Experimental Parasitology 157 (2015) 68e77

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Host origin determines pH tolerance of Tritrichomonas foetus isolates from the feline gastrointestinal and bovine urogenital tracts a, *  Victoria Morin-Adeline a, Stuart T. Fraser b, Colin Stack c, Jan Slapeta a

Faculty of Veterinary Science, University of Sydney, NSW, Australia Disciplines of Physiology, Anatomy & Histology, School of Medical Sciences, University of Sydney, NSW, Australia c School of Science and Health, University of Western Sydney, NSW, Australia b

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Tritrichomonas foetus

 Flow cytometric analysis reveals parasite differences in response to pH in vitro.  The feline Tritrichomonas foetus genotype tolerates weak acidic and alkaline pH.  The bovine Tritrichomonas foetus genotype viability is reduced in weakly acidic pH.  The bovine Tritrichomonas foetus genotype shows cyst-like stages with internalization of flagella in weakly acidic pH.

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Article history: Received 21 January 2015 Received in revised form 2 June 2015 Accepted 30 June 2015 Available online 7 July 2015

The ability for protozoan parasites to tolerate pH fluctuations within their niche is critical for the establishment of infection and require the parasite to be capable of adapting to a distinct pH range. We used two host adapted Tritrichomonas foetus isolates, capable of infecting either the digestive tract (pH 5.3e6.6) of feline hosts or the reproductive tract (pH 7.4e7.8) of bovine hosts to address their adaptability to changing pH. Using flow cytometry, we investigated the pH tolerance of the bovine and feline T. foetus isolates over a range of physiologically relevant pH in vitro. Following exposure to mild acid stress (pH 6), the bovine T. foetus isolates showed a significant decrease in cell viability and increased cytoplasmic granularity (p-value < 0.003, p-value < 0.0002) compared to pH 7 and 8 (p-value > 0.7). In contrast, the feline genotype displayed an enhanced capacity to maintain cell morphology and viability (p-value > 0.05). Microscopic assessment revealed that following exposure to a weak acidic stress (pH 6), the bovine T. foetus transformed into rounded parasites with extended cell volumes and displays a decrease in viability. The higher tolerance for acidic extracellular environment of the feline isolate compared to the bovine isolate suggests that pH could be a critical factor in regulating T. foetus infections and host-specificity. © 2015 Elsevier Inc. All rights reserved.

Keywords: Trichomonads Tritrichomonas foetus pH tolerance pH regulation Hosteparasite interaction Host-switching

1. Introduction

* Corresponding author. McMaster Building B14, Faculty of Veterinary Science, The University of Sydney, NSW 2006, Australia.  E-mail address: [email protected] (J. Slapeta). http://dx.doi.org/10.1016/j.exppara.2015.06.017 0014-4894/© 2015 Elsevier Inc. All rights reserved.

The pH of a given host environment acts as the primary immune defence through altering the resident microbiome environment (Hook et al., 2011; Ng et al., 2004; Urban and Mannan, 2014).

