SHORT COMMUNICATION

Annals of Agricultural and Environmental Medicine 2015, Vol 22, No 4, 677679

www.aaem.pl

Risk factors involved in transmission of

Toxoplasma gondii and Neospora caninum
infection in rabbit farms in Northern Italy
Tereza Machacova1, Eva Bartova1, Kamil Sedlak2, Marie Budikova3, Alessandra Piccirillo4
Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and
Pharmaceutical Sciences, Brno, Czech Republic
2
Department of Virology and Serology, State Veterinary Institute Prague, Czech Republic
3
Department of Mathematics and Statistics, Faculty of Science, Masaryk University, Brno, Czech Republic
4
Department of Comparative Biomedicine and Food Science, University of Padua, Legnaro (Padua), Italy
1

Machacova T, Bartova E, Sedlak K, Budikova M, Piccirillo A.Risk factors involved in transmission of Toxoplasma gondii and Neospora caninum
infection in rabbit farms in Northern Italy. Ann Agric Environ Med. 2015; 22(4): 677679. doi: 10.5604/12321966.1185774

Abstract

Introduction and objective. In Italy, rabbits are frequently reared for meat production. The aim of the study was to find
the seroprevalence of T.gondii and N.caninum parasites, and risk factors of infection in rabbit farms.
Material and methods. Blood samples from 260 apparently healthy breeding rabbits were collected on 13 commercial
farms in Northern Italy. Rabbits were divided into categories according to age, number of births, breed, province and size
of farm. Samples were tested for antibodies to T.gondii and N.caninum using the indirect fluorescence antibody test (IFAT);
samples with a titre 50 were considered positive.
Results. Antibodies to T.gondii and N.caninum were found in 38 (14.6 %) and 3 (1.2 %) rabbits, respectively. A statistically
significant difference (p-value 0.05) was found only in T.gondii prevalence among different rabbit breeds and provinces.
Conclusion. Rabbits from Northern Italy are at risk of T.gondii and N.caninum infection; however, it is lower compared to
seroprevalence noted in other animal species or in humans.

Key words

Toxoplasmosis, neosporosis, rabbits, serological survey, risk factors

Toxoplasmosis is a disease that in sensitive hosts causes

different clinical symptoms, affects their reproduction and
can lead to death [1]. In livestock, it is also connected with
economic losses because sheep, goats and rabbits belong to
the animal species very susceptible to Toxoplasma gondii
infection. In Europe, there are many serological studies
focusing on detection of T. gondii antibodies in rabbits;
however, to our knowledge there is no similar study from
Italy. The only study from Italy was undertaken by Zanet
etal. [2] who isolated the DNA of T.gondii from tissues of
2.1 % of 144 wild rabbits (Sylvilagus floridanus).
The consumption of raw or undercooked meat originated
from meat-producing animals, including rabbits, is a very
important source of T. gondii infection in humans. Since
toxoplasmosis is a zoonosis [3], and rabbits are frequently used
for meat consumption in Italy, it is important to know the risk
factors of T.gondii infection from consumption of rabbit meat
for humans, and the important factors for the transmission
of T.gondii among rabbits. The rabbits could be infected with
T.gondii by the ingestion of food or water contaminated with
T.gondii oocysts from cat faeces [3], or by transmission of
T.gondii to offspring through transplacental infection [4].
Rabbits are usually without clinical symptoms, therefore
detection of antibodies is very important in epizootiological

INTRODUCTION AND OBJECTIVES

Address for correspondence: Eva Brtov, Department of Biology and Wildlife

Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and
Pharmaceutical Sciences, Palackho t. 1/3, 612 42 Brno, Czech Republic
E-mail: bartovae@vfu.cz

Received: 06 November 2014; accepted: 13 February 2015

studies. Rabbits could be infected with T. gondii and

simultaneously with similar parasite N.caninum. This is why
the aim of the presented study was to detect both T.gondii
and N.caninum antibodies in rabbits, and evaluate the risk
factors of infection on rabbit farms.
MATERIALS AND METHODS
During 2009, blood samples were collected from the
auricular vein of 260 apparently healthy breeding rabbits
on 13 commercial rabbit farms in Northern Italy (Veneto
Region). All farms were industrial-cycle type with breeding
and growing units located in the same house, with the
number of breeding and growing does ranging between
3003,000 and between 2,00014,000, respectively. Breeding
does were individually housed in wire cages equipped with
a nest box. Rabbits were fed ad libitum and drinking water
was administered through nipple watering systems. Artificial
insemination by using external semen was practiced on all
farms.
All sampled doe rabbit showed no signs of clinical
symptoms. Data, including age, breed, number of births and
health conditions, were obtained through questionnaires at
the farms. The rabbits were divided into five age categories:
25 months (n=59); 57 months (n=66); 713 months
(n=74); 1330 months (n=26); and rabbits with unknown
age (n=35); five categories according to number of births:
0 birth (n=60); 12 births (n=71); 37 births (n=57); 815
births (n=37); and rabbits with an unknown number of
births (n=35). Four commercial hybrid lines: W (n=40),

