17
Direct Smears

Ryane E. Englar

17.1 Procedural Definition: What Is This Test About?

Direct “wet mount” smears are add‐on diagnostic tests that screen fecal samples for motile organisms, including trophozoites, that may not survive the hypertonic solutions that are involved in the flotation aspect of fecal analysis [1–4]. Trophozoites represent one of many stages in the life cycle of certain protozoa.

Recall from basic biology that protozoa are unicellular eukaryotes. Some are flagellated for the purpose of enhancing movement, feeding, and/or attachment to substrates. Two flagellate protozoans in particular, Giardia spp. and Tritrichomonas foetus, have been implicated as causative agents of gastrointestinal disease of companion animals. These organisms can be identified on wet mounts, contingent upon the examiner’s experience and skill [1, 3].

Direct smears are also an effective tool for the identification of amoebae (e.g. Entamoeba histolytica), nematode larvae (e.g. Strongyloides spp.), spores of Clostridium spp., and motile bacteria, such as Campylobacter [2, 3, 5].

17.2 Procedural Purpose: Why Should I Perform This Test?

Diarrhea is a common clinical presentation among companion animal patients. Protozoal and enteric bacterial pathogens are capable of inducing both acute and chronic disease.

Routine fecal analysis typically includes macroscopic assessment of the sample as well as fecal flotation to identify helminth ova (e.g. those from roundworms, hookworms, and whipworms) and digested skin mites (e.g. Demodex spp. and Cheyletiella) [6] (see Figure 17.1).

However, protozoal cysts, oocysts, and trophozoites from Giardia spp. and Tritrichomonas foetus are notoriously difficult to identify using routine methods. In fact, the solutions that are routinely used for fecal flotation are hypertonic, which tends to distort, if not kill, motile trophozoites. Because of this, trophozoites tend to be misdiagnosed or underdiagnosed, particularly Giardia spp., which are shed intermittently. Cysts from Giardia spp. may be mistaken as yeast because they are sized and shaped similarly.

It is critical that we broaden our diagnostic toolbox to include a combination of methods particularly in diarrheic patients so that we are more likely to diagnose accurately. Expedient diagnosis is vital so that active infections in symptomatic patients do not persist.

17.2.1 The Rationale for Employing Wet Mounts to Diagnose Giardiasis

Giardiasis is a clinical condition that causes intermittent intestinal disease in humans, companion animals, livestock, and wild animals [7–15]. Both symptomatic patients and asymptomatic carriers shed cysts intermittently for indefinite periods. This life cycle promotes recurrent infection and persistence of Giardia spp. within the environment [7].

Infection of dogs with Giardia duodenalis is common [16]. Although prevalence of giardiasis varies regionally within the United States, 15.6% of dogs with diarrhea are infected with Giardia, and prevalence increases among dogs that frequent dog parks [16].

Patients typically become infected through ingestion of cysts found in contaminated food or water [17–21]. Cysts are resistant to cold temperatures and damp environments [18, 19, 21]. Cysts release trophozoites within the small bowel [19, 21]. Trophozoites can be free‐floating throughout the lumen, or they may attach to intestinal mucosa via a ventral sucking disc [19, 21]. As these parasites move toward the colon, they undergo encystation [19, 21].

Photo depicts demodex gatoi, 40x, as observed on routine fecal analysis from a feline patient. — **Figure 17.1** *Demodex gatoi*, 40x, as observed on routine fecal analysis from a feline patient.

*Source:* Courtesy of Dr. Araceli Lucio‐Forster, Cornell University College of Veterinary Medicine.

Patients that are symptomatic exhibit small bowel diarrhea and potentially weight loss, stunted growth, and lack of thriftiness [18, 21–24]. Pediatric patients are more likely to be symptomatic [18, 20–23, 25]. Adult infections may be clinical or subclinical [18, 21, 23, 24]. Diarrhea may become chronic if the patient remains untreated [21, 24].

Cats can also become infected with Giardia.

Diagnosis can be achieved by identification of cysts via zinc sulfate fecal flotation [11, 18, 21, 24, 26] (see Figure 17.2).

Photo depicts giardia cysts in a feline patient, 40x. — **Figure 17.2** *Giardia* cysts in a feline patient, 40x. Concentrated from feces by zinc sulfate centrifugal flotation.

*Source:* Courtesy of Araceli Lucio‐Forster, PhD.

