Cell grazing and Haemonchus contortus control in sheep: lessons from a two-year study in temperate Western Europe.

Managing infections of sheep with anthelmintic resistant gastrointestinal nematodes (GIN) is a major challenge for sheep producers in Western Europe. New methods of grazing management have been poorly explored as a component of an integrated and sustainable control of these parasites. Therefore, the purpose of this study was to evaluate the effect of two different types of grazing systems of sheep (intensive cell grazing versus conventional rotational grazing) on GIN infections over two years in a farm located in a temperate environment of Western France. When considering the whole study, the type of grazing system did not influence significantly the intensity of egg excretions of adult ewes even if the proportion of ewes excreting high numbers of GIN eggs was higher in cell grazing system than in rotational grazing system. The most striking result of this survey was the effect of grazing system on the GIN species composition harbored by ewes and by their lambs: with time, the proportions of H. contortus infections were lower in cell grazing system than in rotational grazing system. In conclusion, the cell grazing system, as implemented in this study, could limit the importance of this highly pathogenic nematode species in sheep.

Stomoxys calcitrans, mechanical vector of virulent Besnoitia besnoiti from chronically infected cattle to susceptible rabbit.

Cattle besnoitiosis caused by Besnoitia besnoiti (Eucoccidiorida: Sarcocystidae) is a re-emerging disease in Europe. Its mechanical transmission by biting flies has not been investigated since the 1960s. The aim of this study was to re-examine the ability of Stomoxys calcitrans (Diptera: Muscidae) to transmit virulent B. besnoiti bradyzoites from chronically infected cows to susceptible rabbits. Three batches of 300 stable flies were allowed to take an interrupted bloodmeal on chronically infected cows, followed by an immediate bloodmeal on three rabbits (Group B). A control group of rabbits and a group exposed to the bites of non-infected S. calcitrans were included in the study. Blood quantitative polymerase chain reaction (qPCR) analyses, and clinical, serological and haematological surveys were performed in the three groups over 152 days until the rabbits were killed. Quantitative PCR analyses and histological examinations were performed in 24 tissue samples per rabbit. Only one rabbit in Group B exhibited clinical signs of the acute phase of besnoitiosis (hyperthermia, weight loss, regenerative anaemia and transient positive qPCR in blood) and was seroconverted. Parasite DNA was detected in four tissue samples from this rabbit, but no cysts were observed on histological examination. These findings indicate that S. calcitrans may act as a mechanical vector of B. besnoiti more efficiently than was previously considered.

Resistance to gastrointestinal nematodes in dairy sheep: Genetic variability and relevance of artificial infection of nucleus rams to select for resistant ewes on farms.

Breeding sheep for enhanced resistance to gastrointestinal parasites is a promising strategy to limit the use of anthelmintics due to the now widespread resistance of parasites to these molecules. This paper reports the genetic parameters estimated for parasite resistance and resilience traits in the Blond-faced Manech dairy sheep breed and the putative impacts of the selection for resistance to gastrointestinal nematodes (GIN) on farms. Two datasets were used. First, the rams of the selection scheme were artificially infected twice with L3 Haemonchus contortus larvae. Faecal egg counts (FEC) and packed cell volume (PCV) loss were measured 30 days after each infection. Secondly, the FEC, PCV and body condition score (BCS) (1-6 measures per ewe) of naturally infected ewes on farms were measured in the spring, summer and autumn over a two-year period. Genetic parameters were estimated for each dataset independently but also globally based on the pedigree connections between the two datasets. For the experimentally infected sires, the FEC following the second infection was moderately heritable (heritability: 0.35) and strongly correlated with FEC after the first infection (genetic correlation: 0.92). For the naturally infected ewes, FEC was also heritable (0.18). Using the two datasets together, a genetic correlation of 0.56-0.71 was estimated between the FEC values of the experimentally infected rams and naturally infected ewes. Consequently, the genetic variability of parasite resistance is similar whatever the physiological status (males or milking/pregnant ewes) and the infection conditions (experimental infection with one parasite or natural infection with several parasites). In practice, when the sire population is divided into two groups based on their genetic value, the FEC of the ewes born to the 50% most resistant sires is half that of the ewes born to the 50% most susceptible sires. Our study shows the feasibility and efficiency of genetic selection for parasitism resistance based on the sires' FEC records to improve parasite resistance in naturally grazing ewes. For breed improvement, and to increase the selection pressure on parasite resistance, it seems more appropriate to measure FEC values on rams after experimental infection rather than on ewes in natural infection conditions because this limits the number and standardizes the conditions of FEC measurements.

