Movable computer ruler (MCR): a new method for measuring the size of Toxoplasma gondii cysts, tachyzoites and other selected parasites.

A new method for measuring the size of parasites and other objects using optical microscopy was developed using a specifically designed movable computer ruler (MCR) derived from digital images of a stage micrometer. Subsequently, MCR can be superimposed on images of parasites to measure their size. MCR derived from the stage micrometer under a particular objective lens can be used to measure the size of an object acquired by the same lens/microscope/camera system. The conditions are fixed for every superimposed image including width, height, pixel number and density. The MCR was tested using selected parasites, and shown to be as accurate as the ocular micrometer disk, screw micrometer eyepiece and image analysis software. The lower technical complexity of the MCR method makes it applicable even in laboratories with limited resources.

Immune response and performance of growing Santa Ines lambs to artificial Trichostrongylus colubriformis infections.

This study was carried out to evaluate the immune response and the impact of Trichostrongylus colubriformis infections on the performance of growing Santa Ines lambs. Thirty male lambs, 3-4 months of age, were maintained in individual pens and restrictively randomised by weight into 3 treatment groups: (1) infected group, artificially infected with 2500 T. colubriformis larvae, three times a week, for 13 weeks, and fed ad libitum; (2) Pair-Fed Group, non-infected and fed with the same amount of food consumed by the infected animal of the same class on the previous day; and (3) control group, non-infected and fed ad libitum. Refused feed was weighed daily to assess the food intake of each lamb. Animals were weighed weekly and blood and fecal samples were collected. At the end of the trial, all lambs were euthanized to determine worm burden and collect intestinal tissues and mucus samples for histological and immunological analysis. The infected group presented eosinophilia, increased number of inflammatory cells in the mucosa, in addition to an increased production of specific immunoglobulins against T. colubriformis, which partially prevented the establishment of infective larvae. As a consequence of parasitism, the infected lambs presented reduced serum albumin concentrations and demonstrated severe small intestine lesions, such as villous atrophy and epithelial erosion, which impaired the digestion and absorption of nutrients, causing a significant loss in performance. The infected group presented a 37% reduction in daily weight gain (107.26 ± 10.8 g/day), when compared with the control group (171.07 ± 7.15 g/day). The infected lambs also demonstrated the worst food conversion, i.e., each animal needed to consume on average 10.05 ± 0.52 kg of food to gain 1 kg live weight. The voluntary hay intake depression in infected animals was small, and statistical difference (P<0.01) was seen only on two occasions (ninth and 12th weeks), in comparison with the control group. In conclusion, Santa Ines lambs infected with T. colubriformis demonstrated a reduction in performance, caused by the damages produced by the adult nematodes in intestinal mucosa, and also by the immunopathological changes associated with the infection.

Development of morphological changes and ileal glucagon gene expression in the small intestine of lambs infected with Trichostrongylus colubriformis.

To investigate the consequences of subclinical Trichostrongylus colubriformis infection on the intestinal mucosa and the associated changes in entero-glucagon gene expression, sheep were infected with 30000 larvae and killed 5, 10, 15 or 20 days after infection. Histological and cytological changes were examined. In the main site of infection, the upper duodenum, villous atrophy associated with crypt hyperplasia developed gradually. Cytological changes in the enterocytes appeared concurrently, characterized by a progressive reduction in brush border and in the number of ribosomes in the cytoplasm, changes in the internal structure of mitochondria, and enlargement of the intercellular spaces. Neither histological nor cytological modifications were found before day 15. At the same time, villous hypertrophy developed distally, beyond the main site of infection; this was interpreted as an adaptive response to parasitism. Enteroglucagon gene expression in the ileum was measured in parallel with the mucosal changes but did not reveal any difference between infected and control sheep. The results indicate that this gastrointestinal hormone does not have a major role in the response to nematode parasitism.

Digital image analysis and identification of eggs from bovine parasitic nematodes.

Computer-assisted microscopy and multivariate statistics were used to establish and evaluate a procedure for identification of bovine strongylid eggs. Ostertagia ostertagi, Cooperia oncophora, Haemonchus placei, Trichostrongylus axei, and Oesophagostomum radiatum eggs were obtained from faeces voided by monospecifically infected calves. Images of single eggs (400 x magnification) were recorded by a CCD camera fitted onto a microscope and digitized on a PC. After separation of eggs from the image background, the pixel (picture element) positions of the egg outline were analysed by algorithms to describe size and shape. A stepwise discriminant analysis was subsequently used to select and rank descriptive features of 4207 eggs according to discriminatory power. Classification criteria were developed by linear discrimination analysis on the basis of selected features, and the criteria evaluated by cross-validation. A maximum average percentage of correct classification of 85.8% resulted when nineteen features were employed in a linear classification criterion. The percentages correct classification for each species were: O. ostertagi 76.3%, C. oncophora 90.8%, O. radiatum 87.8%, H. placei 90.1%, and T. axei 83.8%. Classification based on the five most important features gave an overall correct classification of 81.5%. Images of "unknown' eggs could be identified automatically by the classification criteria after procedural steps performed by PC were linked in a batch program.

