Investigation Into The Prevalence Of Toxoplasma Gondii Infection Biology Essay

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The protozoan parasite Toxoplasma gondii is prevalent worldwide and can infect a remarkably wide range of hosts. It is a member of the phylum Apicomplexa and is distinguished amongst this group for its ability to readily infect almost any mammal, unlike closely related organisms (e.g. Neospora caninum) which are limited in their host range. Toxoplasma is transmitted via three routes; the ingestion of oocysts from the environment, ingestion of tissue cysts in the intermediate host and congenitally through trans-placental crossing from mother to foetus. The importance of each of these routes to the overall prevalence of the parasite is of long debate, and the parasite remains enigmatic in its epidemiology. The definitive host is the cat (felidae family) where the parasite undergoes its sexual stage. The cat sheds millions of infective oocysts containing sporozoites into the environment which are then accidently ingested by an intermediate host. The sporozoites then differentiate into tachyzoites which divide rapidly and cause acute infection. If the intermediate host is pregnant, this stage may cross the placenta transmitting the parasite to the offspring. Tachyzoites convert to bradyzoites, the slowly dividing stage attributed to chronic infection, and form tissue cysts. These tissue cysts can be consumed by prey and in the case of the cat convert into gametes in the enterocytes of the intestine and so the cycle is complete. As another intermediate host can become infected via the ingestion of tissue cysts, and the parasite can propagate vertically through congenital transmission, Toxoplasma can transmit entirely through asexual means (Joynson and Wreghitt, 2001). Despite having a meiotic phase in its lifecycle, and thus having the opportunity of genetic exchange in cats, the genetics of Toxoplasma are highly clonal, comprising of 3 closely related lineages. These lineages, although genetically similar, show a distinct correlation with mouse virulence (Ajzenbery et al., 2002; Sibley and Ajioka, 2008).

Murine models are of particular interest when studying the epidemiology of Toxoplasma as the cat often predates on small members of this group, and therefore parasite transmission and dispersion should be highly evolved and warrants detailed study. High levels of vertical transmission of Toxoplasma has been observed in murine hosts experimentally infected (Beverley, 1959; Owen and Trees, 1998) and also in natural urban populations (Marshall et al., 2004; Murphy et al., 2008). Beverley (1959) observed successive transmission of T. gondii in up to 9 generations in experimentally infected mice. It is recognised that congenital transmission is an important route of transmission in mice, but it is generally considered less important in other species (refs). However, congenital transmission has been observed in successive generation in sheep, which has significant implications for the livestock industry (Duncanson et al., 2001; Morley et al., 2005; Williams et al., 2005).

Prevalence levels of T. gondii in mammals are variable, but generally high, ranging between 30-40% on average, but reaching up to 90% in some populations (Tenter et al., 2000). Previous studies into the prevalence of Toxoplasma in mice include an investigation into congenital transmission of the parasite in an urban population of Mus domesticus by Marshall et al. (2004). In this study 200 mice were captured in houses in Manchester including 16 females. Of the 200 mice obtained, 59% tested positive for Toxoplasma using SAG1 PCR. Congenital transmission was observed in 75% of the pregnancies. A study by Zhang et al. (2004) investigated the prevalence of T. gondii in a wild population of voles that are common in northern and central Eurasia. The study was carried out in Hunan Province, the People's Republic of China using the modified agglutination test (MAT) and they found 29% of the population infected.

The wood mouse Apodemus sylvaticus is a common rodent found in Western Europe that is sometimes considered a pest. They are almost entirely nocturnal, highly adaptable and occupy most habitats that are not too wet. They are common in woodland, arable land, grasslands, sand dunes and urban areas in buildings during harsh winter conditions (Flowerdew, 1991). Woodmice are native to the United Kingdom and are widespread and very common, having an estimated pre-breeding population of approximately 38 million. In deciduous woodland 1-40 individuals per hectare is usual however their densities vary seasonally with early winter peaks and late spring troughs (Harris et al., 1995).

Cat numbers are known to decrease along an urban-rural gradient (Lélu et al., 2010) and so in rural mice, successive transmission of Toxoplasma vertically from mother to pup could facilitate propagation in the absence of the feline. In the current study, a wild population of woodmouse was sampled in a rural area of North Yorkshire. The area around Malham Tarn Field Centre consists of woodland, grazed and ungrazed grassland, moorland and limestone pavements. It is home to numerous small mammals such as rabbits, mice and voles. It is located at an altitude of 381m above sea level and experiences an upland climate. The field centre is situated on the north shore of the tarn and is 5km from Malham village. There are no known feral cats or domestic cats in farms around the surround area Hughes et al., 2008), and given the distance to the nearest town, it is unlikely the area is often frequented by roaming males.

