Giardia and Tritrichomonas infections (Proceedings)

Giardia

Giardia duodenalis (also known as G. intestinalis, G. lamblia) is a pear-shaped, binucleated, flagellated protozoan parasite that infects the small intestine, impairs mucosal absorption, and causes diarrhea. There are two forms: the motile trophozoites that inhabit the small intestine, and the nonmotile infective cysts that pass with the feces into the environment where they can infect new hosts.

The numerous species and strains of Giardia are grouped by genotype in to 7 “assemblages”, denoted by letters A through G. Dogs and cats can be infected by either host-specific genotypes (C and D in dogs; F in cats) or zoonotic genotypes (A and B) that infect humans (Vasilopulos 2007; Thompson, 2008; Payne 2009; Covacin 2011).

Giardia has a worldwide distribution. The overall prevalence in most populations of pet dogs and cats is 1% to 5%, but is higher (10 to 20%) in diarrheic animals. The prevalence is highest in young animals and animals confined together in crowded conditions (e.g., kennels, catteries, shelters, pet shops, puppy mills) (Carlin, 2006; Payne, 2009). Infection rates in group-housed animals can approach 100%.

Life Cycle

Giardia has a direct life cycle and transmission is feco-oral. The usual source of infection is the ingestion of food or water contaminated with cysts. Giardia cysts are environmentally stable and dormant, but upon ingestion by a host they excyst in the duodenum producing two trophozoites from each cyst. The motile trophozoites attach to the brush border surface of the mucosal epithelium by means of ventral cup-shaped adhesion disks, or they swim freely within the adjacent mucus layer. Trophozoites mainly inhabit the duodenum in the dog and the jejunum and ileum in the cat. Clinical signs develop after an incubation period averaging 7 days. As Giardia trophozoites pass into the colon they transform into infective cysts, which are the most typical form in the feces. Cyst excretion begins 5 to 16 days post-infection. Trophozoites are occasionally found in severely diarrheic feces.

Wild animals are potential reservoirs, and water from contaminated streams and ponds may be a source of infection. In cool moist conditions Giardia cysts can remain infectious for months and they are resistant to many disinfectants. Cysts on the haircoat can cause autoinfection during grooming.

Clinical Signs

In most animals Giardia causes relatively mild enterocyte injury, so most infections are asymptomatic, especially in healthy mature animals. The presence and severity of clinical signs are determined by the agent (dose and strain virulence), the host (age, stress, nutrition, and immune status), and the environment (crowded unsanitary conditions) (Payne, 2009). Clinically apparent giardiasis occurs most frequently in young dogs and cats and is characterized by intestinal malabsorption with voluminous foul-smelling, light-colored, watery or “cow pie” diarrhea, steatorrhea, and weight loss. Mucoid diarrhea is occasionally seen in cats. Giardia diarrhea may be acute or chronic, intermittent or continuous, and self-limiting or persistent. The severity of Giardia infection is worsened by concurrent viral, bacterial, protozoal, or helminth infections of the intestines. Corticosteroids can lead to recrudescence of infection in “recovered” animals. Giardia does not typically cause anorexia, vomiting, GI bleeding, or fever.

Diagnosis

Giardiasis should be considered in any dog or cat with unexplained small bowel diarrhea, especially in young and group-housed animals. The diagnosis depends upon fecal tests for the detection of Giardia antigen, cysts, or trophozoites. PCR and sequencing can be used to genotype Giardia (Covacin 2011).

Fecal immunoassays for Giardia antigen include the in-office SNAP Giardia Test (IDEXX) and the microplate ELISA test (ProSpecT/Giardia, Remel), which is offered by commercial labs. Conflicting results have been reported regarding the sensitivity and specificity of the SNAP and microplate ELISA assays for Giardia coproantigen (Mekaru 2007, Rimhanen-Finne 2007, Geurden 2008, Rishniw 2010). The SNAP test may be less sensitive, but this is offset by its convenience, ease of use, and lower cost.

