Serology-based diagnostics for the control of bovine neosporosis

Stefano Guido1, Frank Katzer1, Ian Nanjiani3, Elspeth Milne2, Elisabeth A. Innes1

1 Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian, EH26 0PZ, UK

2 The Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Roslin, Midlothian, EH25 9RG, UK

3 Westpoint Veterinary Group, Dawes Farm, Bognor Road, Warnham, West Sussex, RH12 3SH, UK

Corresponding author: Guido, S. (stefano.guido@moredun.ac.uk; Tel: +44 131 445 5111; Fax: +44 131 445 5111).

Key words: Neospora; neosporosis; diagnostics; cattle; control.

Abstract

The protozoan Neospora caninum is a primary infectious cause of abortion in cattle that

causes significant economic losses worldwide. As effective vaccines and licensed

pharmacological treatments are currently unavailable, control measures rely on biosecurity

and management practice. Serological diagnosis plays a crucial role in the identification of

infected animals and a number of tests have been developed. However, due to the particular

dynamics of the host-parasite interaction and to the characteristics of the currently used

diagnostic tools, a proportion of infected cattle may not be reliably identified and can

potentially undermine efforts towards the control of bovine neosporosis. Here, current

diagnostic methods for N. caninum infection in cattle and the advancements required to

support effective control strategies are discussed.

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Neospora caninum in cattle: why be concerned?

Neospora caninum is a cyst-forming protozoan parasite of the phylum Apicomplexa. It is

regarded as a major reproductive pathogen in cattle causing abortion and perinatal mortality

[1]. Due to its worldwide distribution and efficient transmission, N. caninum impacts the

cattle industry globally resulting in significant economic losses and production inefficiency

[2].

The parasite is characterised by a heteroxenous life-cycle in which dogs and related canids

are definitive hosts and cattle as well as a range of other species can act as intermediate hosts

(Figure 1). Since N. caninum emerged as a major threat to dairy and to a lesser extent beef

herds [2] nearly three decades ago, significant research efforts have been invested in the

development of control measures. At present, vaccination appears the most desirable option

[3] for controlling the disease in herds with high prevalence of infection [4]. However,

despite different experimental vaccination strategies [reviewed by 3] and some promising

results [5, 6] there are currently no commercially available vaccines to help prevent the

disease [7]. Less attractive but also experimentally investigated is the chemotherapeutic

option. Although several compounds showed inhibitory effects against N. caninum both in

vitro and in vivo [8-12], presently there are no licensed therapies for bovine neosporosis. As a

result, current measures aimed at reducing the impact of the disease are restricted to

biosecurity and management practices [13].

Discrimination between infected and uninfected animals is the basis of disease management

and a number of diagnostic tools have been developed for this purpose [reviewed by 14]. This

review discusses current methods used in the diagnosis of N. caninum in live cattle and how

they are currently applied to support disease prevention and control measures.

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In vivo diagnosis of bovine neosporosis

In live cattle, N. caninum infection is primarily diagnosed by serology, namely the detection

of specific antibodies in serum, plasma or milk [15]. Alternative techniques, such as the

detection of parasite DNA by PCR in blood or semen [16, 17] and the assessment of pro-

inflammatory cytokines as markers of exposure to the parasite [18], could also be applied,

however being transitory indicators of infection their use is confined to the research field.

Numerous serological techniques have been developed for the determination of N. caninum

serological status in cattle (Table 1).[19] Routinely, ELISAs (Enzyme-linked Immunosorbent

Assay) represent the technique of choice for high throughput screening hence they are

commonly used at the herd level to support the control of bovine neosporosis. Evaluating and

comparing the performances of diagnostic tests for N. caninum infection is problematic since

there is no true gold standard assay. In a recent comparative study, the performances of ten

commercial ELISAs [15] were assessed and compared based on two definitions of gold

standard: (1) majority of tests (i.e. samples that were classified as positive or negative by the

majority of the tests evaluated were considered as reference positive or negative samples), (2)

pre-test information (i.e. epidemiological, clinical and previous serological data using

experimental ELISAs). Good test agreement and shared high performances in terms of

specificity and sensitivity were observed, leading to the conclusion that serological diagnosis

of N. caninum infection is accurate in measuring tachyzoite specific antibodies in cattle [15].

