Effect of GP19 Peptide Hyperimmune Antiserum on Activated Macrophage during Ehrlichia canis Infection in Canine Macrophage-like Cells

In terms of its veterinary importance, vaccine development against Ehrlichia canis is needed. However, the effect of developing vaccines on humoral immune response against E. canis infection is still unknown. Novel GP194-43 was synthesized according to E. canis GP19 epitope prediction. To restrict any loss and/or illness in the host animal, rabbits were used in this study to produce GP194-43 hyperimmune sera. The effect of GP194-43 hyperimmune sera on neutralization was examined in vitro by determining the inhibition of E. canis infection of the macrophage-like cell line (DH82) in the presence of the sera. Four groups of DH82 cells received differing treatments. These included E. canis experimentally infected DH82 cells, E. canis-infected DH82 cells with control rabbit serum (untreated group), E. canis-infected DH82 cells with GP194-43 rabbit antiserum (treated group) and uninfected cells (negative control group), respectively. The treated group developed a decrease (p < 0.01) in the percentage of E. canis infected cells after 3 days post-infection at 48.57 ± 1.28. In addition, real-time PCR analyses of cytokine mRNA expression involved with the macrophage, humoral, and cellular immune responses were conducted. The findings revealed an upregulated expression of IFNG in the treated group during the infection. This study demonstrated neutralization in the GP194-43 peptide hyperimmune sera of immunized rabbits. Notably, IFN-γ production could be effectively promoted in canine macrophages in relation to the activation of macrophages and adaptive immune responses. The results of this study indicate the potential for the use of this immunogen in further investigations involving immunized and infected dogs as E. canis host species.

Anti- Ehrlichia properties of the dichloromethane extract of Ageratum conyzoides

Ehrlichia canis is an intracellular bacterium that infects hematopoietic cells. It is the causative agent of canine monocytic ehrlichiosis (CME). The disease may be acute, subclinical, or chronic, and is treated with tetracyclines including doxycycline. However, this class of tetracyclines may cause several side effects due to prolonged treatment. Bacterial resistance to antimicrobials has been extensively reported. The present study aimed to assess the anti- Ehrlichia activity of the dichloromethane extract (DCM) of Ageratum conyzoides L. on Ehrlichia- infected DH82 cells. For this purpose, the DCM extract of A. conyzoides collected in the municipality of São Luís, State of Maranhão (MA), northeast Brazil, was obtained from the aerial parts of the plant by exhaustive percolation in H 2 O- CH 2 Cl 2 (2:8) and subsequent extraction of the chemical compound. The chemical composition of these samples was investigated. The anti- Ehrlichia properties of A. conyzoides were confirmed in Ehrlichia- infected DH82 cells at a concentration of 200 μg.mL -1 of its DCM extract. The results of the treatments were evaluated at 18h and 36h after the insertion of the treatments evaluated with A. conyzoides.Based on the results of the chemical analysis of the samples, we may attribute these antirickettsial properties to the compounds from the lignan family that are found in this medicinal plant .

Oxidative Stress in Canine Histiocytic Sarcoma Cells Induced by an Infection with Canine Distemper Virus Led to a Dysregulation of HIF-1α Downstream Pathway Resulting in a Reduced Expression of VEGF-B In Vitro

Histiocytic sarcomas represent malignant tumors which require new treatment strategies. Canine distemper virus (CDV) is a promising candidate due to its oncolytic features reported in a canine histiocytic sarcoma cell line (DH82 cells). Interestingly, the underlying mechanism might include a dysregulation of angiogenesis. Based on these findings, the aim of the present study was to investigate the impact of a persistent CDV-infection on oxidative stress mediated changes in the expression of hypoxia-inducible factor (HIF)-1α and its angiogenic downstream pathway in DH82 cells in vitro. Microarray data analysis, immunofluorescence for 8-hydroxyguanosine, superoxide dismutase 2 and catalase, and flow cytometry for oxidative burst displayed an increased oxidative stress in persistently CDV-infected DH82 cells (DH82Ond pi) compared to controls. The HIF-1α expression in DH82Ond pi increased, as demonstrated by Western blot, and showed an unexpected, often sub-membranous distribution, as shown by immunofluorescence and immunoelectron microscopy. Furthermore, microarray data analysis and immunofluorescence confirmed a reduced expression of VEGF-B in DH82Ond pi compared to controls. In summary, these results suggest a reduced activation of the HIF-1α angiogenic downstream pathway in DH82Ond pi cells in vitro, most likely due to an excessive, unusually localized, and non-functional expression of HIF-1α triggered by a CDV-induced increased oxidative stress.

Cellular Microbiology of Mycoplasma canis

Mycoplasma caniscan infect many mammalian hosts but is best known as a commensal or opportunistic pathogen of dogs. The unexpected presence ofM. canisin brains of dogs with idiopathic meningoencephalitis prompted newin vitrostudies to help fill the void of basic knowledge about the organism's candidate virulence factors, the host responses that it elicits, and its potential roles in pathogenesis. Secretion of reactive oxygen species and sialidase varied quantitatively (P< 0.01) among strains ofM. canisisolated from canine brain tissue or mucosal surfaces. All strains colonized the surface of canine MDCK epithelial and DH82 histiocyte cells and murine C8-D1A astrocytes. Transit through MDCK and DH82 cells was demonstrated by gentamicin protection assays and three-dimensional immunofluorescence imaging. Strains further varied (P< 0.01) in the extents to which they influenced the secretion of tumor necrosis factor alpha (TNF-α) and the neuroendocrine regulatory peptide endothelin-1 by DH82 cells. Inoculation withM. canisalso decreased major histocompatibility complex class II (MHC-II) antigen expression by DH82 cells (P< 0.01), while secretion of gamma interferon (IFN-γ), interleukin-6 (IL-6), interleukin-10 (IL-10), and complement factor H was unaffected. The basis for differences in the responses elicited by these strains was not obvious in their genome sequences. No acute cytopathic effects on any homogeneous cell line, or consistent patterns ofM. canispolyvalent antigen distribution in canine meningoencephalitis case brain tissues, were apparent. Thus, while it is not likely a primary neuropathogen,M. canishas the capacity to influence meningoencephalitis through complex interactions within the multicellular and neurochemicalin vivomilieu.