Testing Strategies for Neospora caninum

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Diagnostic Tests & Testing Strategies for Neospora caninum
ACVIM 2004
Paul Baillargeon, DVM, MSc; Gilles Fecteau, DMV, DACVIM
St-Louis de Gonzagues, QC, Canada; St-Hyacinthe, QC, Canada

INTRODUCTION

Since its first identification in 1984 in a litter of puppies6 and the recognition of its important role in cattle abortion in 1988, N. caninum has been reported in 26 countries all over the world13. Studies have estimated that N. caninum was responsible of 12.5% of abortions in England 11, 15 to 20% in Holland33and as much as 43% in California1,31. The herd sero-prevalence level for most countries ranges between 64% and 83%17,16,21,26,23 and individual sero-prevalence ranges between 10% and 39%3.

Abortion is the dominant clinical feature for animal infected with N. caninum. Despite the fact that the first report of cattle abortion by N. caninum was of the epidemic type (>10% of the females at risk aborting), it is well accepted that a sporadic pattern is more common in endemic herds. Abortion storms however have been observed in endemic herds and are believed to have been induced by factors causing recrudescence of N. caninum in chronically infected animals32. Infected females may abort more than once but this risk is less than 5% at the following pregnancy 14. Hence, this figure does not represent the lifelong risk of re-abortion for an infected female. The presence of serum antibodies does not provide protection even if the risk of re-abortion for an infected animal decreases with the number of pregnancies29.

The majority of new bovine infections are acquired transplacentally in utero30. Up to 93% of the calves born to infected females have developed antibodies at birth28. It has been demonstrated that vertical transmission can maintain the prevalence of infection from generation to generation7. The first evidence of the horizontal transmission (HT) of the parasite was the demonstration, in Québec dairy herds, of the association between farm dogs and the sero-prevalence to N. caninum23. The possibility of HT was further reinforced in 1998 with the demonstration that dogs are a definitive host for N. caninum20. The natural mechanism for HT from dogs to susceptible species has not been clarified yet.

DIAGNOSIS

The identification of N. caninum has triggered an important research effort during the nineties: using the keyword Neospora generates a list of close to 700 scientific papers from the Pubmed journal databank. Diagnostic methods to identify N. caninum in cases of bovine abortions were developed in the early '70. In 1994 and 1995, the development of 2 ELISA tests by Dutch and Californian workers has opened the field of sero-epidemiology. These tests have also helped to refine our understanding of the transmission and of the life cycle of the parasite. Over the last few years, research has focused on the understanding of the pathogenesis and immune mechanisms involved in this infection.

The demonstration of the parasite in the target tissues of an infected animal or foetus is still the only way of confirming an infection by N. caninum12. Tissue sections staining by immunohistochemistry (IHC) and detection of specific antibodies by indirect fluorescent antibody testing (IFAT) have increased both the sensitivity and the specificity of this diagnostic procedure10,5,24. The presence of specific antibodies to N. caninum in the blood of an animal confirms exposure to the parasite. Nevertheless, Dubey has stated that even a low IFAT titer in fetal fluids is specific of the infection12. It is now widely accepted that a high pre-colostral titer is strongly indicative of an in utero acquired infection.

Serological tests have allowed screening of large groups of individuals in order to estimate the prevalence of the disease. Serological testing for N. caninum has been reviewed in 2 different papers in 1998 and 20002,9. An important feature of serology is that test sensitivity(Se) and specificity(Sp) can be adjusted to specific needs by using different cut-offs for interpretation27. Clinicians and epidemiologists are more interested into the predictive value of the results which takes into account the prevalence of the infection in population and the test characteristics. Three types of serologic tests have been used either commercially or for research purposes.

Indirect fluorescent antibody test (IFAT): First serological test used and still used as the reference test to validate other tests. In this procedure, intact tachyzoites are exposed to the sample antibodies and then to fluorescein label-antibodies directed against the animal species specific immunoglobulins of the sample. The evaluation of the results under a fluorescent microscope requires training and experience and, hence may vary from one laboratory to another. Interest in IFAT was related originally to its high specificity (>99%), its cross-reactivity with Toxoplasma gondii and other coccidians being very low.

