Accepted Manuscript Title: Seroprevalence and risk factors associated with Babesia caballi and Theileria equi infections in donkeys from Southern Italy Author: D. Piantedosi, N. D'Alessio, A. Di Loria, F. Di Prisco, U. Mariani, B. Neola, M. Santoro, S. Montagnaro, G. Capelli, V. Veneziano PII: DOI: Reference:
S1090-0233(14)00394-3 http://dx.doi.org/doi: 10.1016/j.tvjl.2014.09.025 YTVJL 4290
To appear in:
The Veterinary Journal
Accepted date:
25-9-2014
Please cite this article as: D. Piantedosi, N. D'Alessio, A. Di Loria, F. Di Prisco, U. Mariani, B. Neola, M. Santoro, S. Montagnaro, G. Capelli, V. Veneziano, Seroprevalence and risk factors associated with Babesia caballi and Theileria equi infections in donkeys from Southern Italy, The Veterinary Journal (2014), http://dx.doi.org/doi: 10.1016/j.tvjl.2014.09.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2 3
Seroprevalence and risk factors associated with Babesia caballi and Theileria equi infections in donkeys from Southern Italy
4 5 6
D. Piantedosia, N. D'Alessio b, A. Di Loria c,*, F. Di Prisco b, U. Mariani b, B. Neola a, M. Santoro d, S. Montagnaro a, G. Capelli e, V. Veneziano a
7 8 9
a
10
c
Department of Health Science, University of Magna Græcia, Catanzaro, Italy
11
d
Department of Public Health and Infectious Diseases, Sapienza University of Rome, Italy
12
e
Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (PD), Italy
Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Italy b Istituto Zooprofilattico Sperimentale del Mezzogiorno, Portici (NA), Italy
13
14 15
* Corresponding author: Tel.: +39 0961 3694284. E-mail address: [email protected] (A. Di Loria).
16
17
Highlights
18 19
This is the first study to estimate the occurrence of donkey piroplasmosis in the Campania region of Southern Italy.
Of 203 serum samples tested, 72 (35.5%) were found to be positive for B. caballi and 90 (44.3%) were positive for T. equi . The overall seroprevalence of EP was 57.1% (116/203) and a proportion of the donkeys were co-infected by both protozoa (46/203 - 22.6%).
Clinical examination of the most seropositive animals failed to show any evidence of abnormalities. Only one male donkey, with a titre of 1:640 against T. equi, showed the acute symptoms of disease.
Our findings showed that EP is widespread in donkeys living in this area of Southern Italy and therefore the establishment of an appropriate and effective control program is of great importance for these equids.
20 21 22 23 24 25 26 27 28 29 30 31 32 33
1 Page 1 of 24
34 35
Donkeys could be chronic carriers and a potential continuous source for maintaining and disseminating the piroplasms in the rural environment.
2 Page 2 of 24
36
Abstract
37
Equine piroplasmosis (EP) has been frequently described in donkeys in subtropical and
38
tropical regions, but published data reflecting large scale surveys are very limited in Europe. The
39
seroprevalence of Babesia caballi and Theileria equi was determined in a donkey population from
40
Campania Region in Southern Italy using a commercial indirect fluorescent antibody test (IFAT),
41
and the risk factors associated with the occurrence of the infection were assessed.
42
43
Of 203 samples, the overall seroprevalence for EP was 57.1% (116/203), with 35.5%
44
(72/203) for B. caballi and 44.3% (90/203) for T. equi. Co-infection was detected in 46 donkeys
45
(22.6%). The distribution of IFAT antibody titres to B. caballi was: 1:80 (n = 67), 1:160 (n = 2), 1:320
46
(n = 3); while, the distribution of IFAT antibody titres to T. equi was: 1:80 (n = 25), 1:160 (n = 42),
47
1:320 (n = 12), 1:640 (n = 8), 1:1280 (n = 3). All examined donkeys were asymptomatic, except one
48
adult male (with a titre of 1:640 against T. equi) that showed clinical signs corresponding to the
49
acute stage of EP, reported for the first time in Italy.
