Interactions of porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus

Porcine circovirus associated disease (PCVAD) has made an economic impact in global swine production, is caused by porcine circovirus type 2 (PCV2) and manifests in forms of multisystemic disease, wasting, pneumonia, diarrhea in growing pigs and reproductive failure in gilts and sows. PCVAD is enhanced by PRRSV co-infection alongside PCV2 infection in pigs and is not very well understood except that PRRSV potentiates PCV2 replication in the host. We characterized apoptosis in gnotobiotic pigs caused by both PCV2a and PCV2b and for the first time established that both PCV2a and PCV2b are able to promote cell death in the hepatocytes of gnotobiotes of clinically affected pigs leading to hepatic failure. We further delineated the role of PRRSV in affecting PCV2a and PCV2b apoptosis in specific pathogen free (SPF) pigs and demonstrated that PRRSV does not cause apoptosis induction in PCV2a and PCV2b infected pigs. We compared and demonstrated that in vitro differences in PCV2 or PRRSV replication or IFN gamma; and IL-10 release in porcine alveolar macrophages (PAMs) inoculated with several PCV2-PRRSV combinations are small and not dependent on ORF1 or ORF2 origin. We analyzed the shedding of both PCV2a and PCV2b in piglets co-infected with PRRSV and ascertained that PRRSV is capable of prolonging PCV2 viremia and subsequent shedding in the nasal, oral secretions and fecal excretion that can increase horizontal transmission of PCV2a and PCV2b in nayve herds. Finally we also verified if prior PRRSV exposure had any detrimental effect on PCV2 vaccines currently available in the US market. We were able to establish that the PCV2 vaccines are able to provide protective immunity to piglets that had prior PRRSV infection

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 \u3c 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

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 µg of GST-Iss, and 1/12 chickens immunized with 50 µg 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.This article is from Avian Diseases 56, no. 1 (2012): 192–199, doi:10.1637/9861-072111-Reg.1.</p

Interactions of porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus

Porcine circovirus associated disease (PCVAD) has made an economic impact in global swine production, is caused by porcine circovirus type 2 (PCV2) and manifests in forms of multisystemic disease, wasting, pneumonia, diarrhea in growing pigs and reproductive failure in gilts and sows. PCVAD is enhanced by PRRSV co-infection alongside PCV2 infection in pigs and is not very well understood except that PRRSV potentiates PCV2 replication in the host. We characterized apoptosis in gnotobiotic pigs caused by both PCV2a and PCV2b and for the first time established that both PCV2a and PCV2b are able to promote cell death in the hepatocytes of gnotobiotes of clinically affected pigs leading to hepatic failure. We further delineated the role of PRRSV in affecting PCV2a and PCV2b apoptosis in specific pathogen free (SPF) pigs and demonstrated that PRRSV does not cause apoptosis induction in PCV2a and PCV2b infected pigs. We compared and demonstrated that in vitro differences in PCV2 or PRRSV replication or IFN gamma; and IL-10 release in porcine alveolar macrophages (PAMs) inoculated with several PCV2-PRRSV combinations are small and not dependent on ORF1 or ORF2 origin. We analyzed the shedding of both PCV2a and PCV2b in piglets co-infected with PRRSV and ascertained that PRRSV is capable of prolonging PCV2 viremia and subsequent shedding in the nasal, oral secretions and fecal excretion that can increase horizontal transmission of PCV2a and PCV2b in nayve herds. Finally we also verified if prior PRRSV exposure had any detrimental effect on PCV2 vaccines currently available in the US market. We were able to establish that the PCV2 vaccines are able to provide protective immunity to piglets that had prior PRRSV infection.</p

A note on enrichment for captive lion-tailed macaques (Macaca silenus)

Two enrichment techniques were devised with the aim of reducing stress and improving welfare for captive lion-tailed macaques in an Indian zoo. In Study A, a log, cotton ropes and a feeding basket were added to the enclosures in different combinations to provide structural and feeding enrichment and were subsequently removed, while in Study B, singly-housed individuals were transferred to a large open-moated enclosure in which they were group-housed. The effects of these two enrichment techniques (social and physical) appeared to significantly improve the welfare of the study individuals by stimulating them to exhibit higher levels of natural behaviours along with the suppression of several abnormal behaviours

Development and Validation of a New TaqMan Real-Time PCR for the Detection of Ornithobacterium rhinotracheale

Ornithobacterium rhinotracheale (ORT) has been associated with poultry respiratory disease worldwide. The organism is fastidious and isolation is challenging. One TaqMan real-time PCR (qPCR) assay has been developed for the detection of ORT. However, during validating the ORT qPCR, the assay performance was suboptimal. During the in silico evaluation, deviations from the basic parameters for primers and probes designs (e.g., presence of stable undesirable primer-dimers) were observed. The suboptimal design led to low efficiency and low sensitivity of the assay. Initially, modification on the probe was carried out to improve the performance of the assay. However, the assay’s performance (efficiency and sensitivity) was still suboptimal. In this manuscript, we describe the development of a new qPCR assay and the comparison of its performance with the currently available assay. A highly efficient, sensitive, and specific qPCR assay was developed with approximately 1000-folds reduction in the limit of detection (from 3 × 106 plasmid DNA copies/mL to 1 × 103 plasmid DNA copies/mL). Additionally, the efficiency of the new assay (E = 98.70%) was significantly better than the current assay (E = 73.18%). The newly developed assay is an improved diagnostic tool for the sensitive and efficient diagnosis of ORT from clinical samples

Correction: Hashish et al. Development and Validation of a New TaqMan Real-Time PCR for the Detection of <i>Ornithobacterium rhinotracheale</i>. <i>Microorganisms</i> 2022, <i>10</i>, 341

The authors would like to make the following corrections to this paper [...

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 This article is from Research in Veterinary Science 92 (2012); 151, doi: 10.1016/j.rvsc.2010.10.013.</p

Development and Validation of Two Diagnostic Real-Time PCR (TaqMan) Assays for the Detection of <em>Bordetella avium</em> from Clinical Samples and Comparison to the Currently Available Real-Time TaqMan PCR Assay

Bordetella avium (BA) is one of many pathogens that cause respiratory diseases in turkeys. However, other bacterial species can easily overgrow it during isolation attempts. This makes confirming the diagnosis of BA as the causative agent of turkey coryza more difficult. Currently, there are two PCR assays for the molecular detection of BA. One is conventional gel-based PCR and the other is TaqMan real-time PCR (qPCR) assay. However, multiple pitfalls were detected in both assays regarding their specificity, sensitivity, and efficiency, which limits their utility as diagnostic tools. In this study, we developed and validated two TaqMan qPCR assays and compared their performance to the currently available TaqMan qPCR. The two assays were able to correctly identify all BA isolates and showed negative results against a wide range of different microorganisms. The two assays were found to have high efficiency with a detection limit of approximately 1 × 103 plasmid DNA Copies/mL with high repeatability and reproducibility. In comparison to the currently available TaqMan qPCR assay, the newly developed assays showed significantly higher PCR efficiencies due to superior primers and probes design. The new assays can serve as a reliable tool for the sensitive, specific, and efficient diagnosis of BA