Julio Benavides

Neospora caninum infection during early pregnancy in cattle: how the isolate influences infection dynamics, clinical outcome and peripheral and local immune responses.

This work studies the influence of Neospora caninum intra-species diversity on abortion outcome, infection dynamics in terms of parasite dissemination and peripheral-local immune responses in pregnant cattle. Animals were intravenously inoculated at day 70 of pregnancy with 107 tachyzoites of two isolates showing marked differences in virulence in vitro and in pregnant mouse models: Nc-Spain7, a high virulence isolate, and Nc-Spain8, a low-to-moderate virulence isolate. After inoculation, pregnancy was monitored, and dams were culled when foetal death was detected. Foetal mortality occurred in all infected heifers between days 24 and 49 post-infection (pi), however, it was detected sooner in Nc-Spain7-infected animals (median day = 34) than those inoculated with Nc-Spain8 (median day = 41) with a trend towards significance (P < 0.11). Similar histological lesions were observed in placentomes and in most of the foetuses from the two infected groups. However, parasites were more frequently detected in the placenta and foetuses by PCR and in the foetal brain by immunohistochemistry in Nc-Spain7-infected animals. Specific antibodies were detected starting at day 13 post-infection in all infected cattle, with higher IgG levels in Nc-Spain7-infected group. IFN-γ and IL-4 profiles also varied between infected groups in PBMC stimulation assays. Infected animals showed significant increases in their cytokine mRNA levels (IFN-γ, IL-4, IL-10, IL-12p40 and TNF-α) in the caruncle at time of foetal death. Differences between the infected groups were also observed for cytokine profiles. These results demonstrate the influence of the N. caninum isolate on foetal death outcome, infection dynamics and immune responses in cattle.

Chronic bovine besnoitiosis: intra-organ parasite distribution, parasite loads and parasite-associated lesions in subclinical cases.

Bovine besnoitiosis caused by Besnoitia besnoiti is a chronic and debilitating disease. The most characteristic clinical signs of chronic besnoitiosis are visible tissue cysts in the scleral conjunctiva and the vagina, thickened skin and a generally poor body condition. However, many seropositive animals remain subclinically infected, and the role that these animals may play in spreading the disease is not known. The aim of the present study was to assess the intra-organ parasite distribution, the parasite load and the parasite-associated lesions in seropositive but subclinically infected animals. These animals were seropositive at the time of several consecutive samplings, had visible tissue cysts in the past and, at time of slaughter, had detectable specific anti-Besnoitia spp. antibody levels, but they did not show evident clinical signs at culling. Thus, histopathological, immunohistochemical and molecular analyses of several samples from the respiratory tract, reproductive tract, other internal organs and skin from six cows were performed. The tissue cysts were located primarily in the upper respiratory tract, i.e., in the rhinarium and larynx/pharynx (four cows), followed by the distal genital tract (vulva/vagina) and the skin of the neck (three and two cows, respectively, out of the four cows with cysts in the respiratory tract). We were unable to detect any parasites in the two remaining cows. Cysts were associated with a significant non-purulent inflammatory infiltrate consisting predominantly of T lymphocytes and activated monocytes/macrophages in two cows. The parasite burden, estimated by quantitative real-time PCR, was very low. It is noteworthy that the only animal that showed a recent increase in the antibody titre had the highest parasite burden and the most conspicuous inflammatory reaction against the cysts. In conclusion, although these cows no longer displayed any visible signs of besnoitiosis, they remained infected. Therefore, cows without visible signs of disease may still be able to transmit the parasite.

High rate of transplacental infection and transmission of Neospora caninum following experimental challenge of cattle at day 210 of gestation

In order to investigate the pathogenesis of neosporosis following a primary infection in late pregnancy, cattle were subcutaneously challenged with 5 × 108Neospora caninum (NC1 isolate) tachyzoites at day 210 of gestation and serial necropsies were then carried out at 14, 28, 42 and 56 days post-infection (dpi). No abortions occurred and all the foetuses were viable at the time of euthanasia. There was a high rate of vertical transmission, as parasites were detected by immunohistochemical labelling and PCR in all the foetuses from 28 dpi. Focal necrotic lesions were observed in the placentomes of the placenta from 28 dpi and showed resolution during later time points, denoted by infiltration of inflammatory cells at 42 dpi and fibrosis at 56 dpi. Foetuses at 28 and 42 dpi showed scarce and isolated lesions which are unlikely to represent a threat to foetal viability. No lesions were observed in the foetuses at 14 or 56 dpi suggesting control of the infection and resolution of the lesions by maternal and foetal immune responses. Once infection was established, it could not be cleared from the host and vertical transmission of the parasite occurred in all infected hosts. Parasite was detected in the placenta at 28 dpi, while in previous experimental infections of cattle at day 70 and 140 of gestation using the same challenge model, it was already present at day 14 post infection. This suggests that a change in the maternal immune response plays a crucial role in limiting the initial infection during the last term of pregnancy.

