Accepted Manuscript Title: Neospora caninum prevalence in dogs raised under different living conditions Author: Muhammad Mudasser Nazir Azhar Maqbool Masood Akhtar Mazhar Ayaz Atif Nisar Ahmad Kamran Ashraf Asif Ali Muhammad Azhar Alam Muhammad Amjad Ali Abdur Rauf Khalid David S. Lindsay PII: DOI: Reference:
S0304-4017(14)00336-7 http://dx.doi.org/doi:10.1016/j.vetpar.2014.05.041 VETPAR 7277
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
25-3-2014 26-5-2014 29-5-2014
Please cite this article as: Nazir, M.M., Maqbool, A., Akhtar, M., Ayaz, M., Ahmad, A.N., Ashraf, K., Ali, A., Alam, M.A., Ali, M.A., Khalid, A.R., Lindsay, D.S.,Neospora caninum prevalence in dogs raised under different living conditions, Veterinary Parasitology (2014), http://dx.doi.org/10.1016/j.vetpar.2014.05.041 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.
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Neospora caninum prevalence in dogs raised under different living conditions
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Muhammad Mudasser Nazir*, Azhar Maqbool*, Masood Akhtar†, Mazhar Ayaz†, Atif
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Nisar Ahmad†, Kamran Ashraf*, Asif Ali*, Muhammad Azhar Alam*, Muhammad Amjad
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Ali†, Abdur Rauf Khalid† and David S. Lindsay‡
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*Department of Parasitology, University of Veterinary and Animal Sciences, Lahore
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54600, Pakistan.
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† Department of Pathobiology, Faculty of Veterinary Sciences, B.Z University, Multan
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60800, Pakistan.
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‡Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional
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College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, 24061, USA.
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Address manuscript correspondence to Dr. M. Mudasser Nazir, Department of
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Pathobiology, Faculty of Veterinary Sciences, Bahauddin Zakariya University, Boson
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Road, Multan, Pakistan 60800. e-mail: [email protected]
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Keywords: Neospora caninum, dog, oocyst, epidemiology
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Abstract:
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Neospora caninum is an important cause of abortion in dairy cattle worldwide.
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Dogs are important in the epidemiology of N. caninum because they act as definitive
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hosts shedding oocysts in the environment. Vertical transmission of the parasite is well
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recognized as an important aspect of the epidemiology of the parasite but the
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importance of horizontal transmission has been less studied. A N. caninum competitive
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ELISA was used to examine serum samples from 600 dogs that were raised under 4
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different living conditions. Samples from 138 dogs living on 24 dairies with a prevalence
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(0-70%) of anti-N. caninum antibodies in the cattle, 294 pet dogs without neurological
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signs, 76 from pet dogs exhibiting neurological signs, and 92 stray dogs were
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examined. The overall seroprevalence of N. caninum was 23.5% (95% CI = ± 2.99) in
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the 600 dogs. Significant (P < 0.05) differences were observed between the 4 different
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populations of dogs. The number of N. caninum positive samples were: 51 (36.9%, 95%
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CI = ± 3.09) of 138 dogs from dairies, 31(10.5%, 95% CI = ± 6.38) of 294 pet dogs
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without neurological signs, disorders, 22 (28.9%, 95% CI = ± 6.70) of 76 pet dogs with
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neurological signs, and 37 (40.2%, 95% CI = ± 2.83) of 92 stray dogs. Seropositivity to
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N. caninum in dogs from dairies was associated with a high prevalence of N. caninum
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antibodies in the cattle. At the 3 dairies where no dogs were present, the
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seroprevalence to N. caninum in the cattle was significantly lower (P < 0.05) than in the
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21 dairies where dogs were present. Seroprevalence was significantly higher (P < 0.05)
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in male dogs (97 of 366; 26.5%, 95% CI = ± 3.40) than in female dogs (44 of 234;
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18.8%, 95% CI = ± 5.65). Seroprevalence in dogs increased with age suggesting
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postnatal exposure to N. caninum infection however, this increase was not significant (P
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> 0.05). The prevalence of N. caninum antibodies was not significantly (P > 0.05)
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different in dogs based on breed. These findings suggest a relationship between N.
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caninum infection of dogs from dairies and cattle on these dairies. However, further
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research is required to determine what is the most important way dogs acquire infection
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and how to prevent dogs from shedding oocysts.
