Neosporosis in Iran; recent evidences and perspectives

Document Type : Review article

Author

Razi Vaccine and Serum Research Institute, Shiraz, Iran

Abstract

Neospora caninum is considered as a cyst forming coccidian parasite nearly related to Toxoplasma gondii. Shortly after discovery of N. caninum,neosporosis has identified as a notable infectious disease of both cattle and dogs worldwide, which it frequently leads to clinical infections in warm-blooded animals such as horses, goats, sheep, camels and deer. More importantly, in cattle industry, it is mentioned one of the important causes of abortion in too many countries. Economic losses from N. caninum infection are associated with abortion, stillbirth, neonatal mortality, increased culling and reduced milk yield in cattle industry in the world. Different diagnostic tools can be used for detection of N. caninum infec­tion including histology, polymerase chain reaction and serology. Because of the intimately biologic relationship of N. caninum to Toxoplasma gondii and since non-human primates had been experimentally infected, an issue of concern is that N. caninum might be zoonotic. Previously, some researchers successfully infected two rhesus monkeys (Macaca mulata) with N. caninum experimentally,which reinforces the concern about the zoonotic potential of this disease. In the one last decade, N. caninum has been extensively investigated in Iran. In this sense, the present paper reviews recent knowledge on biology, life cycle, transmission and zoonotic aspects of N. caninum.  Attention is also paid to presence of N. caninum infection in the last decade in Iran.
 

Keywords


Introduction

In 1984 in Norway was observed an encephalomyelitis and myositis in dogs (Bjerkås et al., 1984) and later in calves with myeloencephalitis (Parish et al., 1987) due to unidentified protozoan parasite which it simulated Toxoplasma gondii but did not respond to the antibodies of T. gondii. The parasite was named and described later as a new discovered genus and species Neospora caninum, which has been ordered in the family Sarcocystidae as a sister group to Toxoplasma

in the phylum Apicomplexa (Dubey et al., 2007). As results of many studies that had been conducted on N. caninum in the past two decades on warm-blooded animals, including many domestic and wildlife species, now it is investigated as a cause of severe canine neuromuscular disease, and neonatal mortality and abortion in cattle, leading to expanding economic losses to the dairy industries (Dubey et al., 2007). Moreover, clinical neosporosis has been known in goats, sheep, white- tailed deer, rhinoceros, water buffaloes, llamas, alpacas, and horses. N. caninum antibodies have also been demonstrated in the serum samples of raccoons, camels, pigs, horses, foxes, cyotes, and felids (Dubey et al., 2002). Previous studies proposed that neosporosis had been presented as a critical disease of dogs and cattle worldwide (Dubey et al., 2007). For this reason, in the last decade, much research has been performed on N. caninum because of its emphasis as a veterinary pathogen in Iran like many countries. Therefore, the present review describes the present knowledge on the biology, life cycle, transmission, and zoonotic aspects of N. caninum and, with especial attention, summarizes the studies of presence specific antibodies, DNA detection, species affected, and its geographical distribution in the last decade in Iran.

 

Life cycle, biology and transmission of N. caninum

Among various hosts of N. caninum as previously mentioned, dogs are assessed as both the intermediate and definitive host for this parasite (Dubey et al., 2007). Previously, grey wolves (Canis lupus) were also affirmed to be natural definitive hosts of N. caninum by shedding of lasting N. caninum oocysts in their feces (Dubey et al., 2011). Besides, coyotes (Canis latrans) (Gondim et al., 2004) and Australian dingoes (Canis lupus dingo) (King et al., 2010), and grey wolves (Canis lupus lupus) (Dubey et al., 2011) have also been experimentally recognized as definitive hosts of N. caninum. Cattle are interestingly the most prevalent intermediate (middle) host of N. caninum; however, a large number of other warm-blooded animals may act as intermediate hosts (fig. 1). Importantly, the presence of birds on dairy farms mentioned as a notable risk factor for this infection and has been related to spread of abortion (Donahoe et al., 2015). Indeed, it has been indicated that chickens may be an admissible intermediate host for N. caninum since parasite DNA was revealed in tissue specimens of outdoor birds (Costa et al., 2008). Recent documents demonstrated high prevalence of N. caninum infection in pigeons and also in free ranging chickens in Iran and thereupon it seems that soil contamination because of the shedding N.

caninum oocysts, since the birds feed from the ground, and determined that the meat from these birds can be a main source for this infection in dogs (Sayari et al., 2014; Bahrami et al., 2016). Moreover, several studies demonstrated susceptibility of different embryonated eggs of domestic birds to N. caninum infection and for this reason, at present, these extensively use for experimental studies on N. caninum (Furuta et al., 2007; Namavari et al., 2011; Mansourian et al., 2015). Tissues of infected animals or feed and water contaminated by these oocysts can infect the intermediate hosts (Donahoe et al., 2015). While definitive hosts become infected by ingesting contaminated tissues of intermediate hosts and can shed oocysts via their faeces (Donahoe et al., 2015; Dubey et al., 2007).

Tachyzoites, bradyzoites (tissue cysts) and oocysts have been identified as the infective stages of the parasite (Dubey et al., 2007). All three mentioned infectious stages are implicated in the transmission of the parasite. Tissue cysts and tachyzoites are asexual stages of the parasite, which found in different cell types and organs of infected hosts (intermediate and definitive host), frequently in the spinal cord and brain (Dubey et al., 2007).

Tachyzoites had also been indicated in the placenta of pregnant cattle. Those are lunate-shaped, show a central nucleus without amylopectin granules and measure approximately 2×6 µm. They propagate rapidly within cells and can contaminate various cell types, such as neural cells, myocytes, renal cells, vascular endothelial cells, dust cells, hepatocytes, and placental trophoblasts (Dubey et al., 2007). Tissue cysts can differ substantially in size, belong to the number of bradyzoites within them. Tissue cysts were found in dogs up to 4 µm thick with a cyst wall up to 107 µm in diameter (Dubey et al., 2007). Bradyzoites replicate slowly (unlike tachyzoites) encysted stages of the parasite, which are slender, have a terminally placed nucleus, and measure approximately 6.5×1.5 µm, and possess a few amylopectin granules, which react with the periodic acid Schiff (PAS) and stain red (Dubey et al., 2004). Dogs as definitive hosts excrete N. caninum oocysts in the unsporulated form in their faeces, which measure approximately10×12 µm. After sporulation, each oocyst comprises two sporocysts, each of which includes four sporozoites, exclusively 6.5×2 µm (Dubey et al., 2007).

