Saturday, January 19, 2013
Phylum Aschelminthes
Phylum Aschelminthes
Six different classes, dominated by the Class Nematoda, represent Aschelminthes as a phylum. The nematodes (Nema, thread) or roundworms are ubiquitous small worms that dwarf in numbers all other groups of multicellular animals except insects and mites.
Aschelminthes are pseudocoelomate, unsegmented worms that are covered with a cuticle. An unusual aschelminth trait is parthenogenesis (development of an unfertilised egg). In the classes Nematoda, Rotifera and Gastrotricha, males may be lacking and successive generations of females are produced parthenogenetically
Still another remarkable characteristic of the phylum is the consistency of cell numbers (eutely), in which a precise and relatively small number of cells, remaining unvaried throughout the animal’s life, comprise both specific organs and the entire animal. The number, constant not only for the species but also for the taxonomic group, can be used as part of its morphological definition (for example, the central nervous system in Ascaris and many other nematodes consists of 162 cells)
Class Nematoda
The class Nematoda is very important to us among the group as it consists of parasites of medical and veterinary importance. The class is characterized by the presence of a complete digestive system, consisting of a mouth, intestine, anus and absence of cilia.
Nematodes are found practically in every ecological setting. They live in animals and plants as parasites and in mud, marine, fresh and brackish waters, soil and mud as free-living organisms. They vary in size from a few microns to slightly over a meter in length.
Morphology
Nematodes are cylindrical in shape, tapering at both ends of the body. The digestive system is a long tube that runs from the buccal cavity to the anus. In hookworms, the mouth is armed with teeth or cutting plates. Some nematodes are provided with lips, papillae or a leaf crown around the external opening of the mouth. The pharynx (oesophagus) is a strong muscular tube with walls that can contract and expand, creating a pump-like suction mechanism. The neuromuscular system consists of a circumesophageal ring or nerve ring around the oesophagus and dorsal and ventral nerve cords, which give rise to motor and sensory nerves. The excretory organs lie within the lateral lines and open via a pore ventral to the oesophagus.
Internal structure of a nematode
The gonads lie in the pseudocoele, a space between the intestine and the body wall that is filled with a fluid. The rugged cuticle is not only an extraordinary effective protection against harmful external substances but also serves as an exoskeleton to which muscles are attached.
Female nematodes have a pair of ovaries consisting of cells (oogonia) that produce the eggs. The uteri are usually packed with eggs. The eggshell material is produced by the uterine cells. The uteri unite to form the vagina, which may contain a seminal vesicle for storing male sperm. The vagina opens to the outside by a pore or vulva at the middle of the body, though it may also be near either end of the body.
The male genital system consists of one testis and a seminal vesicle, which continues posteriorly as a muscular ejaculatory duct that opens into the rectum or cloaca. The cloaca serves as a conduit for sperm and digestive wastes. Copulatory spicules protrude through the cloaca opening. The spicules, usually two in number, unite and form a tube through which spermatozoa are injected into the vulva and vagina of the female.
Generalized structure of male (A) and female (B) nematode
Nematodes are classified into two subclasses: Phasmidia (have caudal sensory organs) and Aphasmidia (lack caudal sensory organs). The phasmids include most soil nematodes, most parasites of insects and vertebrates. The aphasmids are mainly aquatic forms and a few parasitic ones.
Life cycle
Life cycles differ greatly among the many nematodes that are parasites of man. Mature female worms produce eggs, which pass out of the body with faeces. Most of the eggs are produced when they are not embryonated and become embryonated in the soil. The embryonated eggs are ingested and under the action of the gastric juices of the host, the larvae are liberated from the eggs. In some parasites, such as Ascaris, the larvae penetrate the wall of the small intestine and migrate to the lungs via the blood. From the lungs, they reach the pharynx and finally settle in the small intestine, where they will attain maturity, mate and produce eggs.
In other nematodes, such as hookworms, the larvae hatch from eggs in the soil and after going through several moults become infective. These third stage infective forms enter the body through the skin. Trichinella has a unique life cycle in nematodes in that the entire life cycle is spent in the host. Transmission from one host to another is through predation, cannibalism or carrion feeding.
Microscopic Techniques
Microscopic Techniques
Most parasitic agents occur within the gastrointestinal region, body tissues and blood system. Most helminth parasites (nematodes, trematodes, cestodes) and some protozoa inhabit the intestine as well as the bile and pancreatic ducts that empty into the intestinal lumen. The helminths produce characteristic eggs, which pass out in faeces that are used for identification. Either the protozoa exist as active trophozoites or as inactive cysts and their presence in a stool sample is an indication of an infection.
