Neosporosis: quest for better diagnostics and interventions

Apicomplexan protozoan parasites include important pathogens of humans and animals, such as Plasmodium species (the causative agent of malaria), Cryptosporidium parvum (the causative agent of a water-borne enteric infection, cryptosporidiosis), Eimeria species (the causative agent of coccidiosis, a serious enteric disease in almost all vertebrate animals), Toxoplasma gondii and Neospora caninum.

Both N caninum and T gondii share a similar life cycle, except the former uses dogs and other canids as definitive hosts and affects mainly cattle herds, while the latter uses felids as definitive hosts and is more pathogenic towards humans, sheep and goats (Dubey et al, 2007). N caninum has a worldwide distribution and is a major cause of abortion and stillbirth in cattle. Dogs act as the final host, shedding oocysts in the environment (McAllister et al, 1998), while cattle (Figure 1) and other mammals are natural intermediate hosts (Dubey et al, 2007).

The life cycle (Figure 2) is characterised by three distinct stages: tachyzoites, tissue cysts and oocysts. Tachyzoites and bradyzoites occur in the tissues of infected hosts (intermediate and definitive), whereas sporozoites are present in oocysts excreted in the faeces of the definitive host.

Tachyzoites are lunate-shaped, measure approximately 2μm×6μm and have a central nucleus, but lack amylopectin granules (Figure 3).

They divide rapidly within cells and may infect many cell types including neural cells, vascular endothelial cells, myocytes, hepatocytes, renal cells, alveolar macrophages and placental trophoblasts. Bradyzoites are slowly-replicating dormant/encysted stages of the parasite. Tissue cysts may vary in size, depending on the number of bradyzoites within them.

Bradyzoites can be distinguished from tachyzoites by immunohistochemical labelling with a bradyzoite-specific antibody. The environmentally resistant stage of the parasite, the oocyst, is excreted in the faeces of dogs and coyotes in an unsporulated stage. Oocysts sporulate outside the host in about 24 hours.

Nothing is known about the survival of N caninum oocysts in the environment. Likewise, nothing is known about the possibility dogs produce oocysts after ingesting sporulated oocysts. Furthermore, there is no good animal model to study the different phases of the life cycle of N caninum, especially the tissue cyst development.

Epidemiology

N caninum is one of the most important infectious agents that causes abortion and stillbirth in cattle worldwide (Dubey et al, 2006). Cows may also experience reproductive defects, such as oestrum repetition and infertility. Economic losses are mainly caused by fetal death and lower milk production (Hernandez et al, 2001). N caninum is estimated to cause losses in cattle industries in excess of US$1b (£650m) worldwide and up to US$139.5m (£91m) in Australia, annually (Reichel et al, 2013).

N caninum can be horizontally transmitted by ingestion of tissues infected with tachyzoites or bradyzoite-containing cysts, or by ingestion of food or drinking water contaminated by oocysts shed in the faeces of dogs. Several epidemiological studies have identified the presence of dogs in farms as a risk factor for bovine neosporosis and have provided evidence for the occurrence of horizontal transmission in cattle (Dubey et al, 2007). Seropositive cows (that is, with antibodies to N caninum) are more likely to abort than seronegative cows, and most of the live-born calves from those seropositive dams may be clinically normal, but remain congenitally infected (Dubey and Schares, 2006).

The main transmission route in endemically infected cattle farms is vertical or endogenous (that is, transplacental transmission from seropositive dams to fetus during pregnancy). This route may cause abortions or lead to the birth of calves chronically infected or with neurological symptoms.

Abortion or congenital infection occurs depending on the time during gestation when the fetus becomes infected with N caninum. Fetuses dying in utero between three and eight months of gestation are usually expelled showing moderate autolysis, but fetuses dying before five months’ gestation may be mummified and retained in the uterus for several months. Those dying at an early stage of gestation may be reabsorbed, with repeat breeding as an outcome.

Natural and experimental infections have been demonstrated in a broad range of intermediate hosts including domestic, wildlife and laboratory animal species (Dubey et al, 2007). In animals such as cats, dogs, mice, gerbils, rats, sheep and goats experimentally infected with N caninum, tissue cysts with thick walls were found only in neural tissues. In non-human primates, fetal infection was demonstrated after experimental N caninum inoculation into pregnant females, with lesions similar to those found in congenital toxoplasmosis (Barr et al, 1994), raising the possibility humans could act as hosts.

In England, serological testing of 3,232 serum samples from the population and 518 serum samples from a high-risk group showed no evidence of human exposure to N caninum (McCann et al, 2008). However, serological evidence of exposure to the parasite was demonstrated in humans by others (Tranas et al, 1999), with higher seropositivity to N caninum in immuno-compromised patients compared with healthy subjects (Lobato et al, 2006).

Diagnostic methods

The economic impact of N caninum to the worldwide bovine industry justifies the need for the availability of sensitive and specific methods for accurate diagnosis of N caninum (Dubey, 2005). Different diagnostic methods have been developed and used to detect N caninum infection. These include histologic, serologic, immuno-histochemical, and molecular methods (Dubey et al, 2007).

