If you own a dog or cat, you will have to control fleas. Even light flea infestations are annoying to pets, and some dogs and cats develop skin problems because they are allergic to flea bites. Heavy flea infestations can cause pets to be unthrifty and cause anemia in puppies and kittens. Fleas may also host tapeworms, and pets become infected when they ingest infected fleas while grooming. The dog tapeworm may rarely infect humans if they accidentally eat infected fleas (primarily only a problem in children). Other than potential infection with tapeworm, fleas are generally not vectors of any human diseases east of the Mississippi River. Fleas also bite people, and heavy infestations in the home or yard can make life miserable for pet owners and their family and friends. Although there are many different species of fleas in the world, the cat flea, Ctenocephalides felis, is the species that most commonly occurs on dogs and cats in the United States.
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To control fleas successfully, you need to control them in all areas where they occur: on the pet, in the house, and in the yard. Not allowing pets inside the house is the surest way to avoid having fleas inside the house, but not all pet owners favor this method. Whether or not pets are allowed inside, the first step in flea control is to treat the pet(s) with an effective and appropriate on-pet treatment.
Fortunately, there are several highly effective treatments that can be applied to pets for preventive flea control. Good, on-pet flea prevention, combined with frequent cleaning of pet bedding areas, can keep fleas from becoming established in the house or yard. But if pets are infested with adult fleas, the house and yard will also be infested with immature fleas, and these areas will need to be treated, too.
To control fleas effectively, you need to have a basic understanding of flea biology. Only adult fleas live on the animal and suck blood. Female adult fleas lay eggs on their hosts, but, because the small, white eggs are not sticky, they roll off the host and fall to the floor, accumulating in those areas where the pet sleeps or rests. In 2 to 6 days, the eggs hatch into slender, dirty-white-looking larvae that feed on dander, dried excrement of adult fleas, dried blood, and other organic material that falls from the host or accumulates from other sources. Notice that this &#;flea food&#; is also concentrated in areas where pets sleep or rest&#;right where the developing flea larvae need it to be.
Fleas have a complete life cycle and can complete a generation in as short a time as 3 weeks. Only adult fleas bite pets or people.Flea larvae are only about one-fourth of an inch long when fully mature and can be difficult to find, even when present in large numbers. But if your pet is infested with adult fleas, the larvae are there and will quickly develop into more blood-sucking adults. In an established flea infestation, adult fleas represent only a fraction of the total flea population. The eggs, larvae, and pupae far outnumber the adults, and you will not control fleas unless you control these immature stages.
Because flea larvae require high humidity and are repelled by sunlight, they usually move into cracks and crevices or burrow deep in carpet or rugs. The larvae mature in 1 to 3 weeks and then spin a small cocoon in which they develop into adults. This cocoon, or pupal, stage can be just a week long or several months long. You must wash your pet&#;s bedding and vacuum carpets regularly to complete your flea control regimen.
Newly developed adult fleas that are still inside their cocoons can sense whether or not host animals are present in the area, based on vibrations and carbon dioxide concentrations. When no hosts are present, they will delay emergence from the cocoon for up to several months. This is why heavy flea infestations can emerge suddenly in homes that have been vacant for weeks or months. A new homeowner might exclaim, &#;We just moved in and the house is full of fleas! No one has lived here for over 3 months and we don&#;t even have a pet!&#; What has usually happened in such situations is that the previous occupants did have indoor pets. After they moved, the flea eggs and larvae that were left behind developed to the pre-adult stage and waited for a new host to arrive. They emerged as soon as they sensed renewed activity, and, in the absence of a dog or cat, began biting people.
Fleas can be present year-round, but they become much more plentiful in the spring and summer. Protecting pets from these parasites also helps protect humans and helps prevent infestations in the home and yard. Fortunately, there are several effective products that can be applied directly to the animal to control fleas. Be sure to read the label carefully and understand what you are buying. Some of these products work on both ticks and fleas, while some work only for fleas or only for ticks. Some control internal parasites while others do not. Some of these products may be used on dogs or cats, while some may not be used on cats.
Table 1 provides general information about on-pet flea treatments. Note that this table focuses on flea products and flea and tick products. It does not address products that are used primarily for internal parasite control. When using on-pet treatments, it is important to be sure the product is labeled for use on the type of animal being treated, as some of these products should not be used on cats. Never use a product that contains either permethrin or amitraz on a cat. Be sure the dose and frequency of use are appropriate for the weight of the animal. Many of these products are available in different sized packages that are for animals in a specific weight range. Some of the listed products require a prescription and may be purchased only through a veterinarian, but many are available over-the-counter or through online sources. Flea collars are generally ineffective and are not recommended. However, effective tick collars are available for dogs.
Table 1. Some widely available oral and topical treatments for flea and tick control on dogs and cats.
