Evaluation of Anthelmintic Resistance and Exhaust Air Dust PCR as a Diagnostic Tool in Mice Enzootically Infected with Aspiculuris tetraptera

Abstract

The entry of infectious agents in rodent colonies occurs despite robust sentinel monitoring programs, strict quarantine measures, and stringent biosecurity practices. In light of several outbreaks with Aspiculuris tetraptera in our facilities, we investigated the presence of anthelmintic resistance and the use of exhaust air dust (EAD) PCR for early detection of A. tetraptera infection. To determine anthelmintic resistance, C57BL/6, DBA/2, and NCr nude mice were experimentally inoculated with embryonated A. tetraptera ova harvested from enzootically infected mice, followed by treatment with 150 ppm fenbendazole in feed, 150 ppm fenbendazole plus 5 ppm piperazine in feed, or 2.1 mg/mL piperazine in water for 4 or 8 wk. Regardless of the mouse strain or treatment, no A. tetraptera were recovered at necropsy, indicating the lack of resistance in the worms to anthelmintic treatment. In addition, 10 of 12 DBA/2 positive-control mice cleared the A. tetraptera infection without treatment. To evaluate the feasibility of EAD PCR for A. tetraptera, 69 cages of breeder mice enzootically infected with A. tetraptera were housed on a Tecniplast IVC rack as a field study. On day 0, 56% to 58% of the cages on this rack tested positive for A. tetraptera by PCR and fecal centrifugation flotation (FCF). PCR from EAD swabs became positive for A. tetraptera DNA within 1 wk of placing the above cages on the rack. When these mice were treated with 150 ppm fenbendazole in feed, EAD PCR reverted to pinworm-negative after 1 mo of treatment and remained negative for an additional 8 wk. The ability of EAD PCR to detect few A. tetraptera positive mice was investigated by housing only 6 infected mice on another IVC rack as a field study. The EAD PCR from this rack was positive for A. tetraptera DNA within 1 wk of placing the positive mice on it. These findings demonstrate that fenbendazole is still an effective anthelmintic and that EAD PCR is a rapid, noninvasive assay that may be a useful diagnostic tool for antemortem detection of A. tetraptera infection, in conjunction with fecal PCR and FCF.

Figure 1.

Anatomic location of Aspiculuris tetraptera worms. (A) A. tetraptera worms in the proximal colon. Bar, 2 mm. (B) A. tetraptera worms embedded in the crypts of colon. Bar, 1 mm. Magnification, 6.25×.

Figure 2.

Parts of macerated female Aspiculuris tetraptera worms filled with embryonated ova on day 3 of culture in distilled water at room temperature. Bar, 150 µm. Magnification, 40×.

Figure 3.

Slide set-up for culturing Aspiculuris tetraptera worms at room temperature. A square was drawn in the center of a 75 × 25 mm glass slide by using a wax pencil, and 6 to 8 drops of distilled water were placed within the square. The gravid female worms were placed in the distilled water and macerated to release eggs. Each slide with worms was placed on 2 wooden sticks in a Petri dish with folded Kimwipe saturated with water in the bottom.

Figure 4.

Embryonated Aspiculuris tetraptera ova on day 3 of culture in distilled water at room temperature. (A) 2 live ova. (B) 2 live and 1 dead ova. Embryonated Aspiculuris tetraptera ova on day 5 of culture in distilled water at room temperature. (C) 2 live ova. (D) Multiple live ova with one dead ovum. Bar, 50 µm. The unembryonated ova prior to day 3 of culture have a round nucleus inside an ellipsoidal outer shell. The embryonated ova have an elongated embryo which can be seen moving inside the outer shell. The embryonated ova look very similar to each other from day 3 to day 5 of the culture. Magnification, 40×.

Figure 5.

View of the underside of the exhaust prefilter in the air handling unit. A 4 × 18 cm strip of 3M Filtrete 1900 filter paper, with 2 × 2-cm marked squares, was affixed with tape on the underside of the exhaust prefilter.

Figure 6.

Schematic diagram of pattern of flow of exhaust air and HEPA-filtered supply air through (A) Tecniplast Green line IVC rack and (B) Tecniplast Green line IVC cage. Blue arrows indicate supply airflow, and red arrows indicate exhaust airflow. Image courtesy of Tecniplast USA (West Chester, PA).

Figure 7.

The 4 sites for exhaust air dust (EAD) swabbing on Tecniplast Smartflow air-handling unit (AHU) and Green line IVC rack are shown as: EH, the inside of the exhaust hose connected to the exhaust plenum of the IVC rack leading to AHU; EP, the inside of exhaust plenum at the bottom of the IVC rack; PF, the underside of the exhaust prefilter; and SSD, the stainless steel drawer below the exhaust prefilter.

Figure 8.

Schematic diagram showing the features of Tecniplast Smartflow air handling unit.(A) Components inside the air-handling unit: 1, environment air inlet prefilter; 2, supply air HEPA filter; 3, exhaust air prefilter; 4, exhaust air HEPA filter; 5, dust collection tray; 6, exhaust air blower motor; 7, supply air blower motor; 8, exhaust air inlet; 9, exhaust air outlet to the environment; and 10, supply air outlet to rack. (B) Supply and exhaust air flow through the air handling unit. Image courtesy of Tecniplast USA (West Chester, PA).

Figure 9.

After 4 wk of treatment with fenbendazole-medicated feed in study 2, the dirty IVC rack was autoclaved followed by washing at 82 °C (180 °F) for 30 min and a second autoclave cycle. The image shows the adhered dust that remained (A) inside the exhaust hose connected to the exhaust plenum of the IVC rack; and (B) inside the exhaust plenum on the IVC rack. PCR analyses from swabs of this adhered dust were negative for Aspiculuris tetraptera DNA.