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1. Introduction
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Neospora caninum, a protozoan parasite, is recognized as one of the most important infectious agents responsible for abortion in cattle worldwide (Dubey and
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Lindsay, 1996). Vertical transmission is considered as the major route of transmission in
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cattle, whereas the epidemiological importance of the horizontal transmission through
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the ingestion of oocysts (McAllister et al., 1998) seems to vary geographically. Dogs
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(and other Canis species) can act as definitive hosts shedding oocysts in the
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environment (McAllister et al., 1998; Lindsay et al., 1999a; Dijkstra et al., 2001; Gondim
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et al., 2004). Cattle, dogs, sheep, goats, and deer are natural intermediate hosts (Dubey
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et al., 2007). Oocysts are excreted not sporulated but sporulate in the external
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environment within 1 to 2 days depending on temperature and humidity (Lindsay et al.,
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1999b). Sporulated oocysts of N. caninum are orally infectious for cattle (De Marez et
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al., 1999). Numerous epidemiological studies have recognized the presence of dogs on
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cattle farms as a risk factor for bovine neosporosis and have provided evidence for the
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occurrence of horizontal transmission in cattle (McAllister et al., 1996; Pare et al., 1998;
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Wouda et al., 1999; Dubey et al., 2007). There are only a few reports of dogs naturally
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shedding N. caninum oocysts (Basso et al., 2001; Mc Garry et al., 2003; Schares et al.,
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2005). Diagnosis of N. caninum oocyst excretion by dogs is problematic due to their
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structural similarity to Hammondia heydorni, the need for PCR and gene sequencing,
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and the absence of clinical signs in most dogs shedding oocysts (McAllister et al., 1998;
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Basso et al., 2001). In puppies or young dogs (under 1 year of age) clinical neosporosis
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is characterized by various clinical signs including ascending paralysis, hind limb ataxia,
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myocarditis, dermatitis, pneumonia, or encephalitis (Dubey and Lindsay, 1996; Dubey et
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al., 2005). Clinical signs of disease have not been observed in experimentally infected
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dogs shedding N. caninum oocysts (Lindsay et al., 2001; Gondim et al., 2005).
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Experimentally infected dogs usually pass few oocysts in their feces and not all dogs
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that shed oocysts develop antibodies to N. caninum. Puppies excrete significantly fewer
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oocysts after being fed N. caninum tissue cysts than do adult dogs (Gondim et al.,
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2005). Immunity to intestinal oocyst infection to N. caninum begins to decreases after
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18 to 20 months and dogs can re-shed oocysts after challenge with tissue cysts
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(Gondim et al., 2005). In the present study we addressed several aspects of the
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epidemiology of N. caninum in dogs. We examined the seroprevalence of N. caninum
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based on living conditions, sex, age and breed in 600 dogs from a study area in
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Pakistan.
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2. Materials and methods
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2.1. Dog populations sampled
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Four groups of dogs raised under different living conditions, from upper Punjab,
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Pakistan were evaluated for antibodies to N. caninum (Tables 1 & 2). The first group
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was 138 dogs from 24 dairies (Table 2) located in 5 districts of Punjab. Groups 2 and
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three consisted of 370 dogs sampled at the University Pet Centre, University of
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Veterinary and Animal Sciences, Lahore and private veterinary practices located in
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Lahore. Group 2 contained 294 dogs not showing neurological signs and Group 3
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contained 76 dogs with various neurological signs. Group 4 consisted of 92 stray dogs
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acquired from environment after the consent of the Department of Epidemics and Public
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Health, Lahore. Owners, practitioners, and researchers provided epidemiological
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information on dog age, sex, and breed at the time of sampling (Tables 3 & 4).
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2.2. Sample collection and cELISA
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Blood samples were obtained aseptically from the cephalic vein. The serum was collected following centrifugation and stored at −20 °C until further processing at the
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Department of Parasitology, University of Veterinary and Animal Sciences, Lahore,
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Pakistan. Serum reactivity to N. caninum was evaluated using a commercially available
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competitive inhibition enzyme-linked immunosorbent assay kit (cELISA, VMRD,
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Pullman, Washington, USA). The sensitivity and specificity of this cELISA test for dog
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samples were affirmed by the manufacturer to be 91.4% and 99.4%, respectively. The
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samples were used undiluted as suggested by the manufacturer. Positive and negative
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control samples were provided with the kit.
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Results were expressed as percentage of inhibition (PI).
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PI = 100- [(sample O.D. ×100)/ (mean negative control O.D.)].
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Samples were considered positive when ≥30% inhibition was observed.
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Each dog’s sex was recorded as male or female regardless if the animal had
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been neutered or spayed and the dogs were placed in one of 5 different age groups
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(Table 3). Age groups were 6 months to 3 years (N=172), 3 to 6 years (N=96), 6-8 years
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(N=109), greater than 8 years (N=109) and dogs with no age recorded (N=105).
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2.4. Dog breeds
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Dog breed was determined using pedigree standards of the Kennel Club of
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Pakistan (Table 4). Dog breeds were Bully (N=58), Alsatian (N=87), Bullterrier (N=79),
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German shepherd (N=61), Pug (N=44), Labrador retriever (N=102), crossbreed (N=73)
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and mongrel (N=96). A dog was placed in the crossbred group if it possessed the
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phenotype of two distinct breeds and dogs were placed in the mongrel group if no
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defined breed phenotype was apparent.
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2.5. Statistical examination The sample means and 95% confidence intervals were determined using Microsoft Excel 2007 software and Pearson’s x2 test was used to determine if
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differences in prevalence were present in the 4 groups of dogs based on living
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conditions in relation to dairy cattle and by age, breed and sex using (SPSS for
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Windows, version 17.0; SPSS, Chicago, Illinois), and P < 0.05 was considered
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significant prior to conduct of the test.