The lack of a perfect reference assay has been addressed by applying non gold-standard

Bayesian modelling using field data. The performances of two commercial antibody ELISAs

were evaluated within two different scenarios: diagnosis in aborting cows and testing of

purchased animals. The tests showed comparable high accuracy and were classified as “fit for

purpose” when applied to the designated purpose [20]. All currently commercially available

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ELISAs are based on tachyzoite antigens. However, would these tools also work to identify

persistently infected animals when the parasite is relatively quiescent within tissue cysts? In

the author’s opinion this point represent one the main criticalities of current commercial

serological assays. Although the problem was addressed by complementing diagnostic

ELISAs based on tachyzoite antigens with assays based on bradyzoite-specific antigens [21]

(Table 1), none of these tests is currently marketed.

True or false seronegative cattle?

Reports of N. caninum serologically negative dams giving birth to seropositive calves [22-24]

or aborting foetuses in which parasite DNA was detected [23] as well as post-mortem

evidence of N. caninum infection found in tissues of seronegative non-aborting cows [25]

expose a relevant issue that can be encountered when approaching the diagnosis of bovine

neosporosis. The presence of these serologically elusive animals may be attributable to the

variations in tachyzoite-specific antibody titres observed as a result of the changing dynamics

of the host-parasite interaction and the characteristics of the diagnostic tests used.

Detectable specific antibody responses after N. caninum exposure are represented by the

early onset of IgM antibodies which peak at two weeks before declining at four weeks post-

infection followed by the appearance of IgG antibodies. Specific IgG concentrations rise for 3

to 6 months and are believed to persist for life in infected animals [14]. However, fluctuations

occur depending on the physiological status of the dam [26-28] and the activity of the

parasite [29]. Marked rises in antibody titres are observed during the second half of gestation

and are associated with vertical transmission in experimental and field studies [26, 30, 31]. In

persistently infected individuals this may reflect recrudescence of a persistent infection [32]

in which bradyzoites, residing within tissue cysts, reconvert into the more active tachyzoites

that spread throughout the body boosting the host immune responses. Conversely, in some

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cases tachyzoite-specific antibody titres may drop below the detection limits of serological

assays [25, 33] so that previously serologically positive animals may become seronegative

(Figure 1, Key Figure). This may occur in both aborting and non-aborting persistently

infected cows at any stage during gestation [34]. A recent prospective serological study in

dairy cattle found that more than one third of cows, with low antibody titres (1:200) during

pregnancy, became serologically negative at the end of gestation [22]. In addition, two studies

conducted in Argentina reported that 5% [35] and 3% [36] of dams that were seronegative at

calving gave birth to pre-colostrally seropositive calves. Besides reporting fluctuations in the

antibody titres, these findings highlight the limitations of some serological techniques.

Most if not all the work reporting fluctuations in specific N. caninum antibody concentrations

in cattle refers to those humoral responses recognising the tachyzoite stage of the parasite

since they are targeted by the vast majority of commercial and in house diagnostic tests.

Concerns about the sensitivity of tests based only on tachyzoite antigens were raised

following an interesting observation of a cow whose antibody response was able to recognise

bradyzoite-specific and not tachyzoite-specific antigens [25]. This animal was kept under

experimental conditions as a negative control for a N. caninum infection experiment and

tested repeatedly negative with commercial and experimental tachyzoite-based ELISAs;

however, parasite DNA was detected in several tissues post-mortem and further serological

analysis carried out with a bradyzoite-specific (SAG4) antigen-based ELISA highlighted

seropositivity to antigens related to the quiescent bradyzoite stage of N.caninum.

In experimentally infected animals, humoral responses to N. caninum bradyzoite-specific

antigens show individual variability [21]. Indeed, antibody responses against bradyzoite

antigens will depend on the intensity and duration of specific antigen exposure during the

host-parasite interaction in cattle. In addition, rupture of tissue cysts may also be required to

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enhance detectable host immune responses against bradyzoite antigens. As suggested for

some bradyzoite-specific antigens of the closely related apicomplexan Toxoplasma gondii,

antigens specific to the quiescent stage may hardly be exposed [37]. There is no conclusive

information on the extent these antigens are actually exposed to the host immune system as

this may be difficult to assess and subject to individual variability in immunocompetence.

