Recombinant Iss as a potential vaccine for avian colibacillosis

Avian pathogenic Escherichia coli (APEC) cause colibacillosis, a disease which is responsible for significant losses in poultry. Control of colibacillosis is problematic due to the restricted availability of relevant antimicrobial agents and to the frequent failure of vaccines to protect against the diverse range of APEC serogroups causing disease in birds. Previously, we reported that the increased serum survival gene (iss) is strongly associated with APEC strains, but not with fecal commensal E. coli in birds, making iss and the outer membrane protein it encodes (Iss) candidate targets for colibacillosis control procedures. Preliminary studies in birds showed that their immunization with Iss fusion proteins protected against challenge with two of the more-commonly occurring APEC serogroups (O2 and O78). Here, the potential of an Iss-based vaccine was further examined by assessing its effectiveness against an additional and widely occurring APEC serogroup (O1) and its ability to evoke both a serum and mucosal antibody response in immunized birds. In addition, tissues of selected birds were subjected to histopathologic examination in an effort to better characterize the protective response afforded by immunization with this vaccine. Iss fusion proteins were administered intramuscularly to four groups of 2-wk-old broiler chickens. At 2 wk postimmunization, chickens were challenged with APEC strains of the O1, O2, or O78 serogroups. One week after challenge, chickens were euthanatized, necropsied, any lesions consistent with colibacillosis were scored, and tissues from these birds were taken aseptically. Sera were collected pre-immunization, postimmunization, and post-challenge, and antibody titers to Iss were determined by enzyme-linked immunosorbent assay (ELISA). Also, air sac washings were collected to determine the mucosal antibody response to Iss by ELISA. During the observation period following challenge, 3/12 nonimmunized chickens, 1/12 chickens immunized with 10 microg of GST-Iss, and 1/12 chickens immunized with 50 microg of GST-Iss died when challenged with the O78 strain. No other deaths occurred. Immunized chickens produced a serum and mucosal antibody response to Iss and had significantly lower lesion scores than nonimmunized chickens following challenge, regardless of the challenge strain. This study expands on our previous report of the value of Iss as an immunoprotective antigen and demonstrates that immunization with Iss can provide significant protection of chickens against challenge with three different E. coli strains.     

A visual health assessment of captive Asian elephants (Elephas maximus) housed in India

A visual health assessment and survey questionnaire was conducted on 81 Asian elephants (Elephas maximus) housed in 10 animal facilities throughout India between November 2004 and February 2005. The survey questionnaire consisted of 10 questions that evaluated the health of the elephants, and they were completed after visually assessing each individual elephant. The information collected was ranked on a scale that was used to statistically compare the health among the study subjects. This study documented that 43.21% of the captive elephants surveyed exhibited hyperkeratosis. A significant proportion of the elephants owned by tourist camps had poor skin condition when compared with elephants from zoos and at a forest camp. Similarly, captive-born individuals were found to have better skin condition than animals that were caught from the wild. Sixty (74.1%) of the captive elephants that were observed during this study had fissures in their footpads, 20% of which were severe. The prevalence of foot fissures was significantly higher in females. A greater proportion of elephants owned by tourist camps displayed vertical and horizontal toenail cracks in comparison with the forest camp and zoo elephants. It was noted that 76.9% of the wounded animals and 80% of those having abscesses were housed at temples and tourist camps. Also, approximately 8.5% of the captive elephant population observed during this study had eye-related problems, and they were all housed at temples and tourist camps. In conclusion, it was evident that elephants housed at temples or tourist camps exhibited poor skin condition with wounds and abscesses. These findings suggest that the overall condition of the elephants housed at tourist camps was poor compared with elephants housed at zoos and at the forest camp.     

