PROTOZOA I

There are over 50,000 species of protozoa, of which a fifth are parasitic, some 10,000 species. They infect vertebrates and invertebrates and some are even parasitic in plants. Parasitic protozoa are, in general, small, have short generation times, high rates of reproduction and a tendency to induce immunity to reinfection in those hosts that survive. Structurally a protozoan is equivalent to a single eukaryotic cell. Among the unique features in protozoa are the mega- and micronucleus found in Ciliates and the kinetoplast, a DNA containing structure in the mitochondrion of kinetoplastid flagellates. Parasitic protozoa are in no way simple or degenerate and adaptations to parasitism frequently include complex life cycles and specialized ways of entering and maintaining themselves in their hosts.

The protozoa are now usually regarded as a separate kingdom divided in to seven phyla.

Kingdom Protista:

Some authorities recognize more phyla and molecular biology is leading to a major reapprisal. The protista are not a natural grouping, they do not have a common ancestor, some of them are almost certainly descended from multi-cellular organisms and others are probably fungi. The Apicomplex contain an organelle that seems to be a vestigial plastid, and are now sometimes included in the Dinoflafellates.

Examples of parasitic protozoa.

Sarcomastigophora these include the flagellates and the amoebae. Chief amongst the flagellates are the trypansomes. With one exception trypanosomes are transmitted by vectors, usually by insects. The exception is Trypanosoma equiperdum, a disease of horses, which is spread by sex. Trypanosomes are divided into two types stercorarian and salivarian. In stercorarian trypanosomes the trypanosomes are taken up in the blood meal and grow and divide in the hindgut of the insect vector. Infection of the vertebrate host is by contamination, when the insect feeds it defecates and the trypanosomes gain entry through the feeding site or by the skin being scratched. The most important stercorarian trypanosome is Trypanosoma cruzi, this infects 12-24 million people in central and south America, it is spread by triatomid bugs, and as well as man there are a large number of wild and domestic animals which can also act as reservoir hosts. T. cruzi is essentially a tissue parasite, the time spend in the blood is very short, and the trypanosomes enter various cells in the body particularly muscle and nerve cells, where they multiply to produce trypomastigote forms which then invade new cells or are picked up by the insect vector. Infection of heart muscles can lead to heart failure, damage to the sympathetic nervous system of the alimentary canal can lead to enlarged oesphagus and inability to swallow. The disease is called "Chagas" disease, and it is thought that Charles Darwin may have contracted it during his visit to South America and this accounts for his chronic ill health in later life. The triatomid bugs which are vectors for Chaga's disease usually bite at night, when the victim is asleep and often round the face, often called kissing bugs. In the day time the bugs hide in cracks in the walls and a very effective part of the control campaign is to plaster the walls of houses, so there is nowhere for the bugs to hide.

In contrast to the stercorarian trypanosomes, the salivarian trypanosomes develop in the mid gut of the vector, which is a fly and are injected via the salivary glands when the fly feeds. There are two species which infect man

Trypanosoma brucei rhodesiense -acute sleeping sickness

Trypanosoma brucei gambiensis - chronic sleeping sickness

Both parasites are spread by Tsetse flies and both can infect a wide range of mammals as well as man. The symptoms of sleeping sickness are caused by the parasite invading the nervous system.

Domestic livestock are also susceptible to trypanosome infections, where they cause a disease known as nagana. The tsetse belt in Africa - some 10 million square kilometres is effectively closed to cattle farming (could support 7 million cattle). Currently there is a lot of effort going into trying to breed resistant cattle.

Kinetoplastids can have a number of different forms in their life cycles; with differing positions of flagellum and kinetoplast.

In Leishmania, there are two forms in the life cycle, amastigotes found in the macrophages of the host and promastigates in the gut of the vector which is a sandfly. Leishmania causes serious diseases in man. The typical infection is cutaneous causing sores which can be slow to heal - local names Baghdad boils or Aleppo buttons, it occurs around the Mediterranean, you can get cutaneous leishmaniasis in Spain, France, Italy, Bulgaria. There are at least 6 species of Leishmania infecting man, with 15-16 subspecies. Some strains invade the subcutaneous tissues and can cause destruction of the mucous membranes of the mouth and nose. The most serious disease is kala azar, which is a visceral form of the disease involving internal organs such as the liver and spleen. An intriguing problem is how can the amastigote of Leishmania live in macrophages without being killed, because, after all that is the function of macrophages, to endocytose and kill bacteria and so on. When the Leishmania parasite is taken up into a phagolysosome, it does not break out into the cytoplasm of the macrophage, but produces a range of, as yet, poorly characterised factors which disrupt the macrophages normal killing mechanisms. The macrophage has two main killing mechanisms, the production of reactive oxygen intermediates such as superoxide anions or hydrogen peroxide, this seems to be down regulated by the parasite; and the production of lysosomal enzymes such as glycosidases and acid phosphatases is also reduced. The parasites may produce specific inhibitors for these enzymes, the parasite also alters the vacuolar pH, causing it to become more alkaline, the lysosomal enzymes have acidic pH optima. The parasite also inhibits the phagolysosome from fusing with secondary lysosomes.

