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Study of the risk factors associated with  Neospora caninum  seroprevalence

in Algerian cattle populations

Farida Ghalmi a,⇑, Bernard China b, Asma Ghalmi a, Darifa Hammitouche a, Bertrand Losson c

a Ecole Nationale Supérieure Vétérinaire d’Alger, Algiers, Algeriab Institut Scientifique de Santé Publique, Brussels, Belgiumc Université de Liège, Faculté de Médecine Vétérinaire, Liège, Belgium

a r t i c l e i n f o

 Article history:

Received 13 May 2011

Accepted 12 December 2011

Keywords:

Neospora caninum

Cattle

Risk factors

Seroprevalence

Algeria

IFAT

a b s t r a c t

Bovine abortions due to  Neospora caninum  infection were reported worldwide. The situation in Algeria

was unknown. Forthe evaluation of theprevalence of N. caninum and its associated risk factors, 799 cattle

belonging to 87 farms of the north and northeast of Algeria were analyzed. The cattle were divided into

imported cattle, local cattle and improved cattle corresponding to breeding between imported and local

cattle. Sera were examined for the presence of  N. caninum  antibodies by indirect fluorescence antibody

test. The overall seroprevalence for the 87 farms was 52.87% (41.28–62.71%). The overall animal sero-

prevalence was 19.64% (16.82–22.45%). The seroprevalence of N. caninum in local cattle (34.28%) was sig-

nificantly higher ( p < 0.05) than in modern (16.04%) and improved (18.64%) cattle. The risk factors

analysis indicated that cattle population, geographical location, dog presence, season, global farm

hygiene or the presence of abortion were significantly associated with seroprevalence.

 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Neospora caninum is an obligate intracellular protozoan parasite

which has emerged as a cause of infectious abortion in cattle

worldwide (Dubey et al., 2007). In many countries  N. caninum   is

the most frequently diagnosed cause of bovine abortion (Dubey

and Lindsay, 1996).

Thebiological cycle of N. caninum is heteroxenous. Dogs, coyotes

and gray wolf (Canis lupus) are the only species recognized as defin-

itive hosts, in which the sexual phase of  N. caninum  cycle occurs,

resulting in the shedding of oocysts in the feces (Mc Allister et al.,

1998; Gondim et al., 2004; Dubey et al., 2011). It has been estab-

lished that bovinesare the major intermediate hosts of the parasite.

The routes of infection in cattle for N. caninum consist of vertical or

transplacental transmission, and horizontal or oral transmission

(Dubey, 1999). However, vertical transplacental infection from in-

fecteddams to their offspring appears to be the major natural route

of infection, and congenitally infected calves remain persistently

infected and can infect their offspring (Anderson et al., 1997).

The pathogenesis of neosporosis in cows is complex (Dubey

et al., 2006) and it is not well understood why some animals abort

and others do not. Infected cattle remain carriers of the parasite for

life and there are no clear signs of protective immunity ( Dubey,2003).

No fully effective vaccine or treatment is available to prevent or

cure the infection (Dubey et al., 2006). The study of risk factors of 

herd infection by N. caninum is important for the development and

implementation of measures to control bovine neosporosis (Dubey

et al., 2007).

For epidemiological studies, serology is the most used tech-

niques. The Indirect Fluorescence Antibody Test (IFAT) is often used

as a reference serological testfor thedetection of N. caninum (Dubey

and Lindsay, 1996). Many serologic surveys of  N. caninum  infection

in cattle worldwide and the risk factors for N. caninum in cattlehave

been discussed broadly in the literature (Dubey et al., 2007).

In Algeria, the cattle population is estimated to 1.6 million,

including 53.55% dairy cattle and representing an important food

source (MADR, 2008). Despite the massive imports of dairy cows

with high genetic potential, the milk production remains low. It is

estimated at 1.38 million tons in 2000 (Ghozlane et al., 2003) either

0.26% of the world production. This production is in total inade-

quacy with the continued strong growth of the population because

it covers only 40% of the needs (MADR, 2008). Although the

neosporosis has been diagnosed in the main dairy and beef cattle-

producing countries, no previous study has been published in the

literature about the disease in Maghreb. In Algeria, there is only

one published report available on its occurrence that indicated

20.47% seropositivity in 781 dogs in Algiers (Ghalmi et al., 2009b).