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3. Results
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Antibodies to N. caninum were found in 141 (23.5%, 95% CI = ± 2.99) of the 600
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dogs examined in the present study (Tables 1 and 2). Significant differences (P < 0.05)
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in prevalence were observed among different groups of dogs. The number of N.
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caninum positive samples were: 51 (36.9%, 95% CI = ± 3.09) of 138 dogs from dairies
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in Group 1, 31(10.5%, 95% CI = ± 6.38) of 294 pet dogs without neurological signs in
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Group 2, 22 (28.9%, 95% CI = ± 6.70) of 76 pet dogs with neurological signs in Group 3,
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and 37 (40.2%, 95% CI = ± 2.83) of 92 stray dogs in Group 4. Statistically, a significant
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difference (P < 0.05) was observed between male (97of 366, 26.5% 95% CI = ± 3.40)
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and female (44 of 234; 18.8%, 95% CI = ± 5.65) dogs for seroprevalence (Table 3).
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There was no significant (P > 0.05) difference between dogs based on age but
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prevalence was greater (30 of 109; 27.5%, 95% CI = ± 5.97) in animals older than 8
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years of age (Table 3).
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Three of the 24 dairies examined did not have a resident population of dogs
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(Table 2) and the prevalence of N. caninum positive cows varied from 0 to 25% on
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these 3 dairies (Table 2). Of the 21 dairies that had a resident dog population,
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antibodies to N. caninum were found in dogs from 16 (76.2%, 95% CI = ± 15.84) dairies
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while dogs from 5 (23.8%, 95% CI = ± 15.85) dairies were negative for antibodies to N.
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caninum. The N. caninum prevalence in cows from these dairies ranged from 17 to 70%
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(Table 2). A higher rate of seropositivity to N. caninum was found at dairies with
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seropositive dogs (Table 2), while relatively low prevalence of N. caninum in cows from
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dairies without dog populations and seronegative dogs. This difference was statistically
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significant (P < 0.05).
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N. caninum positive dogs were found in Alsatian (12.6%, 95% CI = ± 11.1), Bully
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(20.6%, 95% CI = ± 10.7), Pug (20.4%, 95% CI = ± 12.36), Bullterrier (18.9%, 95% CI =
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± 9.72), German shepherd (31.4%, 95% CI = ± 6.60), Labrador Retriever (19.6%, 95%
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CI = ± 8.34), Crossbreds (28.7%, 95% CI = ± 6.87) and Mongrel (35.4%, 95% CI = ±
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4.23) breeds (Table 4). No significant (P > 0.05) difference among the prevalence of
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exposure different dog breeds was observed.
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4. Discussion
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The prevalence of N. caninum antibodies in stray dogs and farm dogs was
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significantly higher (P < 0.05) when compared to pet dogs, which were mainly from
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urban areas. A higher prevalence of N. caninum antibodies in farm dogs than in urban
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dogs was reported in a study from Japan (Sawada et al., 1998). Our findings indicate
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that stray and dogs from dairies are at a higher risk of exposure to N. caninum than pet
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dogs. In our study, a significantly higher seroprevalence was found in stray dogs as
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compared to pet dogs and farm dogs. The higher rates of seropositivity to N. caninum in
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stray dogs than farm dogs and pet dogs might be due to dietary habits, as outdoor
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animals have direct contact with infective material from positive cattle or other
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intermediate hosts and they have common access to dairies and contaminated
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environment (Dijkstra et al., 2001, 2002a). The higher seroprevalence of N. caninum in
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dogs from N. caninum positive dairies gives us a clue about the epidemiological
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association between dogs and cattle. Previous studies have demonstrated that the
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presence of dogs on cattle farms, can play an important role in the seropositivity and
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bovine abortion (Bartels et al., 1999; Dijkstra et al., 2002b; Schares et al., 2003). Our
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finding of a significant difference of prevalence between male and female dogs was not
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expected based on previous studies (Trees et al., 1993; Barber et al., 1997; Sawada et
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al., 1998). We found a higher prevalence rate in males than females, while earlier
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reports found females were more likely to be seropositive than males (Wouda et al.,
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1999; Fernandes et al., 2004). The prevalence of antibodies to N. caninum increased
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with the age of animals but the increase was not significant (P > 0.05). These findings
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agree with the observations made by others (Souza et al., 2002) and suggests postnatal
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infection. The role of dog breed in relation to N. caninum prevalence is not clear. Most
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of the reports find no specific breed susceptibility or a higher seroprevalence in mixed
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breed dogs (Fernandes et al., 2004).
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Our findings reinforce the importance of dogs in the epidemiology of N. caninum
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and support our hypothesis that “The prevalence of Neospora caninum antibodies is
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different in dogs raised under different living conditions”.
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Acknowledgments
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The current study was financially supported by a grant from the Higher Education
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Commission, Islamabad, Pakistan. The authors would like to thank Dr. Zia ullah Mughal
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from the Pet Center, UVAS, Lahore, Dr. Muhammad Oneeb, Dr. Ahsan Mustafa,
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Noman Nazir, Umer bacha and Muhammad Kashif Nazir for their technical help during
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serum sampling.