The trypansomes and their relatives - kinetoplastida - live in blood or tissues and are transmitted by various vectors. The remaining flagellate parasites are mostly intestinal and are transmitted as cysts or other resistant stages that typically contaminate water or food. A number of flagellates are ubiquitous and common parasites of man, few of them cause much harm, but occasionally they are associated with outbreaks of disease, some of the more important ones are:

Giardia intestinalis, was discovered in 1681 by Leeuwenhoek, common in the small intestine, prevelance rates vary 1-30%. Parasite has a pair of adhesive suckers which gives it a characteristic appearance.

It attaches to the cells of the gut using the suckers and divides by binary fission, in this way huge numbers can build up in the intestine. They are spread by resistant cysts, each of which contains a pair of parasites, it is highly contagious. Causes diarrhoea, vomiting and loss of weight, the parasite does not break down host cells, but the dense layer of parasites over the surface of the intestine probably interferes with absorption and triggers the onset of disease. Giardia is a traveller's disease, widespread in Eastern Europe, also outbreaks in the USA, Aspen in Colorado, the ski resort. A number of animals may be able to act as reservoir hosts, particularly beavers and these are though to be responsible for several outbreaks in N. America Trichomonas. These flagellates have a characteristic axostyle. The trichomonads are aerotolerant anaerobes, that is they breakdown carbohydrates to organic acids and CO₂ whether oxygen is present or not, under anaerobic conditions their metabolism produces hydrogen from specialized organelles called hydrogenosomes. The trichomonads do not have mitochondria. Three species of Trichomonas are found in man.

A related parasite is found in cattle, Trichomonas foetus, it is an important cause of abortion in cattle, in cows the infection is usually self-limiting, i.e. they recover and are immune to re-infection. Bulls remain infective, and since the parasite can survive freezing in liquid nitrogen, it is a problem in artificial insemination. There is no satisfactory way to treat bulls and they usually have to be destroyed.

Histomonas is a cosmopoliton parasite of chickens and turkeys and causes a disease called blackhead. Histomonas looks more like an amoeba than a flagellate, it invades the tissues of the caecum and liver. Like all flagellates it divides by binary fission and huge numbers can be built up in a short time, there are no sexual stages and no cysts in the life cycle. Trophozoites (feeding stages expelled in the faeces rapidly die and although trophozoites may occasionally survive to be eaten and cause a new infection, the most important mode of transmission from fowl to fowl is hidden within the egg of a parastic nematode Heterakis gallinarum, which is also parasitic in the birds caecum. Heterakis is an ascarid and feeds on gut contents, if a histomonad is ingested the flagellates enter the nematodes intestinal cells where they multiply and then break out into the pseudocoelom of the worm and invade the germinative region at the tip of the ovary and eventually penetrate the developing oocytes. As the egg passes out of the female and then out with the faeces, the egg developes into an L1 larva, and the Histomonas continues to grow and divide and invades the tissues of the intestine and reproductive system. The reproductive system of the male nematode is also infected by Histomonas so possibly the parasite can be transmitted to female nematodes during copulation.

The eggs of Heterakis can survive for at least 2 years in the soil, if they are picked up by a suitable bird host, the eggs hatch in the small intestine and juvenile nematodes migrate to the caecum where they become adults. Histomonads relased from the nematodes intestine then infect the bird host. An infected female Heterakis not only infects the bird host, but her own eggs are also infected.

The life cycle can become even more complex if an earthworm eats an infected Heterakis egg. If this happens, the egg hatches and the 2nd stage juvenile penetrates the earthworms gut and becomes dormant in the earthworm's tissues and can survive for at least a year like that, until the earthworm is eaten by a suitable host. Earthworms live for a long time and feeding in contaminated soil they can accumulate vast number of juvenile Heterakis giving massive infections to the unlucky bird that eats them. Opilina. There are about 150 sp of Opilina, they live in the intestines of amphibia and are of no medical or economic importance, their zoological interest comes from the observation that their reproductive cycles are controlled by host hormones. They look like large ciliates.

During Summer, Autumn, Winter the Opalinids reproduce asexually by binary fission. But in the Spring they produce cysts, which pass out in the faeces and are eaten by tadpoles, male and female gametes excyst, then fuse to form a zygote, this is followed by a reduction division and the parasite resumes asexual reproduction. The stimulus for cyst production appears to be one of the host steroid sex hormones. The parasite integrating its life cycle with that of its host.