Fig. 1. Life cycle of N. caninum


N. caninum
can be transmitted horizontally (also termed postnatally or laterally) and also can be transmitted vertically (also termed transplacentally or congenitally). Two forms of vertical transmission were previously indicated: exogenous transplacental transmission and endogenous transplacental transmission (Williams et al., 2009). Horizontal transmission results through eating of tissues contain tachyzoites and/or tissue cysts (bradyzoites) or by consumption of food or drinking water contaminated with sporulated oocysts. While vertical transmission happens when tachyzoites from the dam pass the placenta (Dubey et al., 2007), which preserve spreading in a herd for several years. Exogenous transplacental transmission occurs subsequent eating of sporulated oocysts by ordinary cattle and is related with epizootic abortion storms within a herd (Williams et al., 2009). Endogenous transplacental transmission accompanies recrudescence

infection in a persistently contaminated cow during pregnancy. Horizontal transmission of neonatal animals after birth is significantly needed to retain infection within a herd. While vertical transmission alone cannot sustain infection within herds, which is suggested the main route of transmission in cattle and other domesticated Bovidae species such as the water buffalo (Bubalus bubalis) (Chryssafidis et al., 2011). Domestic dogs and some wild canids, as the only known definitive host of N. caninum, become infected by consuming tissues or placenta from infected cattle with N. caninum and shed the unsporulated oocysts in their faeces during two weeks after that, and sporulate outside the host within 24 hours (Dubey et al., 2007Gondim et al., 2004).

 

Economic impact

In general, less is known about economic losses of neosporosis in cattle industry in the world but losses are computed in milliards of dollars. Reported rates of congenital neosporosis differ, with report of 40.7% up to 95% (Reichel et al., 2013). A previous study indicated a congenital infection rate in heifers, in second, third and fourth parity cows 80%, 71%, 67%, 66%, respectively (Dijkstra et al., 2003). It is believed that dairy cattle generally show a higher rate of infection with N. caninum than beef cattle (Reichel et al., 2013). Therefore, the economic losses will link to the direct price and cost of fetuses lost which is variable accordingly to the age and genetic potential of the dam and also the productive potential of the progeny (Dubey et al., 2007). Moreover, the diagnostic methods of neosporosis-associated abortions are tough and costly (Ortega-Mora et al., 2006). Indirect costs additionally involve professional price and costs related with rebreeding, feasible loss of milk production, and renewal costs of aborted cows (Dijkstra et al., 2003). Neosporosis can result of other economic losses, such as stillbirth or birth of weak calves (Trees et al., 1999). Regarding to the lack of clinical neosporosis in calves more than two months of age, to date, there is no clear document of N. caninum-related incidence in adult cows (Dubey et al., 2007). In Iran, seroepidemilogical reports have shown the high prevalence of neospora infection, especially in dairy cattle (33%, 37%, 46%) (Namavari et al., 2010; Hajikolaei et al., 2007; Razmi et al., 2006), and dogs (Malmasi et al., 2006; Haddadzadeh et al., 2007; Khordadmehr et al., 2012). Also, N. caninum infection was detected as a notable causative agent of bovine abortion in dairy farms in Iran (Razmi et al., 2006; Sadrebazzaz et al., 2007; Salehi et al., 2009; Nematollahi et al., 2013; Gharekhani and Yakhchali, 2019). As indicated recently, the transplacental transmission rate of neosporosis infection in dairy cattle had

estimated as 52% in Iran (Mashhad area- north east of Iran) (Razmi et al., 2013). Therefore, it seems that there are large direct and indirect economic losses (such as congenital infection, abortion, stillbirth or birth of weak calves, loss of milk production, and substitution costs for culled aborted cows) due to neosporosis to cattle industry in Iran. Although, the economic important of the infection has not been established in Iran yet.   

 

Zoonotic Aspects of N. caninum

Until 1988, most of the neosporosisinfection had been misdiagnosed as toxoplasmosis (Dubey et al., 2007). Later, major differences were subsequently indicated that investigate the two parasites regarding to their natural host, virulence factors, antigenicity, and pathogenicity (Dubey et al., 2007). Application of comparative genomics and transcriptomic analyses had also been proposed for differential diagnosis of these two similar parasites(Reichel et al., 2013). In comparison between neosporosis and toxoplasmosis, T. gondii is known as a main disease of sheep and humans, and not of cattle, but neosporosis is considered as a severe disease in cattle, not of sheep, and to date, there is no strong reports for human infection. Previously, some researchers successfully infected the rhesus monkeys (Macaca mulata) with N. caninum experimentally(Barr et al., 1994), which reinforces the concern about the zoonotic potential of this disease. However, only low levels of antibodies have been observed (particularly in immunocompromised populations), and neither the parasite nor its DNA were observed in human tissues. Seroprevalences findings of N. caninum in humans are summarized in Table 1. Although, these results are not frequently comparable because of various serologic assays and different cut-off values used. Recently, immunoglobulin G antibodies to N. caninum was predominantly determined in patients with HIV infection (38%) and patients with neurological disorders (18%), while newborns (5%) and healthy persons (6%) presented lower seropositivity rates. Apparently, seropositivity to N. caninum was markedly related with seropositivity to T. gondiiin both HIV-infected patients and patients with neurological illnesses (Lobato et al., 2006). Older literature reported low level IFAT antibodies in sera from blood donors in California and people (women with repeated abortions and farm workers) in England (6.7% and 0.4%, respectively) (Tranas et al., 1999; Trees and Williams, 2000). Currently, nothing is recognized about the seroprevalences of N. caninum in humans in Iran.

Country

Source of sample

No. tested

Test

%

positive

Ref.

Korea

Blood donors

172

IFAT

ELISA

IB

6.7

Nam et al., 1998

Denmark

Repeated miscarriage

76

IFAT

ELISA

IB

0

Petersen et al., 1999

North of Ireland

Blood donors

247

IFAT

8

Grahamet al., 1999

United States

Blood donors

1029

IFAT

IB

6.7

1.55

Tranas et al., 1999

United Kingdom

Farm workers and women with miscarriage

500

IFAT

0

Trees et aland Williams, 2000

 

Farm workers

General papulation

518

3232

ELISA

 

3

0.57

McCann et al., 2008

Brazil

AIDS

Neurologic disorder

Newborns

61

50

91

ELISA, IFAT, IB

ELISA, IFAT, IB

ELISA, IFAT, IB

38

18

5

Lobato et al., 2006

Egypt

Pregnant women

101

ELISA

7.92

Ibrahim et al., 2009

Seroprevalence, Prevalence, and Isolation Studies of Neospora caninum in Iran

In 2006, seroepidemiology of N. caninum infection was reported in dairy cattle herds in Iran (Khorasan Province, Mashhad area) using ELISA and interestingly, 46% of the examined animals were seropositive for neosporosis infection (Razmi et al., 2006). They believed that abortion was remarkably associated with seropositivity of cattle. Also, their results indicated that neospora infection is widespread in Iran like as many countries. After that, many studies have been conducted on N. caninum to date especially in dairy cattle (because of economic important) and dogs (as a confirmed definitive host) and are summarized in Table 2 and Table 3. Recorded rates of infection vary with observation of 7.8% (Heidari et al., 2014) up to 33.3% (Namavari et al., 2012) in cattle by ELISA. While, infection rates of the dogs are10.6% (Khanmohammadi et al., 2011) up to 44.4% (Khordadmehr et al., 2012) by IFAT and NAT, respectively. In the most reports, no sex predisposition was detected in the examined animals, but it seems age and living places are the important risk factors for N. caninum infections. For example, significant difference was detected regarding infection in industrial (43.9%) and rural cattle (25.8%) (Youssefi et al., 2009). Another study reported that house hold dogs had a lower rate of infection (8.65%) than stray and shepherd dogs (43.35%) (Hosseininejad and Hosseini, 2011). Although, in horse, it was found higher in

