Use of a multiplex polymerase chain reaction assay in the antemortem diagnosis of toxoplasmosis and neosporosis in the central nervous system of cats and dogs

OBJECTIVE: To develop a multiplex polymerase chain reaction (PCR) assay for the detection of Toxoplasma gondii and Neospora caninum DNA in canine and feline biological samples. SAMPLE POPULATION; Biological samples from 7 cats with systemic (n = 4) or CNS (3) toxoplasmosis, 6 dogs with neospora- or toxoplasma-associated encephalitis, and 11 animals with nonprotozoal disease. PROCEDURE: Primers for T gondii, N caninum, and the canine ferritin gene (dogs) or feline histone 3.3 gene (cats) were combined in a single PCR assay. The DNA was extracted from paraffin-embedded brain tissue, CSF, or skeletal muscle. The PCR products with positive results were cloned, and sequence identity was confirmed. RESULTS: Of 7 cats and 4 dogs with immunohistochemical or serologic evidence of toxoplasmosis, PCR results were positive for all cats and 3 dogs for T gondii, and positive for T gondii and N caninum for 1 dog. Another dog had negative PCR results for both parasites. Of 2 dogs with immunohistochemical or serologic evidence of neosporosis, PCR results were positive for 1 for N caninum and positive for the other for T gondii. All negative-control samples yielded negative results for T gondii and N caninum on the PCR assay. CONCLUSIONS AND CLINICAL RELEVANCE: Standard tests for toxoplasmosis or neosporosis associated with the CNS rely on serologic, histologic, or immunohistochemical analysis and can be difficult to interpret. The multiplex PCR assay with built-in control reactions could be a complementary clinical tool for the antemortem diagnosis of toxoplasmosis or neosporosis associated with the CNS.     

Diagnosis and treatment of Neospora caninum infection in a dog

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.     

Clinical neosporosis in the dog: a review

Neospora caninum is an intracellular protozoan parasite that was first recognized in dogs in 1988. N. caninum may cause neuromuscular disease in dogs. Later, it was discovered that N. caninum has a wide host range and is an important cause of abortion in cattle. In this article, the literature on N. caninum in the dog is reviewed, with emphasis on clinical signs, pathology, diagnosis, treatment, and prognosis.     

Nematode eggs observed in cytology of cerebrospinal fluid diagnostic for intramedullary Spirocerca lupi spinal cord migration

Spinal spirocercosis due to aberrant Spirocerca lupi nematode migration is an emerging etiology for acute myelitis in dogs in Israel, causing severe, mostly nonsymmetrical hind limb paresis or paralysis, and sometimes tetraparesis or tetraparalysis. So far, incidental identification of parasites during spinal surgery or at necropsy provides the only definite diagnosis, while antemortem diagnosis of this condition has been uncertain. Specifically, antemortem diagnosis is based on the typical clinical presentation of acute, progressive, asymmetrical hind limb paresis or paralysis, with moderate to severe eosinophilic to mixed cerebrospinal fluid (CSF) pleocytosis and increased CSF protein concentration. Exclusion of other differential diagnoses also requires using spinal cord imaging. In this novel report, we document a case of an intradural spinal spirocercosis in a dog, diagnosed antemortem, by detecting S lupi eggs in the CSF, and subsequent treatment, resulting in the resolution of the clinical signs.     

Repeated transplacental transmission of Neospora caninum in dogs

Four litters of German Shorthaired Pointers from one owner developed a toxoplasmosis-like illness. According to the records, 29 of 39 dogs had hind limb paralysis. Six dogs from 2 litters were necropsied and had generalized encephalomyelitis. Tachyzoites and tissue cysts of Neospora caninum were found in the brain and spinal cord of each dog. Lesions were found in the eyes, extraocular muscles, or both in all of the dogs, and N caninum was detected microscopically in the eyes (retina and choroid in 1 dog), extraocular muscles, or both in 5 of the 6 dogs. Ocular lesions consisted of focal retinitis, choroiditis, mild nonspecific iridocyclitis, and myositis of extraocular muscles. Organisms stained with anti-N caninum serum, but not with anti-Toxoplasma gondii serum in an immunohistochemical test, except in 1 dog. In one dog, aged thick-walled N caninum tissue cysts reacted mildly with anti-T gondii serum.     

Leishmania infantum and Neospora caninum simultaneous skin infection in a young dog in Italy.

