australia is home to a vast array of endemic marsupial species ( superorder australidelphia ) . of an estimated 162 marsupial species present at the time of european settlement in 1788 , twelve species have since become extinct , and many others have suffered major contraction of distribution and substantial to severe population declines ( burbidge et  al . , 2009 , woinarski , 2015 ) .
the primary drivers of these population declines are believed to be : anthropogenic habitat loss ; habitat destruction associated with climate change , altered fire regimes and the introduction of competing feral herbivores and exotic weeds ; and predation by introduced feral species , particularly foxes and cats ( dickman , 1996 , fisher et  al .
, 2003 , mckenzie et  al . , 2007 , burbidge et  al . , 2009 ,
there has also been speculation as to the role of introduced infectious disease as a contributing factor to population declines of australian marsupials ( e.g. , freeland , 1994 , abbott , 2006 , thompson et  al . , 2010 ) .
toxoplasma gondii is arguably the most broadly implicated infection , prompted by reports of cases and outbreaks of severe clinical toxoplasmosis in captive populations of australian marsupial species . from the published literature
, there appears to be a perception that marsupials are particularly susceptible to infection with t.  gondii and to toxoplasmosis .
for example :  toxoplasmosis is a significant disease of australian marsupials commonly causing mortality in captive and free - ranging populations  
( obendorf et  al . , 1996 ) ;  marsupials and new world monkeys are among the most susceptible animals for developing the clinical disease toxoplasmosis   ( skerratt et  al . , 1997 ) ;  australasian marsupials , especially wallabies , are highly susceptible to acute toxoplasmosis 
( dubey and crutchley , 2008 ) ;  australian marsupials are among the most susceptible hosts for t. gondii   ( parameswaran et  al . , 2009a ) ;  t.  gondii is of concern for australian native marsupials , which appear to be particularly susceptible to acute infection   ( hollings et  al . , 2013 ) .
we review estimates of the frequency of t.  gondii infection in free - ranging australian marsupial populations , and then review the extent to which the infections are associated with acute toxoplasmosis or with other effects ( such as behavioural changes and reduced reproductive success ) that may threaten population viability .
t. gondii is a protozoan parasite , which can infect a wide range of endothermic vertebrates .
cats ( felidae ) are the definitive host - infected cats shed environmentally resistant oocysts in the faeces .
oocysts become infective in the environment , and if ingested can infect both intermediate hosts ( including australian marsupial species ) and other definitive hosts .
following ingestion , sporozoites excyst from oocysts , invade the gut epithelium and transform into tachyzoites .
tachyzoites multiply asexually and may colonise many host tissues , evoking a strong immune response .
tachyzoites differentiate into bradyzoites , which produce tissue cysts that are resistant to the immune response .
bradyzoites may be transmitted to a definitive host , or another intermediate host , upon ingestion of infected tissues .
in addition , these hosts may also be infected via vertical transmission from infected mother to foetus / suckling young ( dubey , 1998 , dubey , 2010 ) . in most intermediate host species , including people , t.  gondii infection tends to be subclinical ; toxoplasmosis ( clinical disease caused by t.  gondii infection ) is usually associated with complicating factors such as immunosuppression ( montoya and leisenfeld , 2004 , dubey , 2010 ) .
clinical toxoplasmosis may follow recent infection with t.  gondii , or result from a recrudescent infection .
recrudescence may be prompted by concurrent illness or immunosuppression ( ruskin and remington , 1976 , lappin et  al .
no published studies have investigated the incidence of t.  gondii infection in free - ranging populations of australian marsupials .
surveys have provided estimates infection prevalence and seroprevalence ; these are summarised in table  1 , table  2 .
evidence of t.  gondii infection has been found in free - ranging populations of red kangaroos ( macropus rufus ) , western grey kangaroos ( macropus fuliginosus ) , common wallaroos ( marcopus robustus ) and woylies ( bettongia penicillata ) ( table  1 ) .