50
51
The unique risk factor associated with a higher B. caballi seroprevalence was the presence
52
of horses in the farms, while risk factors associated with a higher T. equi seroprevalence were poor
53
body condition, presence of ruminants in the farms and milk production. The results indicate a
54
high level of exposure in donkeys living in Southern Italy and suggest that donkeys may be an
55
important reservoir of EP.
56
57
Keywords: Donkey; Piroplasms; Babesia caballi; Theileria equi; Tick-borne disease 3 Page 3 of 24
58
Introduction
59
Equine piroplasmosis (EP), also called horse tick fever, is a disease of horses, donkeys,
60
mules and zebras. The infection is caused by two species of intra-erythrocytic apicomplexan
61
protozoa, namely, Babesia caballi and Theileria equi. The parasites are naturally transmitted from
62
host to host via tick vectors and mixed infection with both organisms has been frequently
63
reported in equids (Wise et al., 2013). Throughout the world, ixodid ticks of the genera Boophilus,
64
Dermacentor, Haemaphysalis, Hyalomma and Rhipicephalus have been identified as important
65
vectors for the transmission of these protozoa (Wise et al., 2013).
66
67
EP causes serious economic loss to the equid sector and for this reason some disease-free
68
countries, including Australia, Canada, Japan and the USA, have established regulatory import
69
restrictions in an attempt to prevent the entry of seropositive animals. The disease is endemic in
70
horse populations in many tropical and subtropical regions (Uilenberg, 2006; Kumar et al., 2009;
71
Divan Baldani et al., 2010; Abutarbush et al., 2012; Vieira et al., 2013; Abedi et al., 2014; Rapoport
72
et al., 2014), but temperate areas can be affected as well. Within Europe, EP is more prevalent in
73
France (Fritz, 2010), Portugal (Bachiruddin et al., 1999; Ribeiro et al., 2013), Spain (Camacho et al.,
74
2005; Garcìa-Bocanegra et al., 2012), Turkey (Karatepe et al., 2009), Greece (Kouam et al., 2010)
75
and Italy (Moretti et al., 2010; Grandi et al., 2011; Laus et al., 2013).
76
77
Serological assays, such as the indirect fluorescent antibody test (IFAT) (Sigg et al., 2010)
78
and competitive-inhibition enzyme-linked immunosorbent assay (cELISA) (Mujica et al., 2011; Seo
79
et al., 2011), have been applied in large scale studies and used to monitor the latent stage of the
80
disease, characterized by mild parasitaemia. 4 Page 4 of 24
81
82
In some European countries, including Italy, there has been an increasing interest in
83
donkeys in recent years with their greater use as a pet animal, in leisure activities, for onotherapy
84
(a new instrument in animal assisted therapy) and the rediscovery of donkey milk as a food source
85
for children affected with cow milk allergy (Veneziano et al., 2011).
86
87
Very little information is available regarding parasitic diseases of donkeys, particularly EP.
88
Seroepidemiological and clinical evidence of EP has been reported in India (Kumar et al., 1997;
89
2009), Pakistan (Hussain et al., 2014), Brazil (Kerber et al., 1999;Machado et al., 2012; Vieira et al.,
90
2013), Turkey (Balkaya et al., 2010), some Arabian countries (Turnbull et al., 2002), Africa (de Waal
91
et al., 1994; Segwagwe et al., 1999; Rhalem et al., 2001; Mekibib et al., 2010; Tamera et al., 2011;
92
Salim et al., 2013; Gizachew et al., 2013)and China (Chahan et al., 2006). In Europe, a
93
seroprevalence study in donkeys and other equids was recently conducted in Spain (Garcìa-
94
Bocanegra et al., 2012).
95
96
Because the information on the epidemiology of donkey piroplasmosis in Italy is very
97
limited, the aim of the present study was to determine the seroprevalence of EP in adult donkeys,
98
born and raised in the Campania region of Southern Italy, as well to determine the risk factors
99
associated with the occurrence of the infection.
