Development of trypanosomatids in the phytophagous insect Dysdercus peruvianus (Hemiptera: Pyrrochoridae): a light and eletron microscopic study

Experimental infections of the phytophagous Hemiptera Dysdercus peruvianus with different trypanosomatids were studied for up to 55 days by light microscopy while the course of infection with Leptomonas seymouri and the Leptomonas isolate 49/553G.O. was analyzed by electron microscopy. Rates of infection of D. peruvianus varied according to the infecting flagellate. The lower part of the midgut was found to be the preferential site of colonization where most flagellates were found isolated or arranged in clumps or rosettes. Specialized junctional structures with host cells were never observed. Flagellates could also be seen inside midgut cells within a parasitophorous vacuole. Infection of haemocoele and salivary glands was also observed.

Inter-specific and developmental differences on the array of antennal chemoreceptors in four species of triatominae (Hemiptera: Reduviidae)

The aim of the work was to investigate the pattern of chemoreceptor sensilla in adults and fifth stage nymphs of Rhodnius prolixus, R. neglectus, Triatoma infestans and T. sordida in order to study differences and similarities between genera and species. Three types of sensilla were analyzed by light microscopy: thin-walled trichoidea, thick-walled trichoidea and basiconica. The number of sensilla of each three types were counted. The length of the antennal segments were also used as a variable for the analysis. The statistical analysis showed that the number of these antennal chemoreceptors had significant differences between species and between adults and nymphs of each species. Discriminant analysis separates incompletely the fifth stage nymphs of the four species and showed similarity between them. Discriminant analysis performed with 12 variables of the antennae, allowed a complete separation of the adults of the four species.

Correlation between koilocytes and human papillomavirus detection by PCR in oral and oropharynx squamous cell carcinoma biopsies

The purpose of this study was to compare the histopathological analysis with polymerase chain reaction (PCR) methods to predict the presence of human papillomavirus (HPV) infection in oral squamous cell carcinoma biopsies. Eighty-three paraffin-embedded tissue specimens from patients with oropharynx and mouth floor squamous cell carcinoma were submitted to histopathological analysis under light microscopy, specifically for the determination of the presence of koilocytes. Subsequently, DNA was purified from the same paraffin-embedded specimens and submitted to PCR. Fisher's exact test showed no statistically significant correlation between the two methods. The results suggest that the presence of koilocytes is unreliable for the detection of HPV presence in oral and oropharynx squamous cell carcinoma.

Lanfrediella amphicirrus gen. nov. sp. nov. Nematotaeniidae (Cestoda: Cyclophyllidea), a tapeworm parasite of Rhinella marina (Linnaeus, 1758) (Amphibia: Bufonidae)

The family Nematotaeniidae, tapeworms commonly found in the small intestines of amphibians and reptiles, includes 27 recognised species distributed among four genera: Bitegmen Jones, Cylindrotaenia Jewell, Distoichometra Dickey and Nematotaenia Lühe. The taxonomy of these cestodes is poorly defined, due in part to the difficulties of observing many anatomical traits. This study presents and describes a new genus and species of nematotaeniid parasite found in cane toads (Rhinella marina) from eastern Brazilian Amazonia. The cestodes were collected during the necropsy of 20 hosts captured in the urban area of Belém, Pará. The specimens were fixed and processed for light microscopy, scanning electron microscopy (SEM) and three-dimensional (3D) reconstruction. Samples were also collected for molecular analyses. The specimens presented a cylindrical body, two testes and paruterine organs. However, they could not be allocated to any of the four existing nematotaeniid genera due to the presence of two each of dorsal compact medullary testes, cirri, cirrus pouches, genital pores, ovaries and vitelline glands per mature segment. Lanfrediella amphicirrus gen. nov. sp. nov. is the first nematotaeniid studied using Historesin analysis, SEM and 3D reconstruction, and it is the second taxon for which molecular data have been deposited in GenBank.

