Investigation Into The Prevalence Of Toxoplasma Gondii Infection Biology Essay

The protozoon parasite Toxoplasma gondii is prevailing worldwide and can infect a unusually broad scope of hosts. It is a member of the phylum Apicomplexa and is distinguished amongst this group for its ability to readily infect about any mammal, unlike closely related beings ( e.g. Neospora caninum ) which are limited in their host scope. Toxoplasma is transmitted via three paths ; the consumption of oocysts from the environment, consumption of tissue cysts in the intermediate host and congenitally through trans-placental crossing from female parent to foetus. The importance of each of these paths to the overall prevalence of the parasite is of long argument, and the parasite remains puzzling in its epidemiology. The unequivocal host is the cat ( felidae household ) where the parasite undergoes its sexual phase.

The cat sheds 1000000s of morbific oocysts incorporating sporozoites into the environment which are so accidently ingested by an intermediate host. The sporozoites so distinguish into tachyzoites which divide quickly and do acute infection. If the intermediate host is pregnant, this phase may traverse the placenta conveying the parasite to the progeny. Tachyzoites convert to bradyzoites, the easy dividing phase attributed to chronic infection, and organize tissue cysts. These tissue cysts can be consumed by quarry and in the instance of the cat convert into gametes in the enterocytes of the bowel and so the rhythm is complete. As another intermediate host can go septic via the consumption of tissue cysts, and the parasite can propagate vertically through inborn transmittal, Toxoplasma can convey wholly through nonsexual agencies ( Joynson and Wreghitt, 2001 ) . Despite holding a meiotic stage in its lifecycle, and therefore holding the chance of familial exchange in cats, the genetic sciences of Toxoplasma are extremely clonal, consisting of 3 closely related line of descents.

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These line of descents, although genetically similar, demo a distinguishable correlativity with mouse virulency ( Ajzenbery et al. , 2002 ; Sibley and Ajioka, 2008 ) .Murine theoretical accounts are of peculiar involvement when analyzing the epidemiology of Toxoplasma as the cat frequently predates on little members of this group, and hence parasite transmittal and scattering should be extremely evolved and warrants detailed survey. High degrees of perpendicular transmittal of Toxoplasma has been observed in murine hosts by experimentation infected ( Beverley, 1959 ; Owen and Trees, 1998 ) and besides in natural urban populations ( Marshall et al. , 2004 ; Murphy et al. , 2008 ) . Beverley ( 1959 ) observed consecutive transmittal of T.

gondii in up to 9 coevalss in by experimentation septic mice. It is recognised that inborn transmittal is an of import path of transmittal in mice, but it is by and large considered less of import in other species ( refs ) . However, inborn transmittal has been observed in consecutive coevals in sheep, which has important deductions for the farm animal industry ( Duncanson et al.

, 2001 ; Morley et al. , 2005 ; Williams et al. , 2005 ) .Prevalence degrees of T. gondii in mammals are variable, but by and large high, runing between 30-40 % on norm, but making up to 90 % in some populations ( Tenter et al. , 2000 ) . Previous surveies into the prevalence of Toxoplasma in mice include an probe into inborn transmittal of the parasite in an urban population of Mus domesticus by Marshall et Al.

( 2004 ) . In this survey 200 mice were captured in houses in Manchester including 16 females. Of the 200 mice obtained, 59 % tested positive for Toxoplasma utilizing SAG1 PCR. Congenital transmittal was observed in 75 % of the gestations.

A survey by Zhang et Al. ( 2004 ) investigated the prevalence of T. gondii in a wild population of field mouses that are common in northern and cardinal Eurasia. The survey was carried out in Hunan Province, the People ‘s Republic of China utilizing the modified agglutination trial ( MAT ) and they found 29 % of the population infected.The wood mouse Apodemus sylvaticus is a common gnawer found in Western Europe that is sometimes considered a plague. They are about wholly nocturnal, extremely adaptable and occupy most home grounds that are non excessively wet. They are common in forest, cultivable land, grasslands, sand dunes and urban countries in edifices during rough winter conditions ( Flowerdew, 1991 ) . Woodmice are native to the United Kingdom and are widespread and really common, holding an estimated pre-breeding population of about 38 million.

In deciduous forest 1-40 persons per hectare is usual nevertheless their densenesss vary seasonally with early winter extremums and late spring troughs ( Harris et al. , 1995 ) .Cat Numberss are known to diminish along an urban-rural gradient ( L & A ; eacute ; lu et al. , 2010 ) and so in rural mice, consecutive transmittal of Toxoplasma vertically from female parent to whelp could ease extension in the absence of the felid.