100
101
Materials and methods
102
Animals and sample size 5 Page 5 of 24
103
Campania region is located in Southern Italy and overlooks the Tyrrhenian Sea. It has a
104
territory of 13,595 square km (41° 00’ 00’’ N - 14° 30’ 00’’ E), with a typical Mediterranean
105
temperate climate along the coast which becomes progressively continental in the inland and
106
mountainous areas. The survey was conducted on 203 donkeys born and raised in the study area.
107
The sample size was calculated using the formula proposed by Thrusfield (1995) inserting the
108
following information: study population (9,991 donkeys; data supplied by Italian Association of
109
Breeders, 2011), expected prevalence of piroplasmosis (15%) based on the results of a recent
110
survey in a large horse population from the Central Italy (Moretti et al., 2010), confidence interval
111
(95%) and desired absolute precision (5%).
112
113
The study protocol was approved by the Ethical Committee of the Department of
114
Veterinary Medicine and Animal Production of the Naples University Federico II. Blood samples
115
were collected in autumn 2011 from 21 donkey farms in 16 municipalities in the Campania region.
116
A questionnaire was collected in order to obtain a complete history of the examined animals,
117
including gender, age, breed, use, period of grazing during year, presence of other species (horses,
118
dogs and ruminants) in the farms and ectoparasite control practices.
119
120
Overall, the mean age of the donkey population was 10 years (range, 14 months to 24
121
years). Animals were divided by age into four groups: < 2 years, 2-5 years, 6-7 years, and > 7 years.
122
There were 186 females and 17 males including 93 cross-breeds, 49 Martina Franca, 61 other
123
breeds (37 Amiata, 12 Sicilian Grey, 6 Ragusano and 6 Sardinian). The donkeys were used by
124
farmers for milk production (n = 165), meat production (n = 1), breeding (n = 13) or as pet animals
6 Page 6 of 24
125
(n = 24). One hundred and seventy-six of the donkeys grazed all year, 18 grazed only during the
126
spring/summer season and 9 were boxed throughout the year.
127
128
Clinical examination
129
A complete clinical examination was performed on each animal. Particular attention was
130
directed to clinical signs more frequently reported with EP, such as the presence of pale mucous
131
membranes with or without petechial lesions, fever, jaundice and haemoglobinuria. Body
132
condition score (BCS) of each donkey was determined using the donkey BCS chart as proposed by
133
The Donkey Sanctuary (2003). Briefly, a score of 1-5 was applied (1 = poor, 2 = moderate, 3 = ideal,
134
4 = fat and 5 = obese). The BCS procedure was performed by the same operator (VV) throughout
135
the study.
136
137
Sera preparation and serological test
138
Blood samples were collected from the jugular vein of each donkey into sterile vacuum
139
tubes (Vacutainer; Becton Dickinson) without an anticoagulant agent. Sera were obtained by
140
centrifugation for 10 min at 358 g; they were then stored at –20 °C and thawed at +37 °C
141
immediately before testing.
142
143
Sera were screened at a dilution of 1:80 for antibodies against B. caballi and T. equi using
144
an IFAT, according to the manufacturer’s instructions (Fuller Laboratories). Positive sera were
145
tested using two-fold dilutions from 1:80 up to a final titre of 1:1280. Ten microlitres of diluted
146
serum were placed in wells on masked slides, which contained fixed horse erythrocytes infected 7 Page 7 of 24
147
with B. caballi and T. equi. Positive and negative controls were supplied from the manufacturer.
148
The slides were incubated in a humid chamber at +37°C for 30 min, successively rinsed and soaked
149
in buffer for 10 min. Ten microlitres of fluorescein-conjugated rabbit anti-horse IgG were applied
150
to each well and then incubation and rinse-soak steps were repeated. Finally the slides were dried
151
and mounted with 50% glycerol and 50% carbonate-bicarbonate buffer (pH 9). The slides were
152
observed under an epi-fluorescent microscope (Leica, DM 2500) using a xenon light source. A
153
positive reaction appeared as peripheral clusters of distinct apple-green inclusion bodies within
154
the infected erythrocytes.