V. Morin-Adeline et al. / Experimental Parasitology 157 (2015) 68e77

Within a given host, various organ niches can be distinguished by a specific pH range. During colonization, pH is a major physiological stress which parasites must counteract if they are to establish a successful infection (Beckwith-Cohen et al., 2012; Brosey et al., 2000; Corbeil et al., 1989). Even minor environmental pH fluctuations have been shown to modulate important cellular processes, including virulence, acting to either induce or diminish pathogenicity (Cobo et al., 2004; Fiori et al., 1996; Foster, 1999; Gao et al., 2011). Tritrichomonas foetus is an extracellular protozoan pathogen with a worldwide distribution. Originally described as a parasite restricted to the urogenital tract of cattle, it has more recently been shown to infect the gastrointestinal tract of domestic cats (Levy  et al., 2003; Parsonson et al., 1976; Slapeta et al., 2012). In cattle, T. foetus is a sexually transmitted pathogen with a disease characterized by varying degrees of vaginitis, placentitis and invasion of foetal tissues resulting in foetal loss (Clark et al., 1983; Corbeil et al., 1989). In contrast, infection in cats results in chronic diarrhoea of large bowel origin (Yaeger and Gookin, 2005). Current evidence suggests that both the bovine and feline isolates of T. foetus represent different genotypes of a generalist parasite (Morin Adeline et al., 2014; Reinmann et al., 2012; Slapeta et al., 2010, 2012). Furthermore, the two isolates display a remarkably low level of amino-acid sequence divergence between them across their transcriptomes, despite the large difference in the number of years the isolates have been kept in axenic cultures (Morin-Adeline et al., 2014). The broad host range of isolates, the dramatically different sites of infection/niches and the extent of genetic similarities between isolates makes T. foetus a favourable model to study parasite adaptation. The motile T. foetus parasite exists as pear-shaped trophozoites possessing three anterior flagella. These parasites multiply rapidly by binary fission and under in vitro culture conditions, have a doubling time of approximately 6 h at 37  C (Lun et al., 2000). In contrast to a number of protozoan parasites, there is no cystic environmentally resilient stage of T. foetus isolated from cattle, though an internalized-flagella form, referred to as a pseudocyst has been isolated from fresh bull preputial samples naturally infected with T. foetus (Pereira-Neves et al., 2011). The pseudocysts forms can be induced under in vitro laboratory conditions following incubation at 4  C, however, the physiological relevance of low temperature induction is unknown given that these parasites are constantly at >30  C within their respective hosts. The fact that T. foetus is restricted to two different organ systems within its two hosts makes these pathogens attractive from an adaptation point of view (Beckwith-Cohen et al., 2012; Brosey et al.,  2000; Slapeta et al., 2012). Focussing on the pH between these systems alone, T. foetus parasites have to contend with pH changes of at least 1e2 units which equates to a 10- to 100- fold change in free hydrogen ion activity. Counteracting extracellular protons for the maintenance of cytoplasmic pH within a neutral range is fundamental for the survival of any cell (Jiang et al., 1994; Maroulis et al., 2003; Messerli et al., 2005; Vanderheyden et al., 1996). Slight pH changes can have profound detrimental effects, not only on RNA, protein structure and functionality, but on cellular protonation-deprotonation events that enable metabolic reactions to occur (Knitt and Herschlag, 1996; Whitten et al., 2005). Being a generalist parasite with the ability to colonize very different environments, it is expected that irrespective of the origin of the T. foetus isolate, a similar level of pH tolerance will exist between the T. foetus isolates. In this study, we focus on the initial in vitro adaptive capacities (24 h) of the bovine and feline T. foetus isolates upon introduction into media buffered to the pH range they are likely to encounter within the respective niches they inhabit. In light of the vast pH