Multiple-resistance to ivermectin and benzimidazole of a Haemonchus contortus population in a sheep flock from mainland France, first report.

Anthelmintic resistance was explored by fecal egg count reduction test in a sheep flock from the French Pyrenees at the request of the veterinary practitioner after a poor response to anthelmintics was noted. The FECRT confirmed the suspicion with a mean percentage of reduction in egg excretions of 45% (CI 95%: - 40 to 78.5) and 0% (CI95%: - 162 to 49) within the ivermectin and the benzimidazole groups respectively. Haemonchus contortus was shown to be the IVM and BZ resistant species after morphological and molecular characterizations whereas Teladorsagia circumcincta was probably resistant to BZ only. The H. contortus population was still susceptible to moxidectin, closantel and levamisole. As this sheep flock is a transhumant flock, the spread of this multiple-resistant Haemonchus contortus population to the other sheep flocks sharing the same pastures in Pyrenean Mountains is highly likely. From the knowledge of the authors, this is the first report of multi-resistance to ivermectin and benzimidazole of a Haemonchus contortus population in mainland France.

A real-time PCR approach to identify anthelmintic-resistant nematodes in sheep farms.

Resistance to fenbendazole, ivermectin, and moxidectin was explored by a fecal egg count reduction test in four meat sheep flocks in southwestern France where anthelmintic resistance was suspected. The FECR test results of the present study confirmed the presence of benzimidazole resistance in three out of the four farms and the presence of ivermectin resistance in one flock. In addition, a suspicion of moxidectin resistance was shown in this latter farm. Both conventional morphological and molecular identifications were performed on larval cultures before and after the treatment in the studied farms. A high positive correlation was found between the number of larvae counted under binocular microscope and the number of larvae estimated by the qPCR analysis (R 2 = 0.88) and a high Cohen's Kappa value (0.91) in the detection of strongylid larvae in larval cultures. According to qPCR results, Trichostrongylus species demonstrated high levels of BZ resistance and Teladorsagia circumcincta was involved in the IVM resistance in one farm. The molecular procedures used in this study have the potential to be beneficial for anthelmintic resistance surveillance in sheep industry.

Pyopneumothorax and peritonitis due to perforated duodenal ulcer and associated pleuroperitoneal communication.

Pleuroperitoneal communication is an anatomic entity that is typically asymptomatic but sometimes responsible for hydrothorax. This pleural manifestation can be explained by progressive transdiaphragmatic passage of intra-abdominal fluid because of abdominal hyperpressure. The object of this report is to present a hitherto unreported association of concomitant pleural effusion and acute infectious abdominal disease, due to perforated duodenal ulcer. This underscores that pleural effusion associated with acute abdominal pain may reveal the existence of a communication of this type, and requires surgical management.

Does bovine besnoitiosis affect the sexual function of chronically infected bulls?

Bovine besnoitiosis is a reemerging disease in Europe. The clinically Besnoitia besnoiti infection in bulls is characterized by fever, nasal discharge, and orchitis in the acute phase and by scleroderma in the chronic phase. However, in many bulls, B besnoiti infection remains at a subclinical stage. Bull infertility is an economically relevant consequence of besnoitiosis infection. It is not clear, however, if semen quality returns to normal levels when infected animals have clinically recovered. The aim of this study was to examine the relationship between chronic besnoitiosis and bull sexual function in a region of eastern France, where the disease is reemerging, by comparing semen quality and genital lesions in 11 uninfected, 17 subclinically infected, and 12 clinically infected bulls. The presence of anti-B besnoiti antibodies was detected by Western blot test. Semen was collected by electroejaculation. Bulls clinically infected with B besnoiti showed significantly more genital tract alterations than uninfected or subclinically infected bulls. No relationship was evidenced between besnoitiosis infectious status and semen quality, whereas a significant relationship was noted between genital lesions and semen score. This means that in the absence of moderate to severe genital lesions, chronic bovine besnoitiosis is unlikely to alter semen quality. However, as the presence of infected animals could lead to spread of the disease, culling or separation of clinically infected bulls from the remaining healthy animals is strongly recommended.

