John McGarry

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.

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.

Novel ELISA for detection of Neospora-specific antibodies in cattle

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Neospora species in cattle was developed. Whole formalin-fixed Neospora caninum (NC-Liverpool) tachyzoites were used as antigen and a monoclonal antibody to bovine immunoglobulin light chain and an anti-mouse horseradish peroxidase conjugate were used to reveal bound antibody. A panel of 46 sera, negative by the immunofluorescent antibody test (IFAT), were used in the ELISA at a serum dilution of 1:500 to calculate the negative cut-off value of OD405 = 0.77. There was a 95 per cent agreement between the results of the ELISA and the IFAT with 104 serum samples. The specificity and sensitivity of the ELISA were 96 per cent and 95 per cent, respectively, when compared with the IFAT. No significant cross-reaction was observed with sera from cattle infected experimentally with Toxoplasma gondii, Cryptosporidium parvum, Babesia divergens, Sarcocystis cruzi, Eimeria alabamensis or E bovis. A significantly modified version of the test is now commercially available.

First demonstration of protective immunity against foetopathy in cattle with latent Neospora caninum infection.

The parasite Neospora caninum is an important cause of abortion in cattle world-wide. Chronically infected dams transmit the parasite transplacentally and infected foetuses may be aborted or born chronically infected but clinically normal. Chronically infected cows repeatedly transmit the parasite to foetuses in several pregnancies and some may abort more than once suggesting that the immune response in these cattle is compromised during pregnancy. To investigate the nature of the immune response in chronically infected cattle, five naturally, chronically infected cows were challenged with N. caninum tachyzoites at 10 weeks of gestation. No foetopathy occurred and all five delivered live calves at full-term. In four naive pregnant cows challenged at the same time, all four foetuses died within 3-5 weeks of challenge. Of the five live calves born to the chronically infected challenged cows, three were transplacentally infected with N. caninum. The kinetics of the maternal anti-N. caninum antibody responses during gestation suggested that these transplacental infections were not the result of the superimposed challenge, but the result of the recrudescence of the maternal chronic infection-which occurred concurrently in non-challenged, chronically infected pregnant controls. These data provide the first experimental evidence that protective immunity occurs in neosporosis. They also suggest that whilst immunity to a pre-existing infection will protect against an exogenous challenge, this protective immunity will not prevent transplacental infection. This implies that a subtle form of concomitant immunity exists in chronically infected cattle and has important implications for vaccine development.

Neospora caninum: oocyst challenge of pregnant cows.

Three pregnant cows were each orally challenged at 10 weeks of gestation with 600 sporulated oocysts of Neospora caninum. The number of oocysts was limited by those available. In concurrent bioassays, one oocyst per os infected each of two gerbils. Challenged cattle developed Neospora-specific antibody, cell proliferation and gamma-interferon responses. N. caninum specific PCR demonstrated persisting infection in the brains of cows 4 months after calving. Abortion was not induced and there was no evidence of transplacental infection in the healthy calves born at full-term. This experiment suggests that the dose threshold for induction of abortion exceeds 600 oocysts.

Protracted shedding of oocysts of Neospora caninum by a naturally infected foxhound.

Feces from 15 dogs at 2 different foxhound kennels in the U.K. were examined microscopically for the presence of oocysts of Neospora caninum. One sample containing approximately 400 candidate oocysts per gram was positive in a polymerase chain reaction (PCR) using N. caninum–specific primers. In a sample taken 4 mo later from the same hound, N. caninum oocysts were again detected visually and by PCR. This is the third reported case of a dog naturally excreting oocysts of N. caninum and suggests that oocyst excretion can occur over a relatively long period of time in some circumstances or that reshedding may occur.

Identification of first-stage larvae of metastrongyles from dogs.

By examining larvae from dogs in which the adult stages had been identified, the morphology of Angiostrongylus vasorum, Filaroides osleri, Filaroides hirthi and Crenosoma vulpis from samples of faeces or bronchoalveolar lavage was compared. The tail morphology of the four species was distinctive: A vasorum had a typical cuticular indentation and projection on the dorsal surface of the tail (referred to as the dorsal notch and dorsal spine, respectively) and a smaller indentation on the ventral surface. The tails of the other species are described. A vasorum was significantly longer (mean [sd] 358·4 μm [10·28], range 334 to 380 μm) than the other species (range 229 to 281 μm).

Serological investigation of aborted sheep and pigs for infection by Neospora caninum

Serology for Neospora caninum was undertaken using direct ELISAs on sera from 660 aborted sheep and 454 breeding sows, which had aborted or were considered infertile. All ovine sera were further tested by indirect fluorescent antibody test (IFAT) for N. caninum, and a latex agglutination test (LAT) for Toxoplasma gondii was performed on 423 of the samples, including all those positive by ELISA. ELISA-positive porcine sera were tested by IFAT and an inhibition ELISA for antibodies to N. caninum and by LAT for T. gondii. Only 3 (0.45%) of the ovine sera were seropositive for N. caninum by both ELISA and IFAT whereas although 40 porcine sera were seropositive by ELISA all were negative by IFAT. The results suggest that environmental exposure to N. caninum occurs rarely in sheep and pigs.

Emerging canine angiostrongylosis in northern England: five fatal cases

Angiostrongylus vasorum is a metastrongylid nematode parasite that resides in the pulmonary arteries and right ventricle of dogs and other canids. Since its discovery in France in 1866 by Baillet ([Rosen and others 1970][1]), A vasorum has been reported in dogs in Europe (Denmark, France, Germany,

Lack of serologic evidence of Neospora caninum in humans, England.

Retrospective testing of 3,232 serum samples from the general population and 518 serum samples from a high-risk group showed no evidence of human exposure to Neospora caninum in England. Results were obtained by using immunofluorescence antibody testing and ELISA to analyze frequency distribution.