Bilateral, perivulval cuticular pores in trichostrongylid nematodes.

A new hypodermal gland was discovered in female nematodes of the family Trichostrongylidae. Because the new structure appears to be associated with the vulva, it was named the perivulval pore. It is similar, based on light and scanning electron microscopy, to phasmids that are located laterally on the tails of nematodes of the class Secernentea. Like phasmids, perivulval pores are paired and bilateral, with cuticular ducts to the surface in the areas of the lateral chords. They are located slightly posterior to the vulva in Haemonchus contortus, Haemonchus placei, Haemonchus similis, Mecistocirrus digitatus, Mazamastrongylus pursglovei, Ostertagia ostertagi, and Cooperia oncophora, but in Trichostrongylus colubriformis they are slightly anterior to the vulva. Because of the location near the vulva and the similarity in structure to phasmids, which are, at least in part, secretory, the perivulval pores should be considered as a possible source of a female attractant for males.

Comparative ultrastructure of the cuticle of trichostrongyle nematodes.

The ultrastructure of the cuticle was examined in Austrostrongylus victoriensis, Patricialina birdi and Woolleya monodelphis (Herpetostrongylidae) from marsupials, Paraustrostrongylus ratti (Herpetostrongylidae) from rodents, Nippostrongylus magnus and Odilia bainae (Heligmonellidae) from rodents, Cooperia oncophora and Camelostrongylus mentulatus (Trichostrongylidae) from ruminants, and Nematodirus spathiger (Molineidae) from ruminants. The principal cuticular layers described previously were present in all species investigated. Major differences in the shape and composition of cuticular struts were observed as well as differences in components of the median zone of the cuticle, including the fluid-filled regions present in several species. Several different types of strut were observed. Although strut structure within the Heligmonellidae appeared to be constant, there were variations within both the Herpetostrongylidae and Trichostrongylidae. In Nem. spathiger the cuticular ridges lacked struts. The diversity of structures found in the species examined suggests that more extensive comparative studies of the trichostrongyle cuticle are warranted.

Freeze-substitution techniques for preparing nematodes for scanning electron microscopy.

The effect of different substitution times, temperatures and the incorporation of fixatives on the preservation of three species of nematode for scanning electron microscopy by freeze substitution with methanol, followed by critical point drying, is investigated. Hammerschmidtiella diesingi adults and Trichostrongylus colubriformis infective juveniles were successfully preserved using methanol at 253 K as the substitution medium. Preservation deteriorated with long substitution times, suggesting the extraction of material and that substitution times should be kept as brief as possible. Panagrolaimus davidi was not successfully preserved using pure methanol, but preservation was improved by using fixatives in the substitution medium, the best results being obtained with 1% OsO4/3% glutaraldehyde in methanol. A substitution temperature of 193 K did not give any improvement in preservation. The differences in the quality of preservation between the three species may be due to the relative ability of the cuticle to withstand collapse during critical point drying. Chemical fixation using cold fixative resulted in the retention of a natural posture but poor preservation, whereas hot fixatives resulted in good preservation but the loss of a natural posture. Freeze substitution in methanol may prove useful in the preparation of specimens possessing cuticles or cell walls which have sufficient strength to withstand the drying process (e.g. arthropods, plants, fungi, nematodes). More delicate specimens may require the incorporation of fixatives into the substitution medium or conventional fixation.

Capture and penetration processes of the free-living juveniles of Trichostrongylus colubriformis (Nematoda) by the nematophagous fungus, Arthrobotrys oligospora.

The nematode-trapping fungus Arthrobotrys oligospora traps and invades all the free-living juvenile stages of the trichostrongyle nematode Trichostrongylus colubriformis. The processes of capture and invasion of the 1st-stage juveniles are described using differential interference contrast optics and 3-D reconstruction techniques. The adhesive responsible for capture is well preserved using a freeze-substitution technique for scanning electron microscopy. The invasion process of the ensheathed 3rd-stage juvenile of T. colubriformis takes much longer than in the 1st- or 2nd-stage juvenile and involves the formation of secondary infection pegs between the sheath and the cuticle which appear to penetrate the cuticle by physical pressure.

The formation of the first-stage cuticle within the egg of Trichostrongylus colubriformis.

The ultrastructure of the formation of the first-stage cuticle within the egg of Trichostrongylus colubriformis is described. The first indication of cuticle formation was the formation of the epicuticle external to the epidermal plasma membrane. This becomes thickened to form the epicuticle of the fully formed cuticle. Microtubules and microfilaments associated with the epidermal plasma membrane were observed during the early phases of cuticle formation. The basal zone was secreted into the space between the epicuticle and the epidermal plasma membrane, followed by the formation of the cortical zone. The basal zone had a striated appearance during the earliest stages of its formation.

Primary and secondary infection of the domestic chicken with Trichostrongylus tenuis (Nematoda), a parasite of red grouse, with observations on the effect on the caecal mucosa.