Product name
Adulticide1
IGR2
Admin
Controls ticks
Active ingredients
For use on
Advantage
yes
no
topical
no
imidacloprid
dogs or cats
K9 Advantage
yes
no
topical
yes
imidacloprid/permethrin
dogs only
Advantage Multi
yes
no
topical
no
imidacloprid/moxidectin
dogs or cats
Bravecto
yes
no
oral
yes
fluralaner
dogs or cats
Capstar
yes
no
oral
no
nitenpyram
dogs or cats
Comfortis
yes
no
oral
no
spinosad
dogs or cats
Frontline Top Spot
yes
no
topical
yes
fipronil
dogs only
Frontline Plus
yes
yes
topical
yes
fipronil/methoprene
dogs or cats
Nexgard
yes
no
oral
yes
afoxolaner
dogs only
Preventic collar
no
no
topical
yes
amitraz
dogs only
Program
no
yes
oral
no
lufenuron
dogs or cats
Program Plus
yes
no
oral
no
milbemycin oxime/lufenuron
dogs only
Sentinel spectrum
yes
no
oral
no
milbemycin oxime/lufenuron/praziquantel
dogs only
Promeris
yes
no
topical
yes
metaflumizone/amitraz
dogs only
Promeris for Cats
yes
no
topical
no
metaflumizone
cats
Proticall
yes
no
topical
yes
permethrin
dogs only
Revolution
yes
no
topical
yes3
selamectin
dogs or cats
Seresto
yes
no
topical
yes
imidacloprid + flumethrin
dogs or cats
For more information, please visit Can You Use Fish Tapes Praziquantel for Tapeworms in Dogs.
Trifexis
yes
no
oral
no
spinosad + milbemycin oxime
dogs only
Vectra 3D
yes
yes
topical
yes
dinotefuran/pyriproxyfen/permethrin
dogs only
Vectra for Cats and Kittens
yes
yes
topical
no
dinotefuran/pyriproxyfen
cats
1 Kills adult stage.
2 Insect growth regulator. Kills larval stage.
3 Kills only the American dog tick (Dermacentor variabilis).
There are many options for on-animal flea control in Mississippi. Following is a brief review of a few of the more popular flea control products for use on dogs and cats.
Advantage, K9 Advantix, and Advantage Multi all contain the active ingredient imidacloprid, which kills adult fleas. K9 Advantix also contains permethrin, which kills ticks but renders the product unsafe for cats. Advantage Multi contains imidacloprid plus moxidectin, which enables it to kill adult fleas and prevent heartworms and a number of other intestinal parasites in dogs.
Capstar is an oral flea-control product popular in veterinary clinics because it kills adult fleas within 30 minutes and can be used for both dogs and cats. However, its effectiveness lasts only about 24 hours, so it is not a good choice for long-term control of fleas.
Comfortis is an oral product for dogs and cats that begins killing adult fleas within 30 minutes and remains effective for approximately 30 days. The primary disadvantage is that the flea has to bite the dog or cat for the product to take effect. The dosages are different for dogs and cats, so be sure to read the label carefully. This product must be given with a meal.
Frontline Plus is applied topically to cats and dogs and kills adult fleas, flea larvae, and flea eggs. It also kills chewing lice and all stages of a number of tick species. Although it is applied topically, the manufacturer claims resistance to bathing or swimming and good efficacy for a 30-day period. Fipronil-resistant flea strains have been reported.
Promeris provides control of existing flea and tick infestations in dogs and protects against reinfestation. The formulation for cats contains metiflumizone but no amitraz and claims up to 7 weeks of flea control.
Revolution kills adult fleas, prevents flea eggs from hatching for 1 month, prevents heartworm disease, and is used for the treatment and control of ear mite infestations. Revolution is also indicated for the treatment and control of sarcoptic mange and for the control of tick infestations due to the American dog tick in dogs and roundworm and hookworm infections in cats.
Seresto is a collar with a unique polymer matrix of two active ingredients: imidacloprid to control flea infestations and flumethrin to repel and kill ticks. The active ingredients spread from the site of direct contact over the skin surface of the cat or dog. It works similarly to a monthly topical, but, as the active ingredients wear off over time, a new supply is continuously replenished in low concentrations. The active ingredients spread from the site of direct contact over the skin surface to slowly and continuously release the active ingredients over 8 months. It is water resistant, so it is not necessary to remove the collar before the pet is immersed in water. The manufacturer states that, in order to remain effective for the 8-month period, dogs must not be bathed more than once per month. Also, for dogs that swim once a month or more, the control duration is reduced to 5 months.
