Canine neosporosis: perspectives on pathogenesis and management.

Canine neosporosis is a worldwide disease caused by the obligate intracellular parasite protozoan Neospora caninum, manifesting mainly neurological symptoms. N. caninum has a heteroxenous life cycle and affects a wide range of warm-blooded animals. The domestic and wild canids are the definitive host of the parasite. They shed oocysts after ingestion of tissue cysts from infected intermediate hosts (ovine, equine, bovine, canine, and many other species), containing bradyzoites, or oocyst-contaminated water and food. The presence of dogs in farms is considered a risk factor for production animals. A wide range of diagnostic methods are currently available, but the most used is serology, ie, indirect fluorescent antibody test specific to the antibody detection in blood serum samples. No vaccine is available, but control strategies should be focused on the vertical and horizontal transmission of the parasite, ie, avoid feeding dogs with raw or undercooked meat, and taking care with water for human and animal consumption. No medicines to control the transplacental transmission are available yet.

Introduction

Neospora caninum is an obligate intracellular parasite protozoan that causes neosporosis in a wide range of warm-blooded animals, including domestic and wild animals. Neosporosis is a worldwide emergent disease,1–3 and is usually associated with reproductive (ie, neonatal mortality in animals, particularly in dogs and cattle) and neurological (ie, neuromuscular degeneration) disorders and presents progressive evolution, being more severe in young animals.4,5 For cattle, it is considered one of the most important causes of infertility, abortion, and neonatal mortality. Sheep can also develop reproductive and neonatal disease, but the economic importance of N. caninum in sheep is less clear when compared with cattle.6

In Norway, Bjerkås et al7 diagnosed a disease similar to toxoplasmosis, another apicomplexan parasitic disease, in a litter of seven boxer puppies, which presented Toxoplasma gondii-like tissue cysts with no specific antibodies. The new parasite was firstly classified N. caninum by Dubey et al8 in the US. These authors isolated and described the disease caused by a protozoan infecting a puppy with clinical signs similar to those reported by Bjerkas et al.7 Afterwards, this protozoan was recognized as the causative agent of the disease in dogs, and identified in bovine aborted and mummified fetuses and in calves with neonatal paralysis.9 McAllister et al10 defined the domestic dog (Canis lupus familiaris) as the definitive host of N. caninum, and Gondim et al2 reported the coyote (C. latrans) as a wild definitive host of the parasite by confirming the sexual stage of the parasite, and oocysts being shed in feces. The absence of information concerning the epidemiology of the parasite has limited the introduction of objective and effective measures to prevent and control the infection significantly over time.

Epidemiology

Three infective stages characterize the N. caninum life cycle: sporozoites within sporulated oocysts (sporulated oocysts present two sporocysts with four sporozoites each), rapidly dividing tachyzoites, and slowly proliferating bradyzoites within tissue cysts (Table 1).11–13 Oocysts are the environmentally resistant stage of the parasite shed in the definitive host feces, which can survive for months to years in the environment. Tissue cysts have a huge cyst wall that protects the bradyzoites from the hostile extracellular environment formed during the host immune response. This form is observed mainly in muscle and the central nervous system (CNS). Each tissue cyst may have 20–100 bradyzoites. Bradyzoites survive at 4°C for over 14 days,13,14,18 and are also resistant to pepsin and trypsin digestion. Tachyzoites can be found free in organic fluids and in a parasitophorous vacuole (PV) placed at the cytoplasm of the host cell, but are particularly vulnerable to the harmful effects of extracellular maintenance and rapidly lose their capacity for invasion.4 They can actively penetrate membranes of a wide range of nucleated cells, which suggests low host-cell specificity.18,19 Tachyzoites multiply by endodyogeny,20 and promote the expansion of the PV up to the cell lysis.