This demonstrates the wide diffusion of  N. caninum  in this area. A

study of significant factors that influence the distribution of 

0034-5288/$ - see front matter    2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.rvsc.2011.12.015

⇑ Corresponding author. Address: Ecole Nationale Supérieure Vétérinaire d’Alger,

BP 161 Hacène Badi, El Harrach, Algiers, Algeria. Tel.: +213 (0)21 525132; fax: +213

(0) 21 824481.

E-mail address:   fghalmi@yahoo.fr (F. Ghalmi).

Research in Veterinary Science 93 (2012) 655–661

Contents lists available at SciVerse ScienceDirect

Research in Veterinary Science

j o u r n a l h o m e p a g e :   w w w . e l s e v i e r . c o m / l o c a t e / r v s c

http://dx.doi.org/10.1016/j.rvsc.2011.12.015mailto:fghalmi@yahoo.frhttp://dx.doi.org/10.1016/j.rvsc.2011.12.015http://www.sciencedirect.com/science/journal/00345288http://www.elsevier.com/locate/rvschttp://www.elsevier.com/locate/rvschttp://www.sciencedirect.com/science/journal/00345288http://dx.doi.org/10.1016/j.rvsc.2011.12.015mailto:fghalmi@yahoo.frhttp://dx.doi.org/10.1016/j.rvsc.2011.12.015

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N. caninum in this area, could be useful to betterknowthe epidemi-

ology of  N. caninum  infection in Algerian cattle. Therefore, the aim

of this study was mainly to identify the risk factors associated with

N. caninum seroprevalencein different cattle populations in Algeria.

2. Material and methods

 2.1. Studied area

A cross-sectional study was designed to study the relationship

between the N. caninum serological status of dairy cattle and vari-

ous potential risk factors. The populations of interest were dairy

farms in two distinct regions of Algeria. The geographical location

of the farms is presented in Fig. 1. These farms were located either

in the northern regions (Algiers County) or in the eastern regions

(Bejaia and Setif). The sampled region represented around

25, 000 square kilometers.

Region-I was located in North Algeria (36   490 N; 3   00 E) and

contain about 727 cattle farms characterized mainly by imported

cattle with high genetic potential and region II was in the west

of the country (36 100 N; 5 240 E). The choice of region-II was jus-

tified by the fact that more than half of the local breed cattle arelocated in the eastern part of the country.

The distance between the two regions is approximately 400 km.

 2.2. Sampling procedures

The animals that were studied belonged to 87 dairy farms in

above cited regions. In the region of Algiers 72 farms were sampled

representing 10% of the farms present (n = 727).

A total of 799 serum samples were assessed. They represented

7% of the cattle population (n = 11,500) in the studied region

(INMV, 2009).

The minimum number of cattle to be tested on each farm was

established as 10 (Cannon and Roe, 1982), corresponding to the

probability of detecting at least one seropositive animal per farm.

On farms with less than 10 bovines, all of them were tested.

A stratified sampling was performed each stratum was a cattle

population. Three cattle populations were considered: imported

cattle (IPC), improved cattle (IMC) and local catte (LC). For each

population, the relative precision was similar: 25.5%, 22.2% and

27.2% for IPC, IMC and LC respectively.

 2.3. Collection of blood and epidemiological data

Sera from 799 bovines belonging to 87 farms were collected.

324 cattle from 30 farms were IPC, 370 cattle from 43 farms were

IMC and 105 cattle from 14 farms were LC.

The study period ranged from September 2006 to January 2009.

Blood samples were collected by coccygeal venipuncture, using

identified dry tubes. The serum was removed after centrifugation

at 2700 g  for 10 min and stored at  20 C until analysis.Simultaneously, blood was collected from dogs (n = 107) pres-

ent in 67 out of the 87 farms. Data for  N. caninum   seroprevalence

in those dogs were published previously (Ghalmi et al., 2009b).

An epidemiological questionnaire was fulfilled by farmers who

were asked to answer the questionnaire in order to obtain informa-

tion on the risk factors such as: farm location, herd size, cattle

breed, cattle age, presence of abortion in the five last years, pres-

ence of pregnant cows, stadium of pregnancy, presence of dogs,

Fig. 1.  Map of the analyzed regions. The regions in gray were those where the sampled farms are situated. The Algiers region has been divided into 4 sub-regions, Zeralda,Rouiba, Baraki, Birtouta. The regions of Bejaia and Setif constitute the eastern regions.