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Number cELISA negative 87
Pets with no nervous signs Pets with nervous signs Stray dogs
294
31
263
76
22
54
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CI 95%
36.9
±3.09
92
37
Total dogs
600
141
10.5
±6.38
28.9
±6.70
55
40.2
±2.83
459
23.5
±2.99
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Farm dogs
Percent positive
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Number cELISA positive 51
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Number examined
All four groups were significantly different (P < 0.05) from each other
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Table 1. Prevalence of Neospora caninum antibodies based on how dogs were raised.
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Table 2: Seroprevalence of N. caninum in dogs raised on dairies. Number of cows tested (n)
Positive cattle (n)
Percentage (CI 95%)
Number of dogs at farm (n)
Number of dogs positive (n)
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Number Number of cows in of herds the herd (n)
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* Dairies with no dog population
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† Dairies with dog population
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†1 407 68 31 46(±1.34) 9 3 †2 963 41 13 32(±7.79) 6 0 †3 318 65 29 45(±1.71) 4 2 †4 74 23 4 17(±19.07) 2 0 8 3 †5 382 35 19 54(±1.87) *6 194 19 0 0(±6.04) 0 0 †7 289 61 33 54(±1.41) 4 2 †8 724 27 16 59(±4.80) 8 5 †9 267 49 18 37(±5.14) 5 1 †10 923 12 3 25(±20) 9 0 †11 348 6 4 66(±18.10) 13 5 †12 875 83 47 57(±2.12) 8 4 †13 219 53 20 38(±4.56) 4 1 †14 86 37 26 70(±9.11) 7 5 †15 694 62 14 23(±9.50) 2 0 †16 1047 119 52 44(±1.52) 9 3 †17 658 94 43 46(±1.14) 11 4 *18 417 38 5 13(±16.63) 0 0 †19 376 17 7 41(±6.05) 6 2 †20 113 7 2 28(±23.04) 3 0 †21 264 48 27 56(±2.40) 7 4 †22 49 5 2 40(±12.39) 5 2 †23 310 27 18 67(±9.06) 8 5 *24 22 4 1 25(±34.64) 0 0 138 51 Total 10019 1000 434 43.4(±0.57) Significant difference P < 0.05 of seropositivity at diaries where dogs were present
Seropositive dogs(CI 95%) 33(±15.7) 0(±3.39) 51(±1.38) 0(±1.95) 37(±12.7) 0 51(±1.38) 62(±11.7) 20(±37.8) 0(±4.15) 38(±9.22) 52(±1.96) 25(±34.3) 71(±22.0) 0(±1.95) 33(±15.7) 36(±11.7) 0 33(±19.2) 0(±2.40) 57(±7.3) 40(±12.3) 63(±12.7) 0 37(±3.06)
269 270 271
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Table 3 Seropositivity of dogs based on gender and age group
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Number tested/ positive males
%age male (CI 95%)
172/33
19.2(±6.50)
101/25
24.7(±6.97)
3–6 years
96/18
18.7(±8.85)
49/13
26.5(±9.30)
6–8 years
118/29
24.6(±6.48)
68/22
>8 years
109/30
27.5(±5.97)
87/19
No age record
105/31
29.5(±5.54)
61/18
Total
600/141
23.5(±2.99)
97 of 366 male dogs examined
b
44 of 234 female dogs examined
%age female (CI 95%)
13.5(±13.17)
15.1(±16.83)
41/7
17.1(±14.24)
21.8(±8.38)
52/11
21.1(±11.10)
29.5(±7.27)
49/13
26.5(±9.30)
26.5/±3.40
234/44b
18.8(±5.65)
an
33/5
32.3(±5.94)
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59/8
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6 months3 years
366/97a
Number tested/positive females
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Seropositive (CI 95%)
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Age group†
Number tested/ number positive
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Page 15 of 16
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percent positive (%) 20.6
CI 95%
Bully
Number tested/number positive 58/12
Alsation
87/11
12.6
Bullterrier
79/15
18.8
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Table 4: Prevalence of Neospora caninum antibodies in various dog breeds
German shephard
61/19
31.4
Pug
44/9
20.4
Labrador Retriever
102/20
278 279
73/21
Non-descript (Mongrel)
96/34
M
Crossbreds
cr
±6.60
±12.36
19.6
±8.34
19.9
±4.01
28.8
±6.87
35.4
±4.13
d
P > 0.05
±9.72
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431/86
±11.11
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Total Purebred
±10.70
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Breed
an
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Page 16 of 16
S0304-4017(14)00336-7 http://dx.doi.org/doi:10.1016/j.vetpar.2014.05.041 VETPAR 7277
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
25-3-2014 26-5-2014 29-5-2014
Please cite this article as: Nazir, M.M., Maqbool, A., Akhtar, M., Ayaz, M., Ahmad, A.N., Ashraf, K., Ali, A., Alam, M.A., Ali, M.A., Khalid, A.R., Lindsay, D.S.,Neospora caninum prevalence in dogs raised under different living conditions, Veterinary Parasitology (2014), http://dx.doi.org/10.1016/j.vetpar.2014.05.041 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.