Table 1

Structural and morphological characteristics of Neospora caninum stages

Stage	Structure	Size (length × width)	Reference	Morphology	Comments
Oocyst (sporulated)	–	∼10.6–12.4×10.5–12 μm	Dubey et al14	Thin, colorless, ovoid, or ellipsoidal	Environmentally resistant form of the parasite
	Oocyst wall	∼0.6–0.8 μm thick	Dubey et al14	Colorless, bilayered, smooth-surface walls	The oocyst wall encompasses two sporocysts
	Sporocyst	∼7.4–9.4×5.6–6.4 μm	Dubey et al14		Each sporocyst contains four sporozoites and a residuum
	Sporozoite	5.8–7×1.8–2.2 μm	Dubey et al14	Elongated
Tissue cyst	–	∼30–107 μm (diameter)	Dubey et al15Barr et al16Dubey et al17	Round and oval	Bradyzoites and tachyzoites have conoids, numerous micronemes (size 232×58 nm), rhoptries (145 nm wide), amylopectin, and nucleus
	Cyst wall	∼0.5–4 μm thick	Dubey et al14Speer et al18	Smooth, irregular, wavy contour in tissue sections, but without protrusions. Septa and a secondary tissue cyst wall are absent
	Bradyzoite	∼4.5–8×1.2–1.9 μm	Barr et al16Speer et al18Dubey et al14Dubey et al17	Slender, elongated with a subterminal nucleus, slightly longer than tachyzoites
Tachyzoite	Tachyzoite	∼3–7×1–2 μm	Dubey et al14Speer et al18	Ovoid, lunate, or globular

N. caninum is a heteroxenous parasite.21 Dog (C. lupus familiaris) is considered the definitive nonwild host of the parasite,10,22 whereas coyote (C. latrans),2 Australian dingo (C. lupus dingo),23 and gray wolf (C. lupus)24 are the definitive wild hosts. A wide range of intermediate hosts has been reported (Table 2).25

Table 2

Definitive and intermediate hosts in neosporosis

Epidemiological classification	Common name	Scientific name	Area	References
Definitive host	Dog	Canis lupus familiaris	Non-W	3, 6–8, 10–13, 39, 20–22, 26–32, 34–38, 40
	Coyote	Canis latrans	W	2, 6, 39, 41
	Australian dingo	Canis lupus dingo	W	23, 39
	Gray wolf	Canis lupus	W	6, 24, 39
Intermediate host	Dog	Canis lupus familiaris	Non-W	3, 6, 10, 12, 13, 20–22, 27, 35, 41
	African wild dog	Lycaon pictus	W	39
	Golden jackal	Canis aureus	W	39
	Hoary fox	Pseudalopex vetulus	W	39, 42
	Red fox	Vulpes vulpes	W	6, 39, 41
	Raccoon	Procyon lotor	W	39, 41
	Coyote	Canis latrans	W	2, 6, 39, 41
	Gray wolf	Canis lupus	W	6, 24
	Cattle	Bos taurus	Non-W	5, 6, 11, 15, 16, 32, 34, 43, 44, 46–59, 92
	Buffalo	Bubalus bubalis	Non-W	6, 26, 45, 59–65
	European bison	Bison bonasus bonasus	Non-W	39
	Sheep	Ovis aires	Non-W	6, 63, 66–71
	Goat	Capra hircus	Non-W	6, 63, 67, 69, 70, 72–74
	Pig	Sus scrofa	Non-W	6
	Horse	Equus caballus	Non-W	75, 76
	Donkey	Equus asinus	W	77
	Camel	Camelus dromedarius	W	6
	Capybara	Hydrochoerus hydrochaeris	W	6
	Rat	Rattus norvegicus	Non-W	6, 39, 41
	House mouse	Mus musculus	Non-W	6, 39, 41
	Vole	Microtus arvalis	W	6
	Water vole	Arvicola terrestris	W	6
	Rabbit	Oryctolagus cuniculus	W	6, 54
	Hare	Lepus europaeus	W	6, 39
	Egyptian mongoose	Herpestes ichneumon	W	39
	Cat	Felis catus	Non-W	6, 78
	Feral cat	Felis silvestris	W	6
	Eurasian lynx	Lynx lynx	W	6, 39
	Iberian lynx	Lynx pardinus	W	6
	Diverse deer species	–	W	6, 39, 41
	White rhinoceros	Ceratotherium simum	W	39, 41
	Llama	Llama glama	Non-W	6
	Alpaca	Vicugna pacos	Non-W	79
	Sea otter	Enhydra lutris neresis	M	6, 39
	Spotted seal	Phoca largha	M	6, 39
	Sparrow	Passer domesticus	M	6
	Chicken	Gallus gallus domesticus	Non-W	6
	Pigeon	Columba livia	Non-W	80
	Common raven	Corvus corax	Non-W	6, 39
	Hooded crow	Corvus cornix	Non-W	81
	Western jackdaw	Corvus monedula	Non-W	81

Abbreviations: W, wild area; non-W, nonwild area; M, marine area.

Many aspects of the N. caninum life cycle are still unknown, mainly concerning the sexual stage of the parasite evolution.39 More studies are necessary to clarify the entire enteroepithelial cycle and to elucidate the interaction between the dog and the parasite in natural, not experimental, infection.

