Recovery of putative taeniid eggs from silt in water associated with an outbreak of bovine cysticercosis

Abstract

Degenerate taeniid-like eggs consistent with Taenia saginata were recovered from sediment in the water supply of a beef feedlot under quarantine for Cysticercus bovis. Nine degenerate eggs in total were recovered from 482 modified flotation assays. Flotation controls of sediment spiked with known numbers of T. saginata eggs had poor egg recoveries, supporting the need for more sensitive assays for environmental samples.

Cysticercus bovis is the intermediate stage of the beef tapeworm Taenia saginata. The larval cyst stage (cysticercus) is found in muscle tissue in cattle, while the adult tapeworm resides in the human intestine. Infective T. saginata eggs from human feces can contaminate livestock feed or water and result in bovine cysticercosis, a federally reportable condition in Canada. The Canadian Food Inspection Agency (CFIA) oversees the quarantine and investigation of infected cattle herds; however, when cysticercosis outbreaks occur, the source of contamination is often not determined (1–4). This is due, in part, to the lack of reliable methods for the detection of taeniid eggs in various livestock feedstuffs or water. Failure to determine the source of infection compromises control and prevention in an infected herd and results in prolonged epidemiological investigations and quarantines. This situation, in turn, increases financial hardship for producers and resource expenditure by regulators.

In April 2000, an outbreak of cysticercosis was detected in a feedlot in southwestern Alberta, which eventually resulted in 2944 cattle being slaughtered under federal licence, of which 67 were identified by carcass inspection to be infected. All infections were light and distributed throughout the feedlot, suggesting dissemination of eggs in water or feed. The animals were fed homegrown barley silage, rolled barley, and heat-treated pelleted supplement, with no history of sewage irrigation. Thus epidemiological investigation suggested that the livestock water supply was the most likely source of the infective eggs and it was decided to evaluate environmental water samples for the presence of taeniid eggs (4). The feedlot used water collected from a local creek via a perforated pipe and then pumped to several holding cisterns for further distribution throughout the feedlot. Since cestode eggs have a relatively high specific gravity (5), they quickly settle out of aqueous suspension and into the sediment layer. Therefore, samples consisting of water and sediment (silt) from the main holding cistern were collected. A total of 20 samples were collected on 3 separate occasions between July 19 and August 22, 2000, by gently agitating the superficial layers of sediment at the bottom of the cistern and immediately siphoning the suspension into a 0.5-L plastic bottle. Once settled, each sample consisted of 50% to 75% silt by volume. Samples were maintained at 4°C and shipped to the Centre for Animal Parasitology at the CFIA Laboratory in Saskatoon for analysis.

From each sample, approximately 5 mL of silt was removed and thoroughly mixed with 35 mL of Sheather’s flotation solution (1.30 sp gr). The mixture was then split equally between 2, 20-mL glass tubes. Each tube was filled to the top with additional Sheather’s solution, had a coverslip placed on the convex meniscus, and was then spun at 325 × g for 10 min in a centrifuge equipped with a swing-out rotor. The coverslip was then removed and placed on a glass microscope slide, which was then examined under a compound microscope at 100 × magnification. This procedure was repeated until approximately 25% of the silt in any given sample had been processed. Observance of any helminth eggs or coccidia oocysts was recorded, and any positive or suspect findings were photographed. A fresh wet mount reference slide consisting of several T. saginata eggs in 1.30 sp gr Sheather’s solution was prepared daily from confirmed reference material and served as a positive control for viewing and photography.

Prior to the processing of the diagnostic samples, trial flotations were similarly performed on “spiked” samples consisting of known numbers of fresh T. saginata eggs, with or without silt obtained from the diagnostic samples. A total of 4 flotations were performed with T. saginata eggs only, with recoveries of 40.2% (35/87), 60.6% (63/104), 55.6% (50/90), and 65.7% (65/99); mean recovery was 55.5%. At 100 × magnification, the recovered eggs appeared uniform and similar to those in the reference sample (Figure 1A), with a relatively smooth thick shell and translucent contents revealing a hexacanth embryo. A total of 6 flotations were conducted on samples of approximately 100 or more T. saginata eggs added to approximately 1.25 or 2.5 g of silt, with recoveries of 4, 0, 1, 1, 4, and 2 eggs each. These eggs varied in appearance; 2 appeared similar to those in the reference sample, while the remainder were less distinct, with a dark roughly circular central region (Figure 1B). Due to this variable appearance and the large amount of debris on the slide, eggs were much more difficult to detect than those in the previous flotations without sediment.

Figure 1.

Wet mount Taenia saginata reference specimen (A) in Sheather’s solution. Positive control T. saginata egg (B) recovered from spiked flotation with silt. Degenerate taeniid-like egg (C) recovered from Sheather’s flotation of environmental silt implicated in an outbreak of bovine cysticercosis. All specimens of equal scale.