Some important stains for blood films
Method for cleaning microscope slides
Slides used in laboratories for identification purposes should be clean from dust and grease. Films do not stick well if the slides are dirty and greasy and stained artefacts may give the wrong results. There are several ways of cleaning blood slides but the most practical method is the one that uses Decon
Make up a 2 – 3 % solution of Decon in hot water.
Place slides into the solution individually.
Allow slides to stand in solution for at least ½ hour (1 – 2 hours is better).
Rinse thoroughly in running tap water for an hour.
Rinse briefly in distilled water.
Store slides in ethanol – dry as required or dry and store in clean boxes.
Giemsa’s Stain
This stain can be used in different dilutions. A one in ten dilution is convenient, as it gives rapid staining, although the time varies according to the quality of the stain.
The stain can be used for thin films and thick films.
Method for the thin films
Pour 2 ml of Giemsa into a measuring cylinder and make up to 20 ml with distilled water. Empty the dilute stain into a staining jar.
Fix the blood film by immersing it in methyl alcohol for 2 – 3 minutes.
Wash the slide containing the film in distilled water.
Drain off the water and transfer the slide into a staining jar containing Giemsa.
Stain for 30 – 60 minutes.
Remove the slide and rapidly wash it in distilled water.
Wipe off stain from the back of the slide and leave it in an upright position to dry.
Examine in immersion oil at 100 % magnification.
Method for thick films
Prepare the final stain as for the thin film.
Without any fixation, place the slide in the stain.
Leave for 15 – 30 minutes in the stain (by agitating the staining jar gently from time to time, haemoglobin is removed from the area of the film).
Remove the slide from the stain and wash in distilled water.
Let it dry
Leishman’s Stain
Method for thin films
Place the slide on a staining rack over sink or dish, with the film facing uppermost.
Cover the film with 12 drops of the stain and leave for 1½ minutes (the methyl alcohol in the stain fixes the film).
Add double the quantity of freshly distilled water.
Mix the solution thoroughly with a pipette or by rocking very gently the slide, taking care not to spill the stain.
Allow to stain for 15 to 20 min, the actual time depending on the quality of the stain. The film should be quite a strong pink colour, when sufficiently stained. The colour is then reduced or the film differentiated by washing with distilled water.
Flood the slide with distilled water, then tip and allow the water to run off. Continue to wash with distilled water until the film is a rather pale pink.
Stand the slide in an upright position to drain and dry. Heat must not be used to dry films but the film can be gently dabbed with blotting paper and waved in the air to expedite drying.
Examine in immersion oil at 100% magnification.
Method for thick films
Thick films must be treated very gently at all stages, as they are not firmly attached to the slides.
Place the slide film side downwards in a dish (or stand it upright in ajar) containing tap or distilled water.
Leave until all the haemoglobin has been removed (2 to 3 min) and the film has become completely white.
Stand the slide in an upright position to drain and dry.
Proceed as for staining thin films.
Field’s stain for thick films
Field’s stain is used for rapid diagnostic and identification purposes. For thin films, fix in methyl alcohol for 2 to 3 minutes prior to staining.
Preparation
A. Methylene blue (medicinal) 0.4 g
Azur 0.25 g
Buffered water (B), pH, and 6.8 – 7.0. 250 ml
B. Disodium hydrogen phosphate 10.0g
Potassium dihydrogen phosphate 12.5 g
Distilled water 1000 ml
C. Eosin 0.5 g
Buffered water (B) 250 ml
Method
Dip slide in A 1- 2 sec
Rinse in B 2- 3 sec
Dip in C 1- 3 sec
Rinse gently in tap water 2- 3 sec
Place slide upwards to drain and dry
Examination of Stool for Intestinal Protozoa
Direct Method
Place a fresh sample of stool, about the size of a bean seed, on a slide, add a drop or two of physiological saline, cover with a cover slip, and examine at a magnification 400 - 500X. This technique should reveal the motile forms of intestinal flagellates.
To identify the cysts and the nuclei of Amoeba, one or two drops of a 4% Lugo
Examination of Stool for Worm Eggs
a) Direct Examination
A sample of faeces, about the size of a bean seed, is placed on a slide, mixed with tap water or physiological saline, covered with a cover slip, and examined at low microscope magnification (100 to 200X).
