Parasitology Notes Collated 2024 PDF

Course Notes Semester 2, 2024 Associate Professor Ryan O’Handley. Parasitologist Assessment Dates Assessment Due Date Submit Monday, August Midterm (20%)...

Course Notes Semester 2, 2024 Associate Professor Ryan O’Handley. Parasitologist Assessment Dates Assessment Due Date Submit Monday, August Midterm (20%) In class 19th 1:10pm Monday Creative Work in Parasitology (20%) September 30th In person/electronic 5:00pm Practical Log Book (Formative Assessment Friday, Nov 1 Directly to Ryan (10%) O’Handley University Final Exam (50% - HURDLE) Examination Written Exam Period 1 Introduction to Parasitology What is Parasitology? Parasitology is the study of a biotic relationship between two organisms. One organism, the parasite, lives at the expense (or detriment) of the other organism – the host. For this instructor, Parasitology is the understanding of this relationship, and how it impacts upon the host, that is the fundamental concept behind the study of parasitology (not the taxonomic classification of organisms that happen to be parasites). Thus, Parasitology is the study of infectious diseases at the most fundamental level. Technically, the pathogenic viruses, bacteria and fungi are all parasites! How is the field of Parasitology organised? Although any organism that lives to the detriment of its host is a parasite, the field of parasitology focuses on all pathogenic organisms that are not bacteria, viruses or fungi. These organisms include the following groups: The Protists (or Protozoa)- Single celled eukaryotic organisms – not a taxonomic group The Cestodes – Tapeworms The Trematodes – Flukes The Nematodes – Roundworms The Arthropods – Ticks, Mites, Insects As you can appreciate from the above list, Parasitology is a vast and diverse field. In fact, Parasitology is larger than Bacteriology and Virology put together and each one of the above groups could be taught as stand-alone courses. Fortunately, in veterinary or medical parasitology there are usually only 10-12 important and common parasites for each of the host species. These important parasites will be the primary focus of this course. How important is parasitology to veterinary medicine and animal science? Parasitology is a fundamental aspect of veterinary medicine and animal science. It is a topic that is likely to come up every single day of your career. It is estimated that the global market for parasiticides reached USD $10 billion in 2022 and will grow 6.5% annually to 2032 where it will be valued at USD $19 billion. (https://www.gminsights.com/industry-analysis/animal-parasiticides-market) From the sheep industry, where resistance to antiparasitic drugs is emerging as a major threat, to the horse owner that needs to know how frequently to de-worm her horse, to the cat owner who has been told to get rid of her cat because she wants to start a family, and field veterinarian who finds ticks on cattle in South Australia – Parasitology is a major part of practice. A colleague of mine once told her class, “if you aren’t a good hip surgeon, no one will fault you – but if you don’t know parasitology you are guilty of malpractice” (Susan Little, Oklahoma State University) Parasitology will keep the lights on in your clinic! 2 How do I handle the vast amount of material in this field? Parasitology is a massive field. Fortunately, because parasitology involves a host-parasite relationship, the host is the first thing to consider when dealing with the amount of information that confronts you. While some parasites can infect multiple host species, many are host specific or only cause disease in a specific host, and this makes it easier to handle the large amount of information. As you attend lectures and study for this course always consider the host and clinical signs / location of disease. This will almost always shrink the potential parasitic causes of disease from many to 1 or 2! I do not expect you to know everything about Parasitology in this course. I expect you to have a good understanding of the major parasites of the common species of importance to veterinary and animal science (know about the common parasites in the common hosts). However, I fully expect that following this course, you will understand the terminology and diagnostic methods specific to parasitology to acquire information on parasites (even uncommon parasites) in the less common host species that you may encounter during your career and make an appropriate decision regarding diagnosis, treatment and control. Do I need to know all those Life cycles? Yes! The effective treatment and control of parasitic diseases is based on understanding parasite transmission. To understand parasitic disease transmission, you need to understand the life cycle. For example, consider this case: Case and Questions 1: You receive a call from your client who owns a small feedlot in rural NSW. The abattoir has just condemned 60 of 90 steer carcases from his feedlot due to beef measles (Cysticercus bovis). What is the source of the infection? How does he prevent this situation from happening again? What drugs are used to treat parasitic diseases? There are numerous drugs used to treat parasitic diseases. However, these constantly change and what I tell you today may not be correct in 3 years time. While I will often discuss some of the major drugs used to treat parasitic infections, I will often refer to drug classes as well. For the most part, I will expect you to know the major drugs used to treat parasites and you must know how to acquire this information on your own. To obtain up to date information on approved antiparasitic drugs, please consult the Australian Government Registered Product Database (PUBCRIS). This site lists all products approved for treating diseases of animals in Australia. The link is: https://portal.apvma.gov.au/pubcris 3  Basic Concepts In order to properly practice the field of parasitology, you need to have an understanding of some basic concepts. You may already be familiar with many of these concepts from previous courses but need to understand how they specifically apply to parasitology. Epidemiology Epidemiology is the study of disease within a population. In parasitology, epidemiology involves not only the host population, but the parasite population as well. These populations can be broken down into categories or classes (subpopulations) for specific study. Understanding these subpopulations is usually important with respect to understanding parasite transmission, when clinical disease will develop and in which host the clinical disease will develop. Parasite populations The parasite population is comprised of the parasite stages that reside in (or on) the host(s) AND the parasite stages that are in the environment. For many parasites, developmental or juvenile stages occur in the environment or in other hosts. All of these stages must be taken into account when studying parasite populations. Parasite numbers per host Parasites are almost always over-dispersed within a host population. This means that in a population, many hosts will harbour parasites, but only a few of these hosts will harbour large numbers of parasites. Thus, the majority of parasites in a host population are found in a few individuals. This over-dispersion is very evident in production animals on pasture, such as sheep and cattle, and is very important when considering how manage these parasites on the farm. As the majority of parasites are found in only a few individuals, a large sample size is needed in order to obtain an accurate picture of parasite abundance in the sheep flock or cattle herd. A small sample size will yield misleading results. Cases and Questions 2: In the entire population of dogs in Australia – which population of dogs harbour the vast majority of parasites? 4 Multiple Species Infections A single parasite infection is usually rare under natural (or semi-natural) conditions. A single host can be infected with many different parasite species – a parasite community. These parasites may interact with one another or cause disease in the host synergistically. Parasite population dynamics and parasite control As a population, parasites must exhibit higher reproductive rates than mortality rates, in addition to suitable transmission rates, in order to perpetuate otherwise the parasite will die out. These factors combined comprise the transmission threshold and are important with respect to implementing control practices. However, it is not always possible or practical to increase parasite mortality (using drugs for example) or to reduce transmission rates (changes in management) to levels that cause a parasite (or infection) to die out. In production animal situations, for example, infection levels need only to be reduced below the economic injury threshold (the number of parasites above which production losses occur). Parasite population dynamics differ considerably depending on the type of parasite involved. Generally, parasites can be classified into microparasites and macroparasites when studying population dynamics. Microparasite Microparasites reproduce asexually within the host. Therefore, contact with one infectious stage leads to the development of many individual parasites within the host. Most protozoa are microparasites. Macroparasite Macroparasites do not multiply asexually within a host. Therefore, contact with one infectious stage results in the development of a single individual parasite. Most helminths are macroparasites. You need to understand if a parasite is a microparasite or a macroparasite to implement an effective control program. In addition, you need to know what kind of host you are dealing with and what other hosts or vectors may be involved in the parasite’s life cycle in order to understand parasite population dynamics. Definitive host The definitive host is the host in which the parasite undergoes sexual reproduction (Example: the mosquito is the definitive host for malaria) 5 Intermediate host Host in which the parasite must undergo development other than sexual reproduction in order to complete its life cycle (Example: humans are intermediate hosts for malaria) Paratenic host Host which is involved in transport of an infectious stage but in which no development occurs. Often accidental hosts. Parasite Transmission Parasites must increase their chances of encountering new hosts. In order to