Trifexis is a once-monthly tablet that kills fleas, prevents heartworm disease, and treats and controls adult hookworm, roundworm, and whipworm infections. It is beef-flavored and can be offered as a treat. It combines two active ingredients that are safe and effective. One of its active ingredients, spinosad, is the active ingredient in Comfortis, which begins killing adult fleas within 30 minutes and remains effective for approximately 30 days. The second active ingredient, milbemycin oxime, prevents heartworms and intestinal parasites. Even though Comfortis is also labeled for cats, Trifexis is not. Similar to Comfortis, a disadvantage of this product is that the flea has to bite the dog for it to take effect. However, it is especially appealing to dog owners who want to give one tablet to prevent heartworms, kill fleas, and control the common intestinal parasites listed.
Vectra 3D kills through contact; parasites don&#;t have to bite to die. It begins reducing flea feeding in 5 minutes and kills fleas in 6 hours. Vectra 3D repels and kills fleas, ticks, mosquitoes, biting and sand flies, lice, and mites (excluding mange mites). A repelled vector does not attach to or bite the dog. It kills adult fleas, prevents the development of all immature stages of fleas (eggs, larvae, and pupae), and remains effective after bathing and swimming. It protects dogs for 1 month and may be used on puppies as young as 7 weeks of age. There is a separate product for cats and kittens.
Vectra for Cats and Kittens is a once-a-month topical treatment for use on cats against all flea life stages (eggs, larvae, pupae, and adult fleas). It is fast-acting, killing fleas in 6 hours. It kills by contact, so fleas do not have to bite. It is quick-drying and non-greasy, controls development of all flea stages for 1 month, controls and stops flea infestations, and prevents reinfestation. It can be used on cats as young as 8 weeks of age.
NexGard kills adult fleas and is indicated for the treatment and prevention of flea infestations by the most common flea in dogs (Ctenocephalides felis). It is also used to treat and control the four most common tick species in dogs, black-legged tick, American dog tick, Lone Star tick, and brown dog tick. It can be used on dogs and puppies 8 weeks of age and older that weigh at least 4 pounds. The manufacturer claims protection lasts for 1 month. NexGard should not be used in dogs with a history of seizures or neurologic disorders.
Veterinarians usually recommend controlling pests on the animals as well as in the environment and selecting flea-control products based on the individual animal&#;s needs. It is important to emphasize that some flea products are deadly to cats. If you intend to use a product on cats, always check the label to make sure the product is approved for use on cats, and always follow the manufacturer&#;s directions. Do not use products that contain amitraz or permethrin on cats!
Use on-pet flea treatments properly. Be sure to apply only at the recommended time intervals, and use the dose and frequency that is appropriate for the size animal you are treating. If a product that previously worked well on your pet appears to lose its effectiveness, change to another product that uses a different active ingredient. Also, with heavy flea infestations, it may be necessary to use more than one method of application. For example, Capstar may be used in both dogs and cats to kill all fleas on the pet within 30 minutes. However, this approach would not provide long-term control and would not control fleas in the environment. Heavy infestations usually require multiple approaches starting with a quick knock-down, followed by a long-term product and treatment of the environment.
It is very difficult to treat a newborn puppy or kitten for fleas safely because of their age and size. It is, therefore, wise to treat a pregnant animal for fleas before she gives birth. Use bedding that can be washed frequently, and keep the whelping area flea-free if at all possible. Do not use any flea treatments directly on newborn puppies or kittens!
Only adult fleas live on pets. Eggs fall off the pet and accumulate in bedding areas. Flea larvae feed on the feces of adult fleas, dried blood, and dander, which also falls off the pet and accumulates in bedding areas.Given the biology and habits of immature fleas, it is easy to see why indoor flea infestations are usually concentrated in areas where pets rest and why infestations are often more severe in rooms that are not cleaned regularly and in rooms with carpet or rugs. It is also easy to see why vacuuming and other methods of cleaning pet bedding and floors play such an important role in indoor flea management. Weekly cleaning of pet bedding and the surrounding area removes many eggs and immature fleas before they become adults, and it also removes much of the dander, dried blood, and other organic accumulations on which immature fleas feed. Cleaning is essential to successful indoor flea control!
Not allowing pets indoors is the best way to avoid indoor flea infestations. If pets are allowed indoors, designating special areas for pets to sleep, rest, and spend most of their time can let you concentrate your frequent cleaning efforts on these areas. If pets are allowed on furniture, keep in mind that immature fleas will occur under seat cushions and in other cracks and crevices within the furniture, and vacuum these areas regularly. You will also need to move furniture and vacuum underneath. When you finish vacuuming, remove the vacuum bag, seal it in a plastic garbage bag, and discard it.
When using insecticide sprays to treat established indoor flea infestations, it is important to target both the adult and immature stages. There are many products labeled for indoor control of adult fleas that can be applied as directed sprays. These contain active ingredients such as permethrin, deltamethrin, or pyrethrins. While these products also have activity against immature fleas, flea larvae are difficult to control with traditional adulticide type insecticides because of their habit of burrowing deep into cracks and crevices where they are difficult to reach with insecticides.