findings suggestive of t.  gondii infection ( histopathological evidence without confirmatory testing ) have also been obtained from long nosed bandicoots ( perameles nasuta ) , eastern barred bandicoots ( perameles gunnii ) , southern brown bandicoots / quenda ( isoodon obesulus ) , quokka ( setonix brachyurus ) , brushtail possums ( trichosurus vulpecula ) , brush - tailed phascogales ( phascogale tapoatafa ) and kowari ( dasyuroides byrnie ) ( table  1 ) .
prevalence estimates are all limited by uncertain external validity , due to the use of non - proportionate sampling methods : reliance on culled animals , road kill or trapping for study subjects may entail selection bias .
surveys involving small sample sizes have low power to detect the presence of infection , and marked imprecision in prevalence estimates ( table  1 ) . commonly , the use of diagnostic methodology that is known to be of poor sensitivity and/or specificity in other species leaves apparent prevalence estimates subject to misclassification bias . in marsupial surveys ,
the most commonly used diagnostic test has been the mouse bioassay , which lacks sensitivity ( piergili fioretti , 2004 ) .
none of the surveys of australian marsupials using this technique also used immunohistochemistry or pcr to confirm identification of t.  gondii bradyzoites .
thus , the specificity of the mouse bioassay may be compromised , as t.  gondii bradyzoites can appear very similar to those of neospora caninum under light microscopy ( dubey et  al . , 2009 ) . as with the mouse bioassay ,
sample inoculation into cell culture ( in this case 13-day old chick embryos ( table  1 ) ) lacks sensitivity , often because of laboratory error ( piergili fioretti , 2004 ) .
histopathological examination of host tissues , without confirmatory immunohistochemistry or pcr , was used in a number of marsupial surveys ( table  1 ) . however , this is also an insensitive screening tool , particularly in identifying low burden t.  gondii infections ( piergili fioretti , 2004 ) .
specificity of these results might also be compromised by misidentification of other protozoan parasites as t.  gondii ( dubey et  al . , 2009 ) .
pcr amplification of t.  gondii dna in tissue samples is generally considered a sensitive indicator of infection ( burg et  al . , 1989 ,
pcr has only been used in two surveys of australian marsupials , which collectively sampled four species ( parameswaran et  al . , 2010 ,
pan et  al . , 2012 ) . a high proportion these animals tested positive for t.  gondii by this methodology . though these findings are limited by small sample sizes , they sharply contrast to collective findings of similar species surveyed histopathologically , where infection was rarely identified ( table  1 ) . as the latter studies were based on different species in different locations
however , such discordant results do lend support to the hypothesis that histopathological surveys are insensitive in estimating prevalence of infection in marsupial populations showing no clinical signs of toxoplasmosis .
pcr techniques have been demonstrated to have high specificity ( burg et  al . , 1989 ) : given the use of appropriate negative controls and sequencing in both studies , it seems unlikely that false positives would have substantially influenced the findings of the pcr surveys .
t. gondii serosurveys have found t.  gondii antibodies in populations of western grey kangaroos , eastern grey kangaroos ( macropus giganteus ) , bennett 's wallabies ( macropus rufogriseus ) , bridled nailtail wallabies ( onychogalea fraenata ) , tasmanian pademelons ( thylogale billardierii ) , brush tailed rock wallabies ( petrogale penicillata ) , quokkas , woylies , tasmanian devils ( sarcophilus harisii ) , common wombats ( vombatus ursinus ) , eastern quolls ( dasyurus viverrinus ) , spotted - tail quolls ( dasyurus maculatus ) , chuditch ( or western quolls , dasyurus geoffroii ) , brushtail possums , western ringtail possums ( pseudocheirus occidentalis ) , southern brown bandicoots , eastern barred bandicoots , long - nosed bandicoots and bilbies ( macrotis lagotis ) ( table  2 ) .
seroprevalence surveys are prone to limitations regarding inferring infection prevalence , similar to those affecting surveys of t.  gondii . in particular ,
while the use of serological surveys avoids invasive tissue sampling ( which usually necessitates euthanasia or opportunistic sampling of otherwise dead animals in wildlife populations ) , it is important that the serological test(s ) have been validated at the cut off titre used to differentiate infected and non - infected animals , in the species being studied . identifying whether or not the serological test accurately reflects the infection status of the tested host is essential in estimating the true prevalence of infection from apparent seroprevalence data .
