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Veterinary Parasitology 182 (2011) 349– 351

Contents lists available at ScienceDirect

Veterinary Parasitology

jo u rn al hom epa ge : www.elsev ier .com/ locate /vetpar

hort communication

eroprevalence of Neospora caninum in free-range chickens (Gallusomesticus) from the Americas

. Martins, O.C.H. Kwok, J.P. Dubey ∗

nited States Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Parasitic Diseases Laboratory,uilding 1001, Beltsville, MD 20705-2350, USA

r t i c l e i n f o

rticle history:eceived 15 April 2011eceived in revised form 16 May 2011ccepted 17 May 2011

eywords:eospora caninum

a b s t r a c t

Neospora caninum and Toxoplasma gondii are biologically and morphologically similar coc-cidians with canids as definitive hosts for N. caninum and felids for T. gondii. Feral chickenshave been used as indicators of soil contamination with T. gondii oocysts because they feedfrom ground. In the present study we studied seroprevalence of N. caninum in free rangechickens from different countries in America as an indicator of soil contamination due toN. caninum oocysts. Antibodies to N. caninum were found in sera of 524 (39.5%) of 1324chickens using indirect fluorescent antibody test (IFAT, titer 1:25 or higher). Seropositive

hickens

allus domesticusmericaseroprevalence

chickens from different countries were: 18.5% of 97 from Mexico, 7.2% of 97 from USA,39.5% of 144 from Costa Rica, 71.5% of 102 from Grenada, 44% of 50 from Guatemala, 83.6%of 98 from Nicaragua, 58.1% of 55 from Argentina, 34.3% of 358 from Brazil, 62.3% of 85 fromChile, 11.2% of 62 from Colombia, 38.7% of 80 from Guyana, 18% of 50 from Peru and 21.7%

la. The

of 46 from Venezue

. Introduction

Neospora caninum is an important cause of illness in cat-le and dogs, and occasionally in other animals (Dubey andindsay, 1996). Canids (dogs, coyote, dingo) are its defini-ive hosts and excrete environmentally resistant oocystsn feces (McAllister et al., 1998; Gondim et al., 2004; Kingt al., 2010). How dogs become infected with N. caninum isot fully understood (Dubey et al., 2007d).

Age-related prevalence data indicate that most dogsecome infected after birth; higher prevalences haveeen documented in older versus younger dogs (Dubeyt al., 2007d). The ingestion of infected tissues is theost likely source of infection for carnivores. Theoreti-

ally, tissues of any animal containing tissue cysts can

e a source of infection for dogs. Tissues of infected preyf dogs may represent a logical source of infection; butiable parasite has not been isolated from potential dog

∗ Corresponding author. Tel.: +1 301 504 8128; fax: +1 301 504 9222.E-mail address: Jitender.dubey@ars.usda.gov (J.P. Dubey).

304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.vetpar.2011.05.023

results indicate widespread exposure of chickens to N. caninum.© 2011 Elsevier B.V. All rights reserved.

prey such as birds, rodents or lagomorphs (Dubey andSchares, in press). Recently, infected birds were suggestedas possible source of infection for N. caninum infectionin dogs. Antibodies to N. caninum were found in 23.5%of 200 free-range and 1.5% of 200 indoor chickens fromBrazil, and N. caninum DNA was detected in 6 of 10seropositive chickens (Costa et al., 2008). Experimentally,chickens older than one week inoculated with N. caninumtachyzoites intraperitoneally developed transient infec-tion. Parasites or antibodies were not demonstrable 60days p.i. However, inoculation of chicken with embry-onated eggs produced patent infection and the infectedchorioallantoic membranes of these eggs induced oocystshedding when fed to a dog (Furuta et al., 2007). Thesusceptibility of chicken eggs for N. caninum infectionwas recently confirmed by Mansourian et al. (2009) usingboiler chicken embryonated eggs. Pigeons (Columba livia)were also successfully infected with N. caninum tachyzoites

and are also putative natural reservoirs for N. caninum(Mineo et al., 2009). Gondim et al. (2010) found N. can-inum DNA in 3 of 40 sparrows (Passer domesticus) fromBrazil.

350 J. Martins et al. / Veterinary Parasitology 182 (2011) 349– 351

Table 1Serological prevalence of Neospora caninum in free-range chickens from different countries using the immunofluorescent antibody test (IFAT).