Direct Agglutination test (DAT): This test is a modification of the commercial test used for the detection of T.gondii22. Sensitivity and specificity of the test, now commercially available, compared well with IFAT in that study. The interest of the test, in comparison with IFAT and ELISA, is that it can be used across a large number of animal species without modification.

Enzyme linked immunosorbent assay (ELISA) testing has become the most widely used diagnostic method because it can be automated, hence allowing for screening of large number of samples and for a more objective interpretation of the test results. Five variations of the ELISA tests have been developed: 1. Crude antigen (whole tachyzoites) lysates by sonication or detergent solubilisation 2. Fixed tachyzoites 3. Immuno Stimulating Complex (ISCOM): an IgG avidity ELISA has been developed in 19998 4. Recombinant protein5.Antigen Capture.Commercial kits available have reported Se ranging between 87% and 90% and Sp ranging between 92 and 100%.2

Several diagnostic kits are available commercially and results of different laboratories can not be compared easily. All serological tests perform well with serum containing high antibody levels. At low levels, the ELISA results present more variability and discrepancies may even be observed2. Serological testing works at its best with groups making it the instrument of choice for the control of the infection into populations. Individual testing remains a challenge for results interpretation. Adjusting cutoff levels to the purpose of the investigation would help but is not expected to become routinely used by veterinary practitioners.

COMPARISON OF RESULTS BEFORE AND AFTER MODIFICATIONS TO A COMMERCIAL CRUDE ANTIGEN ELISA DETECTION KIT FOR N. caninum

The serum bank

In order to compare the test results of a modified commercial ELISA kit for N. caninum antibody detection with results obtained before the modification, the serum bank built during the course of a research project on the efficacy of embryo transfer for the prevention of vertical transmission of N. caninum was used.4 This bank was composed of the monthly serum samples of 93 cows and heifers used as recipients for the embryos implanted during the trial. Eighty-seven females were included in the study results4 and 6 were not included because their pre-implantation test result was higher than 0,40 and lower than 0,80. The commercial ELISA kit for the detection of N. caninum antibodies used for these tests has a reported Se of 88.4% and Sp of 99% at a cut-off of 0.6 by the manufacturer (Biovet, St-Hyacinthe, Qc).

The infectious status for N. caninum of these recipients was determined by the serologic profile of their monthly serum tests and by the ELISA result of a pre-colostral serum sample on a live calf or the tissue analysis results of the aborted fetuses or stillborn calves. Eight of the 93 recipients were serologically positive before implantation and, using the 0.6 S/P cutoff ratio, consistently maintained this status along pregnancy. Two of these recipients gave birth to calves that did not present signs of infection at birth or abortion (one was serologically negative on its pre-colostral serum sampling and one was IHC negative at tissue analysis). All other females (n=79) included in the study results, consistently maintained their serological negative status, using the 0.6 cutoff level, and gave birth to either sero-negative on the pre-colostral serum sample or IHC negative calves at birth or abortion. For the 6 females that were not included in the final study results because they did not meet with inclusion criteria, 6 of the 54 monthly serum samples from 3 different animals, using the same cutoff level, were inconsistent with their original negative serological status. Nevertheless, they gave birth to 5 sero-negative calves on a pre-colostral serum sample and one IHC negative stillborn calf at tissue analysis.

The serum samples (n=207) from 25 Neospora negative recipients were randomly selected from the serum bank and resubmitted to the same laboratory who did the original testing. All sera from Neospora positive recipients (n=61) and from the 6 Neospora negative recipients (n=53) that were excluded from the study results were also resubmitted. These samples (n=321) were tested with the modified commercial ELISA kit for N. caninum antibody detection.

Results of the retest

Confidence intervals (CI 95%) for ELISA S/P ratios of the pregnancy monthly serum samples are presented in Table 1 for the non infected and infected groups. The 2003 monthly results are significantly lower for the non infected group of females when compared with the results of the 2000 test. In addition, 11 of the 261 samples (4%) in 2000 would have misclassified these non infected animals in comparison of none in the retest of 2003. These results are in agreement with the limitations reported for ELISA serology with samples containing low levels of antibodies. Bjorkman et al., in a review on serological diagnosis of N. caninum raises the point that "the demonstrable test result of the antigen-antibody reaction is the summation of several molecular interactions." Secondary antibodies may have different affinity for a particular immunoglobulin antigen with sizable effect on test results9. This effect is also known as "background noise" and is best illustrated by testing pre-colostral serum samples. In the embryo transfer study, 60 calves (83%) from the non infected dams had no detectable level of antibody before colostral intake. All of the other calves (n=12) were below 0.20 on their pre-colostral serum3.

