Transgenic chloroplasts are potential bioreactors for recombinant protein production, especially for achievement of high levels of protein expression and proper folding. Production of therapeutic proteins in leaves provides transgene containment by elimination of reproductive structures. Therefore, in this study, human Insulin like Growth Factor-1 is expressed in transgenic chloroplasts for evaluation of structural identity and function.

Purpose: Vaccinia virus has strong potential as a novel therapeutic agent for treatment of pancreatic cancer. We investigated whether arming vaccinia virus with interleukin-10 (IL10) could enhance the antitumor efficacy with the view that IL10 might dampen the host immunity to the virus, increasing viral persistence, thus maximizing the oncolytic effect and antitumor immunity associated with vaccinia virus. Experimental Design: The antitumor efficacy of IL10-armed vaccinia virus (VVLΔTK-IL10) and control VVΔTK was assessed in pancreatic cancer cell lines, mice bearing subcutaneous pancreatic cancer tumors and a pancreatic cancer transgenic mouse model. Viral persistence within the tumors was examined and immune depletion experiments as well as immunophenotyping of splenocytes were carried out to dissect the functional mechanisms associated with the viral efficacy. Results: Compared with unarmed VVLΔTK, VVLΔTK-IL10 had a similar level of cytotoxicity and replication in vitro in murine pancreatic cancer cell lines, but rendered a superior antitumor efficacy in the subcutaneous pancreatic cancer model and a K-ras-p53 mutant-transgenic pancreatic cancer model after systemic delivery, with induction of long-term antitumor immunity. The antitumor efficacy of VVLΔTK-IL10 was dependent on CD4⁺ and CD8⁺, but not NK cells. Clearance of VVLΔTK-IL10 was reduced at early time points compared with the control virus. Treatment with VVLΔTK-IL10 resulted in a reduction in virus-specific, but not tumor-specific CD8⁺ cells compared with VVLΔTK. Conclusions: These results suggest that VVLΔTK-IL10 has strong potential as an antitumor therapeutic for pancreatic cancer. Clin Cancer Res; 21(2); 405–16. ©2014 AACR.

A 1-year study of the ecological cycle of Francisella tularensis was performed in an enzootic area during an inter-epizootic period. The study was based on multiple sampling of all major constituents of the disease cycle. Seroprevalence of tularemia in the European brown hare (Lepus europaeus) population was 5.1% (10/197) with low antibody titers (1/10 and 1/20), and F. tularensis ssp. holarctica was isolated from four hares. F. tularensis was not detected in the 38 common voles (Microtus arvalis), 110 yellow-necked mice (Apodemus flavicollis), or 15 stripped field mice (Apodemus agrarius) trapped during the study, or the by-catch of 8 Eurasian pygmy shrews (Sorex minutus) or 6 common shrews (Sorex araneus). A total of 1106 Ixodes ricinus and 476 Haemaphysalis concinna ticks were collected from vegetation, and 404 I. ricinus, 28 H. concinna ticks, and 15 Ctenophtalmus assimilis and 10 Nosopsyllus fasciatus fleas were combed off small mammals. One H. concinna female and one nymph collected from the vegetation was found infected with F. tularensis ssp. holarctica by TaqMan polymerase chain reaction, thus resulting a 0.42% (2/476) prevalence. F. tularensis–specific DNA was not detected in environmental water samples, and the examined 100 sheep, 50 cows, and 50 buffalos grazed at the study area were all seronegative. During inter-epizootic periods, F. tularensis ssp. holarctica seems to persist only in the European brown hare—H. concinna cycle at the studied habitat. H. concinna may not serve exclusively as an arthropod vector, but it may also harbor bacteria for 3–4 years through multiple life stages and act as an important reservoir of F. tularensis. Rodent species probably do not serve as true reservoir hosts of tularemia.