riding club samples (42.2%) rather rural samples (40%) (Gharekhani et al., 2013). In 2007, N. caninum associated bovine abortion was identified in Iran, which was primarily diagnosed by PCR and then confirmed by histopathology and IHC methods. These Iranian researchers observed a thick- walled (2µm) cyst of N. caninum with 50 µm diameter in one of the IHC- positive brain. Therefore, based on their findings, they stated that neosporosis is a main cause of abortion in dairy cattle of Iran (Razmi et al., 2007). Later, N. caninum was isolated from an aborted fetus in seropositive cattle, which was determined as Nc-Iran that recorded under the accession number FJ655914 in the GenBank database (Salehi et al., 2012). Recently, Pouramini et al. reported the presence of N. caninum in CSF (26.2%), brain (19%), and skeletal muscle (13.42%) of asymptomatic infected stray dogs in Tehran, Iran (Pouramini et al., 2017). Importantly, it was also proposed that close contact to infected farm dogs, carnivores, rodents and poultry could be important risk factors for the occurrence of N. caninum-associated abortion in dairy cattle (Gharekhani and Yakhchali, 2019).

Table 2. Seroprevalence, prevalence, and isolation studies of Neospora caninum in dogs in Iran.

Location (Province)

No. tested

Tissue/source

Test

%positive

Ref.

Tehran

100

Serum

ELISA

33

Malmasi et al., 2006

Tehran

 

Tehran

103

Serum

 

CSF

Brain

Skeletal muscle

IFAT

19.4

 

26.2

19

13.42

Haddadzadeh et al., 2007

Pouramini et al., 2017

Khorasan

174

Feces

PCR

1.1

Razmi, 2009

Ardebil (Meshkin- Shahr)

171

Serum

ELISA

30.4

Sharifdiniet al., 2011

Chaharmahal va Bakhtiari

Isfahan

Khuzestan

248

200

100

Serum

Serum

Serum

ELISA

ELISA

ELISA

29

Hosseininejadet al., 2011

East Azarbaijan (Sarab district)

384

Serum

IFAT

10.6

Khanmohammadi et al., 2011

Fars

180

Serum

ELISA

MAT

54.62

44.44

Khordadmehr et al., 2012

Lorestan

428

Feces

PCR

2.1

Dalimi et al., 2014

Hamedan

270

Serum

IFAT

27

Gharekhani et al., 2014

 

185

Serum

ELISA

8.65

Gharekhani and Yakhchali, 2019

 

Table 3. Seroprevalence, prevalence, and isolation studies of Neospora caninum in different intermediate hosts in Iran.

Host

Location

No. tested

Tissue/source

Test

%positive

Ref.

Cattle

Khorasan

337

Serum

ELISA

46

Razmi et al., 2006

 

Khorasan

100

Brain of aborted fetuses

PCR

13

Razmi et al., 2007

 

Khorasan

12

Brain of aborted fetuses

PCR

IFAT

33

33

Sadrebazzaz et al., 2007

 

Khorasan

151

151

Brain of aborted fetuses

Fetal fluid

PCR

ELISA

11.9

9.9

Razmi et al., 2010

 

Tehran

12

7

Brain of aborted fetuses

Placentas of seropositive dams

PCR

PCR

100

71.4

Salehi et al., 2009

 

Kerman

285

Serum

ELISA

12.6

Nourollahi-Fard et al., 2008

 

Mazandaran

237

Serum

ELISA

32

Razmi et al., 2007

 

Fars

135

Serum

ELISA

33.3

Namavari et al., 2012

 

East Azarbaijan

266

Serum

ELISA

10.5

Nematollahi et al., 2011

 

East Azarbaijan

76

14

Serum

Brain of aborted fetuses

ELISA

PCR

18.4

42.8

Nematollahi et al., 2013

 

Hamedan

1046

 

476

Serum

 

Serum

ELISA

 

ELISA

17.4

 

24.8

Gharekhani et al., 2014

Gharekhani and Yakhchali, 2019

 

Kurdistan

368

Serum

ELISA

7.8

Heidari et al., 2014

 

Various regions

395

Brain of aborted fetuses

PCR

45%

Kamali et al., 2014

 

Various regions

175

Semen

PCR

17.14

Sharifzadeh et al., 2012

 

Various regions

57

Semen

 

PCR

10.53

Doosti et al., 2015

 

Neishabour

100

Serum

ELISA

26

Nourollahi-Fard et al., 2017

 

Sistan

184

Serum

ELISA

3.8

Noori et al., 2019

Water buffalo (Babalus bubalis)

 

Khuzestan

 

West Azarbaijan

181

 

83

Serum

 

Serum

ELISA

 

ELISA

PCR

37

 

19.27

39.75

Hajikolaei et al., 2007

Rezvan et al., 2019

Sheep

Lorestan

586

Serum

ELISA

2.8

Ezatpour et al., 2013

 

Hamedan

358

Serum

ELISA

2.2

Gharekhani et al., 2013

 

Khuzestan

550

Serum

ELISA

38

Gharekhani et al., 2018

Goat

Hamedan

450

Serum

ELISA

6.2

Gharekhani et al., 2016

 

Khuzestan

108

Serum

ELISA

10.8

Gharekhani et al., 2018

Horse

(Equus caballus)

Hamedan

120

Serum

MAT

40.8

Gharekhani et al., 2013

 

Fars

200

serum

MAT

32

Moraveji et al., 2011

Donkey

(Equus africanusasinus)

Hamedan

100

serum

MAT

52

Gharekhani et al., 2013

Camel

(Camelus dromedarius)

Yazd

254

serum

NAT

3.94

Hamidinejat et al., 2013

Cat

Khuzestan

100

serum

MAT

19

Hamidinejat et al., 2011

Chicken

(Gallus domesticus)

Fars

150

serum

MAT

17.33

Sayari et al., 2014

Pigeon

Khuzestan

102

102

Serum

Brain

NAT

PCR

30.39

9.8

Bahrami et al., 2016

Sparrow

(Passer domesticus)

Khuzestan

210

brain

PCR

2.8

Bahrami et al., 2015

Diagnostic assays for identification of N. caninum and neosporosis infection

Many diagnostic tools had been used with different rating of consequences for tracing of N. caninum and its infection in domestic animals, wildlife species, birds and humans (Dubey et al., 2011).