There is no published report on the cause of this background noise. Cross-reactivity with other coccidians, particularly T.gondii, is negligible13. Antibody levels for infected animals vary with stage of pregnancy25. Anecdotal reports have mentioned current vaccination programs as a likely cause of temporarily non specific serologic reactions for N.caninum negative animals. The results of the 2003 retest of our serum bank sampling demonstrates the improved ability of the ELISA test used to interact more specifically with specific N.caninum antibodies.

There does not appear to be any significant change in the ability of the ELISA test to detect N. caninum antibodies in the group of infected recipients and this is in agreement with the observations already published2,9.

Table 1. Confidence Intervals (CI 95%) for ELISA s/p ratios on 322 sera collected monthly during the pregnancy of 39 recipients

Click on the table to see a larger view






Strategies for interpretation of N. caninum ELISA test results

Despite this significant improvement in the serological diagnosis of N. caninum, clinicians will occasionally be puzzled when interpreting serology results. Retesting is not always an option and when performed, may still be influenced by the background noise effect. Two strategies will assist in the interpretation of suspicious or contradictory results.

Pre-colostral serum sampling is an effective way of accurately determining serological status of individual animals (foetus and dam). As already mentioned, more than 80% of the calves from non infected dams are born without detectable levels of N. caninum antibodies. These pre-colostral serum samples were not retested with the test kit used in 2003. We postulate that the proportion of calves without detectable levels would be increased with this new test. On the other hand, calves from infected dams all had S/P ratios above 1.00 except for one. One limitation though, is that up to 8% of infected calves may be born with a low antibody level to serologically positive dams and will eventually seroconvert during the first month of their life. This is apparently caused by late pregnancy fetal infections that do not have time to seroconvert before birth15.

The high level of efficiency of vertical transmission of N. caninum, greater than 90% according to many reports, may be used to predict the serological status of a given individual with a Se and Sp similar to serological testing. Building the familial flowchart of a suspicious animal as in cases presented in Figure 1. helps at identification of discrepancies and will eventually confirm infectious status from progeny or ancestors testing. In this case, cow 265 was not infected and it can be predicted that heifer 807 is not infected neither. Cow 626 in the second flow chart had always been classified as infected in this herd since her March 1999 test. It was obvious when serological status of her progeny became available that her own status needed to be reevaluated. Retesting in March 2003 confirmed that she was indeed serologically negative to N.caninum. This case exemplifies a potentially significant cause for contradictory results on a laboratory report. It is more than likely that misclassification of this animal was an error in sample handling or an improper identification of the animal at the time of the first test.

Click on the table to see a larger view Figure 1. Familial flowchart for assessment of N. caninum serological status






CONCLUSION

The cornerstone of a neosporosis control program in a herd is the serological testing of all animals of unknown infectious status. Herd eradication of N.caninum can be achieved with the assistance of a "Test and Cull" strategy. Recent improvements in detection kits provide results that improve our ability to properly classify individual animals as the results presented in this article tend to demonstrate.

Culling of infected animals early after birth is a more economical way for herd control of N. caninum. Pre-colostral blood sampling and familial flow chart analysis will improve overall predictive value of serological testing in such programs.

Embryo transfer using the washing and trypsin treatment protocol recommended by IETS remains the only proven way to prevent vertical transmission between N. caninum infected dams and their progeny4,18. Biosecurity strategy should include minimizing access to feed bunks and feed storage areas to dogs and wild canids. Serological testing of all new entries in the herd is highly recommended biosecurity measure since prevalence appears to be higher in purchased animals than in the general population19,3.

