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.

Anaemia in Ruminants Caused by Plant Consumption.

Plant toxicology has affected animals throughout evolution. Plants have adapted themselves to the environment. This adaptation has led to the development of defensive strategies to avoid being consumed. Plants have several chemical compounds, which can cause deleterious effects on people or animals that consume them, causing a wide variety of clinical signs. Plants from various latitudes, both cultivated for human and animal feeding or decorative purpose and even wild growth plants are able to generate anaemia in ruminants. Coumarins or ptaquiloside predispose bleeding and haemorrhages, causing a haemorrhagic disease in affected animals. In this group, some important fodder plants, such sweet clover (Genus Melilotus spp.), or other weeds distributed worldwide, such as bracken fern (Pteridium aquilinum) of giant fennel (Ferula communis), are included. On the other hand, sulfur-containing chemicals (e.g., n-propyl disulfate and S-propyl cysteine sulfoxides (SMCOs)) may cause severe direct damage to the erythrocyte and their membrane, leading to their destruction and causing haemolytic anaemia in the animal. This review presents the most frequent intoxication by plants causing anaemia in ruminants. Toxic compounds, clinical signs, diagnosis and possible treatments are also presented.

Comparative Study of the Use of Doxycycline and Oxytetracycline to Treat Anaplasmosis in Fattening Lambs.

Lamb icteric carcasses condemnation due to Anaplasma ovis is causing relevant economic losses. A comparative study was developed on the effects of different antibiotics to treat ovine anaplasmosis in fattening lambs. A total of 100 A. ovis naturally infected lambs were selected and randomly divided into four groups of 25 lambs: Group ID, treated with injectable doxycycline; Group OD, oral doxycycline; Group O, injectable oxytetracycline; and Group C, untreated animals for the control group. Clinical, haematological, and molecular analyses were performed before the treatment and 12 and 45 days after the beginning of the treatments, and carcass condemnation was followed after slaughter. The A. ovis bacterial load was high before the treatments in the four groups and decreased significantly 45 days after treatment in the ID and O Groups (p < 0.001). The parameters that were related to haemolysis showed similar results. At the abattoir, 15 out of the 47 examined carcasses were condemned; 7 of C Group, 6 of OD Group, 2 of O Group, and 0 of ID Group. It can be concluded that injectable doxycycline and oxytetracycline significantly reduce A. ovis bacterial load in blood and carcass condemnation at the abattoir. Further studies are needed in order to confirm these encouraging findings.

Squamous cell carcinoma of the vagina and cervix in sheep - case report.

This report describes the clinical and histopathological characteristics of a squamous cell carcinoma infiltrating the cervix and the vaginal wall, producing reproductive symptoms and subnormal fertility in an adult ewe. Necropsy showed a large (15-cm-long) neoplastic mass infiltrating the vaginal wall and the cervix. Histopathological examination revealed atypical squamous epithelial cords invading the basal membrane and dermis, round anaplastic cells, focal areas of necrosis, keratinisation of isolated cells, and pronounced infiltration by mononuclear cells around the cords. No squamous cell carcinoma of such localisation has been reported from sheep before. In humans, this tumour is the most common gynaecological malignancy in the world.

Effect of a Topical Formulation on Infective Viral Load in Lambs Naturally Infected with Orf Virus.

INTRODUCTION: Orf is a highly contagious eruptive viral disease of the skin and mucosa of sheep and goats. Although vaccination with live or attenuated orf virus is the preferred option for disease control, the vaccine is unavailable in many countries. Treatment of orf lesions involves standard hygiene and in numerous cases, management of presumptive secondary infections with antibiotics, increasing risks of antimicrobial resistance (AMR). The wound dressing formulation Tri-Solfen® containing two local anaesthetics (lignocaine and bupivacaine), adrenaline and an antiseptic (cetrimide) in a gel formulation was developed for pain relief in sheep undergoing surgical husbandry procedures in Australia. Recently, TS therapy was found to reduce suffering and enhance recovery in cattle and buffalo with oral and skin lesions due to foot-and-mouth disease (FMD) virus infection. It was noted that TS has a low pH and is potentially viricidal, potentially aiding disease control. METHODS: One-month-old lambs (n=14), naturally infected with orf, were recruited from a farm during a natural outbreak of the disease. The animals were selected at the early stages of the infection and randomly divided into two cohorts: Group A (n=11) treated with the topical wound gel formulation (TS); and Group B (n=3) an untreated control group. Swabs were obtained before treatment (T0) and on days one (T1), 3 (T2) and 5 (T3) post-treatment, then submitted to direct DNA extraction with real-time PCR quantification, plus incubation with primary tissue cultures from ovine skin fibroblasts (OSF) and T-immortalized goat embryonic fibroblasts (TIGEF). RESULTS: Although no significant differences were found in the clinical progression of the lesions and PCR quantification (p=0.722) between these small cohorts, there was a significant difference (p<0.05) in reduction in infective viral load between the groups when assessed in OSF cell cultures between T0 and T3. CONCLUSION: These preliminary findings suggest that treatment of early stage lesions with this TS may reduce the infective viral load present in orf lesions.