Colectomy in patients with previous colectomy or occlusive vascular diseases: Pitfalls and precautions.

Two principal branches from the aorta provide the colonic blood supply: the superior and inferior mesenteric arteries. There are numerous anatomical variations, which the surgeon must fully understand before embarking on any colonic surgery. A good knowledge of these variations is particularly important when the patient has already undergone colectomy or presents with occlusive vascular disease. The aim of this review is to summarize the standard anatomy and the main variations of the colonic blood supply as they apply to colorectal surgery in this setting.

Effectiveness of intraoperative cholangiography using indocyanine green (versus contrast fluid) for the correct assessment of extrahepatic bile ducts during day-case laparoscopic cholecystectomy.

INTRODUCTION: During cholecystectomy, intraoperative cholangiography using contrast fluid (IOC-CF) is still the "gold standard" for biliary tract identification but has many associated pitfalls. A new IOC technique using indocyanine green (IOC-IG) appears to be promising. Here, we studied the effectiveness of IOC-IG (vs IOC-CF) during day-case laparoscopic cholecystectomy. MATERIALS AND METHODS: Over a 6-month period, we included 23 patients (with no cirrhosis or risk factors for choledocholithiasis) scheduled for day-case laparoscopic cholecystectomy. The primary efficacy criterion was the "analyzability rate" (i.e., the ability to identify the cystic duct, the cystic duct-hepatic duct junction, and the common bile duct) for the IOC-CF and IOC-IG procedures after dissection. Indocyanine green was infused under general anesthesia. The same near-infrared laparoscopic imaging system was used for IOC-IG and conventional visual inspection. IOC-CF was always attempted after dissection. Each patient served as his/her own control. Cholecystectomies were video-recorded for subsequent off-line, blind analysis. RESULTS: The analyzability rate was 74 % for IOC-IG after dissection, 70 % for IOC-CF (p = 0.03), 26 % for conventional visual inspection, and 48 % for IOC-IG before dissection. When each IOC modality (conventional visual inspection, IOC-IG before and after dissection) was considered as a diagnostic test, the accuracy for simultaneous identification of the three anatomic elements was respectively 48, 52, and 74 %. No adverse events occurred during the IOC-IG procedure. CONCLUSION: IOC-IG was feasible and safe. Our results suggest that this technique is more effective than IOC-CF for biliary tract identification after dissection and may constitute a powerful diagnostic test for the detection of extrahepatic ducts.

No detection of Besnoitia besnoiti DNA in the semen of chronically infected bulls.

Bovine besnoitiosis is a chronic and debilitating disease observed in many European countries that may cause important economic losses in cattle. The recent widespread of the parasite in Europe had led the European Food Safety Authority to declare bovine besnoitiosis as a re-emerging disease in Europe. Many aspects of the epidemiology of bovine besnoitiosis such as the main routes of transmission are still unclear and need to be further studied. Among the different hypotheses, a sexual transmission has not yet been investigated. Therefore, the aim of this study was to evaluate the presence of Besnoitia besnoiti DNA in the semen of naturally infected bulls by using a highly sensitive method (real-time qPCR). Both pre-sperm and sperm fractions of 40 bulls, including seronegative (n = 11), seropositive subclinically (n = 17), and seropositive clinically (n = 12) infected animals, were collected by electroejaculation and analyzed by real-time qPCR. No B. besnoiti DNA was detected in 27 pre-sperm and 28 sperm fractions of the 40 examined bulls, suggesting that the transmission of B. besnoiti infection by the semen of chronically infected bulls is very unlikely.

Millimeter-scale alkalinity measurement in marine sediment using DET probes and colorimetric determination.