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Neospora caninum prevalence in dogs raised under different living conditions
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Muhammad Mudasser Nazir*, Azhar Maqbool*, Masood Akhtar†, Mazhar Ayaz†, Atif
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Nisar Ahmad†, Kamran Ashraf*, Asif Ali*, Muhammad Azhar Alam*, Muhammad Amjad
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Ali†, Abdur Rauf Khalid† and David S. Lindsay‡
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*Department of Parasitology, University of Veterinary and Animal Sciences, Lahore
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54600, Pakistan.
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† Department of Pathobiology, Faculty of Veterinary Sciences, B.Z University, Multan
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60800, Pakistan.
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‡Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional
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College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, 24061, USA.
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Address manuscript correspondence to Dr. M. Mudasser Nazir, Department of
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Pathobiology, Faculty of Veterinary Sciences, Bahauddin Zakariya University, Boson
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Road, Multan, Pakistan 60800. e-mail: [email protected]
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Keywords: Neospora caninum, dog, oocyst, epidemiology
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Abstract:
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Neospora caninum is an important cause of abortion in dairy cattle worldwide.
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Dogs are important in the epidemiology of N. caninum because they act as definitive
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hosts shedding oocysts in the environment. Vertical transmission of the parasite is well
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recognized as an important aspect of the epidemiology of the parasite but the
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importance of horizontal transmission has been less studied. A N. caninum competitive
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ELISA was used to examine serum samples from 600 dogs that were raised under 4
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different living conditions. Samples from 138 dogs living on 24 dairies with a prevalence
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(0-70%) of anti-N. caninum antibodies in the cattle, 294 pet dogs without neurological
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signs, 76 from pet dogs exhibiting neurological signs, and 92 stray dogs were
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examined. The overall seroprevalence of N. caninum was 23.5% (95% CI = ± 2.99) in
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the 600 dogs. Significant (P < 0.05) differences were observed between the 4 different
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populations of dogs. The number of N. caninum positive samples were: 51 (36.9%, 95%
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CI = ± 3.09) of 138 dogs from dairies, 31(10.5%, 95% CI = ± 6.38) of 294 pet dogs
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without neurological signs, disorders, 22 (28.9%, 95% CI = ± 6.70) of 76 pet dogs with
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neurological signs, and 37 (40.2%, 95% CI = ± 2.83) of 92 stray dogs. Seropositivity to
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N. caninum in dogs from dairies was associated with a high prevalence of N. caninum
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antibodies in the cattle. At the 3 dairies where no dogs were present, the
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seroprevalence to N. caninum in the cattle was significantly lower (P < 0.05) than in the
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21 dairies where dogs were present. Seroprevalence was significantly higher (P < 0.05)
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in male dogs (97 of 366; 26.5%, 95% CI = ± 3.40) than in female dogs (44 of 234;
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18.8%, 95% CI = ± 5.65). Seroprevalence in dogs increased with age suggesting
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postnatal exposure to N. caninum infection however, this increase was not significant (P
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> 0.05). The prevalence of N. caninum antibodies was not significantly (P > 0.05)
40
different in dogs based on breed. These findings suggest a relationship between N.
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caninum infection of dogs from dairies and cattle on these dairies. However, further
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research is required to determine what is the most important way dogs acquire infection
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and how to prevent dogs from shedding oocysts.
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1. Introduction
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Neospora caninum, a protozoan parasite, is recognized as one of the most important infectious agents responsible for abortion in cattle worldwide (Dubey and
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Lindsay, 1996). Vertical transmission is considered as the major route of transmission in
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cattle, whereas the epidemiological importance of the horizontal transmission through
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the ingestion of oocysts (McAllister et al., 1998) seems to vary geographically. Dogs
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(and other Canis species) can act as definitive hosts shedding oocysts in the
51
environment (McAllister et al., 1998; Lindsay et al., 1999a; Dijkstra et al., 2001; Gondim
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et al., 2004). Cattle, dogs, sheep, goats, and deer are natural intermediate hosts (Dubey
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et al., 2007). Oocysts are excreted not sporulated but sporulate in the external
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environment within 1 to 2 days depending on temperature and humidity (Lindsay et al.,
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1999b). Sporulated oocysts of N. caninum are orally infectious for cattle (De Marez et
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al., 1999). Numerous epidemiological studies have recognized the presence of dogs on
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cattle farms as a risk factor for bovine neosporosis and have provided evidence for the
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occurrence of horizontal transmission in cattle (McAllister et al., 1996; Pare et al., 1998;
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Wouda et al., 1999; Dubey et al., 2007). There are only a few reports of dogs naturally
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shedding N. caninum oocysts (Basso et al., 2001; Mc Garry et al., 2003; Schares et al.,
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2005). Diagnosis of N. caninum oocyst excretion by dogs is problematic due to their
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structural similarity to Hammondia heydorni, the need for PCR and gene sequencing,
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and the absence of clinical signs in most dogs shedding oocysts (McAllister et al., 1998;
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Basso et al., 2001). In puppies or young dogs (under 1 year of age) clinical neosporosis
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is characterized by various clinical signs including ascending paralysis, hind limb ataxia,
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myocarditis, dermatitis, pneumonia, or encephalitis (Dubey and Lindsay, 1996; Dubey et
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al., 2005). Clinical signs of disease have not been observed in experimentally infected
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dogs shedding N. caninum oocysts (Lindsay et al., 2001; Gondim et al., 2005).