Mycoplasma infection is still very common in chicken and turkey flocks. Several low-pathogenicity avian influenza (LPAI) viruses are circulating in wild birds that can be easily transmitted to poultry flocks. However, the effect of LPAI on mycoplasma infection is not well understood. The aim of the present study was to investigate the infection of LPAI virus H3N8 (A/mallard/Hungary/19616/07) in chickens challenged with Mycoplasma gallisepticum. Two groups of chickens were aerosol challenged with M. gallisepticum. Later one of these groups and one mycoplasma-free group were aerosol challenged with the LPAI H3N8 virus. The birds were observed for clinical signs for 8 days, then euthanized, and examined for the presence of M. gallisepticum in the trachea, lung, air sac, liver, spleen, kidney and heart, and for developing anti-mycoplasma and anti-viral antibodies. The LPAI H3N8 virus did not cause any clinical signs but M. gallisepticum infection caused clinical signs, reduction of body weight gain and colonization of the inner organs. These parameters were more severe in the birds co-infected with M. gallisepticum and LPAI H3N8 virus than in the group challenged with M. gallisepticum alone. In addition, in the birds infected with both M. gallisepticum and LPAI H3N8 virus, the anti-mycoplasma antibody response was reduced significantly when compared with the group challenged with M. gallisepticum alone. Co-infection with LPAI H3N8 virus thus enhanced pathogenesis of M. gallisepticum infection significantly.

Brucella spp. were isolated from an abortion case submitted for laboratory examination 8 months after the first clinical symptoms appeared in a kennel consisting of 31 dogs. Pathological investigations revealed the parallel presence of necrotic placentitis and the strong immunostaining of trophoblast cells by immunohistochemistry (IHC) using hyperimmune rabbit anti-Brucella canis primary antibodies. The rapid slide agglutination test was positive in 7 of 31 (23%) cases. The organism B. canis was successfully cultured from the blood, tissues, or vaginal swabs of only 3 of 31 (10%) cases. The isolated strains were identified as B. canis based on their colony morphology and agglutination with R sera. The strains were initially misidentified as B. suis with the “Bruce-ladder” method, and were subsequently correctly identified as B. canis with a single nucleotide polymorphism (SNP) typing test. Three culture-positive cases and 3 culture-negative cases with histories of reproductive disorders were selected and examined for the presence of B. canis infection using histopathology, IHC, and polymerase chain reaction (PCR) assays. Characteristic histologic lesions were found in all of the 6 animals, whereas IHC and PCR yielded positive results only in single cases from both groups. The results imply that all cases of canine abortion should be examined for brucellosis by bacterial culture of aborted fetuses and placentas. Immunohistochemical examination of placentas is also recommended because it is a quick and sensitive technique compared with bacterial culture. Multiple methods (i.e., serology, blood, and genital bacterial cultures) should be applied simultaneously and repeatedly for the reliable screening of B. canis infection in live individuals.

Chickens were infected under experimental conditions with Mycoplasma gallisepticum and low pathogenic avian influenza (LPAI) strain A/mallard/Hungary/19616/07 (H3N8). Two groups of chickens were aerosol challenged with M. gallisepticum strain 1226. Seven days later, one of these groups and one mycoplasma-free group was challenged with LPAI H3N8 virus; one group without challenge remained as negative control. Eight days later, the birds were euthanized and examined for gross pathologic and histologic lesions. The body weight was measured, and the presence of antimycoplasma and antiviral antibodies was tested before the mycoplasma challenge, before the virus challenge, and at the end of the study to confirm both infections. Chickens in the mycoplasma-infected group developed antibodies against M. gallisepticum but not against the influenza virus. Chickens of the group infected with the influenza virus became serologically positive only against the virus, while the birds in the coinfected group developed antibodies against both agents. The LPAI H3N8 virus strain did not cause decrease in body weight and clinical signs, and macroscopic pathological lesions were not present in the chickens. The M. gallisepticum infection caused respiratory signs, airsacculitis, and peritonitis characteristic of mycoplasma infection. However, the clinical signs and pathologic lesions and the reduction in weight gain were much more significant in the group challenged with both M. gallisepticum and LPAI H3N8 virus than in the group challenged with M. gallisepticum alone.