Constrained DET (Diffusive Equilibration in Thin films) probes equipped with 75 sampling layers of agarose gel (DGT Research(©)) were used to sample bottom and pore waters in marine sediment with a 2 mm vertical resolution. After retrieval, each piece of hydrogel, corresponding to 25 µL, was introduced into 1 mL of colorimetric reagent (CR) solution consisting of formic acid and bromophenol blue. After the elution/reaction time, absorbance of the latter mixture was read at 590 nm and compared to a calibration curve obtained with the same protocol applied to mini DET probes soaked in sodium hydrogen carbonate standard solutions. This method allows rapid alkalinity determinations for the small volumes of anoxic pore water entrapped into the gel. The method was assessed on organic-rich coastal marine sediments from Thau lagoon (France). Alkalinity values in the overlying waters were in agreement with data obtained by classical sampling techniques. Pore water data showed a progressive increase of alkalinity in the sediment from 2 to 10 mmol kg(-1), corresponding to anaerobic respiration in organic-rich sediments. Moreover, replicates of high-resolution DET profiles showed important lateral heterogeneity at a decimeter scale. This underlines the importance of high-resolution spatial methods for alkalinity profiling in coastal marine systems.

Development of a protocol testing the ability of Stomoxys calcitrans (Linnaeus, 1758) (Diptera: Muscidae) to transmit Besnoitia besnoiti (Henry, 1913) (Apicomplexa: Sarcocystidae).

Cattle besnoitiosis due to the cyst-forming coccidian parasite Besnoitia besnoiti has recently been reported in expansion in Europe since the end of the twentieth century. The B. besnoiti life cycle and many epidemiological traits are still poorly known. Hematophagous flies, including the worldwide-distributed Stomoxys calcitrans, could be mechanical vectors in the contamination of mouthparts after the puncture of cutaneous cysts or ingestion of infected blood. In this study, a protocol is presented to assess more deeply the role of S. calcitrans, reared in laboratory conditions, in parasite transmission. A preliminary trial showed that stable flies could transmit tachyzoites from bovine artificially parasite-enriched blood to B. besnoiti-free blood using glass feeders. Evidence of transmission was provided by the detection of parasite DNA with Ct values ranging between 32 and 37 in the blood recipient. In a second time, a B. besnoiti-infected heifer harboring many cysts in its dermis was used as a donor of B. besnoiti. An interruption of the blood meal taken by 300 stable flies from this heifer was performed. Immediately after the blood meal was interrupted, they were transferred to a glass feeder containing B. besnoiti-free blood from a non-infected heifer. Quantitative PCR and modified direct fluorescence antibody test (dFAT) were used to detect B. besnoiti DNA and entire parasites, respectively, in the blood recipient, the mouthparts, and the gut contents of S. calcitrans at two time intervals: 1 and 24 h after the interrupted blood meal. Parasite DNA was detected at both time intervals (1 and 24 h) in all samples (blood recipient, mouthparts, and gut contents of stable flies) while entire parasites by dFAT were only found in the abdominal compartment 1 h after the interrupted blood meal. Then, S. calcitrans were able to carry B. besnoiti from chronically infected cattle to an artificial recipient in the conditions of the protocol.

An inter-laboratory comparative study of serological tools employed in the diagnosis of Besnoitia besnoiti infection in bovines.

Bovine besnoitiosis is considered an emerging chronic and debilitating disease in Europe. Many infections remain subclinical, and the only sign of disease is the presence of parasitic cysts in the sclera and conjunctiva. Serological tests are useful for detecting asymptomatic cattle/sub-clinical infections for control purposes, as there are no effective drugs or vaccines. For this purpose, diagnostic tools need to be further standardized. Thus, the aim of this study was to compare the serological tests available in Europe in a multi-centred study. A coded panel of 241 well-characterized sera from infected and non-infected bovines was provided by all participants (SALUVET-Madrid, FLI-Wusterhausen, ENV-Toulouse, IPB-Berne). The tests evaluated were as follows: an in-house ELISA, three commercial ELISAs (INGEZIM BES 12.BES.K1 INGENASA, PrioCHECK Besnoitia Ab V2.0, ID Screen Besnoitia indirect IDVET), two IFATs and seven Western blot tests (tachyzoite and bradyzoite extracts under reducing and non-reducing conditions). Two different definitions of a gold standard were used: (i) the result of the majority of tests ('Majority of tests') and (ii) the majority of test results plus pre-test information based on clinical signs ('Majority of tests plus pre-test info'). Relative to the gold standard 'Majority of tests', almost 100% sensitivity (Se) and specificity (Sp) were obtained with SALUVET-Madrid and FLI-Wusterhausen tachyzoite- and bradyzoite-based Western blot tests under non-reducing conditions. On the ELISAs, PrioCHECK Besnoitia Ab V2.0 showed 100% Se and 98.8% Sp, whereas ID Screen Besnoitia indirect IDVET showed 97.2% Se and 100% Sp. The in-house ELISA and INGEZIM BES 12.BES.K1 INGENASA showed 97.3% and 97.2% Se; and 94.6% and 93.0% Sp, respectively. IFAT FLI-Wusterhausen performed better than IFAT SALUVET-Madrid, with 100% Se and 95.4% Sp. Relative to the gold standard 'Majority of test plus pre-test info', Sp significantly decreased; this result was expected because of the existence of seronegative animals with clinical signs. All ELISAs performed very well and could be used in epidemiological studies; however, Western blot tests performed better and could be employed as a posteriori tests for control purposes in the case of uncertain results from valuable samples.

