Production of an Antibody Fragment (scFv) Targeting PcrV Protein of Pseudomonas aeruginosa in Fed-Batch Cultivation Mode

Background: Pseudomonas aeruginosa is one of the opportunistic pathogens causing frequent hospital-acquired life-threatening infections in mechanically ventilated patients. The most significant virulence factor of P. aeruginosa is T3SS. PcrV is an important structural protein of the T3SS. Methods:In the current investigation, a recombinant scFv mAb against the PcrV protein was expressed in EnBase® (fed-batch) cultivation mode. The pETiteTM N-His SUMO Kan vector, including anti-PcrV scFv gene, was transformed into Escherichia coli (BL21) cells. The expression and solubility of anti-PcrV scFv protein were investigated at two different temperatures (25 °C and 30 °C) and at different induction times (4, 6, 8, 12, and 24 hours). Results:Increased efficiency was achieved by EnBase® compared to LB broth; owing to the slow release of glucose, the maximum level of solubility and total protein expression was observed in EnBase® cultivation system at 30 °C and 24 h post induction. Furthermore, IC₅₀ for anti-PcrV scFv protein was determined to be approximately 7 μg/mL. Conclusion:Anti-PcrV scFv produced in this study showed promising in vitro results, protecting RBC from lysis by P. aeruginosa (exoU+). Key Words: Fed Batch, recombinant protein, Pseudomonas aeruginosa, scFv     

Prevalence of Bovine Viral Diarrhoea Virus antibodies among the industrial dairy cattle herds in suburb of Mashhad-Iran

Mashhad is a major dairy production in Iran. The subject of this study was to survey the seroprevalence of Bovine Viral Diarrhea Virus (BVDV) infection using an indirect Enzyme-linked immunosorbent assay (ELISA) test in industrial dairy cattle herds in suburb of Mashhad-Iran. Totally, 141 serum samples were tested. None of the herds had been vaccinated against BVDV. Commercial indirect ELISA kit was used. The herds divided to 3 sizes as cow population. They were included: small, medium and large herds. Data were analyzed using Chi-square test. Ninety-seven (68.79%) cows were ELISA seropositive. However, the true BVDV seroprevalence was 72.25%. All of the herds were antibody positive against BVDV. The prevalence ranged from 66 to 100% within the herds. There were no significant differences between the presence of antibodies to BVDV and the herd size (P > 0.05). The prevalence in animals lower than 2 years old differed significantly with cows higher than 2 years old (P < 0.05). According to the results, it is concluded that it is likely the presence of persistently infection (PI) animal(s) within the herds in suburb of Mashhad-Iran, which is responsible for the presence antibody.     

Induction of Immunogenic Response in BALB/c Mice by Live and Killed Form of Recombinant Lactococcus lactis Displaying EG95 of Echinococcus granulosus

Background:CE is a zoonotic parasitic infection caused by Echinococcus granulosus worldwide and is associated with economic losses among livestock animals. EG95 is an immunogenic antigen from the E. granulosus. Lactococcus lactis has been prested as a safe vehicle for antigen delivery. The goal of this study was to design a novel L. lactis strain displaying EG95 as a vaccine delivery system. Methods:The eg95 encoding gene fragment fused to the M6 anchoring protein was cloned into the pNZ7021 vector, and L. lactis NZ9000 displaying recombinant EG95 was constructed. The expression of an approximately 32-kDa EG95 protein was confirmed by Western blotting and immunofluorescence analysis. The immune responses were evaluated in BALB/c mice immunized orally and subcutaneously with the live and killed recombinant L. lactis, respectively. Results:Total IgG level in mice immunized with heat-killed recombinant L. lactis (pNZ7021-eg95) significantly increased compared to the control group. sIgA was significantly higher in mice received live recombinant L. lactis (pNZ7021-eg95) compared to the control mice. Splenic lymphocytes from immunized mice represented the high levels of IFN-γ and the low-levels of IL-4 and IL-10. Conclusion:Our results indicate that immunization with EG95-expressing L. lactis can induce both specific humoral and cellular immune responses in mice. Key Words: Echinococcus granulosus, Lactococcus lactis, Immunization, Vaccines     

Effect of dietary GroBiotic®‐A supplementation as a prebiotic on the intestinal microflora,growth performance,haemato‐serological parameters,survival rate and body composition in juvenile beluga (Huso huso Linnaeus, 1754)