The limited immunogenicity observed for the N. caninum bradyzoite-expressed BRS4 and

SRS9 antigens was ascribed to a possible late up-regulation of expression during persistent

infection with only a transient antigenic exposure to the host immune system [38]. What

emerges is the need for more reliable diagnostic tests perhaps using a combination of antigens

relevant for different stages of the disease that could enable the reliable identification of

serologically elusive animals [34].

An alternative explanation for those animals that, despite harbouring N. caninum infection do

not show any detectable immunological response, is the occurrence of acquired or innate

immunotolerance i.e. unresponsiveness of the host immune system to certain pathogen-

specific antigens [23, 39, 40]. This is supported by the fact that not all N. caninum infected

calves born from seropositive dams are pre-colostrally seropositive indicating that congenital

infection may occur without seroconversion [39]. This may be due either to infection of the

foetus prior to reaching immunocompetence with the establishment of immunotolerance or to

infections at a very late stage during pregnancy where the foetus has not had time to develop

a serological response. Immunotolerance is well documented following in utero infections

with ruminant pestiviruses that may result in seronegative yet infected offspring [41];

however, its occurrence during N. caninum infection has not yet been clarified. With regards

to diagnosis of bovine neosporosis for disease control purposes, immunotolerant animals

would be a potential source of infection that would currently be difficult to identify.

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Overall, the frequency, hence the epidemiological impact of serologically elusive animals, is

difficult to assess due to their intrinsic evasiveness to current diagnostic tests. Nevertheless,

the possibility of false negative results should be considered when undertaking serological

testing for bovine neosporosis.

Individual and herd testing

In cattle, the serological status with regards to N. caninum may be assessed in individual

animals through serum, plasma or milk sampling or in groups of lactating cows by bulk milk

testing [42].

Individual serology is a good indicator of the relative risk of abortion. In several studies, the

risk of abortion observed in serologically positive cows was 2 to 26 times higher than in

serologically negative [43, 44]. High antibody titres were also correlated to increased risk of

abortion in parous cows [44] but not in heifers in which the lower antibody titres observed

compared with older cows could not be associated with an increased risk of abortion [45].

These findings may be explained by the higher chance of repeated exposure to the parasite,

either by secondary horizontal infection or reactivation over subsequent pregnancies, of older

cows compared to heifers in which primary infections with a lower antigenic stimulus is more

likely to occur [33].

The time of testing plays a role in the reliable identification of infected animals. In general,

animals should be tested with serology when older than six months of age as there is evidence

of colostral antibodies persisting for several months that may interfere with serological assays

[46]. Most importantly, as antibody fluctuates sampling should be undertaken when there is

the highest chance of detecting most of the infected animals which is likely to be during the

second half of gestation when antibody titres are higher [34] or following an abortion.

However, drawing conclusions about the effective serostatus of individual animals based on

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one testing period only may lead to the wrong conclusions given the potential occurrence of

false negative results, as previously discussed [47]. Therefore, repeat sampling over

subsequent pregnancies to confirm positive results is highly recommended.

Pre-colostral serology in newborn calves can be used to assess vertical transmission and

indirectly assess infection status of the dam [46]. In ruminants, transplacental transfer of

maternal immunoglobulins does not occur because of the syndesmochorial placenta. Passive

immunity is transferred from dam to calf after birth through colostrum. Consequently,

specific antibody responses in pre-colostral calves derive from the activity of the foetal

immune system following exposure to a pathogen in utero. In the majority of calves born

from N. caninum infected dams, the timing of in utero infection will determine the

development of specific antibodies [48]. The absence of specific antibodies in stillborn or

newborn pre-colostral calves would suggest that N. caninum infection is unlikely [14].

Effectively, a detectable pre-colostral antibody response may depend on the stage of

pregnancy hence the maturity of the foetal immune system at the time of infection [29].

Obtaining and testing pre-colostral sera from calves is not a practical diagnostic option,

although it is very informative for research purposes.