Maedi-Visna: the Meningoencephalitis in Naturally Occurring Cases

Lesions were examined at different levels of the central nervous system (CNS) in 64 sheep with natural maedi-visna (MV) meningoencephalitis. All animals showed lesions in more than one of the CNS locations examined; the lesions in the cranial regions were periventricular, while those in the spinal cord affected the white matter funicles. Lesions were found particularly in the cerebellar peduncles (non-suppurative meningoencephalitis), followed by the corpus callosum, hippocampus and thoracic spinal cord. Vascular, infiltrative and malacic histopathological patterns were recognized. One pattern predominated in each section examined, although mixed forms occurred. Vascular lesions occurred with similar frequency at all CNS levels, but infiltrative and malacic lesions predominated at rostral and caudal levels, respectively. Cells consistent with macrophages and shown immunohistochemically to be associated with MV virus were seen in malacic and infiltrative lesions, at the periphery of damaged areas.

Duration of protective immunity and antibody responses in cattle immunised against alcelaphine herpesvirus-1-induced malignant catarrhal fever

Protection of cattle from alcelaphine herpesvirus-1 (AlHV-1)-induced malignant catarrhal fever (MCF) has been described previously, using an attenuated virus vaccine in an unlicensed adjuvant. The vaccine was hypothesised to induce a protective barrier of virus-neutralising antibody in the oro-nasal region, supported by the observation of high titre neutralising antibodies in nasal secretions of protected animals. Here we describe further analysis of this vaccine strategy, studying the effectiveness of the vaccine formulated with a licensed adjuvant; the duration of immunity induced; and the virus-specific antibody responses in plasma and nasal secretions. The results presented here show that the attenuated AlHV-1 vaccine in a licensed adjuvant protected cattle from fatal intranasal challenge with pathogenic AlHV-1 at three or six months. In addition, animals protected from MCF had significantly higher initial anti-viral antibody titres than animals that succumbed to disease; and these antibody titres remained relatively stable after challenge, while titres in vaccinated animals with MCF increased significantly prior to the onset of clinical disease. These data support the view that a mucosal barrier of neutralising antibody blocks infection of vaccinated animals and suggests that the magnitude of the initial response may correlate with long-term protection. Interestingly, the high titre virus-neutralising antibody responses seen in animals that succumbed to MCF after vaccination were not protective.

Neospora caninum infection as a cause of reproductive failure in a sheep flock

Neospora caninum has been detected only sporadically in cases of ovine abortion, and it has therefore traditionally been considered as an unimportant parasite in small ruminants. This study was carried out with the aim of identifying the pathogen causing serious reproductive problems on a commercial sheep farm. Sera from all rams and ewes tested negative for antibodies against Border disease virus, Schmallenberg virus and Coxiella burnetii, and infections by these agents were therefore ruled out. Nevertheless, seropositivity to N. caninum and/or Toxoplasma gondii was detected, although the seroprevalence was higher in the case of N. caninum. The percentage of lambings and the number of lambs per dam were significantly lower in ewes that were seropositive to N. caninum while no effect on these parameters was detected in ewes that were seropositive to T. gondii. There was also no evidence of infection by T. gondii in the foetal/lamb tissues analyzed by PCR and/or immunohistopathological techniques. On the contrary, the DNA of N. caninum was detected in 13 out of 14 foetuses/lambs descendant from dams seropositive to this parasite. Characteristic lesions caused by N. caninum and/or its antigen were also detected. Genotyping of the N. caninum DNA revealed only two closely related microsatellite multilocus genotypes. The results clearly demonstrate that infection by N. caninum was the cause of the low reproductive performance of this sheep flock.