The aim of the current study was to investigate the prevalence of Toxoplasma gondii in a rural, wild population of wood mouse (Apodemus sylvaticus) in North Yorkshire over a ten year period. Furthermore, as the area is largely free of cats this provides the opportunity to consider the importance of transmission routes that bypass the definitive host.

3.2. Objectives

To extract DNA from brain tissue of a cohort of Apodemus sylvaticus

To confirm PCR suitability using primers specific for mammalian tubulin

To detect the presence of Toxoplasma gondii using nested PCR

To analyse prevalence data with respect to biological parameters

To compare prevalence with an urban population of rodents that were collected from an area populated with cats (Marshall et al., 2004)

To catalogue and compare prevalences with other published studies on Apodemus

3.3. Methods and results

The woodmice (Apodemus sylvaticus) were sampled from the surrounding area of Malham Tarn, North Yorkshire over a 10 year period as part of an ongoing study of parasites in the area. Longworth traps were set and left overnight in the woodland area around the field centre (see figure 1) in Autumn 2008.

Figure 3.1. Area surrounding Malham Tarn Field Centre, North Yorkshire (Taken from Digimap, 2000)

A total of 21 mice were killed by chloroform inhalation. The brain tissue samples were dissected out and stored at -20°C in lysis buffer.

DNA was extracted using phenol/chloroform procedure. The extracted DNA was tested for mammalian tubulin using generic primers to α-tubulin to ensure viability to undergo PCR. The presence of Toxoplasma gondii was detected using PCR amplification of the P30 gene and resolved by gel electrophoresis. These techniques are described in full in chapter 2.

Date collected from these 21 mice were combined with previously collected data. Other researchers that carried out these assays during the previous years are as follows: Elizabeth Wright, Denise Thomasson, Omar Gerwash, Muftah Abushahma and Andrew Cox.

Statistics were carried out as described in the materials and methods. Mice weighing less than 14g were considered juveniles (Higgs and Nowell, 2000).

3.4. Results

The first stage in the analysis was to extract DNA from the brain tissue of Apodemus sylvaticus for the screening of Toxoplasma gondii. The DNA was extracted from 21 mice collected in 2008 and the DNA was run on a 1% agarose gel as described in chapter 2. Figure 3.2 shows a typical gel image of the extracted DNA.

M 1 2 3 4 5 6

Figure 3.2. Tissue DNA extraction of mice 275 - 280. DNA visualised on 1% (w/v) agarose gel. M = 1kb plus Invitrogen marker, lane 1 = 275, lane 2 = 276, lane 3 = 277, lane 4 = 278, lane 5 = 279, lane 6 = 280.

DNA was successfully isolated from all 21 mice obtain in 2008 as can be seen from figure 3.2. All extractions showed a high DNA yield with a range of molecular sizes visible. Many of which consisted predominantly of high molecular weight DNA such as mouse 280 in the above figure. This indicates successful extraction of DNA from the brain tissue with high quantities of genomic DNA that isn't excessively degraded. This was true for all mouse extractions in 2008. Each mouse was weighed, measured and sexed at the time of collection, details of which are presented in table 3.1.

A summary of biological parameters and PCR results from the 2008 cohort of mice can be seen in table 3.1. For details of the mice from other year cohorts see Appendix 1. Over the 10 year study, mouse samples ranged between 8 and 37 mice, and in 2008 a total of 21 were captured. Over the period a total of 206 mice were collected. Of these, 21 mice 9 were male and 12 females and ranged between 7 and 10cm in length, and weighed between 13 and 25g.

Mouse No.

Sex

Length (cm)

Weight (g)

275

F

8

17

276

M

7.2

15

277

F

7.9

18

278

M

10

25

279

F

7.7

13

280

F

8.3

20

281

F

7.5

15

282

M

7.8

14

283

F

8.5

18

284

M

8.2

18

285

M

7.5

18

286

F

7

14

287

M

10

24

288

M

7.6

15

289

M

7.7

15

290

M

7.7

15

291

F

7.8

16

292

F

8.5

19

293

F

7.4

18

294

F

8

18

296

F

8

20

Table 3.1. Summary of Apodemus sylvaticus collected in 2008 from Malham

Tarn, North Yorkshire.

Prior to testing the samples for Toxoplasma the DNA, known to consist largely of mammalian DNA, was tested for the presence of mammalian α-tubulin. This was to ensure the DNA was of a viable quality for undergoing PCR and eliminate the possibility of obtaining a false negative due to PCR inhibitors present in the sample. Figure 3.3 shows the gel electrophoresis image of mice 285-292 tested for α-tubulin. The positive control used was mouse DNA from a previous year. As can be seen from figure 2 all mice tested positive for tubulin. All samples shown a high amplification of the α-tubulin gene which is indicative of successful DNA extraction. This confirms that the quality of the DNA extracted was sufficient to undergo PCR successfully and this was true for all 21 mice of the 2008 cohort.