However, sensitivity of microscopic identification is low [11, 17, 21, 23, 27, 28]. Giardia cysts are small and are frequently mistaken for yeast [11]. Cysts are also shed intermittently, which means that serial fecal testing may be necessary to increase the sensitivity of detection in this manner [7, 11].

The trophozoite stage of Giardia is better suited for visualization via direct smear. The concave underside of the trophozoite’s tear‐shaped body makes it appear cupped or spoon‐like as it moves through space in a pattern that resembles a falling leaf [18, 20, 21].

Although zoonotic transmission is possible in human patients that are immunocompromised, for instance, those with human immunodeficiency virus (HIV), genotypes of Giardia that commonly infect dogs and cats are not typically the same as those that infect people [9, 17, 21, 29].

17.2.2 The Rationale for Employing Wet Mounts to Diagnose Trichomoniasis

Trichomoniasis was first described in cattle as a venereal disease that caused infertility, abortion, and endometriosis [30–32]. In the late 1990s and early 2000s, Tritrichomonas foetus was linked to chronic diarrhea in cats [30, 33–36]. Cats become infected via the fecal–oral route [30].

Young cats are at increased risk for infection [30, 35, 37]. Prevalence is also increased amidst crowded populations [30].

Because many cats that are affected with Tritrichomonas foetus have concurrent coinfections, it is unclear if Tritrichomonas foetus alone causes diarrhea or if clinical signs are precipitated by other enteropathogens [30, 32, 33, 38–40].

Affected cats exhibit intermittent or chronic large bowel diarrhea [21, 30, 41]. Large bowel diarrhea is suggestive of colitis and is characterized by the presence of fresh blood and/or fecal mucus in samples that range from semi‐solid to liquid consistency [21, 30]. Patients may be flatulent and/or fecal incontinent [30]. Defecation often involves tenesmus [30]. If tenesmus is severe, patients may experience perianal irritation, inflammation, and/or rectal prolapse [30] (see Figures 17.3 and 17.4).

Affected cats do not typically exhibit weight loss because diarrhea is localized to the large bowel, meaning that nutrient absorption across the small bowel is not impaired [30]. However, during diarrheic bouts, patients may demonstrate depression, anorexia, and fever [30]. Affected cats may also vomit [30].

Tritrichomonas foetus cannot be identified via routine fecal analysis through flotation because its trophozoite phase is destroyed during fecal flotation due to hypertonic solutions [30]. Therefore, direct examination via wet mount is indicated as a first pass attempt to detect Tritrichomonas foetus [30, 42]. The progressive movement of Tritrichomonas foetus is distinct from that of Giardia spp. because trichomonads propel forward in an erratic fashion [41, 42].

Photo depicts perianal irritation. — **Figure 17.3** Perianal irritation.

*Source:* Courtesy of Dr. Jule Schweighoefer.

Photo depicts rectal prolapse in a cat. — **Figure 17.4** Rectal prolapse in a cat.

*Source:* Courtesy of Frank Isom, DVM.

The use of direct smears to diagnose Tritrichomonas foetus is clinically challenging because of the low specificity and sensitivity that are attributed to this test [30, 41]. In spite of these challenges, wet mounts offer a simple and inexpensive starting point [30, 42].

17.2.3 The Rationale for Employing Wet Mounts to Diagnose Campylobacter spp.

Campylobacter spp. are transmitted through the fecal–oral route [43]. Sources of transmission include [43]:

undercooked or raw food
unpasteurized milk
water that has become contaminated with infected feces
ingestion of infected feces
indirect contact with other animals, as through the environment, vectors, and fomites.

Campylobacter spp. have been implicated as causative agents of bacterial diarrhea among companion animal patients in addition to the following pathogens [21, 22, 44, 45]:

Clostridium spp.
Escherichia coli
Salmonella spp.
Yersinia enterocolitica.

The primary challenge associated with isolating bacteria in cases involving acute or chronic diarrhea is determining whether the bacteria are in fact causing pathology or are simply present within the gastrointestinal tract [21, 22]. Colonization of the gut with Campylobacter spp. can be normal [21, 46].

As an added complication, many bacteria are opportunistic: they will not cause disease in and of themselves, but they may cause disease if the intestinal milieu is disrupted [21, 22, 44].

Both healthy and diarrheic patients may produce feces that contain Campylobacter spp. [21, 46–51] Campylobacter spp. are particularly abundant in stool samples from young patients and those patients that are stressed [21, 43, 46]. Campylobacter spp. are also more prevalent among those housed in groups, as is true of shelter or kennel settings [43].