REFERENCES

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21. Ould-Amrouche, A. et al, Veterinary Research,1999;30,(5):531-538;

22. Packham, A. E. et al, Clin.Diagn.Lab Immunol.,1998;5,(4):467-473;

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28. Schares, G. et al, Vet Parasitol,1998;80,(2):87-98;

29. Thurmond, M. C. et al, Am J Vet Res,1997;58,(12):1381-1385;

30. Thurmond, M. C. et al, Current Vet. Therap, 1999;4,(9):425-431;

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32. Wouda, W. et al, Theriogenol, 1999;52,():233-245;

33. Wouda, W. et al, J Vet Diag Invest,1997;9,(2):180-185.

Speaker Information
(click the speaker's name to view other papers and abstracts submitted by this speaker)
Paul Baillargeon, DVM, MSc
Clinique Veterinaire St-Louis-Embryobec
St-Louis De Gonzague
CO. Beauharnois, QUE., CANADA


Gilles Fecteau, DMV, DACVIM

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Lycalopex gymnocercus and Cerdocyon thous from Brazil.
Vet Parasitol 123[3-4]:275-7 2004 Sep 2

Canon-Franco WA, Yai LE, Souza SL, Santos LC, Farias NA, Ruas J, Rossi FW, Gomes AA, Dubey JP, Gennari SM
Domestic dog (Canis domesticus) and the coyote (Canis latrans) are the only known definitive hosts for the protozoan Neospora caninum that causes abortion in dairy cattle. In the present study, antibodies to N. caninum were sought in three species of wild canids, Cerdocyon thous, Lycalopex gymnocercus and Dusicyon vetulus from Brazil. Antibodies to N. caninum were assayed by the indirect fluorescent antibody test (IFAT) and the Neospora agglutination test (NAT). N. caninum antibodies were found in five of 12 L. gymnocercus with IFAT titers of 1:50 in three, 1:100 in one, and 1:1600 in one, and NAT titers of 1:40, 1:80, 1:160, 1:320, and 1:640 in five animals. Antibodies to N. caninum were found in four of 15 C. thous with IFAT titers of 1:50 in one, and 1:100 in three, and NAT titer of 1:40 in one animal. All 30 D. ventulus were seronegative by IFAT and NAT.

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Horner's syndrome associated with Neospora infection.
J Small Anim Pract 41[12]:571-2 2000 Dec
Boydell P, Brogan N
A working collie cross was presented with a three-month history of vague neurological signs and a right-sided Horner's syndrome. Denervation hypersensitivity testing suggested a first order syndrome. There was a significant positive titre to Neospora and clinical signs resolved completely following treatment.

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J Am Anim Hosp Assoc. 2002 Sep-Oct;38(5):415-9. Related Articles, Links


Cutaneous neosporosis during treatment of pemphigus foliaceus in a dog.

Ordeix L, Lloret A, Fondevila D, Dubey JP, Ferrer L, Fondati A.

Department de Medicina i Cirurgia Animal, Veterinary School, Universitat Autonoma de Barcelona, Bellaterra, Spain.

A 4-year-old, intact male rottweiler was presented with a 10-day history of papulonodular dermatitis. At the time of presentation, the dog was receiving prednisone and azathioprine to treat pemphigus foliaceus. Cutaneous neosporosis was diagnosed by immunohistochemistry on skin biopsy specimens and a high serum antibody titer to Neospora caninum by Neospora agglutination test. Electron microscopy examination of skin specimens further supported the diagnosis. Clindamycin therapy, together with withdrawal of immunosuppressive medication, resulted in prolonged clinical remission. This report documents cutaneous neosporosis in an adult dog and suggests that immunosuppressive therapy might be a predisposing factor.

Publication Types:
Case Reports

PMID: 12220024 [PubMed - indexed for MEDLINE]

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1: J Small Anim Pract. 2002 Feb;43(2):76-9. Related Articles, Links


Neosporosis with cerebellar involvement in an adult dog.

Lorenzo V, Pumarola M, Siso S.

Prado de Boadilla Veterinary Practice, Boadilla del Monte, Madrid, Spain.

A case of an adult dog with multifocal, progressive neurological signs caused by Neospora caninum is reported. Pathological studies showed cerebellar lesions due to the parasite, which was also present in other parts of the nervous system and muscle. Cerebellar atrophy related to Neospora infection has been rarely reported in veterinary medicine, and has been shown to affect ruminants and dogs. The cerebellar involvement and the age of the present dog make this case uncommon.