In dairy herds, the detection of N. caninum antibodies in bulk milk is a useful tool for

measuring the within herd seroprevalence [49, 50], namely an estimate of the seroprevalence

within the group of animals that contribute to the milk sample. Several studies showed good

correlation of bulk milk results with herd seroprevalence as assessed through testing of

individual serum or plasma samples [51-53]. However, specific considerations are required

for the interpretation of results. In milk, antibodies appear later and at lower concentration,

about 30 times less, than in serum [54]. In addition, besides the number of serologically

positive animals in a herd, bulk milk antibody concentrations are dependent on the stage of

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lactation and the milk yield [55]. Although several ELISA tests have been adapted for use on

bulk milk samples, sensitivity is reported to be limited. A minimum of 10 - 15% serologically

positive animals appears to be required to produce positive bulk milk testing results [51, 53,

56]. Therefore, bulk milk testing may underestimate the proportion of infected herds, and

misclassify those with low seroprevalence as negative. Nevertheless, ELISAs on bulk milk

are considered cost-effective non-invasive indicators of the herd status that can be applied for

control and surveillance purposes.

Application of serology-based diagnostics to the control of bovine neosporosis

At present, serological techniques represent a useful tool for approaching the control of N.

caninum infection in cattle that may be applied at different phases and for different purposes

during control programmes.

Initial assessment

In the preliminary phases of control programmes, serology is employed to assess whether N.

caninum is directly related to the abortion cases, which is the predominant route of infection

and what is the within-herd seroprevalence [13]. Collectively this information is required to

shape strategies and take action according to the specific situation encountered that may vary

substantially between farms.

In individual cases, positive post-abortion serology is highly suggestive of infection;

however, it is not sufficient to prove that the parasite caused the abortion. Persistently

infected cows may show detectable N. caninum antibody titres that are not necessarily related

to the recent abortion event [14]. In order to justify costs and efforts of a control programme

for bovine neosporosis, the infection-abortion relationship should be investigated. This can be

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approached by comparing the seropositivity rates in aborting and non-aborting cows at

calving. If the former group have a significantly higher seropositivity rate than the latter, a

relationship between parasite infection and occurrence of abortion can be confirmed [23, 57].

Serology may also help in the investigation of the predominant route of transmission (vertical

or horizontal) by testing serum samples from dams and their offspring and, where possible,

from pre-colostral calves [58]. In herds in which the transmission is predominantly vertical,

the distribution of seropositive animals is uniform across age groups with both dams and their

offspring having specific N. caninum antibodies. However, if the mode of transmission is

mainly horizontal there is no association between the serological status of dams and

offspring; serologically positive animals are in age clusters and may have either seronegative

dams or seronegative offspring [59].

N. caninum abortions may follow an epidemic pattern characterised by abortion storms

defined as the abortion of more than 10% of the cattle at risk (i.e. pregnant) within a period of

12 weeks or an endemic pattern in which the abortion problem persists for several months or

years within a herd [60]. Epidemic abortions are believed to be due to a primary horizontal

infection of a group of naive animals whereas endemic abortions occur as a result of recurrent

transplacental transmission within family lines [61]. The abortion pattern can be investigated

by estimating the odds ratio: a parameter that expresses the risk of abortion in the population

at risk [62]. Endemic patterns of abortion are associated with odds ratios of around 2,

whereas in cases of epidemic abortions higher odds ratios are found. Associating information

about the abortion pattern with serology using avidity tests can provide further information

on the predominant transmission route. It is recommended to determine avidity values on

samples obtained immediately after abortion from a representative number (8 to 10) of

seropositive aborting cows. High avidity observed in the presence of an endemic pattern of

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abortion would suggest that the vertical route is the predominant route of infection whereas

low avidity antibodies associated with an epidemic pattern are indicative of recent exposure

by the horizontal route [62-64].

Once the infection-abortion relationship and the predominant route of infection are

established, the measurement of the within-herd seroprevalence and an economic analysis to

estimate the losses attributable to bovine neosporosis are required in order to determine the

best course of action.

Applying management-based control options

A number of strategies for the reduction of reproductive losses due to bovine neosporosis

have been proposed based on knowledge of the parasite life cycle and epidemiology. These

control strategies are currently restricted to management techniques aimed at minimising the

risk of post-natal horizontal infection from dogs to cattle, thus the risk of exogenous vertical

transmission, and preventing endogenous vertical transmission [reviewed by 13].

Exogenous infection from infected canids can be reduced by preventing oocyst contamination

of feedstuff, water, pastures and cattle areas by controlling the access of dogs and wild

canids. Dogs should be prevented from becoming infected by limiting their access to

potentially infected foetal material and placentas [65] as well as by avoiding raw ruminant

tissues as dog food since they may contain N. caninum tissue cysts [66]. .