N. caninum can be transmitted in two ways: vertically and horizontally. Vertical transmission happens during terminal stages of gestation (transplacental transmission with fetal infection) or postnatally, by the transmission of tachyzoites via milk (transmammary transmission).3,52 In horizontal transmission, the infection occurs postnatally after ingestion of water containing sporulated oocysts, or of infected tissues (raw or undercooked meat, fetal membranes) of intermediate hosts with tissue cysts or contaminated with sporulated oocysts.20 Fecal transmission (oocysts) appears to be less important than carnivorism.6

After ingestion, sporulated oocysts or tissue cysts excyst and release sporozoites and bradyzoites, respectively, into the duodenum lumen. In the intestinal epithelium, sporozoites and bradyzoites transform into tachyzoites, undergo a phase of multiplication, possibly in the mesenteric lymph nodes,11 reach the bloodstream, and disseminate to gravid uterus,11 and many cell types, ie, CNS cells, vascular endothelial cells, myocytes, hepatocytes, renal cells, alveolar macrophages, and placental trophoblasts,14,43 invade the cells and start the multiplication, causing severe lesions on the affected tissues. If the host immune response is active, the extracellular environment turns hostile to the parasite; tachyzoites differentiate to bradyzoites and form tissue cysts (asexual stage) in intermediate hosts (chronic stage of the infection).20,25,27,82

The recrudescence of the disease for any suppressive factor promotes alterations in host immunity, causing immunomodulation or immunosuppression, the conversion of bradyzoites to tachyzoites, and the rupture of the tissue cysts. This process releases bradyzoites, which reactivate the infection.32,83 This is well documented to occur in pregnant animals, causing fetal infection.20,82,84

In definitive hosts, tachyzoites transform in merozoites, ie, macrogametes and microgametes, into the intestinal epithelium, undergo merogony (sexual stage), and form a zygote. Zygotes, or nonsporulated oocysts, are released at the intestinal lumen, and shed up to 1 million to the environment with the host feces20,25 2–30 days after ingestion.3,40 The amount and duration of oocysts shed may be highly variable, due to parasite load ingested, strain, host immunity, and infective stage of the parasite. The nonsporulated oocysts become sporulated and orally infectious outside the host in proper environmental conditions (oxygen concentration, humidity, and temperature) in 1–5 days.10,85

No breed predisposition or differential sex susceptibility to neosporosis in dogs is known,33 but age can be a risk factor. Adult dogs shed fewer oocysts than puppies following primary exposure, and puppies may also develop reexcretion after new challenge.36

Some important factors to be considered when reporting N. caninum infection and disease are the infective form of the parasite, strain virulence, parasite load, and immunity status of the host. N. caninum can cause serious disease in cattle and dogs, and occasionally in other animals.6

N. caninum antibodies have been detected in dogs from North, Central, and South America, Europe, South Africa, Asia, and Oceania.27,51 The frequency ranges from 5.8% (six of 104)29 to 12.9% (21 of 163)28 in England, 5.5% (19 of 344) to 23.6% (36 of 152) in the Netherlands,32 7.1% (14 of 198) to 31.3% (15 of 48) in Japan,31 and 0 to 19.2% in the Czech Republic, according to the use of these dogs (4.7% army, 0 police, 2.6% private, and 19.2% shelter dogs).37 These data emphasize the epidemiological importance of canine neosporosis, and highlight neosporosis as a differential diagnosis in pet clinics for dogs affected by neurological diseases, since the parasite can stay in the environment where the definitive host sheds the oocysts in their feces.37

Wouda et al32 suggested that once dogs and cattle live in the same farm, the chances of the infection in cattle increase. Neither Liu et al63 nor Paiz et al71 observed the pasturing system to be a significant risk factor for goats. In herbivorous creatures, N. caninum is the major cause of abortion in both dairy and beef cattle,6 but lactogenic transmission can also occur and be important,49,50 and should be considered and demonstrated in further studies under natural circumstances in calves or dogs.41

Low levels of horizontal transmission in cattle were also observed by Hietala and Thurmond47 and Davison et al,46 but the importance of this route should not be underestimated, mainly due to the maintenance of the parasite in regional herds.49 Worldwide seroprevalence ranges a lot in beef and dairy cattle and buffaloes. In buffaloes, it is ∼48% compared to 16.1% for dairy cattle and 11.5% for beef cattle.65 This high seroprevalence has ranged from 1.5% (three of 200) in southern Vietnam45 and 3.8% (four of 105) in the Philippines53 to 88% (169 of 192) in Brazil62 and 88.3% (424 of 480) in Australia.59

In South America, seroprevalence in dairy cattle has ranged from 10.62% (66 of 121) in Brazil56 to 20.3% in Argentina.58 In addition, Fort et al58 observed that dairy cattle had a 3.1-fold higher risk of contracting the parasite through horizontal transmission than beef cattle in Argentina.