Publication Types:
Case Reports

PMID: 11873953 [PubMed - indexed for MEDLINE]

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Cutaneous neosporosis in two adult dogs on chronic immunosuppressive therapy.

La Perle KM, Del Piero F, Carr RF, Harris C, Stromberg PC.

Department of Veterinary Biosciences, College of Veterinary Medicine, Ohio State University, Columbus 43210, USA.

Antemortem diagnosis of generalized ulcerative and pyogranulomatous dermatitis with numerous intralesional tachyzoites was made from skin biopsy specimens from 2 adult dogs on chronic immunosuppressive therapy. A 9-year-old Italian Greyhound was on long-term corticosteroid therapy for the treatment of a lupus-like systemic autoimmune disorder, and a 7-year-old Labrador Retriever had received several months of chemotherapy for lymphosarcoma. The tachyzoites were identified as Neospora caninum by immunoperoxidase immunohistochemistry. Both dogs were treated with clindamycin. Lesions in the Greyhound resolved; however, the Labrador Retriever was euthanized because of evidence of neuromuscular disease, despite improvement of the skin lesions. These 2 cases indicate that cutaneous neosporosis can occur in adult dogs on chronic immunosuppressive therapy. The disease may result from reactivation of a congenital infection and/or a recently acquired primary infection.

Publication Types:
Case Reports

PMID: 11482605 [PubMed - indexed for MEDLINE]

[malernee]

Vet Parasitol. 1998 Jul 31;78(2):79-85. Related Articles, Links


Neospora caninum infection in a Bernese cattle dog from Italy.

Poli A, Mancianti F, Carli MA, Stroscio MC, Kramer L.

Department of Animal Pathology, School of Veterinary Medicine, University of Pisa, Italy. apoli@vet.unipi.it

A cutaneous nodule associated with Neospora caninum infection was diagnosed in a 5-year-old male Bernese cattle dog from Italy. The ulcerative lesion was 2-3 cm wide located in the skin of the tarsal region. Haematological values were normal and the dog did not show any neurological abnormalities. The dermal lesion consisted of a diffuse necrotic dermatitis with a dense infiltrate of mostly neutrophils and macrophages, surrounded by a fibrous wall. Histological sections revealed numerous tachyzoites of N. caninum scattered throughout the tissue. Diagnosis was confirmed both by immunohistochemical staining and electron microscopic examination. The dog had a 1:640 IFAT titre to N. caninum. Four weeks after surgical excision new subcutaneous nodules reappeared. The cutaneous lesions resolved following 21 days of therapy with clindamycin hydrochloride. These observations demonstrate the presence of N. caninum in Italy and confirm that neosporosis should be considered in the differential diagnosis of pyogranulomatous dermatitis in dogs. Clindamycin may be an effective treatment for cutaneous neosporosis.

Publication Types:
Case Reports

PMID: 9735914 [PubMed - indexed for MEDLINE]

[malernee]

J Am Vet Med Assoc. 1995 Apr 1;206(7):1000-1. Related Articles, Links


Neospora caninum pneumonia in an adult dog.

Greig B, Rossow KD, Collins JE, Dubey JP.

Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Minnesota, St Paul 55108, USA.

Neospora caninum was identified in a lung aspirate specimen from an adult dog with severe pneumonia. Neosporosis was tentatively diagnosed by cytologic examination of a Wright-Giemsa-stained smear of the aspirate specimen, using light microscopy. The infection was confirmed by immunohistochemical examination of a section of lung tissue obtained at necropsy. Neosporosis is usually a fatal ascending neurologic disease of dogs less than 1 year of age. Neospora caninum infections are uncommon in adult dogs and do not have a consistent clinical course. The disease in the dog of this report was unique because the dog had clinical respiratory tract disease and because preliminary diagnosis was made by cytologic examination.

[malernee]

J Am Vet Med Assoc. 1990 Jul 1;197(1):87-9. Related Articles, Links


Diagnosis and treatment of Neospora caninum infection in a dog.

Hay WH, Shell LG, Lindsay DS, Dubey JP.

Virginia-Maryland Regional College of Veterinary Medicine, Department of Small Animal Clinical Sciences, Virginia Polytechnic Institute and State University, Blacksburg.