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Experimentally infected dogs usually pass few oocysts in their feces and not all dogs
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that shed oocysts develop antibodies to N. caninum. Puppies excrete significantly fewer
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oocysts after being fed N. caninum tissue cysts than do adult dogs (Gondim et al.,
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2005). Immunity to intestinal oocyst infection to N. caninum begins to decreases after
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18 to 20 months and dogs can re-shed oocysts after challenge with tissue cysts
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(Gondim et al., 2005). In the present study we addressed several aspects of the
75
epidemiology of N. caninum in dogs. We examined the seroprevalence of N. caninum
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based on living conditions, sex, age and breed in 600 dogs from a study area in
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Pakistan.
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2. Materials and methods
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2.1. Dog populations sampled
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Four groups of dogs raised under different living conditions, from upper Punjab,
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Pakistan were evaluated for antibodies to N. caninum (Tables 1 & 2). The first group
82
was 138 dogs from 24 dairies (Table 2) located in 5 districts of Punjab. Groups 2 and
83
three consisted of 370 dogs sampled at the University Pet Centre, University of
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Veterinary and Animal Sciences, Lahore and private veterinary practices located in
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Lahore. Group 2 contained 294 dogs not showing neurological signs and Group 3
86
contained 76 dogs with various neurological signs. Group 4 consisted of 92 stray dogs
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acquired from environment after the consent of the Department of Epidemics and Public
88
Health, Lahore. Owners, practitioners, and researchers provided epidemiological
89
information on dog age, sex, and breed at the time of sampling (Tables 3 & 4).
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2.2. Sample collection and cELISA
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Blood samples were obtained aseptically from the cephalic vein. The serum was collected following centrifugation and stored at −20 °C until further processing at the
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Department of Parasitology, University of Veterinary and Animal Sciences, Lahore,
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Pakistan. Serum reactivity to N. caninum was evaluated using a commercially available
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competitive inhibition enzyme-linked immunosorbent assay kit (cELISA, VMRD,
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Pullman, Washington, USA). The sensitivity and specificity of this cELISA test for dog
97
samples were affirmed by the manufacturer to be 91.4% and 99.4%, respectively. The
98
samples were used undiluted as suggested by the manufacturer. Positive and negative
99
control samples were provided with the kit.
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Results were expressed as percentage of inhibition (PI).
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PI = 100- [(sample O.D. ×100)/ (mean negative control O.D.)].
102
Samples were considered positive when ≥30% inhibition was observed.
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Each dog’s sex was recorded as male or female regardless if the animal had
104
been neutered or spayed and the dogs were placed in one of 5 different age groups
105
(Table 3). Age groups were 6 months to 3 years (N=172), 3 to 6 years (N=96), 6-8 years
106
(N=109), greater than 8 years (N=109) and dogs with no age recorded (N=105).
107
2.4. Dog breeds
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Dog breed was determined using pedigree standards of the Kennel Club of
109
Pakistan (Table 4). Dog breeds were Bully (N=58), Alsatian (N=87), Bullterrier (N=79),
110
German shepherd (N=61), Pug (N=44), Labrador retriever (N=102), crossbreed (N=73)
111
and mongrel (N=96). A dog was placed in the crossbred group if it possessed the
112
phenotype of two distinct breeds and dogs were placed in the mongrel group if no
113
defined breed phenotype was apparent.
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2.5. Statistical examination The sample means and 95% confidence intervals were determined using Microsoft Excel 2007 software and Pearson’s x2 test was used to determine if
117
differences in prevalence were present in the 4 groups of dogs based on living
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conditions in relation to dairy cattle and by age, breed and sex using (SPSS for
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Windows, version 17.0; SPSS, Chicago, Illinois), and P < 0.05 was considered
120
significant prior to conduct of the test.
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3. Results
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Antibodies to N. caninum were found in 141 (23.5%, 95% CI = ± 2.99) of the 600
123
dogs examined in the present study (Tables 1 and 2). Significant differences (P < 0.05)
124
in prevalence were observed among different groups of dogs. The number of N.
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caninum positive samples were: 51 (36.9%, 95% CI = ± 3.09) of 138 dogs from dairies
126
in Group 1, 31(10.5%, 95% CI = ± 6.38) of 294 pet dogs without neurological signs in
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Group 2, 22 (28.9%, 95% CI = ± 6.70) of 76 pet dogs with neurological signs in Group 3,
128
and 37 (40.2%, 95% CI = ± 2.83) of 92 stray dogs in Group 4. Statistically, a significant
129
difference (P < 0.05) was observed between male (97of 366, 26.5% 95% CI = ± 3.40)
130
and female (44 of 234; 18.8%, 95% CI = ± 5.65) dogs for seroprevalence (Table 3).