very few serological tests have been adequately validated for use in australian marsupial species ( supplementary tables  13 ) . factors that may impact the validity of using serological survey data to estimate infection prevalence include : 1 ) an antibody response may reflect exposure to an infection , but not necessarily establishment of infection in the host ; 2 ) if the sampled animal is acutely infected , it may not yet have a detectable antibody titre ; 3 ) if an infected animal has developed an igm titre , but not yet igg , serological tests that do not detect t.  gondii igm antibodies ( eg the modified agglutination test , or some elisas ) will not classify these animals as infected ; 4 ) in animals with long term chronic t.  gondii infections , serological antibody titres may drop to levels that are below the cut off titre for differentiating infected vs uninfected animals , compromising sensitivity  waning serological titres have been demonstrated in chuditch ( haigh et  al . , 1994 ) , brushtail possums ( eymann et  al
. , 2006 ) , woylies ( a. worth , murdoch university - unpublished results ) and in eastern grey kangaroos clinically suspected of t.  gondii infection ( miller et  al . , 2003 ) ; 5 ) non - specific agglutination , which is known to occur with the direct agglutination test in other species ( dubey , 2010 ) , may compromise test specificity ; 6 ) anti - complementary sera require exclusion from serosurveys that use the complement fixation test , which may bias findings ( e.g. , pope et  al . , 1957a , cook and pope , 1959 ) ; and 7 ) the complement fixation test , the sabin feldman dye test and elisas require species - specific reagents .
however , in studies where the sabin - feldman dye test or complement fixation test were used , there was no description of the test being adapted for the marsupial species surveyed ( pope et  al .
, 1957a , cook and pope , 1959 , gibb et  al . , 1966 , munday , 1972 ) . beyond the potential impact of misclassification and sample size on inferring infection prevalences from apparent prevalences / seroprevalences obtained via surveys , prevalence data are prone to substantial limitations as a measure of infection frequency , including in australian marsupial species
when infection is stable in a population , prevalence approximates the product of the incidence of infection , and mean duration of infection .
many surveys identified low apparent prevalences or seroprevalences . if not impacted by misclassification , these findings could reflect t.  gondii infection being relatively uncommon in the surveyed populations .
alternatively , they may be consistent with t.  gondii infection being relatively common but associated with high short term mortality rates in these populations , resulting in infected animals having a low probability of inclusion in prevalence surveys .
conversely , high prevalence / seroprevalence of t.  gondii infection has been identified in some marsupial populations , including those subject to culls due to overpopulation .
if not impacted by misclassification , such findings suggest , but do not provide conclusive evidence , that t.  gondii infection does not substantially impact population viability .
it has been postulated that other factors , particularly concurrent stressors , may interact with t.  gondii infection to produce adverse outcomes ( e.g. , obendorf and munday , 1983 , johnson et  al . , 1988 , miller et  al . , 2000 ) .
a high prevalence of t.  gondii infection in a stable population may therefore suggest that the population could be at risk of disastrous population impacts , if the population is subject to such factors .
cats were introduced into australia from europe in the 1800s ( denny and dickman , 2010 ) .
it is thus assumed that the introduction and spread of t.  gondii across australia commenced around this time .
this presumptive short history of exposure to t.  gondii infection has been widely suggested to have resulted in increased virulence and pathogenicity of t.  gondii in australian marsupial species , compared to other intermediate hosts worldwide ( johnson et  al . , 1988 , canfield et  al . , 1990 ,
, 1993 , obendorf et  al . , 1996 , barrows , 2006 , eymann et  al . , 2006 , adkesson et  al . , 2007 , hollings et  al . , 2013 ) . however , there is no evidence available to support this claim , and there is no reason to assume that the virulence of a parasite will always be greater in new host species ( ebert and herre , 1996 ) .
