Alexander J. Trees

Estimation of vertical and horizontal transmission parameters of Neospora caninum infections in dairy cattle.

Transmission parameters of Neospora caninum infections in dairy cattle were determined in six herds with a history of Neospora-associated abortions, using an antibody-detection ELISA to detect evidence of infection. A total of 124 seropositive dams and their calves were tested at calving to estimate vertical transmission, and 154 seronegative heifers were monitored prospectively from birth for evidence of post-natal infection. The probability of vertical transmission was very high; 95.2% (95% confidence interval: 89.8%, 98.2%) of the seropositive dams produced calves that were seropositive prior to consumption of colostrum. In the prospective study, three heifers seroconverted, by 1, 8 and 16 months of age, and the overall incidence rate of seroconversion was 1.9 per 100 heifer-years at risk, ranging from 0 to 4.1 per 100 heifer-years at risk within herds. If heifers with inconclusive ELISA results were included in the analysis, the incidence rate of seroconversion was 3.8 per 100 heifer-years at risk, ranging from 0 to 8.3 per 100 heifer-years at risk within herds. This study showed that vertical transmission was the major route involved in the spread of N. caninum in these herds, and that there was only a low detectable level of horizontal transmission. However, further studies are needed to provide parasitological evidence of infection in those heifers that seroconverted.

Neospora caninum -associated abortion in cattle: the time of experimentally-induced parasitaemia during gestation determines foetal survival

The parasite, Neospora caninum is an important cause of abortion in cattle. It is transmitted vertically or horizontally and infection may result in abortion or the birth of a live, healthy but infected calf at full-term. Only a proportion of infected cattle abort and the pathogenesis of abortion is not understood. Groups of cattle were infected with 10(7) N. caninum tachyzoites intravenously at different times relative to gestation. Intravenous inoculation was chosen to reproduce the putative haematogenous spread of N. caninum following either recrudescence of endogenous infection or de novo infection. In all cattle, infection was accompanied by high gamma-interferon and lymphoproliferative responses, and a biased IgG2 response indicating that N. caninum infection is accompanied by a profound Th1 helper T cell-like response. Infection at 10 weeks gestation resulted in foetopathy and resorption of foetal tissues 3 weeks after infection in 5 out of 6 cows. Infection at 30 weeks gestation resulted in the birth of asymptomatic, congenitally-infected calves at full term in all 6 cows, whereas the 6 cows infected before artificial insemination gave birth to live, uninfected calves. These results suggest that the reason some cows abort is related to the time during gestation when they become infected or an existing infection recrudesces.

Redescription of Neospora caninum and its differentiation from related coccidia

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums.

Towards evaluating the economic impact of bovine neosporosis

In spite of the global importance of neosporosis as a cause of bovine abortion, there is very little information about its economic consequences. The economic costs are a product of estimations of the quantity of the effects attributable to Neospora infection, and the particular unit costs of those effects. In this brief review, which arose from a workshop on the economics of coccidiosis held at the COST 820 meeting, Toledo 1998, we discuss the possible effects of neosporosis which are of economic significance and summarise the available estimates of their magnitude to provide a basis for further economic analysis. Neospora infection has been associated with abortion, increased culling and reduced milk yield. In addition, it has been diagnosed in cases of stillbirth and neonatal mortality, it is likely to contribute to early foetal death and resorption and it is responsible for a reduction in the value of female breeding cattle. In quantifying the role of Neospora, it is important that epidemiologically based, case-controlled studies are conducted because, given the extreme efficiency with which bovine Neospora infection is vertically transmitted, demonstration of prevalence of infection in affected animals (including foetuses) is not a true indicator of the significance of this disease. Relatively few epidemiological studies have been conducted, but in investigations in the USA, Holland and Britain, infected cows have been shown to be about three times more likely to abort than non-infected cattle. In the UK this approach has been used to estimate the proportion of abortions in the national dairy population which may be attributable to Neospora caninum.

CLINICAL ASPECTS OF 27 CASES OF NEOSPOROSIS IN DOGS

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 ≥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.

Comparative Genomics of the Apicomplexan Parasites Toxoplasma gondii and Neospora caninum: Coccidia Differing in Host Range and Transmission Strategy

Toxoplasma gondii is a zoonotic protozoan parasite which infects nearly one third of the human population and is found in an extraordinary range of vertebrate hosts. Its epidemiology depends heavily on horizontal transmission, especially between rodents and its definitive host, the cat. Neospora caninum is a recently discovered close relative of Toxoplasma, whose definitive host is the dog. Both species are tissue-dwelling Coccidia and members of the phylum Apicomplexa; they share many common features, but Neospora neither infects humans nor shares the same wide host range as Toxoplasma, rather it shows a striking preference for highly efficient vertical transmission in cattle. These species therefore provide a remarkable opportunity to investigate mechanisms of host restriction, transmission strategies, virulence and zoonotic potential. We sequenced the genome of N. caninum and transcriptomes of the invasive stage of both species, undertaking an extensive comparative genomics and transcriptomics analysis. We estimate that these organisms diverged from their common ancestor around 28 million years ago and find that both genomes and gene expression are remarkably conserved. However, in N. caninum we identified an unexpected expansion of surface antigen gene families and the divergence of secreted virulence factors, including rhoptry kinases. Specifically we show that the rhoptry kinase ROP18 is pseudogenised in N. caninum and that, as a possible consequence, Neospora is unable to phosphorylate host immunity-related GTPases, as Toxoplasma does. This defense strategy is thought to be key to virulence in Toxoplasma. We conclude that the ecological niches occupied by these species are influenced by a relatively small number of gene products which operate at the host-parasite interface and that the dominance of vertical transmission in N. caninum may be associated with the evolution of reduced virulence in this species.