The course of primary and secondary infections with Trichostrongylus tenuis in the domestic chicken was investigated. Primary infections were established after the administration of single and trickle doses of infective-stage larvae. The worm burden in the caeca was highest after a single dose of 500 infective-stage larvae; this gave a mean of 87 nematodes per bird on days 89 of infection, 20 nematodes on day 14 and 0 on day 28 of infection. Following trickle doses of 60, 100, 200, 300, 400 or 500 infective-stage larvae, there was a rise and then a fall in nematode egg output in all groups. In chickens given a primary dose of 500 infective-stage larvae followed 30 days later by a single secondary dose of 500 infective-stage larvae, the mean worm burden during the secondary infection rose to 57 nematodes on day 9 of infection and then fell rapidly to 18 nematodes on day 15 and to 2 on day 30. Scanning electron microscopy showed changes in the caeca of infected birds, with the caecal surface being covered in a layer of mucus from 12 days after infection. Ball of blood-stained mucus containing nematodes were observed in the caecal droppings from day 9 of infection onwards. It is concluded that chickens rapidly expel an established infection of T. tenuis, unlike the normal host, the red grouse.

Primeros casos de trichostrongilosis humana en la Argentina: diagnóstico diferencial; ciclo evolutivo de las larvas de Trichostrongylus / First cases of human trichostrongylosis in the Argentine: Differential diagnosis. Evolution cycle of the Trichostrongylus larvae

Se informa de los primeros casos de infestaciones humanas por Trichostrongylus sp. en la Argentina. Con el objeto de identificarlas se realizaron mediciones micrométricas referentes a tamaños de huevos, larvas rhabditoideas y larvas strongyloideas encapsuladas. Se hicieron tomas microfotográficas de preparaciones de heces de pacientes infestados y de los cultivos de larvas de esas muestras. Se muestran, el huevo característico del género Trichostrongylus, algunas anomalías, huevos en estado de mórula avanzado y con el embrión formado. Además de los caracteres relevantes de la larva rhabditoidea, su cavidad bucal, el bulbo esofágico, luz intestinal en zigzag, dilatación del extremo caudal y la larva Strongyloidea infestante. Se señalan las precauciones que debieran tomarse para no confundir los huevos de Trichostrongylus sp. con huevos grandes de Uncinarias y huevos de Heteroderidae y Tiroglyphus. En 65.000 muestras analizadas se encontraron once casos de Trichostrongylosis (AU)

Primeros casos de trichostrongilosis humana en la Argentina: diagnóstico diferencial; ciclo evolutivo de las larvas de Trichostrongylus / First cases of human trichostrongylosis in the Argentine: Differential diagnosis. Evolution cycle of the Trichostrongylus larvae

Se informa de los primeros casos de infestaciones humanas por Trichostrongylus sp. en la Argentina. Con el objeto de identificarlas se realizaron mediciones micrométricas referentes a tamaños de huevos, larvas rhabditoideas y larvas strongyloideas encapsuladas. Se hicieron tomas microfotográficas de preparaciones de heces de pacientes infestados y de los cultivos de larvas de esas muestras. Se muestran, el huevo característico del género Trichostrongylus, algunas anomalías, huevos en estado de mórula avanzado y con el embrión formado. Además de los caracteres relevantes de la larva rhabditoidea, su cavidad bucal, el bulbo esofágico, luz intestinal en zigzag, dilatación del extremo caudal y la larva Strongyloidea infestante. Se señalan las precauciones que debieran tomarse para no confundir los huevos de Trichostrongylus sp. con huevos grandes de Uncinarias y huevos de Heteroderidae y Tiroglyphus. En 65.000 muestras analizadas se encontraron once casos de Trichostrongylosis

The structure of the cuticle and sheath of the infective juvenile of Trichostrongylus colubriformis.

The infective third-stage juvenile of Trichostrongylus colubriformis is surrounded by its own cuticle as well as the incompletely moulted cuticle of the second-stage juvenile, which is referred to as the sheath. The sheath comprises an outer epicuticle, an amorphous cortical zone, a fibrous basal zone and an inner electron-dense layer. The basal zone of the sheath consists of three layers of fibres; the fibres are parallel within each layer, but the fibre direction of the middle layer is at an angle to that of the inner and outer layers. The cuticle comprises a complex outer epicuticle, an amorphous cortical zone and a striated basal zone. The lateral alae of the cuticle and the sheath are aligned and overlie the lateral hypodermal cords. The lateral alae of the sheath consist of two wing-like expansions of the cortical zone with associated specializations of the inner electron-dense layer which form a groove. The cuticular lateral alae consist of two tube-like expansions of the cortical zone. The lateral alar complex of the cuticle and the sheath may maximise locomotory efficiency and prevent rotation of the juvenile within the sheath.

Tubulin and benzimidazole-resistance in Trichostrongylus colubriformis (Nematoda).

Benzimidazole treatment produced greater effects on microtubule-dependent acetylcholinesterase secretion, the presence of microtubules in intestinal cells, and colchicine binding in susceptible compared with benzimidazole-resistant Trichostrongylus colubriformis. In addition, the binding of benzimidazoles was markedly reduced in preparations from the latter strain, indicating that the mechanism of resistance to benzimidazoles in this nematode involves a reduced affinity of tubulin for benzimidazoles.