A number of very elaborate flagellates occur as symbionts in the gut of termites, where they breakdown the wood which termites feed on but cannot digest on their own.

Ruminants such as cattle, sheep and goats harbour massive number of flagellates and ciliates in their rumen. In ruminants, ciliates are the most important constituent of the rumen fauna, making up 10% of the rumen volume. Exactly what these ciliates do is unclear, defaunated ruminants are said to do as well as untreated ones, but the ciliates can provide up to a fifth of the daily protein requirement and also help to breakdown cellulose. Similar ciliates are found in the caecum and colon of horses and other equines.

Finally a few amoebae. Six species of amoebae are common in man, the most important is Entamoeba histolytica. The trophozoite or feeding stage lives in the lower small intestine or colon where it multiples by binary fission and forms characteristic 4 nucleated cysts. The cysts pass out in the faeces and infection is through contaminated water and food. The prevalence of Entamoeba varies from 1-40%. In amoebic dysentery the amoebae start to invade the mucosa causing ulcers, then they may get into the blood stream and be transported to other sites in the body such as the liver causing further amoebic abscesses. Three-quarters of the people infected with Entamoeba are asymptomatic carriers and only a quarter show pathological symptoms. There seem to be complex environmental factors which cause a non-pathogenic strain of Entamoeba to suddenly switch and become pathogenic, there is also some evidence that infection of the amoebae with a virus may influence virulence. There is also evidence that there may be two strains of Entamoeba.

Naegleria, this is an interesting group of amoebae that can exist in either a flagellated or an amoeboid form. They are primarily free-living found particularly in warm pools or lakes. In the amoeboid form there are sucker-like structures called amoebastomes, which seem to function as a cytostome for phagocytosis. Very occasionally this protozoan is responsible for a form of amoebic meningoencephalitis. The flagellated form probably gets forced into the nasal passages during diving, the parasite then migrates via the olfactory nerve into the brain, death rapidly follows from brain destruction.

Finally, Acanthamoeba, normally free-living like Naeglaeria but an increasing number of cases have been found in people who wear contact lenses and do not clean them properly and can result in ulceration of the cornea.

PROTOZOA II, MALARIA

Human malaria is one of the most important diseases in the world and has a long history. In the middle ages it was known as the ague, or the sweating sickness or the shakes. It was very early on thought, and with good reason, that the disease was associated with swamps and the Italians referred to the bad air in fever-producing areas as mal'aria, from which the word malaria was derived.

Malaria is characterized by severe intermittent fever occurring every 48 or 72 hours, depending on the species. The 48 hour fever is called tertian because it occurs every third day - fever on day 1, no fever on day 2, fever on day 3 and so on. The 72 hour fever is called quartan, because it returns on every fourth day. Malaria is one of those diseases that has influenced the course of history, Alexander the Great died of malaria, the allied invasion of Sicilly in 1943 was almost halted by an outbreak of malaria and the french troops on Haiti were again decimated by malaria during the rebellion at the beginning of the ninteenth century.

Perhaps the first important step in the control of malaria was the discovery of the Peruvian fever tree, Cinchona, by Jesuit missionaries in the early 17 century, the bark of the tree contains the alkaloid quinine, which is an effective antimalarial drug.

Demand for Cinchona, bark soon exceeded supply and attempts to establish Cinchona plantations elsewhere in the world failed, not least because the collection and export of the seeds was banned by the local governments. The solution came in 1861 when Charles Ledger a trader in Peru, sent his servant Manuel Incra Macrami to collect seeds from a group of Cinchona trees that he had discovered on the slopes of the Andes in Bolivia. Manuel camped out in forest for three years, each year the seeds were destroyed by late frosts, but in the fourth year he was lucky and there was a heavy crop of seeds. He carried the seeds back across the mountains to Ledger, who send them to his brother in London. The British government were not interested and the seeds were eventually bought by the Dutch consul, the seeds were then sent to Dutch plantations in Java, where by selective breeding they were able to increase the quinine yield and eventually give the Dutch the monopoly in the quinine market. It was the loss of these plantations to the Japanese in the Second World War that stimulated the rush to look for new synthetic antimalarial compounds. Charles Ledger died in poverty in Australia, Manuel went back to find more seeds was caught by the Bolivian police and died after three weeks of beating.