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presence of aborted cows. Additional information such as the date

of collection and the general hygiene of the farm were also col-

lected. A farm was considered as positive if at least one cow was

positive.

 2.4. Variables

Several factors that could influence the seropositivity of cattle

to   N. caninum  were analyzed. Considering the age of the cattle,

the cattle were divided in age classes (7 years).

For breeds, we considered the imported breeds (Holstein, Prim’

Holstein, Friesan, Montbeliarde, Fleckvieh), the improved cattle

which are crossed breeds and the local cattle that include Algerians

breeds (mainly Brown of Atlas) (Table 2).

For the areas agro-ecological, four classifications were per-

formed. The sampling areas were situated either in the seaside

region, in the Mitidja region, Tellian Atlas or Highlands. The Mitidja

is a great wet plain (1300 km2) in the south east of Algiers. In our

study the Mitidja is represented by the regions of Birtouta and

Blida (Fig. 1). The Tellian Atlas consists of a chain of mountains

along the coast of Mediterranean Sea (region of Bejaia).

The agricultural subdivisions (corresponding approximately to

Daïra) were considered (Table 2): Birtouta, Baraki, Rouiba and

Zeralda. In Algeria, Daïra includes several municipalities.

The gestation status of 461 cows were analyzed, 269 (58.35%)

were gestating and 192 (41.65%) were non-gestating.

The presence of dogs in farms and the seasons (the samples

were taken during the four seasons) were also considered.

The global hygiene of the visited farms was also taken into

account. We observed mainly the maintenance of the premises

and animals and we have classified the farms into three categories

(good, average and bad). We were inspired by European regulation

(EC 852/2004/BEHL15,  European parliament, 2004).

 2.5. Serological test 

Serum samples were analyzed by IFAT to determine the pres-

ence of   N. caninum   specific antibodies, as previously described

(Ghalmi et al., 2009a). In order to determine the IFAT cut off, a val-

idation step has been performed on 100 sera by comparing IFAT to

the validated ELISA herdcheck (IDEXX LABORATORIES) (Wu et al.,

2002) largely used in cattle for the determination of seropreva-

lence against   N. caninum   (Dubey et al., 2007). The sera were

titrated in a twofold dilution from 1/100 to 1/1600 and the speci-

ficity, the sensitivity and the coefficient of Kappa were calculated

using the ELISA has the reference method. The results indicated

that the best agreement was found when the 1/200 dilution was

used (sensitivity = 90%; specificity = 100% and Kappa = 0.82).

Therefore, a positive sample in IFAT was only considered at the

dilution 1/200 or upper.Reactions with tachyzoites presenting a total peripheral fluores-

cence were considered as positive. Positive and negative sera were

included on each slide. All the IFAT positive samples were con-

firmed by immunoblot as previously described (Ghalmi et al.,

2009b).

 2.6. Data analysis

Statistical differences in proportions were compared using the

 x2-test (Yates corrected) or Fisher’s exact test. The strength of the

association between serological status and the epidemiological fac-

tors assessed here was estimated by computing the odds ratio (OR)

for infection occurrence when the factor was present. A multiple

regression was also performed. Specificity, sensitivity, Kappa indexandthe 95%confidenceinterval (CI) were alsocalculated.A multiple

correspondence analysis(ACM) wasperformed. Theused software’s

were Winepiscope 2.0, MedCalc and XLstat. The level of statistical

significanceused was5%. If the p valueswere lower, it wasindicated

in the text.

3. Results

 3.1. Seroprevalence

The overall herd prevalence of N. caninum antibodies among the

87 farms was 52.87% (CI 95%: 41.28–62.71%). Antibodies to

N. caninum  were detected in 157 cattle out of 799, with a general

seroprevalence of 19.64%. When the different cattle populations

were considered, the seroprevalence values were 16.04%, 18.64%

and 34.28% for IPC, IMC and LC, respectively (Table 1).

The seroprevalence in LC was significantly ( p < 0.01) higher

than in the two others populations. All the positive samples were

confirmed as positive by immunoblot. Four profiles were detected

(Fig. 2), among the 157 tested sera, 19 (12.9%) 71 (48.3%), 41

(27.9%), 16 (10.9%) showed profile 1, profile 2, profile 3 and profile

4 respectively.