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difference of the cattle reproductive tract (pH 7.4 and pH 7.8) and the feline lower digestive tracts (pH 5.3 to pH 6.6), we use pH as a niche-specific factor in an effort to understand how these parasites interact with their respective hosts (Beckwith-Cohen et al., 2012; Brosey et al., 2000; Corbeil et al., 1989). Specifically, we explore whether pH can act as a barrier to host switching of the bovine and feline T. foetus isolates. In addition, we provide the first comprehensive ultrastructural data for the feline T. foetus isolate and compare it to the well-documented ultrastructure of the bovine T. foetus. 2. Materials and methods 2.1. Maintenance cultures and media Four Tritrichomonas foetus isolates were used throughout this study and kept in axenic cultures that were passaged on a 48 h basis. Two isolates from a bovine host origin; Kv-1 (ATCC 30924) and BP-4 (ATCC 30003) were purchased from the American Type Culture Collection (ATCC). A further two isolates, Sydney-G08/1 (G08) and Sydney-G10/1 (G10), were axenized from feline clinical  faecal samples from domestic cats (Hale et al., 2009; Slapeta et al., 2010). All isolates were maintained in trypticase, yeast-extract and maltose media (TYM), supplemented with 10% (v/v) heatinactivated lamb serum (Life Technologies, Victoria, Australia). A final concentration of 100 mg ml1 PenStrep/Fungizone ml1 (Gibco, Life Technologies, Victoria, Australia) was added to safe-guard against the growth of biological contaminants. Media was buffered to pH 7 using a potassium phosphate buffer made through a combination of 1 M solutions of K2HPO4 and KH2PO4 to a final concentration of 0.1 M. Cultures were maintained at 37  C. 2.2. Transmission electron microscopy (TEM) The feline isolate G10 and the bovine isolate BP-4 were grown to exponential phase and fixed in 2.5% (v/v) glutaraldehyde (PST, Queensland, Australia) in a 0.1 M sodium cacodylate buffer (pH 7.2) and post-stained in 1% (w/v) OsO4 in 0.1 M sodium cacodylate buffer (pH 7.2). An ethanol dehydratation series was then carried out at room temperature and samples were infiltrated and embedded in Epon resin (PST, Queensland, Australia). Ultrathins were obtained using a diamond knife and mounted on grids which were stained with 2% uranyl acetate and 1% lead citrate. The grids were viewed and images were collected on a JOEL JEM-1400 TEM using 120 Kv equipped with an ES500W Erlangshen CCD camera (Gatan, California, USA). Samples of the bovine T. foetus kept for 24 h in pH 6 buffered media were prepared in a similar manner for TEM. 2.3. pH treatment media and experimental set-up To investigate the pH tolerance of the four T. foetus isolates, three batches of TYM-media were prepared as described for the maintenance media but instead buffered to the three pH values chosen for investigation; pH 6, pH 7 and pH 8. Buffering to the desired pH was achieved by the adjustment of individual volumes of 1 M solutions of K2HPO4 and KH2PO4 (Sambrook et al., 1989), ensuring that the final concentration of 0.1 M phosphate buffer was maintained. All media were supplemented with 10% (v/v) heatinactivated lamb serum and a final concentration of 100 mg PenStrep/Fungizone ml1 (Gibco, Life Technologies, Australia) as per the maintenance TYM-media. Prior to inoculation of T. foetus into different pH-buffered media, exponential phase cultures of each T. foetus isolates were counted using a Neubauer improved haemocytometer (PST, Queensland,

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Australia), and analysed for cell size, granularity and viability using flow cytometry. For each experiment, the feline T. foetus G10 cultures were revived from cryopresered stock that had been passaged 80 times prior to storage, and sub-cultured through two passages before inoculation into buffered media. Feline G08 T. foetus cultures were cryopreserved after 18 passages and revived through two passages before inoculation. A final concentration of 1  105 T. foetus cells ml1 was inoculated into 5 ml of media buffered at either of the pH under investigation (pH 6, 7 and 8) and incubated at 37  C. Subsequent to inoculation, flow cytometric analysis was performed at 24 h intervals for up to 72 h post pH exposure. Triplicates were prepared for the various isolates at each pH and the experiment was repeated independently 3 times. Initial inquiry into effects of parasite passage number on the behaviour of T. foetus in buffered media using G08 feline T. foetus at 6 passages and at 16 passages after axenization showed no interference of passage number on the ability of T. foetus to tolerate various pH (data not shown). To ensure that the results obtained were not influenced by the parasite changing the pH of the media, pH readings of the cultures at 1 h, 2 h and 24 h post T. foetus inoculation were recorded. All pH measurements were taken using a portable pH meter (JENCO Instruments, INC, CA). As controls, pH measurements were recorded for the same intervals for parasites inoculated into 5 ml PBS (pH 7.2) and media kept at 37  C without T. foetus parasites. Cultures

2.4. Flow cytometry assay and fluorescent viability dyes Flow cytometery analysis was performed on a BD FACScan (3colour, 5-parameter) flow cytometer (Becton Dickinson, San Jose, California), equipped with 488 nm laser excitation and BD CellQuest software (V3.3) (Becton Dickinson, San Jose, California). Discrimination of cells was based on light-scattering properties and fluorescent staining. The acquisition template was created and optimized for all four axenic isolates using exponential phase cultures, death phase cultures and a mixture of exponential/dead phase, independently. All analysis were carried out using 200 ml of the cultures and a minimum of 10,000 gated events were collected for each measurement throughout all

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were visualized using bright field microscopy using an Olumpus IX73 inverted microscope (OLYMPUS, Australia). Aliquotes exposed to 24 h of each pH treatments were fixed in 2.5% (v/v) glutaraldehyde (PST, Queensland, Australia) in a 0.1 M sodium cacodylate buffer (pH 7.2) on a slide and stained with the nuclear stain DAPI (Life technologies, Australia). Differential interference contrast (DIC) images were obtained using microscopy using the same microscope set-up equipped with an X-cite series 120 fluorescence illuminator (Excelitas Technologies Corp) and a Olympus XM10 camera (OLYMPUS, Australia).