effectively control parasites, we must understand their modes of transmission. Parasite transmission strategies include: Shedding millions of infective stages into environment (eggs/cysts/oocysts) Encysting in host tissues (predator – prey relationship, hypobiosis) Use of a Vector1 A host is susceptible to a parasite if it cannot eliminate the parasite before the parasite becomes established (the parasite is thus termed infective) A host is resistant if its physiological status prevents survival of the parasite (the parasite is thus termed non-infective) All previously unexposed hosts are generally susceptible to parasite infection / infestation. Parasites, however, can display host specificity (i.e. Eimeria, lice) or infect a range of different host species (i.e. Giardia). Obviously, those parasites that infect a wide range of host species are more difficult to control and manage as there are numerous reservoirs for infection. Understanding modes of transmission and identifying the potential hosts at risk of infection are important in treating and controlling parasitic infections. Direct Transmission Involves direct contact (i.e. sexual activity) by a susceptible host with an infected host or contact with a specialized infectious stage excreted by an infected host. Infectious stages can be picked up by a susceptible host through inhalation, ingestion or even penetration of the skin. Indirect Transmission Indirect transmission involves other host organisms such as vectors (i.e. mosquitoes) or intermediate hosts in which the parasite undergoes some form of development prior to becoming transmissible to a susceptible host. It can also involve a paratenic host in which the parasite remains infective but does not undergo development. Indirect transmission 1 Any agent that transmits an infective organism (ie: insects) 6  from a vector usually involves the vector acquiring the parasite through feeding on an infected host, then transmitting the parasite by feeding on a susceptible host. Indirect transmission through an intermediate host or a paratenic host usually involves the predator prey life cycle (intermediate host is preyed upon or scavenged upon to transmit the parasite). These factors are also extremely important with regard to Zoonoses2 because animals can act as reservoirs for human infections. Cases and Questions 3: Echinococcus granulosis is a tapeworm of dogs. Do dogs acquire the tapeworm via direct or indirect transmission? Parasite Immunology For More information See Tizzard, Veterinary Immunology Parasites are often difficult to control by the host as they are more complex than bacteria and viruses, and different life cycle stages are often antigenically distinct. This is why there are relatively few vaccines against parasites (and no vaccines against human parasites) Innate Immunity Physical and chemical barriers that prevent parasite invasion of the host. Infection is established when innate immunity is breached. Acquired immunity Specialized system of non-self-recognition. Acquired immunity regulates the multiplication and elimination of the parasite. In general, cellular immunity controls intracellular parasites and humoral immunity controls extracellular parasites. Helminth infections, for example, display a TH2 immune response with an increase in eosinophils. Immune Evasion Parasites have developed many strategies to evade the immune defences of the host. These strategies include: 2 Diseases communicable from animals to humans under natural conditions 7  Life Cycle Strategies o multiple stages o anatomic exclusion (intracellular location) o encystment within host tissues (quiescence) o infection of naïve hosts (periparturient rise / vertical transmission) Immune Response Modification o divert immune response (from TH1 to TH2) o immunosuppression (destroy T-cells or macrophages) Antigenic Strategies o antigenic variation o masking with host antigens General Principles of Immunoparasitology All parasites will elicit immune responses (it may or may not be protective) Absence of an appropriate immune response results in greater susceptibility and more severe disease manifestations (i.e.: immunosuppression, immature immune system) Persistence of parasites in hosts is due to the parasite ability to evade host defence mechanisms Specific immune responses develop slowly and usually fail to eliminate the parasite completely Responses to secondary infection are usually efficient and often result in resistance to infection or reduction in clinical disease Pathophysiology The pathogenic effects of parasitism are many and varied and specific pathology will be discussed with each parasite but may include: Trauma / tissue destruction anaemia inflammation malabsorption / maldigestion 8 Pathogenic factors are contributed by the parasite, the host, and the environment and can work synergistically to produce disease. PATHOGENIC FACTORS Parasite Factors Toxins Proteases Host Factors Level of nutrition Hypersensitivity / inflammation Disease Processes Environmental Factors Stress Overcrowding Temperature General Diagnostic Procedures There are many diagnostic procedures used for parasites. Often the test used will depend on the level of clinical signs in the host, and the practicality of the test. For a description of specific tests, consult the following: - See Veterinary Clinical Parasitology (Zajac and Conboy pages 1-40) - Seriously, read Zajac and Conboy pages 1-40! - Australasian Parasites Inside and Out (Chapter 5) Direct faecal smear Advantages - Quick and easy - Positive result is valid - can observe delicate forms such as trophozoites 9 Disadvantages - Poor sensitivity - Negative findings inconclusive Faecal flotation Advantages - Concentrates cysts/oocysts - Clarifies sample - Increased sensitivity Disadvantages - Not as fast as direct smear - destroys delicate forms such as trophozoites Sedimentation Used for trematode eggs and some tapeworm eggs that are too dense to float in solutions Time consuming and can often be labour intensive Baermann Technique Used for lung worms and any parasites that produce larvae that are passed in the faeces ELISAs and Other tests There are many ELISAs and other tests available. It is important to understand that antibodies indicate exposure but not necessarily infection. Antigen tests are usually superior for diagnosis of current infections. Molecular based tests (PCR) detect parasite DNA and also have strengths and limitations. An ocular micrometer is VITAL for diagnostic parasitology. Cases and Questions 4: Using sucrose flotation on the faeces of a dairy calf, you observe 4+ Cryptosporidium parvum oocysts per high power field of view. On a different calf you detect Cryptosporidium parvum by PCR. Do these results differ? Why or why not? Treatment, Control and Management To implement successful parasite control and management strategies, it is vital to first determine the goal of the parasite control/management strategy. Is the goal of the control program to cure an animal of parasites after infection has occurred? Is the goal to prevent infection from occurring? Is the goal to simply control the level of infection to prevent clinical disease and/or production loss 10 from occurring? Different animal husbandry and management systems will have different goals with respect to parasite control, and therefore utilise different techniques or combinations of techniques to achieve these goals. Therefore, once the goal of the control program is established, the techniques can be selected and implemented. By understanding the basic concepts and principles of a parasitic infection, treatment and control programs can be designed and successfully implemented. This may include: There are 3 basic parasite control techniques, but many options for each technique. Management Techniques Management techniques are utilised to reduce or prevent parasite transmission. They can be very effective in controlling parasites and involve a variety of specific methods. Sanitation For parasites transmitted directly, sanitation is one of the most important methods that can be employed to reduce parasite transmission. In the dairy industry, for example, good levels of sanitation in calf rearing areas can effectively reduce the level of transmission of Cryptosporidium to levels where clinical disease does not occur. In contrast, poor sanitation results in calves being exposed to oocyst levels that result in severe clinical disease and even death. Good sanitation is also a key compo need in controlling coccidia in poultry, where all bedding material is completely removed from a barn when one batch of chickens has grown out prior to a new batch of chickens entering the barn. For horses, keeping foaling stalls clean can reduce exposure to the equine ascarid Parascaris equorum amongst foals and therefore reduce changes of clinical disease occurring. Cleaning up after dogs and proper disposal of faeces is highly effective at removing infectious stages of nematodes from the environment thus eliminating chance of exposure in other dogs or humans. Food safety For parasites that utilise trophic transmission (predator-prey or scavenging), techniques that destroy the transmissible stages in meat or meat products can be very effective at eliminating parasites. For example, transmission of the tapeworm, Echinococcus granulosis, to dogs can be prevented by not allowing dogs to eat offal or scavenge sheep carcasses or by freezing sheep offal prior to feeding it to dogs. As infected dogs can infect humans, leading to hydatid disease, this is a very important control method. 