The objectives of the present study were to investigate of GroBiotic^®‐A (GBA) on growth, autochthonous intestinal microbiota and haemato‐serological parameters of beluga juvenile. A total of 180 fish (40.82 ± 5.81 g) were fed diets containing graded levels of GBA (0, 5, 10 and 20 g kg^?1 diet) for 8 weeks. No significantly differences in body composition, total viable aerobic bacteria, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, neutrophils, eosinophils, monocytes, albumin, glucose, triglyceride, cholesterol, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and lactate dehydrogenase were detected between treatment groups. The fish fed on 10 and 20 g kg^?1 GBA significantly showed higher lactic acid bacteria, final weight, body weight increases, weight gain and lower feed conversion ratio compared with the control and 5 g kg^?1 groups. The group fed 20 g kg^?1 GBA showed a highly significant difference in condition factor, survival rate, final length, total red and white blood cells, lymphocytes, haematocrit, haemoglobin, total protein, total immunoglobulin and lysozyme activity. The specific growth rate of the treatment groups was significantly elevated compared with the control groups. These results indicated that GBA at level 20 g kg^?1 improved growth, welfare and survival of beluga juvenile.     

Comparison of the effects of gonadotropin-releasing hormone, human chorionic gonadotropin or progesterone on pregnancy per artificial insemination in repeat-breeder dairy cows

Three different treatments were compared to improve pregnancy per artificial insemination (P/AI) in repeat-breeder (RB) dairy cows. All cows (n = 103) were assigned to one of four groups: (1) gonadotropin-releasing hormone (GnRH); (2) human chorionic gonadotropin (hCG); (3) once-used controlled internal drug release (CIDR) device; and (4) control. All treatments performed 5-6 days after artificial insemination (AI) and milk samples were collected just before treatment for progesterone assays. There were no significant differences in milk fat progesterone concentration among trial groups. Cows were observed for estrus signs thrice daily. Pregnancy per AI on day 45 in hCG and CIDR groups were significantly higher than GnRH and control groups (60.0% and 56.0% vs. 26.9% and 29.6%, respectively), but there were no differences in P/AI between GnRH and control groups. There were also no significant differences between hCG and CIDR groups. Milk fat progesterone concentrations were compared between pregnant and non-pregnant cows in each group and only in the hCG group it was significantly lower in pregnant cows. In conclusion, treating repeat-breeder cows with hCG or once-used CIDR 5-6 days after AI improved P/AI.     

Dietary sour lemon (Citrus limon) peel essential oil supplementation for reduction of deltamethrin‐induced stress in rainbow trout (Oncorhynchus mykiss)

The purpose of this study was to investigate the dietary effects of using essential oil from sour lemon peel (Citrus limon) on growth performance, carcass composition, blood and serum parameters, and liver enzymes of juvenile rainbow trout (Oncorhynchus mykiss) exposed to deltamethrin (DMN) pesticide. Treatments with no lemon peel essential oil (LPEO) and without DMN (control), as well as four treatments with 10% of 96‐h lethal concentration of DMN were prepared with dietary supplementation of LPEO (zero, 200, 400, and 600 mg/kg). Trout juveniles (95.12 ± 0.53 g) were randomly allocated to the treatments (each with three replicates). The results showed that dietary LPEO inclusion improved fish growth parameters in the DMN‐containing treatments. The highest fat content was found in zero LPEO treatment with no DMN while it was lowest in DMN treatments containing 400 and 600 mg/kg of LPEO. Carcass protein content was the highest in DMN treatment with 400 mg/kg of LPEO. Blood and serum biochemical parameters showed statistical differences between no LPEO treatment with DMN exposure and those detected in the control fish (p < .05). Overall, the results indicate that dietary inclusion of LPEO at 400 mg/kg level can decrease some negative effects of DMN exposure.     

Effects of supplementary fennel (Foeniculum vulgare) essential oil in diet on growth and reproductive performance of the ornamental fish,Convict cichlid (Cichlasoma nigrofasciatum)