A young French army doctor (Alphonse Laveran) in Algeria discovered the malaria parasite in red blood cells in 1880. The idea that malaria might be spread by mosquitoes had been around for some time, but it was not until 1897 that a British Army doctor Ronald Ross, working in India finally found the mosquito stage of the parasite. A feat for which he was later awarded the Nobel Prize. Further understanding of the life cycle of malaria took a long time, the liver stage was not demonstrated until 1948, and the 'hypnozoite', the stage responsible for delayed relapses in some species was not discovered until 1979.

Well, the discovery of the role of the mosquito as a vector for the malaria parasite opened up the 'golden age' of parasite control by public health measures, control the mosquito and you can control the disease. Pilot mosquito control campaigns were successful and by 1970 malaria eradication had succeeded in the whole of Europe (in the 1800's malaria was prevalent all round the Mediterranean, Italy, Greece, Yugoslavia, Bulgaria, Turkey, and also occurred in Holland as a Summer epidemic and was widespread in southern Britain), malaria had also been eradicated in USSR, North America, several middle east countries, large parts of South America, the Caribbean, Australia, Japan, and Taiwan, Transmission rates in Africa and India had been severely reduced and the disease had been almost eliminated in Sri-Lanka. In India for example there were 75 million cases in 1950, by the beginning of the 1970's it was down to 100,000 cases. The situation today is not so encouraging. Malaria is estimated to kill between 1.5-2.7 million people annually - one every 12 seconds, often a child under 5. 300-500 million people have malaria and a third of the world population live in zones where they risk catching the disease. Nine out of ten cases of malaria occur in Sub-Saharan Africa and two-thirds of the rest are concentrated in just six countries. In decreasing order of prevalence India, Brazil, Sri-Lanka, Vietnam, Colombia and the Solomon Islands. In India, cases are back up to 2.85 million, and malaria is resurging in many other countries where it had previously been eliminated or sharply reduced. The development of drug resistant strains of malaria, the emergence of insecticide resistant mosquitoes and war and civil unrest leading to the collapse of control programmes have all contributed to the present problems.

There are four species of malaria which infect man,

In P. vivax and P. ovale dormant stages called hypnozoites can persist in the liver for several years and cause relapses when you think everything is over. In P. malariae the parasite can persist for as long as 30 years.

P. falciparum is the most common of the malarial parasites and also the most dangerous. Infected red blood cells develop surface 'knobs' which cause them to stick to endothelial cells (cells lining blood vessels), this causes blockages and brain and intestinal damage, often resulting in death, which can occur within a few days of infection. P. falciparum is especially dangerous to small children and to travellers from non-malarious areas. Adults from areas where malaria is endemic develop a form of partial immunity. This partial immunity develops slowly and only in response to repeated infections. In partially immune people, malaria parasites can often be found in the blood, but without clinical symptoms. People loose their immunity if exposure is not maintained. (after 6 months).

The life cycle has four phases, one sexual and three asexual. Each phase ends with the production of an invasive form, which is mobile and able to enter cells for the next phase. These invasive forms all have three structural features:

1. they have an apical complex for the invasion of cells, this includes an apical ring, small vesicles and duct like structures called rhoptries
2. thick, three layered pellicle
3. a cytoskeleton of microtubules

Infection begins when the mosquito injects sporozoites directly into the blood stream, within a few minutes they reach the liver, where they invade cells and become a hepatic trophozoite (feeding stage), this grows quickly and divides internally to give an hepatic schizont which contains many thousands of tiny, invasive merozoites. Division in Plasmodium is different from that usually seen in cells with nuclei. Usually in cells, the nucleus and cytoplasm divide in two, then into two again and so on. In Plasmodium the DNA and cytoplasm increase and the numerous progeny are created in a single parallel segmentation sharing cytoplasm and genetic material between all the individuals. No chromosomes are formed in this division, so nuclear events are almost impossible to follow. The merozoites are the smallest and shortest lived form of the life cycle, and within a few minutes they invade a red blood cell. So the asexual schizogony is followed by the production of an invasive form. The apical complex of the merozite is specialized to recognise and attach to specific molecules on the surface of the red blood cell. In Plasmodium vivax the recognition molecule is the Duffy blood group antigen. Fy⁴ a particular allele for the Duffy group appears to provide protection against vivax malaria, it is not recognized by the merozites. Frequences of Fy⁴ are high in central Africa reaching 100% in pygmies, but is only 3% in Europeans. The merozoite penetrates the erythrocyte within 20 to 30 seconds and develops into a trophozoite (feeding stage). The malaria parasite is not free within the cytoplasm of the rbc, but is contained within a parasitophorous vacuole. As the trophozoite develops, the vacuole enlarges and its surface area is increased by the development of diverticulae which penetrate the erythrocyte cytoplasm. This membrane probably has an active role in transporting metabolites and nutrients to and from the parasite. There is controversy as to whether the parasitophorous vesicle retains its connection to the outside. The parasite produces proteins that become incorporated into the parasitophorous membrane and also into the surface membrane of the rbc, so infected rbc's express on their surface parasite derived proteins. The growing trophozoite is amoeboid and ingests haemoglobin which is broken down to give an iron haem pigment called haemozoin which accumulates in the food vacuoles. The trophozoites have mitochondria without cristae and are essentially lactate fermentors, i.e. break down glucose to lactic acid, with no TCA cycle. (mammalian rbcs also have no TCA cycle; in birds rbcs have mitochondria and a TCA cycle, and bird malarias have a TCA cycle). After about 30-40 hours growth in the red blood cell, schizogony begins (2nd asexual stage), and the trophozoites divide to give 8-16 merozoites which are released when the rbc ruptures. It is the synchronous rupture of red blood cells which gives the periodic fever, and in particular it is thought that the haemozoin or malarial pigment which is released when the rbc's rupture is responsible for the fever. For the first few cycles the fever can be irregular, then the parasites synchronise their cycles.