it has also been suggested that various stressors may be relevant in the clinical manifestation of t.  gondii infections - both acute and recrudescent - in marsupials ( obendorf and munday , 1983 , johnson et  al . , 1988 , miller et  al .
some reports of toxoplasmosis amongst captive marsupial populations have noted exposure to potential stressors prior to the occurrence of disease .
for example , immediately prior to an outbreak associated with high mortality , a population of captive sugar gliders ( petaurus breviceps ) was exposed to repeated episodes of social disruption , suboptimal nutrition and suboptimal temperature ( barrows , 2006 ) .
other cases or outbreaks of toxoplasmosis in captive wallabies have occurred following relocations ( wilhelmsen and montali , 1980 , dubey and crutchley , 2008 , bermdez et  al . , 2009 ) or social isolation ( adkesson et  al . , 2007 ) .
a study of t.  gondii infection in dasyurids ( 65 wild - caught , 103 laboratory reared ) , using histopathology without confirmatory testing , found the widest dissemination of organisms consistent with t.  gondii in two fat - tailed false antechinus ( pseudantechinus macdonnellensis ) with leukaemia ( attwood and woolley , 1973 ) .
cohort studies are the method of choice for comparing survivability in infected vs uninfected marsupial hosts , but are difficult to complete to an acceptable standard in free - ranging populations .
key challenges include : 1 ) the vast resources required to precisely follow up cohorts of a statistically adequate sample size ; 2 ) the high susceptibility of wildlife cohorts to bias , particularly due to loss to follow - up , and when considering survivability , the typical requirement for a proxy for death ( such as failure to retrap ) which can not differentiate the outcome from loss to follow - up ; 3 ) intermittent surveillance of wildlife cohorts ( to monitor exposure and outcome status ) entails a low probability of sampling infected animals that die acutely of toxoplasmosis , and the seropositive cohort is therefore more likely to involve animals that survive with chronic infection than those which succumb to acute infection ; and 4 ) putative confounding factors , such as age , can be very difficult to accurately measure in wild animals , and therefore accurately account for in statistical analyses , leaving study findings prone to confounding . these challenges likely explain why only two such studies have been undertaken thus far .
the first was on eastern barred bandicoots at two sites in southern tasmania 's huon valley .
both the direct agglutination test ( without 2-mercaptoethanol ) ( dat ) and modified agglutination test ( that includes 2-mercaptoethanol ) ( mat ) were used .
one hundred and thirty three bandicoots were negative on both serological tests , and 68% of these were recaptured at least once .
ten bandicoots were positive on both the mat and dat ( both titres 64 ) ; five were not retrapped , while five had antibodies on two consecutive occasions ( three months apart ) and were not recaptured subsequently ( obendorf et  al . , 1996 ) .
these findings may reflect reduced survival times in eastern barred bandicoots infected with t.  gondii , but there are substantial limitations in drawing such a conclusion .
the generalities listed above apply , and lack of validation of the diagnostic test used is a potential influence on findings .
additionally , the small seropositive cohort size means that chance effects on the results can not be confidently excluded , and confounding ( by age , in particular ) can not be ruled out , as the cumulative risk of t.  gondii infection has been demonstrated to increase with age in other species ( dubey , 2010 ) .
a further seven bandicoots tested positive on the direct agglutination test ( titre 64 ) in this study , but were negative on the modified agglutination test ( titre < 64 ) ; none of these seven bandicoots were recaptured ( obendorf et  al . , 1996 ) . again , while this may reflect a lower survival time in bandicoots acutely infected with t.  gondii , this is not sufficient evidence to draw such a conclusion .
the direct agglutination test has not been validated in eastern barred bandicoots , and it is known to have relatively poor specificity in other species ( dubey , 2010 ) .
therefore , misclassification can not be excluded , along with chance effects due to the small sample size .
survival times of eastern quolls that were seropositive for t.  gondii were compared to those that were seronegative , in a location where the quoll population was classified as 
there was no evidence of a difference in survival time between seropositive and seronegative quolls ( fancourt et  al . , 2014 ) .
however , a lack of power can not be excluded as an influence on study findings , due to the relatively small sample size involved . regarding the use of these data to compare the survival time of t.  gondii infected vs uninfected marsupials , the generalities described above apply , as does a lack of validation in the diagnostic methodology used to infer host t.  gondii infection status from host serological data .