Giardia cysts (oval; 8-12 μm x 7-10 μm) are present in feces much more consistently than motile trophozoites (pear-shaped; 12-17 μm x 7-10 μm x 2-4 μm). Giardia cysts are not usually detected by standard flotation solutions, so zinc sulfate centrifugation-flotation is the preferred method for recovery of Giardia cysts from feces (Dryden, 2006). For this procedure, 2 gm of feces are mixed with 15 ml of 33 percent zinc sulfate solution (1.18 specific gravity) and strained, then the mixture is centrifuged and a drop of the surface layer (meniscus) is transferred to a microscope slide and examined. The pattern of cyst excretion in the feces can be intermittent, so a single fecal specimen may overlook the diagnosis in some cases. To increase sensitivity and maximize the chances of detecting Giardia cysts, it is recommended that at least 3 fresh fecal samples be examined over 3 to 5 days.

The most sensitive and specific technique for detecting Giardia cysts in feces is the direct immunofluorescence assay (IFA; MeriFluor; Meridian Diagnostics), which is considered to be the “gold standard” for diagnosis of Giardia in dogs and cats (Mekaru 2007; Rimhanen-Fenne 2007; Geurden 2008; Rishniw 2010). The IFA is only available through reference labs that have fluorescent microscopy.

Examination of fecal smears for trophozoites is the least reliable method for diagnosing Giardia. Motile Giardia trophozoites can occasionally be identified in wet mounts of fresh diarrheic feces suspended in saline. Giardia trophozoites move in a tumbling or “falling-leaf” motion. A drop of Lugol iodine can be added to kill and stain the trophozoites, which resemble a “monkey face” appearance formed by the two nuclei, the axonemes, and the median body. Trophozoites are found more readily in duodenal specimens (endoscopic aspirates, washings, brushings, or mucosal biopsy impression smears). Duodenal sampling is not practical as a routine diagnostic test for Giardia, but it might be appropriate in patients undergoing gastroduodenoscopy for other reasons.

None of the diagnostic tests for Giardia are 100% reliable and some studies show considerable discordance between assays that detect fecal antigen or cysts (Geurden 2008; Rishniw 2010). Also, the presence of Giardia may be masked temporarily by barium, antibiotics, antacids, antidiarrheals, laxatives, and enemas. The chances of detecting Giardia are improved when fecal antigen testing (e.g., SNAP or ELISA) is combined with zinc sulfate centrifugation-flotation (combined sensitivity of 98%), and when more than one fecal specimen is analyzed. Regardless, some Giardia infections escape detection and negative fecal examinations do not exclude a diagnosis of Giardia. For this reason, a therapeutic response trial of fenbendazole (Panacur) may be appropriate when diagnostics are negative and “occult” giardia is suspected.

Treatment

The safest and most effective treatments for Giardia are fenbendazole (Panacur; 50 mg/kg PO q24hr for 3 to 5 days) and febantel-pyrantel-praziquantel (Drontal Plus; dogs - 25 to 35 mg/kg PO q24h for 3 to 5 days; cats - 50 mg/kg PO q24h for 5 days)(Zajac, 1998; Barr, 1994; Barr, 1998; Payne, 2002; Montoya, 2008; Bowman 2009). Fenbendazole is a benzimidazole that disrupts glucose uptake and energy metabolism by Giardia trophozoites. Febantel is metabolized to fenbendazole and oxfendazole.

Metronidazole (25 mg/kg PO q12h for 7 days) is usually effective, but up to one-third of infections may be metronidazole-resistant. Side effects of metronidazole can include anorexia, vomiting, and reversible neurotoxicity (weakness, ataxia, disorientation, seizures, and blindness). Cats tolerate a liquid formulation of metronidazole benzoate better than bitter-tasting metronidazole USP tablets (Scorza 2004). Albendazole is effective (Barr, 1993), but is not recommended because it has been associated with severe bone marrow toxicity. Furazolidone suspension and quinicrine have also been used to treat Giardia, but are not recommended because they are less effective than other drugs and have a high incidence of side effects (anorexia, lethargy, vomiting).