The smear should not have lumps of faeces to obscure the eggs. A drop of Lugo
b) Thick Smear Technique (Kato and Miura)
Preparation of the Kato stain
Distilled water 500 ml
Glycerine 500 ml
Malachite green, 3% in water 5 ml
Place about 100 mg of faeces on a slide and cover it with a cellophane strip (size 26 x 28 mm), which had been soaked in the Koto Stain for at least 24 hr. Press over the strip with a spatula to have a thin uniform spread of the faeces sample. Allow the preparation to stay at room temperature for ½ to 1 hr. Examine the stool. The glycerine clears the faeces and helminth eggs can be seen at a magnification of 100x.
c) Concentration Method
Place 1 g of stool in a test tube and thoroughly mix it with a concentrated salt solution. Remove any plant particles floating on the surface and allow the mixture to remain undisturbed for 20 to 30 min.
Helminth eggs float on the surface because they are lighter than the salt solution, Transfer the eggs to a microscope slide using a wire loop. Examine at a magnification of 100-200x.
d) Universal Concentration Method
Place 1 g of faeces in a small beaker, add 7 ml of 50% hydrochloric acid and the same amount of ether. Mix the mixture to form a homogeneous solution. Pass the mixture into a centrifuge tube over two layers of muslin placed in a funnel. Centrifuge the solution for a minute or two.
Four layers are formed: the uppermost layer is a yellowish zone of ether below which is a zone consisting of food particles and below this is a zone of hydrochloric acid. The bottommost zone consists of small particles and worm eggs. Carefully insert a pipette into the bottom layer, suck out liquid, and examine it for eggs. Be careful, ether is very volatile and can explode!
Sedimentation Concentration Stool Examination Technique
Add an estimated 5 g of stool to 100 ml of formol glycerol solution (5 ml formaldehyde, 10 ml glycerol, 985 ml of water) in a flask and mix with a glass rod.
Sieve through a mesh into another flask.
Add more formol glycerol up to 3 cm below the top and allow to sediment for a minimum of 20 min at room temperature.
Pour off the supernatant to leave approximately 25 to 30 ml of fluid containing sedimented deposit
Resuspend in formol glycerol and sediment for 20 minutes.
Gently pour off supernatant to leave 15 to 20 ml of suspended deposit.
Use straw to transfer about 0.1 ml of the deposit to each of 3 microscope slides and cover each slide with a cover slip.
ExamineCoccidiosis
Coccidiosis
The family Eimeriidae consists of hundreds of sporozoan organisms that are parasitic to a variety of vertebrates. Coccidiosis is a common name for the diseases due to these sporozoan parasites. Coccidiosis has been reported in chickens, geese, ducks, turkeys, cattle, sheep, rabbits, pigs, fish, reptiles, horses, dogs, and cats, including man.
Eimeria is characterised by the presence of four sporocysts in each spore. The sporocysts occur primarily in the intestinal cells of the host.
Life cycle
Infection is due to ingestion of oocysts by birds. The oocysts are extremely resistant to adverse conditions and will remain viable in the soil for a long time, sometimes even up to more than 15 months. They are minute in size, measuring about 10 to 30 microns. Oocysts are so light that they can be dispersed by wind.
Following ingestion by a bird, the oocysts rupture in the digestive tract and liberate sporocysts that contain sporozoites. The sporozoites invade the cells of the host’s intestinal epithelium where they divide by schizogony to form merozoites.
The infected epithelial cells rupture, releasing more merozoites, which in turn invade more epithelial cells. The process continues until the intestinal wall is severely eroded by the infection.
Some of the merozoites become sex cells. Fusion of these sex cells or gametocytes leads to the formation of oocysts that are passed out in the faeces.
Pathogenesis
E. tenella is a parasite of the caecum of mainly young birds that are under four weeks old. Older birds are increasingly resistant to infection. The severity of the infection depends on the initial dose of the oocysts ingested by the bird. The greater the dose the more serious is the infection. A dose of 200000 oocysts is enough to kill 1 to 2 week old chicks. Death is usually due to severe haemorrhage of the caecal epithelium evident by the fifth day of the infection.
Infection in the young nonimmune chicks is rapid and serious. Spots of blood appear in the faeces around day 5 after infection. By day 7, the faeces are watery and contain oocysts. The birds show signs of loss of appetite and energy, looking very drowsy. About 90% of them die during the first 7 days of infection. E. necatrix causes a milder, chronic infection than E. tenella.