( 2006 ) trapped brushtail possums in various locations in sydney , on four different occasions over two and a half years . trapped possums had their t.  gondii serological status measured using the modified agglutination test , and were considered positive if reacting at a titre of 1:25 .
while this study did not aim to compare survivability of infected vs uninfected hosts , and thus the use of these data for such a purpose entails many limitations , it is of interest that though 5/9 seropositive possums were not retrapped subsequent to testing seropositive for t.  gondii ( 55.6% ; jeffrey 's 95% ci 26.281.3% ) , the proportions of seronegative possums retrapped were similar ( varying from 43 to 65% over subsequent sessions ) .
relatively high morbidity and mortality rates have been reported in outbreaks of toxoplasmosis ( miller et  al . , 1992 , barrows , 2006 , basso et  al . , 2007 , dubey and crutchley , 2008 ) or likely toxoplasmosis ( boorman et  al . , 1977 , jensen et  al . , 1985 ) in captive populations involving a number of australian marsupial species , including sugar gliders , bennett 's wallabies , tammar wallabies ( macropus eugenii ) , common wallaroos , eastern grey kangaroos , red kangaroos and long - nosed potoroos ( potorous tridactylus )
. however , these outbreaks , and case studies of toxoplasmosis in captive marsupials , can not be presumed to be representative of morbidity and mortality rates following t.  gondii infection in free - ranging populations .
thus , they can not be presumed to be indicative of marsupial species ' inherent susceptibility to t.  gondii infection or to toxoplasmosis , despite widespread citations of such studies to these ends in the literature ( e.g. , miller et  al . , 1992 ,
, 1993 , miller et  al . , 2003 , hartley and english , 2005 , barrows , 2006 , bermdez et  al . , 2009 ,
firstly , cases of subclinical t.  gondii infection in captive populations are likely to go unnoticed , and hence uninvestigated and unpublished .
this may lead to an inaccurate preconception that t.  gondii infections typically manifest as clinical disease .
seropositive marsupials have been identified amongst captive populations not showing clinical signs of toxoplasmosis ( riemann et  al . , 1974 ,
, 2000 , de camps et  al . , 2008 ) and subclinical infection consistent with t.  gondii ( not confirmed by further testing ) has been demonstrated histopathologically in captive dasyurids ( attwood et  al . , 1975 ) .
secondly , a substantial number of factors that may be associated with t.  gondii morbidity and mortality may differ between captive and free - ranging populations of australian marsupial species .
these may include differences in : stress - induced immunosuppression ( for example , as a result of housing circumstances , population density , nutrition and interaction with humans ) ; average life expectancy ( captive populations may include substantially older animals than would be found in the wild ) ; the presence of co - morbidities in captive populations that may compromise an animal 's immunity ( and predispose the animal to clinical toxoplasmosis ) but would rarely be compatible with survival in the wild ; and mechanisms of exposure to t.  gondii ( particularly via dietary sources , including levels of infection and strains of t.  gondii present in meat products that are unlikely to be common in diets of free - ranging australian marsupials ) .
these factors may substantially bias pertinent statistics , such as morbidity and mortality rates following t.  gondii infection and/or the rate of recrudescence of latent infection to clinical disease .
experimental studies under laboratory conditions are very useful for investigating the pathological processes of t.  gondii infection and clinical toxoplasmosis in australian marsupial species , and evaluating diagnostic methods .
however , caution is required in extrapolating morbidity and mortality rates observed in such studies to free - ranging populations .
for example , experimental infections undertaken in eastern barred bandicoots demonstrated the ability of t.  gondii to cause fatal disease in this species ( 2/2 infected bandicoots died ) ( bettiol et  al . , 2000 ) .
these findings may reflect eastern barred bandicoots being particularly susceptible to fatal toxoplasmosis following t.  gondii infection .
