Molecular cloning and expression analysis of pig CD81

CD81, also known as TAPA-1 (target of antiproliferative antibody 1), is a member of the tetraspanin family of proteins and a component of the B cell co-receptor complex. Several studies have shown that CD81 plays significant roles in a variety of immune responses, including activation of B cells and T cells. In this study, we cloned pig Cd81 cDNA using RT-PCR coupled with rapid amplification of cDNA ends (RACE)-PCR and determined the complete cDNA sequence of pig Cd81. Pig Cd81 cDNA contains an open reading frame (711 bp) encoding 236 amino acids. The identity of pig CD81 with those of human, cattle, rat, and mouse are 90.30%, 92.26%, 86.22%, and 86.22%, respectively. Alignment of the CD81 amino acid sequence with those of mammalian species showed that the large extracellular loop (LEL) is the most divergent, whereas other domains are largely conserved. Pig Cd81 mRNA was detected by RT-PCR in a broad range of tissues, including lymphoid tissues as well as nonlymphoid tissues, indicated variety of cellular functions of CD81 in most pig tissues. Flow cytometry analyses demonstrated that human CD81 antibody recognizes a pig CD81 on the cell surface. Further, immunohistochemistry analysis using human CD81 antibody on pig spleen was revealed that CD81 expression is widely diffused in spleen tissue. Future study will be focused on defining the functional role of CD81 during the course of pig infectious diseases.     

Basosquamous carcinoma with systemic metastasis in a miniature Pinscher

Basosquamous carcinoma (BSCC) is a rare malignancy, primarily composed of basal cells with foci of squamous differentiation. It is considered to be histologically an intermediate type between basal cell carcinoma and squamous cell carcinoma, and is known to have aggressive behaviors. BSCC occurred in a 17-year-old female minipin with a history of surgical excision for a mammary tumor. The right upper hindlimb was severely enlarged to 8 x 5 cm. Cross-section showed a homogenous white to yellow-white mass compressing the surrounding muscular tissues. The tumor metastasized also to the lungs, heart, abdominal cavity, liver and salivary gland. Microscopically, basaloid cells were crowded into solid nests or lobules separated by well-developed fibrous tissues with occasional keratinizations. Since there was no skin lesions, the tumor is assumed to be originated from the formerly present tumor in mammary gland. To our literature review, this case is the first BSCC with systemic metastasis in a dog.     

An antimicrobial peptide-A3: effects on growth performance,nutrient retention,intestinal and faecal microflora and intestinal morphology of broilers

1. The present study investigated the effects of dietary supplementation with an antimicrobial peptide-A3 (AMP-A3) on growth performance, nutrient retention, intestinal microflora and intestinal morphology of broilers.

2. A total of 320-d-old chicks (Ross 308, average BW 44.0 ± 3.4 g) were randomly allotted to 4 dietary treatments on the basis of initial body weight (BW). The dietary treatments were negative control (NC; basal diet), positive control (PC; basal diet + 15 mg avilamycin/kg diet) and AMP-A3 (basal diet supplemented with 60 or 90 mg/kg AMP-A3). The NC diet was considered as 0 mg/kg AMP-A3 treatment. Experimental diets were given in two phases: starter phase (d 0–21) and finisher phase (d 22–35).

3. The overall BW gain and retention of dry matter (DM), gross energy (GE; d 19–21) and crude protein (CP; d 19–21 and d 33–35) were greater in birds fed on the PC and 90 mg/kg AMP-A3 diets than in birds fed on the NC diet. Also, an increase in dietary AMP-A3 linearly improved BW gain and retention of DM, GE (d 19–21) and CP (d 19–21 and d 33–35).

4. Birds fed on the PC and 90 mg/kg AMP-A3 diets had fewer excreta coliforms (d 21 and d 35), total anaerobic bacteria (TAB) and Clostridium spp. (d 35) and ileum and caecum coliforms (d 35) than birds fed on the NC diet. In addition, birds fed on the diet supplemented with increasing levels of AMP-A3 had linearly reduced excreta TAB (d 35), Clostridium spp. and coliforms (d 21 and d 35) and ileum and caecum coliforms (d 35).

5. Birds fed on the PC and 90 mg/kg AMP-A3 diets had greater villus height of the duodenum, jejunum and ileum than birds fed on the NC diet. Moreover, birds fed on increasing levels of AMP-A3 diet had increased (linear) villus height of the duodenum, jejunum and ileum.

6. These results indicate that 90 mg/kg AMP-A3 has the potential to improve growth performance, nutrient retention and intestinal morphology and to reduce harmful microorganisms in broilers and can be used as a potential antimicrobial growth promoter.     

Serodiagnostic comparison of enzyme-linked immunosorbent assay and surface plasmon resonance for the detection of antibody to Porcine circovirus type 2

This paper describes the cloning and expression of the capsid protein of Porcine circovirus type 2 (PCV2) in an Escherichia coli expression system that was used to produce a fusion protein for subsequent immunologic studies: enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Polymerase chain reaction was used to amplify the gene encoding the capsid protein from the DNA of PCV2. The protein was then cloned into a pRSET prokaryotic expression vector. Western blot analysis revealed that the recombinant protein gave strong signals on a polyvinylidene difluoride membrane when exposed to the serum from a pig infected with PCV2. The expressed protein was purified and used as an antigen for the ELISA and SPR study. A protein chip based on SPR was developed, and the diagnostic potential of SPR was compared with that of ELISA with the use of 70 serum samples obtained from 6 pig farms. There was a strong positive correlation between the ELISA and SPR titers (r = 0.877, P < 0.01). Therefore, this recombinant capsid protein can be used as an antigen for serologic studies, and the SPR, a label-free method, appears to be a valuable and reproducible tool in the serodiagnosis of a PCV2 infection.     

Increased humoral antibody response of foot-and-mouth disease virus vaccine in growing pigs pre-treated with poly-γ-glutamic acid

This study was conducted to determine if humoral antibody response of foot-and-mouth disease (FMD) vaccine improved in 8-week-old growing pigs born to well-vaccinated sows pre-treated with 60 mg of poly-γ-glutamic acid (γ-PGA) three days before vaccination. Antibody against FMD virus serotype O was measured 0, 2, 4 and 6 weeks post-vaccination, using a PrioCHECK FMDV type O ELISA kit. The results showed that positive antibody reactions against FMDV serotype O antigen among a component of the vaccine significantly increased in response to pre-injection with γ-PGA.     

Comparison of antibody titers in rabbits following immunization with inactivated influenza virus via subarachnoidal or subcutaneous route

Rabbits were immunized with inactivated influenza virus via the subarachnoidal (SA) or subcutaneous (SC) route, and the antibody titers in cerebrospinal fluid (CSF) and serum were assayed. There were no nervous signs or morphological lesions related to SA immunization. In the SC group, the antibody titer was elevated in serum, but not elevated in CSF. In the SA group, the antibody titer was significantly elevated in serum and even in CSF, and their antibody titers were much greater than in the SC group. The present results suggest that intrathecal immunization is more effective than SC immunization at inducing a protective immune response against the transneural spread of viruses.