Epidemiological Study Related to the First Outbreak of Ovine Anaplasmosis in Spain.

Ovine anaplasmosis is a vector-borne disease caused by Anaplasma ovis and mainly transmitted through tick bites. In Spain, the first outbreak of ovine anaplasmosis occurred in 2014. An epidemiological study in fifty-one farms was carried out associated with this outbreak in the affected geographical area. An epidemiological questionnaire was performed. In addition, whole blood samples were taken for molecular analysis in 47 of these farms to determine the prevalence of infection of Anaplasma ovis. A. ovis was present in 44 out of 47 PCR-analysed farms (93.6%). However, only 40.4% of the studied farms showed severe clinical signs. The clinical signs affected mainly young animals, which showed severe anaemia, weakness, anorexia, cachexia and epiphora. The early culling of young animals was more frequently reported by severely affected farms than the analysed farms without clinical signs (71.4% vs. 12.5%, p < 0.001). The geographical area where the farm is located seems to be relevant for the presence of clinical signs of the disease. Ovine anaplasmosis is an emerging disease in Europe that spreads rapidly through tick bites and is capable of causing significant economic losses when it spreads in a naive area and causes an epidemic.

A short-term treatment with BKI-1294 does not protect foetuses from sheep experimentally infected with Neospora caninum tachyzoites during pregnancy.

The Neospora caninum Calcium-dependent protein kinase 1 (NcCDPK1) inhibitor BKI-1294 had demonstrated excellent efficacy in a pregnant mouse model of neosporosis, and was also highly efficacious in a pregnant sheep model of toxoplasmosis. In this work, we present the efficacy of BKI-1294 treatment (dosed 5 times orally every 48 h) starting 48 h after intravenous infection of sheep with 105 Nc-Spain7 tachyzoites at mid-pregnancy. In the dams, BKI-1294 plasma concentrations were above the IC50 for N. caninum for 12-15 days. In treated sheep, when they were compared to untreated ones, we observed a minor increase in rectal temperature, higher IFNγ levels after blood stimulation in vitro, and a minor increase of IgG levels against N. caninum soluble antigens through day 28 post-infection. Additionally, the anti-NcSAG1 and anti-NcSAG4 IgGs were lower in treated dams on days 21 and 42 post-infection. However, BKI-1294 did not protect against abortion (87% foetal mortality in both infected groups, treated and untreated) and did not reduce transplacental transmission, parasite load or lesions in placentomes and foetal brain. The lack of foetal protection was likely caused by short systemic exposure in the dams and suboptimal foetal exposure to this parasitostatic drug, which was unable to reduce replication of the likely established N. caninum tachyzoites in the foetus at the moment of treatment. New BKIs with a very low plasma clearance and good ability to cross the blood-brain and placental barriers need to be developed.

Absence of SARS-CoV-2 Antibodies in Natural Environment Exposure in Sheep in Close Contact with Humans.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the zoonotic causative agent of coronavirus disease 2019 (COVID-19) that has caused a pandemic situation with millions of infected humans worldwide. Among domestic animals, there have been limited studies regarding the transmissibility and exposure to the infection in natural conditions. Some animals are exposed and/or susceptible to SARS-CoV-2 infection, such as cats, ferrets and dogs. By contrast, there is no information about the susceptibility of ruminants to SARS-CoV-2. This study tested the antibody response in 90 ovine pre-pandemic serum samples and 336 sheep serum samples from the pandemic period (June 2020 to March 2021). In both cases, the animals were in close contact with a veterinary student community composed of more than 700 members. None of the serum samples analyzed was seroreactive based on an enzyme-linked immunosorbent assay (ELISA) using the receptor-binding domain (RBD) of the spike antigen. In this sense, no statistical difference was observed compared to the pre-pandemic sheep. Our results suggest that it seems unlikely that sheep could play a relevant role in the epidemiology of SARS-CoV-2 infection. This is the first study to report the absence of evidence of sheep exposure to SARS-CoV-2 in natural conditions.