1)      Serological techniques

 Most of the research had employed serological techniques, which are beneficial diagnostic methods to identified animals for presence of N. caninum disposal. In this regard, it was stated that serum samples of inspected cases are the most commonly used specimens for identification of N. caninum antibodies in adult animals.  Mostly, because of high antibody levels in aborted cows with neospora-infected fetuses (Wouda et al., 1998), identification of antibodies to neospora in the serum of acute abortion cases, particularly when sera taken within two weeks of the abortion, can be a noteworthy diagnostic tool. Moreover, evaluation of fetal serum or fetal body fluid (especially peritoneal fluid) for neospora antibodies can assist in diagnosing the infection in five months and older fetuses (Wouda et al., 1998). Serological test may be used on newborn calves before feeding colostrum to identify whether they are congenitally infected. Evaluation of dams and their offspring seroprevalences are useful to compute the frequency of transplacental transmission of infection (Dubey et al., 2007). In an abortion storm, taking blood samples immediately of all animals can be advisable for detection of endemic infection. Since, most abortions arise several weeks behind an acute infection, it is more helpful when the paired serology samples are taken at abortion and also three weeks later (Wouda et al., 1998). Also, individual and bulk milk samples of dairy cows can be applied as further samples for either screening or diagnosis of the infection (Varcasia et al., 2006).

Some serologic assays can be used to identified N. caninum antibodies, such as various types of ELISAs, IFAT (indirect fluorescent antibody test), NAT (neosporaagglutination test), and immunoblot (IB) is useful for detecting N. caninum-specific antigen/antibody with a high sensitivity and specificity. Besides, IgG avidity ELISAs had been considered to differ between chronic and acute neosporosis (Björkman et al., 2005). It is believed that the seroprevalence results are not analogous between different studies because of the use of various techniques, variation in research object, methodology, sample size, samples source, and data commentary (Dubey et al., 2011). In serological examinations, titer and absorbance values are dependent on some factors, such as antigen composition, secondary antibodies, and other using reagents. Additionally, cut-off levels may be indiscriminately designated to assign sensitivity and specificity demanded for a special application (Dubey et al., 2007).

In the recent years, numerous determination of N. caninum seroprevalence have been performed in domestic animals and birds in Iran (Table 2, 3). In these studies, the most frequent used serological methods were ELISA and NAT. Recently, a disperse dye immunoassay method (DDIA) was provided and evaluated for rapid detection of antibodies against N. caninum in cattle and no marked differences were found between DDIA and ELISA, which provides an economic, modest, rap­id, and authentic test for diagnosis of infection in cattle (Selahi et al., 2013). Moreover, Iranian researchers developed an indirect ELISA assay using N. caninum surface antigen (P38) for the sensitive and specific detection of infection in dog colonies. Their findings demonstrated that a favorable sensitivity (100%) and specificity (97.9%) were assessed for SIn cut-off point of 0.23 (Hosseininejad et al., 2010). Also, it was shown that two protein bands with 45 and 41 kDa molecular weight are the most important antigens investigated in Western blotting, in seropositive aborted cows (Nematollahi et al., 2010). Another research also indicated that the LAT with recombinant N. caninum surface antigen 1 (rNcSAG1) might be a rapid, easy, relatively inexpensive, and adequate diagnostic test for detection of specific antibodies under field conditions (Moraveji et al., 2012). In addition, they believed that sanitation of rNcSAG1 purification may decrease possible false positive results and so enhance the agreement rating between the LAT and ELISA. The results of another study after cloning and expression of N. caninum dense granule protein 7 (NcGRA7) in E. coli approved that recombinant NcGRA7 with pMAL-c2X vector might be appropriate for expanding of diagnostic procedures (Kefayat et al., 2012). New literature demonstrated that NcGRA7-based ELISA suggesting utilized a novel fragment of genomic DNA is a suitable tool for epidemiological and screening purposes on cattle and water buffaloes herds (Hamidinejat et al., 2015).

1)      Polymerase chain reaction (PCR)

 It is presented as a reliable sensitive and specific laboratory technique for identification of N. caninum DNA in a variety of tissues from aborted bovine fetuses, such as liver, heart, spinal cord, brain, placentas, and amniotic fluid of infected cattle (Salehi et al., 2009; Nematollahi et al., 2013; Salehi  et al., 2012). Moreover, PCR had been used to determined oocysts in faeces of dogs (Gondim et al., 2004; Razmi, 2009; Dalimi et al., 2014). N. caninum DNA can be surprisingly identified through PCR in formalin fixed and paraffin-embedded aborted brain tissue (Dubey et al., 2011). The Nc5 gene and ITS1 region (the internal transcribed spacer 1) of the rRNA gene of the parasite are the most frequent markers used for common PCR-based N. caninum finding (Dubey et al., 2011). Recently, quantitative PCR (qPCR) is used in academic research on N. caninum (Collantes-Fernandez et al., 2009), which has greater sensitivity and authorizes both discovery and quantitative assessment of the parasitein biological specimens in comparison with conventional and nested PCR.

2)      Histopathological examinations and immunohistochemistry (IHC)

Histopathologic evaluation of the bovine aborted fetus is essential for a deterministic diagnosis and fetal brain is the most systematically affected organs. Since most aborted fetuses might be autolyzed at the time of sampling, even autolyzed semi-liquid brain tissue could be fixed in buffered neutral formalin for histopathologic diagnosis of H&E (hematoxylin and eosin) stained sections (Dubey et al., 2007). The most important feature of brain lesion is focal non-suppurative encephalitis associated with liquefactive necrosis (Nematollahi et al., 2013). Recent study reported the lesions of the brains and spinal cords of aborted fetuses of dairy cattle which included severe congestion, perivascular and perineuronal edema, status spongiosis, perivascular cuffing, focal gliosis, neurophagy, and focal necrosis (Nematollahi et al., 2013; Kamali  et al., 2014). In most aborted fetus extensive cellular infiltrations and focal necrosis are also found in the heart, liver, and skeletal muscle.  Moreover, lesions can be observed in the placenta, which are of little diagnostic value (Dubey et al., 2011). In placentas, severe congestion, vascular thrombosis, perivascular infiltration of mononuclear cells, focal placentitis, and necrotic foci in cotyledons were observed recently (Nematollahi et al., 2013). To date, most researchers believe that histopathology remains an extremely valuable diagnostic tool, and the sensitivity and specificity of fetal histopathology is high. However, immunohistochemistry (IHC) is required because there are generally a few parasitespresent in autolyzed tissues that frequently not visible in common H&E stained sections. So, demonstration of N. caninum by immunohistochemical method in tissue lesions is the best deposition for etiology of abortion presently. A preference of IHC is the presence of the parasites can be linked to the lesions; but, this method is effortful and proportionally insensitive. However, a recent experimental study indicated a reliable compromise between PCR and IHC in discrimination of neospora antigen in the affected tissues (Khodakaram-Tafti et al., 2012).

 

Novel Experimental Studies on N. caninum in Iran

Confirmed recognition of intermediate host species to neospora infection implicates isolation of viable parasites via bioassays in cell culture and/or animal models. In recent years, some interesting studies have been carried out by Iranian researchers on cell culture and animal models for isolation of lasting parasites.

Fig. 2. Diagnostic assays for identification of N. caninum and neosporosis infection.