You can improve control of immature fleas in indoor settings by using a product that includes an insect growth regulator (IGR) product, such as methoprene or nylar (pyriproxyfen), in your flea treatment. These IGR products work by disrupting the growth of immature fleas, causing them to die before they reach adulthood, and/or by interfering with the female flea&#;s ability to produce viable eggs. The greatest strength of these IGR products is that, when used indoors where they are not exposed to sunlight, they last several months and provide long-term control of immature fleas. Because these IGR products will not control adult fleas, it is best to apply a combination treatment containing an adult flea control product and one of the IGRs when attempting to control established indoor flea infestations. In situations where there is no established infestation of adult fleas and the objective is to simply apply a preventive treatment in areas frequented by pets, the IGR products may be effective when used alone.
Most flea treatments for indoor use by homeowners are sold as pre-diluted, ready-to-use (RTU) sprays. These products usually contain an adulticide, such as permethrin, bifenthrin, cyfluthrin, or deltamethrin, and some treatments contain both an adulticide and one of the IGR products. Another option is to buy adulticides and IGR products separately, as concentrates, and mix them together in pump-up type household sprayers. If you use a pump-up type sprayer, be sure to choose one that applies a fine spray pattern or a small pin-stream spray. Many pump-up sprayers apply spray patterns that are too heavy for indoor use.
Indoor flea treatments are also sold as total-release aerosol foggers that contain an adulticide, an IGR, or both. However, the insecticide fog they release does not penetrate well underneath furniture, floor coverings, or seat cushions, and into the cracks and crevices where most immature fleas live. Most of the insecticide applied by total-release foggers settles in places where it is more likely to contact people and pets than fleas. Whether you use one of the ready-to-use pre-mixes or buy concentrates and mix your own spray, directed sprays will provide much better flea control.
Table 2. Some indoor flea control sprays (not for use on pets).
Active ingredient
Brand name (examples)
Pre-mixed, ready-to-use sprays
bifenthrin + zeta-cypermethrin
Ortho Home Defense Max Insect Killer (RTU)
deltamethrin
Enforcer BugMax Home Pest Control (RTU)
dinotefuran
Alpine Flea & Bed Bug (aerosol)
nylar + permethrin
Enforcer Flea Spray for Homes (RTU)
methoprene + permethrin
Zodiac Fleatrol Carpet & Upholstery Pump (RTU), Adams Home Flea & Tick Spray (RTU)
nylar + tetramethrin + sumithrin
Enforcer Flea Spray for Carpets & Furniture (aerosol)
nylar + permethrin + linalool
Bio Spot Inverted Carpet Spray (aerosol)
permethrin + pyriproxyfen
Sentry Home and Carpet Spray
Insecticide concentrates
dinotefuran (40% concentrate)
Alpine WSG
permethrin (13.3% concentrate)
Martins Multipurpose Insecticide
permethrin (10% concentrate)
Hi-Yield Indoor/Outdoor Broad Use Insecticide
Insect growth regulators
nylar (pyriproxyfen)
Martins IG Regulator
methoprene
Precor IG Concentrate
When applying insecticides in indoor situations, it is especially important to carefully read and follow all label directions. Do not apply insecticides to areas where they are not approved for use. For example, some flea treatments may not be applied in areas where food is prepared, some may not be applied directly to furniture, and few flea products are labeled for broadcast application to carpets and floors. Do not apply any insecticide directly to pets unless the label says you can. Do not apply any insecticide as a broadcast spray to carpets or floors or to exposed surfaces of furniture unless the label clearly allows such use. Also, be sure to observe the re-entry period specified on the label. Most products require that people and pets be kept out of the area until spray has dried, but some may have longer re-entry restrictions. Do not apply more insecticide than the maximum rate indicated on the label. Increasing the rate or concentration will not improve control, but it will increase pesticide exposure to your family and pets.
The key to successful indoor flea control is to be thorough in cleaning and treating all areas in the house where fleas occur, giving special attention to areas where pets sleep or rest. A cursory, hastily applied flea treatment is not likely to produce satisfactory results, but a well-planned, carefully conducted treatment will provide good control. Clean and vacuum floors and pet bedding. Move furniture and vacuum underneath, and vacuum cracks and crevices in seats and under seat cushions. Then make a thorough application of an adulticide plus an IGR according to label directions.
Keep in mind that fleas that are already in the pupal stage are especially difficult to control, and newly emerged adult fleas will hop onto a host as soon as possible. This means adult fleas may continue to appear for a few weeks following treatment, even when the treatment was properly applied using effective products, and follow-up treatments are sometimes required. When attempting to control heavy indoor flea infestations, it may be necessary to treat two or three times at 2-week intervals, keeping in mind that the cleaning and vacuuming is as important as the spraying. If you properly clean and treat all of the areas that harbor immature fleas and treat all pets with an effective on-pet treatment, you will be successful in controlling fleas. After you have gotten the fleas under control, you can maintain control by preventive use of on-pet treatments, weekly cleaning of bedding and other areas frequented by pets, and application of IGR treatments at appropriate intervals.