In small ruminants, prevalence ranges according to region and species. In goats, prevalence has ranged from 0.4% (four of 1,060) in Poland73 and 0.9% (four of 464) in South Korea74 to 19.7% (182 of 923) in São Paulo, Brazil72 and 23.6% (21 of 89) in the Philippines.53 In sheep, it has ranged from 0.6% (four of 640) in New Zealand55 and 1.8% (seven of 409) in Rio Grande do Norte, Brazil66 to 59.2% (353 of 596) in São Paulo, Brazil71 and 63% (214 of 339) in the north of Jordan.67 This observed scenario corroborates with the comparisons between species predispositions made by Nasir et al,69 Anastasia et al,70 and Liu et al,63 which indicated sheep to be more likely susceptible to N. caninum infection than goats. In addition, the presence of dogs and bad conditions of hygiene are important risk factors that contribute to the infection in these species.63,71

Pathogenesis

The pathogenesis of neosporosis depends on the balance between the capacity of the tachyzoite to penetrate and multiply inside the cells and the host to impede the parasite proliferation. The cell-invasion process has two different steps – the adhesion to the host-cell surface and penetration to the cell86 – which may take no more than 5 minutes.43 Adhesion, the initial step, is mediated by low-affinity contact, which induces the secretion of microneme-content proteins. In this way, a more specific ligation to the host-cell surface occurs and finally the invasion. For that, adequate receptors at the host-cell surface are required to provide enough signals for the invasion of the parasite into the cell cytoplasm.86

In order to initiate host-cell invasion, tachyzoites reorient themselves perpendicularly to the host cell-surface membrane and enter the cytoplasm by advancing anterior end first, until they are located in the cytoplasm, enclosed by the PV. Invasion is an active process requiring metabolic energy solely on the part of the parasite, but not on the part of the host cell.83

N. caninum tachyzoites interact with the host cell by highly conserved apicomplexan mechanisms. In contrast to other genera, N. caninum can recognize one or more host cell-surface receptors responsible for the initial adhesion and invasion.86 The surface of N. caninum tachyzoites exhibits considerable differences from T. gondii with regard to surface carbohydrate content,83 as well as the interaction of host organelles and scavenging of nutrients.4

Once inside, the parasite induces metabolic alterations in the host, which favor its maintenance.43 Both parasites reorganize the host microtubular cytoskeleton and attract endoplasmic reticulum, mitochondria, lysosomes, multivesicular bodies (temporary storage compartments enriched in sphingolipids and cholesterol at the intersection of the endocytic and exocytic pathways), and Golgi vesicles to their PV, but in N. caninum infection the host endoplasmic reticulum gathers around the PV, not physically associating with the vacuolar membrane. The parasite attracts the host Golgi apparatus to its PV, surrounding it, but induces less fragmentation into ministacks than T. gondii.4,87 Based on that, they scavenge cholesterol from organelles, store it in lipid bodies, and salvage sphingolipids from host Golgi vesicles, sequestering them into their PV; they also attract host mitochondria to their PV, retain these organelles close to the vacuolar membrane, and use the host mitochondria to obtain energy for their benefit; both parasites manipulate the host cell and exploit mammalian resources. These processes are highly conserved in both parasites.4

The parasite must invade the host cell to avoid the host immune response. One of these immune mechanisms is the CD8+ T-cell response. Jordan and Hunter88 emphasized that the CD8+ T-cell population participates in a protective mechanism used by the host in infections caused by apicomplexan protozoa. CD8+ T cells can work as a cytotoxic T lymphocyte or as cytokine-secreting cells. Many factors influence the generation of CD8+ T cell responses, including such cytokines as IL-2 and IL-12, which contribute to T-cell expansion, survival, and the acquisition of effector function.89 The participation of this immune cell in host protection is well known to the T. gondii infection, but recently Correia et al90 observed that the production of IFNγ is also a predominant host-protective mechanism conferred by CD8+ T cells in the course of neosporosis, as observed in toxoplasmosis. This response and the humoral immune response work together on containing the infection.