131
There was no significant (P > 0.05) difference between dogs based on age but
132
prevalence was greater (30 of 109; 27.5%, 95% CI = ± 5.97) in animals older than 8
133
years of age (Table 3).
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Three of the 24 dairies examined did not have a resident population of dogs
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(Table 2) and the prevalence of N. caninum positive cows varied from 0 to 25% on
136
these 3 dairies (Table 2). Of the 21 dairies that had a resident dog population,
Page 6 of 16
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antibodies to N. caninum were found in dogs from 16 (76.2%, 95% CI = ± 15.84) dairies
138
while dogs from 5 (23.8%, 95% CI = ± 15.85) dairies were negative for antibodies to N.
139
caninum. The N. caninum prevalence in cows from these dairies ranged from 17 to 70%
140
(Table 2). A higher rate of seropositivity to N. caninum was found at dairies with
141
seropositive dogs (Table 2), while relatively low prevalence of N. caninum in cows from
142
dairies without dog populations and seronegative dogs. This difference was statistically
143
significant (P < 0.05).
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N. caninum positive dogs were found in Alsatian (12.6%, 95% CI = ± 11.1), Bully
145
(20.6%, 95% CI = ± 10.7), Pug (20.4%, 95% CI = ± 12.36), Bullterrier (18.9%, 95% CI =
146
± 9.72), German shepherd (31.4%, 95% CI = ± 6.60), Labrador Retriever (19.6%, 95%
147
CI = ± 8.34), Crossbreds (28.7%, 95% CI = ± 6.87) and Mongrel (35.4%, 95% CI = ±
148
4.23) breeds (Table 4). No significant (P > 0.05) difference among the prevalence of
149
exposure different dog breeds was observed.
150
4. Discussion
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significantly higher (P < 0.05) when compared to pet dogs, which were mainly from
153
urban areas. A higher prevalence of N. caninum antibodies in farm dogs than in urban
154
dogs was reported in a study from Japan (Sawada et al., 1998). Our findings indicate
155
that stray and dogs from dairies are at a higher risk of exposure to N. caninum than pet
156
dogs. In our study, a significantly higher seroprevalence was found in stray dogs as
157
compared to pet dogs and farm dogs. The higher rates of seropositivity to N. caninum in
158
stray dogs than farm dogs and pet dogs might be due to dietary habits, as outdoor
159
animals have direct contact with infective material from positive cattle or other
Page 7 of 16
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intermediate hosts and they have common access to dairies and contaminated
161
environment (Dijkstra et al., 2001, 2002a). The higher seroprevalence of N. caninum in
162
dogs from N. caninum positive dairies gives us a clue about the epidemiological
163
association between dogs and cattle. Previous studies have demonstrated that the
164
presence of dogs on cattle farms, can play an important role in the seropositivity and
165
bovine abortion (Bartels et al., 1999; Dijkstra et al., 2002b; Schares et al., 2003). Our
166
finding of a significant difference of prevalence between male and female dogs was not
167
expected based on previous studies (Trees et al., 1993; Barber et al., 1997; Sawada et
168
al., 1998). We found a higher prevalence rate in males than females, while earlier
169
reports found females were more likely to be seropositive than males (Wouda et al.,
170
1999; Fernandes et al., 2004). The prevalence of antibodies to N. caninum increased
171
with the age of animals but the increase was not significant (P > 0.05). These findings
172
agree with the observations made by others (Souza et al., 2002) and suggests postnatal
173
infection. The role of dog breed in relation to N. caninum prevalence is not clear. Most
174
of the reports find no specific breed susceptibility or a higher seroprevalence in mixed
175
breed dogs (Fernandes et al., 2004).
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Our findings reinforce the importance of dogs in the epidemiology of N. caninum
177
and support our hypothesis that “The prevalence of Neospora caninum antibodies is
178
different in dogs raised under different living conditions”.
179
Acknowledgments
180
The current study was financially supported by a grant from the Higher Education
181
Commission, Islamabad, Pakistan. The authors would like to thank Dr. Zia ullah Mughal
182
from the Pet Center, UVAS, Lahore, Dr. Muhammad Oneeb, Dr. Ahsan Mustafa,
Page 8 of 16
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Noman Nazir, Umer bacha and Muhammad Kashif Nazir for their technical help during
184
serum sampling.
185
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and Hammondia hammondi faeces collected from dogs in Germany. Int. J.
249
Parasitol. 35, 1525-1537.
250
Souza, S.L.P., Guimarães Jr., J.S., Ferreira, F., Dubey, J.P., Gennari, S.M., 2002. Prevalence of Neospora caninum antibodies in dogs from dairy cattle farms in
252
Paraná, Brazil. J. Parasitol. 88, 408–409.
cr
Trees, A.J., Guy, F., Tennant, B.J., Balfour, A.H., Dubey, J.P., 1993. Prevalence of
us
253
ip t
251
antibodies to Neospora caninum in a population of urban dogs in England. Vet.
255
Rec. 132, 125–6.
