Canine neosporosis: perspectives on pathogenesis and management | VMRR - Dove Medical Press

Rodrigo C Silva,¹ Gustavo P Machado<sup>2

1</sup>Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, Starkville, MS, USA; ²Department of Internal Medicine and Surgery of Small Animals, Dr Munhoz Veterinary Hospital, Itápolis, Brazil

Abstract: 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

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 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 feces^20,25 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 bioassay, 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 (&gt;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 antiprotozoal 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 (&lt;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, IgG_2a, 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 immunocompetent^54,125,126 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.

Author contributions

RCS and GPM participated in all bibliography searches and contributed equally on writing and discussing the manuscript. The manuscript was written, reviewed by RCS and GPM, and edited by RCS. Both authors read and approved the final manuscript.

Disclosure

The authors report no conflicts of interest in this work.

---

References

Popular posts from this blog

Strays welcome - Ways you can reach out to help lonely and neglected animals

- May 12, 2024

racoon distemper :: Article Creator Distemper Virus Cases Surging Among Skunks, Raccoons In St. Thomas Breadcrumb Trail Links News Local News An explosion of distemper cases among St. Thomas' wildlife has prompted the city to take a more proactive role dealing with sick or injured skunks and raccoons. Author of the article: Brian Williams  •  Local Journalism Initiative reporter Published May 08, 2024  •  3 minute read Join the conversation You can save this article by registering for free here. Or sign-in if you have an account. File photo Article content An explosion of distemper cases in skunks and raccoons has prompted St. Thomas officials to change how they respond to sick and injured animals. St. Thomas city council voted to expand the role of the city's animal services in picking up sick or injured skunks and raccoons after an animal-rehabilitation clinic asked for help due to widespr...

Read more

Best Pet Insurance Companies & Plans 2024 | U.S. News

- November 07, 2024

smalls cat food :: Article Creator The Healthiest Wet Cat Food From Top Brands In 2024 When you buy through our links, Business Insider may earn an affiliate commission. Learn more Our cats might not know what a love language is, but they sure love it when we feed them tasty food. The best wet cat food recipes come in a wide range of flavors and textures to please every cat. Ultimately, the healthiest canned food for your cat is one that's nutritionally complete and balanced for their life stage and any existing health issues. We asked veterinarians how to choose nutritious wet food for kittens, adult cats, and seniors. Based on their advice, we narrowed down our picks from top brands. One of the best overall wet cat foods is Wellness Gravies Salmon Entree for adult cats, which contains a healthy balance of protein from salmon, turkey, and egg whites.  All of our recommendations meet the standards set by the Association of American Feed ...

Read more

Binghamton Plaza Shop Owners Concerned About Eminent Domain ...

- September 10, 2023

bullmastiff puppies for sale :: Article Creator MUTT MASTIFF 125 Mutt Motorcycles desired to make the Mastiff the king of the Mutts, so with that in mind they decided to add a little love here and there to make him a little different yet still retain that unmistakable outline of a Mutt Motorcycle. First up is that monster tank. This tank was specifically designed for the two newest models the Mastiff and the GT-SS. They desired to keep the classic tank shape but add a helping of steroids, so they boosted the size to 15 litres. Sitting on the Mastiff and looking down at the huge tank is a beautiful sight. The tank is topped off with a neat Monza style chrome cap for that classic British vibe. Up front we have a super wide deep tread tyre wrapped around a black rim 18'' with stainless spokes, cropped brushed aluminium mudguard, headlight grill and our beautiful stainless steel mini speedometer topped off with a set of anodised black Mutt bar...

Read more

Powered by Blogger

Artificial intelligence

Loading...