Leishmania infantum, the agent of canine leishmaniasis in Mediterranean countries, and Neospora caninum, a recently recognized protozoal pathogen in dogs, were diagnosed in a 9-month-old Argentine Dogo dog. Both skin lesions and neurological signs were present. Histopathology of cutaneous lesions revealed a suppurative, diffuse dermatitis with numerous intracellular protozoa. Serology was positive for both L. infantum (1:640) and N. caninum (1:800). Double-label immunohistochemical staining of skin samples with hyperimmune serum from L. infantum-infected dogs was positive for protozoa within macrophages, while the polyclonal antibody specific for N. caninum showed positive reactions for protozoa in endothelial cells and fibroblasts. Transmission electron microscopy (TEM) confirmed the infection with both protozoa. This is, to the authors' knowledge, the first case of simultaneous infection with L. infantum and N. caninum in a dog. It is possible that the immunosuppressive effects of Leishmania infection or long-term steroid therapy may have been a contributing factor to the development of N. caninum in this dog.     

Failure of dogs to shed oocysts after being fed bovine fetuses naturally infected by Neospora caninum

Neospora caninum is a protozoan that causes abortion in cattle. The dog has recently been identified as a definitive host for N. caninum. To verify if bovine fetuses can infect dogs, nine 2-4-month-old dogs were fed bovine fetuses naturally infected by N. caninum. None of the dogs excreted oocysts, seroconverted, had clinical signs or lesions compatible with N. caninum infection. Additional studies will be necessary to determine the natural mode of infection of dogs by N. caninum.     

The role of the dog in the epidemiology of neosporosis in cattle

Neospora caninum is an intracellular protozoan parasite that was discovered in a dog in 1988. Since then, N. caninum has been demonstrated in a variety of animal species and it has been recognized as an important cause of abortion in cattle. An infection with N. caninum can be maintained in cattle herds for several generations by transplacental transmission from cow to calf. Recently, it was demonstrated that dogs can act as definitive hosts of N. caninum and therefore may be a source of infection for other species by shedding oocysts. Further evidence of a role of the dog in spreading the infection to cattle has been derived from epidemiological studies. The present state of knowledge is reviewed in this paper.     

Seroprevalence and risk factors associated with Neospora caninum infection in different dog populations in Spain

In this study, Neospora caninum seroprevalence and some associated risk factors were investigated in four different dog populations in Spain. N. caninum seropositivity was significantly higher in farm dogs (51%, 51/100) (P<0.001) and lower in household dogs (2.9%, 3/102) (P<0.0001). The seroprevalence in hunting (23%, 23/100) and stray (24.5%, 23/94) dogs was moderate, and no significant differences were observed between these two populations (P>0.05). A significantly higher number of dogs showed titres of 1:50-1:100 (68%, 68/100) than >or=1:200 (33%, 33/100) titres (P<0.0001). N. caninum antibodies were more often detected in mixed breed than pure breed dogs (P<0.01), but when data were stratified by dog type a significant association was not found (P>0.05). A significantly higher prevalence of N. caninum was observed in dogs over 1 year old (P<0.01), indicating that horizontal transmission may be the most important route of infection. The presence of N. caninum antibodies was significantly more frequent in Leishmania infantum-seropositive hunting (P<0.05) and stray dogs (P<0.00001). This study confirms that farm, stray and hunting dogs can be considered at-risk dog populations for N. caninum infection in Spain.     

In vitro activity of neem (Azadirachta indica) and cassava (Manihot esculenta) on three pre-parasitic stages of susceptible and resistant strains of Teladorsagia (Ostertagia) circumcincta

Canines are definitive hosts of Neospora caninum (Apicomplexa). For horizontal transmission from canines to occur, viable oocysts of N. caninum must occur in the environment of susceptible intermediate hosts. Canids in Australia include wild dogs and Aboriginal community dogs. Wild dogs are those dogs that are not dependent on humans for survival and consist of the dingo, feral domestic dog and their hybrid genotypes. Aboriginal community dogs are dependent on humans, domesticated and owned by a family, but are free-roaming and have free access throughout the community. In this study the extent of N. caninum infection was determined in a total of 374 dogs (75 wild dogs and 299 Aboriginal community dogs) using a combination of microscopic, molecular and serological techniques. Oocysts of N. caninum were observed in the faeces of two juvenile Aboriginal community dogs (2/132; 1.5%). To estimate N. caninum prevalence, a new optimised cut-off of 18.5% inhibition for a commercial competitive ELISA was calculated using a two-graph receiver-operating characteristic (TG-ROC) analysis and IFAT as the gold standard resulting in equal sensitivity and specificity of 67.8%. Of the 263 dog sera tested the true prevalence of N. caninum antibodies was 27.0% (95% confidence limit: 10.3-44.1%). The association between the competitive ELISA results in dogs less than 12 month old and older dogs was significant (P=0.042). To our knowledge this is the first large scale parasitological survey of the Aboriginal community dogs and wild dogs from Australia. The high prevalence of N. caninum infection in Aboriginal community dogs illustrates that horizontal transmission of N. caninum is occurring in Australia. These results demonstrated that N. caninum in dogs is widespread, including the semi-arid to arid regions of north-western New South Wales and the Northern Territory. The populations of free-ranging dogs are likely to be important contributors to the sylvatic life cycle of N. caninum.     

