The Chronology of Angiostrongylus vasorum (Baillet, 1866), Kamensky, 1905: Infection in Biomphalaria glabrata (Say, 1818).

The intermediate hosts of the French heartworm Angiostrongylus vasorum are aquatic and terrestrial gastropods. The present work is aimed at clarifying the sites of penetration and the migratory routes of A. vasorum in Biomphalaria glabrata snail tissues and evaluating their perilarval reaction with regard to the cellular composition and histological alterations involved in the gastropod response to infection. Biomphalaria glabrata snails were individually infected with 1000 first-stage larvae (L1) of A. vasorum each and killed at predetermined times after infection. Percutaneous infection occurred simultaneously with oral infection. Despite larval tropism to the fibromuscular tissue, some larvae were located in different tissues and organs. A perilarval reaction was observed around the larvae in a fibromuscular layer, appearing later around the larvae located in the viscera. The number of hemocytes surrounding the larvae increased gradually, forming a pregranuloma. Larval death and degeneration were not observed. No defined migratory pattern occurred, and larval development was apparently not associated with particular tissues or organs. In addition, the infection by A. vasorum induces a systemic mobilization of hemocytes in perilarval reaction.

Efficacy of carvacrol on Rhipicephalus (Boophilus) microplus engorged female ticks (Canestrini, 1887) (Acari: Ixodidae): effects on mortality and reproduction.

The activity of carvacrol was evaluated in R. microplus female ticks by estimating the mean lethal concentration (LC50) and the acaricidal efficacy. Ticks were subjected to the Adult Immersion Test (AIT) to calculate LC50. From this value, the AIT was performed again at the concentrations of 20%, 40%, 60%, 80% and 100% of the LC50. Two control groups were established: one treated with distilled water and one with the solvent (ethanol 50%). The following parameters were evaluated: female weight before oviposition, egg mass weight, pre-oviposition and incubation period, hatching percentage, egg production index, fecundity rate, estimated reproduction, reduction in oviposition and hatching, and product efficacy. The obtained LC50 was 20.11 mg/mL. Egg mass weight, egg production index, pre-oviposition and fecundity rates of groups treated with 80% and 100% of LC50 were statistically different when compared to control groups. The product efficacy in groups treated with 100% of LC50 was 61.10%.

Sublethal concentrations of acetylcarvacrol strongly impact oocyte development of engorged female cattle ticks Rhipicephalus microplus (Canestrini, 1888) (Acari: Ixodidae).

Rhipicephalus microplus, commonly known as southern cattle tick, causes huge economic losses in the cattle industry. Its infestation affects the production of meat and milk and causes discomfort to hosts. In addition, it is the vector of Babesia spp. and Anaplasma spp. The most frequent method used to control these parasites is through synthetic acaricides. However, their indiscriminate use can be toxic for hosts and environment as well as cause selection of resistant ticks. Plant extracts and essential oils emerge as promising alternatives to manage tick infestation. Carvacrol, an aromatic monoterpene extracted from plants, has recognized antimicrobial, antioxidant, insecticidal, repellent and acaricidal activities. Acetylation of carvacrol is believed to enhance its nematicidal and acaricidal activities and to decrease its toxicity to hosts. Thus, the aim of this study was to evaluate the effect of different concentrations of acetylcarvacrol in the morphology of ovaries of engorged R. microplus ticks. The most remarkable morphological alterations found in the female germ cells were irregular and thicker chorion, decreasing in size and irregular shape of female germ cells (oocytes), cytoplasmatic vacuolization as well as ring-shaped nucleoli. These alterations were analyzed through a semi-quantitative method proposed in this study for ixodid ticks. Treatment group IV, which was exposed to 4.5 µL/mL of carvacrol acetate, showed the most significant alterations, and it was also statistically different when compared to control groups. Therefore, sublethal concentrations of acetylcarvacrol demonstrated to impact the reproductive system of R. microplus by causing several damages in the female germ cells. This would hinder the generation of new individuals, probably contributing for a long-term control of tick infestation.

Morphological alterations in salivary glands of Rhipicephalus sanguineus ticks (Acari: Ixodidae) exposed to neem seed oil with known azadirachtin concentration.