After several blood cycles, a proportion of the trophozoites develop not into merozites, but into gametocytes, these take about 4 days to mature, but can then stay viable in the blood for prolonged periods. Nothing happens to the gametocytes unless they are taken up by a mosquito in a blood meal. However, within a few minutes of ingestion by a mosquito dramatic changes take place in the gametocytes. The stimuli for development are uncertain, but a drop in temperature (below 30^oC) and a rise in pH will trigger them in vitro. Both gametocytes swell and burst out of the erythrocyte. The male gametocyte produces 8 microgametes, which consist of a flagellum with an attached nuclear mass. The micro and macrogametes fuse forming a zygote. This is the only stage in the Plasmodium life cycle that is diploid, all the other stages are haploid. A difficulty with Plasmodium genetics is that chromosomes are not visible during cell division. Chromosome like molecules of DNA can be separated by pulse gel electrophoresis and genes allotted to DNA molecules. There are at least 10 such chromosomes and there is great variability between different strains in terms of number and size.

Over a period of 5 to 10 hours the zygote differentiates into a cigar-shaped motile invasive ookinete. This can penetrate either between or through the intestinal cells of the mosquito and comes to rest between the mid-gut cells and the basement membrane. During the differentiation of the ookinete the diploid genome divides as the first step in a two stage meiosis, the second stage takes place at the start of sporogony.

The embedded ookinete becomes an oocyst which grows rapidly and divides internally into sporozoites - the third asexual phase. The oocyst is the longest phase in the life cycle and is very dependent on the temperature, 8-35 days. The mosquito has to survive long enough for the oocyst to mature before it can infect anyone. So only elderly mosquitoes can pass on malaria and in the wild of course many mosquitoes never reach old age. This is where temperature matters, the warmer the weather, the faster the oocyst can develop in the mosquito. Mosquito survival is probably the single most important factor in malaria transmission.

Each oocyst produces up to 1000 sporozoites, when the oocyst bursts the sporozoites are released into the body cavity (haemocoel) of the mosquito. They migrate anteriorly to the salivary glands, where they penetrate the basement membrane, pass through the cells and accumulate in the salivary ducts. So again the asexual phase is followed by a mobile phase. The cycle is completed when the mosquito bites a susceptible host.

Chronic malaria poses more than a threat to individuals. A number of serious genetic disorders involving red blood cells increase in populations stressed by endemic malaria. Of these sickle cell anaemia and thelassaemia are the most common. Each of these is caused by the presence of abnormal haemoglobins in the patients' red blood cells.

In sickle cell anaemia, normal haemoglobin is replaced by haemoglobin S (Hb^S). Hb^S is much less soluble than normal haemoglobin when in the deoxygenated state and tends to precipitate, distorting the rbcs. This is caused by a single amino acid substitution where a glutamic acid in normal haemoglobin is replaced by a valine. A person who is heterozygous Hb^A/Hb^S has a mixture of normal and haemoglobin S, their blood appears normal unless exposed to low oxygen when there is reversible sickling. Heterozygotes that show sickle cell trait appear normal and do not appear to be disadvantaged. The homozygotes on the other hand Hb^S/Hb^S suffer from a devastating illness and many die in infancy. The heterozygotes Hb^S/Hb^A are protected against P. falciparum malaria, the reason why is not clear, but it is probably because sporozoites are unable to infect the red blood cells because the cells are fragile and burst when infected.

In areas with endemic malaria, children who are heterozygous are more likely to survive than children who have normal haemoglobin, i.e. Hb^A/ Hb^S have a selective advantage over Hb^A/Hb^A. As long as the advantage of Hb^A/ Hb^S is greater than the disadvantage of Hb^S/Hb^S the proportion of Hb^A/ Hb^S will increase.

