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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. ([email protected]; 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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