Influence of the gestational stage on the clinical course, lesional development and parasite distribution in experimental ovine neosporosis

Neospora caninum is considered one of the main causes of abortion in cattle, yet recent studies have also emphasised its relevance as an abortifacient in small ruminants. In order to gain deeper insight into the pathogenesis of ovine neosporosis, pregnant ewes were intravenously inoculated with 106 tachyzoites of the Nc-Spain7 isolate at days 40, 90 or 120 of gestation. Infection during the first term resulted in the death of all foetuses between days 19 and 21 post-infection, showing mainly necrotic lesions in foetal liver and the highest parasite DNA detection and burden in both placenta and foetal viscera. After infection at day 90, foetal death was also detected in all ewes, although later (34–48 days post-infection). In this group, lesions were mainly inflammatory. Foetal livers showed the lowest frequency of lesions, as well as the lowest parasite detection and burden. All ewes infected at day 120 delivered viable lambs, although 3 out of 9 showed weakness and recumbency. Neospora DNA was detected in all lambs but one, and parasite burden was similar to that observed in day 90 group. Lesions in this group showed more conspicuous infiltration of inflammatory cells and higher frequency in foetal brain and muscle when compared to both previous groups. These results highlight the crucial role that the stage of gestation plays on the course of ovine neosporosis, similar to that reported in bovine neosporosis, and open the doors to consider sheep as a valid model for exogenous transplacental transmission for ruminant neosporosis.

Vaccination of pigs with the S48 strain of Toxoplasma gondii - safer meat for human consumption

As clinical toxoplasmosis is not considered a problem in pigs, the main reason to implement a control strategy against Toxoplasma gondii (T. gondii) in this species is to reduce the establishment of T. gondii tissue cysts in pork, consequently reducing the risk of the parasite entering the human food chain. Consumption of T. gondii tissue cysts from raw or undercooked meat is one of the main sources of human infection, with infected pork being considered a high risk. This study incorporates a mouse bioassay with molecular detection of T. gondii DNA to study the effectiveness of vaccination (incomplete S48 strain) in its ability to reduce tissue cyst burden in pigs, following oocyst (M4 strain) challenge. Results from the mouse bioassay show that 100% of mice which had received porcine tissues from vaccinated and challenged pigs survived compared with 51.1% of mice which received tissues from non-vaccinated and challenged pigs. The presence (or absence) of T. gondii DNA from individual mouse brains also confirmed these results. This indicates a reduction in viable T. gondii tissue cysts within tissues from pigs which have been previously vaccinated with the S48 strain. In addition, the study demonstrated that the main predilection sites for the parasite were found to be brain and highly vascular muscles (such as tongue, diaphragm, heart and masseter) of pigs, while meat cuts used as human food such as chop, loin, left tricep and left semitendinosus, had a lower burden of T. gondii tissue cysts. These promising results highlight the potential of S48 strain tachyzoites for reducing the number of T. gondii tissues cysts in pork and thus improving food safety.

Immunization of lambs with the S48 strain of Toxoplasma gondii reduces tissue cyst burden following oral challenge with a complete strain of the parasite

This study evaluates the influence of immunizing lambs with the incomplete S48 strain of Toxoplasma gondii, on parasite dissemination following a live oral challenge with a complete strain of T. gondii (M4). Lambs were culled at 14, 28 and 42 days post challenge. Parasite DNA was detected at significantly (p<0.0001) lower levels in samples from the vaccinated/challenged group (0% in heart and 5.9% in skeletal muscles), when compared to the non-vaccinated/challenged animals (75% heart, 87.9% skeletal muscle). S48 T. gondii DNA was found in muscle or lymph nodes until 42 days post infection, suggesting that parasite DNA or tachyzoites could persist longer after immunization than previously thought. Non-vaccinated/challenged animals showed more frequent lesions in muscles and central nervous system than the vaccinated animals. These results demonstrate that vaccination of lambs with the incomplete S48 T. gondii strain, can protect against establishment of tissue cysts following challenge with a complete strain of T. gondii. Consumption of undercooked meat containing T. gondii cysts is a major route of transmission to people, therefore vaccination of food animals may improve the safety of meat for human consumption.

Experimental ruminant models for bovine neosporosis: what is known and what is needed

At present, bovine neosporosis is an important worldwide concern because of its wide geographic distribution and economic impact. Abortion is the main clinical sign of bovine neosporosis in both dairy and beef cattle. Ruminant challenge models are critical to evaluate potential vaccine candidates to help tackle bovine neosporosis and to study pathogenesis and host responses to infection. Several research groups have developed ruminant models of Neospora caninum infection independently of others, resulting in a high degree of variability due to the use of different species of animals, breeds, strains/isolates of N. caninum, doses, routes and times of inoculation. Standardization is greatly needed to advance research in a more collaborative, timely and efficient manner. In the absence of widely accepted international guidelines, this manuscript serves to summarize and discuss the different models and parameters currently in use. Parameters essential for the development of non-pregnant and pregnant ruminant models are outlined and the main knowledge gaps are identified. This information could act as the basis to develop a consensus for international standard guidelines for ruminant models of neosporosis that would be helpful for researchers in this field worldwide.