256
an
254
Wouda, W., Dijkstra, T., Kramer, A.M.H., van Maanen, C., Brinkhof, J.M., 1999. Seroepidemiological evidence for a relationship between Neospora caninum
258
infections in dogs and cattle. Int. J. Parasitol. 29, 1677–1682.
d te Ac ce p
259
M
257
Page 12 of 16
13 259 260
265
Number cELISA negative 87
Pets with no nervous signs Pets with nervous signs Stray dogs
294
31
263
76
22
54
us
CI 95%
36.9
±3.09
92
37
Total dogs
600
141
10.5
±6.38
28.9
±6.70
55
40.2
±2.83
459
23.5
±2.99
an
Farm dogs
Percent positive
cr
138
Number cELISA positive 51
M
Number examined
All four groups were significantly different (P < 0.05) from each other
d
264
Group
te
263
Table 1. Prevalence of Neospora caninum antibodies based on how dogs were raised.
Ac ce p
262
ip t
261
Page 13 of 16
14 265
Table 2: Seroprevalence of N. caninum in dogs raised on dairies. Number of cows tested (n)
Positive cattle (n)
Percentage (CI 95%)
Number of dogs at farm (n)
Number of dogs positive (n)
ip t
Number Number of cows in of herds the herd (n)
267
* Dairies with no dog population
268
† Dairies with dog population
Ac ce p
te
d
M
an
us
cr
266
†1 407 68 31 46(±1.34) 9 3 †2 963 41 13 32(±7.79) 6 0 †3 318 65 29 45(±1.71) 4 2 †4 74 23 4 17(±19.07) 2 0 8 3 †5 382 35 19 54(±1.87) *6 194 19 0 0(±6.04) 0 0 †7 289 61 33 54(±1.41) 4 2 †8 724 27 16 59(±4.80) 8 5 †9 267 49 18 37(±5.14) 5 1 †10 923 12 3 25(±20) 9 0 †11 348 6 4 66(±18.10) 13 5 †12 875 83 47 57(±2.12) 8 4 †13 219 53 20 38(±4.56) 4 1 †14 86 37 26 70(±9.11) 7 5 †15 694 62 14 23(±9.50) 2 0 †16 1047 119 52 44(±1.52) 9 3 †17 658 94 43 46(±1.14) 11 4 *18 417 38 5 13(±16.63) 0 0 †19 376 17 7 41(±6.05) 6 2 †20 113 7 2 28(±23.04) 3 0 †21 264 48 27 56(±2.40) 7 4 †22 49 5 2 40(±12.39) 5 2 †23 310 27 18 67(±9.06) 8 5 *24 22 4 1 25(±34.64) 0 0 138 51 Total 10019 1000 434 43.4(±0.57) Significant difference P < 0.05 of seropositivity at diaries where dogs were present
Seropositive dogs(CI 95%) 33(±15.7) 0(±3.39) 51(±1.38) 0(±1.95) 37(±12.7) 0 51(±1.38) 62(±11.7) 20(±37.8) 0(±4.15) 38(±9.22) 52(±1.96) 25(±34.3) 71(±22.0) 0(±1.95) 33(±15.7) 36(±11.7) 0 33(±19.2) 0(±2.40) 57(±7.3) 40(±12.3) 63(±12.7) 0 37(±3.06)
269 270 271
Page 14 of 16
15
Table 3 Seropositivity of dogs based on gender and age group
274
Number tested/ positive males
%age male (CI 95%)
172/33
19.2(±6.50)
101/25
24.7(±6.97)
3–6 years
96/18
18.7(±8.85)
49/13
26.5(±9.30)
6–8 years
118/29
24.6(±6.48)
68/22
>8 years
109/30
27.5(±5.97)
87/19
No age record
105/31
29.5(±5.54)
61/18
Total
600/141
23.5(±2.99)
97 of 366 male dogs examined
b
44 of 234 female dogs examined
%age female (CI 95%)
13.5(±13.17)
15.1(±16.83)
41/7
17.1(±14.24)
21.8(±8.38)
52/11
21.1(±11.10)
29.5(±7.27)
49/13
26.5(±9.30)
26.5/±3.40
234/44b
18.8(±5.65)
an
33/5
32.3(±5.94)
M
d
te
a
59/8
us
6 months3 years
366/97a
Number tested/positive females
Ac ce p
273
Seropositive (CI 95%)
ip t
Age group†
Number tested/ number positive
cr
272
Page 15 of 16
16
percent positive (%) 20.6
CI 95%
Bully
Number tested/number positive 58/12
Alsation
87/11
12.6
Bullterrier
79/15
18.8
ip t
Table 4: Prevalence of Neospora caninum antibodies in various dog breeds
German shephard
61/19
31.4
Pug
44/9
20.4
Labrador Retriever
102/20
278 279
73/21
Non-descript (Mongrel)
96/34
M
Crossbreds
cr
±6.60
±12.36
19.6
±8.34
19.9
±4.01
28.8
±6.87
35.4
±4.13
d
P > 0.05
±9.72
te
277
431/86
±11.11
Ac ce p
276
Total Purebred
±10.70
us
Purebred
Breed
an
275
Page 16 of 16