Studies on serological cross-reaction of Neospora caninum with Toxoplasma gondii and Hammondia heydorni

The enzyme-linked immunosorbent assay (ELISA) was used to examine cross-reactivity of Neospora caninum with Toxoplasma gondii and Hammondia heydorni. Anti-T. gondii mouse and cat sera cross-reacted with N. caninum soluble antigen (NLA), but not with the recombinant surface antigen (NcSRS2). Anti-H. heydorni dog sera showed no cross-reactivity with either the NLA antigen or the NcSRS2. Lack of cross-reactivity between anti-H. heydorni sera and N. caninum antigens, and the cross-reactivity of anti-T. gondii sera with the NLA suggest that N. caninum has common antigens to T. gondii except for NcSRS2 based on serology. In light of several studies suggesting a closer relationship between N. caninum and H. heydorni than with T gondii, examination of serological cross-reactivity with N. caninum may be necessary to further classify the parasites in addition to molecular and morphological studies and clarification of the life cycle.     

Isolation of Neospora caninum from the brain of a naturally infected dog, and production of encysted bradyzoites in gerbils

Neospora caninum was isolated from the brain of an adult dog in Brazil. Cerebral tissue from the dog was inoculated into Mongolian gerbils. Gerbils were euthanized 3-4 months later and bradyzoite-containing tissue cysts were observed in their brains. N. caninum (designated NC-Bahia) was isolated in cell culture after inoculation with tissue cysts from the gerbils. The identity of the parasite was confirmed by immunohistochemical examination and polymerase chain reaction (PCR). Gerbils may be a useful alternative to immunosuppressed mice for isolation of N. caninum and for production of encysted bradyzoites.     

Point source exposure of cattle to Neospora caninum consistent with periods of common housing and feeding and related to the introduction of a dog

Eight dairy herds with evidence of post-natal transmission of Neospora caninum were used to test the hypothesis of a point source exposure by a retrospective analysis of the housing and feeding of infected age-groups. The first N. caninum-associated abortion or birth of N. caninum-seropositive offspring from the post-natally infected age-group was considered as the first indication of the infection. In seven of the eight dairy herds, a point source exposure to N. caninum of the infected age-groups was found during a limited period of common housing and feeding. In all herds studied, the analysis indicated that the cattle had been infected shortly before the first abortions occurred. In all, except one herd, the post-natal infection was more directly related to housing than to feeding. Therefore, it appeared that the feed was contaminated in the feeding alley. In one herd, the total mixed ration was found to be the probable path of infection. In all farms studied, a new dog (young, adult dog or litter) had been introduced within a period of 1.5 years prior to the first indication of N. caninum infection in the cattle. As there was evidence in all herds of vertical transmission of neosporosis for years, it is hypothesized that the newly introduced dog was infected with N. caninum by materials from already infected cattle and subsequently transmitted the infection to other cattle by shedding of oocysts.     

Experimental infection of dogs (Canis familiaris) with sporulated oocysts of Neospora caninum

Neospora caninum is widely distributed in the world and this parasite is one of the major causes of abortion in cattle. Dogs and coyotes are definitive hosts of N. caninum and several species of domestic and wild animals are intermediate hosts. Dogs can become infected by the ingestion of tissues containing cysts and then excrete oocysts. It is not yet known whether sporulated oocysts are able to induce a patent infection in dogs, i.e. a shedding of N. caninum oocysts in feces. The objective of this study was to experimentally examine the infection of dogs by sporulated oocysts. The oocysts used in the experiment were obtained by feeding dogs with brain of buffaloes (Bubalus bubalis) positive for anti-N. caninum antibodies by indirect fluorescent antibody test (IFAT ≥200). Oocysts shed by these dogs were confirmed to be N. caninum by molecular methods and by bioassay in gerbils, and sporulated N. caninum oocysts were used for the oral infection of four dogs. The dogs were 8 weeks old and negative for antibodies to N. caninum and Toxoplasma gondii. Dogs 1 and 4 received an inoculum of 10,000 sporulated oocysts each; dog 2 an inoculum of 5000 sporulated oocysts and dog 3 received 1000 sporulated oocysts of N. caninum. The total feces excreted by these dogs were collected and examined daily for a period of 30 days. No oocysts were found in their feces. The dogs were monitored monthly for a 6-month period to observe a possible seroconversion and when this occurred the animals were eliminated from the experiment. Dogs 1 and 4 seroconverted 1 month after the infection with titer, in the IFAT, of 1600 and 800, respectively; the other two dogs presented no seroconvertion during the 6-month period. Dogs 1 and 2 were euthanized 180 days after infection and were examined for the detection of N. caninum in tissues (brain, muscle, lymph node, liver, lung, heart and bone marrow) by immunohistochemistry and PCR with negative results in both techniques. Bioassay in gerbils with brain of these dogs was also performed and again the results were negative. In conclusion, dogs infected with sporulated oocysts of N. caninum were not able to shed oocysts in feces. However, a higher dose of infection stimulated the production of antibodies against N. caninum in the dogs.     