A genome scan for QTL affecting resistance to Haemonchus contortus in sheep.

Gastrointestinal nematodes are one of the main health issues in sheep breeding. To identify loci affecting the resistance to Haemonchus contortus, a genome scan was carried out using 1,275 Romane × Martinik Black Belly backcross lambs. The entire population was challenged with Haemonchus contortus in 2 consecutive experimental infections, and fecal egg counts (FEC) and packed cell volumes were measured. A subgroup of 332 lambs with extreme FEC was necropsied to determine the total worm burden, length of female worms, sex ratio in the worm population, abomasal pH, and serum and mucosal G immunoglobulins (IgG) responses. Pepsinogen concentration was measured in another subset of 229 lambs. For QTL detection, 160 microsatellite markers were used as well as the Illumina OvineSNP50 BeadChip that provided 42,469 SNP markers after quality control. Linkage, association, and joint linkage and association analyses were performed with the QTLMAP software. Linkage disequilibrium (LD) was estimated within each pure breed, and association analyses were carried out either considering or not the breed origin of the haplotypes. Four QTL regions on sheep chromosomes (OAR)5, 12, 13, and 21 were identified as key players among many other QTL with small to moderate effects. A QTL on OAR21 affecting pepsinogen concentration exactly matched the pepsinogen (PGA5) locus. A 10-Mbp region affecting FEC after the 1st and 2nd infections was found on OAR12. The SNP markers outperformed microsatellites in the linkage analysis. Taking advantage of the LD helped to refine the locations of the QTL mapped on OAR5 and 13.

A longitudinal study of Besnoitia besnoiti infections and seasonal abundance of Stomoxys calcitrans in a dairy cattle farm of southwest France.

Bovine besnoitiosis, caused by the cyst-forming apicomplexan Besnoitia besnoiti, is commonly reported in some restricted regions of South-Western Europe, and in larger regions of Africa and Asia. This infection is thought to be transmitted by blood feeding insects and is responsible for major economic losses in cattle production. A recent emergence in Europe, notified in the Centre of France, Spain and Germany, has attracted more attention to this disease. Clinical signs could appear in some animals; however, many infected cattle remain asymptomatic or show scleral-conjunctival cysts (SCC) only. Recent development of serological methods allows carrying out seroepidemiological field studies. In this respect, a long-term investigation was performed in a dairy cattle farm localized in an enzootic area of besnoitiosis of South-western France between March 2008 and May 2009. The objective was to estimate the seasonal pattern of B. besnoiti infections based on the presence of SCC and serology (ELISA and Western blot). In parallel, an entomological survey was conducted to describe population dynamics of Stomoxys calcitrans and Tabanidae species. The seroprevalence determined by Western blot in a cohort of 57 animals continuously present during the whole survey increased from 30% in March 2008 to 89.5% in May 2009 and was always higher than the prevalence based on clinically assessed SCC. New positive B. besnoitia seroconversions occurred throughout the year with the highest number in spring. In addition, many seroconversions were reported in the two months before turn-out and could be associated with a high indoors activity of S. calcitrans during this period.

Eosinophils in Haemonchus contortus-infected resistant and susceptible breeds of sheep: abomasal tissue recruitment and in vitro functional state.