Prevention

Reexposure and recurrence of infection is often mistaken as failure to respond to treatment, especially in groups of animals confined together. Cysts remaining in the environment and on the haircoat in treated animals can be a source of reinfection. Control measures should include (1) treatment of all contact animals that are housed together; (2) cleaning and decontamination of the environment, including disinfection with quarternary ammonium (Roccal), and (3) bathing to clean cysts from the haircoat.

An adjuvanted vaccine that contains killed Giardia trophozoites is available (GiardiaVax; FortDodge), but it is not recommended. It has not been proven to cure or prevent infection and it does not reliably lessen cyst shedding (Stein 2003; Anderson, 2004).

Zoonotic transmission of Giardia from pets to humans is possible but considered uncommon. Surveys have shown that some dogs and cats in the U.S. harbor zoonotic genotypes of Giardia (A and B), and that some of these animals are asymptomatic shedders (Vasilopulos 2007; Covacin 2011).

Tritrichomonas Infection in Cats

Tritrichomonas foetus has emerged as an important cause of infectious large intestinal diarrhea and chronic colitis in cats (Gookin 1999, Gookin 2001, Levy, 2003, Bell 2010). Trichomonads are pear-shaped, flagellated protozoa with a characteristic undulating membrane along their body. They are similar in size to Giardia, but they lack a cyst stage and are transmitted directly between hosts as trophozoites. Moist, warm, anaerobic conditions are optimal for trichomonads. In addition to colonizing the colon and distal ileum in cats, T. foetus causes venereal infection in cattle. The prevalence of T. foetus infection is highest in densely housed young cats in crowded catteries and shelters. In a study of purebred show cats, infection was identified in 31% of 117 cats from 89 catteries (Gookin, 2004).

Clinical Signs

Tritrichomonas foetus causes mild to severe lymphoplasmacytic and neutrophilic colitis associated with waxing and waning large bowel diarrhea, which is typically semiformed or “cow pie” in consistency and foul smelling (Gookin 1999). Diarrhea sometimes contains fresh blood or mucus. Severely affected kittens sometimes develop painful anal irritation with dribbling of feces or rectal prolapse. The diarrhea often improves transiently in response to antibiotics. Affected cats generally remain otherwise healthy and in good body condition. Diarrhea is often exacerbated by concurrent enteric infections or parasites, especially Giardia and Cryptosporidium (Gookin 2004).

Diagnosis

The diagnosis of Tritrichomonas foetus infection can be confirmed by direct fecal microscopy, fecal culture, fecal polymerase chain reaction (PCR) assay, or colonic mucosal biopsy. Testing is most reliable in cats that have been off antibiotics for 2 weeks or more. This is because antibiotics can temporarily decrease the number T. foetus and cause false negative test results.

Motile trophozoites T. foetus of can be identified in fresh wet smears of diarrheic feces taken directly from the rectum in about 14% of cases. A drop of feces mixed with a drop of saline is coverslipped and examined under low light at 200X to 400X magnification. The likelihood of detecting trophozoites is lower in formed or dried feces, and in cats recently treated with antibiotics. Trichomonads, which are similar in size and shape to Giardia, are identified by their distinctive undulating membrane and rapid, jerky, “jitterbug” motility compared with the “falling leaf” motility of Giardia.

Protozoal fecal culture is more sensitive than microscopy for diagnosis of T. foetus (Gookin, 2003). Culture requires 0.05 g (size of rice grain) of freshly voided feces inoculated into commercially available protozoal media (Feline In Pouch TF™; Biomed Diagnostics) and incubated at 37o C for 48 hr or at room temperature (25o C) for up to 12 days. The pouch should be examined daily with a microscope to avoid missing a positive result. The sensitivity (detection limit) is 1000 or more trichomonads per sample. Use the wettest part of the stool to obtain viable trichomonads. If voided stool is dry or contaminated with litter, collect a rectal specimen with loop or swab. Rectal mucus on swab is sufficient for culture, but not PCR. An excessively large inoculum of feces into the pouch can promote overgrowth of bacteria, which impairs performance of the culture system. Clouding of the liquid media and the formation of gas bubbles are indicative of objectionable bacterial overgrowth in the culture. Do not refrigerate specimens as temperatures below 60o F rapidly kill T. foetus.

