Morphological and molecular detection of canine dirofilarial species of veterinary and medical importance in Morogoro municipality, Tanzania.

A descriptive cross-sectional survey of Dirofilaria infections in dogs was carried out from January to March 2015 in Morogoro municipality, Tanzania. One hundred and fifty two blood samples were collected from healthy dogs aged more than 6 months living in different areas of Morogoro, and analyzed by modified Knott's technique for circulating microfilariae. Microfilaraemic samples were further analyzed by Polymerase chain reaction (PCR) and PCR products were sequenced for molecular identification. Microfilariae were detected by microscopy in 9 samples (5.92%), of which 6 tested positive by PCR. The 5.8S-internal transcribed spacer 2 (ITS2)-28S ribosomal DNA (rDNA) sequences generated were 97% identical to Dirofilaria immitis and 86% to 90% identical to D. repens, confirming the presence of D. immitis in Tanzania and showing the presence of D. repens, not previously observed.

Ethanolic extract of Azadirachta indica (A. Juss.) causing apoptosis by ROS upregulation in Dirofilaria immitis microfilaria.

Dirofilaria immitis is the causative agent of cardiopulmonary dirofilariasis in the Canine family. The aim of the study was to evaluate the efficacy of the ethanolic extract of Azadirachta indica leaves (EEA) against the microfilaria (mf) of D. immitis in vitro. EEA was evaluated for different compound classes through HPTLC. Relative motility, mortality and morphological alterations were observed in the mf after exposure to EEA. The effect of EEA on redox status in the treated mf was evaluated by some key enzymatic and non-enzymatic parameters. An enhanced reactive oxygen species (ROS) level in the treated mf along with altered redox status was evident. DNA fragmentation and terminal-deoxynucleotidyl-transferase dUTP nick end labeling (TUNEL) confirmed apoptosis. In addition, western blotting revealed down-regulation of anti-apoptotic protein and up-regulation of pro-apoptotic proteins. Taken together, the microfilaricidal activity of EEA can be attributed to its capacity to inflict oxidative stress culminating in apoptosis.

Wolbachia infection and mitochondrial diversity in the canine heartworm (Dirofilaria immitis).

BACKGROUND AND AIMS: Many species of filarial nematodes are infected with Wolbachia pipientis, a maternally inherited endosymbiont. In addition to manipulating host reproduction, these bacteria also affect the evolution of the mitochondrial DNA with which they are transmitted. Selective sweeps can establish a single mitochondrial lineage within a Wolbachia-infected population and purge genetic diversity. While this phenomenon has been studied in insect model systems, it has not been thoroughly examined in a filarial nematode. MATERIALS AND METHODS: Patterns of mitochondrial diversity were examined in Dirofilaria immitis, a Wolbachia-infected species. RESULTS: The levels of genetic diversity observed in canine heartworm were much lower than those in related species not known to be hosts of Wolbachia. CONCLUSION: RESULTS suggest that a maternally inherited endosymbiont can depress mitochondrial diversity in a filarial host.

Dirofilaria immitis: immunohistochemical localization of acid proteinase in the adult worm.

The distribution of Dirofilaria immitis acid proteinase in adult worm tissue was examined biochemically and immunohistochemically. About 45% of the total proteinase activity of 700g supernatant, which was obtained from the 0.25 M sucrose homogenate of live adult worms, was found in the 100,000g supernatant by subcellular centrifugation analysis. The distribution pattern of the proteinase activity observed by Percoll density gradient centrifugation coincided with that of glucose-6-phosphatase, a marker cytosolic enzyme, suggesting that the acid proteinase was present in vivo in a membrane-free form, possibly in the cytosol or secretory fluid. Immunostaining for the proteinase in the parasite tissue using the IgG1 (kappa-type) monoclonal antibody, H-1, revealed immunoreactive enzyme primarily inside the cell as small grains, but not on the cell surface, and immunoreactivity was distributed widely in worm tissues such as lateral cords, dorsal and ventral median cords, the anterior end of the parasite, and intestinal epithelial cells in granular form. In the male reproductive system, the testicular wall and germ cells were labeled with the antibody, and in the female, uterine walls, fertilized eggs, and developing eggs as well as microfilaria were labeled. In conclusion, D. immitis acid proteinase is widely distributed in the parasite tissue, possibly functioning not only in nutrition metabolism but also in production of sperm and microfilariae, etc.

Quantitative buffy coat analysis of blood collected from dogs, cats, and horses.

Using quantitative buffy coat analysis (QBCA), rapid and accurate measurements can be made of the erythrocyte PCV, total WBC count, and platelet count, and the leukocyte population can be differentiated into total granulocytes (including quantitation of eosinophils), and lymphocytes and monocytes. The QBCA is performed by placing a blood sample (50 to 111 microliters) into a high-precision-bore microhematocrit tube that contains a freely moving, closely fitting, cylindrical plastic float. After centrifugation for 5 minutes, the buffy coat components separate by density. The plastic cylinder floats in the buffy coat, thereby expanding the lengths of the buffy coat layers. The layers are measured in a manner that is similar to that used for measuring PCV. Results of QBCA of blood samples from dogs, cats, and horses indicated that the hematologic values obtained correlated with results obtained by use of conventional methods. The accuracy and ease of use of QBCA and the availability of results while the animal is still being examined make QBCA a useful tool for hematologic evaluation of animals.