Another genetic disease which occurs in a much higher frequency in malarial regions is thalassaaemia. Haemoglobin consists of two sub-units, 2 alpha and 2 beta. In thalassaemia one of the subunits is not made, so the individuals lack either a-subunit, in a-thalassaemia or b-subgroups in b-thalassaemia. This causes abnormal shaped blood cells that again may confer increased resistance to infection with malaria.

Glucose-6-phosphate dehydrogenase deficiency in red blood cells is another genetic blood disease associated with malaria. This is an X-chromosome linked trait, so men tend to be more affected than women. Glucose-6-phosphate dehydrogenase is involved in protecting the red blood cells from damage by free radicals and in particular reactive oxygen intermediates. This means that cells lacking glucose-6-phosphate dehydrogenase are susceptible to oxidative stress induced for example by certain drugs such as sulphonamides or eating Vicia fava beans 'favism' red blood cells burst. This increased sensitivity makes them burst when infected with Plasmodium falciparum.

Southeast Asian Ovalcytosis (SAO) protects against cerebral P. falciparum in the heterozygote, homozygotes are not found and are presumed to die in utero.

PROTOZOA III

The malaria parasites of man, with the exception of P. malariae are not transmissible to other animals. So a lot of the experimental work on malaria has been done with species of malaria found in non-human primates and rodents. As well as mammals malaria also occurs in birds and lizards.

Bird malaria is interesting in that in contrast to mammalian rbcs, bird rbcs have a nucleus, contain mitochondria and have a a tricarboxylic acid cycle. Bird malaria unlike mammalian malaria has mitochondria with cristae and have a TCA cycle. It is thought that P. falciparum is related to bird malaria, the other species of human malaria are related to primate malaria.

As well as malaria there is another group of parasitic protozoa that live in rbcs of vertebrates and these are the Piroplasms. They differ from the malarial parasite in that they divide by binary fission not schizogony and they do not produce haemozoin (malarial pigment). The vectors are ticks, not mosquitoes. An important Piroplasm found in cattle, with related species in other vertebrates is Babesia, it causes a serious disease called babesiosis that can be fatal. It has a rather interesting life cycle. The cycle in the mammal begins when the infective forms injected into the blood stream by the tick enter the red blood cell, where they multiply by binary fission. Another major difference from malaria is that Babesia although it starts off in a parasitophorous vacuole, emerges from the vacuole into the cytoplasm and so is in direct contact with the red cell cytoplasm. So the parasite escapes from the vacuole. Blood stages which are probably gametocytes are taken up by the tick when it feeds, these become gametes which fuse to give a zygote which then enters the epithelial cells of the gut. The zygote divides in the gut cells of the tick which burst to release merozoites which enter various cells in the body where another round of multiplication takes place. If the tick is a mature female, the merozoites can also enter the egg where they again multiply so that when the larva hatches from the egg it is already infected and there is another round of multiplication in the gut before the merozoites enter the salivary glands where they undergo yet further division, eventually giving rise to small round infective sporozoites, which are injected into the mammalian host.

This is transovarian transmission, it is important because one host ticks would be extremely poor vectors otherwise, because they go through all their life cycle stages on the same host. One-host ticks do not feed on successive hosts so would not transmit parasites from host to host, so only by transmitting the parasite to the next generation can new hosts become infected.

Tick life cycle involves four stages

egg --> larva --> nymph --> adult

In one-host ticks the larva develops to adult on the same host. In two host ticks, there is a change of host between nymph and adult, in three host ticks there is an additional host change between larva and nymph.

In piroplasm infections the stage of tick which picks up the infection never becomes infective itself. Depending on the life cycle of the tick.

Babesiosis is a serious disease - Texas fever or red water fever, due to haemoglobin in the urine. The haemoglobin of course comes from the breakdown of red blood cells. Control is aimed at controlling the tick by regular dipping of cattle and also the development of an anti-tick vaccine directed against components of the tick salivary gland, which prevents the tick from feeding. In Europe there have been a number of cases of cattle Babesia in humans, but always in people who have had their spleens removed - frequently fatal infection.

Related to Babesia is another Piroplasm called Theileria, causes serious disease in cattle in Africa, East Coast fever. It differs from Babesia in that it has a multiplication phase in macrophages before it invades the red blood cell. So infective stages introduced by the tick bite first invade lymphocytes, the parasite induces the lymphocytes to divide and the parasite also divides and is distributed between the two daughter cells and in this way a massive infection can be built up before, after a predetermined number of divisions, microschizonts are produced which then infect the red blood cells. With Theileria there is no transovarian infection (it is spread primarily by 2 and 3 host ticks), so in the tick, the infection goes

larva --> nymph

nymph --> adult

Control is again primarily by tick control and quarantine. Animals which recover from Theileria infections are immune to further infections. There has been some success in inducing immunity by controlled infection, followed by chemotherapy.