however , the widespread citation of this study as providing conclusive evidence of the high susceptibility of australian marsupial species to toxoplasmosis is premature , for several reasons .
firstly , the study was restricted to eastern barred bandicoots , and thus assumptions that the results are applicable to all australian marsupial species are not appropriate .
secondly , the small number of bandicoots infected is marked limitation in using the findings of this study to estimate the morbidity and mortality rates of toxoplasmosis post infection in this species .
thirdly , the inoculation dose and strain of t.  gondii used in the experiment may not reflect the typical dose of infection , and virulence and pathogenicity , of the strains of t.  gondii to which free - ranging populations of australian marsupial species are commonly exposed .
the bandicoots were orally infected with 100 cysts of the p89 strain of t.  gondii , which is highly virulent in mice ( dubey et  al . , 1995 ) .
the authors noted that a previous attempt to induce infection in bandicoots with a lower dose of 10 oocysts of the same strain was unsuccessful ( bettiol et  al .
finally , the morbidity and mortality rates observed in this study may have been biased by circumstances related to the experiment . in particular , the bandicoots studied may have been under stress .
they were captured from the wild for the purposes of the study , then housed in captivity , fed a novel diet , and subjected to handling and procedures which included an oral inoculation procedure , repeated rectal temperature monitoring and repeated blood tests .
if stress - induced immunosuppression does indeed act as a causal complement in toxoplasmosis , stress may have facilitated the development of clinical disease in these bandicoots .
studies undertaken in marsupial populations habituated to laboratory conditions prior to experimental infection may be more accurate in estimating morbidity and mortality rates associated with t.  gondii infection in free - ranging populations , by moderating the potential impact of stress .
experimental infections undertaken in four eastern grey kangaroos , as part of a validation study for t.  gondii serological tests , did not result in clinical toxoplasmosis .
three kangaroos were infected orally with the pork i strain of t.  gondii ( 5 , 50 and 500 oocysts administered , respectively ) and one kangaroo was infected via intramuscular injection of 250 oocysts of the same strain .
three of the four kangaroos seroconverted during the 48 days post infection ( the kangaroo dosed with 5 oocysts did not ) ; none of the kangaroos developed clinical signs of disease within this period ( johnson et  al .
the interpretation of these results is limited , however , by a lack of tests to confirm the animals developed infection post inoculation ( eg histopathology or pcr of body tissues ) .
in contrast , in experimental infections of tammar wallabies , undertaken as part of a vaccination trial , 7/9 wallabies orally dosed with of 500 t.  gondii oocysts ( me-49 strain ) died 1114 days post infection ; 1/1 wallabies dosed with 1000 t. 
gondii oocysts ( me-49 strain ) orally died 12 days post infection ; 1/1 wallabies dosed with 1000 t.  gondii oocysts ( pt-12 strain ) orally died 15 days post infection ; and 1/1 wallabies dosed with 10  000 t.  gondii oocysts ( pt-12 strain ) orally died 9 days post infection ( reddacliff et  al . , 1993 ) .
as part of another vaccination trial , six tammar wallabies were dosed with the attenuated s48 strain of t.  gondii intramuscularly ( two with 62  000 , two with 125  000 , and two with 250  000 tachyzoites ) .
ten days after inoculation , both wallabies dosed with 125  000 tachyzoites and one wallaby dosed with 250  000 tachyzoites died acutely , and one wallaby dosed with 62  000
the remaining two wallabies survived without clinical illness ( lynch et  al . , 1993 ) .
the differences in the clinical outcome of infection between and within these studies may reflect differences in : the virulence and pathogenicity of the strains of t.  gondii used in each study ; the t.  gondii inoculation dose ( two strains of t.  gondii have been demonstrated to have a dose - dependent pathogenicity in rats ( dubey , 1996 , de champs et  al . , 1998 ) ) ; the susceptibility of the marsupial species studied to clinical toxoplasmosis ( which may or may not interact with t.  gondii strain type or inoculation dose ) ; or stress - induced immunosuppression of the study subjects , possibly due to inherent differences in the susceptibility of different marsupial species to stress in captivity , or to differences in management practices between the experimental settings .