Epidemiology of Oestrus ovis infection of sheep in northeast Spain (mid-Ebro Valley).

Slaughterhouse surveys to determine the prevalence and intensity of larval Oestrus ovis Linnaeus (Diptera: Oestridae) in sheep, were conducted monthly for 1 year in northeastern Spain. The prevalence of O. ovis in 120 sheep in northeast Spain was 84.2%. The monthly prevalence ranged from 62.5% in January to 100% in June and October. Of the 3,831 larvae collected, 3,005 were first instar larvae (78.4%), 744 second instar larvae (19.4%), and 82 third instar larvae (2.1%). The mean larval burden per infected sheep was 37.9, throughout the year although infection was higher in October. The high number of first stage larvae in winter (especially October, November, and December) may be due to a period of slower larval maturation. Third-stage larvae were always present but sometimes in very low numbers compared to the total. According to the results, oestrosis appears to be a serious problem in the region.

The Relevance of Caseous Lymphadenitis as a Cause of Culling in Adult Sheep.

Four hundred and ninety-eight culled sheep received at the Ruminant Clinical Service of the Veterinary Faculty of Zaragoza, Spain, were examined in life and after humanitarian sacrifice in order to reach the final diagnosis of the cause of culling and to evaluate the presence of caseous lymphadenitis (CLA) lesions. One hundred and forty-seven of the 498 studied animals (29.52%) showed CLA compatible lesions that were subsequently confirmed by Corynebacterium pseudotuberculosis isolation. One hundred and seven of the 147 CLA affected animals presenting the visceral clinical form of the disease (72.79%), while only 32 animals were affected by the superficial form (21.77%). In addition, eight animals were found to be affected in both the visceral and the superficial presentations (5.44%). Eighty-four of the 147 CLA-affected animals (57.14%) did not show any concurrent disease, considering, in this case, CLA the main cause of culling (84/498: 16.87%). In the superficial presentation, the retropharyngeal lymph node, as a sole lesion, was the most frequently affected (13/32: 40.63%). Further, in the visceral form of the disease, 85.06% of the affected animals had the lesions located in the respiratory system (91/107: 85.06%). CLA was revealed as an important cause of culling in sheep production.

Cervical artificial insemination in sheep: sperm volume and concentration using an antiretrograde flow device.

There has been development of an antiretrograde flow device (DARIO), for sheep cervical artificial insemination (CAI). There, however, needs to be optimization of sperm volume and concentration of insemination doses when the DARIO is used for CAI. Objectives were to compare fertility rates (proportion of ewes lambing as a result of CAI) when there was use of the DARIO for CAI: two sperm volumes containing equal numbers of spermatozoa: 0.25 mL of 1,600 × 106 spermatozoa/mL and 0.50 mL of 800 × 106 spermatozoa/mL (Test 1 group), and two sperm volumes with a different number of spermatozoa/AI dose: 0.25 mL and 0.50 mL of 1,600 × 106 spermatozoa/mL (Test 2 group). There were 335 ewes from seven farms assigned to 60 batches (equally divided into a Control and Test 1 group). For the Test 2 group, 462 ewes from nine farms were assigned to 88 batches (equally proportioned into Control group and Test 2 groups). For the Test 1 group, proportion of ewes lambing as a result of CAI were 0.701 ±â€¯0.2679 and 0.595 ±â€¯0.2393 for the Control and Test 1 groups, respectively (P = 0.163). For the Test 2 group, proportions of ewes lambing were 0.550 ±â€¯0.2598 and 0.658 ±â€¯0.2412 for the Control and Test 2 group, respectively (P = 0.041). An inclusion of a larger number of spermatozoa per insemination in a 0.50 mL dose volume resulted improved proportion of ewes lambing as a result of CAI when there was used of the DARIO.

Prevalence of Salmonella enterica subsp. diarizonae serotype 61:k:1:5:(7) in nasal secretions and stool of sheep flocks with and without cases of chronic proliferative rhinitis.