155
156
Statistical analyses
157
The seroprevalence according to the various characteristics (gender, age, breed,
158
management, nutritional status, presences of other animal species, use and milking) was
159
calculated with an associated 95% confidence interval (CI). Differences in prevalence between
160
these various groups were assessed by the two-sided chi-square t test. A P value
S1090-0233(14)00394-3 http://dx.doi.org/doi: 10.1016/j.tvjl.2014.09.025 YTVJL 4290
To appear in:
The Veterinary Journal
Accepted date:
25-9-2014
Please cite this article as: D. Piantedosi, N. D'Alessio, A. Di Loria, F. Di Prisco, U. Mariani, B. Neola, M. Santoro, S. Montagnaro, G. Capelli, V. Veneziano, Seroprevalence and risk factors associated with Babesia caballi and Theileria equi infections in donkeys from Southern Italy, The Veterinary Journal (2014), http://dx.doi.org/doi: 10.1016/j.tvjl.2014.09.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2 3
Seroprevalence and risk factors associated with Babesia caballi and Theileria equi infections in donkeys from Southern Italy
4 5 6
D. Piantedosia, N. D'Alessio b, A. Di Loria c,*, F. Di Prisco b, U. Mariani b, B. Neola a, M. Santoro d, S. Montagnaro a, G. Capelli e, V. Veneziano a
7 8 9
a
10
c
Department of Health Science, University of Magna Græcia, Catanzaro, Italy
11
d
Department of Public Health and Infectious Diseases, Sapienza University of Rome, Italy
12
e
Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (PD), Italy
Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Italy b Istituto Zooprofilattico Sperimentale del Mezzogiorno, Portici (NA), Italy
13
14 15
* Corresponding author: Tel.: +39 0961 3694284. E-mail address: [email protected] (A. Di Loria).
16
17
Highlights
18 19
This is the first study to estimate the occurrence of donkey piroplasmosis in the Campania region of Southern Italy.
Of 203 serum samples tested, 72 (35.5%) were found to be positive for B. caballi and 90 (44.3%) were positive for T. equi . The overall seroprevalence of EP was 57.1% (116/203) and a proportion of the donkeys were co-infected by both protozoa (46/203 - 22.6%).
Clinical examination of the most seropositive animals failed to show any evidence of abnormalities. Only one male donkey, with a titre of 1:640 against T. equi, showed the acute symptoms of disease.
Our findings showed that EP is widespread in donkeys living in this area of Southern Italy and therefore the establishment of an appropriate and effective control program is of great importance for these equids.
20 21 22 23 24 25 26 27 28 29 30 31 32 33
1 Page 1 of 24
34 35
Donkeys could be chronic carriers and a potential continuous source for maintaining and disseminating the piroplasms in the rural environment.
2 Page 2 of 24
36
Abstract
37
Equine piroplasmosis (EP) has been frequently described in donkeys in subtropical and
38
tropical regions, but published data reflecting large scale surveys are very limited in Europe. The
39
seroprevalence of Babesia caballi and Theileria equi was determined in a donkey population from
40
Campania Region in Southern Italy using a commercial indirect fluorescent antibody test (IFAT),
41
and the risk factors associated with the occurrence of the infection were assessed.
42
43
Of 203 samples, the overall seroprevalence for EP was 57.1% (116/203), with 35.5%
44
(72/203) for B. caballi and 44.3% (90/203) for T. equi. Co-infection was detected in 46 donkeys
45
(22.6%). The distribution of IFAT antibody titres to B. caballi was: 1:80 (n = 67), 1:160 (n = 2), 1:320
46
(n = 3); while, the distribution of IFAT antibody titres to T. equi was: 1:80 (n = 25), 1:160 (n = 42),
47
1:320 (n = 12), 1:640 (n = 8), 1:1280 (n = 3). All examined donkeys were asymptomatic, except one
48
adult male (with a titre of 1:640 against T. equi) that showed clinical signs corresponding to the
49
acute stage of EP, reported for the first time in Italy.
50
51
The unique risk factor associated with a higher B. caballi seroprevalence was the presence
52
of horses in the farms, while risk factors associated with a higher T. equi seroprevalence were poor
53
body condition, presence of ruminants in the farms and milk production. The results indicate a
54
high level of exposure in donkeys living in Southern Italy and suggest that donkeys may be an
55
important reservoir of EP.