further , the results of these studies can not be confidently extrapolated to morbidity and mortality rates in free - ranging marsupial populations , due to factors such as the small numbers of animals infected , the potentially biasing influences of captivity , and inoculation doses and strains of t.  gondii infection which may not reflect those commonly occurring in free - ranging marsupial populations .
in laboratory rats and mice , infection with t.  gondii has been shown to increase activity level and exploratory behaviour and reduce aversion to predator odour ( eg berdoy et  al . , 2000 , vyas et  al . , 2007 ,
the ability to cause these behavioural changes is considered to be an adaptation of the parasite to increase transmission to the definitive host ( mcconkey et  al . , 2013 , vyas , 2013 ) .
it has been suggested that similar behavioural changes in infected marsupials may make them more vulnerable to predation by exotic predators , such as foxes and cats ( obendorf et  al . , 1996 ,
, 2013 , worth et  al . , 2014 document many exceptions to the commonly reported behavioural effects of t.  gondii infection in laboratory rodents , which they suggest are due to the use of different t.  gondii strains , different host species and sexes , and different methodologies to measure behaviour .
before it can be confidently asserted that infection with t.  gondii causes behavioural changes that may increase predation risk in marsupial hosts , data from detailed behavioural studies on these hosts are required . to date
an experimental study found infected eastern barred bandicoots were more likely to be outside their nest boxes during daylight hours , from 10 days post infection ( bettiol et  al . , 2000 ) .
however , the sample size was too small to confidently exclude chance effects or ensure randomisation achieved control of confounding .
additionally , measurement error and observer bias can not be ruled out , as the method of measurement and whether observers were blinded in making these observations were not reported .
another study found t.  gondii seroprevalence to be higher in roadkill pademelons than in culled pademelons , possibly indicating slower reaction times .
however , this may have been a chance finding resulting from the small roadkill sample size ( hollings et  al . ,
from what is known of t.  gondii infection in other species , offspring survival could be influenced by vertical transmission of the parasite .
alternatively , effects of the parasite on the reproductive fitness of mature marsupials could influence the success of breeding .
available evidence strongly suggests that vertical transmission of t.  gondii infection can occur in black - faced kangaroos ( macropus fuliginosus melanops ) , western grey kangaroos and woylies ( dubey et  al .
vertical transmission of t.  gondii from chronically infected western grey kangaroo dams to their young may occur commonly : two out of nine ( 22.2% ; jeffrey 's 95% ci 6.755.6% ) pouch young of chronically infected dams were positive for t.  gondii via pcr ( parameswaran et  al .
no study has investigated the impacts of infection on the survival of vertically infected young . if vertical transmission does occur relatively frequently , and commonly results in adverse impacts on the health of infected young , reproductive success in an infected population may be compromised .
reproductive success of mature marsupials associated with t.  gondii infection has been investigated in one study , of eastern quolls ( fancourt et  al . , 2014 ) .
september was higher in females that were seropositive for t.  gondii , than those that were seronegative .
similarly , testicular volume during the mating season was higher in seropositive males , although the implications of this finding is unclear as a relationship between testicular volume and reproductive capacity in quolls has not been demonstrated .
in addition , there were difficulties in accurately measuring and controlling for putative confounding variables , which complicates interpretation of the data ( fancourt et  al . , 2014 ) .
anecdotally , high prevalence of t.  gondii was observed in populations of western grey kangaroos , red kangaroos and common wallaroos culled due to overpopulation ( parameswaran et  al . , 2010 ,
2012 ) . while this suggests that t.  gondii infection does not substantially impact reproductive capacity in these species ,
t.  gondii may be an important factor in population declines of marsupial species in australia .
however , this remains a hypothesis , as scientifically robust supporting evidence is not yet sufficient to draw any conclusions .
such research should consider:1)validation of diagnostic test(s ) used to detect the presence or absence of t.  gondii infection in the species under study , to enable consideration of potential impacts of misclassification on analytical study findings ( boadella et  al . , 2011 ) .