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Fig. 1. Representative flow cytometry contour plots of the four Tritrichomonas foetus isolates used in this study. Representative forward and side scatter flow cytometry plots of a mid-exponential phase culture of four T. foetus isolates used in this study cultured under maintenance conditions at pH 7. Gated regions on each plot represent forward and side light scattering regions (FSC/SSC) characteristic of healthy parasite trophozoites of each isolate. AeB represents cultures of bovine T. foetus genotype of the Kv-1 and BP-4 isolates, respectively. CeD represents cultures of feline T. foetus genotypes of the G08 and G10 isolates, respectively.

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experiments. The fluorescent viability dyes, propidium iodide (PI) (Life Technologies, Australia) and fluorescein diacetate (FDA) (Life Technologies, Australia) were used as a measure of cell health and viability. Propidium iodide (PI) which enters dying or damaged cells and binds to DNA was used to detect apoptosis. Fluorescein diacetate (FDA) was used to monitor membrane integrity and cellular activity as per manufacturer's instructions. T. foetus cells (1  106 in 200 ml of culture medium) were aliquotted from cultures grown at different pH values and incubated with 5 mg ml1 PI and 10 mg ml1 FDA for 3 min at RT. Flow cytometry was then performed immediately. No washing steps are needed for these fluorescent probes. Forward scatter (size) and side scatter (granularity) were first assessed followed by detection of FDA and PI fluorescence in fluorescent channels 1 (FL1) and FL2 respectively. At least 10,000 cells were analysed per assay point. Data was analysed using the FlowJo 9 software package (Treestar, Ashland, OR). 2.5. Statistical analysis Gated values of all biological replicates and experimental replicates at each pH were averaged at each time point for individual isolates and imported into GraphPad Prism (V6.04) (La Jolla, CA). As no significant differences were obtained between the bovine (n ¼ 2) and the feline (n ¼ 2) T. foetus at each pH, the isolates of each genotype were averaged for further analysis. A two-way ANOVA, followed by a post hoc Tukey's test was carried out to determine significant differences between the three pH investigated at each time point post inoculation to each pH buffered media. 3. Results Comparative flow cytometry of four mid-exponential phase T. foetus isolates (two feline genotypes and two bovine genotypes) kept under maintenance conditions revealed similar genotypespecific patterns of cell size and granularity Fig. 1 (A)e(D). We utilised forward (FSC) and side (SSC) light scattering properties which relates to cell size and granularity (cytoplasmic content),

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respectively, coupled with two viability fluorescent dyes (PI and FDA) to successfully distinguish viable from non-viable T. foetus enabling us to define a healthy trophozoites population Fig. 2(A)e(C). Comparative TEM of the bovine and feline T. foetus genotypes kept under maintenance conditions showed that the four isolates possess highly conserved organelle and microtubule organization Fig. 3 (A)e(G). Localization of hydrogenosomes in the vicinity of the axostyle is evident in both bovine and feline T. foetus isolates Fig. 3(E), (F). The costa, a cytoskeletal structure originating from the anterior region of the cell and runs the length of the cell is present in both genotypes Fig. 3(C), (H). Under maintenance culturing conditions both T. foetus genotypes possess a proportion (bovine 1/5; feline 2/5) of cells with internalised flagella Fig. 3(C), (D). Flow cytometry revealed that 10%e30% of cells in all maintenance T. foetus cultures consisted of parasites that varied in granularity from the gated majority of trophozoites Fig. 1(A)e(D). These populations may consist of parasites with internalized flagella or dividing parasites that contain two nuclei prior to cytokinesis. To investigate the in vitro tolerance of T. foetus to the range of pH they encounter within their host, we used the above developed flow cytometry approach that defined a gated region of healthy trophozoites Figs. 2(A)e(C) and 4(A), (B). As 24 h old T. foetus cultures represent mid-exponential growth phase of T. foetus, we concentrate on reporting the behaviour parasites at this time point (Lun et al., 2000) (data not shown). The parasites within the predefined flow cytometry gate was assessed as the percentage of parasites that retain size/granularity and percentage viability (the number of parasites producing high green fluorescence; FDA staining e live cells, but low red fluorescence; PI staining e dead cells) Fig. 2, population 2. As no significant differences were obtained within genotypes, the feline isolates (n ¼ 2) and bovine isolates (n ¼ 2) were combined for statistical analysis (data not shown). At all pH tested, the feline T. foetus genotypes showed greater ability to remain within the gated region with a high proportion (69%e78%) of viable parasites compared to bovine T. foetus genotype Fig. 4(C), (D) top row, Table 1. At 24 h, there was no significant difference between the different experimental treatments for the feline T. foetus genotype (p-value > 0.05) (Table 1). In