+ve 1 2 3 4 5 6 7 8 -ve M

Figure 3.3. Alpha-tubulin PCR of mice 285-292 visualised on 1% agarose gel. M = 1kb plus Invitrogen marker, +ve denotes mammalian positive control, lane 1 = 285, lane 2 = 286, lane 3 = 287, lane 4 = 288, lane 5 = 289, lane 6 = 290, lane 7 = 291, 8 = 292, -ve denotes water negative control.

In previous years, samples that could not be successful amplified by PCR for tubulin were omitted from the study. This was not necessary for any samples in 2008. Following successful amplification of the α-tubulin gene, the samples were then tested for Toxoplasma using a nested PCR designed to target the P30 gene. Each samples was tested at 4 concentrations of DNA, and this was replicated 3 times per dilution. The 4 concentrations were as follows: 1µl, 1/5 dilution, 1/10 dilution and 2µl in a 25µl PCR reaction.

An example of successful detection of Toxoplasma can be seen from figure 3.4 (sample 283). Following the initial first round of DNA using the external primers a product of 914bp is amplified in a successful PCR of a positive samples. This product is then used in the second round of the PCR assay to give a highly specific product of 522bp (see below).

283 284 285 286 287 288 289 290 -ve +ve M

Figure 3.4. SAG-1 PCR gel electrophoresis image of Malham mice 284-291, 2008 at 1m DNA per reaction, +ve denotes cloned SAG1 positive control, -ve denotes water negative control

A total of 21 mice were captured in 2008 of which 5 tested positive for Toxoplasma by SAG1 PCR, indicating a prevalence of 23.81% (±15.29%) for this year cohort (table 3.2).

Mouse No.

Sex

Tubulin

SAG1

275

F

+

-

276

M

+

+

277

F

+

+

278

M

+

-

279

F

+

-

280

F

+

-

281

F

+

+

282

M

+

+

283

F

+

+

284

M

+

-

285

M

+

-

286

F

+

-

287

M

+

-

288

M

+

-

289

M

+

-

290

M

+

-

291

F

+

-

292

F

+

-

293

F

+

-

294

F

+

-

296

F

+

-

Table 3.2. Summary of tubulin results and Toxoplasma gondii infection tested by SAG1 PCR at Malham Tarn, North Yorkshire in 2008.

Table 3.3 shows the number of mice collected and the number of mice infected each year. The overall prevalence of Toxoplasma gondii over the ten year study period was 40.78% (95% confidence interval 34.07% - 47.79%) of a total of 206 individuals. The number of mice captured each year over the study was variable and ranged between 8 - 37 individuals. The highest prevalence recorded was 69% in 2003. The lowest was 10.5% in 2007.

Year

1999

2000

2001

2002

2003

2004

2005

2006

2007

2008

Total

Total mice

37

24

19

16

29

8

24

9

19

21

206

Infected

17

4

6

10

20

3

13

4

2

5

84

Prevalence (%)

46

17

32

62.5

69

37.5

54

44

10.5

23.8

40.78

Table 3.3. Prevalence of Toxoplasma gondii infection at Malham Tarn, North Yorkshire over the 10 year study period.

Figure 3.5. Prevalence (%) of Toxoplasma infection over the 10 year study period with standard error.

The weight, length and sex of the mice were not recoded prior to 2003 and thus these mice were omitted from analysis involving these parameters. Of the 111 mice with such available data (2003 - 2008) 47 individuals were positive (42.3%). A total of 54 females and 57 males were screened and of which 23 females, and 24 males were positive for Toxoplasma. No significant difference between T. gondii infection and sex was found using Fisher's exact test (P = 1.000). If infection was due to oocyst ingestion then a higher prevalence of infection would be expected in older rather than younger animals i.e. an age prevalence effect would be seen. To test this hypothesis the prevalences in juveniles and adults were compared. A total of 97 adults and 14 juveniles were observed in this cohort of which 44 and 5 were positive for Toxoplasma respectively. No significant difference between adults and juveniles and infection were found (P = 0.5777 by Fisher's exact test). Therefore the null hypothesis that there is no age prevalence effect is accepted.

To gain an impression of the role of cats in infection of a population of rodents, the Apodemus data represented here (cat absent) were compared with an urban population of Mus domesticus (cat present). Marshall et al. (2004) tested a total of 200 Mus domesticus to Toxoplasma using SAG1 PCR and found 59% infected. The prevalences observed in the current study in an area free of cats (40.78%) was compared with the results from Marshall et al. (2004) (59%) and the difference was shown to be highly significant using Fisher's exact test (P = 0.0005). Therefore the null hypothesis that the cat has no role in prevalence is rejected.