Archive

• December 202487
• November 2024102
• October 2024112
• September 2024119
• August 202453
• July 202495
• June 202478
• May 202492
• April 202482
• March 202449

• February 202467
• January 202469
• December 202357
• November 202350
• October 202386
• September 202382
• August 202359
• July 202345
• June 202386
• May 2023106
• April 2023103
• March 2023106
• February 202382
• January 202389
• December 202285
• November 202296
• October 202281
• September 202287
• August 202280
• July 202290
• June 202284
• May 202278
• April 202263
• March 202234
• February 202263
• January 202258
• December 202165
• November 202186
• October 202157
• June 202187
• May 202193
• April 202166
• November 20202
• October 202011
• September 202017
• August 202014
• July 202019
• June 202016
• May 202010
• April 202010
• March 20207
• February 202012
• January 202012
• December 201912
• November 2019212
• October 2019306
• September 2019275
• August 2019267
• July 2019254
• June 2019213
• May 2019206
• April 2019187
• March 2019200
• February 2019187
• January 2019156
• December 2018132
• November 2018207
• October 201894

Show more Show less

Labels

• bowel problems
• gastroenteritis
• gastroenterology
• gastroenterology and gastrointestinal
• gastrointestinal disease
• gastrointestinal virus
• Gastrointestinal+Diseases+from+Animals - Google News
• IFTTT
• stomach diseases
• stomach+diseases - Google News

Popular Posts

Swap Shop Items - WKYK

- June 15, 2021

(If you would like to submit an item for the "Swap Shop" use the form here To submit an item by phone call 828-682-3510 ext 442) (Lost and Found Items page is here) Submitted items stay on the website for one month unless deleted by the person who submitted the item.  All submitted items are in rotation on the radio programs for one week and expire one week from the air date of the section in which the items are listed and can be placed back on the radio by resubmitting the listing. Help Wanted:      Need housekeeper for large old home in Little Switzerland. MUST have EXCELLENT references and no drugs and non smoker. Will be every 2 weeks through September. Wonderful family and good pay. Call 828-467-8311 Help Wanted:      Yard work needed: Need weeds removed from large flower beds. I also need mulch spread in and around beds. Job is immediately and pay will be good and prompt. Must have references and be drug free. Call 838-467-8311. June 15, 2021 F...

Read more

The 14 Best Dogs for Kids with Autism, According to Experts

- November 27, 2024

small dog breeds :: Article Creator 21 Low-Maintenance Dog Breeds Of All Sizes For First Time Pet Parents When you're thinking about adopting a dog, you may not be sure what breed is right for you. There are so many cute breeds, tall breeds, small breeds and everything in between! But if you're a first-time pet owner, you may want to consider the more low-maintenance dog breeds. Full disclosure: Low maintenance does not mean no maintenance! Walking, grooming, feeding and vet visits are all a required part of being a pet parent. But from unconditional love to endless sloppy kisses, loving a dog is one of the best things you'll ever do for yourself! If you're new at pet parenthood or simply looking for a dog that's a good fit for your family's busy lifestyle, there are many great breeds that are lower maintenance because they may not require as much grooming, space, and exercise as others. Some breeds definitely are more ...

Read more

These Adorable Small Dogs Are Just the Right Size for Your Family

- December 01, 2024

royal canin kitten food :: Article Creator The 16 Best Kitten Foods Of 2024, Reviewed With Advice From Vets When you buy through our links, Business Insider may earn an affiliate commission. Learn more Kittens need an animal-based diet rich in protein and essential nutrients to support their growth and development. When it comes to the best kitten foods, your choices come in all types of textures and recipes, from shreds and chunks in gravy to smooth pâté and crunchy kibbles. We consulted veterinarians and animal nutritionists to find out what makes a nutritious dry or wet kitten food. To look for in dry and wet foods to meet the nutritional needs of kittens. Then we researched dozens of foods to make our picks for this guide. One of the best overall canned foods is Purina Pro Plan Kitten Salmon and Ocean Fish Entree. It contains high-quality sources of omega fatty acids to support a kit...