Singular PCV2a or PCV2b infection results in apoptosis of hepatocytes in clinically affected gnotobiotic pigs

Porcine circovirus type 2 (PCV2) is clinically associated with respiratory disease, failure-to-thrive, hepatitis, and diarrhea; however, the precise pathogenesis of PCV2-associated disease and in particular its involvement in apoptosis is still controversial. The objectives of this study were (1) to determine whether PCV2 is associated with apoptosis by examining and comparing hepatic tissues from clinically affected or unaffected gnotobiotic pigs that were experimentally infected with PCV2, (2) to determine if there are differences between PCV2a and PCV2b in inducing hepatocyte apoptosis, and (3) to determine if there are differences between apoptosis detection systems. Forty-eight gnotobiotic pigs were separated into five groups based on inoculation status and development of clinical disease: (1) sham-inoculated, clinically-unaffected (n=4), (2) inoculated with PCV2a, clinically-unaffected (n=10), (3) inoculated with PCV2a, clinically-affected (n=6), (4) inoculated with PCV2b, clinically-unaffected, (n=13) and (5) inoculated with PCV2b, clinically-affected (n=15). Formalin-fixed, paraffin-embedded sections of liver from all pigs were analyzed for signs of apoptosis [presence of single strand DNA breaks in the nucleus by the terminal transferase dUTP nick end labeling (TUNEL) assay or presence of intra-nuclear cleaved caspase 3 (CCasp3) demonstrated by CCasp3 immunohistochemistry (IHC)]. In addition, the liver tissues were also tested for presence of cytoplasmic and intra-nuclear PCV2 antigen by an IHC assay. Specific CCasp3 and TUNEL labeling was detected in the nucleus of hepatocytes in PCV2a and PCV2b infected pigs with significantly (P<0.05) higher levels of apoptotic cells in clinically-affected pigs. Regardless of PCV2 subtype (PCV2a; PCV2b), there were higher levels of PCV2 antigen in clinically-affected pigs compared to clinically-unaffected pigs. There was no significant difference in detection rate of apoptotic cells between the TUNEL assay and CCasp3 IHC. When high amounts of PCV2 antigen were present, the incidence of CCasp3 and TUNEL staining also increased regardless of the PCV2 genotype. This suggests that PCV2-induced apoptosis of hepatocytes is important in the pathogenesis of PCV2-associated lesions and disease.     

ORF1 but not ORF2 dependent differences are important for in vitro replication of PCV2 in porcine alveolar macrophages singularly or coinfected with PRRSV

The objective of this study was to investigate cytokine expression and in vitro replication of porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) in pulmonary alveolar macrophages (PAMs) emphasizing PCV2 open-reading frame (ORF) origin (PCV2a or PCV2b) and PRRSV strain. Chimeric PCV2 viruses composed of different combinations of ORF1 and ORF2 of PCV2a or PCV2b (chimera PCV2a-2b and chimera PCV2b-2a) were constructed and five different PRRSV isolates were utilized: Type 1 (SD 01-08) or type 2 (NC16845b, VR-2332, MN-184, JA-142). PAMs were infected singularly or with combinations of PCV2b, PCV2a, chimera PCV2a-2b, and chimera PCV2b-2a, and one of the five PRRSV isolates. Real-time PCR was used to test PAMs (PCV2 mRNA) and supernatants (PRRSV RNA, PCV2 DNA, PCV2 mRNA) harvested at 24, 48, 72 and 96h post inoculation (hpi). Levels of IFN-γ, TNF-α and IL-10 were determined by quantitative ELISAs. PCV2 replication in PAMs was limited to groups inoculated with PCV2 strains containing ORF1 of PCV2a (PCV2a, chimera PCV2a-2b). Furthermore, in supernatants, PCV2 mRNA was only detected in groups coinfected with PRRSV regardless of strain at 48hpi supporting an enhancing effect of PRRSV on PCV2 infection. Changes in cytokine levels were minimal and associated with PRRSV strain for TNF-α. In summary, in vitro differences in PCV2 replication in PAMs inoculated with different PCV2-PRRSV combinations were independent of PCV2 ORF2 origin with minimal effects of concurrent PRRSV infection perhaps indicating that PCV2-specific changes in ORF1 may be more important than those in ORF2.