56
57
Keywords: Donkey; Piroplasms; Babesia caballi; Theileria equi; Tick-borne disease 3 Page 3 of 24
58
Introduction
59
Equine piroplasmosis (EP), also called horse tick fever, is a disease of horses, donkeys,
60
mules and zebras. The infection is caused by two species of intra-erythrocytic apicomplexan
61
protozoa, namely, Babesia caballi and Theileria equi. The parasites are naturally transmitted from
62
host to host via tick vectors and mixed infection with both organisms has been frequently
63
reported in equids (Wise et al., 2013). Throughout the world, ixodid ticks of the genera Boophilus,
64
Dermacentor, Haemaphysalis, Hyalomma and Rhipicephalus have been identified as important
65
vectors for the transmission of these protozoa (Wise et al., 2013).
66
67
EP causes serious economic loss to the equid sector and for this reason some disease-free
68
countries, including Australia, Canada, Japan and the USA, have established regulatory import
69
restrictions in an attempt to prevent the entry of seropositive animals. The disease is endemic in
70
horse populations in many tropical and subtropical regions (Uilenberg, 2006; Kumar et al., 2009;
71
Divan Baldani et al., 2010; Abutarbush et al., 2012; Vieira et al., 2013; Abedi et al., 2014; Rapoport
72
et al., 2014), but temperate areas can be affected as well. Within Europe, EP is more prevalent in
73
France (Fritz, 2010), Portugal (Bachiruddin et al., 1999; Ribeiro et al., 2013), Spain (Camacho et al.,
74
2005; Garcìa-Bocanegra et al., 2012), Turkey (Karatepe et al., 2009), Greece (Kouam et al., 2010)
75
and Italy (Moretti et al., 2010; Grandi et al., 2011; Laus et al., 2013).
76
77
Serological assays, such as the indirect fluorescent antibody test (IFAT) (Sigg et al., 2010)
78
and competitive-inhibition enzyme-linked immunosorbent assay (cELISA) (Mujica et al., 2011; Seo
79
et al., 2011), have been applied in large scale studies and used to monitor the latent stage of the
80
disease, characterized by mild parasitaemia. 4 Page 4 of 24
81
82
In some European countries, including Italy, there has been an increasing interest in
83
donkeys in recent years with their greater use as a pet animal, in leisure activities, for onotherapy
84
(a new instrument in animal assisted therapy) and the rediscovery of donkey milk as a food source
85
for children affected with cow milk allergy (Veneziano et al., 2011).
86
87
Very little information is available regarding parasitic diseases of donkeys, particularly EP.
88
Seroepidemiological and clinical evidence of EP has been reported in India (Kumar et al., 1997;
89
2009), Pakistan (Hussain et al., 2014), Brazil (Kerber et al., 1999;Machado et al., 2012; Vieira et al.,
90
2013), Turkey (Balkaya et al., 2010), some Arabian countries (Turnbull et al., 2002), Africa (de Waal
91
et al., 1994; Segwagwe et al., 1999; Rhalem et al., 2001; Mekibib et al., 2010; Tamera et al., 2011;
92
Salim et al., 2013; Gizachew et al., 2013)and China (Chahan et al., 2006). In Europe, a
93
seroprevalence study in donkeys and other equids was recently conducted in Spain (Garcìa-
94
Bocanegra et al., 2012).
95
96
Because the information on the epidemiology of donkey piroplasmosis in Italy is very
97
limited, the aim of the present study was to determine the seroprevalence of EP in adult donkeys,
98
born and raised in the Campania region of Southern Italy, as well to determine the risk factors
99
associated with the occurrence of the infection.
100
101
Materials and methods
102
Animals and sample size 5 Page 5 of 24
103
Campania region is located in Southern Italy and overlooks the Tyrrhenian Sea. It has a
104
territory of 13,595 square km (41° 00’ 00’’ N - 14° 30’ 00’’ E), with a typical Mediterranean
105
temperate climate along the coast which becomes progressively continental in the inland and
106
mountainous areas. The survey was conducted on 203 donkeys born and raised in the study area.