11 Cooking or freezing food is also effective at destroying infectious stages parasite transmitted using trophic stages. Cooking meat to 70 degrees Celsius destroys the tissue cysts of Toxoplasma gondii as does freezing meat products. Proper cooking of meat can also prevent transmission of Trichinella. Meat inspection is also an effective way of preventing parasite transmission. Meat inspectors trim or condemn carcasses infected with beef or sheep measles (Taenia saginata and Taenia ovis) thus preventing their transmission to humans and dogs respectively. Vector control For vector transmitted parasites, control of the vector through physical means, chemical means or through elimination of breeding sites can effectively reducer parasite transmission. In heartworm endemic areas, for example, screening in kennels or dog houses to prevent mosquitoes from feeding on dogs at night can greatly reduce exposure to heartworm. Likewise, trimming back vegetation preventing animals from entering bush land can prevent infestations with ticks that are also excellent vectors for many parasites. Chemotherapy – Drugs with efficacy against parasites. o Some drugs are not appropriate for food animals or have withdrawal periods o Praziquantel, Pyrantel, Fenbendazole, Toltrazuril, Metronidazole etc. o See section 3.2 Australasian Parasites Inside and Out Supportive treatment (rehydration therapy) Environmental decontamination Vaccination Chemotherapies (Antiparasitic drugs and compounds) When using antiparasitic compounds, you must understand the parasite, the host, the management aims of the client and the nature of the compound you are using. For food and fibre producing animals you must consider the following: Export Slaughter Interval (ESI) Withholding period (WHP) Wool Harvesting Interval (WHI) It is a legal requirement to read the label. The label contains the most current and up to date information on the product and how it is to be used. Off label use can occur on the advice of a veterinarian only and violations of WHP and ESI need to be considered (for example - backyard chickens being treated off label and their eggs are often sold at a farmer’s market). 12 There are more than 1000 products registered for parasite control and they can be found in the following classes. Anthelmintics Anthelmintics Antiprotozoal chemicals Aminoacetonitriles (AA) Aminoacetonitriles (AA) Antimonials arsenicals (As) Arsenicals (As) Arsenicals (As) Aryl amides and urea Benzene sulphonamides (BS) Benzene sulphonamides (BS) derivatives (UD) Benzimidazoles (BZ) Benzimidazoles (BZ) Benzimidazoles (BZ) Depsipeptides (DP) Depsipeptides (DP) Ionophores (IO) Isoquinolones (IQ) Isoquinolones (IQ) Nitroimidazoles and Macrocyclic lactones (ML) Macrocyclic lactones (ML) heterocycles (NI) Organophosphate compounds Organophosphate compounds Pyridines (PY) (OP) (OP) Phenyl ureas (PU) Piperazines (PI) Piperazines (PI) Pyrimidines (PD) Salicylanilides (SA) Salicylanilides (SA) Sulphonamides (SU) Spiroindoles (SP) Spiroindoles (SP) Tetracyclines (TE) Tetrahydropyrimidines and Tetrahydropyrimidines and Thiamine analogues (TH) imidazothiazoles (TI) imidazothiazoles (TI) Triazones/benzene acetonitrils Triclabendazole (TC) Triclabendazole (TC) (TZ) Parasitic drugs have been widely used for centuries to control parasites in animals and there are many drugs/chemicals available for treating protozoal, helminth and arthropod infections and infestations. However, there have been very few new classes of anti-parasitic drugs developed recently and over-use of some anti-parasitic drugs has led to the development of resistance. Drugs used to Cure Drugs used to cure animals are used following diagnosis of an infection. It is not practical to “cure” a flock of sheep of their parasites. However, there are times when drugs are used to cure animals of parasites. For dogs and cats, diagnosis of a roundworm or tapeworm infection results in prompt treatment of the animal to cure them of infection. In horses or production animals, the development of clinical disease in certain individuals may warrant the use of drugs to cure them of infection or disease. Drugs used for Prophylaxis In many animal management systems, anti-parasitic drugs are used to prevent disease or infection from occurring and are termed prophylactics. In production animals, prophylaxis is used to manage parasite levels within the flock/herd, often to maximise production. For example, in commercial broiler chicken operations, anti-coccidial drugs are administered in the feed to prevent chickens from developing coccidiosis. Sheep are often drenched at specific times of the year to keep parasites at levels where they do not impact on production. In companion animals, prophylaxis is utilised in high-risk groups or in areas where certain parasites are highly endemic. For example, puppies should receive anthelmintic at 2, 4, 6 and 8 weeks of age because they are born with Toxocara and hookworm can be transmitted to them in the milk. In regions where heartworm is endemic, dogs are often given a prophylactic anthelmintic orally or via injection. 13 While prophylaxis can be used effectively to prevent infections, clinical disease or production losses in animals, if it is over-used or not managed properly, drug resistance can develop rapidly. Drug resistance has been widely reported for GINs of sheep, cattle and horses and drug resistant strains of fleas and heartworm have been reported in some parts of the world. Drug Resistance Drug resistance is essentially a form of artificial selection. It evolves like any other genetically determined characteristic evolves by natural selection except that the drug is an artificial driver of evolution. It occurs when a genetic mutation in a parasite population develops that confers resistance to the drug the parasites are regularly exposed to. Parasites that possess the mutation now have a biological advantage over the other parasites in the population and have a greater chance of passing their genetics down to future generations. Over time, the proportion of the parasite population with the resistant genes grows, ultimately to a point where it comprises almost all of the parasite population. While the development of resistance can be complex, it can be summarised in a simple scenario. Picture a flock of 1000 sheep. If these sheep are infected on average (remember parasites are overdispersed) with 5000 Trichostrongylus worms each, that is a parasite population of 5 million. The sheep are then given the drug ivermectin to control their parasites and moved to a clean pasture. With a population of 5 million parasites, it is not inconceivable that a few individual parasites may have mutations that confer resistance to ivermectin. Following treatment, these individual worms will be the only ones left in the parasite population and any offspring they produce (eggs shed in the faeces of the sheep that moult to the L3 stage) will also be resistant to ivermectin. When sheep ingest these worms on pasture, the proportion of resistant worms being carried by the sheep increases. At some point, ivermectin treatment will not be effective as the sheep will be primarily infected with resistant worms. In systems that utilise parasite prophylaxis, resistance must be managed. Ways to manage resistance include rotation of different drug classes through the production system. This will delay the emergence of resistance. Also, management of refugia is important. Refugia is the population of parasites not exposed to the drug. The population of parasites in refugia will contain primarily parasites that are susceptible to the drug. Keeping a portion of parasites in refugia by not treating all of the animals and/or keeping animals on reasonably contaminated pastures, will ensure parasites susceptible to the drug will survive and pass these genes to the next generation. Vaccines Vaccines are one of the best ways to control infectious diseases. However, there are few effective vaccines against parasitic diseases. Protists and helminths are complex eukaryotes, with multiple stages in their lifecycle, and as such have proven difficult with respect to the development of vaccines. There are some vaccines that have been developed for parasites that are effective, but these are live, attenuated or precocious vaccines. For example, coccidiosis vaccines in chickens 14 have been developed by continuously passaging the parasite through chickens and selecting parasites that are of low virulence. These are then administered to chickens in commercial operations to immunise birds to more virulent strains of the parasite. An attenuated strain of Toxoplasma gondii has also been developed into a vaccine. Sheep in New Zealand and Europe receive this live vaccine which prevent abortion due to T. gondii exposure. These live vaccines, while they can be effective, are not ideal as they must be kept in storage conditions that can limit their distribution, such as frozen in liquid nitrogen. In many cases, the vaccine alone is not enough to control the infection and must be used in combination with drugs. Considerations for Control Techniques Once the goals of the parasite control program have been established there are often other considerations with respect to which techniques are implemented. Some of these considerations include the following: Economic Injury Threshold In production animal systems, the economic injury threshold should be factored into any parasite control programs. The economic injury threshold is defined as the minimum number of parasites that result production loss. In theory, when parasite levels reach the EIT, control should be initiated. However, the EIT must also be considered along with the costs to implement parasite control. These costs include the money required to purchase drugs or vaccines and the cost of labour and disturbance costs if animals need to be mustered. Theoretically, the cost benefit in terms of increased animal production from parasite control should exceed the potential cost of production loss combined with the parasite control costs. These costs can be difficult if not impossible to determine exactly in production systems, but consideration must be given to these costs. Spending $20 per sheep to control a parasite that cause $10 per sheep in production loss is not good business. Drug Withholding Periods and Intervals When drugs (and potentially some vaccines) are used as part of parasite control programs for food producing animals, consideration must be given to the withholding periods for the drugs. For all drugs used in food animals, pharmacokinetic studies have been conducted to determine when drug residues and metabolites reach acceptable levels in the meat, milk or fibre. A withholding period for the drug will then be determined. This is the period of time that must elapse following treatment before the animal products can enter the food chain. Depending on if the animal products are for the domestic or export market, the terminology and period of time can be different. Some of these terms are as follows: Meat Withholding Period: Time between drug treatment and slaughter for domestic use Export Slaughter Interval: Time between application of a drug and slaughter for