 


1)      N. caninum and cell culture

Up to now, numerous host cells have been hopefully suggested for the laboratory preservation, multiplication and passage of N. caninum tachyzoites, such as Vero cell (Cadore et al., 2009), bovine mononuclear cell (Tuo et al., 2005), cat and dog fibroblast cell (Lei et al., 2005), cat kidney cell (Lei et al., 2005), rat astrocytes (Pinheiro et al., 2006), human cancer cell lines such as MCF-7 (Lv et

al., 2010), trophoblastic (BeWo) and uterine cervical (HeLa) cells (Carvalho et al., 2010). Among these, the Vero cell line is the most commonly used for the propagation of the parasite in vitro in an attachment surface. Previous data described that MA-104 (African green monkey kidney epithelial- like cell) and SW742 (human colorectal epithelial- like cell) cells display convenience susceptibility to N. caninum in comparison with Vero cells (Khordadmehr et al., 2014).  Moreover, it has been stated that Theileria lestoquardi and Theileria annulata infected lymphoblastoid cell lines as suspension cell culture are liable to Nc-1 tachyzoites and could be used as a suitable host cell line for tachyzoites culture in vitro conditions (Khordadmehr et al., 2014; Kargar et al., 2013; Khordadmehr et al., 2012b). Interestingly, it has been reported that the culture of tachyzoites in J774 cell resulted in a significant increase in the number of multiplicated tachyzoites and led to rapid attenuation of tachyzoites in comparison with Vero cell line which can be used as an appropriate in vitro model to produce of live attenuated vaccine. These findings, for the first time, represented the marked impact of host cell on virulence of N. caninum tachyzoites (Khordadmehr et al., 2013).

1)      N. caninum and animal models

Previous studies have been described that some species of gerbils (Meriones unguiculatus and Meriones tristrami) and sand rats (Psammoomys ubesus) are sentient to N. caninum tachyzoites infection (Dubey et al., 2007; Pipano et al., 2002). Mostly, bioassays implementation in these models are expensive associated with regarding ethical considerations and need populations of immunosuppressed species, such as cortisone-treated outbred mice or IFN-γ gene knockout mice (Dubey et al., 2011). In these senses, embryonated eggs have recently been suggested and approved as a laboratory animal model for experimental infection (Furuta et al., 2007; Khodakaram-Tafti et al., 2012; Khordadmehr et al., 2013; Mansourian et al., 2017) and also for assessment of the virulence of N. caninum tachyzoites (Namavari et al., 2011). In a separate study, experimental N. caninum infection was performed in quail, partridge, broiler and laying chicken embryonated eggs. These findings interestingly showed that among various animal models, the lowest LD50 was belonged to the broiler chickens, which suggested the broiler chicken embryonated egg as the best animal model for experimental neosporosis. Surprisingly, partridge is known as the most susceptible bird to N. caninum infection. Also, these results reinforced that there is genetic sensitivity to N. caninum in chickens like mice (Mansourian et al., 2015). Another recent publication suggests that pigeon embryos may be a suitable choice for the biologic studies and acute infection of N. caninum in living organisms (Bahrami et al., 2016). The results of these studies suggest new insights into application of the inexpensive and available animal models for further N. caninum research.

Conclusion

Neosporosis has identified as a notable infectious disease of both cattle and dogs worldwide, which it frequently leads to clinical infections in warm-blooded animals. Because of the intimately biologic relationship of N. caninum to Toxoplasma gondii and since non-human primates had been experimentally infected, an issue of concern is that N. caninum might be zoonotic. On the other hand, it seems that there are large direct and indirect economic losses (such as congenital infection, abortion, stillbirth or birth of weak calves, loss of milk production and substitution costs for culled aborted cows) due to neosporosis to cattle industry in Iran.

 

Acknowledgments

No applicable

Conflict of interest statement

There is no conflict of interest.