Protozoan diseases of cattle, particularly nagana and Theileria are a major constraint on protein production in Africa.

Coccidia. The Coccidia are parasites of the intestinal tracts of vertebrates. The numbers of different species of coccidian is staggering, there are at least 2,700 species of coccidian in rodents alone, and the vast majority of Coccidia species are probably yet to be described. Coccidia are extremely common in domestic animals and cause millions of pounds of losses every year. Coccidia normally have self limiting infections followed by immunity to re-infection. Under natural conditions animals become infected with small numbers of oocysts and are only mildly affected. Under crowded conditions, things like battery hens, or confining laming ewes in sheds, large numbers of oocysts can be taken up causing severe or fatal infections - especially in young animals. Coccidia are a particular problem in poultry rearing.

In Eimeria, oocysts hatch in the gut and release sporozoites that enter gut cells and undergo a phase of sexual multiplication or schizogony. The released merozoites invade other cells and the cycle is repeated two or three times depending on the species. Eventually the merozoites enter cells and develop into gametocytes and fertilization takes place to produce a zygote. A cyst wall forms round the zygote to give an oocyst which passes out in the faeces. Inside the oocysts the nucleus divides to give eventually 4 sporocysts each containing 2 sporozoites, which is the infective stage. So, a single host life cycle. Eimerids show a very high degree of host specificity, usually restricted to one host species, and often one organ within the host and to specific region of the organ. For example Eimeria tenella in chickens infects only one of the two gastric diverticula. Different species of Eimeria may have specific positions within the host cell. Some species develop below the nucleus, others above it and in a few cases actually within the nucleus itself. A problem for any parasite that invades intestinal cells, is that these cells turn-over rapidly with half lives of the order of 24-48 hours. This means that parasites like Eimeria have to complete their development within the intestinal cell before it is sloughed off, or slow down the rate of cell turnover.

Control of Eimeria infections is by hygiene, not overcrowding the animals and by prophylactic chemotherapy. Once an infection has developed there is not much that can be done about it, but given prophylactically, chemotherapy is very effective. All commercial chicken foods incorporate coccidiostats (anti-coccidial drugs). The big problem is drug resistance, feeding drugs to all the chickens all of the time puts a tremendous selection pressure on the parasite and the commercial life of a coccidiostat is only about 2 years.

It used to be thought that primates were not infected by coccidia and they were not responsible for any human disease. But this is not the case. Cryptosporidium parvum is a parasite that has recently become newsworthy. It is found in a range of vertebrates cows, sheep, rodents, cats, dogs and man. It is not sure if it is the same species of Cryptosporidium that infects all of these mammals, if it is Cryposporidium is a widespread zoonosis. Some workers claim there are two strains, one which is primarily infective to humans, the other infects animals and to a lesser extent man. Its life cycle is like that of Eimeria, only one host, the trophozoites (feeding stage) are very small 2-6mm and they infect the brush border of the intestinal cell (appear to be extra-cellular in location). Infection is by the faeco-oral route. Most people are infected as children and develop a life long immunity. The cysts are so small and resistant that they are not removed by conventional water treatment. There have been a number of outbreaks blamed on contamination of public water supplies by cysts from animals. An outbreak in Aspen (Colorado) (-posh ski resort) was blamed on cysts from beavers. Outbreaks in the UK have been blamed on cysts from calves. Cryptosporidium is a major problem in immuno- suppressed people - AIDS sufferers, transplant patients. Another, increasingly important human coccidian parasite is Toxoplasma. The definitive host of Toxplasma is the cat. In the cat intestine the parasite has a typical coccidian life cycle, the parasite invades the intestinal cells where there may be several cycles of schizogony before gametocytes are produced and eventually oocysts. The oocysts take 3-5 days to sporulate, that is to form sporozoites, before they are infective. An infected cat can produce vast numbers of oocytes, with production peaking at days 5-8 after infection. If a cat eats an infective oocyst, the cycle is repeated. If, however, the oocysts are eaten by another mammal, usually a mouse or a vole or sheep the sporozoites do not stay in the gut, but migrate into the tissues, liver, lungs, muscle and cause a disseminated infection. At the start of an infection of a secondary host the parasite divides rapidly forming tachyzoites, which go on to infect new cells, as the hosts become more resistant, division slows, and a finally dormant cysts are formed containing bradyzoites. If eaten by a cat, both tachyzoites or bradyzoites can initiate a new infection. If eaten by another potential secondary host another disseminated infection occurs.