export 15 Milk Withholding Period: Time between drug treatment and milk harvest for human consumption Wool Harvest Interval: Time between drug treatment and wool harvesting Public Health As many parasitic diseases are zoonotic (transmissible from animals to humans under natural conditions), the risk of parasite infection or infestation to public health must also be considered. When public health is at risk, then control techniques should be implemented irrespective of factors such as clinical disease in the animal, or production losses (the EIT). For example, Identification of a few Toxocara canis eggs on a faecal flotation from a household dog would warrant treatment, even if the dog was not showing any clinical signs of disease, due to the public health risk of the parasite. Integrated Parasite Management Integrated parasite (pest) management (IPM) is broadly defined as a program that utilises all appropriate and practical methods and strategies to control parasites. IPM utilises both chemical and non-chemical methods of control and is a well-developed concept. IPM has a long history of successful implementation and in Australia and it was an IPM program that allowed for the eradication of sheep scab from Australia during the late 1800’s. Unfortunately, due to the development of safe and inexpensive anthelmintics, IPM programs are often not utilised today. Instead, a “one size fits all” approach where animals receive antiparasitic drugs on a schedule (interval dose system) is often used and requires no parasite monitoring or real strategic thinking. This strategy can lead to unnecessary use of drugs leading to overmedication and drug resistance. It can also lead to administration of improper drugs for the parasites in a population or issues with biosecurity due to lack of parasite monitoring. While more difficult to implement, the principles of IPM should be practiced. There are 4 steps to Integrated Parasite Management. 1 Parasite Identification and Monitoring Use of diagnostic testing to identify which parasites occur in animals is an important first step in IPM. As discussed in part 2, the type of test used depends on the animal management system. Nevertheless, some level of periodic monitoring should occur in all animal management systems as parasite populations may change over time and to confirm the efficacy of any antiparasitics being used. As factors favourable to parasitism increase, so should the frequency of parasite monitoring. 16 2 Setting of Action Thresholds Once the parasites that occur in an animal population have been identified, action thresholds can be established for these parasites. The action threshold is a well-defined, preferably quantifiable, guide by which a control decision can be made with respect to a particular parasite. Action thresholds should be set based on what is understood about the biology of the parasite and with consideration of management practices and environmental conditions. As a result, action thresholds may differ between regions for the same types of animals. 3 Prevention Prevention of parasitism is another key to the successful implementation of IPM. The goal of preventative measures is to limit the exposure to the infectious stages of the parasite. Management techniques such as sanitation, reducing stocking densities, pasture spelling, etc can be used to prevent parasite transmission. Ideally, these measures can keep parasite levels below action thresholds. 4 Control. Despite measures being taken to reduce or prevent exposure, often parasite control measures must be implemented. As described above, these measures could involve management, anti-parasitic drugs and vaccines. Usually, a combination of measures is required for successful parasite control. Ideally drug use should be targeted, where possible, to only those that require treatment in order to reduce unnecessary treatments, maintain refugia and prevent drug resistance. 17  Part II - The Protists (Protozoa) General Information Protists are a heterogeneous assemblage of more than 50,000 species. There are numerous free- living, commensal and parasitic species. The word “Protozoa” was once considered a phylum name; however, it is now a common term used to describe from 7 to 30 different phyla (depending on which text you read). Structure Protozoa are single-celled eukaryotic organisms and generally have the typical complement of organelles surrounded by a lipid bilayer plasma membrane typical of all metazoan cells. nucleus, mitochondria, Golgi apparatus, endoplasmic reticulum etc. Protozoa differ from typical metazoan cells (and each other) through the development of many specialized organelles which may aid in the attachment, locomotion, feeding, host cell entry, etc. Examples of these include: Glycosomes – which contain glycolytic enzymes used Kinetoplasts – extra chromosomal DNA Rhoptries – organelles used to mediate cell invasion Flagella – used for locomotion Some parasitic protozoa also possess a glycocalyx, which is a glycoprotein surface coating that functions to provide protection against the host immune system. Physiology and Nutrition Some protozoa are photosynthetic (and the apicomplexa have remnants of a chloroplast), but those relevant to parasitology are heterotrophs. Protozoa can be particle feeders (grazers and predators). Some species have temporary or permanent “mouth” openings (such as the cytostomes of the ciliates). Intracellular protozoa modify the host cell in order to acquire nutrients. 18 Digestion is intracellular within food vacuoles. Food reaches these vacuoles through specialized “mouth” structures, engulfment, phagocytosis or pinocytosis and transport of nutrients across the cell membrane can occur through simple diffusion or be carrier transported Metabolic processes are typical of eukaryotic cells for most (but not all) protozoa Glycolysis, Krebs cycle etc. Excretion: Ammonia is the main nitrogenous waste produced by protozoa and it diffuses across the cell membrane. Water balance is maintained in free living aquatic protozoa using contractile vacuoles, but few parasitic protozoa possess contractile vacuoles (the ciliate Balantidium coli is a notable exception) Locomotion occurs by use of flagella, cilia, pseudopodia or other methods Flagella Ø are whip like structures extending from the kinetosome) or basal body) of the cell. The flagella move in a helical fashion to push fluids along its axis Ø Flagella from some species are bent back along the cell surface to form an undulating membrane Cilia Ø Are structurally similar to flagella but beat back and forth at regular intervals Ø Often highly organised into rows Pseudopodia Ø Temporary extensions of the cell membrane Ø Involves a process termed cytoplasmic streaming to protrude the cell, adhere to a surface, and then contract to created movement. Gliding locomotion Ø Characteristic of the Apicomplexa Ø Ridges on the surface of the parasite called epicytic folds create and undulating wave to provide locomotion (it is pretty cool to watch) Reproduction Protozoa reproduction can be asexual, or a combination of asexual and sexual reproduction. Asexual reproduction occurs in all parasitic protozoa and is the only mode of reproduction in many. 19  Binary fission Ø Cell divides equally into 2 equivalent cells Ø Division occurs orderly Ø Kinetosome – kinetoplast (if present) – nucleus – cytoplasm Budding Ø Fission with 2 unequal cells formed Multiple fission / Schizogony / Merogony Ø Nucleus divides repeatedly followed by simultaneous cytokinesis Ø Creation of 2 or more daughter cells within the mother cell Sexual reproduction is common in many species and involves fusion of identical gametes (isogametes) or gametes that differ in size (anisogametes). For parasitic protozoa, the type of reproduction that occurs within a host is used to define the definitive host from the intermediate host in the parasite’s life cycle. Definitive host is the host in which the parasite reaches sexual maturity Intermediate host is a host where parasite development occurs but not sexual maturity Encystment - Many protozoa encyst by producing a resistant covering to protect their transmissible form from environmental factors. Some protozoa may encyst within the tissues of the host. Life cycles are highly varied. Some parasitic protozoa have a simple lifecycle with direct transmission from host to host. Others have complex lifecycles involving intermediate hosts, definitive hosts and vectors. Host entry – Parasitic protozoa can enter hosts via direct contact, ingestion (of cysts in the environment or in intermediate hosts, or via a vector Taxonomy and Classification Since there is no agreement on the taxonomic classification of protozoa (and because this instructor is a parasitologist not a taxonomist) taxonomic classification will not be a focus of this section of the course. Typically, parasitologists organise protozoa by their mode of locomotion, even though the organisms may not be taxonomically related. They are organised as follows: Flagellates – use flagella for locomotion Giardia, Hexamita, Histomonas, Trichomonas, Tritrichomonas, Trypanosoma and Leishmania (Kinetoplast flagellates) 20 Amoeba – locomotion by pseudopodia Entamoeba, Acanthamoeba, Naegleria Ciliates – utilise cilia for locomotion Balantidium, Ichthyophthirius Apicomplexa – intracellular parasites with asexual and sexual stages (most important group of human and veterinary parasites) Eimeria, Cystoisospora, Cryptosporidium, Sarcocystis, Toxoplasma, Neospora, Babesia, Theileria, Cytauxzoon, Leucocytozoon, Plasmodium (Malaria) Two other groups, the Microsporidia and Myxozoa are also covered traditionally by veterinary protozoology, although they are no longer considered protozoa. (Examples are Encephalitozoon, Myxobolus) As this is a Veterinary Parasitology course – We will be dealing with the protozoa generally by organ system.. 