Bovine coccidiosis is not a serious disease but it can kill calves if the infection is quite heavy. The effects of infection in cattle are weight loss, slow growth and a reduction in milk production.
Clean, uncrowded, dry living quarters are of great importance in controlling the infection in chickens and livestock.
Isospora belli and Isospora natalensis are two eimerian parasites of man. They invade the small intestine and shed sporocysts that are detected in the faeces of an infected person. Symptoms are usually not serious but include abdominal pain, diarrhoea, flatulence, abdominal cramps, nausea, loss of appetite, lassitude, loss of weight and fever.
Theileria
Theileria are tick-transmitted protozoan parasites of domestic and wild ruminants. Theileria parva and Theileria annulata are members of this genus that cause debilitating and often fatal disease in cattle. In eastern, central and southern Africa , the most important species is Theileria parva, which causes East Coast fever. In addition to cattle, T. parva infects African buffalo and waterbucks, both of which serve only as reservoirs of infection.
T. parva threatens the lives of over 25 million cattle and is a severe economic constraint to cattle farming in Burundi Kenya Malawi Mozambique Rwanda Sudan Tanzania Uganda Democratic Republic of the Congo Zambia Zimbabwe
The existence of three subtypes of T. parva – Theileria parva parva, Theileria parva bovis and Theileria parva lawrenceicomplicates the search for better control methods. All three are transmitted by the brown ear tick, Rhipicephalus appendiculatus, but only two – T. p. parva and T. p. lawrencei produce severe disease in cattle.
T. p. parva and T. p. bovis are transmitted between cattle. T. p. lawrencei is transmitted to cattle mainly from buffaloes, which act only as reservoirs and do not show any clinical symptoms of theileriosis.
Life cycle
The life cycle of T. parva is complex. In both the tick and the mammalian host, the parasite undergoes a series of cellular transformations into different forms. Upon ingestion by R. appendiculatus tick, the parasite undergoes differentiation first in the tick gut, leading to the formation of forms called kinetes. The kinetes migrate to the tick’s salivary glands, where they differentiate into infective sporozoites.
As the tick feeds on cattle, the sporozoites are injected into the blood along with tick saliva. In the host, the parasites attach to and enter lymphocytes. Within two to three days of invading the lymphocytes, the sporozoites develop into intracellular forms called schizonts. The infected lymphocytes grow bigger and begin to divide. These enlarged lymphocytes are known as lymphoblasts. The division of the lymphocytes ensures that each daughter cell is infected. In the end, large numbers of lymphocytes are infected so that the infection spreads throughout the lymphatic system, leading to the widespread destruction of the lymphatic cells.
In the later stages of the infection, some of the schizonts differentiate into merozoites that are released into the bloodstream where they invade red blood cells. In the red cells, the parasites change into forms that are called piroplasms, which are infective to ticks. As the ticks feed on infected animals, they ingest red blood cells that are infected with piroplasms, and this completes the life cycle. The incubation period is usually 10 to 25 days
Pathogenesis and symptomatology
Typical clinical symptoms associated with theileriosis include inflammation and swelling of the lymph nodes, followed by generalized lymphadenopathy, fever, anorexia, and rapid deterioration of condition. Other symptoms include lacrimation, nasal discharge and corneal opacity, an increased respiratory rate and diarrhoea. Death is usually due to pulmonary oedema, severe dyspnoea and discharge. Some animals develop a fatal disease called ‘turning sickness’, in which infected cells block capillaries in the CNS and cause neurological signs. Some animals recover from the infection and become asymptomatic carriers; others may have poor productivity while others may become stunted in growth.
Post-mortem examination shows extensive haemorrhages in different tissues and body organs, they lymphoid system is greatly swollen but may be shrunken in chronic cases, the liver and the spleen are enlarged. The lungs are reddened and both the trachea and bronchi are filled with fluid and froth.
The main effect of theileriosis on the host is the extensive destruction of the lymphatic cells by multiplying schizonts.Morbidity and mortality vary with host’s susceptibility and the strain of parasite. The mortality can reach 100% in susceptible cattle from nonendemic areas in three to four weeks of infection. However, mortality is low in indigenous zebu cattle in endemic areas. Although animals that recover from the infection acquire immunity that may last for a long time, some of the animals act as reservoirs of infection, or show low productivity and appear stunted.