Salmonella enterica subsp. diarizonae serotype 61: k: 1,5, (7) (SED) is a microorganism well adapted to sheep; however, it has also been described producing chronic proliferative rhinitis (CPR) in ovine. CPR causes a proliferative inflammation of the ventral nasal turbinates that may totally obstruct the nasal cavity. The main objective of the present study was to investigate the prevalence of SED in nostrils and stool of sheep without CPR clinical signs in commercial sheep farms of Spain with and without previous clinical cases of CPR. Five samplings were performed in 10 commercial sheep farms for one year. Samples from nostrils and faeces were taken from four animals without CPR visible clinical signs that belonged to four different age ranges at each farm visit. The prevalence of positive animals was 45.3 %, and the number of positive samples in nostrils was higher than in faeces (38.5 % vs 22.5 %). Only on one farm was no positive result obtained in the entire study. In almost all positive farms, sheep belonging to the youngest age ranges accounted for more than 50 % of positive isolates. Finally, farms with a previous diagnosis of CPR were 1.784 times more likely to have an animal with positive isolation than farms without a previous diagnosis. This could suggest that the infection pressure in the farm might favour the occurrence of clinical cases of the disease. However, further studies will be necessary to unravel why this saprophytic bacterium is able to cross the epithelial barrier causing severe rhinitis in certain animals.

Anaplasmosis Outbreak in Lambs: First Report Causing Carcass Condemnation.

In spring and summer 2020, six outbreaks of condemnation of jaundiced lamb carcasses were diagnosed in different farms in Aragón region, Spain. Anaplasma ovis was identified in all affected farms. Four hundred and ninety-two lambs from two affected farms were more closely examined. Clinical examination, haematologies, biochemistries, histopathology and microbiological and molecular analyses were performed. After slaughter, 34.84% of the lambs showed jaundiced carcasses and 79.64% presented splenomegaly at the abattoir. All tested lambs with icteric carcasses showed positive A. ovis PCR, although 72.72% of the unaffected lambs also tested positive. However, the bacterial load was significantly higher in the animals that showed jaundiced carcasses (Cq: 25.00 vs 26.16; p = 0.004). Moreover, all the tested lambs that showed severe anaemia were PCR positive. On the contrary, the PCR negative lambs did not show anaemia. Lambs that presented icteric carcasses displayed severe regenerative anaemia with significantly lower erythrocyte count (7.18 vs. 11.97), haematocrit (26.89 vs. 34.82) and haemoglobin (8.50 vs. 11.10) than unaffected lambs. Reticulocyte count (18.80 vs. 5.65) was also significantly increased in affected animals. This article describes a new disorder caused by Anaplasma ovis that is producing significant economic losses associated with the carcass condemnation of apparently healthy lamb.

Management of Risk Factors Associated with Chronic Oral Lesions in Sheep.

Oral disorders constitute a significant cause of weight loss in sheep. In a study of disorders of the oral cavity of 36,033 sheep from 60 meat sheep flocks in Spain, we looked for management risk factors associated with chronic oral lesions. Mandibular and maxillary disorders were assessed as an external manifestation of oral lesions by palpation, searching for tissue swellings, fistulae, or open wounds. The prevalence of flocks containing sheep with jaw disorders was 98.3%, with an average individual prevalence of 5.5%. The majority of lesions were located in in the mid-region of the mandible, and the most relevant risk factor was increasing age. Use of acidic diets based on silage and inclusion of hard foods, such as maize straw or hay, was associated with the occurrence of jaw inflammation. It was concluded that hard diets containing plant material with edges and sharp areas, plus acidic foods including silage, are more likely to cause lesions of the gingiva, enabling entry of pathogens to the oral mucosa that eventually establish locally in bony tissues, usually as mandibular osteomyelitis. It was also observed that mineral supplementation appears to prevent the occurrence of these lesions.

Impact of a Topical Anaesthesia Wound Management Formulation on Pain, Inflammation and Reduction of Secondary Infections after Tail Docking in Lambs.

We examined several procedures for surgical tail docking; with and without general anaesthesia (GA), including the use of a topical wound gel formulation to provide pain relief (PR) and improve healing after surgery, containing local anaesthetics lignocaine and bupivacaine, with cetrimide and adrenalin. Forty-four lambs were recruited into four equal cohorts: Groups A and C, the tail was excised with a scalpel without anaesthesia or stitches; Groups B and D, the tail was surgically excised and stitched under GA; Groups C and D wounds were immediately sprayed with PR. Behavioural observations identified that Groups A and C displayed significantly less pain-related behaviours than Groups B and D shortly after the procedure, especially if treated with PR. Similarly, the mean of days when animals showed no signs of wound infection was longer in the groups not undergoing stitching. Finally, treatment with PR appeared to reduce the cortisol response and avoided the elevation of serum amyloid A in lambs where the tail was excised without general anaesthesia. In conclusion, surgical tail-docking without GA but where wounds are immediately sprayed with PR, appears as an affordable and more welfare-appropriate method for conducting tail docking in lambs.