Neem (Azadirachta indica) has attracted the attention of researchers worldwide due to its repellent properties and recognized effects on the morphology and physiology of arthropods, including ticks. Therefore, this study aimed to demonstrate the effects of neem seed oil enriched with azadirachtin on salivary glands of Rhipicephalus sanguineus ticks, targets of great veterinary interest because of their ability to transmit pathogens to dogs. For this, R. sanguineus semi-engorged females were subjected to treatment with neem seed oil, with known azadirachtin concentrations (200, 400 and 600ppm). After dissection, salivary glands were collected and evaluated through morphological techniques in light microscopy, confocal scanning laser microscopy and transmission electron microscopy, so that the possible relation between neem action and further impairment in these ectoparasites feed performance could be established. Neem oil demonstrated a clear dose-dependent effect in the analyzed samples. The agranular (type I) and granular acini (types II and III) showed, particularly in individuals treated with the highest concentrations of the product, cells with irregular shape, intense cytoplasmic disorganization and vacuolation, dilation of rough endoplasmic reticulum lumen, besides alterations in mitochondrial intermembrane space. These morphological damages may indicate modifications in salivary glands physiology, demonstrating the harmful effects of compounds present in neem oil on ticks. These results reinforce the potential of neem as an alternative method for controlling R. sanguineus ticks, instead of synthetic acaricides.

Morphological effects of neem (Azadirachta indica A. Juss) seed oil with known azadirachtin concentrations on the oocytes of semi-engorged Rhipicephalus sanguineus ticks (Acari: Ixodidae).

The concern about the harmful effects caused by synthetic pesticides has led to the search for safe and ecological alternatives for pest control. In this context, the neem tree (Azadirachta indica) stands out due to its repellent properties and effects on various arthropods, including ticks. For this reason, this study aimed to demonstrate the potential of neem as a control method for Rhipicephalus sanguineus ticks, important vectors of diseases in the veterinary point of view. For this, R. sanguineus semi-engorged females were subjected to treatment with neem seed oil enriched with azadirachtin, its main compound, and ovaries were assessed by means of morphological techniques in conventional light microscopy, confocal laser scanning microscopy, and transmission electron microscopy. Neem demonstrated a clear dose-dependent effect in the analyzed samples. The observed oocytes presented, especially in the groups treated with higher concentrations of neem oil, obvious signs of cytoplasmic disorganization, cellular vacuolization, nuclear and nucleolar irregularity, dilation in mitochondrial cristae, alterations in mitochondrial matrix, and swelling of rough endoplasmic reticulum. Intracellular microorganisms were observed in all analyzed groups, reinforcing the importance of ticks in the transmission of pathogens. A greater quantity of microorganisms was noted as the concentration of neem increased, indicating that the damaged oocytes may be more susceptible for their development. Such morphological alterations may promote future damages in reproductive performance of these animals and demonstrate the potential of neem seed oil for the control of R. sanguineus ticks, paving the way for new, cheaper, and safer methods of control.

Morphological alterations in the synganglion and integument of Rhipicephalus sanguineus ticks exposed to aqueous extracts of neem leaves (Azadirachta indica A. JUSS).

Currently, the necessity of controlling infestation by ticks, especially by Rhipicephalus sanguineus, has led researchers and public health managers around the world to search for new and more efficient control methods. This way, we can highlight neem (Azadirachta indica A. Juss) leaf, bark, and seed extracts, which have been very effective on tick control, and moreover causing less damage to the environment and to the host. This study showed the potential of neem as a control method for R. sanguineus through morphological and morphometric evaluation of the integument and synganglion of females, in semiengorged stage. To attain this, routine techniques of optical microscopy, scanning electron microscopy and morphometry of the cuticle and subcuticle of the integument were applied. Expressive morphological alterations were observed in both organs, presenting a dose-dependent effect. Integument epithelial cells and nerve cells of the synganglion showed signs of cell vacuolation, dilated intercellular boundaries, and cellular disorganization, alterations not previously reported in studies with neem. In addition, variations in subcuticle thickness were also observed. In general, the effects of neem are multiple, and affect the morphology and physiology of target animals in various ways. The results presented in this work are the first evidence of its effects in the coating and nervous system of ticks, thus allowing an indication of neem aqueous extracts as a potential control method of the brown dog tick and opening new perspectives on acaricide use.