A comparative experimental study was performed between Barbados Black Belly (resistant) and INRA-401 (susceptible) breeds of sheep in which primary infection with Haemonchus contortus was terminated on day 16. Measurements of parasite burden, abomasal tissue eosinophilia, IL-4, IL-5 and IL-13 mRNA transcripts in abomasal mucosa, and in vitro larval killing abilities of blood eosinophils were performed. The results show that: (1) worm burden was significantly lower and blood eosinophilia higher in the Black Belly than in the INRA breed. (2) Abomasal cytokine expression was noticed but no difference existed between the two breeds. (3) Three out of four Black Belly sheep had higher tissue eosinophil numbers compared to the INRA sheep (more eosinophils observed in the pyloric than in the fundic region in both breeds). (4) No significant difference was observed in the in vitro larval immobilizing potential of eosinophils between the two breeds. Collectively, abomasal eosinophil number and larval killing abilities of blood eosinophils do not seem to explain the difference in worm burden between the two breeds.

Specific IgG antibody responses in Oestrus ovis L. (Diptera: Oestridae) infected sheep: associations with intensity of infection and larval development.

Larvae of Oestrus ovis (Diptera: Oestridae) are ubiquitous parasites of nasal and sinusal cavities of sheep and goats. According to the chronobiology of O. ovis infections in Sardinia and the seasonal pattern of the IgG response, the optimal period to investigate the relationships between O. ovis larval populations and intensity of local and systemic IgG antibody responses was mid-July in the summer season. Sarda x Lacaune ewes (n=186), divided into three ram-families were used in the study. Systemic and local IgG responses were measured by ELISA tests using second stage larval crude extracts (L2CE) and L2 (L2SGC) and L3 (L3SGC) salivary gland contents as coating antigens. The number of larval instars, larval length of L1, L2 and L3 larvae, and larval weight of L2 and L3 larvae were individually recorded after ewe necropsy. Negative correlations among larval establishment and/or larval development on the one hand and intensity of local or systemic IgG responses on the other hand were found in two out of three studied ram-families.

Immune response to Haemonchus contortus infection in susceptible (INRA 401) and resistant (Barbados Black Belly) breeds of lambs.

The immune responses to Haemonchus contortus were compared in studies in resistant Barbados Black Belly (BBB) and susceptible INRA 401 (INRA) breeds of lambs. The cytokine patterns indicated a Th2-biased response in both breeds. A more persistent and elevated Th2 cytokine mRNA transcription and blood eosinophilia were noted in the BBB lambs. However, at days 4 and 30 post-infection, abomasal recruitment of eosinophils and mast cells were similar between the two breeds. Following primary infections, the BBB demonstrated a substantially lower faecal egg count compared to the INRA lambs. Furthermore, worm counts at 4 and 30 days post-infection, and adult female worm size and in utero egg counts 30 days after the first infection were significantly lower in the BBB than in the INRA breed. In the INRA breed, re-infection caused a significant reduction in most parasitological parameters compared with those observed after the primary infection. A similar response was not observed in the BBB sheep. In conclusion, while the major driving force in the response to H. contortus infection is a Th2-biased immunity in which the BBB showed its maximal performance during the primary infection, the INRA breed performed better after re-infection compared to its response to first exposure.

Effects of immunization of Pelibuey lambs with Oestrus ovis digestive tract protein extracts on larval establishment and development.

Larval midgut proteins of hematophagous parasites contain strong antigens that can be used for host immunization. This concept has been applied for immunization of Pelibuey sheep against Oestrus ovis L. (Diptera: Oestridae). The aim of this study was to examine the effect of immunization on larval establishment (LE) and development. Immunized lambs (I, n = 6) received two injections of crude gut membrane protein extracts (GMPE) from third instar larvae with Freund's incomplete adjuvant (FIA) on days 0 (Day of first immunization) and 21 (0.4 and 0.45 mg GMPE/lamb, respectively). The control group (C, n = 5) received physiological saline with FIA. Lambs were challenged with first instars on Day 29 (20 larvae) and Day 43 (25 larvae). Blood samples were collected biweekly and IgG titers were analyzed by ELISA. All lambs were slaughtered on Day 90 and number of larvae recovered, larval stage and larval weight were recorded at necropsy. No significant effect of immunization on LE (C = 28.9%; I = 31.0% P > 0.05) was observed. Antibody titers were higher in the immunized group on Day 28 (P < 0.05), but subsequently similar in both groups. Larval physiological age and weight were also significantly (P < 0.05) affected by immunization. Immunization of Pelibuey lambs with GMPE did not affect LE but did delay O. ovis larval development.