21 Protozoa – GI and Mucosal Surfaces There are numerous protozoa that infect the gastrointestinal tract and other mucosal surfaces of animals and humans. They share many of the same fundamental characteristics of infection and disease as they must interact with the epithelial cells lining the GI tract, Urinary Tract, and respiratory tract. General Pathogenesis and Clinical Signs The pathogenesis and clinical signs associated with protozoal infections of mucosal surfaces depend on the following factors: Parasite Factors: Ability to attach to and/or invade the mucosa Host factors: Age, immune status, immune response Environmental Factors: Overcrowding, temperature, etc. Pathology induced by parasite: - Blockage of absorptive surface - Lysis and destruction of host tissue - Invasion of cells Pathology induced by host: - Mucosal inflammation - Hypersensitivity These parasite and host factors contribute to disturbances in: - Digestive Capacity - Absorptive Surface - Motor Functions 22 These Lead to local and systemic clinical signs which include: Local: Diarrhoea and dysentery Systemic: Anorexia Steatorrhea (fat in stool) Weight loss Abdominal pain Wasting Allergic disease (pruritus) Some Gastrointestinal Protozoa by Host Species Parasite Genera Location Comments Host Dog Giardia Small Intestine Zoonotic Cryptosporidium Small Intestine Zoonotic Cystoisospora Small Intestine Sarcocystis Small Intestine Definitive host Neospora Small Intestine Definitive host Entamoeba Colon Rare Balantidium Colon Rare Cat Giardia Small Intestine Zoonotic Cryptosporidium Small Intestine, Zoonotic Cystoisospora Small Intestine Sarcocystis Small Intestine Definitive host Toxoplasma Small Intestine Definitive host, Zoonotic Besnoitia Small Intestine Definitive host Entamoeba Colon Rare Tritrichomonas Ileum, cecum, colon Pet Birds Trichomonas Crop Giardia Small Intestine Cryptosporidium Small Intestine Eimeria Intestines, Cecum Poultry Trichomonas Crop Cryptosporidium Small Intestine Eimeria Small Intestine, Large Intestine, Many species of great Cecum veterinary importance Hexamita Intestine 23  Histomonas Cecum, Liver Cattle Cryptosporidium Abomasum, Small intestine, Zoonotic Large intestine Giardia Small Intestine Zoonotic? Eimeria Small Intestine, Large Intestine Sheep and Giardia Small Intestine Zoonotic Goats Cryptosporidium Small & Large intestine Zoonotic Eimeria Small Intestine, Large Intestine Swine Giardia Small Intestine Zoonotic? Cystoisospora Small Intestine Eimeria Small Intestine Balantidium Colon Zoonotic? Pathogenic? Entamoeba Colon Rare Horses Giardia Small Intestine Eimeria Small Intestine Rodents and Rabbits Giardia Small Intestine Cryptosporidium Small Intestine, Stomach Eimeria Intestines and Liver Encephalitozoon Kidney, Liver, Brain Reptiles and Amphibians Giardia Small Intestine Entamoeba Intestine Major pathogen of Snakes Cryptosporidium Intestine Fish Spironucleus GI Tract (Hexamita) 24 Giardiasis (Giardia spp) Giardiasis is a common intestinal disease of mammals, birds, reptiles, and amphibians caused by various species of Giardia Giardia duodenalis (= intestinalis = lamblia) - Most important species of Giardia to veterinary medicine - Infects wide range of mammals including dogs, cats, cattle, sheep, and Humans - Public health concern as animals can act as a reservoir for human infections - It is the most common intestinal parasite of dogs in Australia Giardia muris - rodents Giardia microti - muskrats and voles Giardia ardeae and G. psittaci - birds Morphology 2 forms, Trophozoite and Cyst Trophozoite – motile feeding stage, non-infectious - piriform (pear-shaped), measuring 12-17 X 7-10 um - Binucleate with 4 pairs of flagella and bilaterally symmetrical - attaches to intestinal mucosa using bi-lobed ventral adhesive disk and feeds via vacuoles on dorsal surface 25 Cyst – environmentally resistant infectious stage - oval shaped measuring 9-13 um in length - contains 2-4 nuclei - internal structures may be visible (nuclei, axostyles, median bodies) Life Cycle Giardia has a simple, direct life cycle involving an infectious cyst stage and motile trophozoite stage Cysts ingested by the host excyst releasing trophozoites into the duodenum Trophozoites reproduce by binary fission and colonize small intestine Trophozoites encyst as they pass through the GI tract in response to increasing concentrations of bile Cysts are then passed in the faeces (up to 106 per gram of faeces are commonly excreted) Prepatent period: 4 – 10 days 26 Epidemiology Transmission - Direct transmission through faecal-oral route is most important - Highly contagious with ingestion of as few as 10 cysts required for infection - Water-borne transmission common in human outbreaks - Cysts are susceptible to desiccation but remain viable in cool moist conditions for months - Cysts are resistant to conventional water disinfectants (i.e.: chlorination) Prevalence - Highly prevalent and ubiquitous (found everywhere) - Infections are most common in young animals (and humans) and in confinement where contact occurs frequently (kennels, catteries, dairy, day-care, etc.) Dogs and Cats: - Very common parasite in dogs in Australia!!! (9.3 % prevalence overall; 5.5% household dogs – most infections in puppies). 12% prevalence in racing greyhounds (why?) 2% prevalence Australian cats Livestock: - in calves and lambs, up to 100% reported with infections most common in calves older than 30 days of age Birds: - Can be very common in pet birds, especially in bird colonies Pathophysiology and Pathogenesis - Highly variable as both parasite products and host factors contribute - Diffuse shortening of intestinal microvilli which reduces mucosal absorptive surface area of the small intestine - Decreased intestinal enzyme activity - Villous atrophy may or may not occur 27  - Increased intestinal motility (decreased transit time through gut) - results in malabsorption and maldigestion Clinical signs Many (perhaps most) infections with Giardia are asymptomatic. Many dogs infected with Giardia do not have diarrhoea (question – why?). When clinical signs are present, they range from acute to chronic and reoccurring - Diarrhoea (pale and foul smelling) is the most common clinical sign - Steatorrhea (fat in the stool), anorexia, abdominal pain, vomiting - Extraintestinal manifestations such as urticaria and pruritis (allergic disease) are reported in dogs and humans. Feather picking is reported in birds with Giardia Diagnosis Giardia is the “most commonly mis-, under- & over diagnosed parasite in vet practice” Zinc Sulfate Centrifugal Flotation Faecal Exam Multiple samples (3 consecutive) may be needed, as cyst excretion can be intermittent Cysts are best observed by ZnSO4 flotation which can result in collapse of cytoplasm in cyst creating a distinctive crescent shaped refractile artifact Direct Smear Trophozoites can be demonstrated in fresh diarrheic samples (observe motion) Cysts may be observed if in high numbers Iodine can be used to stain both trophozoites and cysts Antigen Detection Many commercially available and detect Giardia-specific antigen in faeces. Good sensitivity and specificity but can be expensive. 28 Treatment and Control Dogs: Febantel is registered for treating Giardia in dogs. It is metabolised to fenbendazole which has activity against Giardia. Must be administered once per day over 3-day period Cats and other species: no approved treatment in Australia and must use drugs off label. Febantel, Fenbendazole, Metronidazole (Cats), Dimetridazole (Birds) Control: - Involves sanitation to limit exposure to infectious cysts - Useful disinfectants include bleach, Lysol and sterinol with high contact times (15 – 30 min) to ensure cysts are inactivated Cases and Questions 5: You diagnose Giardia in a 7-month-old dog. The dog is not displaying any gastrointestinal signs and is in general good health. Do you treat the dog for the Giardia infection? Why or why not? Trichomoniasis Many species of non-pathogenic trichomonads occur in gastrointestinal tract of various domestic animals and tend to multiply in the presence of diarrhoea. However, trichomonads are responsible for some important diseases in veterinary medicine. Bovine Genital trichomoniasis – Tritrichomonas foetus Feline intestinal trichomoniasis – Tritrichomonas blagburni (some debate on name) - Recent evidence indicates this to be a separate species from bovines Avian Trichomoniasis – Trichomonas gallinae (In Humans Trichomonas vaginalis is a sexually transmitted infection) Morphology Trichomonads have only a single trophozoite form (do not produce a cyst) They are characteristically pear-shaped, with 3-5 flagella, an undulating membrane and trailing flagella 29 T. foetus – 6-25 µm in length, 3 flagella (1) T. gallinae – 5-9 µm in length, 4 flagella (2) (1) (2) (drawings not to scale) Bovine Genital Trichomoniasis – Tritrichomonas foetus Life Cycle Venereal disease of cattle transmitted during natural service Trophozoites reproduce by binary fission - Bulls: Trophozoites reside in the prepuce, penis, epididymis, vas deferens - Cows/Heifers: Trophozoites reside in the vagina, cervix, and uterus 30 Epidemiology Cows/heifers: - Trophozoites are transferred to cows and heifers from infected bull during copulation - Infections can persist for weeks-months - usually recover after infection Bulls: - Trophozoites transferred from infected cows/heifers to bulls during copulation - Infected bulls are often infected for life, but there is an association between age and infection - Younger bulls (< 2 years of age) may be refractory to infection. Mature bulls remain infected due to markedly deeper epithelial crypts in the skin of their penis and prepuce, providing a favourable environment for T. foetus. Artificial Insemination: - Semen is not infective unless contaminated with preputial fluid during collection 31  - contaminated semen will remain infectious through the addition of diluents, antibiotics, and the freezing process Epidemiology Can be common wherever natural service is common. In Australia, a recent study (2022) found 11%-15% of bulls infected at abattoir - which is not a decline from 65% herds and 11% of bulls infected in the NT in a study from 1988. Irons PC. et al Prevalence of Tritrichomonas foetus in beef bulls slaughtered at two abattoirs in northern Australia. Aust Vet J. 2022 May;100(5):201-204. Pathogenesis - Invasion of uterus leads to placentitis which results in detachment, death, and abortion of foetus - Tritrichomonas may also invade foetal tissues Clinical Signs Cows/Heifers - Infertility and abortion up to 5 months after breeding (due to small size of passed fetes, abortions often go unnoticed) - Vaginitis - Mucopurulent discharge, pyometra - Retention of fetes/membranes leading to endometritus and sterility Bulls: - Show no sign of infection 32 Diagnosis Alterations in calving patterns and females returning to service are indicators of infection. Diagnosis confirmed by demonstration of trophozoites in preputial scrapings or washings (smegma), vaginal secretions, vaginal washings, or aborted foetuses. Care must be taken to avoid contamination with gastrointestinal trichomonads. Tritrichomonas foetus has 3 anterior flagella and 1 trailing flagellum and displays a characteristic “rolling” motion. If it is not observed in the washings, they should be cultured. DX may require culture: Commercially available InPouch test used to culture the organisms or Modified Diamond’s Media can be used. PCR is used for confirmation Repeated examination may be necessary to determine infection status Treatment and Control Cows/heifers - No treatment available - 4 months sexual rest will usually clear reproductive tracts of T. foetus Bulls - No approved drugs - Metronidazole administered I.V. at 75 mg/kg may be attempted - Treatment is difficult and should only be used for exceptionally valuable animals Control - Cull infected bulls and use non-infected or virgin bulls - Artificial insemination - Running younger bulls can reduce transmission (replace every 2 years) - Vaccine available in USA for female herd (TrichGuardtm) but efficacy is questionable. 