Morphology of the midgut of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) adult ticks in different feeding stages.

The intestinal epithelial cells of ticks are fundamental for their full feeding and reproductive success, besides being considered important sites for the development of pathogens. Rhipicephalus sanguineus ticks are known for their great medical and veterinary importance, and for this reason, the knowledge of their intestinal morphology may provide relevant subsidies for the control of these animals, either by direct acaricidal action over these cells or by the production of vaccines. Therefore, this study aimed to describe the midgut morphology of male and female R. sanguineus ticks in different feeding stages, by means of histological analysis. Significant differences were observed between the genders, and such alterations may refer mainly to the distinct demands for nutrients, much higher in females, which need to develop and carry out the egg-laying process. In general, the midgut is coated by a thin muscle layer and presents a pseudostratified epithelium, in which two basic types of cells can be observed, connected to a basal membrane-generative or stem and digestive cells. The latter was classified as follows: residual, deriving from the phase anterior to ecdysis; pinocytic, with vesicles containing liquid or pre-digested components of blood; phagocytic, with entire cells or remnants of nuclear material inside cytoplasmic vesicles; and mature, free in the lumen. Digestion is presumably intracellular and asynchronous and corresponds to a process which starts with the differentiation of generative cells into pinocytic digestive cells, which subsequently start to phagocytize intact blood cells and finally detach from the epithelium, being eliminated with feces.

Histochemical and ultrastructural analysis of hepatic glycogen and collagen fibers in alloxan-induced diabetic rats submitted to long-term physical training.

Alterations in liver functions are common among diabetic patients, and many symptoms in the liver have been reported, including changes in glycogen stores and in the amount of collagen fibers. The practice of physical training and its morphological effects in this organ, however, are scarcely studied. In order to observe the morphological effects of alloxan-induced diabetes and the alterations arising from the practice of long-term chronic physical training in the liver, samples were collected and processed, and then analyzed by means of the histochemical techniques Periodic Acid-Schiff and Picrosirius-Hematoxylin, and ultrastructural cytochemical test of Afzelius. Through evaluation of the tissue, it was observed a drastic reduction in hepatic glycogen stores of sedentary diabetics, recovered in trained diabetic rats. Furthermore, it was detected a decrease in the content of perisinusoidal collagen fibers in the diabetic liver, also recovered due to the development of a training protocol. On ultrastructural level, cytochemical analysis confirmed the loss of glycogen and the recovery obtained by training. In conclusion, the practice of a long-term chronic physical training protocol may be considered an important assistant in the treatment of diabetes, mitigating the occurrence of possible damages to liver tissue.

Collagen and reticular fibers in left ventricular muscle in diabetic rats: physical exercise prevents its changes?

Diabetic cardiomyopathy contributes to the high incidence of mortality in both types of diabetes. We aimed to investigate the histochemical aspects of collagen and reticular fibers in the cardiac muscle and evaluate the influence of physical exercise on these aspects. Wistar rats were divided in 4 groups: sedentary control (SC), trained control (TC), sedentary diabetic (SD) and trained diabetic (TD). Diabetes was induced with alloxan (35 mg/kg). Training program consisted of swimming 1h/day with a load of 4.8% of body weight for TD and 5.2% for TC, during 8 weeks. At the end of the experiment, samples of the left ventricle were collected, fixed in Bouin and processed on historesin. Sections were stained with periodic acid of Schiff, picrosirius-hematoxylin and ammoniacal silver. The PAS technique shows that individuals of group SD presented more intense reaction that the other groups. Picrosirius-hematoxylin technique showed a possible deposition of collagen fibers in SD. The TD group presented a reaction a lot similar to the controls' for both techniques', showing a possible prevention of this deposition. These results indicate that physical exercises might have an important role on the prevention of some negative alterations caused by experimental diabetes.