Brazilian Angiostrongylus cantonensis haplotypes, ac8 and ac9, have two different biological and morphological profiles

Angiostrongylus cantonensis is the etiologic agent of eosinophilic meningoencephalitis in humans. Cases have been recorded in many parts of the world, including Brazil. The aim of this study was to compare the differences in the biology and morphology of two different Brazilian haplotypes of A. : ac8 and ac9. A significantly larger number of L1 larvae eliminated in the faeces of rodents at the beginning of the patent period was observed for ac9 haplotype and compared to the total of L1 larvae eliminated, there was a significant difference between the two haplotypes. The ac9 haplotype showed a significant difference in the proportion of female and male specimens (0.6:1), but the same was not observed for ac8 (1.2:1). The morphometric analysis showed that male and female specimens isolated from ac8 haplotype were significantly larger with respect to body length, oesophagus length, spicule length (male) and distance from the anus to the rear end (female) compared to specimens from ac9. The morphological analysis by light microscopy showed little variation in the level of bifurcations at the lateral rays in the right lobe of the copulatory bursa between the two haplotypes. The biological, morphological and morphometric variations observed between the two haplotypes agree with the observed variation at the molecular level using the cytochrome oxidase subunit I marker and reinforce the possible influence of geographical isolation on the development of these haplotypes.

Can sibling species of the Drosophila willistoni subgroup be recognized through combined microscopy techniques?

ABSTRACT In several arthropod groups, male genitalia is the most important feature for species identification, especially in cryptic species. Cryptic species are very common in the Drosophila genus, and the Neotropical Drosophila willistoni species group is a good example. This group currently includes 24 species divided into three subgroups: alagitans, bocainensis and willistoni. There are six sibling species in the willistoni subgroup – D. willistoni, D. insularis, D. tropicalis, D. equinoxialis, D. pavlovskiana and D. paulistorum, which is a species complex composed of six semispecies – Amazonian, Andean-Brazilian, Centroamerican, Interior, Orinocan and Transitional. The objective of this study was to characterize male genitalia of the willistoni subgroup, including the D. paulistorum species complex, using scanning electron microscopy and light microscopy. We also tried to contribute to the identification of these cryptic species and to add some comments about evolutionary history, based on male genitalia characters. Despite being cryptic species, some differences were found among the siblings, including the Drosophila paulistorum semispecies.

Histochemical and ultrastructural study of Caesalpinia peltophoroides Benth. (Leguminosae-Caesalpinoideae) seeds

The objective of this work was to correlate data on light microscopy observations through histochemical analysis and polarized light techniques and investigations in transmission electron microscopy (TEM) to characterize the reserve materials in C. peltophoroides Benth. (Leguminosae-Caesalpinoideae) cotyledons, popularly known as "sibipiruna", a tropical tree species with wide distribution in Brazil. The cotyledon mesophyll, especially in the abaxial face, is rich in unsaturated neutral lipids contained in numerous lipid bodies dispersed in the cytoplasm. Proteins, more concentrated in the adaxial face of the cotyledons, occur in all the mesophyll and are stored in protein bodies containing globoids, with variable number and size, responsible for accumulation of mineral reserves. Calcium oxalate druses have distribution restricted to the cotyledons adaxial face and are associated with protein bodies. Starch, also distributed all over the cotyledon mesophyll, occurs in small amounts in plastids with developed lamellar system. Secretory cavities rich in phenolic compounds occur among procambial strands.