This study evaluated the effects of diet containing 0 (C: control), 75 (D₁), 100 (D₂), 125 (D₃) and 150 (D₄) mg kg^?1 F. vulgare essential oil on growth and reproductive performance of C. nigrofasciatum. A total of 225 Convict cichlids female with mean weight (1.65 ± 0.02 g) were distributed into 15 glass aquaria (15 fish tank^?1) in triplicate treatments. During 40 days of experimental period, fish fed at 3% of their body weight daily. At the end of experiment, growth indices, gonadosomatic index and reproductive indices were determined. The best feed conversion ratio was obtained in D₄ (1.19 ± 0.03, P < 0.05). The addition of F. vulgare essential oil did not have any statistical effects on other growth indices (specific growth rate, weight gain and condition factor). Total survival rates in all treatments and control were uniformly high, ranging from 93% to 97% (P > 0.05). F. vulgare essential oil has no negative effect on survival rate of C. nigrofasciatum. There were no significant differences among protein content of fish body (P > 0.05). The lowest value of fat content (26.5 ± 0.5%) was observed in D_2, and it had significant difference with control and other groups (P < 0.05). The ash percentage in treatments fed with F. vulgare essential oil were higher than control fish (P < 0.05). The highest moisture content was detected in D₄ (72.68 ± 0.19%) and D₂ (71.23 ± 2%) groups. Among the used dosage, D₄ was the most effective dosage that could significantly increase GSI (11.06 ± 1.55%), fecundity (340 ± 21) and hatching ratio (92.33 ± 1.63%). There were no significant differences in diameter of eggs among various treatments (P > 0.05).     

Age-Specific Gastric Cancer Risk Indicated by the Combination of Helicobacter pylori Sero-Status and Serum Pepsinogen Levels

Background:

Serologic screening of gastric cancer (GC) by serum pepsinogens (sPG) levels and Helicobacter pylori (Hp) sero-status, though highly informative, has provided heterogeneous results. Here, we have evaluated the modifying effects of demographic factors on the risk impact of Hp sero-status/sPG levels in gastric cancer, with particular emphasis on age.

Methods:

A cross-sectional study was carried out on 1341 individuals (GC = 578, healthy = 763), who were stratified into two age groups: 35-59 years (middle-aged, n = 830) and ≥ 60 years (60 years-plus, n = 511). Demographic factors and serological states (Hp sero-staus and sPG levels) were recorded by subject interview and serum ELISAs, respectively. Covariate-specific odds ratios were calculated by multivariable logistic regression.

Results:

Hp infection was consistently associated with increased sPGI and sPGII levels in the 60 year-plus, but not the middle-aged group. The joint examination of the variable states of the three serum biomarkers (Hp serology, sPGI, and sPGI/II ratio), in the 60 year-plus age group, demonstrated a stepwise escalation of risk from the single (sPGI_low; OR = 2.6), to double (sPGI_low/sPGI/II_low; OR = 3.55, and Hp_positive/sPGI_low; OR = 5.0) and ultimately triple (Hp_positive/PGI_low/PGI/II_low; OR = 10.48) positive states, in reference to the triple negatives. However, this pattern was not exhibited in the middle-aged subjects.

Conclusion:

Age was clearly identified as a modifying factor on the risk projection of the combined states of Hp serology and sPG levels in gastric cancer screening, reflected by the augmented (~10.5 fold) risk of GC in the triple positive (Hp_positive/sPGI_low/sPGI/II_low) 60 year-plus subjects, which was not evident in the middle-aged group. Key Words: Biomarkers, Demography, Age Distribution     

Effects of Pediococcus pentosaceus supplementation on growth performance,intestinal microflora and disease resistance of white shrimp,Litopenaeus vannamei

Litopenaeus vannamei is economically important shrimp species in worldwide aquaculture. This study was conducted to assess the effect of different levels of probiotic Pediococcus pentosaceus (PP) on growth performance, feed utilization, digestive enzyme activity, intestinal microflora count and body composition of L. vannamei. Four diets containing different concentrations [0 (PP0), 10⁶ (PPI), 10⁷ (PPII) and 10⁸ (PPIII) CFU/g] of PP were formulated. After 8 weeks feeding trial, the experimental shrimps were challenged with Vibrio anguillarum and noted the surveillance. At the end of the feeding trial, the obtained results revealed a significant increase (p < .05) in final body weight, final length, weight gain (WG), survival rate, protease and amylase activities, lactobacillus sp. and Bacillus sp. intestinal count, total haemocyte counts (THC) and lysozyme activity in PPIII group when compared with the other groups. Similarly, WG, amylase activity, Bacillus sp. count, THC and lysozyme activity were significantly enhanced in case of PPII compared to the control group (p < .05). Interestingly, FCR and Vibrio sp. counts were significantly decreased in case of PPIII group when compared to the other groups (p < .05). Also, significant level of surveillance was noted in the challenging test with V. anguillarum. These results suggested that the P. pentosaceus improved the growth performance, digestive enzyme activity, immunity and tolerance against V. anguillarum of L. vannamei.