Fecal PCR assay is the most accurate test (high sensitivity and specificity) for detecting T. foetus (Gookin 2002). Feces for PCR (180 to 220 mg) should be free of litter and is best preserved in 3 to 5 ml of isopropyl rubbing alcohol for shipping at room temperature. The sensitivity limit of PCR is 10 trichomonads per 200 mg fecal sample.

In colonic mucosal biopsies from infected cats, trichomonads are sometimes identified in the superficial mucus and mucosal crypts, accompanied by an infiltrate of lymphocytes, plasma cells, and neutrophils (Yaeger, 2005). Fluorescence in situ hybridization (FISH) can also identify T. foetus.

Treatment

Treatment of feline trichomoniasis is often unsuccessful. In untreated cats the longterm prognosis is good based on the finding that diarrhea resolves spontaneously in 88% of infected cats within 2 years (median, 9 months; range 5 months to 2 years) (Foster, 2004). However, subclinical infection persists for years in over half of the cats after resolution of the diarrhea (median, 3 years; range 2 to 5 years (Foster, 2004). Some cats seemingly remain infected for life. Clinical disease may be prolonged in cats living under dense housing conditions, possibly attributable stress. Treatment with diet changes and conventional antibiotics may prolong clinical signs and delay spontaneous resolution in some cats.

Tritrichomonas foetus is resistant to most antibiotics and is extremely difficult to eradicate (Gookin 2001). Numerous antibiotics have been evaluated. Some antibiotics reduce the number of organisms and improve the diarrhea without eliminating the infection, so diarrhea relapses whenever antibiotics are stopped. Diarrhea is typically refractory to corticosteroids.

The most successful treatment for eliminating T. foetus is ronidazole (30 mg/kg PO, once or twice daily for 14 days). Ronidazole is a nitroimidazole related to metronidazole. (Gookin, 2006). The side effects in some cats include lethargy, decreased appetite, and neurotoxicity (agitation, trembling, weakness, hyperaesthesia, ataxia, seizures) (Rosado, 2007). Cats with neurotoxic signs usually improve when the drug is stopped, but recovery can take 1 to 4 weeks. Ronidazole should not be used in pregnant and nursing queens or in very young kittens. Ronidazole is not approved for veterinary or human use, but some pharmacies compound chemical grade ronidazole for veterinary use. Because of its bitter taste, ronidazole compounded in gel caps is better tolerated than flavored suspension. When prescribing ronidazole obtain informed consent and instruct owners to wear protective gloves when handling it.

Ronidazole is not effective in every case. Relapse of infection can occur up to 20 weeks after completion of treatment; thus, follow-up PCR testing is recommended to confirm infection is eliminated at 1 to 2 weeks after completion of treatment and again after 20 weeks. A cure can only be proven by repeated negative PCR tests for 20 weeks or more after treatment.

Infected cats should be isolated during treatment to prevent reinfection, which is a common problem in catteries. To prevent auto-reinfection during treatment the litter should be replaced frequently and the box disinfected. In infected catteries, control of T. foetus requires repeated testing to identify infected cats, which can then be isolated and treated. Other measures include reducing housing density, reducing stress, improving diet, and treating concurrent infections such as Giardia or Cryptosporidium. Concurrent infection with Giardia is common, so T. foetus-infected cats should be routinely tested for Giardia and even treated empirically with fenbendazole to eliminate “occult” Giardia and helminth co-infections (Gookin, 2004). In catteries, older cats can be asymptomatic carriers and infect younger cats.

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