all of the tested bacteria. Immunolocalization showed abundance in S. iniae-infected fish tissues. Quantitative RT-PCR analysis revealed that high doses raised mRNA expression levels compared to low doses and expression levels increased in the infected fish, particularly after 24 h, indicating that TP4 exerts potent bactericidal activity against some fish pathogens and plays an essential role in fish immunity.

Seroprevalence, isolation, molecular detection and genetic diversity of Toxoplasma gondii from small ruminants in Egypt.

Toxoplasmosis is an infectious zoonotic disease caused by protozoan Toxoplasma gondii. Detection of T. gondii infection with touchy and particular strategies is a key advance to control and prevent toxoplasmosis. Genotyping can explain the virulence, epidemiology and setting up new methodologies for diagnosis and control in human and animals. The point of this study was to assess the seroprevalence of T. gondii in sheep and goat in Egypt and to comprehend the genetic variety of T. gondii isolates circling in Egypt. Blood samples were gathered from 113 ewes and 95 she-goats from three Egyptian governorates (Cairo, Giza and Al-Sharkia). Also blood and tissue samples were gathered from 193 sheep and 51 goats from Cairo and Giza abattoirs. All samples were assayed serologically utilizing ELISA and OnSite Toxo IgG/IgM Rapid test cassettes (OTRT) tests and the tissue samples of the seropositive animals were digested and microscopically examined then bio-assayed in mice as viability test. All the T. gondii isolates undergo molecular identification using PCR and genotyped utilizing nPCR/RFLP analysis of SAG2 gene. The total seropositivity of live sheep and goat was 47.15 and 39.2% utilizing ELISA and OTRT respectively. Concerning abattoirs, seropositivity, positive microscopic examination, mice viability from sheep samples were 47.1%, 37.3% and 44.1% respectively while that of goats were 45.5%, 33.3% and 48.6% respectively. Eighteen T. gondii isolates were affirmed utilizing PCR. Genotyping confirmed 10 isolates (55.5%) as type II, 6 (33.3%) as type III and 2 (11.1%) as atypical genotypes. Type II and III are the genotypes mostly circling among small ruminants in Egypt and this is most significance for the public health in Egypt.