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Tereza Machacova, Eva Bartova, Kamil Sedlak, Marie Budikova, Alessandra Piccirillo. Risk factors involved in transmission of Toxoplasma gondii and Neospora caninum

X(n=160), Y (n=40), and Z (crossbred of New Zealand White

x Californian, n=20), marked similarly as in the study of
Lonardi etal. [5], were included in the current study. Doe
rabbits were also divided according to the provinces in
which they were bred: Padova (n=20), Rovigo (n=20), Treviso
(n=160) and Verona (n=60); and the size of the farms: small
farms (<1,000 does, n=120) and large farms ( 1,000 does,
n=140). Data are summarized in Table 1.
Each blood sample was centrifuged and removed serum
was frozen at -20oC until testing. Antibodies to T. gondii
and N. caninum were detected by indirect fluorescence
antibody test (IFAT) using a commercially available T.gondii
antigen and N.caninum antigen (VMRD, Pullman, USA),
respectively, and anti-rabbit IgG FITC conjugate (Sigma
Aldrich, Czech Republic). Positive and negative control sera
from an experimental study of domestic rabbits with T.gondii
oocysts [6] were included in the tests. Sera were diluted with
PBS two-fold, starting at 1:50. In the present study, cut-off
titre 50 was used for both parasites, which is higher than in
experimental studies (cut-off titre 20), to minimize the risk
of false positive samples due to cross-reactivity.
Table 1. Characteristics of rabbits and results of Toxoplasma gondii and
Neospora caninum seroprevalence (IFAT)
Characteristics
Gender
Female (does)
Age categories (months)

No. of rabbits
tested

260

T. gondii
Positive (%)

N. caninum
Positive (%)

38 (14.6 %)

3 (1.2 %)

25

59

4 (6.8 %)

0 (0 %)

57

66

8 (12.1 %)

2 (3 %)

713

74

10 (13.5 %)

0 (0 %)

1330

26

3 (11.5 %)

1 (3.8 %)

not known

35

13 (37 %)

0 (0 %)

No of births

60

4 (6.7 %)

0 (0 %)

12

71

6 (8.5 %)

2 (2.8 %)

37

57

9 (15.8 %)

0 (0 %)

815

37

6 (16.2 %)

1 (2.7 %)

many but not known

35

13 (37 %)

0 (0 %)

Breed

40

3 (7.5 %)

0 (0 %)

160

23 (14.4 %)

3 (1.9 %)

40

12 (30 %)

0 (0 %)

20

0 (0 %)

0 (0 %)

Padova

20

0 (0 %)

0 (0 %)

Rovigo

20

3 (15 %)

0 (0 %)

Treviso

160

11 (6.9 %)

3 (1.9 %)

Verona

60

24 (40 %)

0 (0 %)

Provincie

Size of farm

Small farms (< 1000 does)

120

19 (15.8 %)

1 (0.8 %)

Large farms ( 1000 does)

140

19 (13.6 %)

2 (1.4 %)

In case of T. gondii, the results of chi-square test proved (with the risk of error not more than
5%) dependence on breed (2=12.64, df=3, p-value=0.005) and on provinces (2=42.08, df=3,
p-value < 0.05). Multiple comparison method based on arcsine transformation proved that there
is difference in prevalence between breeds X and Z and between Y and Z; and also between
two provinces Padova and Rovigo, two provinces Padova and Verona and two provinces
Treviso and Verona. In case of N. caninum, statistical analysis could not be done because of
small number of positive samples.