Serological tests can be applied antemortem and can provide information on the stage of infection (acute versus chronic). Several assays are available for detecting antibodies to N caninum in cattle (Wapenaar et al, 2007). These include the direct agglutination test and the immunofluorescent antibody test, which use whole parasite antigen or whole tachyzoite (Figure 4), respectively, and the ELISA, which uses tachyzoite lysate antigens and recombinant antigens (Jenkins et al, 2002).

However, cross-reactivity has been reported between sera from animals infected with N caninum, T gondii or Sarcocystis species (Baszler et al, 1996). On the other hand, ELISAs with recombinant antigens can be produced in large quantities and better standardised for the serological assays. Several recombinant N caninum antigens, such as NcSAG1t, NcGRA6, NcGRA7, NcSRS2 and NcSAG1, of potential diagnostic value have been used in serodiagnostic assays for bovine neosporosis (Dubey and Schares, 2006; Hiasa et al, 2012).

In a more recent study, the N caninum subtilisin-like serine protease-1 (NcSUB1), which contains five copies of tandem repeats (protein regions that are highly antigenic/immunogenic), with high number of epitopes in these tandem repeats, may facilitate B-cell activation and result in higher antibody production and greater antigenicity. This NcSUB1 antigen can be used to complement or enhance serodiagnosis of neospora infection (Ybañez et al, 2013).

Different genetic targets and numerous PCR assays have also been developed for the diagnosis of N caninum infection. PCR protocols are used to detect N caninum DNA in the body tissues of aborted fetuses or other intermediate hosts. Also, other samples such as amniotic fluid, cerebrospinal fluid and oocyst-contaminated dog or coyote faeces have been examined by PCR for the presence of N caninum DNA. A multiplex PCR was developed and shown to be able to distinguish among strains and genotypes of N caninum, thus can be a valuable aid in studies on the molecular epidemiology N caninum in different host species or geographic regions (Al-Qassab et al, 2009).

Treatment options

Although the clinical signs of disease in dogs and cattle have now been recognised for more than two decades, economically viable treatment and control options are still not available (Reichel et al, 2014). For dogs, a number of drugs are available for the treatment of the clinical cases (clindamycin, potentiated sulphonamides and pyrimethamine), which may result in the resolution of lesions (Reichel et al, 2007). However, treatment needs to be applied as early as possible before clinical signs become irreversible. Coccidiostatic drugs have been found to be efficacious against some stages of the parasite life cycle in vitro (Lindsay et al, 1994) and experimentally in vivo (Kritzner et al, 2002).

Test-and-cull approaches have been implemented to control the disease in cattle (Hall et al, 2005). N caninum-infected cows were culled from the herd or not re-bred, and a reduction in the within-herd prevalence of infection was achieved.

However, this approach is expensive and economically non-justified to controlling neosporosis (Reichel and Ellis, 2006), especially if a large part of the herd is infected. Chemotherapeutic drugs cannot prevent either abortions or infections in cattle (Dubey et al, 2007; Reichel et al, 2014); however, frequent doses of toltrazuril could cure experimental tachyzoite infection (Kritzner et al, 2002). Vaccination can be the most cost-effective approach in controlling neosporosis.

An inactivated vaccine (Bovilis Neoguard) against neosporosis has been available commercially for a number of years, but appears to be partially successful in preventing abortion in cattle (Weston et al, 2012). Live vaccination, using tachyzoites of an attenuated strain of N caninum NC-Nowra isolate (Williams et al, 2007) or N caninum NcIs491 isolate (Mazuz et al, 2015), has demonstrated good efficacy as indicated by the reduction in the abortion rates due to N caninum.

However, it may suffer as a commercial product from high production cost and limited shelf-life, although experts argue vaccination remains the most feasible control strategy and investment should continue in the development of a live vaccine against neosporosis if we are to maintain cattle productivity worldwide (Reichel et al, 2015).

Future needs

After 25 years of studying N caninum and neosporosis, many questions have been addressed and more than 1,800 studies have been published. However, many aspects of N caninum are still unknown – there is still a long way to reach a complete understanding of the parasite’s ecology, epidemiology, host range and environmental aspects involved, as well as its pathogenesis.

Increased understanding of N caninum infection mechanism, in particular the process of brain invasion, is needed.

Because the ingestion of oocysts is an important route of infection, future research should address the development of new methods to detect and inactivate oocysts in faeces, soil and water. Studies evaluating the survival or inactivation of the organism in dog litters would be also very valuable. Such information could provide insight into management strategies to diminish environmental contamination caused by outdoor dogs.

Furthermore, future projects should try to improve communication between veterinarians, dog owners and the public about neosporosis risk factors and mechanisms of prevention. Such integrated efforts, and a means of maximising their impact, might lead to a decline in neosporosis.

Acknowledgements

Due to space limitation, the author was unable to comprehensively cite many worthy contributions to the field.