Neospora caninum in dogs: detection of antibodies by ELISA using an iscom antigen

An indirect enzyme linked immunosorbent assay (ELISA) for detection of antibodies to Neospora caninum in serum from dogs is described. Extracted tachyzoite proteins incorporated into immunostimulating complexes (iscoms) were used as coating antigen. A mixture of a monoclonal antibody to dog immunoglobulin G and a horse radish peroxidase conjugated antibody to mouse Ig was used to detect bound antibody. When the iscom preparation was analysed by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis it appeared to consist of a restricted number of proteins compared with whole parasite homogenates. In immunoblot analysis, using N. caninum positive sera from rabbits and dogs as probes, the major antigens recognized had approximate molecular weights between 30 and 45 and 17 to 19kDa. Compared with an ELISA using a crude solubilized tachyzoite antigen, the iscom ELISA substantially improved the sensitivity and specificity (to 97·6% and 95·6%, respectively, against an immunofluorescence test, IFAT, as indicator of true status). There was a statistically significant positive correlation between IFAT titres and iscom ELISA OD450 values. The iscom ELISA absorbances (and the IFAT titres) of dogs with proven clinical infections were not higher than those from nonclinically affected, putatively infected dogs.

Prevalence of antibodies to Neospora caninum in different canid populations

A total of 1,554 dogs from 5 countries on 3 continents were tested for antibodies to Neospora caninum using an indirect fluorescent antibody test. In Australia, overall, 42/451 (9%, 95% confidence interval [CI] 6-12%) dogs were seropositive (Melbourne 11/207 [5%, 95% CI 2-9%]; Sydney 18/150 [12%, 95% CI 7-18%]; Perth 13/94 [14%, 95% CI 8-22%]). Antibodies to N. caninum were also detected in dogs in South America (Uruguay [20%, 95% CI 16-24%, n = 414]) and sub-Saharan Africa (Tanzania [22%, 95% CI 12-36%, n = 49]). In contrast, only 1 of 500 dogs tested from the Falkland Islands and none of 140 dogs from Kenya was seropositive. Of wild canids, 1/54 (2%, 95% CI 0-10%) British foxes and 15/169 (9%, 95% CI 5-14%) Australian dingoes had antibodies to N. caninum.

Neospora caninum in persistently infected, pregnant cows: spontaneous transplacental infection is associated with an acute increase in maternal antibody

Nine cows which were naturally and persistently infected with Neospora caninum were housed and observed intensively throughout pregnancy. No recrudescence of a latent infection was detected by PCR tests on maternal blood but fetal infection, implying a recrudescence of maternal parasitosis, was associated with a marked increase in maternal antibody. The increase occurred in the second half of pregnancy in five cows which infected their calves, and before mid-pregnancy in one cow which aborted. There was no change in the avidity of the antibody, which remained high and characteristic of long-term infection. In three infected cows that gave birth to uninfected calves there was no marked increase in maternal antibody. Antigen-specific interferon gamma responses of peripheral blood mononuclear cells were observed in all the infected cattle but they did not vary significantly either during pregnancy, or whether the cows did or did not infect their calves, although the responses were consistently higher in the latter. There was no change in the plasma concentrations of cortisol or acute phase proteins associated with the recrudescence of the parasite. Three uninfected cows housed with the infected cows remained uninfected throughout the experiment. No immunosuppressive event was detected which might have provoked parasite recrudescence but the acute antibody rise associated with transplacental infection provides a valuable, non-invasive marker for further studies to investigate the cause and consequences of parasite recrudescence in N caninum infection in cattle.

Analysis of gene expression from the Wolbachia genome of a filarial nematode supports both metabolic and defensive roles within the symbiosis

The α-proteobacterium Wolbachia is probably the most prevalent, vertically transmitted symbiont on Earth. In contrast with its wide distribution in arthropods, Wolbachia is restricted to one family of animal-parasitic nematodes, the Onchocercidae. This includes filarial pathogens such as Onchocerca volvulus, the cause of human onchocerciasis, or river blindness. The symbiosis between filariae and Wolbachia is obligate, although the basis of this dependency is not fully understood. Previous studies suggested that Wolbachia may provision metabolites (e.g., haem, riboflavin, and nucleotides) and/or contribute to immune defense. Importantly, Wolbachia is restricted to somatic tissues in adult male worms, whereas females also harbor bacteria in the germline. We sought to characterize the nature of the symbiosis between Wolbachia and O. ochengi, a bovine parasite representing the closest relative of O. volvulus. First, we sequenced the complete genome of Wolbachia strain wOo, which revealed an inability to synthesize riboflavin de novo. Using RNA-seq, we also generated endobacterial transcriptomes from male soma and female germline. In the soma, transcripts for membrane transport and respiration were up-regulated, while the gonad exhibited enrichment for DNA replication and translation. The most abundant Wolbachia proteins, as determined by geLC-MS, included ligands for mammalian Toll-like receptors. Enzymes involved in nucleotide synthesis were dominant among metabolism-related proteins, whereas the haem biosynthetic pathway was poorly represented. We conclude that Wolbachia may have a mitochondrion-like function in the soma, generating ATP for its host. Moreover, the abundance of immunogenic proteins in wOo suggests a role in diverting the immune system toward an ineffective antibacterial response.