33 Feline Trichomoniasis This is a recently discovered disease and occurs in young cats and cats housed in crowded conditions (shelters and colonies). It is caused by Tritrichomonas blagburni. Life cycle - Cats are infected through direct contact and ingestion of trophozoites (no cyst stage) Epidemiology - High prevalence in USA in show cats and in cats housed in crowded conditions - In Australia, disease is reported but prevalence in shelters is low so far. Pathogenesis - Colonisation of ileum, cecum, and colon resulting in a large bowel type diarrhoea. - Pathogenesis uncertain, but loss of goblet cells and infiltration if inflammatory cells observed. Clinical signs - Chronic large bowel diarrhoea (cow pie like stools) - Blood and mucus may be present Diagnosis - Observation of flagellates in direct smears but must differentiate from Giardia trophs (different locomotion) (no cyst stage so flotation will be ineffective) - Culture in MDM or in pouch with confirmation by PCR is also used. Treatment and Control - No treatment approved - Ronidazole (30 mg/kg twice daily for 2 weeks) is reported in literature to resolve diarrhoea and may eliminate infection - Sanitation, hygiene and eliminate overcrowding is important 34 Avian Trichomoniasis – Trichomonas gallinae A condition known as “Canker” in pigeons and “Frounce” in birds of prey It is primarily a disease in pigeons, doves and raptors, but can occur occasionally in turkeys and chickens and pet birds (reported in budgerigars in Australia). Recent research from Europe indicated the parasite has a much wider host range and now includes finches. Life Cycle Direct, reproduces by binary fission Pigeons - Trichomonads are transferred from the crop of adults to squabs via feeding “pigeon milk” (regurgitated food) - Infected pigeons can also transmit trichomonads via contaminating surface water or water feeders with their oral secretions Birds of Prey - T. gallinae is transferred to birds of prey when they feed themselves or their young with pigeons Poultry and other birds - Can contract the disease by direct contact or consuming water contaminated by pigeons 35 Pathophysiology and Pathogenesis - Trophozoites invade the mucosa of the upper intestinal tract (buccal cavity, sinuses, pharynx, oesophagus, crop) - Liver and other organs are occasionally invaded - Raised yellow caseous lesions first appear in mouth spread to the upper digestive tract. The lesions make closing the mouth impossible - Lesions can enlarge, become confluent, and build-up of caseous material can occlude the oesophagus - In liver, lesions appear on surface as solid, white to yellow circular masses Clinical Signs - Birds will have difficulty closing their mouth. They will drool and make continuous swallowing movements - Greenish to yellow fluid in oral cavity or dripping from beak - Ruffled and emaciated appearance Diagnosis - The clinical signs and gross lesions restricted to upper portion of digestive tract are suggestive of trichomoniasis - Microscopic observation of large numbers of T. gallinae in direct smears from oral fluids or lesions in the mouth, crop, or digestive tract is confirmatory - Trichomoniasis should be considered in any birds with regurgitation or lesions in their upper GI tract. Control and Treatment - Eliminate infected birds and suspected carriers from the flock - Avoid feeding pigeons to Birds of Prey - Practice high level of sanitation and ensure there is a source of clean fresh water (prevent pigeons from contaminating water and food in poultry operations) - Ronidazole, Carnidazole, dimetridazole - See PUBCRIS for approved chemotherapies 36 Histomoniasis (Blackhead) Histomoniasis is a disease affecting primarily turkeys, but also other birds such as chickens, peafowl, grouse, quail, peacocks etc. The causative agent is the flagellate Histomonas meleagridis Turkeys - Highly susceptible to infection and most infected turkeys die - Disease also known as “Blackhead” Chickens - Easily infected but usually milder form of disease Morphology Two forms: Flagellate Stage: - Is found in the lumen of the cecum - nearly spherical 8-12 µm in diameter with a Single flagellum Amoeboid Stage: - Is found in the tissues. - Pleomorphic 8-21 µm Life Cycle Life cycle is unique as it involves the common caecal nematode Heterakis gallinarium Heterakis worms ingest Histomonas trophozoites within the cecum and Histomonas is incorporated into the caecal worms’ eggs Birds become infected by ingesting Heterakis eggs or earthworms that have ingested Heterakis eggs The histomonad is released from the egg after it hatches in the intestine of the host Histomonas reproduces by binary fission and invades the caecal wall. 37  Direct transmission can occur and is important in large outbreaks. Transmission in these cases can be through ingestion or “cloacal drinking” the parasite is taken in through the cloaca by reverse peristalsis. Epidemiology - Common wherever turkeys and chickens are raised especially in backyard flocks - High prevalence in areas favouring coexistence of Heterakis gallinarium and earthworms (backyard flocks) - Histomonas can remain infective within the Heterakis egg for long periods (1-2 years) - 3- 12-week-old turkeys and 4-6 week old chickens most susceptible to infection 38  - Chickens are a MAJOR reservoir for the disease due to the high prevalence of the caecal worm in chickens Clinical Signs - Clinical signs appear 15-21 days after infection and are characterized by Brilliant- yellow (sulphur coloured) faeces - Depression, drooping wings, anorexia and emaciation follow - Head may (or may not) be cyanotic (black head) - Mortality is high in turkeys (50 –100%) and younger birds are most susceptible - Generally low mortality in chickens, but up to 30% reported Pathophysiology and Pathogenesis - Certain bacteria are required to be present for Histomonas to attain virulence (E. coli, Clostridium perfringes, Bacillus subtilis). This relationship is not completely understood. - Disease results when Histomonas penetrates the caecal wall, enter the bloodstream and parasitize the liver - Ceca becomes enlarged and thickened with pinpoint caseous lesions - Liver lesions are composed of circular depressed areas of necrosis which measure 1-2 cm and are yellow to grey in colour (are apparent by day 10 of the infection) - In heavy infections the lesions coalesce and involve large areas of the liver Diagnosis - The brilliant yellow faeces combined with the caecal and liver lesions are pathognomonic, but the caecal lesions in chickens can be confused with coccidiosis. - Histomonads may also be demonstrated histologically where the organisms occur in clusters in liver sections. Control and Treatment - Good sanitation must be practised. 39  - Turkeys (and other birds) MUST be raised separately from chickens (and not be raised where chickens were raised for about 3 years) - Control Heterakis gallinarium in birds and limit access to eggs and earthworms - Prophylactic and therapeutic treatment for caecal worm can be used – dimetridazole can be used to treat blackhead, but NOT ALLOWED for treating histomoniasis in chickens or turkeys. Amebiasis (zoonosis–Entaboeba histolytica) There are numerous pathogenic and non-pathogenic species of amebae that can be found in animals and humans. Pathogenic species include obligative parasites as well as free-living amebae Pathogenic Obligate Parasites – Intestinal Amebiasis o Entamoeba histolytica – mammals - ZOONOTIC o Entamoeba invadens – Causes severe disease and death in captive reptiles o Recently identified species pathogenic to cane toads Pathogenic Free-Living Amebae – Primary Amebic Meningoencephalitis Naegleria fowleri, Acanthamoeba spp. Intestinal Amebiasis Entamoeba histolytica (mammals) Entamoeba invadens (reptiles) Entamoeba spp (cane toads) Morphology Trophozoites and Cysts occur 40 Trophozoite - Trophozoites are ameboid, 12-60 µm in size (average 20 µm) - E. histolytica has a single nucleus with central endosome surrounded by fine ring of chromatin - May contain erythrocytes which differentiates them from other amebae Cyst - Cysts are round, 10-20 µm in size and contain 4 nuclei with “rounded” chromatid bar Life Cycle Simple, Direct Cysts are ingested and release trophozoites in the lower ileum Trophozoites colonize the large intestine and divide by binary fission Trophozoites may invade the intestinal wall, enter the circulation and invade other tissues, especially the liver Cysts are formed in the lower colon and are passed in the faeces 41 Epidemiology - Entamoeba histolytica is primarily a parasite of humans and non-human primates. Humans can act as a reservoir for animals and vice versa (Zooanthroponotic and Zoonotic transmission) - Infections have also been reported in, dogs, cats, pigs, rats, and possibly cattle, but prevalence in domestic animals is unknown. - Transmission is usually by the direct faecal-oral route, but waterborne transmission can also occur. - Cysts not as resistant as Giardia cysts, killed by temp > 55oC, and super chlorination - Entamoeba invadens is a severe pathogen of snakes, lizards, and giant tortoises, but turtles are quite tolerant and act as a reservoir. 