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Population 2 35.2%

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cell size (forward scatter) Fig. 2. Determination of Tritrichomonas foetus viability using flow cytometry. Cell size (FSC) and granularity (SSC), combined with the viability dyes PI and FDA of T. foetus cultures enable the successful determination of viable and non-viable T. foetus cell populations. A. Representative plot of cell size/granularity (FSC/SSC) of a T. foetus culture consisting of mixed 72 h old parasites with 24 h old parasites. Gating each of the populations individually in A. for B. red fluorescence (PI positive e dead cells) and C. green fluorescence (FDA positive e live cells) confirms the viability of each population. Population 1 is representative of non-viable cells showing greater red fluorescence than population 2 which shows greater green fluorescence. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3. Comparative transmission electron microscopy of the bovine and feline Tritrichomonas foetus. Transmission electron microscopy (TEM) of T. foetus maintained under maintenance culture conditions (pH 7) show that the bovine and feline genotypes possess highly conserved organelle and microtubule organization. A, C, G, E represent bovine T. foetus (BP-4); B, D, H, F represent feline T. foetus (G10). A, B. A highly-ordered array of microtubules forming the axostyle (black arrowheads) runs central along the length of cell of both genotypes. G, F. The axosyle structure is widest in the anterior region of the cell, where it meets with the pelta (white arrowhead) at the cell anterior. C, H. The costa (black arrow), a cytoskeletal structure origination from the anterior region of the cell runs the length of the cell is present in both genotypes E, F Moving towards the posterior region of the cell, the axosyle (black arrowheads) narrows and protrudes from the cell forming a distinct tip covered by the cell membrane. E, F Localization of hydrogenosomes (white arrow) in the vicinity of the axostyle (black arrowhead) is evident in both bovine and feline T. foetus isolates. C. D Internalization of flagella (grey arrowheads) is apparent in both T. foetus genotypes. Scale bars: 1 mm.

contrast, a significant decrease in the frequency of bovine T. foetus within the gated region was obtained when comparing pH 6 and pH 7 as well as pH 6 and pH 8, compared to between pH 7 and pH 8 (Tukey's test, p-value < 0.0002), Fig. 4(C), Table 1. The most extreme difference was seen in bovine T. foetus cultures with 96% of ungated cells at pH 6 after 24 h Fig. 4(C) compared to feline T. foetus cultures which had 15% of ungated cells at pH 6 after 24 h Fig. 4(D). Microscopic observations of the bovine T. foetus at 24 h in pH 8 showed a larger population of parasites compared to bovine T. foetus at pH 7 within the same time frame, which accounts for the shorter life of the culture by 48 h Fig. 4(C) pH measurements taken at 1 h, 2 h and 24 h post-inoculation of T. foetus into buffered media or PBS revealed that at a concentration of 1  105 parasites ml1, T. foetus does not alter the pH of its environment. Furthermore, no change in pH was noted for buffered media kept at 37  C without parasites. Light microscopy analysis confirmed that while the feline T. foetus maintained a ‘tear-dropped’ morphology at all pH values tested, this was not the case for bovine T. foetus at pH 6 Fig. 5. We then inquired into the population of ungated bovine T. foetus cells exposed to 24 h of pH 6-buffered media Fig. 4(C), top left;