validation of tests in various marsupial species would also help to retrospectively clarify previous research findings.2)the impact of sample sizes on results - ideally planning and undertaking studies where an adequate sample size for statistical extrapolations to a population level is realistically achievable , or where the novelty of descriptive findings would justify a study of only descriptive value .
measures of sampling error / precision should be included in results and considered in the interpretation of study findings if making analytical inferences ( o'brien et  al . , 2009 , boadella et  al . , 2011).3)sampling strategies , in an attempt to minimise the potential influence of selection bias ( nusser et  al . , 2008 , anderson , 2001 , boadella et  al . ,
proportionate sampling is ideal , though often not realistically achievable in observational studies of wildlife.4)performing studies that involve temporal sequence on the same individuals ( cohort studies ) , to compare morbidity and mortality rates of t.  gondii infected vs non infected animals in free - ranging populations .
studies comparing findings in both stable populations and populations subject to stressors ( such as habitat fragmentation ) would be valuable in investigating the possibility of interaction between stressors and the impact of t.  gondii infection on population health.5)controlling for confounding variables when making statistical extrapolations ( for example , controlling for sex and age if using a poisson regression to compare survivability of t.  gondii infected and uninfected hosts ) . in most marsupial species , further research aimed at identifying and validating measures of potential confounders - particularly age
- is required.6)potential influences of data clustering , which is potentially common in wildlife studies but rarely considered when making analytical inferences .
validation of diagnostic test(s ) used to detect the presence or absence of t.  gondii infection in the species under study , to enable consideration of potential impacts of misclassification on analytical study findings ( boadella et  al . , 2011 ) .
validation of tests in various marsupial species would also help to retrospectively clarify previous research findings .
the impact of sample sizes on results - ideally planning and undertaking studies where an adequate sample size for statistical extrapolations to a population level is realistically achievable , or where the novelty of descriptive findings would justify a study of only descriptive value .
measures of sampling error / precision should be included in results and considered in the interpretation of study findings if making analytical inferences ( o'brien et  al . , 2009 , boadella et  al . , 2011 ) .
sampling strategies , in an attempt to minimise the potential influence of selection bias ( nusser et  al . , 2008 , anderson , 2001 , boadella et  al . ,
proportionate sampling is ideal , though often not realistically achievable in observational studies of wildlife .
performing studies that involve temporal sequence on the same individuals ( cohort studies ) , to compare morbidity and mortality rates of t.  gondii infected vs non infected animals in free - ranging populations .
studies comparing findings in both stable populations and populations subject to stressors ( such as habitat fragmentation ) would be valuable in investigating the possibility of interaction between stressors and the impact of t.  gondii infection on population health .
controlling for confounding variables when making statistical extrapolations ( for example , controlling for sex and age if using a poisson regression to compare survivability of t.  gondii infected and uninfected hosts ) . in most marsupial species , further research aimed at identifying and validating measures of potential confounders -
potential influences of data clustering , which is potentially common in wildlife studies but rarely considered when making analytical inferences .
 it has often been asserted that australian marsupial species are particularly susceptible to toxoplasma gondii infection and to clinical toxoplasmosis following infection . 
 this implicates t.  gondii as a potential threat to marsupial population viability , and contrasts to what is known of t.  gondii in populations of several other host species . 
 we reviewed the literature , and found a lack of scientifically robust evidence addressing the occurrence of t.  gondii infection in free - ranging populations of australian marsupial species , and the impacts of the infection on population health . 
 key limitations included a lack of studies in free - ranging marsupial populations , study findings susceptible to substantial chance influences , and selection , misclassification and confounding biases . 
 the lack of scientifically robust data available on this topic indicates that assertions that free - ranging populations of australian marsupials are particularly susceptible to t.  gondii infection and to toxoplasmosis are premature . 
 the threat of t. gondii to the viability of free - ranging marsupial populations should therefore be regarded , at this stage , as a hypothesis . 
Introduction
The frequency of 
Frequency of disease (toxoplasmosis) following infection with 
Other possible effects of 
Conclusions