Chromosomal abnormalities in couples with recurrent first trimester abortions

PURPOSE: To investigate the prevalence of chromosomal abnormalities in couples with two or more recurrent first trimester miscarriages of unknown cause. METHODS: The study was conducted on 151 women and 94 partners who had an obstetrical history of two or more consecutive first trimester abortions (1-12 weeks of gestation). The controls were 100 healthy women without a history of pregnancy loss. Chromosomal analysis was performed on peripheral blood lymphocytes cultured for 72 hours, using Trypsin-Giemsa (GTG) banding. In all cases, at least 30 metaphases were analyzed and 2 karyotypes were prepared, using light microscopy. The statistical analysis was performed using the Student t-test for normally distributed data and the Mann-Whitney test for non-parametric data. The Kruskal-Wallis test or Analysis of Variance was used to compare the mean values between three or more groups. The software used was Statistical Package for the Social Sciences (SPSS), version 17.0. RESULTS: The frequency of chromosomal abnormalities in women with recurrent miscarriages was 7.3%, including 4.7% with X-chromosome mosaicism, 2% with reciprocal translocations and 0.6% with Robertsonian translocations. A total of 2.1% of the partners of women with recurrent miscarriages had chromosomal abnormalities, including 1% with X-chromosome mosaicism and 1% with inversions. Among the controls, 1% had mosaicism. CONCLUSION: An association between chromosomal abnormalities and recurrent miscarriage in the first trimester of pregnancy (OR=7.7; 95%CI 1.2--170.5) was observed in the present study. Etiologic identification of genetic factors represents important clinical information for genetic counseling and orientation of the couple about the risk for future pregnancies and decreases the number of investigations needed to elucidate the possible causes of miscarriages.

Experimental poisoning by Senecio brasiliensis in calves: quantitative and semi-quantitative study on changes in the hepatic extracellular matrix and sinusoidal cells

Extracellular matrix plays an important role in chronic hepatic lesions and has been studied in experimental intoxication models. However in cattle, studies on chronic disease have focused on the hepatocellular damage and extracellular matrix (ECM) changes are usually overlooked. There are no specific studies on the hepatic ECM in either normal or chronically damaged bovine liver. Thus an experimental model of hepatic toxicity model using Senecio brasiliensis poisoned calves was designed. Senecio brasiliensis contains pyrrolizidine alkaloids which cause either acute or chronic progressive dose dependent liver damage. Five calves were orally fed with 0.38g of dry leaves of S. brasiliensis/kg/day for 24 days. Liver needle biopsy specimens were obtained every 15 days for 60 days. Clinical signs of digestive complications appeared at 3rd week. One calf died on 45th day and four were evaluated up to 60th day. Biopsy samples were processed for routine light microscopy, immuno-histochemistry and transmission electron microscopy. From 30th day on progressive liver damage characterized by hepatocellular ballooning, necrosis, apoptosis and megalocytosis, centrilobular, pericellular and portal fibrosis were seen by light microscopy. Quantitative and semi-quantitative measurements of hepatic ECM components were performed before and after the onset of lesions. Morphometric analysis of total collagen and elastic fiber system was conducted. Total collagen and I and III collagen types progressively increased in throughout the liver of affected calves. Changes in location, amount and disposition of the elastic fiber system were also observed. Then numbers of Kupffer cells were significantly increased at 30th day and total numbers of sinusoidal cells were significantly increased at 45th and 60th days. Liver damage was progressive and irreversible even after the exposure to the plant was discontinued. Severe fibrotic lesions occurred mainly in portal tracts, followed by veno-occlusive and pericellular fibrosis. Collagen types I and III s were present in every normal and damaged liver, with predominance of type I. In affected calves the increase of total collagen and elastic fibers system paralleled the number of total sinusoidal cells.

Sperm morphological and morphometric evaluation in captive collared peccaries (Pecari tajacu)

The aim of this study was to compare different staining methods for the evaluation of sperm morphology by light microscopy and also to describe the morphometry of the entire sperm in collared peccaries (Pecari tajacu). Semen from 10 males was obtained by electroejaculation and evaluated for sperm motility, vigor, and concentration. Semen smears were prepared through three different staining methods: Bengal rose, brome-phenol blue, and eosin-nigrosin. Smears were evaluated under light microscopy and sperm morphologic alterations were determined in percentage. In addition, sperm morphometric analysis was conducted by light microscopy coupled to image analyzer software. The smears stained with Bengal Rose provide the best results for the visualization of the sperm tail, midpiece, and head. The use of eosin-nigrosin stain did not allow an adequate impregnation, and some sperm presented a few contrasts with the background. A higher incidence of bent coiled tails was verified in the use of brome-phenol blue staining (P<0.05). Through morphometric evaluation, it was observed that the tail occupies the greatest proportion (89%) of the sperm which presents a discretely elongated head. According to the results, the use of the Bengal Rose stain is recommended for the morphologic evaluation of the collared peccary sperm.