Usually, a prepatent period of 5–8 days after ingestion of tissue cysts can be observed in the intestinal form of the disease, but little is known about cysts’ localization in the organ or about the affected structures.21 N. caninum can produce necrotic lesions, which are detectable in a few days; it causes cell death by the active multiplication of the tachyzoites, and can induce neuromuscular diseases in dogs and other species by the destruction of a large number of neuronal cells, ie, cranial and spinal nerves, which affect the nerve-impulse conductibility among the cells.1

Clinical signs and diagnosis

In general, neosporosis is asymptomatic in adult and older dogs.21,40 More severe and frequent infections occur in young dogs (age less than 6 months old). The clinical signs are usually general and the same as observed in toxoplasmosis, but neurological and muscular abnormalities predominate,33 as forelimbs paralysis. In younger puppies, it is normal to observe clinical signs starting at 3–9 weeks old. Forelimb atrophy and gradual muscular rigidity are the most important clinical signs that differentiate neosporosis from those other disorders causing paralysis; even so, hind limbs are more severely affected than forelimbs. The paralysis progresses to rigid contracture of the muscle from the affected limbs. This arthrogryposis results in a scar formation on these muscles, due to injury at the lower motor neuron and myositis. Articular deformation and joint curvature (genu recurvatum) can occur in some puppies,30 followed by cervical weakness, dysphagia, megaesophagus, and death.8,25,26 Dogs remain in an alert status, paralyzed, and require special care for months.25 In addition, severe diarrhea and incoordination, multifocal pulmonary consolidation and necrosis to the necropsy (necrotic and purulent bronchopneumonia), fibrinohemorrhagic enteritis (villous atrophy and crypt hyperplasia in the jejunum and ileum), myocarditis, and nonpurulent encephalitis may occur in young puppies.40

It is more probable that dogs over 6 months old can develop the disease by the reactivation of a latent chronic infection. Adult dogs typically develop polymyositis and/or meningoencephalomyelitis, with clinical signs related to the multifocal lesions in the CNS, mainly in the cerebellum; on the other hand, myocarditis, dermatitis, pneumonia, diffuse peritonitis with peritoneal effusion, and multifocal dissemination can occur but are less common, as a result of higher tachyzoite dissemination.25 These dogs may be obtunded and develop ataxia, circling, head tilt, nystagmus, hypermetria, head tremors, delayed placing reactions, abnormal cranial nerve function, anisocoria, depressed segmental reflexes, tetraparesis, cervical hyperesthesia, and seizures.20 In the chronic stage, most animals remain asymptomatic. However, females may have immunosuppression and reactivate the infection during gestation. In this way, bradyzoites switch back to tachyzoites, cross the placenta, and infect the fetus.82,84

In buffaloes, naturally occurring disease is rare, but abortion has been reported in animals that have contact with dogs (60.3%),60 which highlights the role of dogs in the epidemiological chain of neosporosis for production animals.

Neosporosis should be considered when hyperesthesia, muscle swelling, or atrophy is observed at the clinical exam. Dogs of any age can die by CNS or muscular inflammation.25 Neurological disorders in dogs have many causes, and neosporosis should be included on the differential diagnosis. Focal lesions have been reported in the brain and spinal cord of a 6-year-old dog with severe mononuclear cell infiltration (large numbers of T lymphocytes and B lymphocytes, and small numbers of macrophages) of the nerve roots of the cauda equina and of the lumbar nerve roots. Necrotizing cerebellitis and hepatitis were also observed.38

Epidemiological history and clinical findings can help the diagnosis of suspected neosporosis, as well as the control and prevention of the disease. The findings observed on complete blood count and serum biochemistry panel are also suggestive, but should not be used to confirm the infection. Normal complete blood count with mild nonregenerative anemia, mild eosinophilia, or monocytosis can be observed; nucleated cells are predominantly nondegenerated to mildly degenerated neutrophils. Myositis can cause high alanine aminotransferase values, and hyperglobulinemia can also be observed. The number of nuclear cells in cerebrospinal fluid may be over 1,000 cells/μL. Protein concentration can also be increased.20

Image diagnosis can also help with the extent of the lesions, ie, radiography (diffuse interstitial pattern in dogs with pneumonia), magnetic resonance imaging, and ultrasonography (cerebellar atrophy in dogs with cerebellar signs),20 but with limited contribution. No pathognomonic findings have been reported for neosporosis.

Parasite detection by direct and/or indirect methods is of high importance for the diagnosis and prognosis of the disease and prevention and control of the infection in urban, rural, and wild areas. The available methodologies allow clinicians and researchers to amplify knowledge about classical and molecular epidemiology and pathogenesis of the disease, evaluate the efficacy of available and future drugs used to treat the clinical signs, and adopt measures to avoid dissemination of the parasite.