The disease is primarily a disease of kittens, adult cats are usually immune. Humans usually acquire their infections from infected cats or from infected meat. The disease is usually symptomless in man and after the first infection you develop a life long immunity although you may still be harbouring bradyzoites. However, if the first infection occurs during pregnancy, there is a danger that the parasite will invade the foetus where it can cause severe damage. The chances of a maternal infection being transmitted to the foetus are about 45%. Of the infants so infected, 60% will have a symptomless sub-clinical infection, the remainder will have symptoms ranging from mild to severe. In Britain the incidence of congenital toxoplasmosis is about 1 per 2000 line births (or less than 20 per year). In countries such as France or Austria where meat is eaten rare, the number of cases of congenital toxoplasmosis is much higher, 8 per 2000 in France, and in Paris, 50% of the population tested positive for Toxoplasma antibodies. In countries, such as France, where the level of congenital toxoplasmosis is high, routine serological tests are carried out at antenatal clinics during pregnancy, and transplacental infection can be very effectively stopped by drug therapy.

It is debateable, with the low levels of congenital toxoplasmosis found in the UK, whether a screening programme on the scale of that carried out in France is justified. But more could be done to educate the general public and particularly pregnant women about the dangers of Toxoplasma. The leaflets handed out at pre-natal clinic do not usually say much about Toxoplasma or what measures to take to avoid it. Relatively simple precautions like not eating raw or undercooked meat when pregnant, washing hands after preparing raw meat, avoid cross contamination on kitchen utensils between raw meat and other food all considerably reduce the chances of infection. If you own a cat, do not feed it raw meat and if pregnant get someone else to empty the litter tray. Or since the oocysts take a minimum of three days to sporulate, empty the litter tray every two days.

Toxoplasma can become a serious disease in people who are immunosuppressed, since the disseminated phase of the disease can continue unchecked. If immunity is impaired dormant bradyzoites can become reactivated. Some of the dementia associated with terminal aids is due to brain damage caused by tachyzoites.

Toxoplasma can also occur in sheep and is a major cause of abortion in Welsh hill flocks, sometimes leading to catastrophic outbreaks, where 30% of the ewes may loose their lambs. The disease of course cannot be directly transmitted from sheep to sheep, but animals become infected by eating grass, straw or concentrates that have become contaminated with cat faeces. Like humans, once infected, sheep develop a life-long immunity.

Two final phyla of parasitic protozoa - the Microspora or Microsporidea as they are sometimes called and the Myxozoa. Both groups form spores with polar filaments.

The polar filaments are tube like and coiled inside the spore and look very much like the nematocysts of cnidarians. When a host eats the spore the filaments are expelled. In the Myxozoa the filaments are held in polar capsules and their expulsion helps to anchor the spores. In the Microspora, there are no separate polar capsules and the filaments penetrate the intestinal cells of the host and the amoeba like sporoplasm (nucleus and cytoplasm inside the spore) travel down the tube into the cell. So in the Microspora the filament is a method of penetrating the cell. Penetration is fast <2 sec, and is powered by osmotic forces (cf nematocysts).

Myxozoa, mostly parasites of fish, a few in amphibians and reptiles, none from mammals or birds. Most people consider the Myxozoa to be metazoans not protozoans(posibly related to Cnidaria, but more likely related to Rotifer/Nematode groups), because the spores are multi-cellular. They can be economically important in fish farming - Myxosoma cerebralis causes whirling disease in salmonids, causes high mortality in hatchery reared trout. The parasite invades the cartilage of the head and spine. The spores are released when the fish dies or is eaten by a predator. The freshly released spores are not infective and it is probable that  tubificid annelids act as intermediate hosts. Recent reports suggest fresh-water bryozoans may be involved in other Myxozoan life cycles.

The Microspora are principally intracellular parasites of invertebrates-protozoa, platyhelminths, nematodes, bryozoa, rotifers, annelids and arthropods, also in lower vertebrates - fish, amphibia, reptiles and occasionally mammals. Nosema common parasite of honey bees, also an important parasite of silk worms in mid 1800's Nosema almost destroyed french silk industry - Pasteur spent a lot of time working on its control.

Minchinia is a pathogen of oysters is also economically important.

Occasionally opportunistic infections of Microspora in AIDS patients. Most Microsporidia develop in the cytoplasm, a few are surrounded by a parasitophorous vacuole. The Microspora have no functional mitochondria (probably a secondary loss as there is what may be a mitochondrial remnant), atypical golgi, no peroxisomes, prokaryote like ribosomal RNA and ribosomes and a very small genome. Molecular studies indicate that they may be closer to the fungi than other protozoa. Pneumocystis cerainly similarly seems more closely related to fungi.