Ethical approval

No applicable

       References
   Bahrami S., Hamidinejat H., Mayahi M. and Ahmadi Baloutaki M. (2015). A Survey of Neospora caninum infection in sparrows (Passer domesticus) in Khuzestan Province, Iran. Archives of Razi Institute, 70 (4), pp. 279-281
Bahrami S., Boroumand Z., Alborzi A.R., Namavari M. and Mousavi S.B. (2016). A molecular and serological study of Neospora caninum infection in pigeons from southwest Iran. Veterinarski Arhive, 86(6), pp. 815-823.
Bahrami S., Rezaie A., Boroomand Z., Namavari M. and Ghavami S. (2016). Embryonated pigeon eggs as a model to investigate Neospora caninum infection. Laboratory Animals, 51 (2), pp. 191-203.
Barr B.C., Conrad P.A., Sverlow K.W., Tarantal A.F. and Hendrickx A.G. (1994). Experimental fetal and transplacental Neosporainfection in the nonhuman primate. Laboratory Investigation Journal, 71, pp. 236-242.
Bjerkås I., Mohn S.F. and Presthus J. (1984). Unidentified cyst-forming sporozoon causing encephalomyelitis and myositis in dogs. Zeitschrift für Parasitenkunde, 70, pp. 271-274.
Björkman C., Gondim L.F., Naslund K., Trees A.J. and McAllister M.M. (2005). IgG avidity pattern in cattle after ingestion of Neospora caninum oocysts. Veterinary Parasitology, 128(3-4), pp. 195-200.
Cadore C.C., Vogel F.S., Flores E.F., Sangioni L.A. and Camillo G. (2009). Susceptibility of cell lines and primary cell cultures to Neospora caninum. Ciencia Rural, Santa Maria, 39 (5), pp. 1581–1585.
Carvalho J.V., Alves C.M., Cardoso M.R., Mota C.M., Barbosa B.F., Ferro E.A., Silva N.M., Mineo T.W., Mineo J.R. and Silva D.A. (2010). Differential susceptibility of human trophoblastic (BeWo) and uterine cervical (HeLa) cells to Neospora caninum infection. International Journal of Parasitology, 40, pp. 1629–1637.
Chryssafidis A.L., Soares R.M., Rodrigues A.A.R., Carvalho N.A.T. and Gennari S.M. (2011). Evidence of congenital transmission of Neospora caninumin naturally infected water buffalo (Bubalus bubalis) fetus from Brazil.  Parasitology Research, 108(3), pp. 741-743.
Collantes-Fernandez E., Zaballos A., Alvarez-Garcia G. and Ortega-Mora L.M. (2002). Quantitative detection of Neospora caninum in bovine aborted fetuses and experimentally infected mice by real-time PCR. Journal of Clinical Microbiology, 40, pp. 1194–1198.
Dalimi A., Sabevarinejad Gh., Ghafarifar F. and Forouzandeh-Moghadam M. (2014). Molecular detection of Neospora caninum from naturally infected dogs in Lorestan province, West of Iran. Archives of Razi Institute, 69 (2), pp. 185-190.
Dijkstra T., Barkema H.W., Eysker M., Hesselink J.W. and Wouda W. (2003). Evaluation of a single serological screening of dairy herds for Neospora caninum antibodies. Veterinary Parasitology, 110, pp. 161–169.
Costa K.S., Santos S.L., Uzeˆda R.S., Pinherio A.M., Almeida M.A.O., Araujo F.R., McAllister M.M. and Gondim L.F.P. (2008). Chickens (Gallus domesticus) are natural intermediate hosts of Neospora caninum. International Journal of Parasitology, 38, pp. 157-159.
Donahoe S.L., Lindsay S.A., Krockenberger M., Phalen D. and Šlapeta J. (2015). A review of neosporosis and pathologic findings of Neospora caninum infection in wildlife. International Journal of Parasitology, 4, pp. 216-238.
Doosti A., Khamesipour F., Nekoei Sh. and Lutvikadic I. (2015). Survey for the presence of Neospora caninum in frozen bull’s semen samples by PCR assay. Asian Pacific Journal of Tropical Biomedicine, 5(1), pp. 7-12.
Dubey J.P., Sreekumar C., Knickman E., Miska K.B., Vianna M.C.B., Kwok O.C.H., Hill D.E.M., Jenkins C., Lindsay D.S. and Greene C.E. (2004). Biologic, morphologic and molecular characterization of Neospora caninum isolates from littermate dogs. International Journal of Parasitology, 34, pp. 1157-1167.
Dubey J.P., Schares G. and Ortega-Mora L.M. (2007). Epidemiology and control of neosporosis and Neospora caninum. Clinical Microbiology Reviews, 20, pp. 323–367.
Dubey J.P., Jenkins M.C., Rajendran C., Miska K., Ferreira L.R., Martins J., Kwok O.C.H. and Choudhary S. (2011). Gray wolf (Canis lupus) is a natural definitive host for Neospora caninum. Veterinary Parasitology, 181, pp. 382-387.
Dubey J.P. and Schares G. (2011). Neosporosis in animals-the last five years. Veterinary Parasitology, 180, pp. 90-108.
Ezatpour B., Alirezaei M., Hassanvand A., Zibaei M., Azadpour M. and Ebrahimzadeh F. (2013). The first report of Neospora caninum prevalence in aborted and healthy sheep from west of Iran. Comparative Clinical Pathology, 24, pp. 19-22.
Furuta P.I., Mineo T.W.P., Carrasco A.O.T., Godoy G.S., Pinto A.A. and Machado R.Z. (2007). Neospora caninum infection in birds: experimental infections in chicken and embryonated eggs. Parasitology, 34, pp. 1931–1939.
Gharekhani J., Tavoosidana G. and Akbarein H. (2014). Serological study of Neospora caninum infection in dogs and cattle from west of Iran. Comparative Clinical Pathology, 23 (5), pp. 1203-1207.
Gharekhani J., Tavoosidana G.R. and Naderisefat G.R. (2013). Seroprevalence of Neospora infection in horses and donkeys in Hamedan province, Western Iran. Veterinary World, 6(9), pp. 620-622.
Gharekhani J., Tavoosidana G.R. and Zandieh M. (2013). Seroprevalence of Neospora caninum in sheep from Western Iran. Veterinary World, 6(10), pp. 709-710.
Gharekhani J., Esmaeilnejad B., Rezaei H., Yakhchali M., Heidari H. and Azhari M. (2016). Prevalence of anti-Neospora caninum antibodies in Iranian goats. Annals of Parasitology, 62(2), pp. 111–114.
Gharekhani J., Yakhchali M., Esmaeilnejad B., Mardani K., Majidi G., Sohrabi A., Berahmat R. and Hazhir Alaei M. (2018). Archives of Razi Institute, 73 (4), pp. 305-310.
Gharekhani J. and Yakhchali M. (2019). Neospora caninum infection in dairy farms with history of abortion in West of Iran. Veterinary and Animal Sciences, 8, pp. 100071
Gondim L.F.P., McAllister M.M., Pitt W.C. and Zemlicka D.E. (2004). Coyotes (Canis latrans) are definitive hosts of Neospora caninum. International Journal of Parasitology, 34, pp. 159-161.
Graham D.A., Calvert V., Whyte M. and Marks J. (1999). Absence of serological evidence for human Neospora caninum infection. Veterinary Record, 144, pp. 672-3.
Haddadzadeh H.R., Sadrebazzaz A., Malmasi A., Talei-Ardakani H., Khazraeii-Nia P. and Sadreshirazi N. (2007). Seroprevalence of Neospora caninum infection in dogs from rural and urban environments in Tehran. Parasitology Research, 101 (6), pp. 1563-1565.
Hajikolaei M.R.H., Goraninejad S., Hamidinejat H., Ghorbanpour M. and Paryab R. (2007). Occurrence of Neospora caninum antibodies in water buffaloes (Bubalus bulalis) from the south-western region of Iran. Bulletin of Veterinary Institute Pulawy, 51, pp. 233- 35.
Hamidinejat H., Mosalanejad B., Avizeh R., Razi Jalali M.H., Ghorbanpour M. and Namavari M. (2011). Neospora caninum and Toxoplasma gondii antibody prevalence in Ahvaz feral cats, Iran. Jundishapur Journal of Microbiology, 4(4), pp. 217-222.