In the current survey, a wild population of woodmouse was sampled in a rural country of North Yorkshire. The country around Malham Tarn Field Centre consists of forest, grazed and ungrazed grassland, moorland and limestone pavings. It is home to legion little mammals such as coneies, mice and field mouses. It is located at an height of 381m above sea degree and experiences an highland clime. The field Centre is situated on the north shore of the tarn and is 5km from Malham small town. There are no known ferine cats or domestic cats in farms around the surround country Hughes et al. , 2008 ) , and given the distance to the nearest town, it is improbable the country is frequently frequented by rolling males.

The purpose of the current survey was to look into the prevalence of Toxoplasma gondii in a rural, wild population of wood mouse ( Apodemus sylvaticus ) in North Yorkshire over a 10 twelvemonth period. Furthermore, as the country is mostly free of cats this provides the chance to see the importance of transmittal paths that bypass the unequivocal host.3.2. AimsTo pull out DNA from encephalon tissue of a cohort of Apodemus sylvaticusTo corroborate PCR suitableness utilizing primers specific for mammalian tubulinTo observe the presence of Toxoplasma gondii utilizing nested PCRTo analyze prevalence informations with regard to biological parametric quantitiesTo compare prevalence with an urban population of gnawers that were collected from an country populated with cats ( Marshall et al. , 2004 )To catalogue and compare prevalences with other published surveies on Apodemus3.

3. Methods and consequencesThe woodmice ( Apodemus sylvaticus ) were sampled from the environing country of Malham Tarn, North Yorkshire over a 10 twelvemonth period as portion of an on-going survey of parasites in the country. Longworth traps were set and left overnight in the forest country around the field Centre ( see figure 1 ) in Autumn 2008.Figure 3.1. Area environing Malham Tarn Field Centre, North Yorkshire ( Taken from Digimap, 2000 )A sum of 21 mice were killed by trichloromethane inspiration. The encephalon tissue samples were dissected out and stored at -20 & A ; deg ; C in lysis buffer.

Deoxyribonucleic acid was extracted utilizing phenol/chloroform process. The extracted DNA was tested for mammalian tubulin utilizing generic primers to ?-tubulin to guarantee viability to undergo PCR. The presence of Toxoplasma gondii was detected utilizing PCR elaboration of the P30 cistron and resolved by gel cataphoresis. These techniques are described in full in chapter 2.Date collected from these 21 mice were combined with antecedently collected information. Other research workers that carried out these checks during the old old ages are as follows: Elizabeth Wright, Denise Thomasson, Omar Gerwash, Muftah Abushahma and Andrew Cox.Statisticss were carried out as described in the stuffs and methods. Mice weighing less than 14g were considered juveniles ( Higgs and Nowell, 2000 ) .

3.4. ConsequencesThe first phase in the analysis was to pull out DNA from the encephalon tissue of Apodemus sylvaticus for the showing of Toxoplasma gondii. The Deoxyribonucleic acid was extracted from 21 mice collected in 2008 and the Deoxyribonucleic acid 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. Deoxyribonucleic acid 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.Deoxyribonucleic acid was successfully isolated from all 21 mice obtain in 2008 as can be seen from figure 3.2. All extractions showed a high DNA output with a scope of molecular sizes seeable. Many of which consisted preponderantly of high molecular weight DNA such as mouse 280 in the above figure.

This indicates successful extraction of Deoxyribonucleic acid from the encephalon tissue with high measures of genomic Deoxyribonucleic acid that is n’t overly degraded. This was true for all mouse extractions in 2008. Each mouse was weighed, measured and sexed at the clip of aggregation, inside informations of which are presented in table 3.1.A sum-up of biological parametric quantities and PCR consequences from the 2008 cohort of mice can be seen in table 3.1. For inside informations of the mice from other twelvemonth cohorts see Appendix 1. Over the 10 twelvemonth survey, mouse samples ranged between 8 and 37 mice, and in 2008 a sum of 21 were captured.

Over the period a sum 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.Sexual activityLength ( centimeter )Weight ( g )275F817276Meter7.215277F7.918278Meter1025279F7.713280F8.320281F7.

515282Meter7.814283F8.518284Meter8.218285Meter7.518286F714287Meter1024288Meter7.615289Meter7.

715290Meter7.715291F7.816292F8.

519293F7.418294F818296F820Table 3.1. Summary of Apodemus sylvaticus collected in 2008 from MalhamTarn, North Yorkshire.Prior to proving the samples for Toxoplasma the DNA, known to dwell mostly of mammalian DNA, was tested for the presence of mammalian ?-tubulin. This was to guarantee the DNA was of a feasible quality for undergoing PCR and extinguish the possibility of obtaining a false negative due to PCR inhibitors present in the sample. Figure 3.3 shows the gel cataphoresis image of mice 285-292 tested for ?-tubulin.

The positive control used was mouse DNA from a old twelvemonth. As can be seen from figure 2 all mice tested positive for tubulin. All samples shown a high elaboration of the ?-tubulin cistron which is declarative 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 H2O negative control.