Control measures aimed at limiting endogenous transmission include the removal of

serologically positive cows from the herd (test-and-cull regime) or the exclusion of breeding

of heifers born from seropositive dams (selective breeding). These are based on the evidence

that serologically positive animals are at an increased risk of abortion compared to those

animals that are serologically negative; therefore the reduction of the within-herd

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seroprevalence would limit the occurrence of abortions. The culling of N. caninum

seropositive dams was shown to reduce the within-herd prevalence over time [67][70, 73,

74]. However, due to the high costs involved, this measure may be economically sustainable

only in herds with low seroprevalence of the disease in which a small proportion of animals

would have to be removed. Decreased rates of infection were also observed in herds in which

heifer calves from seropositive dams were not retained as replacements [67]. The reliable

identification of all N. caninum infected animals is a pre-requisite for a control programme

based either on the removal, or the exclusion from breeding, of infected animals. This

requires serological tests in which the cut-off thresholds are adjusted in order to provide the

maximum level of diagnostic sensitivity [68]. However, as previously discussed, the

possibility of false negative results should be taken into account.

Embryo transfer (ET) from a seropositive dam to a seronegative recipient has been suggested

as an alternative reproductive measure for preventing vertical transmission of N. caninum;

however its application is restricted to cattle of high genetic merit in which the value of the

future calf would justify the high costs [69]. Although not preventing the endogenous

transmission of N. caninum to the foetus, the use of beef bull semen to inseminate N.

caninum seropositive cows was shown to reduce the risk of abortion; this might be due to the

favourable effect of cross-breeding on foetal health and placental function [70]. In addition,

this technique ensures that breeding replacements from infected cattle are removed as female

dairy x beef crosses are not normally retained for milk production.

A non-interventionist (“live with the disease”) option has also been postulated. Economical

modelling showed that in some herds with a seroprevalence below 18-21% the costs related

to the identification of infected animals may exceed the benefits of controlling the disease

[71]. In this situation, implementing suitable biosecurity measures aimed at managing the

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host-pathogen interaction can yield high returns and be beneficial for the control of other

infectious diseases of cattle.

Monitoring herd prevalence and avoiding reintroduction

Once control options are implemented, monitoring and maintaining the achievement of the

improved status is advisable as the risk of reintroducing the disease cannot be eliminated

completely. Periodically testing a representative number of animals with individual serology

or monitoring bulk milk for the presence of N. caninum-specific antibodies in dairy herds can

be applied in both herds that are free from N. caninum to test the conservation of the free

status and herds that are progressing towards the reduction of the seroprevalence.

As replacement cows and heifers represent a risk of disease reintroduction, replacement stock

should be purchased from N. caninum-free herds with outstanding reproductive performances

and serologically tested at the farm of origin. In general, cut-offs of the diagnostic tests

employed should be adjusted in order to favour high sensitivity [20]. Because of the

fluctuations in antibody titres in infected animals with the possibility of false negative results

occurring, repeating testing may be advisable. Some authors recommend repeating sampling

after a period of 4-6 weeks following introduction and re-testing doubtful samples using

Western-Blotting [72, 73]. Although not always practical, requiring testing of the mothers

whose daughters are prospective purchases may help in identifying false negative individuals

and systematically avoiding animals from family lines in which N. caninum is likely to be

transmitted through the vertical route.

Concluding remarks

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Effective and economically sustainable control measures for bovine neosporosis are urgently

required as the disease continues to threaten animal welfare and efficient productivity in

cattle operations worldwide. Advances are anticipated from the development and

commercialisation of effective vaccination or antiprotozoal therapeutic options; however,

these solutions are not currently available and may be absent from the global market for

several years to come.

At present, mitigating the impact of bovine neosporosis can be achieved by implementing

practical management techniques with the overall goal of reducing the prevalence of the

infection in breeding herds (Box 1) . The diagnosis of N. caninum infection in live animals is

a pre-requisite for disease management and currently available serological techniques can be

applied to control programmes; however, they present some limitations for which

improvement may be desirable.

A major challenge is the identification of those animals that despite being infected with the

parasite test serologically negative in tachyzoite antigens-based assays. While the numbers of

these animals are unknown they pose a risk for biosecurity management of the disease in

particular when conducting tests to enable purchase of N. caninum negative animals to bring

onto the farm.