While not allowing pets inside the house is the best way to prevent indoor flea infestations, you might not want to ban indoor pets from the house after a heavy flea infestation has developed and you have treated as described above. Any remaining emerging adult fleas are eager for a blood meal and, in the absence of pets, are more likely to bite people living in the house. Instead, treat the pet with one of the long-lasting on-pet treatments and allow it to remain indoors. Most emerging adult fleas will be attracted to the pet, where they will be controlled by the on-pet treatment. Essentially, you are using the pet as a flea trap.
Of course, you can also hire a professional pest control company to apply indoor flea treatments. The pest control technician will have effective insecticides and growth regulators and the knowledge and equipment to apply them safely and properly. However, you will still need to do the necessary cleaning before the technician arrives, and you will still need to be present at the time of treatment to move furniture and work with the technician to assure an effective treatment. Omitting the cleaning steps and hiring a pest control company to &#;just treat the areas you can reach&#; will not give satisfactory results. You have to do your part with cleaning and making sure the technician has access to all areas that need to be treated.
Occasionally, high numbers of fleas will occur outside, in the yard or landscape. This usually happens when the area is frequented by flea-infested pets or wild animals. When attempting to control fleas in an outdoor situation, keep in mind that you will not have lasting success unless you also control fleas on all animals that frequent the area. On-pet flea treatments are an excellent way to treat pets, but, in cases where the area is being frequented by stray dogs or cats or wild animals, such as opossums or raccoons, treating the animal itself may not be an option. However, it may be possible to prevent or discourage the animals from using the area. Fencing the yard or sealing crawlspaces under buildings and openings to attics (be sure to maintain proper ventilation) are examples of ways to achieve this. If you feed pets outside, avoid feeding free-choice in areas where strays or wild animals can access the food. Otherwise, you will be attracting unwanted animals, and the fleas they carry, onto your property.
Fleas can occur in three places: on the pet, in the house, and in the yard.Table 3. Some treatments to control fleas in the yard.
Active ingredient
Brand name (examples)
Rate/ sq ft
Comments
Treatments applied as sprays
bifenthrin (0.3%) +
zeta-cypermethrin (0.075%)
Ortho BugClear Insect Killer Ready-to-Spray
6 fl oz
Sold in a ready-to-use, hose-end spray bottle. Attach to garden hose and spray per label directions.
dinotefuran (40%)
Alpine WSG
10 g/gal
Apply to lawns, landscape beds, porches, patios, and other areas where pets rest or spend time. Do not apply to animals.
permethrin (38% concentrate)
Hi-Yield 38 Plus Turf, Termite, & Ornamental Insect Concentrate
0.4 to 0.8 fl oz/gal
Apply as a broadcast spray using a hose-end sprayer or other appropriate sprayer.
permethrin (10%)
Hi-Yield Indoor/Outdoor Broad Use Insecticide
1.5 fl oz in 10 gal per sq ft
Apply as a broadcast spray.
Treatments applied as granules
bifenthrin (0.2% granules)
Ortho Bug-B-Gon Lawn Insect Killer
0.6&#;2.3 lb
Water following application.
gamma-cyhalothrin (0.05% granules)
Triazicide Insect Killer for Lawns
0.8 lb
Water following application.
permethrin (0.5% granules)
Hi-Yield Kill-A-Bug II Lawn Granules
2&#;3 lb
Water following application.
In situations where lawns become infested by fleas, broadcast insecticide treatments, applied either as sprays or granules, can aid in reducing the incidence of bites on people who are using the area. But keep in mind that most immature fleas occur in areas where pets sleep or rest, and these areas may not be in the lawn itself. Instead, they may be in some nearby, more protected site, such as under porches, under shrubs, under crawl spaces of homes, and in garages or utility sheds. Remember that cats can climb, and flea breeding sites sometimes occur in overhead areas of garages and sheds. Especially heavy flea infestations often occur in garages, storage sheds, or crawlspaces of houses where litters of kittens or puppies are being raised. Some insecticides labeled for use against fleas in a lawn are not labeled for use in these other situations, and some insecticides labeled for use in these other situations are not labeled for broadcast treatment of lawns. Be sure to check the product label before making applications, and use products that are labeled for the site you are treating.
Successful outdoor flea control depends more on controlling fleas in areas where pets routinely rest than in the open, sunny areas of the yard&#;immature fleas rarely occur in these open, sunny areas. You might need to treat the sunny part of the yard to control adult fleas that have hopped out there, but spend most of your effort on identifying and treating those areas where pets sleep and rest and immature fleas breed.