Neospora caninum, a protozoan organism, caused extensor rigidity of the pelvic limbs in a 12-week-old dog. Diagnosis was based on results of muscle biopsy, neuroelectrodiagnostics, serotesting, and cell culture. Indirect fluorescent antibody (IFA) titer to N caninum was 1:800 at time of admission and 1:3,200 after 4 and 6 weeks. A reciprocal IFA titer of 50 to N caninum was also found in the CSF. Serotesting for T gondii was negative. Treatment with clindamycin followed by sulfadiazine and trimethoprim did not change the pelvic limb extensor rigidity, but other signs of minor neurologic dysfunction improved.

[malernee]

Vet Rec. 1996 Nov 2;139(18):439-43. Related Articles, Links


Clinical aspects of 27 cases of neosporosis in dogs.

Barber JS, Trees AJ.

Department of Veterinary Parasitology, Liverpool School of Tropical Medicine.

Twenty-seven cases of neosporosis in European dogs are described. The disease was confirmed by immunohistochemistry, electron microscopy, or a favourable response to treatment in the dogs with appropriate clinical signs, and by the presence of antibodies to Neospora caninum but not to Toxoplasma gondii. The affected dogs were two days to seven years old, and of 13 different breeds. Both sexes were affected and in most cases littermates remained normal. Twenty-one cases had an initial hindlimb paresis or ataxia, in which muscle atrophy was the most consistent clinical sign. Rigid hyperextension developed in approximately half of the cases. Anorexia and pyrexia were rare. Other clinical signs included forelimb ataxia, head tremors with tetraparesis and sudden collapse due to myocarditis. Titres of > or = 1:800 in the N caninum indirect fluorescent antibody test were detected in the 20 cases from which serum samples were taken. Such high titres are rare in healthy dogs and strongly suggest a diagnosis of neosporosis. Sixteen of the dogs received appropriate antiprotozoal treatment with clindamycin, potentiated sulphonamides and/or pyrimethamine; 10 made a full or functional recovery. Recovery was less likely in peracute cases with severe clinical signs, and when the treatment was delayed.

PMID: 8931299 [PubMed - indexed for MEDLINE]

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Educational Events

Report on November 1997 Seminar
NEOSPOROSIS IN DOGS.