Serologically elusive animals may have a persistent infection in which antibody titres against

the tachyzoite stage may have dropped below the detectable levels of the diagnostic tests. As

currently employed serological tests are based on tachyzoite antigens, they target mainly

those humoral responses that are mounted following acute infection or recrudescence and

might miss those antibody responses that may be induced during persistent infection.

Although some bradyzoite stage-specific antigens have been employed for serological

analysis in cattle, a more complete analysis of antibody responses against N. caninum during

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persistent infection is required. The identification and characterisation of novel stage-specific

antigens, capitalising on the most recent resources coming from the N. caninum genome

annotation, may provide additional antigens for use in serological assays (Outstanding

questions box). These new tests may help determine whether antibody responses during

persistent infection can be more reliably detected and such tests may be used to identify N.

caninum positive animals that currently test negative to tachyzoite-based serological tests.

Within the wider context of integrated high health schemes, applying a wider range of N.

caninum antigens in the diagnostic tests used may enhance the effectiveness of management-

based control programmes providing both animal welfare and economic benefits to cattle

producers.

Figure 1 – Heteroxenous life-cycle of Neospora caninum

(A) In the intermediate host infection may occur horizontally through ingestion of food or

water contaminated with sporozoite-containing oocysts previously shed in the faeces of

acutely infected canids; or (B) vertically from dam to foetus through the placenta [61].

Highly efficient [36], trans-placental transmission may be exogenous when infective oocysts

are ingested during pregnancy; or endogenous due to recrudescence of a persistent infection

[74]. N. caninum is able to invade a number of nucleated cell types where multiplies by a

process called endodyogeny (i.e. a mechanism of asexual reproduction in which two progeny

cells are assembled within the mother cell). Heavily infected cells then rupture, releasing N.

caninum tachyzoites, the rapidly multiplying stage, which disseminate and infect other cells.

(C) Following a phase of rapid proliferation, N. caninum differentiates into the quiescent

bradyzoite stage that resides within intracellular tissue cysts establishing life-long infections

[75]. (D) During pregnancy, modifications of the dam’s immune responses encourage

reactivation of the parasite and reconversion of bradyzoites into tachyzoites which may infect

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and cross the placenta resulting in foetal infection [29]. Recrudescence of a persistent

infection with vertical transmission may occur over consecutive pregnancies and may result

in abortion or in the birth of healthy but congenitally infected calves and if these are heifers

they may vertically transmit the parasite to their progeny [76].

(F) The life cycle is completed when bradyzoite-containing tissue cysts, that are present the

intermediate host tissues, are ingested by a definitive carnivore host.

Figure 2, Key figure – Hypothetical antibody responses following N. caninum infection in

cattle

(A) Ingestion of sporulated oocysts with release of sporozoites may expose the host im-

mune system to sporozoite-specific antigens; there is little information about sporozo-

ite-specific antibody responses during early stages following oocyst infection. Once

sporozoites infect cells they convert to tachyzoites.

(B) Tachyzoite multiply rapidly by endodyogeny inside a number of cell types that then

rupture triggering the development of tachyzoite-specific antibody responses. These

antibody responses are found in most infected animals and are detected by current

diagnostic tools that are based on tachyzoite antigens.

(C) Tachyzoite convert into the quiescent bradyzoite/tissue cyst stage, bradyzoite-specific

antibodies are produced; however, the quantity and duration of these responses are

unknown.

(D)During conversion into the bradyzoite/tissue cyst stage, tachyzoite-specific antibody

responses may decline below the cut-offs of current diagnostic tests. This may result

in a proportion of false negative results.

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(E) Immunomodulation during pregnancy may enable reactivation of bradyzoite into ta-

chyzoites that spread throughout the body thus boosting the tachyzoite specific im-

mune responses.

(F) Bradyzoite/tissue cysts specific antibodies may decline at this stage although there is

little information about the dynamics of these stage-specific humoral responses as dia-

gnostic tests targeting bradyzoite-specific humoral responses are not currently used.

Acknowledgements

The authors wish to acknowledge Prof. Luis M. Ortega-Mora, Dr Thomas Dijkstra, Dr

Caroline Frey and Dr Monica Mazuz for having provided useful information about the

specific measures for the control of bovine neosporosis in Spain, The Netherlands,

Switzerland and Israel. The present review was funded by AHDB Beef and Lamb division of

the Agriculture and Horticulture Development Board (AHDB) and RESAS, Scottish

Government.

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