Identification of molecular biomarkers associated with disease progression in the testis of bulls infected with Besnoitia besnoiti.

Breeding bulls infected with Besnoitia besnoiti may develop sterility during either acute or chronic infection. The aim of this study was to investigate the molecular pathogenesis of B. besnoiti infection with prognosis value in bull sterility. Accordingly, five well-characterized groups of naturally and experimentally infected males were selected for the study based on clinical signs and lesions compatible with B. besnoiti infection, serological results and parasite detection. A broad panel of molecular markers representative of endothelial activation and fibrosis was investigated and complemented with a histopathological approach that included conventional histology and immunohistochemistry. The results indicated the predominance of an intense inflammatory infiltrate composed mainly of resident and recruited circulating macrophages and to a lesser extent of CD3+ cells in infected bulls. In addition, a few biomarkers were associated with acute, chronic or subclinical bovine besnoitiosis. The testicular parenchyma showed a higher number of differentially expressed genes in natural infections (acute and chronic infections) versus scrotal skin in experimental infections (subclinical infection). In subclinical infections, most genes were downregulated except for the CCL24 and CXCL2 genes, which were upregulated. In contrast, the acute phase was mainly characterized by the upregulation of IL-1α, IL-6 and TIMP1, whereas in the chronic phase, the upregulation of ICAM and the downregulation of MMP13, PLAT and IL-1α were the most relevant findings. Macrophages could be responsible for the highest level of gene regulation in the testicular parenchyma of severely affected and sterile bulls, and all these genes could be prognostic markers of sterility.

Treatment with Bumped Kinase Inhibitor 1294 Is Safe and Leads to Significant Protection against Abortion and Vertical Transmission in Sheep Experimentally Infected with Toxoplasma gondii during Pregnancy.

Previous studies on drug efficacy showed low protection against abortion and vertical transmission of Toxoplasma gondii in pregnant sheep. Bumped kinase inhibitors (BKIs), which are ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1), were shown to be highly efficacious against several apicomplexan parasites in vitro and in laboratory animal models. Here, we present the safety and efficacy of BKI-1294 treatment (dosed orally at 100 mg/kg of body weight 5 times every 48 h) initiated 48 h after oral infection of sheep at midpregnancy with 1,000 TgShSp1 oocysts. BKI-1294 demonstrated systemic exposure in pregnant ewes, with maximum plasma concentrations of 2 to 3 µM and trough concentrations of 0.4 µM at 48 h after each dose. Oral administration of BKI-1294 in uninfected sheep at midpregnancy was deemed safe, since there were no changes in behavior, fecal consistency, rectal temperatures, hematological and biochemical parameters, or fetal mortality/morbidity. In ewes infected with a T. gondii oocyst dose lethal for fetuses, BKI-1294 treatment led to a minor rectal temperature increase after infection and a decrease in fetal/lamb mortality of 71%. None of the lambs born alive in the treated group exhibited congenital encephalitis lesions, and vertical transmission was prevented in 53% of them. BKI-1294 treatment during infection led to strong interferon gamma production after cell stimulation in vitro and a low humoral immune response to soluble tachyzoite antigens but high levels of anti-SAG1 antibodies. The results demonstrate a proof of concept for the therapeutic use of BKI-1294 to protect ovine fetuses from T. gondii infection during pregnancy.

Influence of dose and route of administration on the outcome of infection with the virulent Neospora caninum isolate Nc-Spain7 in pregnant sheep at mid-gestation.

Experimental infections in pregnant sheep have been focused on studying the effect of the time of challenge on the outcome of N. caninum infection, whereas the impact of the dose and route of challenge has not been studied in depth. Therefore, clinical outcome, immune responses, parasite detection and burden, and lesion severity in placental tissues and foetal brains were investigated in 90-day-pregnant sheep inoculated intravenously with 105 (G1), 104 (G2), 103 (G3), or 102 (G4) tachyzoites or subcutaneously with 104 (G5) tachyzoites of the virulent Nc-Spain7 isolate and an uninfected group (G6). Comparing challenge doses, G1 was the only group that had 100% abortion. Likewise, IFNγ levels in G1 increased earlier than those in other intravenously infected groups, and IgG levels on day 21 post-infection (pi) were higher in G1 than those in other intravenously infected groups. Concerning vertical transmission, G1 shows a higher parasite burden in the foetal brain than did G2 and G3. Comparing routes of administration, no differences in foetal survival rate or parasite load in the foetal brain were found. Although G2 had higher IFNγ levels than G5 on day 10 pi, no differences were found in humoral immune responses. Because the outcome after intravenous infection with 105 tachyzoites was similar to that observed after intravenous infection with 106 tachyzoites used in a previous work (100% abortion and vertical transmission), we conclude that it may be reasonable to use 105 tachyzoites administered by the intravenous route in further experiments when assessing drugs or vaccine candidates.

Vascular wall injury and inflammation are key pathogenic mechanisms responsible for early testicular degeneration during acute besnoitiosis in bulls.