42 Pathophysiology and Pathogenesis - Entamoeba histolytica trophozoites hydrolyze the host’s large intestinal tissues using cysteine proteases. - Intestinal lesions result which develop into characteristic “flask – shaped” undermining ulcers once the organism penetrates the muscularis mucosa - Penetration through the submucosa and serosa enables trophozoites to be carried by the blood and lymph to extraintestinal sites such as the liver, lung (or other sites) where the parasites can form necrotic abscesses Clinical Signs - Clinical signs can vary greatly, and infections are often asymptomatic (no mucosal invasion) - Diarrhoea, dysentery (blood and mucus in stool) and vomiting along with signs of colitis are the most common clinical signs - Anorexia, weight loss - Hepatomegaly and fever occur during hepatic infection - In reptiles, anorexia, weight loss, green coloured urates, and rapid death are clinical signs (death is the worst clinical sign) Diagnosis - Demonstration of trophozoites or cysts on fresh or stained fixed faecal smears will reveal heavy infections - Faecal flotation is used to demonstrate cysts in formed faeces - Morphology is Important; Stained smears may have to be evaluated by specialized lab as many non-pathogenic amoebae are often confused with pathogenic amoeba Control and Treatment - Good sanitation is important to prevent amebiasis - Snakes, lizards and tortoises should not be housed with turtles or housed where they are at risk of their environment contaminated with turtle faeces 43  - Metronidazole is suggested as the treatment (this is the drug of choice in humans), but very little is known about the treatment of amebiasis in dogs or other domestic animals including reptiles Primary Amoebic Meningoencephalitis Is a rare acute, fulminant rapidly fatal illness caused exposure to water containing the free-living amebae Naegleria fowleri or Acanthamoeba spp. Was discovered in South Australia by Dr Malcolm Fowler, a Pathologist at IMVS in 1965. It was later discovered the organism contaminated the pipeline to Port Augusta and people were contracting the disease through the water When the amebae are forced up the nose in warm water they migrate along the olfactory nerves into the cranium. They rapidly destroy the brain and death occurs within 2 weeks. Humans have been treated using amphotericin B, but unfortunately most cases are diagnosed at autopsy. Balantidiasis (zoonosis) Balantidiasis is disease primarily of primates (including humans) but has been reported in other mammals. It is caused by the ciliated protozoan Balantidium coli which is very common in pigs. The pathogenicity of the parasite in pigs has yet to be determined. Morphology Trophozoite and Cyst forms Trophozoite - Trophozoites are ovoid, covered with cilia, and have a funnel-shaped cytostome - 2 sizes: 42-60 µm X 30-40 µm (Small) and 90-120 µm X 60-80 µm (Large) trophozoites as long as 200 µm reported. - Have a large macronucleus: centrally located, often curved or bean shaped - Micronucleus: Spherical and close to macronucleus Cyst - Cysts are spherical to ellipsoid. They are large, 50-75 µm in diameter and have a thick refractile wall 44 Life Cycle Direct life cycle and conjugation can occur between trophozoites Cysts are ingested by the host Trophozoites released from the cysts divide by binary fission and colonize the large intestine Trophozoites encyst in response to the dehydration of fecal material as it moves through the large intestine (trophozoites can also encysts after feces have been passed Epidemiology - B. coli is ubiquitous with a prevalence in pigs of close to 100% - The prevalence in primates, humans and other animals is unknown - The parasite is transmitted directly through the faecal-oral route, but contaminated food and water are also important sources of infection 45  - The pig is the probable source of infection for humans Pathophysiology and Pathogenesis - Most infections are asymptomatic, and disease is rare (especially in pigs) - When pathogenic, the parasite invades the tissues of the large intestine using the enzyme hyaluronidase - Secondary invasion often takes place following the induction of the intestinal lesion - B. coli rarely invades extraintestinal tissues, but peritonitis, appendix perforation and pulmonary involvement has been reported Clinical Signs - The clinical signs include mild colitis and diarrhoea and occasionally severe “amoebic type” dysentery Diagnosis - Trophozoites can be demonstrated on faecal smears (fresh faeces) - Cysts can be observed on faecal floats Control and Treatment - Hygiene is important in swine operations and primate colonies - No treatment registered for use in pigs (is it even required?) - Tetracyclines for treating primates 46 Protozoa II – Coccidia and Coccidiosis Coccidiosis is a generic term used to describe disease caused intracellular parasites that undergo merogony in the host intestinal cells. There are 16 genera, with the 2 most important being: o Eimeria (1000 species). o Cystoisospora spp (Also known as Isospospora) Coccidia are Apicomplexans (Phylum Apicomplexa) which are all: OBLIGATE INTRACELLULAR PARASITES (They must infect a cell) There are hundreds of species of coccidia and coccidia infect all animals of veterinary importance Each specific species of Eimeria or Cystoisospora tends to be host specific, infecting only a single host species or closely related host species However, each host species can be infected by many different coccidians simultaneously In a host animal, each species of coccidia may have a specific predilection site IN GENERAL, the more distal the site of infection the more severe the clinical signs Prepatent periods (time between infection and appearance of oocysts in the faeces) varies for each species and can be from 3 days to 3 weeks! General Morphology The Zoite (i.e. sporozoite, merozoite) - The zoite is the functional unit of all Apicomplexans, - Zoites are motile, banana-shaped and measure between 2-8 µm X 1-5 µm - Zoites contain specialized structures and organelles (rhoptries, conoid, micronemes) at their apical end collectively termed the Apical Complex which are used for cell invasion and are only visible under an electron microscope 47 Sporozoites - are the infective form of the parasite and are found within sporulated oocysts Merozoites - are produced within host cells through a form of asexual reproduction called merogony (synonym = schizogony) Oocyst - Oocysts are formed as a result of sexual reproduction - Oocysts are round to ovoid and vary in size depending on species - The oocyst is often an environmentally resistant stage that requires further development called sporulation to become infectious - Unsporulated oocysts contain a diploid single cell called a sporoblast or sporont (equivalent to a zygote) - Sporulated oocysts contain haploid sporozoites which may or may not be enclosed in a Sporocyst(s) - The Number of sporozoites/sporocysts depends on Genus Eimeria - 4 sporocysts each containing 2 sporozoites = 8 sporozoites Isospora - 2 sporocysts each containing 4 sporozoites = 8 sporozoites Typical Apicomplexan “Zoite” 48  Unsporulated Oocyst Sporulated Eimeria oocyst Sporulated Cystoisospora oocyst 49 Generalized Coccidian Life Cycle (IMPORTANT!!!!) Many stages occur comprising asexual reproduction (merogony and sporogony) and Sexual reproduction (gametogony). - Sporulated oocysts are ingested by the host (if not sporulated, not infective) - Sporozoites emerge and invade an intestinal cell (epithelial or lamina propria) - Round up as trophozoite inside the cell and form a parasitophorous vacuole - Trophozoite grows to become first generation meront (Also called Schizont) - Merogony (Asexual reproduction AKA Schizogony) to produce first generation merozoites - Merozoites burst from cell and invade fresh cells to become second generation meronts (the number of these asexual generations depends on species of coccidia but 2 or 3 is usually the limit for many species) - After a genetically pre-determined number of asexual repetitions merozoites produced by the last merogony enter a new host cell and develop into a macrogametocyte or microgametocyte (gametogony) - These mature into single cell macrogametes (female sex cell) or undergo repeated division becoming biflagellate microgametes (male sex cells) - Microgametes are released and fertilize Macrogamete to form a diploid zygote - A resistant wall then develops around Zygote to form oocyst - Host cell ruptures releasing oocyst which passes out in faeces - Oocysts undergo sporulation (sporogony) when correct conditions present (moisture, temperature, oxygen etc) producing haploid sporozoites and are now infectious Infection is usually self-limiting in absence of reinfection Oocysts can be very resistant to environmental conditions 50 Epidemiology Prevalence - Highly prevalent especially in crowded conditions (poultry operations, feedlots, catteries, etc) - Animals will often have mixed infections (some highly pathogenic, others not pathogenic) - Transmission by faecal-oral route, contaminated food and water Disease - Often asymptomatic - Disease associated with raising animals in confinement 51  - “Coccidiosis is a disease of the feedlot not of the range” 3 - Disease associated with young animals and stress elements (weaning, adverse weather, shipping, etc) - Adult animals develop degree of immunity that protects from disease but not always re- infection - Immunity is species specific (immunity to E. bovis offers no protection to E. zuernii) and often strain specific Pathophysiology and Pathogenesis - Severity of disease proportional to the number of infective oocysts ingested and location of infection (crypts and colon = more severe disease) - Parasite can destroy epithelial cells causing villous atrophy and intestinal lesions - Crypt hyperplasia can occur resulting in immature epithelial cells along the villi - Denudation of epithelium can result from infection of crypts - Haemorrhaging is seen in severe disease Clinical Signs - General signs of diarrheal enteritis and malabsorption - Blood may or may not be in faeces (depends of parasite species, the severity of infection, and the location of the infection) - Systemic signs of blood loss if the infection has caused haemorrhage - Poor weight gain, emaciation, death Diagnosis - Faecal flotation to demonstrate oocysts, but clinical signs usually start to appear before oocysts are passed - History and clinical signs are also important as the mere presence of oocysts is not proof of coccidiosis 3 Levine, N.D., 1985. Veterinary Protozoology. Iowa State University Press, Ames Iowa, p 139. 