 3.2. Risk factors

Concerning the risk factors associated with seroprevalence, the

results of the statistics are summarized in Table 2. Considering the

age of the cattle, when the seroprevalence results were considered

all together, no significant association ( p > 0.05) between age clas-

ses and seroprevalence was shown. Nevertheless, the seropreva-

lence is significantly ( p < 0.05, OR = 0.64) less important in young

cattle ( 0.05). Nevertheless, The there

was a strong association between local breeds and seroprevalence

(OR = 2.47, p  < 0.05).

There was a significant association between agro-ecological

areas and the seroprevalence ( p < 0.05). The belonging to seaside

and Tellian Atlas areas were considered as a risk factor. When

the high titers were considered, there was a significantly higher

( p = 0.02) number of positive samples in Mitidja area for titer

1600 (data not shown). At the population level, the IPC and the

IMC were considered since the LC were mainly present in the east

part of Algeria. For IPC there was a significant difference ( p < 0.01)

between the seroprevalence and the seaside and Mitidja regionswhich is not the case for IMC ( p = 0.52).

When the regions were considered (Table 2), there was no sig-

nificant association between the farm location and the seropreva-

lence ( p > 0.05). The belonging to Zeralda (west of Algiers, Fig. 1),

Rouiba (east of Algiers) and Bejaia (east of Algeria) region was con-

sidered as a risk factor (OR > 1, p  < 0.05) although the belonging to

Birtouta region (south of Algiers) was shown to be protective

(OR < 1, p  < 0.05).

In our study, the seroprevalence in pregnant and non-pregnant

cows was not significantly different ( p > 0.05). The conclusion was

the same for all cattle populations. Among the 192 pregnant cows,

the pregnancy stage was known for 179 (93.23%) of them. The

seroprevalence was significantly ( p < 0.01) lower for early pregnant

cows (1–3 months) and significantly ( p < 0.05) higher for mid-pregnancy cows (4–6 months). If the cows with an abortion history

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(n = 54) were considered, there was a significant association with

seroprevalence. Interestingly, when the populations were consid-

ered, the OR was 9.77 (3.76–25.41), 9.43 (3.26–27.27) and 1.4

(0.43–4.54) for IPC, IMC and LC, respectively. Therefore, the associ-

ation between the seroprevalence and the fact that cows already

aborted in the past was only significant ( p  < 0.01) for IPC and

IMC but not for LC.

When the seasons were considered, the seroprevalence was sig-

nificantly higher in the spring time and significantly lower in

autumn. This observation was done for each studied populations

( p < 0.01). The distribution of high titers was also significantly dif-

ferent with the season since the highest titers were also observed

in the spring time ( p < 0.01).

The good hygiene status of the farm was a protective factor and

the bad hygiene status of the farm was a risk factor. This observa-

tion is independent of the studied population.

There was a significantly ( p  < 0.01) higher seroprevalence in

cattle in contact with dogs. At the population level, the association

between dogs and seroprevalence in cattle was only significant for

IPC (Table 3).

When a multiple regression analysis was performed only the

presence of dogs, the fact that the cow has aborted and the season

were significant ( p < 0.05).

A multiple correspondence analysis (Fig. 3) allowed visualizing

the factors associated with seropositivity or seronegativity. This

representation indicated that the variables closest to the positive

serology were the presence of abortion, the seaside region, the

bad hygiene of the farm, the spring season and the presence of 

dogs.

 Table 1

Seroprevalence in function in the populations studied.

Populations

of cattle

n (%) Negative Positive Seroprevalence

(%) (CI 95%)

IPC 324 (4.55) 272 52 16.04 (11.92–20.07)LC*

IMC 370 (46.30) 301 69 18.64 (14.55–22.64)LC*

LC 105 (13.14) 69 36 34.28 (25.01–43.54)IPC,IMC*

Total 799 642 157 19.64 (16.82–22.45)

* Seroprevalence significantly different ( p < 0.01) for the indicated populations.

 Table 2

Number of cattle, seroprevalence (%) for  N. caninum antibodies by general characteristics; odds ratio ( OR) for seropositive results, 95% confidence interval (CI) and p-value for thechi-squared test corrected for design effect.

Variable   n   Seroprevalence (CI) (%) OR (CI) Type of effect

 Age (months)   1.01 (0.77–1.33) NS No effect

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4. Discussion

N. caninum is a cause of abortion in cattle. Seroprevalence stud-

ies are the most useful epidemiological tool to investigate cattle

infection by this particular parasite. The identification of factors

that can modify the seroprevalence is a major clue. This identifica-

tion could lead to preventive actions allowing the reduction of cat-

tle infection. The risk factors for   N. caninum   infections were

investigated previously (Hemphill and Gottstein, 2000).