107
The sample size was calculated using the formula proposed by Thrusfield (1995) inserting the
108
following information: study population (9,991 donkeys; data supplied by Italian Association of
109
Breeders, 2011), expected prevalence of piroplasmosis (15%) based on the results of a recent
110
survey in a large horse population from the Central Italy (Moretti et al., 2010), confidence interval
111
(95%) and desired absolute precision (5%).
112
113
The study protocol was approved by the Ethical Committee of the Department of
114
Veterinary Medicine and Animal Production of the Naples University Federico II. Blood samples
115
were collected in autumn 2011 from 21 donkey farms in 16 municipalities in the Campania region.
116
A questionnaire was collected in order to obtain a complete history of the examined animals,
117
including gender, age, breed, use, period of grazing during year, presence of other species (horses,
118
dogs and ruminants) in the farms and ectoparasite control practices.
119
120
Overall, the mean age of the donkey population was 10 years (range, 14 months to 24
121
years). Animals were divided by age into four groups: < 2 years, 2-5 years, 6-7 years, and > 7 years.
122
There were 186 females and 17 males including 93 cross-breeds, 49 Martina Franca, 61 other
123
breeds (37 Amiata, 12 Sicilian Grey, 6 Ragusano and 6 Sardinian). The donkeys were used by
124
farmers for milk production (n = 165), meat production (n = 1), breeding (n = 13) or as pet animals
6 Page 6 of 24
125
(n = 24). One hundred and seventy-six of the donkeys grazed all year, 18 grazed only during the
126
spring/summer season and 9 were boxed throughout the year.
127
128
Clinical examination
129
A complete clinical examination was performed on each animal. Particular attention was
130
directed to clinical signs more frequently reported with EP, such as the presence of pale mucous
131
membranes with or without petechial lesions, fever, jaundice and haemoglobinuria. Body
132
condition score (BCS) of each donkey was determined using the donkey BCS chart as proposed by
133
The Donkey Sanctuary (2003). Briefly, a score of 1-5 was applied (1 = poor, 2 = moderate, 3 = ideal,
134
4 = fat and 5 = obese). The BCS procedure was performed by the same operator (VV) throughout
135
the study.
136
137
Sera preparation and serological test
138
Blood samples were collected from the jugular vein of each donkey into sterile vacuum
139
tubes (Vacutainer; Becton Dickinson) without an anticoagulant agent. Sera were obtained by
140
centrifugation for 10 min at 358 g; they were then stored at –20 °C and thawed at +37 °C
141
immediately before testing.
142
143
Sera were screened at a dilution of 1:80 for antibodies against B. caballi and T. equi using
144
an IFAT, according to the manufacturer’s instructions (Fuller Laboratories). Positive sera were
145
tested using two-fold dilutions from 1:80 up to a final titre of 1:1280. Ten microlitres of diluted
146
serum were placed in wells on masked slides, which contained fixed horse erythrocytes infected 7 Page 7 of 24
147
with B. caballi and T. equi. Positive and negative controls were supplied from the manufacturer.
148
The slides were incubated in a humid chamber at +37°C for 30 min, successively rinsed and soaked
149
in buffer for 10 min. Ten microlitres of fluorescein-conjugated rabbit anti-horse IgG were applied
150
to each well and then incubation and rinse-soak steps were repeated. Finally the slides were dried
151
and mounted with 50% glycerol and 50% carbonate-bicarbonate buffer (pH 9). The slides were
152
observed under an epi-fluorescent microscope (Leica, DM 2500) using a xenon light source. A
153
positive reaction appeared as peripheral clusters of distinct apple-green inclusion bodies within
154
the infected erythrocytes.
155
156
Statistical analyses
157
The seroprevalence according to the various characteristics (gender, age, breed,
158
management, nutritional status, presences of other animal species, use and milking) was
159
calculated with an associated 95% confidence interval (CI). Differences in prevalence between
160
these various groups were assessed by the two-sided chi-square t test. A P value