Read more

Top 20 Largest Dog Breeds

- October 07, 2024

toy dog breeds :: Article Creator 10 Teacup Dog Breeds For Compact Living WorldAnimalFoundation.Org is reader-supported. When you buy through links on our site, we may earn an affiliate commission. Learn More Teacup dogs are tiny, lovable companions that weigh between 4 to 6 pounds, often bred from the smallest puppies of a litter. While they aren't officially recognized by American dog breed registries, they fall under the toy dog category and have captured many hearts, especially in recent years. Britannica notes that, unlike traditional toy breeds that have been around for centuries and were adored by royalty, teacup dogs gained popularity around 2005, thanks in part to social media and celebrities showcasing their pint-sized pets. These adorable dogs are not only rare but also incredibly portable, making them perfect for people who love to bring their furry friends along wherever they go, often fitting easily into bags or purses. Though ...

Read more

12 Shepherd Dog Breeds That Are the Talk of the Flock

- December 14, 2024

yorkie puppies :: Article Creator Hysterics At Yorkie's 'Parallel Park' Tactic To Be Picked Up A charming video of a "super smart" Yorkshire terrier puppy executing a "parallel park" move has captured the hearts of millions on TikTok. The clip, shared by user @vedranaweahomes on November 13, has amassed over 1.5 million views and showcases the tiny dog's peculiar habit of aligning itself perpendicularly to its owner when it wants to be picked up. The caption overlaid on the video humorously asks: "Does anyone else's dog parallel park when they want to be picked up?" More From Newsweek Vault: Walmart+ Members Get New Perk Aimed at Pet Owners The footage features the Yorkie, named Rolo, gazing up at the camera while a voice off-screen says, "Ready? Come on." Rolo then swiftly moves into position, as if parking a miniature car. It's unknown exactly why the Yorkie in the viral c...

Read more

Mayo Clinic ranked best hospital for adult gastrointestinal care - Healio

- July 31, 2019

Edward Loftus, Jr. Mayo Clinic tops U.S. News & World Report ’s 2019-2020 Best Hospitals rankings for adult gastroenterology and gastrointestinal surgery. “Receiving recognition as the top hospital for gastrointestinal care for adults is a testament to the tremendous team we have at the Mayo Clinic,” Edward V. Loftus, Jr., MD, of Mayo Clinic, told Healio Gastroenterology and Liver Disease . “I am honored to be a part of this team and look forward to continuing to provide excellent care to all of our patients with myriad of gastrointestinal conditions.” Other hospitals ranked in the top 10 for adult gastroenterology care and gastrointestinal surgery are: Cedars-Sinai Medical Center; Johns Hopkins Hospital; Cleveland Clinic; Massachusetts General Hospital; UCLA Medical Center; Mayo Clinic-Phoenix; UPMC Presbyterian Shadyside; Mount Sinai Hospital; and, New York-Presbyterian Hospital-Columbia and Cornell. Additionally, the Mayo Clinic ranked first in ...

Read more

These Adorable Small Dogs Are Just the Right Size for Your Family

- December 06, 2024

funny monkey :: Article Creator These Jokes About The Escaped Monkeys In South Carolina Are, Unfortunately, Very Funny Yahoo is using AI to generate takeaways from this article. This means the info may not always match what's in the article. Reporting mistakes helps us improve the experience.Generate Key Takeaways If you've been avoiding the internet this past week, you might not know about the escaped lab monkeys in South Carolina. The State / TNS / Getty Images Yes, escaped lab monkeys. Anadolu / Anadolu via Getty Images Forty-three rhesus macaque monkeys escaped from a research facility in Yemassee, South Carolina, on Wednesday after an employee accidentally left a door open. According to the AP, "Outside of rats and mice, rhesus macaques are one of the most studied animals on the planet." Abhishek Mittal / Getty Images/iStockphoto Yemassee Police Chief Gregory Alexander told the AP, "They are n...