In old old ages, samples that could non be successful amplified by PCR for tubulin were omitted from the survey. This was non necessary for any samples in 2008. Following successful elaboration of the ?-tubulin cistron, the samples were so tested for Toxoplasma utilizing a nested PCR designed to aim the P30 cistron.

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 illustration of successful sensing of Toxoplasma can be seen from figure 3.4 ( sample 283 ) . Following the initial first unit of ammunition of DNA utilizing the external primers a merchandise of 914bp is amplified in a successful PCR of a positive samples. This merchandise is so used in the 2nd unit of ammunition of the PCR assay to give a extremely specific merchandise of 522bp ( see below ) .

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

Figure 3.4.

SAG-1 PCR gel cataphoresis image of Malham mice 284-291, 2008 at 1m DNA per reaction, +ve denotes cloned SAG1 positive control, -ve denotes H2O negative controlA sum of 21 mice were captured in 2008 of which 5 tested positive for Toxoplasma by SAG1 PCR, bespeaking a prevalence of 23.81 % ( ±15.29 % ) for this twelvemonth cohort ( table 3.2 ) .

Mouse No.Sexual activityTubulinSAG1275F

+

276Meter

+

+

277F

+

+

278Meter

+

279F

+

280F

+

281F

+

+

282Meter

+

+

283F

+

+

284Meter

+

285Meter

+

286F

+

287Meter

+

288Meter

+

289Meter

+

290Meter

+

291F

+

292F

+

293F

+

294F

+

296F

+

Table 3.2. Summary of tubulin consequences and Toxoplasma gondii infection tested by SAG1 PCR at Malham Tarn, North Yorkshire in 2008.Table 3.3 shows the figure of mice collected and the figure of mice infected each twelvemonth.

The overall prevalence of Toxoplasma gondii over the 10 twelvemonth survey period was 40.78 % ( 95 % assurance interval 34.07 % – 47.79 % ) of a sum of 206 persons.

The figure of mice captured each twelvemonth over the survey was variable and ranged between 8 – 37 persons. The highest prevalence recorded was 69 % in 2003. The lowest was 10.5 % in 2007.Year1999200020012002200320042005200620072008EntireEntire mice372419162982491921206Infected1746102031342584Prevalence ( % )46173262.56937.

5544410.523.840.78Table 3.3. Prevalence of Toxoplasma gondii infection at Malham Tarn, North Yorkshire over the 10 twelvemonth survey period.Figure 3.

5. Prevalence ( % ) of Toxoplasma infection over the 10 twelvemonth survey period with standard mistake.The weight, length and sex of the mice were non recoded prior to 2003 and therefore these mice were omitted from analysis affecting these parametric quantities. Of the 111 mice with such available informations ( 2003 – 2008 ) 47 persons were positive ( 42.3 % ) . A sum of 54 females and 57 males were screened and of which 23 females, and 24 males were positive for Toxoplasma. No important difference between T. gondii infection and sex was found utilizing Fisher ‘s exact trial ( P = 1.

000 ) . If infection was due to oocyst consumption so a higher prevalence of infection would be expected in older instead than younger animate beings i.e. an age prevalence consequence would be seen.

To prove this hypothesis the prevalences in juveniles and grownups were compared. A sum of 97 grownups and 14 juveniles were observed in this cohort of which 44 and 5 were positive for Toxoplasma severally. No important difference between grownups and juveniles and infection were found ( P = 0.5777 by Fisher ‘s exact trial ) . Therefore the void hypothesis that there is no age prevalence consequence is accepted.To derive an feeling of the function of cats in infection of a population of gnawers, the Apodemus information represented here ( cat absent ) were compared with an urban population of Mus domesticus ( cat nowadays ) . Marshall et Al. ( 2004 ) tested a sum of 200 Mus domesticus to Toxoplasma utilizing SAG1 PCR and found 59 % infected.

The prevalences observed in the current survey in an country free of cats ( 40.78 % ) was compared with the consequences from Marshall et Al. ( 2004 ) ( 59 % ) and the difference was shown to be extremely important utilizing Fisher ‘s exact trial ( P = 0.

0005 ) . Therefore the void hypothesis that the cat has no function in prevalence is rejected.Very few surveies on the prevalence of Toxoplasma in Apodemus can be found in the literature. A sum-up of other surveies, along with the method of sensing, is shown in table 3.4.

WritersLocationPrevalence ( % )Testing methodJeon and Hong, 2000Korea1.49 ( ±0.73 )Enzyme-linked-immunosorbent serologic assayAlfornso et al. , 2007France4 ( ±4.17 )MatHejlicek et al. , 1981Czech Republic20 ( ±24.79 )SFRTable 3.4.