Seroprevalence was statistically analysed, considering

the variables of age, number of births, breeds, province
and size of farms. Data analysis was performed by ChiSquare test for independence resp. Fisher exact test for
Tables 22 using STATISTICA Cz 12 [7]. Dependence on
age and seroprevalence was evaluated by Wilcoxon test. Null
hypothesis was tested to ascertain if T.gondii seroprevalence
depended on age, number of births, breeds, province and
size of farms. Multiple comparison method based on arcsine
transformation was used to compare T. gondii prevalence
between breeds and provinces. The differences were
considered statistically significant when p-value was 0.05.
RESULTS AND DISCUSSION
In the presented study, antibodies to T.gondii and N.caninum
were found in 38 (14.6%) and 3 (1.2%) doe breeding rabbits,
respectively. No co-infection between T. gondii and
N.caninum was found. Titres of T.gondii antibodies were
at a low level (50100). To the best of the authors knowledge,
this is the first serological study focused on T.gondii and
N.caninum seroprevalence in rabbits from Northern Italy,
and the first detection of N.caninum antibodies in domestic
rabbits in Europe. It could be concluded that the does from
Northern Italy are at risk of T. gondii and N. caninum
infection; however the seroprevalence is lower compared
to the seroprevalences noted in other animal species, or in
humans.
Similar T.gondii prevalence was obtained by the different
methods used. Antibodies to T. gondii were detected, e.g.
by indirect haemagglutination test in 23.4% (18/77) rabbits
from China [8], by Enzyme-Linked Immuno Sorbent
Assay (ELISA) in 15.5 % (103/1883) rabbits from the Czech
Republic [9] and in 11 % (22/194) rabbits from Egypt [10], or
by Modified Agglutination Test (MAT) in 16.3 % (70/429)
rabbits from Mexico [11].
The results of the present study, based on different risk
factors (age, number of births, breeds, provinces and size
of farm), are summarized in Table 1. Lower T.gondii and
N. caninum prevalence (6.8% and 0%, respectively) were
found in rabbits until 5 months of age, while in older rabbits,
prevalence increased to 13.5 % and 3.8 %, respectively (p-value
> 0.05). These results are in contrast to a study by AlvaradoEsquivel etal. [11] from Mexico; they detected higher T.gondii
seroprevalence 41.9% in young rabbits (age category: 0.31
month) compared to older ones. Similar to Alvarado-Esquivel
etal. [11], high T.gondii seropositivity of young domestic
rabbits was confirmed by Uhlikov and Hbner [4]; they also
discussed the possibility of transplacental transmission of
T.gondii in rabbits. The seroprevalence of T.gondii increased
with the number of births, without statistical significant
difference (p-value=0.4141) however. This increasing trend
of prevalence may be associated with post-natal infection
through ingestion of food or water contaminated with
T.gondii oocysts. The highest prevalence was found in hybrid
line Y (30 % for T.gondii) and X (14.4 % and 1.9% for T.gondii
and N.caninum, respectively), while Z hybrids were negative
for both T.gondii and N.caninum antibodies. The difference
in T.gondii prevalence in breeds was statistically significant
in some of breeds (test statistics=12.64, df=3; p-value=0.005),
which is in contrast with the results of Alvarado-Esquivel
etal. [11], who did not find statistical differences in breeds. The

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Annals of Agricultural and Environmental Medicine 2015, Vol 22, No 4

Tereza Machacova, Eva Bartova, Kamil Sedlak, Marie Budikova, Alessandra Piccirillo. Risk factors involved in transmission of Toxoplasma gondii and Neospora caninum

rabbits came from four provinces; the highest seroprevalence

of T. gondii (40%) and N. caninum (1.9%) antibodies was
found in Verona and Treviso provinces, respectively. In
the case of T.gondii, the results were statistically different
(test statistics=42.08, df=3; p-value < 0.05) between some
of the provinces. Alvarado-Esquivel et al. [11] also found
a statistically significant different T. gondii prevalence in
rabbits from six municipalities in Mexico. This fact was
explained by different climatic conditions. However, in the
present study, there were no strong differences in climatic
conditions of the six provinces, and the samples were
collected on 13 farms. The highest T.gondii seroprevalence
(55%) was found in rabbits from one large farm in Verona
province; the highest prevalence of N.caninum antibodies
(5%) was found on one small and two large farms in Treviso
province. Nevertheless, there was no statistically significant
difference (test statistics=0.26 resp. 0.20, df=1; p-value > 0.05)
between either T.gondii and N.caninum seroprevalence and
size of farms.
Two of 38 (5%) rabbit does positive for T.gondii antibodies
had been treated for respiratory or enteric disorders, which
could be connected with symptoms of T.gondii infection. No
signs of disease were recorded in the other does.
CONCLUSION

It is worth noting that rabbit does from Northern Italy are

at risk of T.gondii and N.caninum infection which could be
transmitted to their offspring that are mainly used for human
consumption. That is why it is very important to implement
good hygienic conditions on farms where the rabbits are
bred. There was no data available about the presence of
cats and dogs on the rabbit farms to study this risk factor.
However, avoidance of the presence of cats and dogs on
the farms is highly recommended to minimize the risk of
contamination of the environment, food and water with
T.gondii and N.caninum oocysts, respectively.

Acknowledgements
The study was funded by the Grant IZSME 05/10 RC
C71J1000012000 from Italy, and Grant IMA 2014-FVHE17 from the Czech Republic.
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