Read more

11 Dog Breeds Similar to the German Shepherd

- December 16, 2024

dogue :: Article Creator The Dogue Days Of Summer When taking a scroll through the archives, one Vogue editor happened upon a February 1897 cover starring a labrador puppy. "His Soliloquy: To be or not to be—a dog show dog?" the cover line read. Another from July 1898 featured a portrait of a handsome collie named Drumtochty, outfitted, appropriately, in a Balmoral Tam'o shanter. Indeed, dogs have always had a place in the pages of Vogue, from Coco Chanel, who graced the April 1931 issue with her Great Dane, Gigot, to Azzedine Alaïa, who was photographed with a Yorkshire terrier in each hand by Arthur Elgort in February 1986. These days, it's just a bit easier for a hound to go viral. When Demi Moore brought her microscopic Chihuahua, Pilaf, to the spring 2024 Versace show in Milan, the pup instantly captivated the world with her bulging eyes and exposed tongue. When she posed in Chopard on the Croisette in Cannes it only cemen...

Read more

These Adorable Small Dogs Are Just the Right Size for Your Family

- December 13, 2024

farm animal diseases :: Article Creator Controlling Infectious Disease Between Wildlife And Livestock On Shared Rangeland The H5N1 bird flu — widespread in wild birds worldwide and the cause of outbreaks in poultry, U.S. Dairy cows and even several recent human cases among agricultural workers – is a prime example of mixed species disease transmission occurring where wildlife and livestock interact. Sapna Chitlapilly Dass, Ph.D., assistant professor in microbial ecology and microbiome interactions, Department of Animal Science, is studying the ongoing threat of emerging pathogens that can necessitate prompt deployment of medical countermeasures for life-saving interventions. And Dass, a Texas A&M AgriLife researcher in the College of Agriculture and Life Sciences, wants to nip it in the bud, so to speak. She wants to address the potential of disease transmission at the rangeland level rather than waiting until it reaches the SARS-CoV-2 vir...

Read more

Top 20 Largest Dog Breeds

- October 10, 2024

felv in cats :: Article Creator A Kitten's Fight Against FIP Soot (left) and Shepard (right). (Photo courtesy of University of Illinois College of Veterinary Medicine and Lauren Bryan) Shepard, a 10-month-old kitten is on a journey to recovery from feline infectious peritonitis (FIP) after his brother and littermate, Soot, passed away, likely of FIP, 3 months before Shepard began showing clinical signs of the disease. Shepard's treatment was overseen by Katelynn Ondek, DVM, PhD, a veterinary neurology and neurosurgery resident, who explained in a University of Illinois College of Veterinary Medicine report1 how the team at the University of Illinois Veterinary Teaching Hospital in Urbana diagnosed and treated the usually fatal disease before treatment for it—compounded GS-441524—was made available in certain states in June 2024.2 Soot and Shepard were both adopted by the same family. In March 2024, Soot began limping on his...