Plasmodium spp. and Haemoproteus spp. infection in birds of the Brazilian Atlantic Forest detected by microscopy and polymerase chain reaction

In recent years haemosporidian infection by protozoa of the genus Plasmodium and Haemoproteus, has been considered one of the most important factors related to the extinction and/or population decline of several species of birds worldwide. In Brazil, despite the large avian biodiversity, few studies have been designed to detect this infection, especially among wild birds in captivity. Thus, the objective of this study was to analyze the prevalence of Plasmodium spp. and Haemoproteus spp. infection in wild birds in captivity in the Atlantic Forest of southeastern Brazil using microscopy and the polymerase chain reaction. Blood samples of 119 different species of birds kept in captivity at IBAMA during the period of July 2011 to July 2012 were collected. The parasite density was determined based only on readings of blood smears by light microscopy. The mean prevalence of Plasmodium spp. and Haemoproteus spp. infection obtained through the microscopic examination of blood smears and PCR were similar (83.19% and 81.3%, respectively), with Caracara plancus and Saltator similis being the most parasitized. The mean parasitemia determined by the microscopic counting of evolutionary forms of Plasmodium spp. and Haemoproteus spp. was 1.51%. The results obtained from this study reinforce the importance of the handling of captive birds, especially when they will be reintroduced into the wild.

The carotid body of the spontaneous insulin-dependent diabetic rat

The carotid bodies from adult spontaneous insulin-dependent diabetic rats (strain BB/S) were perfusion-fixed at normal arterial blood pressure with 3% phosphate-buffered glutaraldehyde and compared with the organs from control rats (strain BB/Sc) prepared in the same way. Serial 5-µm sections were cut, stained, and using an interactive image analysis system, were analysed to determine the volumes of the carotid body and its vascular and extravascular compartments. There was no evidence of systemic arterial disease in the carotid stem arteries in either group of animals, and the microvasculature of the organs appeared normal by light microscopy. The volume of the carotid body was unchanged 3 months after the onset of diabetes but was increased at 6 months. The total vascular volume of the organ was unchanged, but the volume of the small vessels (5-12 µm) was increased. In the control group the small vessels comprised 5% of the total volume of the carotid body, or about 44% of the vascular compartment. The percentage of small vessels increased at 3 months in the diabetic group, but had returned to normal at 6 months. The extravascular volume followed the same pattern as the total carotid body volume and so did not change appreciably when expressed as a percentage of the total volume of the organ. The increase in size of the carotid body in diabetic rats is due, therefore, to an augmented extravascular volume. In one diabetic specimen the carotid sinus nerve showed signs of diabetic neuropathy, axonal swelling and intramyelinic oedema. The clinical implications of these results are discussed.

Probable effect of photoperiod on seasonal variation in the nuclear volume of the adrenal cortex of viscacha (Lagostomus maximus maximus)