52  - Gross intestinal lesions at necropsy and coccidia observed in lesion scrapings or histopathology. Often large meronts observed on intestinal scraping or impression smear Infection with non-pathogenic species of coccidia is common. Diagnosis is dependent on the age of susceptibility and finding oocysts in the feces of animals suffering clinical signs of coccidiosis Treatment and Control - Prevention is the key!!! You do not really treat coccidiosis - Prophylactic drugs are used to prevent the development of disease (often mixed with feed or water) - Treatment with drugs after clinical signs develop is rarely effective - The drugs currently available are effective only on the initial stages of the parasite therefore treatment after disease develops is not effective (disease will resolve on its own, so it gives the impression treatment is effective) - Supportive treatment and control of secondary infections is extremely important - Reducing exposure to oocysts through good management can prevent or reduce the severity of disease - Avian Coccidiosis One of the most common and expensive diseases of poultry production Chickens - 7 species of Eimeria involved § Eimeria acervulina - most frequently encountered § Eimeria tenella - most pathogenic (high mortality) - Site of infection ranges from small intestine to cecum depending on species involved 53  Species Site of infection Prepatent period (hours) E. acervulina Duodenum 89 E. brunette Lower SI, Caeca, rectum 120 E. maxima Mid SI 120 E. mitis SI, Caeca, rectum 91 E. necatrix SI 138 E. praecox SI 84 E. tenella Caecum 132 - Intestinal/Cecal lesions range from minor to rounded whitish plaques to severe necrotic cores and haemorrhage with significant blood loss Clinical Signs - Bloody droppings and hemorrhagic diarrhea beginning 4 days post infection - Acute death loss (mortality rates can be high) - Emaciation, pallor, inappetence Control - Isolate sick birds; treat healthy birds with anticoccidial medication (treating sick birds is futile). - Almost all poultry flocks receive preventative medication - RESISTANCE TO PROPHYLAXYS BECOMING WIDESPRESD - Drugs must be rotated or use “shuttle” program (one drug in starter another drug in grower feed) - Vaccines, comprised of attenuated oocysts are now available (Administer orally or using an eye dropper) - Raise young chickens separate from older birds Turkeys - 7 species of Eimeria infect turkeys o E. adenoeides, E. gallopavonis, E. meleagrimitis are most pathogenic 54  - Lesions are not as spectacular as in chickens, but mortality rates can be high in young birds Clinical Signs - Watery mucoid diarrhea - Anorexia - Ruffled feathers and general signs of illness Control - Preventative medication and control measures as with chickens Ducks and Geese In geese, renal coccidiosis caused by Eimeria truncata and intestinal coccidiosis caused by E. anseris are of pathogenic significance. In ducks, many species of pathogenic coccidia have been described. Treatment and control as for turkeys and chickens Bovine Coccidiosis - at least 12 species infect cattle and mixed infections are the rule - Eimeria zuernii and Eimeria bovis are the most pathogenic - Coccidiosis occurs in calves 3-weeks to 6-months of age, but usually seen in calves 2 – 6 months of age or in older group housed calves - First generation meronts grow to giant size in endothelial cells of lacteals in the villi (E. bovis) or lamina propria (E. zuernii) of the ileum and produce thousands of merozoites - Prepatent period 6 – 24 days depending on the species Clinical Signs - Moderate infections produce diarrhea, listlessness, anorexia 55  - Severe infections produce liquid bloody diarrhea (which may travel 2-3 m), emaciation and tenesmus - Hind quarters can become soiled with faeces - Secondary infections are common (especially pneumonia) - Animals that do not die in 7-10 days will usually recover Winter coccidiosis - Characterized by diarrhea and tenesmus occurs during severe cold weather and other stress factors (weaning, shift from pasture to feedlot) Nervous coccidiosis - Calves suffer muscular tremors, convulsions, blindness and 50% mortality in addition to acute diarrhea - The pathogenesis not understood but majority of cases occur in severe cold (don’t have to worry about it here) Control - Decrease stocking rates, minimize stress, clean housing and feed - Drugs used for treatment and prevention include: o Decoquinate, amprolium, monensin, sulfamethazine, sulfaquinoxaline, lasalocid - During an outbreak, anticoccidial drugs should be given to the calves that are not yet showing clinical signs - Supportive therapy and fluid replacement until gut epithelium is replaced is extremely important in calves with clinical coccidiosis - To prevent successive transmission do not house a new group of calves on a lot where another group of calves were previously housed without cleaning and sanitizing the lot. - 56 Ovine/Caprine Coccidiosis - 11 species of Eimeria infect sheep, 9 infect goats - Prepatent period of about 7-23 days depending on species Sheep - Primarily a disease of feedlot lambs or after shipping, usually occurring 12 d – 3 wks after arrival - Lambs can have watery diarrhea for several days up to 2 weeks (usually not bloody) - Depression, inappetence followed by weight loss - Mortality is seldom more than 10%, but significant weight loss can occur - Soiled wool may attract flies =Fly Strike (myiasis) Goats - More susceptible than sheep - Clinical signs typically follow weaning (Diarrhea) - Heavily infected kids usually die and kids that recover may fail to grow normall Control and Treatment - Treatment and control as in cattle - Raise lambs on slatted floor pens - Good sanitation practices Camelid Coccidiosis - Eimeria macusaniensis is a particularly important coccidian of alpacas. The oocysts are unusual in appearance and resemble Eimeria leukarti of horses. The disease is severe, and animals show signs of lethargy, weight loss, diarrhea and circulatory shock. Mortality rates can be very high in both Llamas and Alpacas. 57 Equine Coccidiosis - Eimeria leuckarti - Coccidiosis is rare in horses - Few reports of diarrhea or weight loss in infected horses Rabbit Coccidiosis - There are several pathogenic species in rabbits, and these are major causes of disease in the commercial rabbit industry - Rabbits can die suddenly from coccidiosis if placed under stress Liver -Eimeria stiedai - Infects the bile duct epithelium - Causes biliary hyperplasia and cirrhosis resulting in diarrhea Intestine - Eimeria intestinalis, Eimeria flavescens - Infect the crypts of the small intestine (E. intestinalis) and cecum (E. flavescens) - Infection causes denudation of the epithelium and severe diarrhea Control - Prophylactic administration anticoccidials - Prevent contamination of feeders/ waterers - Proper management is important 58 Swine Coccidiosis - Coccidiosis is a severe disease of nursing piglets caused by Cystoisospora suis - Ubiquitous where pigs are farrowed in confinement - I. suis is responsible for 15-20 % of piglet diarrhea - Infects enterocytes throughout the small intestine and occasionally cecum and colon - Prepatent period 4- 5 days, patent period 2 weeks - Established I. suis infection interferes with Salmonella typhimurium infection Clinical signs - Yellowish-Gray pasty diarrhea 6 – 14 days of age, piglets covered with diarrhea smell like “soured milk” - Blood not present unless other disease agents involved - Depressed weight gains, high morbidity, moderate mortality Treatment and Control - administration of anticoccidials prophylactically (Toltrazuril registered in Australia) - Nursing piglets do not eat or drink enough for drugs added to feed or water to be effective so have to “drench” piglets - Improved sanitation is required to reduce the number of infectious oocysts in environment Canine and Feline Coccidiosis Dogs Cystoisospora (Isospora) canis 59  - Largest oocyst of canine Isospora species - Develops in lamina propria of distal small intestine and may cause diarrhea associated with weaning stress Cystoisospora (Isospora) ohioensis complex - Isospora ohioensis, Isospora burrowsi and Isospora neorivolta cannot be separated based on oocyst size - I. ohioensis develops in the enterocytes in the S.I , cecum, and colon and can cause diarrhea in puppies and is associated with stress factors Cats Cystoisospora (Isospora) rivolta and Cystoisospora (Isospora) felis - Develop in the enterocytes of small intestine and can cause diarrhea and enteritis in kittens (newborn – 4-week-old) - Stress factors (such as other disease agents) are likely involved Paratenic Host - Isospora infections in dogs and cats can be acquired through a paratenic (transport) host - Mice, cattle, sheep and other herbivores can act as paratenic host when they ingest oocysts and sporozoites invade extraintestinal tissue (Mesenteric lymph nodes, brain, muscle) - Sporozoites become encysted and are released following ingestion of the paratenic host Control - Sanitation to reduce exposure to oocysts (ammonium-based products) - Treatment of clinical cases with anticoccidials may reduce patent period, but response is seldom dramatic. 60  Cases and Questions 6: In South Australia, coccidiosis is often a problem when rangeland goats are mustered for sale or feed lotting. Why are goats so susceptible to coccidiosis and what can be done to prevent it from occurring? Cryptosporidiosis (zoonosis) Cryptosporidiosis is caused by small apicomplexan parasites in the genus Cryptosporidium Many species of Cryptosporidium infect the microvillous region of epithelial cells lining the gastrointestinal and respiratory tracts of many different vertebrates Intracellular, extracytoplasmic parasites of epithelial cells NOTE: Cryptosporidium parvum is zoonotic and infections in vet students are common due to handling of calves with neonatal diarrhea!!! Did you read that – infections in vet students are common! Wash your hands after handling dairy calves. Mammals Cryptosporidium parvum and C. parvum like species - Infect the small intestine of a wide range of mammal - Very important and common cause of neonatal diarrhea in calves and potentially other ruminants Cryptosporidium andersoni - Infects the abomasum of older calves (> month