Microscopy

2.12.1. Morphometric assessments of Entamoeba cysts and trophozoites

Direct microscopic examination of faecal suspensions in saline and wet mounted with 0.9% saline and Lugol's iodine was conducted. Approximately 2 mg of stool sample was picked up using a wooden stick and mixed with a drop of normal saline (0.9%) on a glass slide with applicator stick. The preparation was covered with a cover slip and observed under the microscope. For iodine wet mount preparation, approximately 2 mg of faecal sample was picked up using a wooden stick and mixed with a drop of dilute

Lugol’s iodine. It was covered with a coverslip and observed under the microscope for the presence of cysts and trophozoites. Entamoeba cysts were also concentrated using zinc-sulfate gradient floatation (Faust’s method) (Ramos et al., 2005). In this assay, a fine faecal suspension was made by mixing 1 g of faecal sample and 10 mL of lukewarm distilled water. The coarse particles were removed by straining through a wire gauge. The filtrate was collected in a tube and centrifuged for 1 min at 1,455 × g. The supernatant fluid was poured off and distilled water was added to the sediment. It was shaken well and centrifuged and the procedure was repeated two to three times until the supernatant fluid became clear, which was then poured off. 3-4 mL of a 33% zinc sulphate was added to the sediment. The sediment was then stirred and more zinc sulphate solution was added to fill the tube up to the top and centrifuged again for at least 1 min at 1455 × g. The surface film was then removed by a loop on to a glass slide, covered with a cover slip, and observed under Nomarski contrast with a 100× oil immersion objective lens in combination with an ocular micrometer, on an Olympus DP71 digital micro-imaging camera. The diameters of cysts (n=35) and trophozoites (n=15) were measured and averages and ranges were calculated.

2.12.2. Morphometric assessments of Eimeria spp. oocysts

Morphological characteristics for sporulated oocysts were determined for faecal samples that were qPCR positive (section 2.6.3.). Approximately 2g faeces were placed in 2% (w/v) potassium dichromate solution (K2Cr2O7), mixed well and poured into petri

dishes to a depth of less than 1 cm and kept under close observation at room temperature in the dark to facilitate sporulation. Faecal flotation was conducted using a saturated sodium chloride and 50% sucrose (w/v) solution as previously described (Soulsby, 1982). In this protocol, flotation solutions, NaCl (360 g liter−1; specific gravity, 1.21) and sucrose solution (500 g of sucrose and 6.5 g of phenol in 320 mL of water) were prepared

centrifuged at 500 × g for 10 min. The upper 5 mL of each supernatant was transferred to a 50-mL tube. A sample from the supernatant layer was transferred to a slide covered by a cover slip, and observed under the microscope and observed under Nomarski contrast with a 100× oil immersion objective.

Sporulated oocysts were observed using an Olympus DP71 digital micro-imaging camera and images were taken using Nomarski contrast imaging system with a 100× oil immersion objective lens in combination with an ocular micrometre on an Olympus DP71 digital micro-imaging camera. Morphological features were recorded and measurements were performed on oocysts (n=35) of each identified Eimeria species. All measurements were given in micrometres (μm) as the mean followed by the range in parentheses. Minor shape variations were observed. The number of oocyst cell wall layers was confirmed by crushing individual oocysts with gentle coverslip pressure. Species were differentiated by reference to the descriptions given by Honess (1942), Levine et al., (1962a) and Shah and Joshi (1963).

2.12.3. Isolation and analysis of single Eimeria oocysts using a micromanipulator

A 3-axis hydraulic micromanipulator (MO-102, Nirashige, Japan) was used to select four morphologically similar Eimeria spp. oocysts from each faecal sample. Where multiple morphotypes were observed (i.e. mixed infections), four oocysts of each morphotype were selected and transferred to separate slides.

The morphologically similar oocysts (n=4 per morphotype) isolated from each qPCR positive faecal sample were transferred to a new slide, examined and photographed using microscopy (Olympus DP71 digital micro-imaging camera) to confirm morphological similarity. Measurements were recorded for species identification based on morphological characteristics. These were then transferred into a PCR tube containing

10 µL of lysis buffer (0.005% SDS in TE solution) by washing the coverslip with 100 µL saline. After a brief centrifugation, the tube was frozen in liquid nitrogen and thawed in a 95 °C water bath for four rounds to disrupt the oocyst walls. After the addition of 0.5 µL proteinase K (20 mM), the tube was incubated at 56 °C for 2 h and then at 95 °C for 15 min. The entire lysate of the morphologically similar oocysts was used for two separate PCRs (18S rRNA and COI) as previously described in sections 2.7.14 and 2.7.15).

2.13. Additional sampling of rangeland goats at Beaufort River Meats

abattoir

In order to conduct further microbial analysis on rangeland goats faecal samples during the slaughter process, additional goat faecal samples (n=400) were sampled at Beaufort River Meats abattoir, that processes goats, sheep and occasionally deer and is located 60 km east of Katanning and 254 km south of Perth in the Woodanilling region of Western Australia. The goats included in the present study originated from four consignments; Meedo Station in the Carnarvon region, Tamala Station in the Shark Bay region, Wagga Wagga Station in the Yalgoo region and Wooramel Station in the Wooramel region, which arrived at the abattoir between November and December 2016 as shown in Fig. 2.4. As part of the standard management practice, goats were trapped in yards and supplied with oaten hay and water ad libitum. Goats were loaded in the morning (05:30) and transported in open trailers for approximately 12 hours. On arrival at the abattoir (lairage), goats were kept in a shaded pen with access to water. Slaughter commenced at 10.00 am on the day following transport. Slaughter of goats from different consignments was occurred on different days.

After evisceration, goats’ digestive tracts proceeded to the offal room where the sampling was carried out.

Figure 2.4. Captured and transported rangeland goats arriving at Beaufort River Meats abattoir, Western Australia. Photo provided by Laurence Macri, BRM.

From each of the four consignments, 100 goats were sampled with specimens collected at regular time intervals (approximately two to four minutes) as the consignment was processed, so as to spread the sampling across the entire consignment. After evisceration, goats’ digestive tracts were separated from carcasses for collection of offal. For each sample, approximately 25 g of faecal content was obtained by making a transverse incision of the large intestine, 10 to 20 cm proximal to the anus, using a sterile scalpel blade and then using a sterile-gloved hand to express faecal matter into a sterile polypropylene container. Samples were then immediately labelled, stored on ice or in a refrigerator (4.0°C), while being transported to the laboratory for isolation of Salmonella

Figure 2.5. Removal of digestive tracts from rangeland goats after evisceration at Beaufort River Meats abattoir, Western Australia. Photo provided by Laurence Macri, BRM.