Microscopically, Campylobacter spp. are Gram‐negative, curved bacteria. When viewed under light microscopy, Campylobacter spp. take on a seagull wing S‐shape [21]. This shape is not pathognomonic for Campylobacter spp. [21, 46]. Other bacteria with this characteristic shape include Helicobacter, Arcobacter, and Anaerobiospirillum [21, 46].

Fecal culture can confirm the presence of Campylobacter spp., but this diagnostic test requires additional time and the ability to ship samples to outside diagnostic laboratories [22]. Therefore, a direct smear is an appropriate first step.

Campylobacter spp. are zoonotic [46, 52–57]. Therefore, diagnosis is essential to protect those who are living in close contact with affected patients, particularly those who may be immunocompromised [43].

17.3 Equipment

Performing a direct smear via wet mount requires minimal supplies [2, 3, 5, 20, 30]:

exam gloves for handling biosamples
a microscope with the ability to adjust the condenser diaphragm
microscope slides
newsprint
0.9% (physiologic) saline (NaCl) at room temperature or slightly warmed
wooden applicator stick or comparable tool for mixing feces and saline
cover slip
Lugol’s iodine
a fresh sample of feces.

Fecal samples should be fresh, ideally five minutes old or less [2, 36, 58].

Samples are typically taken directly from the rectum via gloved finger during rectal examination. Mucous and/or diarrheic samples are appropriate specimen because sample size is small – roughly the size of the head of a match (1–2 mm³) [5].

Direct smears can also be made from fecal matter that clings to a rectal thermometer following its insertion into the rectum [4, 59].

Photo depicts supplies for direct smear. — **Figure 17.5** Supplies for direct smear.

*Source:* Courtesy of Jeremy Bessett, Inaugural Class of 2023, University of Arizona College of Veterinary Medicine.

An alternate means of sample collection is via rectal saline lavage [3]. In this method, 6–12 ml of 0.9% saline is inserted into the rectum through a soft rubber catheter to which a water‐based lubricant has been applied [3]. The saline is aspirated and delivery into the rectum is repeated several times [3].

17.4 Procedural Steps [1–5, 20, 30, 60–62]

Gather supplies (see Figure 17.5).
Apply fecal sample to a microscope slide (see Figures 17.6 and 17.7).
Apply one drop of 0.9% saline to the same microscope slide unless the sample was obtained by rectal lavage, in which case you can skip ahead to the next step (see Figure 17.8).
Mix the fecal sample and the saline with a wooden applicator stick or comparable tool to form a slurry that spans approximately 1×1 centimeter (see Figures 17.9 and 17.10).
Check the “thickness” of the slurry against newsprint. You should be able to read newsprint through the wet mount. If you cannot, then the mixture is too dense and will need to be thinned out by removing some of the fecal sample, adding more saline, or both (see Figure 17.11).
Place a coverslip over the slurry (see Figure 17.12).

Figure 17.6 Using a wooden applicator stick to apply the patient’s fecal sample to a microscope slide.

Source: Courtesy of Jeremy Bessett, Inaugural Class of 2023, University of Arizona College of Veterinary Medicine.

Figure 17.7 The patient’s sample has been added to a microscope slide.

Source: Courtesy of Jeremy Bessett, Inaugural Class of 2023, University of Arizona College of Veterinary Medicine.
Place the microscope slide on the stage of the microscope.
Look through the microscope oculars.
Adjust the position of the oculars so that you can look through both oculars at the same time to view a single image.
Adjust the condenser diaphragm so that it is not opened too wide. You do not want illumination to be too bright.
Scan the slide for eggs, cysts, and larvae at low magnification (10×).

Figure 17.8 Adding one drop of 0.9% saline to the slide that contains the fecal sample.

Source: Courtesy of Jeremy Bessett, Inaugural Class of 2023, University of Arizona College of Veterinary Medicine.

Figure 17.9 Getting ready to use a wooden applicator stick to mix the saline and fecal sample.

Source: Courtesy of Jeremy Bessett, Inaugural Class of 2023, University of Arizona College of Veterinary Medicine.
Increase the magnification to 20×, then 40×, and scan the slide with each adjustment to magnifying power, evaluating for motile trophozoites.
Increase the magnification to 100× under oil immersion and scan the slide to search for motile bacteria.
Following thorough scans for motile trophozoites and bacteria, apply iodine to the edge of the cover glass to assist in the identification of organisms. Note that iodine will kill motile organisms; however, it will make them more visible and therefore more readily identified.