Products for treating fleas in lawns are available for application as granules or as liquid sprays. Although they might be easier to apply, granules are generally less effective than liquid sprays, and liquid sprays are better suited for treating under shrubs and porches and other resting areas. When attempting to control heavy outdoor flea infestations, apply a second application in 7 to 10 days. Be sure to observe the restricted entry interval specified on the label of the product you use.
This work is partially supported by Crop Protection and Pest Management, Extension Implementation Program grant no. ---/CRIS Number from the USDA National Institute of Food and Agriculture. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture.
The information given here is for educational purposes only. References to commercial products, trade names, or suppliers are made with the understanding that no endorsement is implied and that no discrimination against other products or suppliers is intended.
Publication (POD-12-23)
Revised by Blake Layton, PhD, Extension Professor, Jerome Goddard, PhD, Extension Professor, and Joe MacGown, Scientific Illustrator, Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology.
The Cyclospora/Cystoisospora/Cryptosporidium parasites are apicomplexan protozoa, and they are intracellular, oocyst-forming parasites. The three genera recognized as causing human diarrhea and thought to be spread via contaminated food and water are Cyclospora, Cystoisospora, and Cryptosporidium. Cyclospora infection has been identified as common in tropical and subtropical regions, usually with a wet season peak. It is considered endemic in Haiti, Peru, Guatemala, Venezuela, Southeast Asia, Nepal, and India (60,&#;62). There have been a series of large outbreaks of foodborne infection in the United States and Canada traced to imported berries and vegetables, including raspberries, mesclun, and basil. Symptoms include an acute watery diarrhea that may extend for several months if untreated (60,&#;62). In AIDS patients, Cyclospora infection can also cause biliary tract involvement manifested by acalculous cholecystitis and cholangitis (62). Cystoisospora has a similar distribution: Latin America, the Middle East, Southeast Asia, and tropical areas in Africa. As with the other Cyclospora/Cystoisospora/Cryptosporidium infections, illness is more pronounced in the very young, travelers, and AIDS patients. In contrast to infection with Cyclospora and Cryptosporidium, Cystoisospora infection in AIDS patients may include systemic spread to lymph nodes, liver, and spleen. Despite appropriate treatment, infections may become chronic in these patients (63).
Why is it necessary to resort to staining or methods other than direct microscopy to identify these organisms? The spherical oocysts of Cryptosporidium are only 4 to 6 μm in diameter. At this size, they are too small to reliably identify in wet preparations by light microscopy. The unsporulated 8- to 10-μm oocysts of Cyclospora are perfectly round in outline and contain greenish inclusions, or morulas. However, despite these properties being quite identifiable, their comparatively small size means that they can be confused with smaller amoebae or pus cells. Although of larger size at 10 to 20 by 20 to 30 μm, the oocysts of Cystoisospora are unsporulated or contain a contracted sporont when passed. These oocysts are hyaline in appearance and can be poorly visualized by direct light microscopy. For all three species, the use of alternative methods other than direct microscopy is required to enhance detection.
Although these parasites are often considered together, there are features of Cryptosporidium that set it apart from other species. It is considered either as basal to all other Apicomplexa or more closely related to the gregarine parasites, with which Cryptosporidium shares many life cycle features. When describing the genus in , Tyzzer (280) chose the name Cryptosporidium to emphasize the difference from other parasites, notably the absence of a sporocyst and the presence of naked sporozoa within the oocyst. These oocysts differ in having no need for sporocyst maturation, they are immediately infectious, and importantly, they are capable of autoinfection (64, 276). The immense significance of Tyzzer's discovery was not appreciated for many decades.
Further impetus for study of Cryptosporidium came from the veterinary world with the description of Cryptosporidium meleagridis in turkey poults in Scotland in (65). saw the recognition of Cryptosporidium associated with scouring in neonatal calves in Iowa (66). As the importance of cryptosporidial illness in calves was becoming recognized, Henriksen and Pohlenz in Denmark made the discovery that oocysts stain acid fast in a Ziehl-Neelsen (ZN) stain; this was a largely empirical finding based on testing a variety of stains from a neighboring bacteriology laboratory (67). The first report of human illness came in with the identification of Cryptosporidium in rectal biopsy specimens of a 3-year-old child (68). A subsequent review of more cases in humans suggested that infection seemed limited to immunosuppressed patients (69). In , first reports describing Cryptosporidium in intestinal biopsy specimens of AIDS patients were seemingly in agreement with this theory. Initial efforts at staining oocysts from fecal specimens included the use of Giemsa and Kinyoun stains (69). Later that year, at a time when there were only an estimated 1,000 AIDS cases worldwide, a comparison study of specimens from eight patients using 15 different staining options confirmed that modified acid-fast staining performed best (70). Prior to the introduction of antiretroviral therapy, the diagnosis of Cryptosporidium was recognized as one of the AIDS-defining illnesses. The often-unrelenting symptoms included chronic diarrhea or severe cholera-like illness and could also have involved biliary tract colonization, cholecystitis, and cholangitis (71). The prediction that infection would be confined to the immunocompromised proved to be erroneous. Cryptosporidium was soon identified in immunocompetent patients as a cause of self-limiting, watery diarrhea of several weeks' duration; infection was also more common among young children than among adults (72). In subsequent years, Cryptosporidium has also been identified as a significant cause of illness in patients with malignancy and other types of immunodeficiency (73).