Prevalence of Toxoplasma gondii infection Apodemus species in other surveies3.5. DiscussionIn the current survey we investigated the prevalence of Toxoplasma gondii in a wild population of Apodemus sylvaticus in North Yorkshire utilizing a PCR-based sensing check. Toxoplasma positiveness was measured by the sensing of the P30 cistron which encodes the Surface Antigen 1 ( SAG1 ) . We found an norm of 40.78 % ( ±13.72 % ) mice infected over the 10 twelvemonth survey, with the annual prevalences runing between 10.5 and 69 % .

As infection from the unequivocal host and carnivory are improbable paths of transmittal in this population, high prevalences of Toxoplasma would hold deductions for perpendicular transmittal. Twenty-one mice were sampled in 2008 of which 5 were positive ( 23.8 % ) . Toxoplasma has been detected every twelvemonth in mice sampled from the country and therefore it seems likely that perpendicular transmittal is playing a important function in keeping the parasite in this population of wood mouse. The prolongation of the Toxoplasma between cat and mouse is thought to be an of import reservoir for infection due to the mouse being the natural quarry of cats.

The beginning of infection of T. gondii in mice is thought be oocysts in the environment ; nevertheless consecutive inborn transmittal has been recorded in by experimentation infected and wild populations of mice ( Owen and Trees, 1998 ; Marshall et al. , 2004 ; Murphy et al. , 2008 ) bespeaking that infection can me maintained in the absence of the cat derived oocysts. Rats are besides considered of import in the epidemiology of Toxoplasma, non least because of their synanthropic being global, but besides for their capacity to move as a reservoir host of T. gondii for hogs and cats. Congenital transmittal has been observed by experimentation in rats, nevertheless non through consecutive coevalss ( Dubey and Shen, 1991 ; Dubey et al.

, 1997 ; Zenner et al. , 1993 ) . This would bespeak that there is considerable variableness between species in their capacity to vertically convey T. gondii over a figure of coevalss and hence in the absence of the unequivocal host. The common rat, Rattus norvegicus, is most celebrated for its lay waste toing grade on human history for disease transmittal, and is considered to be the greatest mammal plague of all clip. Rat-borne diseases such as the cholera, bubonic pestilence, typhus, infective icterus and many more have killed more worlds than all the wars in history.

Rattus norvegicus is enormously successful and widespread inhabiting every continent except Antarctica. Its immense birth-rates and short gestations periods ( 22-24 yearss ) consequence in an mean production of 60 offspring per twelvemonth per female ( Takahashi and Lore, 1980 ) . The prevalences recorded of T. gondii antibodies in Rattus norvegicus are variable, runing between 0 % ( n = 186 ) in India ( Mir et al.,1982 ) to 70 % ( n = 20 ) in Italy ( Genchi et al.1991 ) . Interestingly a survey by Cook and Pope ( 1959 ) found a prevalence of 91 % ( n = 23 ) in the H2O rat Hydromys chrysogaster in Australia.

Toxoplasma uses a unusually broad scope of hosts which is unusual in its category. The prevalence of Toxoplasma in mammals is extremely variable, but it is one of the most common zoonotic diseases global, with an norm of 30-40 % of mammals infected ( Tenter et al. , 2000 ) . Of the 3 paths of transmittal, it remains ill-defined which of these contribute most significantly to the high degrees of infection.

Although this remains a combative issue, it normally regarded that the unequivocal host is premier subscriber to prevalence degree. Cats can let go of 1000000s of oocysts into the environment which can stay feasible for up to a twelvemonth ( Benenson et al. , 1982 ) . However, cats can merely cast morbific oocysts for up to 14 yearss post-infection as a kitty and this drastically reduces the sum of clip the unequivocal host can lend to its epidemiology. Carnivory is besides considered of import in the transmittal of T. gondii nevertheless, herbivores are besides susceptible to high degrees of infection, even in instances where cats are improbable to come into contact with the animate being such as sheep ( Duncanson et al.

, 2001 ; Williams et al. , 2005 ; Morley et al. , 2005 ) . Our consequences show systematically high degrees of Toxoplasma in an natural population of woodmice in an country where infection due to oocysts in the environment are likely to be a rare event. This is an agreement with other surveies that have shown vertically transmittal to be really effectual in mice. It hence seems likely that perpendicular transmittal besides occurs at high frequences in wild populations of mice and it is this manner of transmittal which sustains the infection in the absence of the cat. Initial infection most likely comes from the felid host via oocysts in the environment ingested by contaminated nutrient, which is so sustained via consecutive transmittal from female parent to whelp. This has deductions for the ability of other members of Toxoplasma ‘s overplus of intermediate hosts to move as reservoir hosts via this manner.

To day of the month, no check exists to find the parasite phase responsible for infection which is cardinal to it ‘s puzzling epidemiology, but findings such as these may assist us understand puzzlingly high prevalences of Toxoplasma in certain countries and species that are non normally associated with the unequivocal host.

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