American continent Country No. chicken tested No. positive (%) No. chickens with IFATtitres

25 ≥100

North Mexico (Dubey et al., 2004b) 97 18 (18.5) 11 7USA

Illinois (Dubey et al., 2007b) 10 2 (20.0) 2 0Ohio (Dubey et al., 2003c) 87 5 (5.7) 2 3

Subtotal USA 97 7 (7.2) 4 3Central Costa Rica (Dubey et al., 2006c) 144 57 (39.5) 44 13

Grenada (Dubey et al., 2005d) 102 73 (71.5) 57 16Guatemala (Dubey et al., 2005e) 50 22 (44) 14 8Nicaragua (Dubey et al., 2006a) 98 82 (83.6) 22 60

South ArgentinaLa Plata (Dubey et al., 2003a) 29 15 (51.7) 7 8Santiago (Dubey et al., 2005b) 26 17 (65.3) 13 4

Subtotal Argentina 55 32 (58.1) 20 12Brazil

Amazon (Dubey et al., 2006d) 50 35 (70.0) 13 22Fernando de Noronha (Dubey et al., 2010) 50 7 (14.0) 5 2Pará (Dubey et al., 2007c) 38 9 (23.6) 5 4Paraná (Dubey et al., 2003b) 40 11 (27.5) 9 2Rio de Janeiro (Silva et al., 2003) 115 45 (39.1) 27 18Rio Grande do Sul (Dubey et al., 2007c) 50 13 (26.0) 4 9São Paulo (Dubey et al., 2002) 15 3 (20.0) 2 1

Subtotal Brazil 358 123 (34.3) 65 58Chile (Dubey et al., 2006b) 85 53 (62.3) 33 20Colombia (Dubey et al., 2005c) 62 7 (11.2) 6 1Guyana (Dubey et al., 2007a) 80 31 (38.7) 23 8

1

Peru (Dubey et al., 2004a)

Venezuela (Dubey et al., 2005a)Total (%)

Feral chickens have been used as indicators of soil con-tamination with Toxoplasma gondii oocysts because theyfeed from ground (Dubey, 2010). Because N. caninum is bio-logically similar to T. gondii in the present study we studiedseroprevalence of N. caninum in free range chickens fromdifferent countries in America as indicator of soil contam-ination due to N. caninum oocysts.

2. Materials and methods

2.1. Sera sample

During the course of a study on worldwide prevalenceof T. gondii in free-range chickens, sera from chickens wereobtained from different properties that were at least 1 kmapart (Dubey, 2010). The same sera were examined in thepresent study. A total of these 1324 sera from countries inNorth, Central and South America were tested here for N.caninum antibodies (Table 1).

2.2. Immunofluorescent antibody test (IFAT)

For preparation of antigen slides, cell cultures infectedwith tachyzoites of the NC-1 strain (Dubey et al., 1988)were scraped, disrupted by passage through 27 G needlesand centrifuged at 2000 rpm (1400 × g) for 10 min. The pel-let was resuspended in aqueous 0.85% NaCl (saline), and

the tachyzoite suspension was dispensed on to 12-welledPCR slides (LabSource Inc., Chicago, IL, USA), dried at roomtemperature, fixed with 100% methanol, and after dryingthe slides were stored at −20 ◦C.

50 9 (18.0) 3 646 10 (21.7) 9 1

324 524 (39.5) 311 (23.4) 213 (16.0)

The IFAT procedure was same as previously describedby Dubey et al. (1988). A commercial fluorescein-labeledanti-chicken IgG (KPL, Gaithersburg, MD, USA) was usedas the secondary antibody. Serum from an experimentallyinfected chicken with 104 tachyzoites of the strain of NC-1was used as positive control. A negative control serum wasobtained from a chicken tested previously to N. caninum. Anarbitrary cut off of 1:25 has been chosen. Both positive andnegative controls were used on each slide. Each sample wasscreened using 1:25 dilution and only a bright fluorescenceof the whole tachyzoite surface was considered as a positiveresult. All sera positive at 1:25 dilution were retested at1:100 dilution.

3. Results and discussion

Antibodies to N. caninum were found in 39.5% of 1324chickens from the Americas with highest prevalence inchickens from Nicaragua (Table 1). It is noteworthy that16% of sera were positive at 1:100 serum dilution. Atpresent nothing is known of specificity of any serologi-cal test for the diagnosis of patent N. caninum infectionin chickens because the parasite has not been isolatedfrom naturally-infected birds, and chickens experimen-tally infected with N. caninum developed only intransientinfection and became seronegative by 60 day p.i. (Furuta

et al., 2007). Our results indicate a widespread expo-sure of free range chickens to N. caninum. The role ofchickens in the epidemiology of N. caninum needs furtherinvestigation.

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onflict of interest

The authors have no conflict of interest.

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