In the intervening years, many Cryptosporidium species and possible subspecies have been identified, with many exhibiting a very limited host range. The two species commonly detected in humans are C. parvum and C. hominis. The latter species does not typically infect animals, but C. parvum is commonly infects cattle. This species is recognized as an environmental problem in water catchments in rural communities. In total, there are approximately 20 species or genotypes of Cryptosporidium that have been identified as causing illness in humans, but most of these generally have a more restricted host range (74). The waterborne outbreak in Milwaukee, WI, that affected 400,000 people has been attributed to human feces (C. hominis from the sewage treatment plant) entering the drinking water system after a failure of the water treatment plant filtration system (30, 75).
With the advent of PCR assays, the dimension of illness caused by Cryptosporidium has been dramatically reestimated (76). In an analysis of the global burden of Cryptosporidium, estimates quoted show that 15 to 25% of diarrhea in childhood is attributable to this parasite (77). Furthermore, those most at risk are the immunocompromised, the malnourished, and infants of low birth weight. Review articles have stressed that any form of infection, even asymptomatic or postsymptomatic, can result in significantly reduced growth rates by the age of 6 to 9 years (78, 79). The importance of this infection has been confirmed in the Global Enteric Multicenter Study (GEMS) of the etiology of diarrhea in children (to 59 months) in sub-Saharan Africa and south Asia. Based on detection by immunoassay, results showed that Cryptosporidium infection was the second-most-common infection in infants and was associated with the highest risk of death in toddlers (80). PCR testing in Uganda has also shown concomitant respiratory tract involvement among children with diagnosed intestinal infection (80).
These recent figures illustrate that infection rates in developing countries have been significantly underestimated. In poorly resourced regions, where access to molecular techniques may not be available, it is important that attention is focused on improving existing technology, especially microscopy and staining techniques. The introduction of LED-sourced fluorescence has reduced the ongoing cost and maintenance of fluorescence microscopy, which should enhance the test options for the diagnosis of Cyclospora/Cystoisospora/Cryptosporidium and microsporidian parasites.
In considering alternative approaches for concentrating and staining oocysts of Cryptosporidium, it is essential to have a gold standard method for comparison. It is generally accepted that a fluorescent antibody test meets that requirement. First developed by Sterling and Arrowood (81), the assay was based on a monoclonal antibody to oocyst cell wall antigen. Similar versions of this assay are now marketed commercially by a number of manufacturers (71). Apart from cost, a limitation of this test is the requirement that the fecal smear must be processed or cleared in some way to ensure that fluorescence can easily be detected. This test will be described in more detail below.
Unlike with other species, the oocyst of Cryptosporidium is very small; at 4 to 6 μm, it is too small to reliably identify by light microscopy when unstained. With profuse diarrhea, the number of oocysts can be very high; one estimate is 109/ml. At these numbers, diagnosis is relatively straightforward. However, detection is more difficult when oocyst numbers are low, as in asymptomatic infection or during the period of excretion after the cessation of symptoms. This period may extend to 2 months postinfection (82). Additionally, there is potential for inadvertent spread if convalescent cases are not recognized. Even subclinical infection is thought to have long-term health consequences for young children (77).
There have been two approaches to concentrating specimens. The flotation technique requires application of fecal specimen to a solution of higher density, such as zinc sulfate, Sheather's sucrose-phenol, or saturated sodium chloride solution. Sucrose-phenol has been favored for isolation of Cryptosporidium, with the oocysts recovered from the top meniscus after centrifugation. A disadvantage of this technique is that microscopy must be performed within 15 min of sedimentation. Otherwise, lysis and distortion of the oocysts can occur in the highly osmotic fluid. Additionally, the presence of sucrose may interfere with staining reactions (83, 84). This technique is unsuitable for recovery of some of the heavier helminth eggs and so is not generally regarded as applicable for use in screening for other parasites. Comparison studies of flotation versus the FEA concentration technique have provided varied results.
In one study, 703 stool specimens were processed by both methods during an outbreak of cryptosporidiosis. Despite results being comparable statistically, there was limited overlap of low-positivity results; approximately 20% of positive results were detected by a single method only (84). In contrast, in another study, dilutions of seeded fecal specimens were processed by both methods. In the FEA method, at the lowest positive cutoff level, there was nil recovery by flotation (85).