Direct methods consist in the visualization of oocysts, tissue cysts, or tachyzoites, stained or not, by light microscopy, histopathology, immunohistochemistry, in vitro cell culture and in vivo isolation by gerbil and/or mouse bio assay, fecal flotation of dog feces, and molecular methods, ie, polymerase chain reaction (PCR).40,91,92 Among these, each test can provide some type of information not provided by another, ie, histopathology and immunohistochemistry can determine the extension and severity of the lesion in a tissue, whereas molecular methods can just detect the presence/absence and amount of parasite DNA.

The limit of detection, sensitivity, specificity, and positive and negative predictive values of each test are a crucial part of standardization, and very important to offer an accurate tool for the diagnostic routine. Light microscopy is the cheapest and quickest method that can be used by clinicians, but has limited sensitivity and requires experience in characterizing the infective stages of the parasite, which can be misinterpreted as another apicomplexan. In the same way, cell-culture and gerbil/mouse isolation allow isolation of the parasite, but the results should be confirmed by another technique more specific for identification of the parasite. Both isolation techniques are laborious, require specific technical personnel to handle the animals or cell lines to control other types of infection in a closed system, and require specific infrastructure to maintain the animals and cells under ethical conditions, which all increase the costs. One drawback of both methods is limit of detection. Depending on the sample, the parasite isolation in samples with low parasite load or avirulent or less virulent strains may be difficult. Even if possible, it may still take a minimum of 20–30 days to get results.

Fecal flotation, with sugar or zinc sulfate solutions, is the best technique to isolate N. caninum oocysts in dog feces, but it is laborious and may take some time to run. Another limitation is that dogs presenting clinical signs do not shed oocysts, resulting in negative findings.20 This technique has high epidemiological value. The cost is not high, but the clinical value for a clinician is limited.

Histopathology and immunohistochemistry are highly recommended in reproductive problems, ie, abortion and fetal lesions, and have been used with molecular techniques. Their sensitivity and specificity will depend on a good target, antibodies, microscope, and an experienced technician to provide an accurate evaluation and conclusion. The cost is usually higher than the methodologies reported earlier, but the results are much more conclusive concerning the extent and severity of the lesions.

Molecular methods are considered to have the best sensitivity and specificity, but are too relative. The sensitivity and specificity of these tests are linked to the standardization of the technique, type of sample, stage of the infection, targeted region, and all reagents used. A well-standardized methodology can detect down to just one copy of DNA present in the analytes, but methodologies with problems can fail to detect the parasite or misdiagnose N. caninum infection. Corroborating this, van Maanen et al93 observed low agreement between the majority scores of immunohistochemistry and PCR methods (single or nested) in bovine fetal tissues on an interlaboratory comparison in Europe, with false-positive PCR results indicating contamination problems in some instances. The authors strongly linked this problem to the DNA-extraction methods, which could have been optimized and improved the performance of the test. In this way, quality control is an important point in choosing tests or labs to run the samples. Quality control may increase the price of a test, but it also improves the accuracy of the results.

On the other hand, Baszler et al94 and Sanchez et al95 observed 97% and 85% agreement, respectively, between immunohistochemistry and a well-standardized PCR protocol in formalin-fixed paraffin-embedded brain tissue of bovine fetuses. They also observed 88% agreement for fresh tissues.

The wide range of molecular targets allows a wide range for application of molecular techniques, ie, DNA, parasite-load measurement, parasite detection in very low concentrations, exploration of the population evolution of the parasite, analysis of the likelihood of the detected parasites with those from the same and other areas, and characterization of the isolates. In this way, different methodologies have been developed, ie, quantitative real-time PCR, random fragment-length polymorphism PCR, and the use of multilocus microsatellite typing of ten microsatellites for molecular genotyping, which allowed Campero et al92 to identify the isolate NC-Argentina LP1 as a unique genetic pattern, different from all reported isolates.

Differently from direct methods, indirect methods include serological tests by immunoblotting and detection of the indirect response of the host (specific antibodies) to the parasite challenge.33,96–98 Routine antemortem diagnosis is usually based on and relies on serology, because organisms are not easily found by cytological examination (Romanowsky or unstained slides), and once found can be misdiagnosed with other apicomplexan parasites, ie, T. gondii or Hammondia heydorni. The limitation of serology is related to the physiopathology of the disease. Usually, antibody levels take 2–3 weeks of infection to be detectable. Antibody research is highly recommended for screening infection in a population, mainly in a herd, and in individual patients to analyze the response of the immune system. The presence of N. caninum antibodies in serum samples confirms the parasite exposition, but not the disease. Once detectable, rising titers (usually, fourfold increased titers in 15–30 days’ paired serology) can be identified in acutely, not chronically, infected dogs.20 Usually, dogs shedding oocysts do not present detectable antibodies.