Confirmed clinical Neospora caninum infection in a boxer puppy from Argentina

Generalized neosporosis was diagnosed in a 2-month-old boxer puppy. The dog had a history of progressive paralysis and muscle atrophy, followed by cervical weakness, stiff jaws and dysphagia. The dog had a 1:12,800 antibody titer for Neospora caninum and was negative for antibodies to Toxoplasma gondii by the indirect fluorescent antibody test (IFAT). After euthanasia a complete necropsy was carried out. The puppy had a megaesophagus. Microscopically, tachyzoites and tissue cysts were observed in histologic brain sections. Severe myositis was observed in esophagus and striated muscle sections and several groups of tachyzoites were associated with these lesions. Immunohistochemically, parasites in the brain and striated muscle reacted to anti-N. caninum antiserum. Western blot analysis allowed the identification of three major and four minor antigens of N. caninum tachyzoites corresponding to 30, 37, 45-kDa and 28, 29, 43, 47 and 67-kDa bands, respectively. Cerebral homogenate of the dog was inoculated into four Mongolian gerbils (Meriones unguiculatus). Forty-nine days after inoculation, all the gerbils had positive IFAT titers to N. caninum (1:200, 1:400, 1:100 and 1:400). Genomic DNA was isolated from the brain, lung and striated muscle from the puppy and from the brain of one of the inoculated gerbils. The N. caninum specific primer pair Np 6/21 produced 328 bp amplicons on electrophoretic gels. This is the first confirmed clinical case of generalized canine neosporosis in Argentina.     

Neospora caninum associated with septic peritonitis in an adult dog

A 7-year-old, male neutered Rhodesian Ridgeback dog was referred to the University of California-Davis Veterinary Medical Teaching Hospital with a 4-month history of peritonitis and episodic abdominal discomfort, lethargy, and weakness. Marked abdominal distension with a prominent fluid wave was noted on physical examination. Cytologic analysis of the abdominal fluid indicated a septic exudate with mixed bacteria and many protozoal zoites. Differentials for the identity of the protozoal zoites included Toxoplasma gondii, Sarcocystis neurona, and Neospora caninum. Indirect latex agglutination antigen testing, standard indirect fluorescent antibody testing, and PCR analysis were performed to identify the zoites. The dog's serum antibody titer for N caninum tachyzoites was 1:20,480, known polysera to N caninum reacted against zoites in the abdominal fluid, and PCR analysis of the abdominal fluid was positive for the presence of a known gene of N caninum. Based on the morphologic, immunologic, and molecular findings, the zoites were identified as N caninum. It remains unclear how the tachyzoites gained access to the peritoneal cavity. To the authors' knowledge, there are no reports of free N caninum in abdominal fluid of any species.     