SUMMARY
1. Usually neuromuscular disease (ascending paralysis), but wide range of signs possible.
2. Most commonly reported in puppies and young dogs, but may occur at any age.
3. Worldwide occurrence. No breed or sex predilections.
4. Serology (a type of blood test) is most useful diagnostic test. Examination of biopsy or post-mortem tissues confirms.
5. Treatment results in functional recovery in many cases.
6. Control/prevention is difficult since the lifecycle and modes of transmission of Neospora caninum are incompletely understood.
INTRODUCTION
Neosporosis is a relatively recently recognised disease complex, which has most commonly been reported as an ascending paralysis of young dogs, but which can cause a wide variety of clinical signs in dogs of all ages.
NEOSPORA CANINUM - THE CAUSATIVE ORGANISM
· A microscopic, Toxoplasma gondii-like parasite, causing muscle weakness, was described in three litters of Boxer puppies in Norway in 1984. In 1988 researchers in the United States named the causative organism Neospora caninum.
· The parasite was isolated in 1988, grown in tissue culture and tests were developed. There followed many reports of neosporosis, particularly as the neuromuscular form of the disease in litters of young pups, from all over the world. Examination of stored tissue samples revealed that neosporosis was not a new disease, but occurred in dogs at least as early as the 1950s.
· N.caninum occurs naturally in cattle (where it is an important cause of abortion), sheep, goats, deer and horses. Experimental infections have been reported in many species, including rats, mice and monkeys but no natural cases have yet been reported in cats or man.
· The lifecycle of N.caninum is not yet fully understood.
WHICH DOGS ARE AT RISK FROM NEOSPOROSIS?
Age
Dogs of any age may develop neosporosis. Most reported cases have involved several littermates, with clinical signs developing at between two and twenty weeks of age, but confirmed cases have occurred in dogs as young as 2 days old and as old as 15 years, and many involve isolated individuals, as well as groups of cases in related dogs. It is also possible that neosporosis may result in stillbirths and abortions/resorptions, but neosporosis has not been confirmed in such cases as yet
Breed
Any breed or type of dog may be affected. Neosporosis has been confirmed in more than 30 breeds, ranging from Yorkshire Terrier, Cavalier King Charles Spaniel and West Highland White Terrier through Border Collie, Springer Spaniel and Husky to Great Dane, Bernese Mountain Dog and Irish Wolfhound. Labradors and Boxers have been well represented, but these are very popular breeds.
Cases have been reported from owners with a single pet through to large breeders with many animals on the premises, and in dogs from both rural and urban areas. No sex predilections have been found.
Other concurrent disease, such as canine distemper, is not commonly found in cases of neosporosis, but immunosuppression may make the disease worse.
CLINICAL SIGNS
Most commonly - a hindlimb weakness, which progresses to paralysis, forelimb weakness and difficulty in chewing, swallowing and then breathing, resulting in death or euthanasia. The course of the disease is variable, with peracute cases dying within a week of the first signs being noticed, to a much more chronic course in which signs gradually progress over several weeks. Owners often initially notice a bunny-hopping type of gait, reluctance to jump up or a splaying out of legs when squatting. One or both hindlimbs may be affected. In about half of cases a rigid extension of stifle and/or hock develops. Incontinence is rare initially but may develop as the disease progresses. Fever and inappetance are rare, with most dogs remaining bright and alert until the later stages.
Dogs may also present with one or more of the following signs:-
· Weakness/paralysis of forelimb(s) only
· Drunken-type or high stepping gait
· Altered behaviour
· Blindness
· Head tilt
· Head nodding / tremors
· Seizures (fits)
· Sudden death due to inflammation of the heart
· Pneumonia (cough, breathlessness)
· Skin abnormalities
Many other conditions may result in similar clinical signs, including :-
· Trauma e.g. bitch must have stood on pup, 'dog fell, etc.
· Intervertebral disc disease including 'slipped discs", wobblers
· Toxoplasmosis
· Other infectious diseases e.g. canine distemper, rabies
· Congenital/inherited defects e.g. progressive axonopathy of Boxers, spina bifida
· Thrombo-embolic diseases (bloodclots)
· Neoplasia (growths/cancer)
· Poisoning e.g. botulism
etc., etc.
DIAGNOSTIC TESTS
X-rays are often used to rule out differential diagnoses, as are routine blood tests (haematology and clinical biochemistry). There are no specific changes in these tests in cases of neosporosis.
Serology (blood tests to measure antibody levels) - the indirect fluorescent antibody test (IFAT) is usually used to measure antibodies to N. caninum. A result (titre) of 1:50 or more (this is the dilution of blood used in the test) is considered positive evidence of exposure to N. caninum, but not necessarily of disease. Virtually all confirmed cases of neosporosis have had high titres (1:800 or more). Although a few clinically normal dogs have had titres up to 1:12800, a titre of 1:800 or more in a dog with clinical signs is good supportive evidence of neosporosis. Most titres fall over a period of weeks following treatment However, antibodies remain detectable for many months or even years. Test titre results depend on many factors and the above information relates mainly to the IFAT used at Liverpool University, and not necessarily to tests carried out by other labs.
Collection of the fluid around the spinal cord (CSF analysis) usually reveals non-specific changes, but parasites may be detected
Electromyography/nerve conduction studies may reveal nerve or muscle damage.