The neuroendocrine system regulates several organic functions such as reproduction, metabolism and adaptation to the environment. This system shows seasonal changes linked to the environment. The experimental model used in the present study was Lagostomus maximus maximus (viscacha). The reproduction of males of this species is photoperiod dependent. Twenty-four adult male viscachas were captured in their habitat at different times during one year. The adrenal glands were processed for light microscopy. Serial cuts were stained with hematoxylin-eosin for the morphometric study, and 100 nuclei of each zone of the adrenal cortex were counted per animal. Data were analyzed statistically by ANOVA and the Tukey test. The cells of the glomerulosa zone are arranged in a tube-shaped structure. The fasciculata zone has large cells with central nuclei and clearly visible nucleoli and with a vacuolar cytoplasm. In the reticularis zone there are two of types of cells, one with a nucleus of fine chromatin and a clearly visible nucleolus and the other with nuclear pycnosis. Morphometric analysis showed maximum nuclear volumes during the February-March period with values of 133 ± 7.3 µm3 for the glomerulosa, 286.4 ± 14.72 µm3 for the fasciculata, and 126.3 ± 9.49 µm3 for the reticularis. Minimum nuclear volumes were observed in August with values of 88.24 ± 9.9 µm3 for the glomerulosa, 163.7 ± 7.78 µm3 for the fasciculata and 64.58 ± 4.53 µm3 for the reticularis. The short winter photoperiod to which viscacha is subjected could inhibit the adrenal cortex through a melatonin increase which reduces the nuclear volume as well as the cellular activity.

Supraorganized collagen enhances Schwann cell reactivity and organization in vitro

We investigated the reactivity and expression of basal lamina collagen by Schwann cells (SCs) cultivated on a supraorganized bovine-derived collagen substrate. SC cultures were obtained from sciatic nerves of neonatal Sprague-Dawley rats and seeded on 24-well culture plates containing collagen substrate. The homogeneity of the cultures was evaluated with an SC marker antibody (anti-S-100). After 1 week, the cultures were fixed and processed for immunocytochemistry by using antibodies against type IV collagen, S-100 and p75NTR (pan neurotrophin receptor) and for scanning electron microscopy (SEM). Positive labeling with antibodies to the cited molecules was observed, indicating that the collagen substrate stimulates SC alignment and adhesion (collagen IV labeling - organized collagen substrate: 706.33 ± 370.86, non-organized collagen substrate: 744.00 ± 262.09; S-100 labeling - organized collagen: 3809.00 ± 120.28, non-organized collagen: 3026.00 ± 144.63, P < 0.05) and reactivity (p75NTR labeling - organized collagen: 2156.33 ± 561.78, non-organized collagen: 1424.00 ± 405.90, P < 0.05; means ± standard error of the mean in absorbance units). Cell alignment and adhesion to the substrate were confirmed by SEM analysis. The present results indicate that the collagen substrate with an aligned suprastructure, as seen by polarized light microscopy, provides an adequate scaffold for SCs, which in turn may increase the efficiency of the nerve regenerative process after in vivo repair.

Experimental model of intervertebral disc degeneration by needle puncture in Wistar rats

Animal models of intervertebral disc degeneration play an important role in clarifying the physiopathological mechanisms and testing novel therapeutic strategies. The objective of the present study is to describe a simple animal model of disc degeneration involving Wistar rats to be used for research studies. Disc degeneration was confirmed and classified by radiography, magnetic resonance and histological evaluation. Adult male Wistar rats were anesthetized and submitted to percutaneous disc puncture with a 20-gauge needle on levels 6-7 and 8-9 of the coccygeal vertebrae. The needle was inserted into the discs guided by fluoroscopy and its tip was positioned crossing the nucleus pulposus up to the contralateral annulus fibrosus, rotated 360° twice, and held for 30 s. To grade the severity of intervertebral disc degeneration, we measured the intervertebral disc height from radiographic images 7 and 30 days after the injury, and the signal intensity T2-weighted magnetic resonance imaging. Histological analysis was performed with hematoxylin-eosin and collagen fiber orientation using picrosirius red staining and polarized light microscopy. Imaging and histological score analyses revealed significant disc degeneration both 7 and 30 days after the lesion, without deaths or systemic complications. Interobserver histological evaluation showed significant agreement. There was a significant positive correlation between histological score and intervertebral disc height 7 and 30 days after the lesion. We conclude that the tail disc puncture method using Wistar rats is a simple, cost-effective and reproducible model for inducing disc degeneration.