Very few studies on the prevalence of Toxoplasma in Apodemus can be found in the literature. A summary of other studies, along with the method of detection, is shown in table 3.4.

Authors

Location

Prevalence (%)

Testing method

Jeon and Hong, 2000

Korea

1.49 (±0.73)

ELISA

Alfornso et al., 2007

France

4 (±4.17)

MAT

Hejlicek et al., 1981

Czech Republic

20 (±24.79)

SFR

Table 3.4. Prevalence of Toxoplasma gondii infection Apodemus species in other studies

3.5. Discussion

In the current study we investigated the prevalence of Toxoplasma gondii in a wild population of Apodemus sylvaticus in North Yorkshire using a PCR-based detection assay. Toxoplasma positivity was measured by the detection of the P30 gene which encodes the Surface Antigen 1 (SAG1). We found an average of 40.78% (±13.72%) mice infected over the 10 year study, with the yearly prevalences ranging between 10.5 and 69%. As infection from the definitive host and carnivory are unlikely routes of transmission in this population, high prevalences of Toxoplasma would have implications for vertical transmission. Twenty-one mice were sampled in 2008 of which 5 were positive (23.8%). Toxoplasma has been detected every year in mice sampled from the area and therefore it seems likely that vertical transmission is playing a significant role in maintaining the parasite in this population of wood mouse. The perpetuation of the Toxoplasma between cat and mouse is thought to be an important reservoir for infection due to the mouse being the natural prey of cats. The source of infection of T. gondii in mice is thought be oocysts in the environment; however successive congenital transmission has been recorded in experimentally infected and wild populations of mice (Owen and Trees, 1998; Marshall et al., 2004; Murphy et al., 2008) indicating that infection can me maintained in the absence of the cat derived oocysts. Rats are also considered important in the epidemiology of Toxoplasma, not least because of their synanthropic existence world-wide, but also for their capacity to act as a reservoir host of T. gondii for pigs and cats. Congenital transmission has been observed experimentally in rats, however not through successive generations (Dubey and Shen, 1991; Dubey et al., 1997; Zenner et al., 1993). This would indicate that there is considerable variability between species in their capacity to vertically transmit T. gondii over a number of generations and therefore in the absence of the definitive host. The common rat, Rattus norvegicus, is most noted for its devastating mark on human history for disease transmission, and is considered to be the greatest mammal pest of all time. Rat-borne diseases such as the cholera, bubonic plague, typhus, infectious jaundice and many more have killed more humans than all the wars in history. Rattus norvegicus is hugely successful and widespread inhabiting every continent except Antarctica. Its huge birth-rates and short gestations periods (22-24 days) result in an average production of 60 offspring per year per female (Takahashi and Lore, 1980). The prevalences recorded of T. gondii antibodies in Rattus norvegicus are variable, ranging between 0% (n = 186) in India (Mir et al.,1982) to 70% (n = 20) in Italy (Genchi et al.1991). Interestingly a study by Cook and Pope (1959) found a prevalence of 91% (n = 23) in the water rat Hydromys chrysogaster in Australia.

Toxoplasma uses a remarkably wide range of hosts which is unusual in its class. The prevalence of Toxoplasma in mammals is highly variable, but it is one of the most common zoonoses world-wide, with an average of 30-40% of mammals infected (Tenter et al., 2000). Of the 3 routes of transmission, it remains unclear which of these contribute most significantly to the high levels of infection. Although this remains a contentious issue, it commonly regarded that the definitive host is prime contributor to prevalence level. Cats can release millions of oocysts into the environment which can remain viable for up to a year (Benenson et al., 1982). However, cats can only shed infective oocysts for up to 14 days post-infection as a kitten and this drastically reduces the amount of time the definitive host can contribute to its epidemiology. Carnivory is also considered important in the transmission of T. gondii however, herbivores are also susceptible to high levels of infection, even in cases where cats are unlikely to come into contact with the animal such as sheep (Duncanson et al., 2001; Williams et al., 2005; Morley et al., 2005). Our results show consistently high levels of Toxoplasma in an natural population of woodmice in an area where infection due to oocysts in the environment are likely to be a rare event. This is an accord with other studies that have shown vertically transmission to be very effective in mice. It therefore seems likely that vertical transmission also occurs at high frequencies in wild populations of mice and it is this mode of transmission which sustains the infection in the absence of the cat. Initial infection most probably comes from the feline host via oocysts in the environment ingested by contaminated food, which is then sustained via serial transmission from mother to pup. This has implications for the ability of other members of Toxoplasma's plethora of intermediate hosts to act as reservoir hosts via this mode. To date, no assay exists to determine the parasite stage responsible for infection which is central to it's enigmatic epidemiology, but findings such as these may help us understand puzzlingly high prevalences of Toxoplasma in certain areas and species that are not usually associated with the definitive host.

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