Fig. 6(A). Flow cytometry plots revealed parasites possessing an increase in cellular granularity (cytoplasmic content) Fig. 6(A). Gating on the extremes of this population for fluorescent viability signals revealed that these cells were predominantly viable (93% showing green fluorescence e FDA positive) Fig. 6(B)e(D). At 48 h post exposure to media buffered at pH 6, bovine T. foetus cells show polymorphic granularity and cell size. A large proportion of cells also begin to resemble the characteristic population shape and position of non-viable T. foetus cells, signifying the death of the culture Fig. 4(C). This was confirmed by using the gating as for ‘population 1’ in Fig. 2 for fluorescence to show low FDA but high PI staining of this population (data not shown). Microscopic examination of the bovine T. foetus parasites exposed to media buffered at pH 6 for 24 h revealed parasites as large rounded, multinucleated cells compared to the same parasite incubated at pH 7 Fig 5(A), (G). In order to better analyse the increase in granularity observed by flow cytometry of the bovine cells buffered at pH 6 for 24 h, ultrastructural features of the parasite were analysed using TEM. The majority of cells demonstrated a high number of cytoplasmic vacuoles and internalized flagella Fig 7(A), (B) compared to healthy

V. Morin-Adeline et al. / Experimental Parasitology 157 (2015) 68e77

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Fig. 4. pH tolerance of the bovine and feline Tritrichomonas foetus. Representative flow cytometry results showing pH tolerance of the bovine and feline T. foetus genotypes in media buffered to pH 6, 7 and 8. The gated regions (blue gates within plots) represent forward and side light scattering regions (FSC/SSC) characteristic of healthy parasite trophozoites. AeB. Plot of cell size/granularity and viability of the maintenance cultures of bovine and feline T. foetus genotypes used to inoculate freshly buffered media at the various pH. Starting cultures of both genotypes show high green fluorescence (FDA staining e live cells) and low red fluorescence (PI staining e dead cells). CeD. Representative plots of the cultures after exposure to pH 6, 7 and 8 buffered media for 24 h (top row) and 48 h (bottom row). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

bovine T. foetus Fig 3(A), (C), (G) and (E). The abnormal cellular characteristics and organelle organization described correlates with the increase in granularity observed by flow cytometry. Furthermore, irregular plasma membranes were observed which resembled membrane blebbing and the presence of heterochromatin condensation was seen in the nucleus of some cells Fig 7(C), (D). These observations have been previously described in trichomonads undergoing apoptosis (Chose et al., 2002, 2003; da Costa and Benchimol, 2004; Mariante et al., 2003; Melo-Braga et al., 2003).

4. Discussion In this study we reveal that the bovine T. foetus has a lower tolerance to acid stress compared to the feline T. foetus isolate. The pH range we tested was chosen as a host/niche-specific trait the parasites are likely to encounter upon initial stages of infection in their host organ or their non-host organ niche. At 24 h post media change, we observe that the viability of the bovine T. foetus is always lower than that of the feline T. foetus, further highlighting the previously described intra-species growth differences of T. foetus

Table 1 Summary of Tritrichomonas foetus exposed to different pH treatments and comparison between 24 h and 48 h post inoculation. Genotype

Parameter

Hours post inoculation

pH 6

pH 7

pH 8

pH 6 vs. pH 7

(%, SEM) Feline T. foetus

Size/granularity Viability

Bovine T. foetus

Size/granularity Viability

24 48 24 48 24 48 24 48

h h h h h h h h

82.02, 67.91, 75.90, 70.91, 27.81, 34.29, 27.57, 26.41,

1.65 3.13 4.87 4.43 4.21 5.49 4.21 7.80

pH 6 vs. pH 8

pH 7 vs. pH 8

0.9152 0.0017* 0.9595 0.9677 0.0002* 0.0304

Host origin determines pH tolerance of Tritrichomonas foetus isolates from the feline gastrointestinal and bovine urogenital tracts.

The ability for protozoan parasites to tolerate pH fluctuations within their niche is critical for the establishment of infection and require the para...
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