Read more

1.	Dubey JP. Recent advances in Neospora and neosporosis. Vet Parasitol. 1999;84(3–4):349–367.
2.	Gondim LF, McAllister MM, Pitt WC, Zemlicka DE. Coyotes (Canis latrans) are definitive hosts of Neospora caninum. Int J Parasitol. 2004;34(2):159–161.
3.	Dubey JP, Schares G, Ortega-Mora LM. Epidemiology and control of neosporosis and Neospora caninum. Clin Microbiol Rev. 2007; 20(2):323–367.
4.	Nolan SJ, Romano JD, Luechtefeld T, Coppens I. Neospora caninum recruits host cell structures to its parasitophorous vacuole and salvages lipids from organelles. Eukaryot Cell. 2015;14(5):454–473.
5.	Anderson ML, Andrianarivo AG, Conrad PA. Neosporosis in cattle. Anim Reprod Sci. 2000;60–61:417–431.
6.	Dubey JP, Schares G. Neosporosis in animals – the last five years. Vet Parasitol. 2011;180(1–2):90–108.
7.	Bjerkås I, Mohn SF, Presthus J. Unidentified cyst-forming sporozoon causing encephalomyelitis and myositis in dogs. Z Parasitenkd. 1984;70(2):271–274.
8.	Dubey JP, Carpenter JL, Speer CA, Topper MJ, Uggla A. Newly recognized fatal protozoan disease of dogs. J Am Vet Med Assoc. 1988;192(9):1269–1285.
9.	Marsh AE, Barr BC, Packham AE, Conrad PA. Description of a new Neospora species (Protozoa: Apicomplexa: Sarcocystidae). J Parasitol. 1998;84(5):983–991.
10.	McAllister MM, Dubey JP, Lindsay DS, Jolley WR, Wills RA, McGuire AM. Dogs are definitive hosts of Neospora caninum. Int J Parasitol. 1998;28(9):1473–1478.
11.	Dubey JP, Buxton D, Wouda W. Pathogenesis of bovine neosporosis. J Comp Pathol. 2006;134(4):267–289.
12.	Dubey JP, Lindsay DS. A review of Neospora caninum and neosporosis. Vet Parasitol. 1996;67(1–2):1–59.
13.	Lindsay DS, Dubey JP, Duncan RB. Confirmation that the dog is a definitive host for Neospora caninum. Vet Parasitol. 1999;82(4):327–333.
14.	Dubey JP, Barr BC, Barta JR, et al. Redescription of Neospora caninum and its differentiation from related coccidia. Int J Parasitol. 2002;32(8):929–946.
15.	Dubey JP, Leathers CW, Lindsay DS. Neospora caninum-like protozoon associated with fatal myelitis in newborn calves. J Parasitol. 1989;75(1):146–148.
16.	Barr BC, Anderson ML, Dubey JP, Conrad PA. Neospora-like protozoal infections associated with bovine abortions. Vet Pathol. 1991;28(2):110–116.
17.	Dubey JP, Sreekumar C, Knickman E, et al. Biologic, morphologic, and molecular characterisation of Neospora caninum isolates from littermate dogs. Int J Parasitol. 2004;34(10):1157–1167.
18.	Speer CA, Dubey JP, McAllister MM, Blixt JA. Comparative ultrastructure of tachyzoites, bradyzoites, and tissue cysts of Neospora caninum and Toxoplasma gondii. Int J Parasitol. 1999;29(10):1509–1519.
19.	Hemphill A, Fuchs N, Sonda S, Hehl A. The antigenic composition of Neospora caninum. Int J Parasitol. 1999;29(8):1175–1188.
20.	Sykes JE. Neosporosis. In: Sykes JE, editor. Canine and Feline Infectious Diseases. Vol 1. 1st ed. St Louis: Elsevier; 2014:704–712.
21.	Lindsay DS, Dubey JP. Canine neosporosis. J Vet Parasitol. 2000;87(1):1–11.
22.	Basso W, Venturini L, Venturini MC, et al. First isolation of Neospora caninum from the feces of a naturally infected dog. J Parasitol. 2001;87(3):612–618.
23.	King JS, Slapeta J, Jenkins DJ, Al-Qassab SE, Ellis JT, Windsor PA. Australian dingoes are definitive hosts of Neospora caninum. Int J Parasitol. 2010;40(8):945–950.
24.	Dubey JP, Jenkins MC, Rajendran C, et al. Gray wolf (Canis lupus) is a natural definitive host for Neospora caninum. Vet Parasitol. 2011;181(2–4):382–387.
25.	Greene CE. Toxoplasmosis and neosporosis. In: Greene CE, editor. Infectious Diseases of the Dog and Cat. 4th ed. St Louis: Elsevier; 2012:821–827.
26.	Cummings JF, de Lahunta A, Suter MM, Jacobson RH. Canine protozoan polyradiculoneuritis. Acta Neuropathol. 1988;76(1):46–54.
27.	Dubey JP, Lindsay DS. Neosporosis. Parasitol Today. 1993; 9(12):452–458.
28.	Trees AJ, Guy F, Tennant BJ, Balfour AH, Dubey JP. Prevalence of antibodies to Neospora caninum in a population of urban dogs in England. Vet Rec. 1993;132(6):125–126.
29.