Hamidinejat H., Ghorbanpour M., Rasooli A., Nouri M., Hekmatimoghadam S.H., Namavari M.M., Pourmehdi-Borojeni M. and Sazmand A. (2013). Occurrence of anti-Toxoplasma gondii and Neospora caninum antibodies in camels (Camelus dromedarius) in the center of Iran. Turkish Journal of Veterinary and Animal Sciences, 37, pp. 277-281
Hamidinejat H., Seifi-Abad-Shapouri M.R., Namavari M.M., Shayan P. and Kefayat M. (2015).Development of an Indirect ELISA Using Different Fragments of Recombinant Ncgra7 for Detection of Neospora caninum Infection in Cattle and Water Buffalo. Iranian Journal of Parasitology,10(1), pp. 69-77.
Heidari H., Mohammadzadeh A. and Gharekhani J. (2014). Seroprevalence of Neospora caninum in slaughtered native cattle in Kurdistan province, Iran. Veterinary Research Forum, 5 (1), pp. 69 -72.
Hosseininejad M., Hosseini F., Mosharraf M., Shahbaz S., Mahzounieh M. and Schares G. (2010). Development of an indirect ELISA test using an affinity purified surface antigen (P38) for sero-diagnosis of canine Neospora caninum infection. Veterinary Parasitology, 171, pp. 337–342.
Hosseininejad M. and Hosseini F. (2011). Seroprevalence of Neospora caninum and Toxoplasma gondii infection in dogs from west and central parts of Iran using two indirect ELISA tests and assessment of associate risk factors. Iranian Journal of Veterinary Research, 12 (1), pp. 46-51.
Ibrahim H.M., Huang P., Salem T.A., Talaat R.M., Nasr M.I., Xuan X. and Nishikawa Y. (2009). Short report: prevalence of Neospora caninum and Toxoplasma gondii antibodies in northern Egypt. American Journal of Tropical Medicine and Hygiene, 80, pp. 263-67.
Kamali A., Seifi H., Movassaghi A.R., Razmi G.R. and Naseri Z. (2014). Histopathological and molecular study of Neospora caninum infection in bovine aborted fetuses. Asian Pacific Journal of Tropical Biomedicine, 4(12), pp. 990-994.
Kargar M., Mojaver S., Namavari M., Sayari M. and Rahimian A. (2013). Suspension culture of Neospora caninum by Theileria annulata- infected cell line. Tropical Biomedicine, 30(2), pp. 394-354.
Kefayat M., Hamidinejat H., Seifiabadshapoori M.R., Namavari M.M., Shayan P. and Gooraninejad S. (2012). Cloning and expression of Neospora caninum dense-granule 7 in E. coli. Journal of Parasitic Diseases, 38 (2), pp. 196-200.
Khanmohammadi M. and Fallah E. (2011). Prevalence of Neospora caninum antibodies in Shepherd dogs in Sarab district, East Azerbaijan Province, Iran. African Journal of Microbiology Research, 5(28), 5062-5066.
Khodakaram-Tafti A., Mansourian M., Namavari M. and Hosseini A. (2012). Immunohistochemical and polymerase chain reaction studies in Neospora caninum experimentally infected broiler chicken embryonated eggs. Veterinary Parasitology, 188, pp. 10–13.
Khordadmehr M., Hosseini S.M.H., Mohsenifar E., Namavari M.M. and Khordadmehr S. (2012). Seroprevalence of Neospora caninum in farm and household dogs determined by ELISA. Online Journal of Veterinary Research, 16 (4), pp. 172-181.
Khordadmehr M., Khodakarm-Tafti A., Namavari M., Mansourian M., Karimiyan A. and Rahimian A. (2012). Effect of host cell on virulence of Neospora caninum. Online Journal of Veterinary Research, 16 (1), pp. 38-48.
Khordadmehr M., Namavari M.M., Khodakaram-Tafti A., Mansourian M., Rahimian A. and Daneshbod Y. (2013). Comparison of use of Vero cell line and suspension culture of murine macrophage to attenuation of virulence of Neospora caninum. Research in Veterinary Science, 95 (2), pp. 515-521.
Khordadmehr M., Namavari M. and Khodakaram-Tafti A. (2014). Susceptibility of various cell lines to Neospora caninum tachyzoites cultivation. Archives of Razi Institute, 69 (1), pp. 57-62.
King J.S., Slapeta J., Jenkins D.J., Al-Qassab S.E., Ellis J.T. and Windsor P.A. (2010). Australian dingoes are definitive host of Neospora caninum. International Journal of Parasitology, 40, pp. 945-950.
Lei Y., Davey M. and Ellis J.T. (2005). Attachment and invasion of Toxoplasma gondii and Neospora caninum to epithelial and fibroblast cell lines in vitro. Parasitology, 131, pp. 583–590.
Lv Q., Li J., Gong P., Xing S. and Zhang X. (2010). Neospora caninum: in vitro culture of tachyzoites in MCF-7 human breast carcinoma cells. Experimental Parasitology, 126, pp. 536-539.
Malmasi A., Hosseininejad M., Haddadzadeh H., Badii A. and Bahonar A. (2006). Serologic study of anti-Neospora caninum antibodies in household dogs and dogs living in dairy and beef cattle farms in Tehran, Iran. Parasitology Research, 100 (5), pp. 1143-5.
Mansourian M., Khodakaram-Tafti A. and Namavari M. (2009). Histopathological and clinical investigations in Neospora caninum experimentally infected broiler chicken embryonated eggs. Veterinary Parasitology, 166, 185–190.
Mansourian M., Namavari M., Khodakaram-Tafti A. and Rahimian A. (2015). Experimental Neospora caninum infection in domestic bird’s embryonated eggs. Journal of Parasitic Diseases, 39(2), pp. 241–244.
McCann C.M., Vyse A.J., Salmon R.L., Thomas D., Williams D.J.L., McGarry J.W., Pebody R. and Trees A.J. (2008). Lack of serologic evidence of Neospora caninum in humans, England. Emerging Infectious Diseases, 14, pp. 978–980.
Moraveji M., Hosseini M.H., Amrabadi O., Rahimian A., Namazi F. and Namavari M. (2011). Seroprevalence of Neospora spp. in horses in South of Iran. Tropical Biomedicine, 28(3), pp. 514–517.
Moraveji M., Hosseini A., Moghaddar N., Namavari M.M. and Eskandari M.H. (2012). Development of latex agglutination test with recombinant NcSAG1 for the rapid detection of antibodies to Neospora caninum in cattle. Veterinary Parasitology, 26, pp. 211-7.
Nam H.W., Kang S.W. and Choi W.Y. (1998). Antibody reaction of human anti- Toxoplasma gondii positive and negative sera with Neospora caninum– specifi c antibodies in goats from Sri Lanka. Korean Journal of Parasitology, 36, pp. 269-75.
Namavari M., Mansourian M., Khodakaram-Tafti A., Hosseini M.H., Rahimiyan A., Khordadmehr M. and Lotfi M. (2011). Application of chicken embryonated eggs as a new model for evaluating the virulence of Neospora caninum tachyzoites. Comparative Clinical Pathology, pp. 1346–1349.
Namavari M., Hosseini M.H., Mansourian M., Shams Z., Amrabadi O., Tahamtan Y. and Moazeni-Jula F. (2012). Testing for infective abortive agents in cattle in Iran. Online Journal of Veterinary Research, 16(3), pp. 147-153.
Nematollahi A. and Jafari-Jozani R. (2010). Study on pattern of Neospora caninum tachyzoite proteins by SDS-PAGE and Western blotting in aborted cows. Iranian Journal of Veterinary Research, 11 (4), pp. 383-6.
Nematollahi N., Jaafari R. and Moghaddam G.R. (2011).Seroprevalence of Neospora caninum Infection in Dairy Cattle in Tabriz, Northwest Iran. Iranian Journal of Parasitolog, 6 (4), pp. 95-98.
Nematollahi A., Moghaddam G.H., Jaafari R., Ashrafi-Helan J. and Norouzi M. (2013). Study on outbreak of Neospora caninum-associated abortion in dairy cows in Tabriz (Northwest Iran) by serological, molecular and histopathologic methods. Asian Pacific Journal of Tropical Biomedicine, pp. 942-946.