Recent advances in understanding the epidemiology of Neospora caninum in cattle

INTRODUCTION: Neospora caninum is the most frequently diagnosed cause of cattle abortion in New Zealand and is an important pathogen worldwide. The parasite has been found in aborted bovine foetuses and in puppies with neurological disease. Recent developments have provided new insights into the epidemiology of the disease that are reviewed in this article. TRANSMISSION: Transplacental infection is of major importance in the spread of N. caninum overseas. Most congenital infections result in the birth of apparently healthy calves. Seroepidemiological studies indicate introduced point-source infections are also likely, as do investigations of abortion outbreaks in cattle herds attributed to N. caninum. Horizontal transmission is an important determinant of the stability of infection prevalence in a herd. Two potential sources of horizontal transmission that have recently been reported are: via a canine definitive host and; cow-to-calf transmission through pooled colostrum or milk. Transmission parameters for the organism have been estimated using mathematical modeling, to explore the epidemiology and options for control of N. caninum infection in dairy cattle in the absence of precise epidemiological data. LIFE-CYCLE: Seroepidemiologic studies, combined with the knowledge that dogs can be a definitive host, provide evidence supporting a dog-cattle life-cycle. The importance of dogs in the epidemiology of bovine neosporosis is not yet clear. It is likely that N. caninum oocysts in dog faeces could serve as a source of infection for cattle and recent studies have shown that the canine and bovine isolatesflare the same organism. Infection of cattle orally exposed to oocysts has been demonstrated experimentally but not in nature. It is not known if other wild carnivores may serve as definitive hosts of N. caninum. CONCLUSION: Despite active research for over a decade, very little is known about the mechanisms of transmission of N. caninum. Vertical transmission is well documented but horizontal spread and the pathogenesis of abortion from this disease need further investigation. It is evident that, even with a very high probability of vertical transmission, some form of horizontal transmission is required for the disease to persist in a herd.     

Detection of Neospora caninum in dog organs using real time PCR systems

Neospora caninum is a parasite responsible for paresis in dogs. The dog can harbour enkysted parasites in several organs. The detection of N. caninum was performed using 3 different real time PCR systems all amplifying the NC5 DNA region. One system was based on Sybrgreen, one on Plexor technology and the last on Taqman probe. Comparison of the three methods indicated that the detection limit was 1 equivalent genome on pure DNA but that this detection limit increased in the presence of foreign DNA using the Sybrgreen and Plexor systems. Therefore, the Taqman system was chosen to detect N. caninum in liver and spleen of naturally infected dogs. The overall prevalence was 32.2%. Comparison between PCR results and serological results using IFAT showed that among the 28 PCR positive dogs only 9 were seropositive and that 8 seropositive dogs were PCR negative. Therefore serology can underestimate the real carriage in dogs. However, PCR methods must be improved in terms of sensitivity and inhibition problems.     

Incidence of Neospora caninum and other intestinal protozoan parasites in populations of Swiss dogs

The protozoan parasite Neospora caninum is one of the most important abortifacient organisms in cattle worldwide. The dog is known to act as definitive host although its potential role as infection source for bovines still remains unelucidated. The aim of the present study was to compile initial epidemiological data on the prevalence and incidence of N. caninum in Swiss dogs acting as definitive hosts. Thus, 249 Swiss dogs were investigated coproscopically in monthly intervals over a period of 1 year. A total of 3289 fecal samples was tested by the flotation technique. Among these, 202 were shown to contain Sarcocystis sp. (6.1%), 149 Cystoisospora sp. (=Isospora sp.; 4.5%) and 25 Hammondia/Neospora-like oocysts (HNlO) (0.7%). All but one sample containing HNlO were from different dogs; one dog shed HNlO at two subsequent time points. Calculation of the yearly incidence for HNlO resulted in the surprisingly high value of 9.2%. Farm dogs exhibited a higher incidence for HNlO than urban family dogs. Thirteen out of the 25 HNlO-samples showed sporulation after 5 days incubation at room temperature. HNlO were further differentiated by species-specific PCR. However, all HNlO-samples were negative for N. caninum, Hammondia heydorni and Toxoplasma gondii. One reason may be the low oocyst density found in most fecal samples, which did not permit us to carry out PCR under optimal conditions. Three out of the 25 HNlO-cases contained enough oocysts to allow further enrichment and purification by the flotation technique. Subsequently, twenty to fifty sporulated HNlO-oocysts were orally administered to Meriones unguiculatus. All gerbils were seronegative for N. caninum at 5 weeks p.i. A N. caninum-seroprevalence of 7.8% was determined by ELISA upon 1132 serum samples collected from dogs randomly selected by veterinarians among their clinical patients.     

Colonic perforation after corticosteroid and surgical treatment of intervertebral disk disease in a dog

Colonic perforation after corticosteroid and surgical treatment of intervertebral disk disease in a dog Perforation of the distal segment of the descending colon, after corticosteroid and neurosurgical treatment for an intervertebral disk herniation, resulted in the death of a 3-year-old, male, Lhasa Apso. Initially, the dog was paraparetic in the hindlimbs, but became paraplegic after 4 days of conservative treatment. Corticosteroids were administered before referral and during decompressive hemilaminectomy. Six days after surgery, the dog had improved neurologically, but was depressed, anorectic, and vomiting. Abdominocentesis revealed septic peritonitis. The dog died shortly after a perforation in the descending colon was surgically corrected.