The indirect fluorescent antibody test (IFAT),8 Neospora-agglutination test,98 and enzyme-linked immunosorbent assay (ELISA)44,96 are the most used techniques for N. caninum-antibody research. IFAT was the first serological test used.8 It has been used extensively for diagnosis of the infection in dogs, is considered a gold-standard test,48 and used in epidemiological studies for domestic and wild animals. ELISA and IFAT are usually used to detect N. caninum infection in adult animals and herds, and both are also considered complementary techniques in the diagnosis of neosporosis.96 Western blotting is usually recommended to confirm uncertain results in valuable samples.99,100 In addition, avidity tests are useful for investigating the route of N. caninum transmission in herds by differing acute and chronic infections.100,101 Again, performance (sensitivity, specificity, and positive and negative predictive values) can range among laboratories, and costs are directly linked to the performance of the tests. Usually, costs for a test or retest are low, but can increase in a periodic control in a herd or large population.

Alvarez-Garcia et al100 compared ten commercial indirect or competitive available ELISA tests for bovine neosporosis and obtained excellent sensitivities (95.8%–100%, except the IDEXX rum iELISA test, with 85.9%) and specificities (93%–100%, except the VMRD cELISA test, with 65%), but almost perfect agreement among all tests (80%–90%), as observed by Campero et al57 comparing IFAT and immunoblotting (considered relative standards of comparison), and p38 ELISA (97%), which resulted in 97.8% sensitivity and 99.5% specificity. Björkman et al101 observed moderate-to-substantial agreement among four European laboratories using IgG-avidity ELISA tests to differentiate acute and chronic bovine neosporosis. On the other hand, Waldner et al102 observed good agreement (76%) between two commercial ELISA kits, but higher than comparing each test to a commercial direct agglutination test (46% and 60%).

In this way, laboratorial findings are of fundamental importance and make a contribution to the diagnosis of the infection and disease, but should be linked to clinical signs to have enough conclusive information for the treatment of each animal and epidemiological information to adopt adequate measures for prevention and control of the infection. Epidemiological, molecular, and physiopathological information obtained from routine tests and research will help to amplify knowledge about the disease, host–parasite interaction, and the disease’s relationship with the environment.

Treatment, prevention, and control

Treatment of neosporosis is usually difficult and temporarily, partially, or completely ineffective. Long periods (>8 weeks) of treatment may be required. The treatment of dogs with neurological signs is too long, and has a poor prognosis. Treatment is most effective in early stages, before muscular contracture has occurred.103 For cutaneous neosporosis, it seems to be more effective. Clindamycin is the primary drug used to treat canine neosporosis.20 It is the only lincosamide that possesses additional antiprotozoal activity. Therefore, it is effective against N. caninum tachyzoites. The combination of clindamycin and sulfonamide is highly effective against neosporosis. In addition, the synergistic action of sulfonamides and pyrimethamine enhances the anti protozoal effects.104,105 Clindamycin affects the multiplication of N. caninum tachyzoites, but is thought to have little or no effect on bradyzoites.106 In this way, tissue cysts may persist with even ∼2 months of treatment, and treatment of chronic neosporosis by exposing bradyzoites to the immune response should be considered and researched.

Usually, most of the protocols used to treat neosporosis focus on the control of the clinical manifestations,107 rather than the parasitological cure. The most used protocols include clindamycin 7.5–15 mg·kg−1 per os (PO [orally]) or subcutaneously every 8 hours for 4–8 weeks, trimethoprim–sulfonamide 15–20 mg·kg−1 PO every 12 hours for 4–8 weeks, or pyrimethamine–sulfonamide 5–30 mg·kg−1 PO every 12 hours for 4–8 weeks.25 Other protocols indicated by Sykes20 consist of the same drugs with different doses and frequency, ie, clindamycin 10–12 mg·kg−1 PO every 8 hours for 4 weeks, trimethoprim–sulfonamide 15 mg·kg−1 PO every 12 hours for 4 weeks, pyrimethamine 1 mg·kg−1 PO every 24 hours for 4 weeks, or ponazuril 20 mg·kg−1 PO every 24 hours for 4 weeks. Treatment should be continued as long as clinical improvement is occurring.20

If one member of littermates has neosporosis, all members should be checked for N. caninum antibodies, and all those testing seropositive should be treated. The use of immunosuppressive drugs should be avoided in seropositive puppies.20 The parasite can be transmitted multiple times from the infected female dog to her offspring, because no preventive treatment is available.12

Dogs should never have access to bovine placental materials, dead calves, fetal membranes, aborted fetuses, or raw/undercooked meat, and should be prevented from defecating around livestock. Also, contact with infected tissues of other intermediate hosts, eg, sheep, goat, horse, or feces of other domestic and wild canids, eg, coyotes or Australian dingoes, should be avoided. Discourage predation.20 Stop dogs from defecating in feeders, drinking from fountains, water sources, pastures, and corrals.

