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Neosporosis is a major cause for abortion in cows, and is also a cause for congenital neurological defects; however, its impact as causative agent of neurological syndromes has been poorly documented. There are few reports of the occurrence of Neospora caninum in congenitally infected calves with neurological signs and in adult cattle (Dubey and Schares 2006).
The aim of this study was to investigate the presence of Neospora—DNA in cattle with neurological signs, which had been diagnosed negative for rabies. All cattle suspected of rabies infection exhibited one or more neurological signs, such as sialorrhea, behaviour alteration, aggressiveness, opisthotonus, blindness, incoordination, paralysis, ataxia and recumbency. The animals died on commercial farms (dairy and beef herds) from 105 municipalities in all regions of Paraná State, southern Brazil. A total of 253 CNS samples were collected from bovines with neurological disease from 236 herds. The animals consisted of 45 calves (<12 months), 76 heifers (12–24 months) and 132 adult bovines (>24 months). Paraná State comprises 199,323.9 km2, and has an estimated cattle population of 9,505,441 animals in 399 municipalities.
The animals were necropsied, and CNS tissues were collected by the veterinarian from the official service of The Agriculture and Supply Secretary of Paraná (SEAB/PR), in order to comply with the National Program for Herbivore Rabies Control of the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA) (BRASIL 2009). The CNS samples were kept in bags and surrounded by ice. All samples were immediately transported by courier service to the approved state veterinary diagnostic laboratory for rabies diagnosis. The samples were submitted to arrive at the laboratory within 48 hours of collection. In the laboratory, tissue was taken from areas of the CNS—brain, cerebellum and medulla oblongata. Samples were collected using clean and sterile instruments. Additionally, samples of brain, cerebellum and medulla oblongata (approximately 100 mg) were also stored at −80°C for DNA extraction.
A total of 253 samples which had been diagnosed negative for rabies were investigated by N caninum PCR. For PCR-based diagnosis of N caninum, the specific genomic Nc5 region was selected as the target sequence for DNA amplification. The Nc5 region has been shown to be highly specific to N caninum (Yamage and others 1996). This method has been shown to be extremely sensitive when used for detection of N caninum DNA in the presence of host DNA in naturally occurring infections (Hughes and others 2006). Primers Np6 and Np21, 328-bp region spanning, were produced according to Yamage and others (1996). As a control to detect contamination, a water blank isolation was always performed alongside the DNA isolation. The sensitivity and specificity were determined for PCR of tissues by using defined Neospora-positive (histological examination and artificial spiking) and Neospora-negative samples. Positive controls (gerbil liver samples experimentally infected with N caninum and confirmed by histology, mouse brain samples spiked with N caninum tachyzoites, and samples from flasks of N caninum-infected Vero cells cultures) and negative controls (brain samples from a non-infected mouse and samples from flasks of non-infected Vero cells cultures) were included in each PCR run. PCR reactions were conducted carefully to minimise contamination as previously described (Dubey and Schares 2006, Hughes and others 2006). Specificity of PCR was 100% for mouse brain and non-infected Vero cells cultures. Sensitivity of N caninum PCR was 91% for experimentally infected samples.
N caninum DNA was successfully amplified from brain tissue from 28 of the 253 samples tested. The PCR assay yielded the expected 328-bp product in the brain samples of five calves (<12 months), six heifers (12–24 months), and 17 adult cattle (>24 months). N caninum DNA was found in both dairy and beef breeds of cattle, as Simmental (two), Holstein (five), Jersey (one), Nelore (four), Girolanda (one), Brown Swiss (four), Canchim (one), and cattle without defined breed (ten). The positive animals identified were from 20 municipalities. These results confirmed the presence of N caninum DNA in the brain of cattle with neurological signs.
The frequency of 11.1 per cent of CNS-positive specimens for N caninum found in this study is higher than that reported by Malaguti and others (2012), who found 0.66 per cent of neosporosis by PCR in CNS of 302 cattle with neurological syndrome that tested negative for rabies in São Paulo State, however, the specimens came from 10 Brazilian states. In the present study, Neospora DNA was found in 23 bovines (>24 months) with neurological signs. Malaguti and others (2012) found only two positive cases by PCR (Nc5 region) in adult cattle in São Paulo, however, fresh and frozen tissue were used for DNA extraction, and additional information about the samples were not reported. In the present study, specimens came only from Paraná State, where the seroprevalence to N caninum is high, and the parasite causes neurological signs (Locatelli-Dittrich and others 2003). The studies in Paraná State have found that 33 per cent (423/1263) of cattle were seropositive for antibodies against N caninum, 77 per cent (59/77) of the farms had seropositive cattle, and seropositive animals were reported in all regions of the state, showing that the parasite is widely distributed in the state (Locatelli-Dittrich and others 2008). The higher prevalence of Neospora DNA in this study may be also associated with the samples (ie, areas of the brain that were selected and amount of the sample used for PCR). There is no evidence as to which tissues are the most suitable for sampling. However, a number of studies indicate that brain tissue is the most suitable for the detection of N caninum DNA by PCR (Dubey and Schares 2006).
There are two reports regarding neurological syndrome associated with N caninum in Paraná state (Locatelli-Dittrich and others 2003, Santos and others 2006). These studies reported cases of neosporosis in calves, however, the disease was not detected in adult cattle. N caninum (BNC-PR1) was isolated from a naturally infected blind calf which was born on a dairy farm in Paraná State. The calf was born weak and unable to rise on its own, and it had shown signs of visual and nervous system defects since birth (Locatelli-Dittrich and others 2003). Encephalitis was reported in a calf infected with N caninum, also in Paraná State (Santos and others 2006).
A bovine older than one year was diagnosed with N caninum-associated encephalitis in Sao Paulo State, Brazil, by PCR and histological examination. The animal showed clinical signs of neurological syndrome (sialorrhea, incoordination, recumbency and opisthotonus), and died in four days (Malaguti and others 2012).
In the present study the detection of N caninum DNA in the brain of cattle could suggest that the neurological signs were caused by the parasites, however, further work is required to establish the possible role of Neospora in relation to neurological disease in cattle. N caninum affects the CNS, resulting in neurological disease in calves, adult dogs and sheep (Bishop and others, 2010; Garosi and others, 2010). In recent years, Neospora is also suggested to play a potential role in neurologic disorders in horses (Dubey and Schares 2011).
Although N caninum-infected calves are usually born healthy but persistently infected, the neurological signs reported in the cattle in this study could be the result of reactivation of latent tissue cysts when the cattle become immunosuppressed, and also maybe associated with the strain of N caninum (Dubey and Schares 2011). Because the Brazilian isolate (BNC-PR1 strain) causes neurological signs in cattle (Locatelli-Dittrich and others 2003), finding of N caninum DNA in the brain of cows and heifers in Paraná State suggests that the parasite might cause clinical neosporosis in adult cattle, however, histological evaluation is required to confirm the diagnosis of neosporosis (Dubey and Schares 2006).
Rabies is an important cause for neurological disease in dairy and beef cattle in Brazil, however, neosporosis should be kept in mind on differential diagnosis of neurological signs in young calves, heifers and adult cattle. This study represents the first report of DNA prevalence of N caninum in cattle with neurological syndrome in southern Brazil, and neosporosis should now be considered in the differential diagnosis of cattle presenting with neurological disorders.
This work was supported by ‘Secretaria de Estado da Ciência, Tecnologia e Ensino Superior’ (SETI) (CV: 037/04).
Provenance: not commissioned; externally peer reviewed
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