The FEA technique has more general application than flotation and has been favored for recovery of oocysts, principally because filtration followed by the extraction of fats by ethyl acetate gives a cleaner product, suitable for use in the direct fluorescent antibody (DFA) microscopy test. In , Weber et al. (86) reviewed the efficiency of this process. They tested the threshold for detection using both a formed and a watery stool, supplemented with C. parvum oocysts at six different concentrations. Using 10% formalin and centrifugation at 500 × g for 2 min, the number of oocysts recovered by FEA represented a loss of 51.2% of expected numbers for liquid stool and 93.2% for the formed stool. The threshold for detection from liquid stool was 10,000 oocysts/g by acid-fast staining and 5,000 oocysts/g by DFA testing. There were high numbers of oocysts detected in the filter gauze and discarded supernatant. These rather-disconcerting findings were followed by a further report from the same research group, advocating a new FEA-flotation procedure. An increased centrifugation time of 10 min at 500 × g (now standard) did not improve yield from five seeded specimens but compacted the deposit, making DFA testing more difficult. In an additional flotation step, the deposit was suspended in water and layered on a solution of saturated sodium chloride. Following centrifugation, a clean, concentrated harvest of oocysts was recovered from the interface between the two solutions. Despite this modification, the limits of detection were still calculated to be 5 to 10,000 oocysts/g of stool. It was also noted that recovery was reduced from stools containing higher levels of fat.
Using a more typical cross-section of specimen types rather than seeded specimens, Clavel et al. (87) compared recoveries of Cryptosporidium oocysts from 73 stored positive specimens. By similarly extending the centrifugation time from 2 to 10 min, they found a 13% increase in the recovery of oocysts. Despite the increased fecal debris after extended centrifugation times, modified acid-fast stains could still be interpreted. A further examination of the FEA process was conducted in Brazil by Pacheco et al. (88), who again using stored positive specimens. In this study, 27 specimens were processed in two ways: by the FEA technique and by sedimentation by centrifugation (SC), without ethyl acetate extraction. Centrifugation was at 400 × g for 2 min. Unfortunately, this centrifugation speed is much lower than the 500 × g for 10 min now commonly used. Despite this discrepancy, the observations reflect the difficulties in recovery of oocysts of small size and low density. In the FEA process, the ring of ethyl acetate-extracted fats was also sampled and oocysts were detected in 93.3% of samples. The numbers in the fatty plug exceeded those in the deposit in 25.9% of these. This finding has bearing on the capacity to diagnose infection when oocyst numbers are low or when the patient has malabsorption and associated high fat levels in feces. Although the quality of the modified acid-fast smears prepared from the SC deposit was lower, with more stain residue, this method was the preferred option, as it avoided having to stain both the deposit and the ethyl acetate-fatty residue generated by the FEA method.
While there are doubts about the efficiency of recovery of oocysts and spores by the conventional formalin ethyl acetate method, at this stage, there are no practical alternatives. There is the added convenience of using only a single approach for concentrating eggs and cysts for microscopy. The effectiveness of this technique is that it produces a much cleaner product, due in large part to the ethyl acetate extraction of fats, which is of particular importance for immunofluorescence assay (IFA) staining of Cryptosporidium to reduce the level of background fluorescence. There may also be validity to the move to perform a modified concentration technique without using formalin (88, 89). Perhaps by removing formalin, subtle changes in the specific gravity of the suspending solution may be sufficient to ensure recovery of oocysts of very low density. In theory, flotation as a means of concentrating oocysts may offer more success in the recovery of structures of low density. However, as mentioned previously, the two methods have been compared directly, with no observed advantage in the use of flotation (84). Furthermore, the more complex nature of this procedure does not lend itself to large-scale processing of specimens.
Commentary about the recovery of Cystoisospora and Cyclospora is more limited than for Cryptosporidium due largely to the lower frequency of detection. In comparing methods for concentrating Cystoisospora oocysts, Pacheco et al. (88) used FEA and sedimentation by centrifugation alone (SC). As they had observed for Cryptosporidium, they found that in the FEA concentration process, there were high losses of Cystoisospora oocysts in the ring of fatty debris and that best yield was obtained with centrifugation alone (89).
Similarly, Kimura et al. (89) screened fecal specimens in Nepal for Cyclospora by three methods: FEA, sucrose flotation, and direct smear fluorescence microscopy. The results using the last two techniques were not statistically different but were significantly higher than those obtained by FEA. Furthermore, the yield in FEA positives was much lower, with 55% containing only one oocyst in a set number of fields examined. They postulate that oocysts must get trapped in the fecal plug, again suggesting a low specific gravity.
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