No effective vaccines to protect susceptible animals and avoid the infection are commercially available,20,33 but some effort and research has been invested in dogs and cattle. An important drawback in designing a vaccine against N. caninum is that the immune response itself is potentially pathogenic to the fetus.108

Vaccine research has been under way for a long time, and most approaches have focused on inactivated whole vaccines (killed tachyzoites),109–111 the use of subunit native (tachyzoite-extract vaccine formulated with a soy lecithin/β-glucan adjuvant)112,113 and recombinant N. caninum antigens,114–117 and live naturally attenuated parasites.118–120

Studies began with the use of a crude lysate of the parasite to prevent infection of offspring, followed by application in cattle and commercialization of a registered vaccine for N. caninum (NeoGuard™) for use in cattle,121 but with low efficacy (<25%).109 Also, live vaccines presented higher efficacy, up to 100% in experimental infections,118,119 producing strong cellular and IFNγ responses correlated with protection against fetopathy, but immunization with whole-tachyzoite lysate using different adjuvants failed to protect against fetal death.118

A preventive vaccine based on a recombinant canine herpesvirus expressing N. caninum surface protein (NcSRS2), similar to the authentic parasite protein for dogs117 and cattle,116 has been tested. Nishikawa et al117 observed immunized dogs producing N. caninum IgG antibodies but no clinical signs, and infectious canine herpesvirus was not recovered from these dogs, whereas Staska et al116 detected parasite-specific CD4+ T lymphocytes and IFNγ response in experimentally infected cattle, which supports further investigations of vaccines incorporating NcSRS2 gene sequences or peptides. Even with these good results, no additional further studies have been undertaken.

In C57BL6 mice, Ramamoorthy et al122 observed protective effects of an attenuated γ-irradiated N. caninum tachyzoite vaccine. No vaccinated mice presented any clinical signs up to the end of the study, but showed significant increases in secreted levels of IFNγ, IL-10, IgG1, IgG2a, and small amounts of IL-4. Though a possibly good candidate in preventing vertical transmission in cattle, an important drawback of this vaccine may be that the irradiated tachyzoites may not persist in the host for as long as a live vaccine,123 requiring booster immunizations.122

In addition, in silica approaches open a new point of view for vaccine-candidate prediction and should be exploited; precise protein targets are paramount, and arguably the most important factor determining the success or failure of this approach.124 The same happens with drugs to prevent transplacental transmission,20,33 and new studies on these directions are required.

Public health impact

The potential of N. caninum to infect humans is unknown. N. caninum antibodies have been detected in the serum samples of immunocompetent54,125,126 and immunosuppressed humans,126,127 but no parasite isolation or detectable DNA has been reported.20 N. caninum may have the potential to infect humans in the presence of HIV128 and be linked to neurological complaints,126 but while the parasite has not been linked directly to human disease, the possibility of clinically apparent N. caninum infections should not be dismissed.128 In this way, N. caninum is not considered zoonotic.20,41

Future perspectives

New insights for neosporosis and epidemiological aspects of the pathogen’s evolution should be focused on the contribution of proteomics and RNA-sequencing and next-generation sequencing techniques, ie, proteogenomics. This has an important role to play in successful annotation of gene models, protein-coding genes, and allowing the validation of important proteins as potential targets for host–parasite interaction and treatment research.129 Also, gene prediction opens wide the way for vaccine candidates by bioinformatic programs.124 These and derivative approaches linked to the conventional and unconventional research, as well as diagnostic methods, will contribute to much better understanding of the parasite’s evolution, biochemical interactions, parasite–host cell interaction, parasite chemical resistance, and host predisposition, as well as intrinsic properties of each strain, clones, and isolates, with regard to virulence and capacity to spread and adapt to new hosts, new targets for treatment, and possible candidates for vaccines.

Final considerations

Neosporosis is an important infection in pet and production animals. Control measures should focus on vertical and horizontal transmission to limit simultaneously environmental contamination and transmission to intermediate hosts. As the treatment response is limited to the stage of the parasite, the success of the clinical treatment increases as how early it starts, which should start before neurological pathology is observed.