The fact that 52.87% of the studied herds have at least one sero-

positive animal to N. caninum suggests that N. caninum infection is

widespread among dairy herds in Algeria.

The study of risk factors allowed identifying some factors play-

ing an important role in the development of N. caninum infection in

cattle. The comparison of our data with data from others countries

is sometimes difficult due to the fact that these countries were

sometimes very different from Algeria regarding to the climate,

the breed or farming methods. But since there is no real data for

the NorthernAfrica countries, our work could be considered as pio-

neer and would constitute a comparison point for further studies.

Considering the age of cattle, the seroconversion risk can in-

crease with time or gestations ( Jensen et al., 1999; Rinaldi et al.,

2005) suggesting that the horizontal transmission is important in

some herds (Dubey et al., 2007). But the literature data are some-

times contradictory. In Spain the risk of infection increased with

age but in Sweden it is the opposite (Bartels et al., 2006). In our

study, age was not significantly correlated with seroprevalence.

At the population level, an effect was observed for imported cattle

but not for improved cattle or local cattle. In IPC, there is an in-

crease in seroprevalence with the age. For IMC and LC the sero-

prevalence was higher for

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agreement with data from the USA (Barling et al., 2001) where the

risk of seropositivity was higher in the spring. This result may be

explained by the fact that the sporulation of   N. caninum   oocysts

is temperature-dependent.These climatic observations can be related to the differences in

seroprevalences observed in the different geographic areas such as

the Mitidja or the seaside regions. It seems that seaside region is

more favorable to cattle infection by   N. caninum. Moreover, the

east of Algeria presented the highest levels of seroprevalence

which can be related either to the climate or to the abundance of 

LC in this region since the LC showed the highest seroprevalence.

Since   N. caninum   is a major abortion factor in cattle, the

parameters linked to abortion have been studied. The gestation

status was not a risk factor in our study since the pregnant and

non- pregnant cows showed the same level of seropositivity.

The same observation was made in all populations. This was sur-

prising since pregnancy leads to a modification of the immune re-

sponse with an increase of humoral immune response and adecrease in cellular response which is in favor of the multiplica-

tion of an intracellular pathogen such as  N. caninum. Pregnancy fa-

vors   N. caninum   reactivation leading to an increase in specific

antibodies production. But no significant difference in end titers

was observed between pregnant and non-pregnant cows. When

the gestation stage was investigated, a significant difference was

observed in seroprevalence between early stage (1–3 months)

and latter stages (>4 months). This indicates that the immune sys-

tem of pregnant cows in middle or late stage of gestation is more

exposed or more responding to N. caninum infection. These differ-

ences were observed in all three studied populations (IPC, IMC

and LC). When the aborted cows were analyzed, a clear associa-

tion between seroprevalence and abortion has been observed with

an odds ratio of 4.52. It is an indirect evidence that   N. caninumcould be involved in abortion in cows in Algeria. Interestingly, this

observation was also made for IPC and IMC but not for LC. This

indicated that LC are probably less susceptible to abort when they

are infected by  N. caninum.

Finally, a clear relationship was showed between the generalhygienic status of the farm and the seroprevalence in cattle. It is

therefore obvious that a better hygiene in the farm can reduce

the risk of infection by  N. caninum.

In conclusion, the analysis of risk factors leading to  N. caninum

infection in several cattle populations in Algeria indicated that

some factors such as the presence of dogs, the aborting cows, the

season, the region or the hygiene of the farm were risk factors

for N. caninum   infection. Taking together, all the above analyzed

risk factors for N. caninum  seroprevalence indicate that horizontal

infectionresulting from the ingestion of oocysts shed by dogs is the

most probable route of  N. caninum   infection in pastured cattle of 

areas in Algiers.

At the prevention level, it seems clear that it is better to avoid

contact between cattle or cattle food and dogs in the farm. It willallow blocking the natural biological cycle of the parasite. The

elimination of aborted fetuses and placentas is also important to

avoid contamination of the dogs.

 Acknowledgments

This work was supported by the Algerian higher education min-

istry and the Belgian technical cooperation. The author’s thank Dr

Anich for his critical reading of the manuscript.

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