Noori M., Rasekhi M., ganjali M. and Nourollahi-Fard S.R. (2019). Seroprevalence of Neospora caninum Infection and Associated Risk Factors in Cattle of Sistan Areas, Southeastern Iran in 2016. Iranian Journal of Parasitology, 14 (2), pp. 340-346.
Nourollahi-Fard S.R., Khalili M., Aminzadeh A. (2008). Prevalence of antibodies to Neospora caninum in cattle in Kerman province, South East Iran. Veterinarski Arhive, 78 (3), pp. 253-259.
Nourollahi-Fard S.R., Khalili M., Fazli O., Sharifi H. and Radfar M.H. (2017). Seroprevalence of Neospora caninum in cattle of Neishabour, Northeast of Iran. Slovenian Veterinary Research, 54 (1), pp. 5-9.
Lobato J., Silva D.A.O., Mineo T.W.P., Amaral J.D.H.F., Segundo G.R.S., Costa-Cruz J.M., Ferreira M.S., Borges A.S. and Mineo J.R. (2006). Detection of immunoglobulin G antibodies to Neospora caninum in humans: high seropositivity rates in patients who are infected by human immunodeficiency virus or have neurological disorders. Clinical Vaccine and Immunology, 13, pp. 84–89.
Ortega-Mora L.M., Ferna´ndez-Garcı´a A. and Go´mez-Bautista M. (2006). Diagnosis of bovine neosporosis: recent advances and perspectives. Acta Parasitologica, 51, pp. 1-14.
Parish S.M., Maag-Miller L., Besser T.E., Weidner J.P., McElwain T., Knowles D.P. and Leathers C.W. (1987). Myelitis associated with protozoal infection in newborn calves. Jornal of American Veterinary Medicine Association, 191, pp. 1599-1600.
Petersen E., Lebech M., Jensen L., Lind P., Rask M., Bagger P., Bjo¨rkman C. and Uggla A. (1999). Neospora caninum infection and repeated abortions in humans. Emerging Infectious Diseases, 5, pp. 278-280.
Pinheiro A.M., Costa S.L., Freire S.M., Almeida M.A., Tardy M., El Bacha R. and Costa M.F. (2006). Astroglial cells in primary culture: a valid model to study Neospora caninum infection in the CNS. Veterinary Immunology and Immunopathology, 113, pp. 243–247.
Pipano E., Shkap V., Kreigel Y., Leibovitz B., Savitsky I. and Fish I. (2002). Babesia bovis and Babesia bigemina persistence of infection in Friesian cows following vaccination with live antibabesial vaccines. Veterinary Journal, 164, pp. 64–68.
Pouramini A., Jamshidi Sh., Shayan P., Ebrahimzadeh E., Namavari M. and Shirian S. (2017). Molecular and serological detection of Neospora caninum in multiple tissues and CSF in asymptomatic infected stray dogs in Tehran, Iran. Iranian Journal of Veterinary Medicine, 11 (2), 105-112.
Razmi G.R., Mohammadi G.R., Garrosi T., Farzaneh N., Fallah A.H. and Maleki M. (2006). Seroepidemiology of Neospora caninum infection in dairy cattle herds in Mashhad area, Iran. Veterinary Parasitology, 135, pp. 187-189.
Razmi G.R., Maleki M., Farzaneh N., Talebkhan Garoussi M. and Fallah A.H. (2007). First report of Neospora caninum-associated bovine abortion in Mashhad area, Iran. Parasitology Research, 100, pp. 755–757.
Razmi G. (2009). Fecal and molecular survey of Neospora caninum in farm and household dogs in Mashhad Area, Khorasan Province, Iran. Korean Journal of Parasitology, 47, pp.  417–420.
Razmi G.R, Zarea H. and Naseri Z. (2010). A survey of Neospora caninum associated bovine abortion in large dairy farms of Mashhad, Iran. Parasitology Research, 106, pp. 1419-1423.
Razmi G.R., Zarae H., Nourbakhash M.F. and Naseri Z. (2013). Estimating the rate of transplacental transmission of Neospora caninum to aborted fetuses in seropositive dams in Mashhad area, Iran. Iranian Journal of Veterinary Medicine, 7(4), pp. 253-256.
Reichel M.P., Alejandra Ayanegui-Alcerreca M., Gondim L.F. and Ellis J.T. (2013). What is the global economic impact of Neospora caninum in cattle-the billion dollar question. International Journal of Parasitology, 43, pp. 133-142.
Rezvan H., Khaki A., Namavari M. and Abedizadeh R. (2019). An investigation of the concurrency of anti-Neospora antibody and parasitemia in water buffalo (Bubalus bubalis) in northwest of Iran. Veterinary Research Forum, 10 (1), 79 – 84.
Sadrebazzaz A., Habibi G., Haddadzadeh H. and Ashrafi J. (2007). Evaluation of bovine abortion associated with Neospora caninum by different diagnostic techniques in Mashhad, Iranian Journal of Parasitology, 100, pp. 1257-1260.
Salehi N., Haddadzadeh H., Ashrafihelan J., Shayan P. and Sadrebazzaz A. (2009). Molecular and pathological study of bovine aboarted fetuses and placenta from Neospora caninum infected dairy cattle. Iranian Journal of Parasitology, 4, pp. 40-51.
Salehi N., Haddadzadeh H., Shayan P. and Koohi M.K. (2012). Isolation of Neospora caninum from an aborted fetus of seropositive cattle in Iran. Veterinarski Arhive, 82 (6), 545-553.
Sayari M., Namavari M. and Mojaver S. (2014). Seroprevalence of Neospora caninum infection in free ranging chicken (Gallus domesticus). Journal of Parasitic Diseases, 40, pp. 845-847.
Selahi F., Namavari M., Hosseini M.H., Mansourian M. and Tahamtan Y. (2013). Development of a disperse dye immunoassay technique for detection of antibodies against Neospora caninum in cattle. Korean Journal of Parasitology, 51(1), pp. 129-32.
Sharifdini M., Mohebali M., Keshavarz H., Hosseininejad M., Hajjaran H., Akhoundi B., Rahimi Foroushani A., Zarei Z. and Charehdar S. (2011). Neospora caninum and Leishmania infantum Co-Infection in Domestic Dogs (Canis familiaris) in Meshkin-Shahr District, Northwestern Iran. Iranian Journal of Arthropod-Borne Diseases, 1(3), pp. 60-68.
Sharifzadeh A., Doosti A. and Ghasemi Dehkordi P. (2012).PCR Assay for Detection of Neospora Caninum in Fresh and Frozen Semen Specimens of Iranian Bulls. World Applied Scientific Journal, 17 (6), pp. 742-749.
Tranas J., Heinzen R.A., Weiss L.M. and McAllister M.M. (1999). Serological evidence of human infection with the protozoan Neospora caninum. Clinical Diagnostic and Laboratory Immunology, 6, pp. 765-767.
Trees A.J., Davison H.C., Innes E.A. and Wastling J.M. (1999). Towards evaluating the economic impact of bo­vine neosporosis. International Journal of Parasitology, 29(8), pp. 1195-1200.
Trees A.J. and Williams D.J.L. (2000). Neosporosis in the United Kingdom. International Journal of Parasitology, 30, pp. 891-893.
Tuo W., Fetterer R. and Dubey J.P. (2005). Identification and characterization of Neospora caninum cyclophilin that elicits gamma interferon production. Infection and Immunity, 73, pp. 5093–5100.
Varcasia A., Capelli G., Ruiu A., Ladu M., Scala A. and Björkman C. (2006). Prevalence of Neospora caninum infection in Sardinian dairy farms (Italy) detected by iscom ELISA on tank bulk milk. Parasitology Research, 98(3), pp. 264-267.
Williams, D.J.L., Hartley C.S., Bjorkman C. and Trees A.J. (2009). Endogenous and exogenous transplacental trasmission of Neospora caninum- how the route of transmission impacts on epidemiology and control of disease. Parasitology, 136, pp. 1895-1900.
Wouda W., Moen A.R. and Schukken Y.H. (1998). Abortion risk in progeny of cows after a Neospora caninum epidemic. Theriogenology, 49(7), pp. 1311-1316.
Youssefi M.R., Arabkhazaeli F. and Hassan A.T.M. (2009). Seroprevalence of Neospora caninum infection in rural and industrial cattle in northern Iran. Iranian Journal of Parasitology, 4, pp. 20-23.