BACKGROUND: Bovine besnoitiosis, caused by the apicomplexan parasite Besnoitia besnoiti, is a chronic and debilitating cattle disease that notably impairs fertility. Acutely infected bulls may develop respiratory signs and orchitis, and sterility has been reported in chronic infections. However, the pathogenesis of acute disease and its impact on reproductive function remain unknown. METHODS: Herein, we studied the microscopic lesions as well as parasite presence and load in the testis (pampiniform plexus, testicular parenchyma and scrotal skin) of seven bulls with an acute B. besnoiti infection. Acute infection was confirmed by serological techniques (IgM seropositive results and IgG seronegative results) and subsequent parasite detection by PCR and histological techniques. RESULTS: The most parasitized tissue was the scrotal skin. Moreover, the presence of tachyzoites, as shown by immunohistochemistry, was associated with vasculitis, and three bulls had already developed juvenile tissue cysts. In all animals, severe endothelial injury was evidenced by marked congestion, thrombosis, necrotizing vasculitis and angiogenesis, among others, in the pampiniform plexus, testicular parenchyma and scrotal skin. Vascular lesions coexisted with lesions characteristic of a chronic infection in the majority of bulls: hyperkeratosis, acanthosis and a marked diffuse fibroplasia in the dermis of the scrotum. An intense inflammatory infiltrate was also observed in the testicular parenchyma accompanied by different degrees of germline atrophy in the seminiferous tubules with the disappearance of various strata of germ cells in four bulls. CONCLUSIONS: This study confirmed that severe acute besnoitiosis leads to early sterility that might be permanent, which is supported by the severe lesions observed. Consequently, we hypothesized that testicular degeneration might be a consequence of (i) thermoregulation failure induced by vascular lesions in pampiniform plexus and scrotal skin lesions; (ii) severe vascular wall injury induced by the inflammatory response in the testis; and (iii) blood-testis barrier damage and alteration of spermatogenesis by immunoresponse.

A model for chronic bovine besnoitiosis: Parasite stage and inoculation route are key factors.

In this work, an experimental model for chronic besnoitiosis in bovine was developed and characterized. Using a previously established calf model, two new variables (parasite stage and inoculation route) were combined and used. Twelve Holstein Friesian 3-month-old male calves were randomly divided into four groups of three animals each. Bradyzoites were obtained from a chronically infected bull and used for inoculation via three different inoculation routes. Three groups were inoculated with 106 bradyzoites by intravenous (G1), subcutaneous (G2) and intradermal (G3) routes, and a non-infected control group (G4) was inoculated with PBS. The trial lasted for 90 days and included daily clinical monitoring as well as weekly skin biopsies and blood sampling. Sera were obtained to analyse both cellular and humoral responses. Once the calves were euthanized, tissues from the skin, eyes, respiratory and reproductive tracts, among others, were collected to study presence of the parasite. Clinically, the infection was classified as mild to moderate for the acute stage since all infected calves showed lymphadenopathy from four days post-infection (pi) and fever from one week pi until 24 days pi. However, the most relevant results were achieved during the chronic stage that was classified as moderate to severe. In fact, pathognomonic conjunctival cysts were observed in all infected calves from 40 days pi onwards and were more abundant in G3. Moreover, one calf from this group developed skin lesions (49 days pi). The microscopic tissue cysts and Besnoitia DNA were detected primarily in skin, reproductive tract and respiratory tissue samples, and parasite load was higher in G3. In conclusion, the parasite stage (bradyzoite) and the inoculation route are key factors that influence the outcome of an infection. In particular, the intradermal route led to more severe clinical signs of the chronic phase in the inoculated calves.

Safety and efficacy of the bumped kinase inhibitor BKI-1553 in pregnant sheep experimentally infected with Neospora caninum tachyzoites.

Neospora caninum is one of the main causes of abortion in cattle, and recent studies have highlighted its relevance as an abortifacient in small ruminants. Vaccines or drugs for the control of neosporosis are lacking. Bumped kinase inhibitors (BKIs), which are ATP-competitive inhibitors of calcium dependent protein kinase 1 (CDPK1), were shown to be highly efficacious against several apicomplexan parasites in vitro and in laboratory animal models. We here present the pharmacokinetics, safety and efficacy of BKI-1553 in pregnant ewes and foetuses using a pregnant sheep model of N. caninum infection. BKI-1553 showed exposure in pregnant ewes with trough concentrations of approximately 4 µM, and of 1  µM in foetuses. Subcutaneous BKI-1553 administration increased rectal temperatures shortly after treatment, and resulted in dermal nodules triggering a slight monocytosis after repeated doses at short intervals. BKI-1553 treatment decreased fever in infected pregnant ewes already after two applications, resulted in a 37-50% reduction in foetal mortality, and modulated immune responses; IFNγ levels were increased early after infection and IgG levels were reduced subsequently. N. caninum was abundantly found in placental tissues; however, parasite detection in foetal brain tissue decreased from 94% in the infected/untreated group to 69-71% in the treated groups. In summary, BKI-1553 confers partial protection against abortion in a ruminant experimental model of N. caninum infection during pregnancy. In addition, reduced parasite detection, parasite load and lesions in foetal brains were observed.

Ulcerative enteritis (quail disease) in lories.

Ulcerative enteritis is found in a wide range of avian hosts but has not been described in psittacine birds. This case report describes ulcerative enteritis in four lories (two Trichoglossus sp. and two Eos sp.) that were found dead without any previous sign of disease. Macroscopically, all four birds showed good body condition. The only remarkable finding was a moderate dilatation of the small intestine with the presence of multiple yellow foci. Histologically, multiple ulcers extended into the submucosa and were filled with necrotic debris; bacteria and fibrin were observed in the intestinal mucosa. The liver and spleen exhibited a multifocal fibrinoid necrosis associated with a very moderate inflammatory reaction. Microbiological isolation revealed colonies of Clostridium colinum and Clostridium perfringens in the intestinal tract of the investigated birds.