CT/MRI scans are not yet widely available enough to have been used in many cases of neosporosis, but these techniques may be able to detect some CNS changes, such as large cysts and areas of inflammation, as occurs in toxoplasmosis in humans.
CONFIRMATION OF NEOSPOROSIS
Pre-death biopsies (e.g. of muscle, or skin in dermatological cases), utilising special staining techniques (immunohistochemistry), may confirm the diagnosis.
Post-mortem findings may confirm the disease in fatal cases. Parasites are most likely to be found in sections of brain, spinal cord and affected muscle, but may also be seen in heart lungs, liver and/or kidney.
DNA tests, such as polymerase chain reaction (PCR) techniques are likely to be more widely available in the near future to identity parasite nucleic add, in CSF samples, biopsy material or tissue sections.
TREATMENT
· Clindamycin (Antirobe) [11-22mg/kg twice daily
· Potentiated sulphonamides (e.g. Tribissen Co-Trimoxazole) [15mg/kg twice daily]
· Pyrimethamine (Daraprim ,anti-malarial drug) [1mg.kg once daily]
Treatment should be instituted as soon as possible when neosporosis is suspected. Since the drugs have few side effects and are relatively cheap, this might even be before serological test results are available. If the dog is going to respond, there should be some improvement within a few days of commencing treatment Treatment should continue until the dog has fully recovered or no further clinical improvement is seen (2-9 weeks). Supportive treatment, e.g. aspirin like drugs, low doses of corticosteroids, plus good nursing care e.g. bladder expression and physiotherapy are also beneficial.
About half of appropriately treated dogs might be expected to make a full or functional recovery, although many are left with an odd gait, muscle wastage or roached back. Rigid hyperextension is the sign least likely to be reversed. If this hyperextension is unilateral, amputation of the affected limb may improve the dogs mobility. Peracute and very chronic cases are the least likely to respond.
There is anecdotal evidence that relapses may occur, but these generally respond well to a further short course of treatment
There is also evidence that some, generally more mildly affected, dogs make a spontaneous recovery.
CONTROL AND PREVENTION
Transmission of the parasite from an infected, but clinically normal bitch to her puppies was for a long time the only confirmed route of infection in dogs. However, the number of puppies infected in each litter varies from none to all of the litter, with overall only about 20% pups seropositive. Fewer than half of these infected pups are ever likely to develop clinical signs of neosporosis. Transmission can occur repeatedly over several consecutive litters. Bitches with an IFAT titre of 1:50 have produced affected puppies, but infections and disease are more likely in pups born to bitches with high titres. Care should be taken in interpreting low IFAT results - the sensitivity and specificity of the tests used are not known exactly, and the risks of producing an affected puppy is actually quite low. Similarly, there may be legal implications if a positive bitch is bred from, regarding whether or not resulting puppies should be tested before sale and whether any results should be disclosed to purchasers when puppies are apparently healthy at the time of the sale. Such matters need careful discussion!
There is only limited data available on the preventative treatment of bitches during pregnancy to prevent prenatal infection of pups, or of seropositive littermates, but such treatments have generally been unsuccessful.
Since congenital infection is far less than 100% efficient and experimentally, cats and mice have been infected by mouth), infection after birth is also suspected to occur, and studies of infection rates in many dogs of all ages have provided evidence that this is so. The route of infection is thought to be through the ingestion of raw meat (especially beef), so it is sensible to advise cooking meat thoroughly before feeding (including scraped beef used for weaning puppies). It is also likely that freezing would destroy the parasite. It is also possible that infection could result from the ingestion of the parasite from the environment after it has been shed by other infected animals, although the identity of such animals is presently unknown.
Finally, although there is evidence that post-natal infection does occur, it is still not known whether disease in adult dogs is due to a relapse of congenital infection or follows from a recent infection in the adult dog.
HOW COMMON IS NEOSPOROSIS?
Neosporosis occurs worldwide. Cases have been reported from Europe, USA, Canada, Australia, South Africa, Japan and Costa Rica. There is also evidence of infection in equatorial Africa and South America.
Prevalence (of infection, not disease) varies from 0.5% to 17% of dogs tested. Studies in the UK have shown a decline in prevalence over the past 10 years. Whether or not such a decline is also happening in other countries is not known.
CONCLUSION
Neosporosis is not a common disease, but it may result in a wide variety of clinical signs. It should be suspected in all neuromuscular disorders where another cause has not been confirmed. Indeed, since treatment is relatively sate and not too expensive, early treatment of suspected cases, even before test results are available, is worthwhile. Neosporosis should also be considered in cases of sudden death in pups and young dogs, in ulcerative dermatitis and in cases of pneumonia where serology and/or histopathology will aid in diagnosis.


ACKNOWLEDGEMENTS
I would like to thank Petsavers for supporting the studies on canine neosporosis carried out at Liverpool University, and Prof A.J.Trees and all the members of the Veterinary Parasitology group there. Also all the vets and dog owners who helped in my research.
Dr Jackie Barber, BVetMed, PhD, MRCVS formerly at :-
Veterinary Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA Phone 01517089393.
Now in private practice in North West England.

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