Virulence in Mice of a Toxoplasma gondii Type II Isolate Does Not Correlate With the Outcome of Experimental Infection in Pregnant Sheep.

Toxoplasma gondii is an apicomplexan parasite that infects almost all warm-blooded animals. Little is known about how the parasite virulence in mice extrapolates to other relevant hosts. In the current study, in vitro phenotype and in vivo behavior in mice and sheep of a type II T. gondii isolate (TgShSp1) were compared with the reference type II T. gondii isolate (TgME49). The results of in vitro assays and the intraperitoneal inoculation of tachyzoites in mice indicated an enhanced virulence for the laboratory isolate, TgME49, compared to the recently obtained TgShSp1 isolate. TgShSp1 proliferated at a slower rate and had delayed lysis plaque formation compared to TgME49, but it formed more cyst-like structures in vitro. No mortality was observed in adult mice after infection with 1-105 tachyzoites intraperitoneally or with 25-2,000 oocysts orally of TgShSp1. In sheep orally challenged with oocysts, TgME49 infection resulted in sporadically higher rectal temperatures and higher parasite load in cotyledons from ewes that gave birth and brain tissues of the respective lambs, but no differences between these two isolates were found on fetal/lamb mortality or lesions and number of T. gondii-positive lambs. The congenital infection after challenge at mid-pregnancy with TgShSp1, measured as offspring mortality and vertical transmission, was different depending on the challenged host. In mice, mortality in 50% of the pups was observed when a dam was challenged with a high oocyst dose (500 TgShSp1 oocysts), whereas in sheep infected with the same dose of oocysts, mortality occurred in all fetuses. Likewise, mortality of 9 and 27% of the pups was observed in mice after infection with 100 and 25 TgShSp1 oocysts, respectively, while in sheep, infection with 50 and 10 TgShSp1 oocysts triggered mortality in 68 and 66% of the fetuses/lambs. Differences in vertical transmission in the surviving offspring were only found with the lower oocyst doses (100% after infection with 10 TgShSp1 oocysts in sheep and only 37% in mice after infection with 25 TgShSp1 oocysts). In conclusion, virulence in mice of T. gondii type II isolates may not be a good indicator to predict the outcome of infection in pregnant sheep.

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.

Polioencephalomalacia in adult sheep grazing pastures with prostrate pigweed.

Polioencephalomalacia was diagnosed in 2 animals from different farms. In apparently healthy animals from same farms, fecal thiaminase and a significant reduction in erythrocyte trans-ketolase activity was observed. The presence of thiaminase in Amaranthus blitoides could have contributed to the development of polioencephalomalacia in sheep grazing on natural pastures.

Ovine congenital myotonia associated with a mutation in the muscle chloride channel gene.

Congenital myotonia (CM) is characterised by a delay in muscular relaxation after sudden contractions. In a recent outbreak of ovine CM affecting 1% of new-born lambs in a Spanish flock of Rasa Aragonesa sheep, a comparative pathology approach was taken: because a mutation in the muscle chloride channel gene (CLCN1) was identified as responsible for CM in goats, the same gene was sequenced in the affected lambs. A non-synonymous single nucleotide variation (SNV) in the second exon of CLCN1 was associated with this pathology. Rams carrying this SNV heterozygously were thereafter identified and replaced by wild-type homozygous young males. No additional CM cases were detected in subsequent lambing seasons.

Clogging transition of many-particle systems flowing through bottlenecks.

When a large set of discrete bodies passes through a bottleneck, the flow may become intermittent due to the development of clogs that obstruct the constriction. Clogging is observed, for instance, in colloidal suspensions, granular materials and crowd swarming, where consequences may be dramatic. Despite its ubiquity, a general framework embracing research in such a wide variety of scenarios is still lacking. We show that in systems of very different nature and scale -including sheep herds, pedestrian crowds, assemblies of grains, and colloids- the probability distribution of time lapses between the passages of consecutive bodies exhibits a power-law tail with an exponent that depends on the system condition. Consequently, we identify the transition to clogging in terms of the divergence of the average time lapse. Such a unified description allows us to put forward a qualitative clogging state diagram whose most conspicuous feature is the presence of a length scale qualitatively related to the presence of a finite size orifice. This approach helps to understand paradoxical phenomena, such as the faster-is-slower effect predicted for pedestrians evacuating a room and might become a starting point for researchers working in a wide variety of situations where clogging represents a hindrance.