A total of 482 flotations were performed on the diagnostic field samples. A single taeniid-like egg, consistent with those of Taenia spp., was recovered from each of 9 flotations. Two, 1, and 6 such eggs were recovered from samples collected on July19, 2000, August 14, 2000, and August 22, 2000, respectively. Much debris was present on the flotation slides, which made detection of eggs difficult. Although morphologically similar to those in the T. saginata reference sample, the outer margin (embryophore) of the recovered eggs had an irregular, eroded appearance, and the central contents were less distinctive, with no visible hooks, consistent with degenerative changes (Figure 1C). As determined from the photographic images, these eggs (n = 8; 1 egg was not photographed) measured 31 to 37 μm (mean 34 μm) by 25 to 30 μm (mean 28 μm). The inner core (oncosphere) dimensions measured 25 to 33 μm (mean 28 μm) by 20 to 25 μm (mean 22 μm). The control eggs (n = 8) measured 34 to 39 μm (mean 36 μm) by 29 to 34 μm (mean 32 μm), with oncosphere dimensions of 24 to 29 μm (mean 27 μm) by 21 to 24 μm (mean 22 μm). Various gastrointestinal nematode eggs (morphologically consistent with Nematodirus sp., strongylids, Strongyloides sp., and Trichuris sp.) and coccidia oocysts (some sporulated and consistent with Eimeria sp.) were also detected in the flotation samples and were in good condition.

The Centre for Animal Parasitology utilizes a standardized flotation method for the detection of parasite eggs and oocysts in feces that relies on differences in specific gravity to concentrate eggs and oocysts (6). However, relatively heavy eggs, such as those of cestodes and trematodes, require flotation solutions of such high specific gravity that eggs are often distorted, and, more importantly, not adequately separated from sample debris. David and Lindquist (5), using sucrose density gradient centrifugation, determined the specific gravity of Taenia sp. eggs to be 1.2251 and suggested that the infrequency with which these eggs are detected on flotation may be due to their relatively small size, high specific gravity, and subsequent tendency to settle at the same level as various pigments and debris. Thus, current flotation methods for separating taeniid eggs from environmental debris are not reliable. Despite this limitation, a decision was made to proceed with a minimally modified method, because the recovery of a single taeniid egg from the submitted samples would be epidemiologically significant.

The trial flotations conducted on spiked samples consisting of fresh T. saginata eggs combined with environmental sediment (silt) yielded minimal egg recoveries. This effect may be due to sediment adhering to eggs and changing their physical properties, such that they do not rise to the uppermost flotation layers, mechanical interference to the flotation process, failure to visualize recovered eggs amongst the debris, or a combination thereof. Onyango-Abuje (7) described a processing of T. saginata eggs from fecal debris in which eggs were treated with an acid-pepsin mixture to remove the outer sticky layer. Although it is possible that such a technique may have merit in reducing the adherence of eggs to surrounding sediment, it was not attempted here.

Although only 9 taeniid-like eggs were recovered from the field samples, the low egg recoveries from the samples spiked with T. saginata eggs and sediment suggest that the actual number of eggs in the field samples may have been much higher. Recovered eggs were degenerate, and it is not known what effect degeneration might have had on egg flotation and recovery. As might be expected, the outer coating and inner content of the recovered eggs demonstrated the greatest loss of integrity. The slightly lower average outer dimensions of the recovered eggs are consistent with degeneration of the outer coat. Although the eggs of other, more common, parasites that were recovered did not appear degenerate, this could have been due to a more recent and frequent influx of these eggs into the water system, an increased resistance to environmental factors, or both. Coccidia oocysts, in particular, are generally regarded to be highly resistant to environmental conditions (8).

The degenerate appearance and distribution of the recovered taeniid-like eggs in sediment from all 3 sampling periods indicate that the eggs may have been in the water, on-farm, or in the creek for a prolonged period of time. It is difficult to speculate on the viability and infectivity of these eggs, as in vitro hatching was not attempted. However, they may have been viable earlier on, as the cattle in this outbreak are presumed to have been exposed to infective eggs in February or early March (4). Eggs of T. saginata can remain infective for at least several months, depending on the environmental conditions, with cool, wet conditions favoring longer survival. In this case, the warm summer temperatures at the time of sample collection may have contributed to more rapid degeneration of the eggs. Although the morphological characteristics of the eggs recovered from the diagnostic samples were consistent with those of taeniids (including T. saginata), due to the degenerative changes they could not be definitively identified as such. As well, eggs of all taeniid species, including Echinococcus spp., are morphologically indistinguishable. The use of molecular techniques (9) to further characterize the recovered eggs might have provided a more specific diagnosis. It is possible that the recovered eggs were pseudoparasites, such as plant material (10); however, the history of cysticercosis and morphological features support our conclusions. The finding of other parasite eggs in the field samples also demonstrates that the water supply was susceptible to contamination with a wide variety of parasites from fecal sources.

Depending on the source of contamination, infective eggs may be sparsely distributed in the water or feed matrix, thus requiring an assay with high sensitivity or a supplemental step(s) to concentrate a sufficient number of eggs to allow for detection. Although flotations on samples of vegetation spiked with T. saginata egg yielded recoveries of only approximately 29% (11), continuous flow centrifugation of similarly spiked samples of soil drainage water and vegetation reported egg recoveries of 84% and 96%, respectively (12). Further evaluation of this or other techniques with various environmental matrices is required to develop a reliable, validated assay and associated quality-assurance system for the routine diagnostic processing of field samples for T. saginata eggs. This will help to reduce the overall impact of bovine cysticercosis on both government disease control resources and affected producers.