Diagnosis
Theileria should be suspected in tick-infested animals that present with fever and enlarged lymph nodes, and where mortality seems to affect mainly calves. Diagnosis is made by identification of the schizonts in thin blood smears, lymph node and liver biopsies. At necropsy, schizonts may be found in impression smears from internal organs.
Polymerase chain reaction (PCR) tests and DNA probes are sometimes used to identify Theileria species. Antibodies to T. parva, can be detected with an enzyme-linked immunosorbent assay (ELISA) and other immunological tests.
In areas where East Coast fever is endemic, dipping or spraying cattle with an acaricide to kill the ticks is a sure way of controlling the disease. The dipping or spraying of the animals must be carried out regularly. Because cattle that are regularly treated with acaricide are not exposed to T. parva, they develop no immunity and thus have no protection against the parasite or other tick-borne diseases if treatment is interrupted. Regular application of the acaricides has its drawbacks- it is environmentally unfriendly and can lead to emergence of insecticide resistant ticks.
Treatment of theileriosis is possible with drugs and vaccines.
Babesia
Piroplasms are protozoan parasites that include Babesia and Theileria. These intracellular tick-borne transmitted diseases are of great economic importance because they are responsible for serious losses in domestic livestock, particularly cattle.
Babesiosis
Babesia bigemina and Babesia bovis are economically important parasites of sheep, cattle, goats, horses, pigs, dogs and cats and are responsible for a disease called babesiosis (redwater fever or tick fever). Babesiosis is transmitted by a number of one-host ticks, such as Boophilus microplus, Boophilus decoloratus, and Boophilus annulatus.
Human babesiosis has been reported mainly in North America and Europe and has been linked to B. bovis, which causes cattle babesiosis. The other parasite linked to human infection is B. microti, a natural parasite of rodents.
Life cycle
The infection begins with a bite by an infected tick, which introduces sporozoites into the blood from it salivary glands. The sporozoites invade the host’s red blood cells and undergo several schizogonic divisions, increasing their numbers enormously. Eventually some of the infected red blood cells rupture, releasing thousands of merozoites and gametocytes into the circulation.
When a tick imbibes blood from an infected animal, the female gamete and male gamete unite in the tick’s gut to produce embryos. Some of these motile embryos or ookinetes migrate through the ticks’ tissues and invade the ovaries where they are incorporated into the developing eggs. Consequently, the ticks lay eggs that are already infected.
The nymphs that arise from such eggs are therefore congenitally infected. Some of the merozoites migrate from the ovaries to the salivary glands of the young ticks and after dividing, give rise to sporozoites. These young ticks are able to pass on the infection directly to a host on taking their first blood meal.
Infected animals show increased body temperature, malaise and loss of appetite, constipation or diarrhoea, vomiting, bloody urine and anaemia. Erythrocytic parasites are present in the lumens of capillaries of all internal organs including the brain. Mortality is high and death may occur in one week.
In human babesiosis, malaise, anorexia and fatigue are usually evident a week after being bitten by an infected tick. These are followed several days later by high fever, chills, drenching sweats, muscle pain and headache. The symptoms may continue for months or abate, then recur. Some infected people do not show any symptoms at all.
Individuals who are at risk are those with reduced immunity, especially the elderly and those who have undergone splenectomy. There is no specific treatment for babesiosis, although antimalarial drugs are sometimes used.
Six-legged, fur-covered, sea-faring and conferences - all packed full of parasites!
It looks like we've made it through another year of parasites, filled with posts on new research that was published this year on all manners of parasitic and infectious organisms. Among many other things, this year we covered some parasitological going-ons in the insect world with Zombee parasitoids, a story of parasitoid wasp, aphids and their symbionts, a wasp that can manipulate the colour of berries, and a cricket-infecting horsehair worm which has abandoned sex.
We also wrote about parasites that are infecting our furry friends including reindeer roundworms, a fleaof desert rodents, echidna gut parasites, anteater parasites, and a caring, maternal bat tick.
There were a lot of parasite action under the sea too, with jellyfish parasites that provide a floating buffet for some fish, a thorny-head worm which infects krill as a way of getting itself into whales, a leech that lives on shrimps, a prickly worm that lives in the stomach of dolphins, and a story of death, sex and fish guts.
Those are just a few example of post from this year; browse through the archives for a lot more parasitological tales.
Also for the first time on this blog, Susan and I had decided to report from conferences that we had attended on our respective continents! I wrote up a series of blog posts from the Australian Society for Parasitology annual conference, and Susan also wrote a few posts reporting from the American Society of Parasitology annual meeting.
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